MAPPING CHROMOSOMAL GENES OF SACCHAROMYCES CEREVISIAE USING AN IMPROVED GENETIC MAPPING METHOD REED B. WICKNER
Laboratory of Biochemical Pharmacologj., Naiiorial Institute of Arthritis, Metabolism, and Digestiue Diseases, National Institutes of Health, Bethesda, Maryland 20205 Manuscript received December 11,1978 Revised copy received March 15,1979 ABSTRACT
A triploid (3n) strain of Saccharomyces cereuisiae was constructed carrying a standard marker on each of chromosomes I through XVZZ in the --/+J+ configuration. This is called a “supertriploid.” Meiotic spores from this strain (n ~ ’ 2were ) mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n n/2) produced in this way resulted in elimination of an average of 4.2 chromosomes as the possible location of the unmapped marker. The distribution of extra chromosomes in the 2n n / 2 ) strains was nearly random. Meiotic segregrants of these crosses carrying the unmapped mutation in the configuration were then crossed with multiply marked haploid strains to further narrow the possible location of the unmapped mutation to a single chromosome. Scoring of markers by complemention tests was simplified by mating spore clones with mixtures of a and a strains, each pair carrying the same set of markers. Using this new, more rapid method (“supertriploid mapping”), eight genes required for the maintenance of the killer plasmid were located on the genetic map of S. cerevisiae.
+-
+-
+
-
-/+
ENETIC mapping in yeast is important for (1) permanently and definitively identifying mutant genes; (2) studying the function, mutation or rearrangement of genes; (3) discovering other roles of the same gene (pleiotropies); and (4)examining the organization of a eukaryotic genome. The chromosomal location of a newly identified gene is difficult to determine because the yeast map is over 3,200 centimorgans long (MORTIMER and HAWTHORNE 19731, there are substantial unmarked regions ( MORTIMER and HAWTHORNE 1973) and 17 chromosomes are already known (LINDEGREN and and MORTIMER 1960, 1968; LINDEGREN, et al. LINDEGREN 1951; HAWTHORNE 1962; OSHIMA,quoted in HWANG, LINDEGREN and LINDEGREN 1963; MORTIMER 1966, 1973). The best current general mapping method is and HAWTHORNE that of MORTIMER and HAWTHORNE (1973). The aneuploid meiotic segregants of a wild-type triploid are crossed with a haploid carrying the unmapped mutations(s) and standard markers on known chromosomes. Markers in the +/configuration in these crosses segregate 2+ : 2- (normal, or 2 : 2 segregation); those in the +/+/- configuration are a mixture of 4 + : 0, 3+ : 1-, and Genetics 92: 803-821 July, 1979.
804
R. B. WICKNER
24- : 2- tetrads (trisomic segregation). Thus, if a standard marker shows normal segregation in a cross in which an unmapped mutation shows trisomic segregation (or the reverse), these two genes must be on different chromosomes. The main disadvantage of this method is that multiply marked haploid strains must be constructed for each unmapped mutation before the collection of data can begin. In the present work, following a suggestion of SHERMANand MORTIMER (MORTIMER and TAVARES 1976). standard markers for each of chromosomes Z through XVZZ were incorporated into the triploid strain, avoiding strain construction for each mutation to be mapped. This marked triploid is called a “supertriploid.” I n combination with simplifications in complementation testing and utilization of the aneuploid strains generated by these crosses, this “supertriploid method” substantially reduces the labor involved in localizing a gene to a particular chromosome. Using this new method, eight genes required for the main1978) have been mapped. tenance of the killer plasmid virus (WICKNER MATERIALS A N D METHODS
Media: YPAD contained 1% yeast extract, 2% peptone 2% dextrose, 2% agar and 0.04% adenine sulfate. YPG medium contained 1% yeast extract, 2% peptone, 2% agar, 0.04% adenine and 3% (v/v) glycerol. SD medium contained 0.67% yeast nitrogen base (Difco), 2% agar and 2% dextrose. Complete minimal medium was SD supplemented with: adenine sulfate, 440 m g per 1; uracil, 24 mg per 1; tryptophan, 24 m g per 1; histidine, 24 mg per 1; arginine, 24 mg per 1; methionine, 24 mg per 1; 36 mg per 1; leucine, 36 mg per 1; isoleucine, 36 mg per 1; lysine, 36 mg per 1; aspartic acid, IOU m g per 1; valine, 150 mg per 1; threonine, 200 mg per 1; and phenylalanine, 60 m g per l: Omission media each lack a single component of complete minimal medium, except that mutants requiring methionine, aspartate or threonine were scored on medium lacking all three of the components (-MAT medium). The mak mutants were scored as previously described (WICKNERand LEIBOWITZ 1976). Presporulation medium contained 0.8% yeast extract, 0.8% peptone, 10% dextrose and 2% agar. Sporulation medium contained 1% potassium acetate, 0.1% yeast extract, 0.05% dextrose and 2% agar. S h a h : Many of the strains of S. cereuisiae used in this study are shown i n Table 1. The makl3, makl7, m k 1 8 , mak19, mak20, mak22, and mak24 mutations were previously described (WICKNZR 1978). Procedui e for performing “supertriploid9 crosses: (1) The supertriploid strain 1252 (or 1234 or 1249) is grown on presporulation medium and transferred to sporulation medium for three to five days at 26”. (2) The sporulated culture is suspended in water and treated for 30 min at room temperature with 0.2 volume of a 1 : l O dilution of gluculase (Endo Laboratories) 1959). (3) The digested spores are mixed as for tetrad dissection (JOHNSTON and MORTIMER with a haploid of mating type a (see legend of Table 3) carrying the mutation(s) to be mapped and an auxotrophic marker different from any of those in strain 1252. This mixture is placed on YPAD for eight to 12 h r at 26” to allow mating to occur. (4) The mating mixture is diluted in water and plated for zygote clones on SD plates. (5) About 40 zygote clones from (4) are grown on presporulation plates and transferred to sporulation plates a t 25” for five to seven days. About 30 of these clones will sporulate. (6) About 15 tetrads of each of these 30 zygote clones are dissected. (7) Sport germination is poor for most of these crosses. For those crosses having three or more complete tetrads (usually 30 to 50% of these crosses), all complete tetrads and enough tetrads with three viable spores are picked to fill one master plate. Spore clones are placed in the pattern of a 48-prong stamping device (WEISSand MILCAREK1974) so that 12 tetrads can be accommodated on each master plate. The prongs of the stamping device are congruent with the wells of a Falcon 96-well microtiter plate (catalog No. 3040). (8) The wells of the microtiter
805
GENETIC MAPPING IN YEAST
TABLE 1 Strains of Saccharomyces cerevisiae Des'gnation
1120 2050-1 7B
Reference
Genotype
a pet8 ilu3 leu1 his2 his6 met2 a r g [KIL-k]* a asp5 his2 his6 arg1 arg4 cdcl4 aro7 pet17
trpl adel gall [KIL-k] 1125 STI 1234 1252 1249 1926-8B 2066-23B 1965-3B 1910-1 9A 1230 1232 1117 1118 1267 1268
a ural ade2 lysl hisl his7 mal-I
[KIL-k] (1120 x 2050-17B) x 11251. ST1 hid$ STI hiss$ ST1 his6$ WICKNER 1978 a leu2 mak18-2 [KIL-o] WICKNER 1978 a ade3 mak17-I [KIL-o] WICKNFX1978 a trp3 makl3-I This work a lys2 ural mak26-I a pet8 met2 argl his7 [KIL-k] a pet8 met2 argl his7 [KIL-k] a hisl ural ade2 lysl mal-I [KIL-k] a hisl ural ade2 lysl rnal-I [KIL-k] a &I pet17 arg4 his2 his6 trpl adel asp5 cdc14 [KIL-k] a gall pet17 argl his2 his6 trpl adel asp5 cdc14 [KIL-k]
* The arginine requirement of strain 1120 is not due to either argl or arg4. iSTI was constructed by mating the diploid 1120 x 2050-17B with the haploid 1125. It was
assumed that a spontaneous mitotic recombinant of 1120 x 2050-17B which had become a/a would mate with 1125 (JAMESand LEE-WHITING 1955; ROMAN 1958), so that ST1 would have the genotype a/a/a for chromogome 111. The fact that almost all of the mating-type a segregants of 12.34, 1249, and 1262 were a and not a/a shows that this was the case (see Table 6 ) . $ ST1 is hisZ/hisZ/+ and his6/his6/+. Mitotic recombination was induced with UV light ( 5 sec, eE erg/sec/mmZ) and his- colonies were picked. Each was sporulated, and the spores mated with his2 and his6 testers. Strain 12.34 is hk6+ hi&; 1249 and 1252 are his& hi&+. Strains 1234, 1249, and 1252 each also segregate an unidentified arginine and methionine requirement. Since argl, arg4, met2 and asp5 are all scored using complementation tests, these undefined mutations do not interfere with the procedure. plates are filled with 0.12 ml each of water using an Accudrop No. 1 dispenser (Dynatech Laboratories, Alexandria, Virginia). Using the stamping device, spore clones are transferred to the microtiter dishes, cell suspensions made, and replicas made onto dropout plates [(i) -isoleucine and valine, (ii) -leucine, (iii) -histidine, (iv) methionine, aspartate, and threonine, (v) -arginine, (vi) -tryptophan, (vii) -phenylalanine, (viii) -adenine, (ix) uracil, and (x) -lysine], YPG, whatever plates are needed for scoring the unmapped mutation(s), and six other YPAD plates. One YPAD plate is incubated at 37". All dropout plates and YPG are incubated at 25". (9) Two of the replicas on YPAD are used to score mating type. Suspensions of each mating-type tester in water are made in petri dishes and small amounts applied to the replica on YPAD using the stamping device. Alternatively, one can lightly spray the replica with a suspension of the mating type-tester, using an aerosol device. The other three replicas on YPAD are used for complementation tests. One is mated (using the stamping device or aerosol) with a suspension in a petri dish of a mixture of strains 1117 and 1118, another with a mixture of strains 1230 and 1232, and the third with a mixture of strains 1267 and 1268. Each of these pairs carry the same markers, but opposite mating types. After two days of growth at 26", these plates are replica-plated to the appropriate media to test particular markers. For example, the replica mated with strains 1117 and 1118 is replica-plated
-
a06
R. B. WICKNER
to -uracil, -lysine, -adenine, -histidine, and YPAD at 37" This method of doing the complementation tests saves work and plates and makes reading the results quicker as well. The analysis of the results will be discussed below. Nomenclature: An otherwise haploid strain is disomic for a particular chromosome if it has 1 two ropies of that chromosome. When such a strain is crossed with a normal haploid, the 2n strairi formed is said to be trisomic for that chromosome. If the disomic strain was for a particular marker on the disomic chromosome, and the haploid was - for that marker, the trisomic 2n 1 strain will be for that marker. On meiosis, segregation of the markers on that chromosome that were in the configuration will result in tetrads of the following phenotypes: 4+:0--, 3+:1--, and 2+:2--. This is called trisomic segregation because it indicates the presence i n the 2n 1 strain of three copies of that chromosome. If the marker in the configuration is tightly centromere-linked, only 4+:& and 2+:&- tetrads will be observed, and these will occur in a ratio of about 2:1 (see Table 2). If the marker is far from its centromere, the most common tetrad type will be 3+:1(SHAFFERet al. 1971; CULBERTSON and HENRY1973; RILEY and MANNEY1978). The expected distribution of tetrad and spore types for centromere markers and centromere-distant markers is shown in Table 2. This distribution assumes completely trivalent pairing. The sporulation of a triploid strain produces spores with chromosome numbers varying between n and 2n. Assuming a binomial distribution of the extra chromosome of each homologous set of three into the spores is equivalent to assuming independent assortment of the chromosomes. This assumption implies that 95% of spores have between five and 12 extra chromosomes ( n = 17). This distribution is designated n+ 42.
