Molec. gen. Genet. 156, 79-85 (1977) © by Springer-Verlag 1977
Mapping of the Two Mitochondrial Antimycin A Resistance Loci in Saccharomyces cerevisiae Georg Michaelis and Elke Pratje Fakult~it flir Biologic der Universit/it Bielefeld, Postfach 8640, D-4800 Bielefeld 1, Federal Republic of Germany
Summary. Retention or loss of the two new mitochondrial antimycin A resistance loci A~ and A~ has been analyzed in a large number of stable cytoplasmic petite mutants. Using these deletion mutants it was possible to localize the two antimycin A resistance loci in the O i - O i l region of mitochondrial DNA. The genetic loci are mapped in the following order: OH-A~-AH-cobl-Ov The mapping relationship of mutants resistant to antimycin A or funiculosin to various cob mutants is described.
Introduction The cytochrome bcl complex of the mitochondrial respiratory chain contains at least two functionally different cytochrome b species, each displaying distinct kinetic and spectral properties (for a review see Rieske, 1976). It was not yet possible to decide whether or not these distinct properties result from different cytochrome b polypeptides or from one type of cytochrome b residing in different membrane environments (for reviews see Rieske, 1976; Weiss, 1976). Cytochrome b is translated on mitochondrial ribosomes as demonstrated for Neurospora crassa by Weiss and Ziganke (1974) and for Saccharomyces cerevisiae by Katan et al. (1976). A distinct region on mitochondrial D N A of Saccharomyces cerevisiae has been identified, in which mutations lead to a selective loss of cytochrome b (Groot Obbink et al., 1976; Slonimski and Tzagoloff, 1976 ; Tzagoloff et al., 1976) or to a double deficiency in cytochrome oxidase and cytochrome b (Foury and Tzagoloff, 1976; Kotylak and Slonimski, 1976; Pajot et al., 1976). These mutants, designated box, cob or cyb, are all respiratory deficient and belong to the general class of mit- mutants. For offprints contact: G. Michaelis
We have successfully used another approach to select for mitochondrial Complex III mutants which are still respiratory competent. This selection procedure takes advantage of the specific inhibitory actions of the antibiotics antimycin A and funiculosin. Respective mitochondrial resistance mutations were isolated and characterized (Michaelis, 1976; Pratje and Michaelis, 1976). The existance of two distinct mitochondrial loci for antimycin A resistance could be demonstrated by allelism tests. All mitochondrial mutations conferring resistance to funiculosin were found to map at the second antimycin A resistance lOCUS A n. Both loci are involved in the determination of component(s) of the cytochrome bc 1 complex (Pratje and Michaelis, 1977). In the present article we report the precise localization of the two antimycin A resistance loci A~ and AII in the Oi - Oil region of the mitochondrial genome.
Materials and Methods 1. Strains. The strains of Saccharomyces cerevisiae used in the present work are listed in Table 1. The petite mutants (GM10-2B/901 to 940) carrying antibiotic resistance markers have been derived from strain GM10-2B by ethidium bromide mutagenesis and subsequent clonal purification. Stable p clones were selected retaining either the antimycin A or funiculosin resistance or both simultaneously. The p- clones derived from the antibiotic sensitive strain D22 were isolated by Tzagoloffet al. (1976). The cob mutants from strains D273-10B, D273-10B/A1, D273-10B/A21 and D273-10B/ A16 have been described previously (Tzagoloffet al., 1976). 2. Media. The standard media have been described in detail by
Pratje and Michaelis (1977) and are only briefly mentioned here. YPglucose=yeast extract, peptone, glucose; WO=minimal glucose ; N3 = yeast extract, peptone, glycerol;N1 =yeast extract, peptone, ethanol. Antibiotic plates contained 0,05 (or 0,1) gg antimycin A, or 5 gg funiculosin, or 25 gg DCMU, or 6 gg oligomycin per ml N1 medium.
80
G. Michaelis and E. Pratje : Mapping of the Two Mitochondrial Antimycin A Resistance Loci
Table 1. Genotype and origin of strains used Strain
Chromosomal genotype Mitochondrial genotype
Origin of strains
D243-4A D243-4A/8 D243-4A/A32 D243-4A/D732 D243-4A/D731 D243-4A/Fun5 GM3-5B GM5-2D GM7-5D GM10-2B D22 D273-10B D273-10B/A1 D273-10B/A16 D273-10B/A21 FFl123-19C
In In In In in In In In In In In In In In in In
Michaelis et al. (1971 Michaelis et al. (1971) Pratje and Michaelis (1977) this study this study Pratje and Michaelis (1977) Meiosis of GM3 (Michaelis, 1976) Meiosis of GM5 (Michaelis, 1976) Pratje and Michaelis (1977) Pratje and Michaelis (1977) Tzagoloff et al. (1976) Tzagoloff et al. (1976) Tzagoloff et al. (1976) Tzagoloff Tzagoloff et al. (1976) Fukuhara
FF1301-17B
In c~ade2
a adel lys2 a adel lys2 a adel lys2 a adel Iys2 a adel lys2 a adel lys2 c~his4 c~his4 lys2 c~his4 a lys2 a ade2 e c~met c~met c~met c~hisl ura3
p+ pO p+ anarl-32 (locus AI) p+ diur2-732 (locus AI) p+ diffl-731 (locus An) p+ funr-5 (locus An) p+ anarl-Gla (locus AI) p + ana'2-G25 (locus An) p+ funr-5 (locus An) p+ anarl-Gla funr-5 p+ p+ p+ p+ eryr-621 olf4-622 pa~-623 p + eryr-624 olirl-625 par~-626 p÷ capr-321 eryr-514 oliri-145 parr-454 p+ cap'-321 eryr-514 olir2-144 par~-454
Fukuhara
The present nomenclature is adapted from the recommendations proposed at the "Interdisciplinary Conference on the Genetics and Biogenesis of Chloroplasts and Mitochondria", Miinchen, August 1976
3. Retention of Antibiotic Resistance Loci in p- Testers. Mutants
resistant to antimycin A, funiculosin, or oligomycin were crossed to various p- mutants in liquid YPglucose medium according to the synchronous mating procedure of Jakob (1962). The diploids were patched on minimal glucose plates, replicated on the same medium after two days, grown for two days and spread on minimal glucose for single colonies. After two days incubation they were replicated on N1 medium with and without antibiotics. The occurrence of antibiotic sensitive colonies was used as the criterion for the presence of the drug-sensitive alleles in the mitochondrial DNA of the p clones. x cob Crosses. Cultures of the cobmutants were spread on minimal glucose plates. Suspensions of the p- clones were dropped on each cob- lawn. The diploids that had formed after two days were replicated on N3 medium and scored for growth on glycerol after an additional two days.