+/+
+
--/+/+ --/+/+
+
+
--/+/+
-
TABLE 2 Theoretical distribution of tetrad and spore types from
-/+/-/-
Tetrad tvDeP
Frequency
Phenotype
Tightly centromerelinked gene*
113
4+:0
Z+:%
trpl/+ trpl/+
trpl trpl
Spore types
Frequency
4/15 4+:0
mal/+ mal/+
1/15
Frequency
+=
1/3
+/+ = 1/6 trpl = 1/6
++ +/+ +/+
Phenotype
Centromereunlinked genet
2/3
trpl/+
Z + : L
8/15 3+:1-
rnal rnal
rnal rnal/rnul mal/+
2/15
Spore types
3+:1-
++ +/+ +/+ +/+ +/+ + ++
= 1/3 Frequency
+ = 10/30
+/+ = 6/30 rnal = 5/30
m a l / + = 8/30 rnal/rnal = 1/30
* The segregation of trpl is independent of recombination events because of its tight centromere linkage. At the first meiotic division, one of the three homologs will migrate alone to one pole. If this one is that carrying the trpl mutation, a 2 trpl :2 tetrad will result. If it is one of the two chromosomes carrying t r p f , a 2 t r p l / f :2+ tetrad will result. Therefore, the ratio of the former to the latter tetrad type is 1:& as shown. f- We assume that ma2 is so far from its centromere, that many crossovers occur between it and its centromere and the distribution of the two ma2 mutant alleles among the six chromatids is random. There are 15 passible distinct arrangements. If they are equally likely, the indicated distribution results. For intermediate distances, the calculations of SHAFFERet al. (1971) and RILEYand MANNEY(1978) predict intermediate frequencies of -/- segregants. Our calculation assumes completely trivalent pairing. Any bivalent-univalent pairing will decrease the overall frequency of -/- segregants.
+/+
807
GENETIC MAPPING I N YEAST
FIGURE 1.-Genotype of "supertriploid" strain 1252.' I
A. B. C.
I1
- +_ _ +_ + - adel +gall ~ + his7 + X
A. B. C.
ilu3
+ +
-
+ ural
- -
V
IV
111
+ +
+ +
_a _ a -trpl cy
+ -++ asp5 XI1
--
+
his1
VI
r a t
VI11
+ ___ + sgz __ +- + leu1 __
xv
XVI
XIV
XI11
VI1
+ + +
his2 his2cdd.l. -
+ +
XVII
p~ e t d + + f f ~ __ f fpet17 argl Aro7 ade2 $-
+
IX
his6 ___ his6~ hisblysl
2
+ -+ -+-f-
* The markers in rows A, B and C are those of strains 1120,2050-17B and 1125, respectively,
the three haploid parents of the supertriploid, except for his6 in row C which was generated by mitotic recombination (see Table 1). -f The assignment of r n a l to Chromosome XZZZ was based on linkage to SUPS by random spore analysis [82 parental spores, 38 recombinant spores (MORTIMER and HAWTHORNE 1973)l. This has now been confirmed by tetrad analysis (PD = 28, NPD = 1, T= 43). RESULTS
As described in Table 1, three triploid strains (1234, 1249 and 1252) were constructed carrying at least one recessive marker in the -/+/+ configuration on each of chromosomes Z through XVZZ. The genotype of strain 1252 is shown in Figure 1. All meiotic segregants (spores) o€ strain 1252 are his6-, and each should contain a random assortment of about n/2 extra chromosomes. For a marker in the -/+/-I- configuration in the triploid, the four most common types and -. This is shown in Table 2 for trpl (chromoof spores are -/+, some ZV) and rnal (chromosome XZZZ). Segregants with the -/- configuration are produced occasionally for markers not tightly centromere-linked and will be considered further below. When segregants from the triploid are crossed with a haploid carrying an unmapped mutation (for example, makl8 in Figure 2), the results can be used
+/+, +,
FIGURE 2.-Use Supei triploid
of aneuploidy to narrow the location of an unmapped mutation. Supertriploid crosses Spores
+
__ trpl
++ +
-
Haploid
trpi segregation
2 f 2-
m a k f d segregation Tnsomic
+/+
x x
makld
+ 4+:0
Noinformation
Noinformation
makld
+ 4+:0
Noinformation
Noinformation
trpl
x
makl8 + 2+:2-
Noinformation*
makldnoton chromosome ZVt
x
makld
makldnoton No information* chromosome IV$ * If both trpl and makld show the same type of segregation, no conclusion can be reached since several chromosomes are trisomic and several are i n the normal diploid state in each sporulating 2n n/2 strain. -f Because t r p l shows 2+:2- segregation in this cross, there is no extra chromosome ZV segregating i n this cross. But makld is showing trisomic segregation (a mixture of 4+:0,3+:1and 2+:2- tetrads), indicating that there is segregating in this cross a n extra chromosome on which makld is located. Therefore, mak18 is not on chromosome ZV. $ Because trpl shows trisomic segregation in this cross, there must be a n extra chromosome ZV. But makld is segregating 2+:2-, so that there is not an extra chromosome on which makld is located. Therefore, makld is not on chromosome ZV. trpl/+
+-
+ trisomic
~
808
R. B. WICKNER
m
5 0
2 0
c
809
GENETIC MAPPING IN YEAST
to eliminate a particular chromosome as the location of the new gene if the standard marker (trpl in Figure 2) segregates 2: 2 and the unmapped mutation shows trisomic segregation, or vice uersa (Figure 2 ) . In any one cross, markers on several chromosomes are segregating, so that each cross eliminates a random assortment of chromosomes as possible locations of the mutation. Crosses of this type are called supertriploid crosses. Mapping of makl8: Data for a series of crosses to map makl8 are shown in Table 3. Each chromosome is eliminated as the location of makld at least once, except for chromosomes V ,VI, VZZZ,and XZZ. To further narrow the location of makl8, one of the crosses in Table 3 was selected in which makl8 showed trisomic segregation and the standard markers for chromosomes V , VZ, VZZZ,and XZZ were absent (cross 2162). A 4+:0 tetrad for makl8 (tetrad 2162-3) is presumed to have two spore clones that are makl8/+ and two spore clones that are Each of the four spore clones of tetrad 2162-3 was crossed with strains carrying markers on chromosomes V , VZ, VZZZ, and XZZ. The results for one of the spore clones, 2162-3C, which proved to be makl8/+, are shown in Table 4.In cross 2226, cdcl4 ( V I )and asp5 (XZZ) segregated 24- : 2-, while makl8 showed abnormal (trisomic) segregation, as did arg4 (VZZZ) . This eliminates chromosomes VZ and XZZ as possible locations for makl8, but, of course, does not prove it is on chromosome VZZZ.Cross 2227 simi-
+.
TABLE 4
“Secondary” crosses to map makl8* Cross 2226:
1267
(a gall pet17 arg4 his2 his6 trpl adel asp5 cdcl4 [KIL-k])
x 2162-3C (a cdel ade2 rna2 met2 makI8-2/+) Marker
Chromosome
mak18-2 cdcl4 asp5 arg4 trpl pet27 Cross 2227:
VI XII VIII IV
xv
4+:0
3 0 0 6
a
0
3+:1--
5 0 0 3 0 0
Segregation 2+:2-
1 9 9 0 9 7
1+:3-
0 0 0 0 0 2
0:4-
-
0 0 0 0 0 0
1118 (a hisl u r d ade2 lys2 mal-I [KIL-%I) X 2162-3C ( a adel ade2 rna2 met2 mak18-2/+)
Marker
maki8-2 hisl lys2 ural
Chromosome
4f:O
4 V IX XI
0 0 0
3+:1-
8 0 0 0
Segregation 2+:2-
0 12 12 10
1+:3-
0 0 0 2
0 : L
0 0 0 0
* Strain 2162-3C is a mak18/+ segregant of cross 2162 (Table 3), a cross between a segregant of the supertriploid and a haploid carrying makl8. The segregation of mak28 in the crosses 2226 and 2227 shows that the chro’mosomecarrying makl8 was trisomic in the zygotes.
810
R. B. WICKNER
larly eliminates chromosome V, and these two crosses confirm the data in Table 3, eliminating chromosomes ZV, ZX, XI, and XV. These crosses, called “secondary crosses,” provide an efficient way to narrow the possible location of the unmapped mutation. Once the “primary” supertriploid crosses have eliminated some chromosomes and provided segregants, the secondary crosses become preferable: one can decide which chromosomes to test in secondary crosses, and the spore germination frequency is USUally good. The crosses in Tables 3 and 4 leave only chromosome VZZZ as a possible location for makl8. A makl8 strain was crossed with a strain carrying several markers on chromosome VZZZ, and tight linkage was found with pet3 [PD (parental ditype) = 56, NPD (nonparental ditype) = 0, T (tetratype) = 21. Thus, makl8 is located on the right arm of chromosome VZZZ (Figure 3). Mapping of makl7: Makl7-1 was similarly localized to chromosomes Z or X by supertriploid crosses (Table 5 ) . From cross 2138, a tetrad that showed 4f:O segregation for makl7 was further analyzed by crossing each spore clone with various mak+ strains marked on chromosomes I or X. The analysis was similar to that shown in Table 4 for makl8. Two spore clones (2138-3A and 2138-3B) were disomic for chromosome X and the chromosome on which makl7 resides, but were monosomic for chromosome 1. The other two spore clones (2138-3C and 2138-3D) were disomic for chromosome Z, but not for either chromosome X o r the chromosome carrying makl7. Thus, makl7 is not on chromosome Z, and only chromosome X remains. No significant linkage was found with ura2 (PD = 7, NPD = 9, T = 34), cdcll (PD = 7, NPD = 4, T = 20), or iZv3 (PD = 2, NPD = 1, T .=6 ) .
+/-
Vlll
-
P 1 ) Sequence not ertabhrhed with genes outride parentherer . . . Mitotic linkage . . Trisomic linkage
..
FIGURE 3.--Map location of mak13, makl7, makld, makl9, mak20, mak22, mak24 and mak26.
GENETIC MAPPING IN YEAST
811
812
GENETIC M A P P I N G I N YEAST
813
ura2 makl7 cdcll makl7 [KIL-k] or+ Diploids were constructed of genotype [KIL-k], and mitotic recombination was induced with UV light (JAMESand LEE-WHITING 1955; HURST and FOGEL 1964). Of 13 clones that had become homozygous for cdcll (right arm of chromosome X), none had become malt-. Therefore, makl7 is not on the right arm of chromosome X. However, of 18 clones homozygous for ura2, all had become m a k . Therefore, makl7 is on the left arm of chromosome X, probably centromere-distal to ura2 (Figure 3). Makl3: Using a combination of "supertriploid" crosses and secondary crosses as above, makl3 was localized to chromosome ZX (Table 6). No meiotic linkage was found between makl3 and either his5 (PD = 12, NPD = 13, T = 421, his6 (PD = 8, NPD = 12, T = 47), or lysl (PD = 18, NPD = 14, T = 86). Mitotic recombination was then used to confirm the location of makl3 on chromosome lysl makl3-I ZX. From two K+ diploids were derived 46 l y s mitotic recombinants. Of these, 38 were also K-. Less than I % of total clones of these diploids (not selecting lys- mitotic recombinants) were K-. None of 42 his- mitotic recomhis5 makl3 binants derived from two diploids were K-. Thus, makl3 is located on the right arm of chromosome ZX (Figure 3). Mak26: The supertriploid method narrowed the location of mak26 to either chromosome XZV or XVZZ (Table 7). Meiotic crosses showed that mak26 is 22 CM from petx (PD = 29, NPD = 0, T = 23), a gene that we had previously shown to be on chromosome XZV (WICKNER and LEIBOWITZ 1976). No linkage was found with pet8, rna2, or 2ys9. Therefore, mak26 is on chromosome XZV near kex2, ski3, and ski4 (Figure 3 ) . Mak24: Similarly, mak24 was narrowed to chromosomes VIZ, VZZZ, or XZ by the supertriploid method (data not shown). Meiotic crosses showed that ma1124 was 12 cM from cyh2 on chromosome VIZ (PD = 51, h T D = 0, T = 16). Linkage data and analysis of individual tetrads showed that the gene order is centromere-trp5-mak24-cyh2-metl3-aro2 (data not shown; Figure 3). Makl9: This gene was localized on chromosome VZZZ by the supertriploid method (data not shown) and found by meiotic linkage to be 43 cM to the right of pet3 (Figure 3). Make0 and mak22: The mak20 and m k 2 2 genes were localized by the supertriploid method to chromosomes VZZZ and XZZ, respectively, and in each case mitotic recombination data showed that they were not on the right arms of their respective chromosomes (data not shown). Since mak7 on VZZZ and makl2 on XZZ have been located on the left arms of these chromosomes, mitotic recombination could not easily be carried out with these markers. No meiotic linkage was found between mak20 and mak7. We tentatively place mak20 on the left arm of VZZZ and mak22 on the left arm of XZZ (Figure 3). -/- segregants from the -/+/-I- triploid: When three homologs are present in meiosis (at least for chromosomes ZZZ and XZ), trivalent pairing is frequent (SHAFFER et ~2.1971;CULBERTSON and HENRY 1973; RILEYand MANNEY 1978) ;
+ +
+ +
+ +
+
814.
R. B. WICKNEE
815
G E N E T I C M A P P I N G IN YEAST
I
TRISOMIC ZYGOTE
FIRST MEIOTIC DIVISION
SECOND MEIOTIC DIVISION
w n -/+/+
FIGURE 4.--Generation of -/-- segregants from a zygote. In a normal meiosis (2 homologs), chromosomes that have recombined with each other separate at the first meiotic division. This is not the case with a trisomic zygote (SHAFFERet d. 1971; CULBERTSONand HENKY 1973; RILEYand MANNEY 1978).
that is, recombination is not restricted to exchanges between a pair of chromosomes (trivalent recombination), and homologs that have recombined with each other may migrate to the same pole in meiotic division I (trivalent segregation). One consequence of this is that -/- meiotic segregants can arise from the -/+/+ starting configuration (or segregants from the -/-/+ configuration). Such an event is diagrammed in Figure 4.Clearly, segregants from the supertriploid that are -/- for a standard marker will confuse the mapping configuration) will segregate mostly procedure; the standard marker (in -J-/+ 2-t:2-, but if the unmapped mutation is on that chromosome, it will be in the +/+/- configuration and show trisomic segregation. It may be falsely concluded that the unknown marker is not on this chromosome. The frequency of -/- disomic segregants from a -/+/+ strain depends on the distance of the marker from the centromere. A tightly centromere-linked marker (such as adel, trpl, leul, arg4, pet8 or ilv3 in the supertriploid) will rarely, if ever, produce such segregants (SHAFFERet al. 1971). For loosely centromere-linked markers (such as hid, crlcll, lysl, petl7, asp5, aro7 and mating type in the supertriploid) ,theoretical calculations (SHAFFERet al. 1971; RILEY and MANNEY 1978; Table 2) indicate an increasing frequency of such segregants, reaching a maximum of 3.3% of total segregants for markers recombining freely with their centromeres (such as his7, ural, m a l , ade2, met2 or argl in the supertriploid). These frequencies have been measured for mating
+/+
816
R. B. WICKNER
817
GENETIC M A P P I N G IN YEAST
type-2.4% (SHAFFERet al. 1971) or 1.3% (RILEYand NIANNEY 1978)-and and HENRY1973)-and are approxifor the fasl locus-3.1% (CULBERTSON mately as expected since mating type is about 25 cM from its centromere, while fasl is more than 85 cM from its centromere. For a standard marker distant from its centromere in the -/+/+ configuraand one-thirtieth -1- (see Table tion, one-sixth of the segregants should be 2). Thus, one-sixth of 2+:2- segregations in supertriploid crosses for standard markers distant from their centromeres (his7, u r d , m a l , a d d , met2, argl) are due to -Jdisomes. Fewer 2472- segregations for loosely centromerelinked markers (hid, cdcl4, lysl, petl7, asp5, aro7) and almost none for tightly centromere-linked markers ( d e l , trpl, leul, arg4, pet8, ilv3) will be due to -/- disomes. Mating type will cause no confusion because disomy is easily recognized, and it occurs with about the expected frequency (four trisomic and 45 2: 2) in crosses of a haploids with spores of a/a/a supertriploids (Table 8). Thus, if a new marker is excluded from a chromosome by a single cross in which it shows trisomic segregation, while a standard marker distant from its centromere shows 2:2 segregation, there is a one-sixth chance that this is an error. Such cases should be confirmed by a second cross. This problem does not affect cases where the standard marker shows trisomic segregation and the unmapped marker segregates 2+:2-, nor is it a consideration in the secondary crosses (the type shown in Table 4). Analysis of disome frequency and co-disomy: An analysis of 49 crosses between the spores of strains 1234, 1249, or 1252 and various haploid strains is -$
1.o
ull 11 0.5
0.2
TRlSO MI C/2:2 FIGURE 5.-Ratio
‘of trisomic to normal segregation in supertriploid crosses.