4. Restoration Test in p
5. Two-Factor Crosses Between Drug Resistance and cob- Mutations.
These crosses were performed in YP10 (1% yeast extract, 1% peptone, 10% glucose) as described by Kotylak and Slonimski (1976). The diploids were left unshaken for three days at 28°C in WO medium containing 10% glucose. They were then spread on minimal glucose plates to yield about 100 p+ colonies. After three days the plates were replicated on N3 and antibiotic media and scored for sensitive p+ recombinants. 6. Abbreviations. A~/A~ =allelic forms of the anal locus conferring
antimycin A resistance/sensitivity. AR/AS~= allelic forms of the ana2 locus conferring antimycin A resistance/sensitivity. FUNR/FUNS = atlelic forms of the fun locus = aria2 locus conferring funiculosin resistance/sensitivity. R S D732/Dv3z = allelic forms of the diu2 locus = anal locus conferring diuron (= DCMU) resistance/sensitivity. OR/Os (O~/OSl)= allelic forms of the oli 1 locus (oli2 locus) conferring oligomycin resistance/sensitivity. C, E, P=symbols for the ribl, rib3, and parl loci respectively. coW/cob + =allelic forms of either the cobl or cob2 loci con-
trolling cytochrome b biosynthesis. The terms box (Kotylak and Slonimski, 1976) and cyb (Groot Obbink et al., 1976) have been used for similar mutations. They all belong to the general class of respiratory deficient mit- mutants which retain mitochondrial protein synthesis (Tzagoloff et al., 1975). p-/p+ =cytoplasmic petite/grande. A superscript zero sign (O) following the gene symbol will indicate the loss of the respective marker. DCMU = 3-(3,4-Dichlorophenyl)-l, 1-dimethylurea
Results T z a g o l o f f et al. (1976) h a v e l o c a l i z e d t h e i r c y t o c h r o m e b d e f i c i e n t c o b - m u t a n t s w i t h i n the O ~ - O u r e g i o n o f m i t o c h o n d r i a l D N A by d e l e t i o n m a p p i n g w i t h p e t i t e clones. 20 o f t h e s e p e t i t e m u t a n t s d i s c r i m i nate between various cob- mutations. We have anal y z e d t h e s a m e 20 p e t i t e testers for r e t e n t i o n o r loss o f the t w o a n t i m y c i n A r e s i s t a n c e loci A~ a n d A n by c r o s s i n g t h e m to the a n t i b i o t i c r e s i s t a n t strains GM3-5B, GM5-2D and GM7-5D. The production o f sensitive p+ d i p l o i d s in s u c h a petite x A R c r o s s is t a k e n as a c r i t e r i o n t h a t t h e r e s p e c t i v e p e t i t e c l o n e h a s r e t a i n e d t h e d r u g s e n s i t i v e allele in its m i t o c h o n d r i a l g e n o m e . S e n s i t i v e d i p l o i d p+ c o l o n i e s w e r e f o u n d in c r o s s e s w i t h all t h r e e a n t i b i o t i c r e s i s t a n c e m u t a t i o n s tested, as i l l u s t r a t e d in T a b l e 2. T h i s result s h o w s t h a t the t w o a n t i m y c i n A r e s i s t a n c e loci are l o c a t e d in the O i - O n s e g m e n t o f the m i t o c h o n d r i a l genome. O n l y t h o s e p e t i t e c l o n e s c a p a b l e o f r e s t o r i n g the c o b 2 m u t a t i o n s M 1 0 - 1 5 2 , M21-71 a n d M 1 7 - 1 6 2 p r o -
G . M i c h a e l i s a n d E. P r a t j e : M a p p i n g
T a b l e 2. R e t e n t i o n
of the two O~l
p- D22/RP
of the Two Mitochondrial
antimycin
A resistance loci and
cob2
Antimycin
cob
A Resistance Loci
markers
(data
taken
81
from
Tzagoloff
cobl
O~
100B
+
+
+
+
+
+
+
+
+
.
123B
-
+
+
+
+
+
+
+
+
.
.
.
.
A~I a
A~2 5
FUN~
+
+
+
+
+
+
+
+
+
+ +
+ +
+ +
+
+
+
+
+
+
+ +
+ +
+ +
+ +
+ +
. .
27B
+
+
+
+
+
+
.
.
.
.
.
.
.
.
+
+
+
45B
+
+
+
+
+
+
.
.
.
.
.
.
.
.
+
+
+
84B
+
+
+
+
+
+
.
.
+
+
+
96B
+
+
+
+
+
+
+
+
+
5B 7B
.
.
-
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ -
-
+ +
+ +
59B 49B 86B
.
95B 104B 40B
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + +
--
+ + + +
+ + + +
. --
.
+
+
+
+
+
+
+
+
+
+
--
+
+
+
+
+
+
+
+
+
+
+
--
-
-
78B 43B 2B
. + -
.
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + -
+ +
+ +
+ +
. -
.
.
.
. -
.
.
. _
.
.
_
. -~
. + +
.
. + +
. + +
+ +
+ +
.
.
.
.
.
.
.
A,
+ +
.
.
An
+
.
.
A~
+
.
.
testers
-
.
.
. .
by p
14B
.
.
.
.
1976)
57B 76B
.
.
.
et al.,
. .
. .
.
.