0.1
818
R. B. WICKNER
o o * a + ~ M i n * o -
0 0 0 0 0 0 0 0 0 0
-1
I
o°C.loo
~ 0 0 0 0 0 0
G E N E T I C M A P P I N G IN YEAST
819
shown in Tables 8 and 9 and Figure 5. In these crosses (excluding 111, where some selection for haploidy was present), 183 cases of disomy and 163 cases of normal segregation were observed (Table 8). Chromosomes V and X I never showed 2+: 2- segregation when their standard markers, hid and u r d , respectively, were present. Possibly, the supertriploids were actually tetrasomic for chromosomes V and/or X I . All other chromosomes showed both disomic and 2:2 segregation in varying proportions (Table 8). Chromosome ZZZ could always be scored, and chromosome X V , because it was marked with two markers (pet17 and ade2), was scored in about two-thirds of the cases. Table 9 shows some evidence that each o€ the standard markers is on a different chromosome. Two pairs of markers were not shown to be on different chromosomes and for several other pairs, data was insufficient. These data might also be taken as evidence that this method will be useful for genes on all chromosomes. Analysis of 49 supertriploid crosses was carried out to determine if disomy (or monosomy) for certain chromosomes was always accompanied by disomy (or monosomy) for other chromosomes. Of 544 [=en( n-I ) ] possible pairwise combinations for chromosomes Z to XVZZ, 426 were observed (data not shown). NO striking correlations were observed in these limited data. PARRY and COX (1970) presented evidence for significant coincidence of disomy among chromosames VZZZ, ZX and ZV in spores from a triploid grown to colonies before they were mated. In supertriploid crosses, the spores are mated as they germinate. Presumably 2n n/2 strains are healthier than n nJ2 strains. In the 49 crosses analyzed in Tables 8 and 9, five showed chromosomes VZZZ and ZX disomic, three showed chromosome VZZZ disomic and chromosome ZX 2:2, one showed chromosome VZZZ 2:2 and chromosome ZX disomic, and none showed both 2:2. All pairwise combinations with chromosomes ZV and VZZZ or ZX were also realized. Therefore, no evidence is observed for coincident disomy of chromosomes ZV, VZZZ and ZX. Perhaps most important, from the point of view of mapping, is the distribution of the ratio of chromosomes showing trisomic to those showing 2: 2 segregation in each supertriploid cross. If each 2n n/2 zygote in a supertriploid cross tended to either lose chromosomes to become 2n or gain chromosomes by nondisjunction to become nearly 3n, the method would not work. In fact, though the theoretical ratio (assuming no chromosome loss) is slightly under two (Table 2), the observed ratio varies around one, the ideal number for mapping purposes (Figure 5). In these 49 crosses, an average of 4.2 chromosomes per cross were eliminated as the location of the unmapped marker involved.
+
+-
I -
+-
DISCUSSION
An improved mapping method described here has been useful in mapping several mak genes. Several improvements could be made in this method. If the haploid strain contained several unmapped mutations, then with no additional work, it would be possible to get several times as much information from each cross. If the super-
820
R. B. WICKNER
triploid were reconstructed with more than one marker on each chromosome (as in the case of chromosome XV here), then the proportion of crosses in which the segregation of each chromosome could be assessed would be increased. Also, additional standard markers could be included in the haploid strain. The use of centromere markers on all chromosomes would avoid the problem of -/- segregants from the supertriploid. Suppressors of the unmapped mutation or the standard markers (frequently found in the case of mak mutants) can cause confusion in this mapping procedure, converting 2+: 2- segregation into apparently trisomic segregation. Such a suppressor of mak27-I resulted in our erroneously thinking that this gene was not located on any of the known chromosomes. I n fact we have since located it on chromosome XZZZ (unpublished results). Our current overall strategy is to do a series of supertriploid crosses to narrow the possible locations of the new mutant gene to about five chromosomes. Then, -/+ disomic strains for the new mutant gene generated in these crosses are crossed with other multiply marked haploids to further narrow the possibilities. These -/+ disomic strains should contain about extra chromosomes. Once the chromosome is determined, mitotic recombination and meiotic linkage are utilized, as in the examples discussed here. We hope that this and other improved mapping methods will encourage the routine mapping of new mutant genes. 11/44
I am grateful to R. K. MORTIMER, F. SHERMAN, S. HENRY, J. GAME,A. TOH-E, and P. GUERRY for valuable discussions during this work, and to R. CONTOPOULOS, J. BASSEL,S. FOGEL, and X. K. MORTIMER of the Yeast Genetic Stock Center for supplying us with numerous strains used i n these studies. The method of combining a and (Y testers in complementation tests was devised by MURRAY S. COHN. Note added in proof: F. HILGER and R. K. MORTIMER (Cold Spring Harbor Meeting on the Molecular Biology of Yeast, 1979, Abstract) have shown that arg2 is located on the left arm of chromosome XV. The work presented here reflects this fact. Using mapping methods involving aneuploidy, it was previously suggested that a r g l was on and TABOR 1978). The results of HILGER and a new eighteenth chromosome (COHN, TABOR MORTIMER, and our own experience with mak27-l (see DISCUSSION) indicate that while aneuploidy is useful in locating genes on the known chromosomes, great caution must be exercised in using aneuploidy to define a new chromosome. LITERATURE CITED
COHN,M. S., C. W. TABOR and H. TABOR, 1978 Isolation and characterization of Saccharomyces cerevisiae mutants deficient in S-adenosylmethionine decarboxylase, spermidine, and spermine. J. Bacteriol. 134: 208-213. CULBEXTSON, M. R. and S. A. HENRY,1973 Genetic analysis of hybrid strains trisomic for the chromosome containing a fatty acid synthetase gene complex (fasl) i n yeast. Genetics 75: 441-458. HAWTHORNE, D. C. and R. K. MORTIMER,1960 Chromosome mapping in Saccharomyces: centromere-linked genes. Genetics 4.5: 1085-1 1 IO. ---, 1968 Genetic mapping of nonsense suppressors in yeast. Genetics 60 : 735-742. H U ~ S D. T , P. and S. FOGEL,1964 Mitotic recombination and heteroallelic repair in Succharoniyces cercuisiae. Genetics 50 : 435-458.
GENETIC MAPPING I N YEAST
821
HWANG, Y. L., G. LINDEGREN and C. C. LINDEGREN, 1963 Mapping the eleventh centromere in Saccharomyces. Can J. Genet. Cytol. 5 : 290-298. JAMES, A. P. and B. LEE-WHITING, 1955 Radiation-induced genetic segregations in vegetative cells of diploid yeast. Genetics 40: 826-831. JOHNSTON, J. R. and R. K. MORTIMER, 1959 Use of snail digestive juice in isolation of yeast spore tetrads. J. Bacteriol. 78: 292. c. c. and G. LINDEGREN, 1951 Linkage relationships in Saccharomyces of genes LIIVDEGREN, controlling +he fermentation of carbohydrates and the synthesis of vitamins, amino acids and nucleic acid components. Indian Phytopathol. 4: 11-20. E. SHULTand Y. L. HWANG,1962 Centromeres, sites of LINDEGREN, C. C., G. LINDEGREN, affinity and gene loci on the chromosomes of Saccharomyces. Nature 194: 260-265. MORTIMER, R. K. and D. C. HAWTHORNE, 1966 Genetic mapping in Saccharomyces. Genetics 53: 165-173. -, 1973 Genetic mapping in Saccharomyces. IV. Mapping of temperature-sensitive genes and use of disomic strains in localizing genes. Genetics 74: 33-54. MORTIMER, R. K. and F. C. TAVARES, 1976 Genetic mapping in yeast. pp. 572-574. In: MicroAmerican Soc. Microbiol., Washington, D.C. biology, 1976. Edited by D. SCHLESSINGER.
PARRY, E. M. and B. S. Cox, 1970 The tolerance of aneuploidy in yeast. Genet. Res. 16: 333-340. 1978 Tetraploid strains of Saccharomyces cerevisiae that are RILEY,M. I. and T. R. MANNEY, trisomic for chromosome ZZZ. tienetics 89 :667-684. ROMAN, H., 1958 A comparison of spontaneous and ultraviolet-induced allelic recombination with references to the outside markers. Cold Spring Harbor Symp. Quant. Biol. 23: 155-160. and G. R. FINK,1971. A stable aneuploid of SacSHAFPER,B., I. BREARLEY,R. LITTLEWOOD charomyces cerevis 'ae.Genetics 67 :483-495. 1974 Mass screening for mutants with altered DNases by microWEISS,B. and C. MILCAREK, assay techniques. Methods Enzymol. 29 : 180-193. WICKNER,R. B., 1978 Twenty-six chromosomal genes needed to maintain the killer doublestranded RNA plasmid of Saccharomyces cerevisiae. Genetics 88: 419-425. WICIINER,R. B. and M. J. LEIBOWITZ,1976 Two chromosomal genes required for killing expression in killer strains of Saccharomyces cerevisiae. Genetics 82 : 429442. Corresponding Editor: F. SHERMAN
Laboratory of Biochemical Pharmacologj., Naiiorial Institute of Arthritis, Metabolism, and Digestiue Diseases, National Institutes of Health, Bethesda, Maryland 20205 Manuscript received December 11,1978 Revised copy received March 15,1979 ABSTRACT
A triploid (3n) strain of Saccharomyces cereuisiae was constructed carrying a standard marker on each of chromosomes I through XVZZ in the --/+J+ configuration. This is called a “supertriploid.” Meiotic spores from this strain (n ~ ’ 2were ) mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n n/2) produced in this way resulted in elimination of an average of 4.2 chromosomes as the possible location of the unmapped marker. The distribution of extra chromosomes in the 2n n / 2 ) strains was nearly random. Meiotic segregrants of these crosses carrying the unmapped mutation in the configuration were then crossed with multiply marked haploid strains to further narrow the possible location of the unmapped mutation to a single chromosome. Scoring of markers by complemention tests was simplified by mating spore clones with mixtures of a and a strains, each pair carrying the same set of markers. Using this new, more rapid method (“supertriploid mapping”), eight genes required for the maintenance of the killer plasmid were located on the genetic map of S. cerevisiae.
+-
+-
+
-
-/+
ENETIC mapping in yeast is important for (1) permanently and definitively identifying mutant genes; (2) studying the function, mutation or rearrangement of genes; (3) discovering other roles of the same gene (pleiotropies); and (4)examining the organization of a eukaryotic genome. The chromosomal location of a newly identified gene is difficult to determine because the yeast map is over 3,200 centimorgans long (MORTIMER and HAWTHORNE 19731, there are substantial unmarked regions ( MORTIMER and HAWTHORNE 1973) and 17 chromosomes are already known (LINDEGREN and and MORTIMER 1960, 1968; LINDEGREN, et al. LINDEGREN 1951; HAWTHORNE 1962; OSHIMA,quoted in HWANG, LINDEGREN and LINDEGREN 1963; MORTIMER 1966, 1973). The best current general mapping method is and HAWTHORNE that of MORTIMER and HAWTHORNE (1973). The aneuploid meiotic segregants of a wild-type triploid are crossed with a haploid carrying the unmapped mutations(s) and standard markers on known chromosomes. Markers in the +/configuration in these crosses segregate 2+ : 2- (normal, or 2 : 2 segregation); those in the +/+/- configuration are a mixture of 4 + : 0, 3+ : 1-, and Genetics 92: 803-821 July, 1979.
804
R. B. WICKNER
24- : 2- tetrads (trisomic segregation). Thus, if a standard marker shows normal segregation in a cross in which an unmapped mutation shows trisomic segregation (or the reverse), these two genes must be on different chromosomes. The main disadvantage of this method is that multiply marked haploid strains must be constructed for each unmapped mutation before the collection of data can begin. In the present work, following a suggestion of SHERMANand MORTIMER (MORTIMER and TAVARES 1976). standard markers for each of chromosomes Z through XVZZ were incorporated into the triploid strain, avoiding strain construction for each mutation to be mapped. This marked triploid is called a “supertriploid.” I n combination with simplifications in complementation testing and utilization of the aneuploid strains generated by these crosses, this “supertriploid method” substantially reduces the labor involved in localizing a gene to a particular chromosome. Using this new method, eight genes required for the main1978) have been mapped. tenance of the killer plasmid virus (WICKNER MATERIALS A N D METHODS
Media: YPAD contained 1% yeast extract, 2% peptone 2% dextrose, 2% agar and 0.04% adenine sulfate. YPG medium contained 1% yeast extract, 2% peptone, 2% agar, 0.04% adenine and 3% (v/v) glycerol. SD medium contained 0.67% yeast nitrogen base (Difco), 2% agar and 2% dextrose. Complete minimal medium was SD supplemented with: adenine sulfate, 440 m g per 1; uracil, 24 mg per 1; tryptophan, 24 m g per 1; histidine, 24 mg per 1; arginine, 24 mg per 1; methionine, 24 mg per 1; 36 mg per 1; leucine, 36 mg per 1; isoleucine, 36 mg per 1; lysine, 36 mg per 1; aspartic acid, IOU m g per 1; valine, 150 mg per 1; threonine, 200 mg per 1; and phenylalanine, 60 m g per l: Omission media each lack a single component of complete minimal medium, except that mutants requiring methionine, aspartate or threonine were scored on medium lacking all three of the components (-MAT medium). The mak mutants were scored as previously described (WICKNERand LEIBOWITZ 1976). Presporulation medium contained 0.8% yeast extract, 0.8% peptone, 10% dextrose and 2% agar. Sporulation medium contained 1% potassium acetate, 0.1% yeast extract, 0.05% dextrose and 2% agar. S h a h : Many of the strains of S. cereuisiae used in this study are shown i n Table 1. The makl3, makl7, m k 1 8 , mak19, mak20, mak22, and mak24 mutations were previously described (WICKNZR 1978). Procedui e for performing “supertriploid9 crosses: (1) The supertriploid strain 1252 (or 1234 or 1249) is grown on presporulation medium and transferred to sporulation medium for three to five days at 26”. (2) The sporulated culture is suspended in water and treated for 30 min at room temperature with 0.2 volume of a 1 : l O dilution of gluculase (Endo Laboratories) 1959). (3) The digested spores are mixed as for tetrad dissection (JOHNSTON and MORTIMER with a haploid of mating type a (see legend of Table 3) carrying the mutation(s) to be mapped and an auxotrophic marker different from any of those in strain 1252. This mixture is placed on YPAD for eight to 12 h r at 26” to allow mating to occur. (4) The mating mixture is diluted in water and plated for zygote clones on SD plates. (5) About 40 zygote clones from (4) are grown on presporulation plates and transferred to sporulation plates a t 25” for five to seven days. About 30 of these clones will sporulate. (6) About 15 tetrads of each of these 30 zygote clones are dissected. (7) Sport germination is poor for most of these crosses. For those crosses having three or more complete tetrads (usually 30 to 50% of these crosses), all complete tetrads and enough tetrads with three viable spores are picked to fill one master plate. Spore clones are placed in the pattern of a 48-prong stamping device (WEISSand MILCAREK1974) so that 12 tetrads can be accommodated on each master plate. The prongs of the stamping device are congruent with the wells of a Falcon 96-well microtiter plate (catalog No. 3040). (8) The wells of the microtiter
805
GENETIC MAPPING IN YEAST
TABLE 1 Strains of Saccharomyces cerevisiae Des'gnation
1120 2050-1 7B
Reference
Genotype
a pet8 ilu3 leu1 his2 his6 met2 a r g [KIL-k]* a asp5 his2 his6 arg1 arg4 cdcl4 aro7 pet17
trpl adel gall [KIL-k] 1125 STI 1234 1252 1249 1926-8B 2066-23B 1965-3B 1910-1 9A 1230 1232 1117 1118 1267 1268
a ural ade2 lysl hisl his7 mal-I
[KIL-k] (1120 x 2050-17B) x 11251. ST1 hid$ STI hiss$ ST1 his6$ WICKNER 1978 a leu2 mak18-2 [KIL-o] WICKNER 1978 a ade3 mak17-I [KIL-o] WICKNFX1978 a trp3 makl3-I This work a lys2 ural mak26-I a pet8 met2 argl his7 [KIL-k] a pet8 met2 argl his7 [KIL-k] a hisl ural ade2 lysl mal-I [KIL-k] a hisl ural ade2 lysl rnal-I [KIL-k] a &I pet17 arg4 his2 his6 trpl adel asp5 cdc14 [KIL-k] a gall pet17 argl his2 his6 trpl adel asp5 cdc14 [KIL-k]
* The arginine requirement of strain 1120 is not due to either argl or arg4. iSTI was constructed by mating the diploid 1120 x 2050-17B with the haploid 1125. It was
assumed that a spontaneous mitotic recombinant of 1120 x 2050-17B which had become a/a would mate with 1125 (JAMESand LEE-WHITING 1955; ROMAN 1958), so that ST1 would have the genotype a/a/a for chromogome 111. The fact that almost all of the mating-type a segregants of 12.34, 1249, and 1262 were a and not a/a shows that this was the case (see Table 6 ) . $ ST1 is hisZ/hisZ/+ and his6/his6/+. Mitotic recombination was induced with UV light ( 5 sec, eE erg/sec/mmZ) and his- colonies were picked. Each was sporulated, and the spores mated with his2 and his6 testers. Strain 12.34 is hk6+ hi&; 1249 and 1252 are his& hi&+. Strains 1234, 1249, and 1252 each also segregate an unidentified arginine and methionine requirement. Since argl, arg4, met2 and asp5 are all scored using complementation tests, these undefined mutations do not interfere with the procedure. plates are filled with 0.12 ml each of water using an Accudrop No. 1 dispenser (Dynatech Laboratories, Alexandria, Virginia). Using the stamping device, spore clones are transferred to the microtiter dishes, cell suspensions made, and replicas made onto dropout plates [(i) -isoleucine and valine, (ii) -leucine, (iii) -histidine, (iv) methionine, aspartate, and threonine, (v) -arginine, (vi) -tryptophan, (vii) -phenylalanine, (viii) -adenine, (ix) uracil, and (x) -lysine], YPG, whatever plates are needed for scoring the unmapped mutation(s), and six other YPAD plates. One YPAD plate is incubated at 37". All dropout plates and YPG are incubated at 25". (9) Two of the replicas on YPAD are used to score mating type. Suspensions of each mating-type tester in water are made in petri dishes and small amounts applied to the replica on YPAD using the stamping device. Alternatively, one can lightly spray the replica with a suspension of the mating type-tester, using an aerosol device. The other three replicas on YPAD are used for complementation tests. One is mated (using the stamping device or aerosol) with a suspension in a petri dish of a mixture of strains 1117 and 1118, another with a mixture of strains 1230 and 1232, and the third with a mixture of strains 1267 and 1268. Each of these pairs carry the same markers, but opposite mating types. After two days of growth at 26", these plates are replica-plated to the appropriate media to test particular markers. For example, the replica mated with strains 1117 and 1118 is replica-plated
-
a06
R. B. WICKNER
to -uracil, -lysine, -adenine, -histidine, and YPAD at 37" This method of doing the complementation tests saves work and plates and makes reading the results quicker as well. The analysis of the results will be discussed below. Nomenclature: An otherwise haploid strain is disomic for a particular chromosome if it has 1 two ropies of that chromosome. When such a strain is crossed with a normal haploid, the 2n strairi formed is said to be trisomic for that chromosome. If the disomic strain was for a particular marker on the disomic chromosome, and the haploid was - for that marker, the trisomic 2n 1 strain will be for that marker. On meiosis, segregation of the markers on that chromosome that were in the configuration will result in tetrads of the following phenotypes: 4+:0--, 3+:1--, and 2+:2--. This is called trisomic segregation because it indicates the presence i n the 2n 1 strain of three copies of that chromosome. If the marker in the configuration is tightly centromere-linked, only 4+:& and 2+:&- tetrads will be observed, and these will occur in a ratio of about 2:1 (see Table 2). If the marker is far from its centromere, the most common tetrad type will be 3+:1(SHAFFERet al. 1971; CULBERTSON and HENRY1973; RILEY and MANNEY1978). The expected distribution of tetrad and spore types for centromere markers and centromere-distant markers is shown in Table 2. This distribution assumes completely trivalent pairing. The sporulation of a triploid strain produces spores with chromosome numbers varying between n and 2n. Assuming a binomial distribution of the extra chromosome of each homologous set of three into the spores is equivalent to assuming independent assortment of the chromosomes. This assumption implies that 95% of spores have between five and 12 extra chromosomes ( n = 17). This distribution is designated n+ 42.