. -
-
Petite x cob - crosses : plus indicates growth on glycerol and minus lack of growth. Petite x A R or FUN R crosses : plus indicates the presence of sensitive p+ diploids, and minus indicates the absence of such colonies. GM5-2D and GM7-5D. For details see Materials and Methods
duce sensitive diploid p+ colonies when crossed to the mutant of locus AI (Table 2). This indicates a close association of a n a r l - G l a with these three cob2 mutations. All petite testers restoring respiration of the cob mutant M18-68 have retained the sensitive alleles of the A l l lOCUS (Table 2). Anar2-G25 and fun r5 gave identical results confirming the arrangement of both mutations causing different phenotypes in the same locus AII. These results permit the AH locus to be placed near the cob mutation M18-68. A second independent deletion map was constructed taking advantage of the possibility to isolate petite testers which have retained either the marker for antimycin A or funiculosin resistance. Since mitochondrial D N A fragments of petite clones can recombine with the D N A of cob- mutants, the formation of respiratory competent wild type recombinants occurs if the cob + allele is present in the petite mutant and is integrated in place of the coballele. The p+ double-resistant anarfun r strain GM102B was mutagenized with ethidium bromide and petite clones were selected retaining either the antimycin A or the funiculosin resistance marker or both. After 3 subclonings 35 stable petite mutants were isolated and crossed to the cytochrome b deficient cobstrains of Tzagoloff et al. (1976). The diploids were tested for growth on glycerol. As demonstrated in
Strains resistant to antimycin
A or funiculosin:
GM3-5B,
Table 3 all cob2 mutants showed growth when crossed to the nine ARFUN R petites. M10-152 is the only cob2 mutation which is restored by all petite clones retaining the anal locus. The twelve A ° F U N R petites restore the three cob mutations M18-68, M8219 and M9-226, indicating the localization of the fun r or ana2 locus within this region. Surprisingly all 35 petite testers have lost the olil and oli2 markers. The arrangement of the cob- mutations is in good agreement when the two deletion maps are compared. The relative order of the three cob2 mutations M10152, M21-71 and M17-162 could not be established although they are well discriminated by some petite testers. According to the results presented, the two antimycin A resistance loci Ar and AH can be placed close to the cob mutations M10-152 and M18-68 respectively. Although the results of the deletion mapping are consistent, these data do not yield much information on the genetic distance between the markers. In general this distance is proportional to the frequency of recombinants which can be measured in two-factor crosses. The results of such two-factor crosses between cob- and antibiotic resistance mutations are summarized in Table 4. Since only cob ÷ diploids can readily be analyzed for drug resistance or sensitivity, the percentage of recombinants in Table 4 refers to
82
G . M i c h a e l i s a n d E. P r a t j e : M a p p i n g
T a b l e 3. R e s t o r a t i o n
of cob-
mutations
by petite clones that retain mitochondrial 011
p-ARFUN
°
GM10-2B/901
R
p- ARFUN R
p+
ARFUN t
-
4.
4.
4.
.
.
903 904
-
4, +
4. 4.
4, .
905 906
-
+ +
. +
907 908
-
4. +
+ .
909 910
-
4, +
. .
.
911
-
+
.
.
912 913
-
4. +
+ +
4,
A Resistance Loci
A and/or funiculosin resistance
.
.
.
. 4.
. . .
.
. .
.
. . .
.
. . .
. . .
.
. .
.
.
.
.
.
.
. m
.
.
.
.
. .
.
.
.
.
.
.
.
.
. . .
. . .
.
.
.
+
+
4"
+
.
917 918
. .
. .
. .
. .
. .
4" +
4" +
4" +
4" +
4" 4,
4" 4.
P +
. +
919 921
. .
. .
. .
.
.
4. 4,
4. +
4, 4.
4, +
4, +
+ +
4, -I-
922 923 924
. . .
. . .
. . .
.
.
4. + +
4. 4. +
4, 4, +
4.
4, . . . .
4. .
925 926
. .
. .
. .
+ 4,
+ 4.
44.
+ 4,
928
.
.
.
930
.
.
.
+ .
.
.
+ .
.
.
.
.
.
+ .
.
. .
.
.
.
. . .
.
4,
m
.
.
. . .
.
.
.
.
.
.
.
m
m
.
.
. .
.
.
.
.
.
.
.
. .
. . .
.
. .
.
. .
. .
. .
.
.
. .
.
.
. .
.
. .
.
.
.
.
.
.
. .
.
.
.
. .
. .
.
.
.
.
.
.
.
.
+
. .
.
.
. .
. .
.
. .
. .
. .
+ .
+
. .
Ot
.
GM10-2B/931
-
Antimycin
cobl
-
GM10-2B/916
GM10-2B
antimycin
cob2
902
914 p- A°FUN
of the Two Mitochondrial
.
.
.
.
. .
.
4, . .
.
4, .
.
.
.
.
.
.
.
+
4.
+
4, 4.
4. 4.
4, +
4. 4.
+
4,
4,
4.
+
+
. .
.
4, .
+ .
. + .
.
4.
4.
+
4-
4,
4.
4,
4.
4,
4,
4.
4.
4,
4.
4.
4.
4.
+
4.
4.
4.
4.
4.
-
+
+
4"
+
4,
+
4.
+
.
.
.
.
.
933 934 935 936
-
+ 4. + +
+ 4, 4. +
+ 4. + +
+ 4. + +
+ 4. + +
+ 4. + +
+ 4. 4. +
4, 4. +
+ 4.
4. 4.
4. 4,
+ 4.
+ 4, -
4, 4, -
+ + -
937 938
--
4, +
4, +
4. 4.
+ +
4. +
+ +
+ +
+ +
4. +
4, +
4, +
4, +
+ +
+ +
+ +
939 940
-
4. 4,
4, 4.
4, 4.
4. 4.
4. 4,
+ 4.
+ 4.
+ 4.
+ 4,
+ 4,
4, 4.
4. 4,
4, 4.
4, 4.
+ +
4,
+
4,
4,
4.
4,
4.
4,
4,
4,
4.
4,
4.
4.
4,
4,
The p- clones, derived from GM10-2B, were dropped on WO plates spread with lawns of the cob- mutants. The diploids grown f o r 2 d a y s a t 28 ° C w e r e r e p l i c a t e d o n N 3 m e d i u m a n d s c o r e d f o r g l y c e r o l g r o w t h a f t e r 2 d a y s . P l u s i n d i c a t e s g l y c e r o l g r o w t h , m i n u s absence of growth and a P some few colonies. Retention with the following two strains FF1123-19C and FF1301-17B
or loss of the two mitochondrial markers a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s
the total number of p+ colonies, assuming that the frequencies of cob + and c o b - colonies are equal as well as the two types of reciprocal recombinants. It is not possible to deduce the order of c o b l , cob2 and antimycin A resistance mutations from the recombination data of Table 4. To ensure that the recombination values in the ana r x c o b - crosses are not affected by the critical antimycin A resistance phenotype (Pratje and Michaelis, 1977) the crosses were repeated with the diuron resistant mutant D243-4A/
O1 a n d
O11 h a s
been
analyzed
D732 which was found to m a p in the anal locus. Resistant and sensitive colonies can well be distingiushed at 25 gg diuron per ml. The crosses c o b - x diur2-732 and c o b - x anar1-32 gave indentical results (Table 4). Although the two-factor crosses do not yield data on the arrangement of the mutations investigated, this type of cross nevertheless confirms the localization of the two antimycin A resistance loci close to the cob mutations M10-152 and M18-68. N o recombinants could be detected in the respective crosses.