+/+
+
--/+/+ --/+/+
+
+
--/+/+
-
TABLE 2 Theoretical distribution of tetrad and spore types from
-/+/-/-
Tetrad tvDeP
Frequency
Phenotype
Tightly centromerelinked gene*
113
4+:0
Z+:%
trpl/+ trpl/+
trpl trpl
Spore types
Frequency
4/15 4+:0
mal/+ mal/+
1/15
Frequency
+=
1/3
+/+ = 1/6 trpl = 1/6
++ +/+ +/+
Phenotype
Centromereunlinked genet
2/3
trpl/+
Z + : L
8/15 3+:1-
rnal rnal
rnal rnal/rnul mal/+
2/15
Spore types
3+:1-
++ +/+ +/+ +/+ +/+ + ++
= 1/3 Frequency
+ = 10/30
+/+ = 6/30 rnal = 5/30
m a l / + = 8/30 rnal/rnal = 1/30
* The segregation of trpl is independent of recombination events because of its tight centromere linkage. At the first meiotic division, one of the three homologs will migrate alone to one pole. If this one is that carrying the trpl mutation, a 2 trpl :2 tetrad will result. If it is one of the two chromosomes carrying t r p f , a 2 t r p l / f :2+ tetrad will result. Therefore, the ratio of the former to the latter tetrad type is 1:& as shown. f- We assume that ma2 is so far from its centromere, that many crossovers occur between it and its centromere and the distribution of the two ma2 mutant alleles among the six chromatids is random. There are 15 passible distinct arrangements. If they are equally likely, the indicated distribution results. For intermediate distances, the calculations of SHAFFERet al. (1971) and RILEYand MANNEY(1978) predict intermediate frequencies of -/- segregants. Our calculation assumes completely trivalent pairing. Any bivalent-univalent pairing will decrease the overall frequency of -/- segregants.
+/+
807
GENETIC MAPPING I N YEAST
FIGURE 1.-Genotype of "supertriploid" strain 1252.' I
A. B. C.
I1
- +_ _ +_ + - adel +gall ~ + his7 + X
A. B. C.
ilu3
+ +
-
+ ural
- -
V
IV
111
+ +
+ +
_a _ a -trpl cy
+ -++ asp5 XI1
--
+
his1
VI
r a t
VI11
+ ___ + sgz __ +- + leu1 __
xv
XVI
XIV
XI11
VI1
+ + +
his2 his2cdd.l. -
+ +
XVII
p~ e t d + + f f ~ __ f fpet17 argl Aro7 ade2 $-
+
IX
his6 ___ his6~ hisblysl
2
+ -+ -+-f-
* The markers in rows A, B and C are those of strains 1120,2050-17B and 1125, respectively,
the three haploid parents of the supertriploid, except for his6 in row C which was generated by mitotic recombination (see Table 1). -f The assignment of r n a l to Chromosome XZZZ was based on linkage to SUPS by random spore analysis [82 parental spores, 38 recombinant spores (MORTIMER and HAWTHORNE 1973)l. This has now been confirmed by tetrad analysis (PD = 28, NPD = 1, T= 43). RESULTS
As described in Table 1, three triploid strains (1234, 1249 and 1252) were constructed carrying at least one recessive marker in the -/+/+ configuration on each of chromosomes Z through XVZZ. The genotype of strain 1252 is shown in Figure 1. All meiotic segregants (spores) o€ strain 1252 are his6-, and each should contain a random assortment of about n/2 extra chromosomes. For a marker in the -/+/-I- configuration in the triploid, the four most common types and -. This is shown in Table 2 for trpl (chromoof spores are -/+, some ZV) and rnal (chromosome XZZZ). Segregants with the -/- configuration are produced occasionally for markers not tightly centromere-linked and will be considered further below. When segregants from the triploid are crossed with a haploid carrying an unmapped mutation (for example, makl8 in Figure 2), the results can be used
+/+, +,
FIGURE 2.-Use Supei triploid
of aneuploidy to narrow the location of an unmapped mutation. Supertriploid crosses Spores
+
__ trpl
++ +
-
Haploid
trpi segregation
2 f 2-
m a k f d segregation Tnsomic
+/+
x x
makld
+ 4+:0
Noinformation
Noinformation
makld
+ 4+:0
Noinformation
Noinformation
trpl
x
makl8 + 2+:2-
Noinformation*
makldnoton chromosome ZVt
x
makld
makldnoton No information* chromosome IV$ * If both trpl and makld show the same type of segregation, no conclusion can be reached since several chromosomes are trisomic and several are i n the normal diploid state in each sporulating 2n n/2 strain. -f Because t r p l shows 2+:2- segregation in this cross, there is no extra chromosome ZV segregating i n this cross. But makld is showing trisomic segregation (a mixture of 4+:0,3+:1and 2+:2- tetrads), indicating that there is segregating in this cross a n extra chromosome on which makld is located. Therefore, mak18 is not on chromosome ZV. $ Because trpl shows trisomic segregation in this cross, there must be a n extra chromosome ZV. But makld is segregating 2+:2-, so that there is not an extra chromosome on which makld is located. Therefore, makld is not on chromosome ZV. trpl/+
+-
+ trisomic
~
808
R. B. WICKNER
m
5 0
2 0
c
809
GENETIC MAPPING IN YEAST
to eliminate a particular chromosome as the location of the new gene if the standard marker (trpl in Figure 2) segregates 2: 2 and the unmapped mutation shows trisomic segregation, or vice uersa (Figure 2 ) . In any one cross, markers on several chromosomes are segregating, so that each cross eliminates a random assortment of chromosomes as possible locations of the mutation. Crosses of this type are called supertriploid crosses. Mapping of makl8: Data for a series of crosses to map makl8 are shown in Table 3. Each chromosome is eliminated as the location of makld at least once, except for chromosomes V ,VI, VZZZ,and XZZ. To further narrow the location of makl8, one of the crosses in Table 3 was selected in which makl8 showed trisomic segregation and the standard markers for chromosomes V , VZ, VZZZ,and XZZ were absent (cross 2162). A 4+:0 tetrad for makl8 (tetrad 2162-3) is presumed to have two spore clones that are makl8/+ and two spore clones that are Each of the four spore clones of tetrad 2162-3 was crossed with strains carrying markers on chromosomes V , VZ, VZZZ, and XZZ. The results for one of the spore clones, 2162-3C, which proved to be makl8/+, are shown in Table 4.In cross 2226, cdcl4 ( V I )and asp5 (XZZ) segregated 24- : 2-, while makl8 showed abnormal (trisomic) segregation, as did arg4 (VZZZ) . This eliminates chromosomes VZ and XZZ as possible locations for makl8, but, of course, does not prove it is on chromosome VZZZ.Cross 2227 simi-
+.