G. Michaelis and E. Pratje: Mapping of the Two Mitochondrial Antimycin A Resistance Loci
Discussion
Table 4. Linkage analysis between mutations of the two antimycin A resistance loci and cob mutations a
AI A32
At D732
Several laboratories have recently described mitochondrial mutations in Saccharomyces cerevisiae which confer resistance to inhibitors acting on the cytochrome bcl complex of the mitochondrial respiratory chain. These mutations include resistance to mucidin (Subik, 1975), diuron (Colson et al., 1977), antimycin A (Michaelis, 1976 ; Burger et al., 1976; Groot Obbink et al., 1977) or funiculosin (Pratje and Michaelis, 1977). Allelism studies indicated the existence of at least two mitochondrial loci for mucidin (Subik et al., 1977), diuron (Colson et al., 1977) and antimycin A resistance (Pratje and Michaelis, 1977) respectively. A genetic analysis is required to allocate the various resistance and mit- mutations to the mitochondrial genetic loci involved in the specification of components of the cytochrome bcl complex. Funiculosin resistance has previously been reported to map at the second antimycin A resistance locus (Pratje and Michaelis, 1977). Preliminary experiments indicate that the two loci for antimycin A resistance are allelic to the two diuron resistance loci (Colson et al., personal communication). In the present work
A~I Fun5
% Rec. total % Rec. total % Rec. total colonies colonies colonies M10-152 M21-71 M17-162 M33-119 M18-68 M8-181 M6-200 M17-231 M15-207 M8-53 M13-101 M24-241 M7-40
0 1.9 0.07 2.4 3.3 2.5 3.4 8.1 3.5 6.9 6.9 1.8 4.7
967 2079 1495 2145 2702 2011 3044 2564 1287 976 1701 883 784
0 2.8 0.07 3.2 3.1 3.2 2.7 6.3 3.9 5 2.4 1.5 1.8
1886 1759 1430 1847 2709 1020 2344 1709 2184 1862 2286 1629 2607
1.3 0.8 0.7 0 0 1.2 3.4 4.8 2.2 2.5 2.1 0.3 2.6
1052 857 1210 1568 930 1065 872 806 679 791 900 1196 574
The c o b - mutants were crossed to D243-4A/A32,/D732,/Fun5 as described in Materials and Methods. After about 10 generations the diploids were spread on WO plates. These WO masters were replicated on N3 and N3 antibiotic media (50 ng antimycin A/ml, 25 pg D C M U / m l or 5 pg funiculosin/ml). Growth on glycerol and the antibiotic media was scored after 2-3 days incubation at 28 ° C
AI
83
All I 1
I I|
I
I
1
I 1
¢xt
C~
!
!
~
I
1
I
C~
C~
1
1
I
I
I
I
I
/
cob 2
cob 1
/
J
J
7 /" f f J
J
Fig. 1. Arrangement of the two antimycin A resistance loci A 1 and A~I on the mitochondrial genome of Saccharomyces cerevisiae. The order of the cob markers follows Tzagoloff et al. (1976). The two antimycin A resistance loci are placed on the map according to the results presented in Tables 2, 3 and 4
84
G. Michaelis and E. Pratje : Mapping of the Two Mitochondrial Antimycin A Resistance Loci
we have mapped the two antimycin A resistance loci AI and A~I in the O i - O n region of mitochondrial DNA, and more precisely in the cob2 locus defined by Tzagoloff et al. (1976). Two independent series of petite clones were used for the deletion mapping. The petite mutants of the first series, isolated and described by Tzagoloff et al. (1976), were characterized by their ability to restore glycerol growth to various cob- mutants. These petite testers were crossed to the antimycin A and funiculosin resistant p ÷ strains, and the diploids were tested for the occurrence of antibiotic sensitive p + cells. Such sensitive p+ colonies are only produced with those petite testers retaining the respective sensitive allele. We have isolated a second series of petite testers, retaining one or both antimycin A resistance loci. These petite strains were crossed to the cob mutants o f T z a g o l o f f e t al. (1976), and the diploids were tested for growth on glycerol. The result of this restoration test is in agreement with the arrangement of cobmutations as determined by Tzagoloff et al. (1976). The two series of petite testers yielded coherent mapping results and allowed the two A g loci to be placed in the cob region. It should be mentioned that the cob2 locus has already been subdivided in four loci, designated box 1, 2, 3 and 4, by Kotylak and Slonimski (1976). Two factor crosses between cob- and antibiotic resistant mutants produced ambiguous results. The cob mutants could not be separated into two groups based on the frequency of recombinants. Never; theless, the very close association of A~ with cob mutation M 10-152 and A R with cob mutation M 18-68 is confirmed since no recombinants could be detected in these crosses. The results of our mapping studies are summarized in Figure 1. In this article we present evidence that the cob region specifies two sites involved in the binding of antimycin A and funiculosin. Several important questions remain to be answered. The number of mitochondrial structural and regulatory genes for the cytochrome bca complex, the number of cytochrome b polypeptide species, and the gene-product relationship for the two A R loci and cytochrome b remain to be established. A biochemical comparison of proteins from antimycin A resistant and sensitive strains, now under investigation in our laboratory, should contribute to the solution of these problems. Acknowledgements. We wish to thank Dr. A. Tzagoloff for kindly providing us with his petite and cob- mutants. Careful reading of the manuscript by Dr. D. Miller is greatly acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft.
References Burger, G., Lang, B., Bandlow, W., Schweyen, R.J., Backhaus, B., Kaudewitz, F. : Antimycin resistance in Saccharomyces ¢ere-
visiae. a new mutation on the mitDNA conferring antimycin resistance on the mitochondrial respiratory chain. Biochem. biophys. Res. Commun. 72, 1201.-1208 (1976) Colson, A.-M., The Van, L., Convent, B., Briquet, M., Goffeau, A.: Mitochondrial heredity of resistance to 3-(3,4-dichlorphenyl)-l,l-dimethylurea, an inhibitor of cytochrome b oxidation, in Saccharomyces cerevisiae. Europ. J. Biochem. 74, 521-526 (1977) Foury, F., Tzagoloff, A.: Assembly of the mitochondrial membrane system XIX. Genetic characterization of mit- mutants with deficiencies in cytochrome oxidase and coenzyme QHzcytochrome c reductase. Molec. gen. Genet. 149, 43 50 (1976) Groot Obbink, D.J., Hall, R.M., Linnane, A.W., Lukins, H.B., Monk, B.C., Spithill, T.W., Trembath, M.K.: Mitochondrial genes involved in the determination of mitochondrial membrane proteins. In: (C. Saccone, A.M. Kroon, eds.), The genetic function of mitochondrial DNA, pp. 163-173. Amsterdam: Elsevier/ North-Holland Biomedical Press 1976 Groot Obbink, D.J., Spithill, T.W., Maxwell, R.J., Linnane, A.W. : Biogenesis of mitochondria 48. Mikamycin resistance in Saccharomyces cerevisiae- A mitochondrial mutation conferring resistance to an antimycin A-like contaminant in mikamycin. Molec. gen. Genet. 151, 127 136 (1977) Jakob, H.: Technique de synchronisation de la formation des zygotes chez la levure Saccharomyces cerebisiae. C.R. Acad. Sci. (Paris) 254, 3909 (1962) Katan, M.B., Van Harten-Loosbroek, N., Groot, G.S.P.: The cytochrome bcl complex of yeast mitochondria. Site of translation of the polypeptides in vivo. Europ. J. Biochem. 70, 409-417 (1976) Kotylak, Z., Slonimski, P.P.: Joint control of cytochromes a and b by a unique mitochondrial DNA region comprising four genetic loci. In: (C. Saccone, A.M. Kroon, eds.), The genetic function of mitochondriai DNA, pp. 143 154. Amsterdam: Elsevier/North-Holland Biomedical Press 1976 Michaelis, G.: Cytoplasmic inheritance of antimycin A resistance in Saccharomyces cerevisiae. Molec. gen. Genet. 146, 133 137 (1976) Michaelis, G., Douglass, S., Tsai, M.-J., Criddle, R.S.: Mitochondrial DNA and suppressiveness of petite mutants in Saccharomyces cerevisiae. Biochem. Genet. 5, 487495 (1971) Pajot, P., Wambier-Kluppel, M.L., Kotylak, Z., Slonimski, P.P.: Regulation of cytochrome oxidase formation by mutations in a mitochondrial gene for cytochrome b. In: (Th. Bticher, W. Neupert, W. Sebald, S. Werner, eds.), Genetics and biogenesis of chloroplasts and mitochondria, pp. 443-451. Amsterdam: Elsevier/North-Holland Biomedical Press 1976 Pratje, E., Michaelis, G.: Two mitochondrial antimycin A resistance loci in Saccharomyces cerevisiae. In: (Th. Bficher, W. Neupert, W. Sebald, S. Werner, eds.), Genetics and biogenesis of chloroplasts and mitochondria, pp. 443 451. Amsterdam: Elsevier/North-Holland Biomedical Press 1976 Pratje, E., Michaelis, G. : Allelism studies of mitochondrial mutants resistant to antimycin A or funiculosin in Saecharomyces cerevisiae. Molec. gen. Genet. 152, 167-174 (1977) Rieske, J.S.: Composition, structure, and function of complex III of the respiratory chain. Biochim. biophys. Acta (Amst.) 456, 195:-247 (1976) Slonimski, P.P., Tzagoloff, A. : Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QHz-cytochrome c reductase. Europ. J. Biochem. 61, 27-41 (1976) Subik, J.: Mucidin-resistant antimycin A-sensitive mitochondrial mutant of Saccharomyces cerevisiae. FEBS Letters 59, 273 276 (I975) SuNk, J., Kovacova, V., Takacsova, G.: Mucidin resistance in yeast. Isolation, characterization and genetic analysis of nuclear
G. Michaelis and E. Pratje : Mapping of the Two Mkochondrial Antimycin A Resistance Loci and mitochondrial mucidin-resistant mutants of Saccharomyces cerevisiae. Europ. J. Biochem. 73, 275 286 (1977) Tzagoloff, A., Akai, A., Needleman, R.B. : Properties of cytoplasmic mutants of Saccharomyces cerevisiae with specific lesions in cytochrome oxidase. Proc. nat. Acad. Sci. (Wash.) 72, 2054-2057 (1975) Tzagoloff, A., Foury, F., Akai, A. : Assembly of the mitochondrial membrane system XVIII. Genetic loci on mitochondrial DNA involved in cytochrome b biosynthesis. Molec. gen. Genet. 149, 33-42 (1976) Weiss, H.: Subunit composition and biogenesis of mitochondrial cytochrome b. Biochim. biophys. Acta (Amst.) 456, 291 313 (1976)
Note Added in Proof
The classification of cob mutations into cobl and cob2 follows Tzagoloff et al. (1976). Slonimski and coworkers have recently presented evidence that the cob- mutations corresponding to the AH locus should rather be regrouped at the terminal part of the cobl region (Colloquium on Genetics and Biogenesis of Mitochondria, Schliersee, August 1977).
85
Weiss, H., Ziganke, B. : Cytochrome b in Neurospora crassa mitochondria. Site of translation of the heme protein. Europ. J. Biochem. 41, 63-71 (1974)
Communicated
by W. Gajewski
Received July 19, 1977
Mapping of the Two Mitochondrial Antimycin A Resistance Loci in Saccharomyces cerevisiae Georg Michaelis and Elke Pratje Fakult~it flir Biologic der Universit/it Bielefeld, Postfach 8640, D-4800 Bielefeld 1, Federal Republic of Germany
Summary. Retention or loss of the two new mitochondrial antimycin A resistance loci A~ and A~ has been analyzed in a large number of stable cytoplasmic petite mutants. Using these deletion mutants it was possible to localize the two antimycin A resistance loci in the O i - O i l region of mitochondrial DNA. The genetic loci are mapped in the following order: OH-A~-AH-cobl-Ov The mapping relationship of mutants resistant to antimycin A or funiculosin to various cob mutants is described.