TABLE 4
“Secondary” crosses to map makl8* Cross 2226:
1267
(a gall pet17 arg4 his2 his6 trpl adel asp5 cdcl4 [KIL-k])
x 2162-3C (a cdel ade2 rna2 met2 makI8-2/+) Marker
Chromosome
mak18-2 cdcl4 asp5 arg4 trpl pet27 Cross 2227:
VI XII VIII IV
xv
4+:0
3 0 0 6
a
0
3+:1--
5 0 0 3 0 0
Segregation 2+:2-
1 9 9 0 9 7
1+:3-
0 0 0 0 0 2
0:4-
-
0 0 0 0 0 0
1118 (a hisl u r d ade2 lys2 mal-I [KIL-%I) X 2162-3C ( a adel ade2 rna2 met2 mak18-2/+)
Marker
maki8-2 hisl lys2 ural
Chromosome
4f:O
4 V IX XI
0 0 0
3+:1-
8 0 0 0
Segregation 2+:2-
0 12 12 10
1+:3-
0 0 0 2
0 : L
0 0 0 0
* Strain 2162-3C is a mak18/+ segregant of cross 2162 (Table 3), a cross between a segregant of the supertriploid and a haploid carrying makl8. The segregation of mak28 in the crosses 2226 and 2227 shows that the chro’mosomecarrying makl8 was trisomic in the zygotes.
810
R. B. WICKNER
larly eliminates chromosome V, and these two crosses confirm the data in Table 3, eliminating chromosomes ZV, ZX, XI, and XV. These crosses, called “secondary crosses,” provide an efficient way to narrow the possible location of the unmapped mutation. Once the “primary” supertriploid crosses have eliminated some chromosomes and provided segregants, the secondary crosses become preferable: one can decide which chromosomes to test in secondary crosses, and the spore germination frequency is USUally good. The crosses in Tables 3 and 4 leave only chromosome VZZZ as a possible location for makl8. A makl8 strain was crossed with a strain carrying several markers on chromosome VZZZ, and tight linkage was found with pet3 [PD (parental ditype) = 56, NPD (nonparental ditype) = 0, T (tetratype) = 21. Thus, makl8 is located on the right arm of chromosome VZZZ (Figure 3). Mapping of makl7: Makl7-1 was similarly localized to chromosomes Z or X by supertriploid crosses (Table 5 ) . From cross 2138, a tetrad that showed 4f:O segregation for makl7 was further analyzed by crossing each spore clone with various mak+ strains marked on chromosomes I or X. The analysis was similar to that shown in Table 4 for makl8. Two spore clones (2138-3A and 2138-3B) were disomic for chromosome X and the chromosome on which makl7 resides, but were monosomic for chromosome 1. The other two spore clones (2138-3C and 2138-3D) were disomic for chromosome Z, but not for either chromosome X o r the chromosome carrying makl7. Thus, makl7 is not on chromosome Z, and only chromosome X remains. No significant linkage was found with ura2 (PD = 7, NPD = 9, T = 34), cdcll (PD = 7, NPD = 4, T = 20), or iZv3 (PD = 2, NPD = 1, T .=6 ) .
+/-
Vlll
-
P 1 ) Sequence not ertabhrhed with genes outride parentherer . . . Mitotic linkage . . Trisomic linkage
..
FIGURE 3.--Map location of mak13, makl7, makld, makl9, mak20, mak22, mak24 and mak26.
GENETIC MAPPING IN YEAST
811
812
GENETIC M A P P I N G I N YEAST
813
ura2 makl7 cdcll makl7 [KIL-k] or+ Diploids were constructed of genotype [KIL-k], and mitotic recombination was induced with UV light (JAMESand LEE-WHITING 1955; HURST and FOGEL 1964). Of 13 clones that had become homozygous for cdcll (right arm of chromosome X), none had become malt-. Therefore, makl7 is not on the right arm of chromosome X. However, of 18 clones homozygous for ura2, all had become m a k . Therefore, makl7 is on the left arm of chromosome X, probably centromere-distal to ura2 (Figure 3). Makl3: Using a combination of "supertriploid" crosses and secondary crosses as above, makl3 was localized to chromosome ZX (Table 6). No meiotic linkage was found between makl3 and either his5 (PD = 12, NPD = 13, T = 421, his6 (PD = 8, NPD = 12, T = 47), or lysl (PD = 18, NPD = 14, T = 86). Mitotic recombination was then used to confirm the location of makl3 on chromosome lysl makl3-I ZX. From two K+ diploids were derived 46 l y s mitotic recombinants. Of these, 38 were also K-. Less than I % of total clones of these diploids (not selecting lys- mitotic recombinants) were K-. None of 42 his- mitotic recomhis5 makl3 binants derived from two diploids were K-. Thus, makl3 is located on the right arm of chromosome ZX (Figure 3). Mak26: The supertriploid method narrowed the location of mak26 to either chromosome XZV or XVZZ (Table 7). Meiotic crosses showed that mak26 is 22 CM from petx (PD = 29, NPD = 0, T = 23), a gene that we had previously shown to be on chromosome XZV (WICKNER and LEIBOWITZ 1976). No linkage was found with pet8, rna2, or 2ys9. Therefore, mak26 is on chromosome XZV near kex2, ski3, and ski4 (Figure 3 ) . Mak24: Similarly, mak24 was narrowed to chromosomes VIZ, VZZZ, or XZ by the supertriploid method (data not shown). Meiotic crosses showed that ma1124 was 12 cM from cyh2 on chromosome VIZ (PD = 51, h T D = 0, T = 16). Linkage data and analysis of individual tetrads showed that the gene order is centromere-trp5-mak24-cyh2-metl3-aro2 (data not shown; Figure 3). Makl9: This gene was localized on chromosome VZZZ by the supertriploid method (data not shown) and found by meiotic linkage to be 43 cM to the right of pet3 (Figure 3). Make0 and mak22: The mak20 and m k 2 2 genes were localized by the supertriploid method to chromosomes VZZZ and XZZ, respectively, and in each case mitotic recombination data showed that they were not on the right arms of their respective chromosomes (data not shown). Since mak7 on VZZZ and makl2 on XZZ have been located on the left arms of these chromosomes, mitotic recombination could not easily be carried out with these markers. No meiotic linkage was found between mak20 and mak7. We tentatively place mak20 on the left arm of VZZZ and mak22 on the left arm of XZZ (Figure 3). -/- segregants from the -/+/-I- triploid: When three homologs are present in meiosis (at least for chromosomes ZZZ and XZ), trivalent pairing is frequent (SHAFFER et ~2.1971;CULBERTSON and HENRY 1973; RILEYand MANNEY 1978) ;
+ +
+ +
+ +
+
814.
R. B. WICKNEE
815
G E N E T I C M A P P I N G IN YEAST
I
TRISOMIC ZYGOTE
FIRST MEIOTIC DIVISION
SECOND MEIOTIC DIVISION
w n -/+/+
FIGURE 4.--Generation of -/-- segregants from a zygote. In a normal meiosis (2 homologs), chromosomes that have recombined with each other separate at the first meiotic division. This is not the case with a trisomic zygote (SHAFFERet d. 1971; CULBERTSONand HENKY 1973; RILEYand MANNEY 1978).
that is, recombination is not restricted to exchanges between a pair of chromosomes (trivalent recombination), and homologs that have recombined with each other may migrate to the same pole in meiotic division I (trivalent segregation). One consequence of this is that -/- meiotic segregants can arise from the -/+/+ starting configuration (or segregants from the -/-/+ configuration). Such an event is diagrammed in Figure 4.Clearly, segregants from the supertriploid that are -/- for a standard marker will confuse the mapping configuration) will segregate mostly procedure; the standard marker (in -J-/+ 2-t:2-, but if the unmapped mutation is on that chromosome, it will be in the +/+/- configuration and show trisomic segregation. It may be falsely concluded that the unknown marker is not on this chromosome. The frequency of -/- disomic segregants from a -/+/+ strain depends on the distance of the marker from the centromere. A tightly centromere-linked marker (such as adel, trpl, leul, arg4, pet8 or ilv3 in the supertriploid) will rarely, if ever, produce such segregants (SHAFFERet al. 1971). For loosely centromere-linked markers (such as hid, crlcll, lysl, petl7, asp5, aro7 and mating type in the supertriploid) ,theoretical calculations (SHAFFERet al. 1971; RILEY and MANNEY 1978; Table 2) indicate an increasing frequency of such segregants, reaching a maximum of 3.3% of total segregants for markers recombining freely with their centromeres (such as his7, ural, m a l , ade2, met2 or argl in the supertriploid). These frequencies have been measured for mating
+/+
816
R. B. WICKNER
817
GENETIC M A P P I N G IN YEAST
type-2.4% (SHAFFERet al. 1971) or 1.3% (RILEYand NIANNEY 1978)-and and HENRY1973)-and are approxifor the fasl locus-3.1% (CULBERTSON mately as expected since mating type is about 25 cM from its centromere, while fasl is more than 85 cM from its centromere. For a standard marker distant from its centromere in the -/+/+ configuraand one-thirtieth -1- (see Table tion, one-sixth of the segregants should be 2). Thus, one-sixth of 2+:2- segregations in supertriploid crosses for standard markers distant from their centromeres (his7, u r d , m a l , a d d , met2, argl) are due to -Jdisomes. Fewer 2472- segregations for loosely centromerelinked markers (hid, cdcl4, lysl, petl7, asp5, aro7) and almost none for tightly centromere-linked markers ( d e l , trpl, leul, arg4, pet8, ilv3) will be due to -/- disomes. Mating type will cause no confusion because disomy is easily recognized, and it occurs with about the expected frequency (four trisomic and 45 2: 2) in crosses of a haploids with spores of a/a/a supertriploids (Table 8). Thus, if a new marker is excluded from a chromosome by a single cross in which it shows trisomic segregation, while a standard marker distant from its centromere shows 2:2 segregation, there is a one-sixth chance that this is an error. Such cases should be confirmed by a second cross. This problem does not affect cases where the standard marker shows trisomic segregation and the unmapped marker segregates 2+:2-, nor is it a consideration in the secondary crosses (the type shown in Table 4). Analysis of disome frequency and co-disomy: An analysis of 49 crosses between the spores of strains 1234, 1249, or 1252 and various haploid strains is -$
1.o
ull 11 0.5
0.2
TRlSO MI C/2:2 FIGURE 5.-Ratio
‘of trisomic to normal segregation in supertriploid crosses.
0.1
818
R. B. WICKNER
o o * a + ~ M i n * o -
0 0 0 0 0 0 0 0 0 0
-1
I
o°C.loo
~ 0 0 0 0 0 0
G E N E T I C M A P P I N G IN YEAST
819
shown in Tables 8 and 9 and Figure 5. In these crosses (excluding 111, where some selection for haploidy was present), 183 cases of disomy and 163 cases of normal segregation were observed (Table 8). Chromosomes V and X I never showed 2+: 2- segregation when their standard markers, hid and u r d , respectively, were present. Possibly, the supertriploids were actually tetrasomic for chromosomes V and/or X I . All other chromosomes showed both disomic and 2:2 segregation in varying proportions (Table 8). Chromosome ZZZ could always be scored, and chromosome X V , because it was marked with two markers (pet17 and ade2), was scored in about two-thirds of the cases. Table 9 shows some evidence that each o€ the standard markers is on a different chromosome. Two pairs of markers were not shown to be on different chromosomes and for several other pairs, data was insufficient. These data might also be taken as evidence that this method will be useful for genes on all chromosomes. Analysis of 49 supertriploid crosses was carried out to determine if disomy (or monosomy) for certain chromosomes was always accompanied by disomy (or monosomy) for other chromosomes. Of 544 [=en( n-I ) ] possible pairwise combinations for chromosomes Z to XVZZ, 426 were observed (data not shown). NO striking correlations were observed in these limited data. PARRY and COX (1970) presented evidence for significant coincidence of disomy among chromosames VZZZ, ZX and ZV in spores from a triploid grown to colonies before they were mated. In supertriploid crosses, the spores are mated as they germinate. Presumably 2n n/2 strains are healthier than n nJ2 strains. In the 49 crosses analyzed in Tables 8 and 9, five showed chromosomes VZZZ and ZX disomic, three showed chromosome VZZZ disomic and chromosome ZX 2:2, one showed chromosome VZZZ 2:2 and chromosome ZX disomic, and none showed both 2:2. All pairwise combinations with chromosomes ZV and VZZZ or ZX were also realized. Therefore, no evidence is observed for coincident disomy of chromosomes ZV, VZZZ and ZX. Perhaps most important, from the point of view of mapping, is the distribution of the ratio of chromosomes showing trisomic to those showing 2: 2 segregation in each supertriploid cross. If each 2n n/2 zygote in a supertriploid cross tended to either lose chromosomes to become 2n or gain chromosomes by nondisjunction to become nearly 3n, the method would not work. In fact, though the theoretical ratio (assuming no chromosome loss) is slightly under two (Table 2), the observed ratio varies around one, the ideal number for mapping purposes (Figure 5). In these 49 crosses, an average of 4.2 chromosomes per cross were eliminated as the location of the unmapped marker involved.
+
+-
I -
+-
DISCUSSION
An improved mapping method described here has been useful in mapping several mak genes. Several improvements could be made in this method. If the haploid strain contained several unmapped mutations, then with no additional work, it would be possible to get several times as much information from each cross. If the super-
820
R. B. WICKNER
triploid were reconstructed with more than one marker on each chromosome (as in the case of chromosome XV here), then the proportion of crosses in which the segregation of each chromosome could be assessed would be increased. Also, additional standard markers could be included in the haploid strain. The use of centromere markers on all chromosomes would avoid the problem of -/- segregants from the supertriploid. Suppressors of the unmapped mutation or the standard markers (frequently found in the case of mak mutants) can cause confusion in this mapping procedure, converting 2+: 2- segregation into apparently trisomic segregation. Such a suppressor of mak27-I resulted in our erroneously thinking that this gene was not located on any of the known chromosomes. I n fact we have since located it on chromosome XZZZ (unpublished results). Our current overall strategy is to do a series of supertriploid crosses to narrow the possible locations of the new mutant gene to about five chromosomes. Then, -/+ disomic strains for the new mutant gene generated in these crosses are crossed with other multiply marked haploids to further narrow the possibilities. These -/+ disomic strains should contain about extra chromosomes. Once the chromosome is determined, mitotic recombination and meiotic linkage are utilized, as in the examples discussed here. We hope that this and other improved mapping methods will encourage the routine mapping of new mutant genes. 11/44
I am grateful to R. K. MORTIMER, F. SHERMAN, S. HENRY, J. GAME,A. TOH-E, and P. GUERRY for valuable discussions during this work, and to R. CONTOPOULOS, J. BASSEL,S. FOGEL, and X. K. MORTIMER of the Yeast Genetic Stock Center for supplying us with numerous strains used i n these studies. The method of combining a and (Y testers in complementation tests was devised by MURRAY S. COHN. Note added in proof: F. HILGER and R. K. MORTIMER (Cold Spring Harbor Meeting on the Molecular Biology of Yeast, 1979, Abstract) have shown that arg2 is located on the left arm of chromosome XV. The work presented here reflects this fact. Using mapping methods involving aneuploidy, it was previously suggested that a r g l was on and TABOR 1978). The results of HILGER and a new eighteenth chromosome (COHN, TABOR MORTIMER, and our own experience with mak27-l (see DISCUSSION) indicate that while aneuploidy is useful in locating genes on the known chromosomes, great caution must be exercised in using aneuploidy to define a new chromosome. LITERATURE CITED
COHN,M. S., C. W. TABOR and H. TABOR, 1978 Isolation and characterization of Saccharomyces cerevisiae mutants deficient in S-adenosylmethionine decarboxylase, spermidine, and spermine. J. Bacteriol. 134: 208-213. CULBEXTSON, M. R. and S. A. HENRY,1973 Genetic analysis of hybrid strains trisomic for the chromosome containing a fatty acid synthetase gene complex (fasl) i n yeast. Genetics 75: 441-458. HAWTHORNE, D. C. and R. K. MORTIMER,1960 Chromosome mapping in Saccharomyces: centromere-linked genes. Genetics 4.5: 1085-1 1 IO. ---, 1968 Genetic mapping of nonsense suppressors in yeast. Genetics 60 : 735-742. H U ~ S D. T , P. and S. FOGEL,1964 Mitotic recombination and heteroallelic repair in Succharoniyces cercuisiae. Genetics 50 : 435-458.
GENETIC MAPPING I N YEAST
821
HWANG, Y. L., G. LINDEGREN and C. C. LINDEGREN, 1963 Mapping the eleventh centromere in Saccharomyces. Can J. Genet. Cytol. 5 : 290-298. JAMES, A. P. and B. LEE-WHITING, 1955 Radiation-induced genetic segregations in vegetative cells of diploid yeast. Genetics 40: 826-831. JOHNSTON, J. R. and R. K. MORTIMER, 1959 Use of snail digestive juice in isolation of yeast spore tetrads. J. Bacteriol. 78: 292. c. c. and G. LINDEGREN, 1951 Linkage relationships in Saccharomyces of genes LIIVDEGREN, controlling +he fermentation of carbohydrates and the synthesis of vitamins, amino acids and nucleic acid components. Indian Phytopathol. 4: 11-20. E. SHULTand Y. L. HWANG,1962 Centromeres, sites of LINDEGREN, C. C., G. LINDEGREN, affinity and gene loci on the chromosomes of Saccharomyces. Nature 194: 260-265. MORTIMER, R. K. and D. C. HAWTHORNE, 1966 Genetic mapping in Saccharomyces. Genetics 53: 165-173. -, 1973 Genetic mapping in Saccharomyces. IV. Mapping of temperature-sensitive genes and use of disomic strains in localizing genes. Genetics 74: 33-54. MORTIMER, R. K. and F. C. TAVARES, 1976 Genetic mapping in yeast. pp. 572-574. In: MicroAmerican Soc. Microbiol., Washington, D.C. biology, 1976. Edited by D. SCHLESSINGER.
PARRY, E. M. and B. S. Cox, 1970 The tolerance of aneuploidy in yeast. Genet. Res. 16: 333-340. 1978 Tetraploid strains of Saccharomyces cerevisiae that are RILEY,M. I. and T. R. MANNEY, trisomic for chromosome ZZZ. tienetics 89 :667-684. ROMAN, H., 1958 A comparison of spontaneous and ultraviolet-induced allelic recombination with references to the outside markers. Cold Spring Harbor Symp. Quant. Biol. 23: 155-160. and G. R. FINK,1971. A stable aneuploid of SacSHAFPER,B., I. BREARLEY,R. LITTLEWOOD charomyces cerevis 'ae.Genetics 67 :483-495. 1974 Mass screening for mutants with altered DNases by microWEISS,B. and C. MILCAREK, assay techniques. Methods Enzymol. 29 : 180-193. WICKNER,R. B., 1978 Twenty-six chromosomal genes needed to maintain the killer doublestranded RNA plasmid of Saccharomyces cerevisiae. Genetics 88: 419-425. WICIINER,R. B. and M. J. LEIBOWITZ,1976 Two chromosomal genes required for killing expression in killer strains of Saccharomyces cerevisiae. Genetics 82 : 429442. Corresponding Editor: F. SHERMAN