Introduction The cytochrome bcl complex of the mitochondrial respiratory chain contains at least two functionally different cytochrome b species, each displaying distinct kinetic and spectral properties (for a review see Rieske, 1976). It was not yet possible to decide whether or not these distinct properties result from different cytochrome b polypeptides or from one type of cytochrome b residing in different membrane environments (for reviews see Rieske, 1976; Weiss, 1976). Cytochrome b is translated on mitochondrial ribosomes as demonstrated for Neurospora crassa by Weiss and Ziganke (1974) and for Saccharomyces cerevisiae by Katan et al. (1976). A distinct region on mitochondrial D N A of Saccharomyces cerevisiae has been identified, in which mutations lead to a selective loss of cytochrome b (Groot Obbink et al., 1976; Slonimski and Tzagoloff, 1976 ; Tzagoloff et al., 1976) or to a double deficiency in cytochrome oxidase and cytochrome b (Foury and Tzagoloff, 1976; Kotylak and Slonimski, 1976; Pajot et al., 1976). These mutants, designated box, cob or cyb, are all respiratory deficient and belong to the general class of mit- mutants. For offprints contact: G. Michaelis
We have successfully used another approach to select for mitochondrial Complex III mutants which are still respiratory competent. This selection procedure takes advantage of the specific inhibitory actions of the antibiotics antimycin A and funiculosin. Respective mitochondrial resistance mutations were isolated and characterized (Michaelis, 1976; Pratje and Michaelis, 1976). The existance of two distinct mitochondrial loci for antimycin A resistance could be demonstrated by allelism tests. All mitochondrial mutations conferring resistance to funiculosin were found to map at the second antimycin A resistance lOCUS A n. Both loci are involved in the determination of component(s) of the cytochrome bc 1 complex (Pratje and Michaelis, 1977). In the present article we report the precise localization of the two antimycin A resistance loci A~ and AII in the Oi - Oil region of the mitochondrial genome.
Materials and Methods 1. Strains. The strains of Saccharomyces cerevisiae used in the present work are listed in Table 1. The petite mutants (GM10-2B/901 to 940) carrying antibiotic resistance markers have been derived from strain GM10-2B by ethidium bromide mutagenesis and subsequent clonal purification. Stable p clones were selected retaining either the antimycin A or funiculosin resistance or both simultaneously. The p- clones derived from the antibiotic sensitive strain D22 were isolated by Tzagoloffet al. (1976). The cob mutants from strains D273-10B, D273-10B/A1, D273-10B/A21 and D273-10B/ A16 have been described previously (Tzagoloffet al., 1976). 2. Media. The standard media have been described in detail by
Pratje and Michaelis (1977) and are only briefly mentioned here. YPglucose=yeast extract, peptone, glucose; WO=minimal glucose ; N3 = yeast extract, peptone, glycerol;N1 =yeast extract, peptone, ethanol. Antibiotic plates contained 0,05 (or 0,1) gg antimycin A, or 5 gg funiculosin, or 25 gg DCMU, or 6 gg oligomycin per ml N1 medium.
80
G. Michaelis and E. Pratje : Mapping of the Two Mitochondrial Antimycin A Resistance Loci
Table 1. Genotype and origin of strains used Strain
Chromosomal genotype Mitochondrial genotype
Origin of strains
D243-4A D243-4A/8 D243-4A/A32 D243-4A/D732 D243-4A/D731 D243-4A/Fun5 GM3-5B GM5-2D GM7-5D GM10-2B D22 D273-10B D273-10B/A1 D273-10B/A16 D273-10B/A21 FFl123-19C
In In In In in In In In In In In In In In in In
Michaelis et al. (1971 Michaelis et al. (1971) Pratje and Michaelis (1977) this study this study Pratje and Michaelis (1977) Meiosis of GM3 (Michaelis, 1976) Meiosis of GM5 (Michaelis, 1976) Pratje and Michaelis (1977) Pratje and Michaelis (1977) Tzagoloff et al. (1976) Tzagoloff et al. (1976) Tzagoloff et al. (1976) Tzagoloff Tzagoloff et al. (1976) Fukuhara
FF1301-17B
In c~ade2
a adel lys2 a adel lys2 a adel lys2 a adel Iys2 a adel lys2 a adel lys2 c~his4 c~his4 lys2 c~his4 a lys2 a ade2 e c~met c~met c~met c~hisl ura3
p+ pO p+ anarl-32 (locus AI) p+ diur2-732 (locus AI) p+ diffl-731 (locus An) p+ funr-5 (locus An) p+ anarl-Gla (locus AI) p + ana'2-G25 (locus An) p+ funr-5 (locus An) p+ anarl-Gla funr-5 p+ p+ p+ p+ eryr-621 olf4-622 pa~-623 p + eryr-624 olirl-625 par~-626 p÷ capr-321 eryr-514 oliri-145 parr-454 p+ cap'-321 eryr-514 olir2-144 par~-454
Fukuhara
The present nomenclature is adapted from the recommendations proposed at the "Interdisciplinary Conference on the Genetics and Biogenesis of Chloroplasts and Mitochondria", Miinchen, August 1976
3. Retention of Antibiotic Resistance Loci in p- Testers. Mutants
resistant to antimycin A, funiculosin, or oligomycin were crossed to various p- mutants in liquid YPglucose medium according to the synchronous mating procedure of Jakob (1962). The diploids were patched on minimal glucose plates, replicated on the same medium after two days, grown for two days and spread on minimal glucose for single colonies. After two days incubation they were replicated on N1 medium with and without antibiotics. The occurrence of antibiotic sensitive colonies was used as the criterion for the presence of the drug-sensitive alleles in the mitochondrial DNA of the p clones. x cob Crosses. Cultures of the cobmutants were spread on minimal glucose plates. Suspensions of the p- clones were dropped on each cob- lawn. The diploids that had formed after two days were replicated on N3 medium and scored for growth on glycerol after an additional two days.
4. Restoration Test in p
5. Two-Factor Crosses Between Drug Resistance and cob- Mutations.
These crosses were performed in YP10 (1% yeast extract, 1% peptone, 10% glucose) as described by Kotylak and Slonimski (1976). The diploids were left unshaken for three days at 28°C in WO medium containing 10% glucose. They were then spread on minimal glucose plates to yield about 100 p+ colonies. After three days the plates were replicated on N3 and antibiotic media and scored for sensitive p+ recombinants. 6. Abbreviations. A~/A~ =allelic forms of the anal locus conferring
antimycin A resistance/sensitivity. AR/AS~= allelic forms of the ana2 locus conferring antimycin A resistance/sensitivity. FUNR/FUNS = atlelic forms of the fun locus = aria2 locus conferring funiculosin resistance/sensitivity. R S D732/Dv3z = allelic forms of the diu2 locus = anal locus conferring diuron (= DCMU) resistance/sensitivity. OR/Os (O~/OSl)= allelic forms of the oli 1 locus (oli2 locus) conferring oligomycin resistance/sensitivity. C, E, P=symbols for the ribl, rib3, and parl loci respectively. coW/cob + =allelic forms of either the cobl or cob2 loci con-
trolling cytochrome b biosynthesis. The terms box (Kotylak and Slonimski, 1976) and cyb (Groot Obbink et al., 1976) have been used for similar mutations. They all belong to the general class of respiratory deficient mit- mutants which retain mitochondrial protein synthesis (Tzagoloff et al., 1975). p-/p+ =cytoplasmic petite/grande. A superscript zero sign (O) following the gene symbol will indicate the loss of the respective marker. DCMU = 3-(3,4-Dichlorophenyl)-l, 1-dimethylurea
Results T z a g o l o f f et al. (1976) h a v e l o c a l i z e d t h e i r c y t o c h r o m e b d e f i c i e n t c o b - m u t a n t s w i t h i n the O ~ - O u r e g i o n o f m i t o c h o n d r i a l D N A by d e l e t i o n m a p p i n g w i t h p e t i t e clones. 20 o f t h e s e p e t i t e m u t a n t s d i s c r i m i nate between various cob- mutations. We have anal y z e d t h e s a m e 20 p e t i t e testers for r e t e n t i o n o r loss o f the t w o a n t i m y c i n A r e s i s t a n c e loci A~ a n d A n by c r o s s i n g t h e m to the a n t i b i o t i c r e s i s t a n t strains GM3-5B, GM5-2D and GM7-5D. The production o f sensitive p+ d i p l o i d s in s u c h a petite x A R c r o s s is t a k e n as a c r i t e r i o n t h a t t h e r e s p e c t i v e p e t i t e c l o n e h a s r e t a i n e d t h e d r u g s e n s i t i v e allele in its m i t o c h o n d r i a l g e n o m e . S e n s i t i v e d i p l o i d p+ c o l o n i e s w e r e f o u n d in c r o s s e s w i t h all t h r e e a n t i b i o t i c r e s i s t a n c e m u t a t i o n s tested, as i l l u s t r a t e d in T a b l e 2. T h i s result s h o w s t h a t the t w o a n t i m y c i n A r e s i s t a n c e loci are l o c a t e d in the O i - O n s e g m e n t o f the m i t o c h o n d r i a l genome. O n l y t h o s e p e t i t e c l o n e s c a p a b l e o f r e s t o r i n g the c o b 2 m u t a t i o n s M 1 0 - 1 5 2 , M21-71 a n d M 1 7 - 1 6 2 p r o -
G . M i c h a e l i s a n d E. P r a t j e : M a p p i n g
T a b l e 2. R e t e n t i o n
of the two O~l
p- D22/RP
of the Two Mitochondrial
antimycin
A resistance loci and
cob2
Antimycin
cob
A Resistance Loci
markers
(data
taken
81
from
Tzagoloff
cobl
O~
100B
+
+
+
+
+
+
+
+
+
.
123B
-
+
+
+
+
+
+
+
+
.
.
.
.
A~I a
A~2 5
FUN~
+
+
+
+
+
+
+
+
+
+ +
+ +
+ +
+
+
+
+
+
+
+ +
+ +
+ +
+ +
+ +
. .
27B
+
+
+
+
+
+
.
.
.
.
.
.
.
.
+
+
+
45B
+
+
+
+
+
+
.
.
.
.
.
.
.
.
+
+
+
84B
+
+
+
+
+
+
.
.
+
+
+
96B
+
+
+
+
+
+
+
+
+
5B 7B
.
.
-
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ -
-
+ +
+ +
59B 49B 86B
.
95B 104B 40B
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + + +
+ + +
--
+ + + +
+ + + +
. --
.
+
+
+
+
+
+
+
+
+
+
--
+
+
+
+
+
+
+
+
+
+
+
--
-
-
78B 43B 2B
. + -
.
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + -
+ +
+ +
+ +
. -
.
.
.
. -
.
.
. _
.
.
_
. -~
. + +
.
. + +
. + +
+ +
+ +
.
.
.
.
.
.
.
A,
+ +
.
.
An
+
.
.
A~
+
.
.
testers
-
.
.
. .
by p
14B
.
.
.
.
1976)
57B 76B
.
.
.
et al.,
. .
. .
.
.
. -
-
Petite x cob - crosses : plus indicates growth on glycerol and minus lack of growth. Petite x A R or FUN R crosses : plus indicates the presence of sensitive p+ diploids, and minus indicates the absence of such colonies. GM5-2D and GM7-5D. For details see Materials and Methods
duce sensitive diploid p+ colonies when crossed to the mutant of locus AI (Table 2). This indicates a close association of a n a r l - G l a with these three cob2 mutations. All petite testers restoring respiration of the cob mutant M18-68 have retained the sensitive alleles of the A l l lOCUS (Table 2). Anar2-G25 and fun r5 gave identical results confirming the arrangement of both mutations causing different phenotypes in the same locus AII. These results permit the AH locus to be placed near the cob mutation M18-68. A second independent deletion map was constructed taking advantage of the possibility to isolate petite testers which have retained either the marker for antimycin A or funiculosin resistance. Since mitochondrial D N A fragments of petite clones can recombine with the D N A of cob- mutants, the formation of respiratory competent wild type recombinants occurs if the cob + allele is present in the petite mutant and is integrated in place of the coballele. The p+ double-resistant anarfun r strain GM102B was mutagenized with ethidium bromide and petite clones were selected retaining either the antimycin A or the funiculosin resistance marker or both. After 3 subclonings 35 stable petite mutants were isolated and crossed to the cytochrome b deficient cobstrains of Tzagoloff et al. (1976). The diploids were tested for growth on glycerol. As demonstrated in
Strains resistant to antimycin
A or funiculosin:
GM3-5B,
Table 3 all cob2 mutants showed growth when crossed to the nine ARFUN R petites. M10-152 is the only cob2 mutation which is restored by all petite clones retaining the anal locus. The twelve A ° F U N R petites restore the three cob mutations M18-68, M8219 and M9-226, indicating the localization of the fun r or ana2 locus within this region. Surprisingly all 35 petite testers have lost the olil and oli2 markers. The arrangement of the cob- mutations is in good agreement when the two deletion maps are compared. The relative order of the three cob2 mutations M10152, M21-71 and M17-162 could not be established although they are well discriminated by some petite testers. According to the results presented, the two antimycin A resistance loci Ar and AH can be placed close to the cob mutations M10-152 and M18-68 respectively. Although the results of the deletion mapping are consistent, these data do not yield much information on the genetic distance between the markers. In general this distance is proportional to the frequency of recombinants which can be measured in two-factor crosses. The results of such two-factor crosses between cob- and antibiotic resistance mutations are summarized in Table 4. Since only cob ÷ diploids can readily be analyzed for drug resistance or sensitivity, the percentage of recombinants in Table 4 refers to
82
G . M i c h a e l i s a n d E. P r a t j e : M a p p i n g
T a b l e 3. R e s t o r a t i o n
of cob-
mutations
by petite clones that retain mitochondrial 011
p-ARFUN
°
GM10-2B/901
R
p- ARFUN R
p+
ARFUN t
-
4.
4.
4.
.
.
903 904
-
4, +
4. 4.
4, .
905 906
-
+ +
. +
907 908
-
4. +
+ .
909 910
-
4, +
. .
.
911
-
+
.
.
912 913
-
4. +
+ +
4,
A Resistance Loci
A and/or funiculosin resistance
.
.
.
. 4.
. . .
.
. .
.
. . .
.
. . .
. . .
.
. .
.
.
.
.
.
.
. m
.
.
.
.
. .
.
.
.
.
.
.
.
.
. . .
. . .
.
.
.
+
+
4"
+
.
917 918
. .
. .
. .
. .
. .
4" +
4" +
4" +
4" +
4" 4,
4" 4.
P +
. +
919 921
. .
. .
. .
.
.
4. 4,
4. +
4, 4.
4, +
4, +
+ +
4, -I-
922 923 924
. . .
. . .
. . .
.
.
4. + +
4. 4. +
4, 4, +
4.
4, . . . .
4. .
925 926
. .
. .
. .
+ 4,
+ 4.
44.
+ 4,
928
.
.
.
930
.
.
.
+ .
.
.
+ .
.
.
.
.
.
+ .
.
. .
.
.
.
. . .
.
4,
m
.
.
. . .
.
.
.
.
.
.
.
m
m
.
.
. .
.
.
.
.
.
.
.
. .
. . .
.
. .
.
. .
. .
. .
.
.
. .
.
.
. .
.
. .
.
.
.
.
.
.
. .
.
.
.
. .
. .
.
.
.
.
.
.
.
.
+
. .
.
.
. .
. .
.
. .
. .
. .
+ .
+
. .
Ot
.
GM10-2B/931
-
Antimycin
cobl
-
GM10-2B/916
GM10-2B
antimycin
cob2
902
914 p- A°FUN
of the Two Mitochondrial
.
.
.
.
. .
.
4, . .
.
4, .
.
.
.
.
.
.
.
+
4.
+
4, 4.
4. 4.
4, +
4. 4.
+
4,
4,
4.
+
+
. .
.
4, .
+ .
. + .
.
4.
4.
+
4-
4,
4.
4,
4.
4,
4,
4.
4.
4,
4.
4.
4.
4.
+
4.
4.
4.
4.
4.
-
+
+
4"
+
4,
+
4.
+
.
.
.
.
.
933 934 935 936
-
+ 4. + +
+ 4, 4. +
+ 4. + +
+ 4. + +
+ 4. + +
+ 4. + +
+ 4. 4. +
4, 4. +
+ 4.
4. 4.
4. 4,
+ 4.
+ 4, -
4, 4, -
+ + -
937 938
--
4, +
4, +
4. 4.
+ +
4. +
+ +
+ +
+ +
4. +
4, +
4, +
4, +
+ +
+ +
+ +
939 940
-
4. 4,
4, 4.
4, 4.
4. 4.
4. 4,
+ 4.
+ 4.
+ 4.
+ 4,
+ 4,
4, 4.
4. 4,
4, 4.
4, 4.
+ +
4,
+
4,
4,
4.
4,
4.
4,
4,
4,
4.
4,
4.
4.
4,
4,
The p- clones, derived from GM10-2B, were dropped on WO plates spread with lawns of the cob- mutants. The diploids grown f o r 2 d a y s a t 28 ° C w e r e r e p l i c a t e d o n N 3 m e d i u m a n d s c o r e d f o r g l y c e r o l g r o w t h a f t e r 2 d a y s . P l u s i n d i c a t e s g l y c e r o l g r o w t h , m i n u s absence of growth and a P some few colonies. Retention with the following two strains FF1123-19C and FF1301-17B
or loss of the two mitochondrial markers a s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s
the total number of p+ colonies, assuming that the frequencies of cob + and c o b - colonies are equal as well as the two types of reciprocal recombinants. It is not possible to deduce the order of c o b l , cob2 and antimycin A resistance mutations from the recombination data of Table 4. To ensure that the recombination values in the ana r x c o b - crosses are not affected by the critical antimycin A resistance phenotype (Pratje and Michaelis, 1977) the crosses were repeated with the diuron resistant mutant D243-4A/
O1 a n d
O11 h a s
been
analyzed
D732 which was found to m a p in the anal locus. Resistant and sensitive colonies can well be distingiushed at 25 gg diuron per ml. The crosses c o b - x diur2-732 and c o b - x anar1-32 gave indentical results (Table 4). Although the two-factor crosses do not yield data on the arrangement of the mutations investigated, this type of cross nevertheless confirms the localization of the two antimycin A resistance loci close to the cob mutations M10-152 and M18-68. N o recombinants could be detected in the respective crosses.
G. Michaelis and E. Pratje: Mapping of the Two Mitochondrial Antimycin A Resistance Loci
Discussion
Table 4. Linkage analysis between mutations of the two antimycin A resistance loci and cob mutations a
AI A32
At D732
Several laboratories have recently described mitochondrial mutations in Saccharomyces cerevisiae which confer resistance to inhibitors acting on the cytochrome bcl complex of the mitochondrial respiratory chain. These mutations include resistance to mucidin (Subik, 1975), diuron (Colson et al., 1977), antimycin A (Michaelis, 1976 ; Burger et al., 1976; Groot Obbink et al., 1977) or funiculosin (Pratje and Michaelis, 1977). Allelism studies indicated the existence of at least two mitochondrial loci for mucidin (Subik et al., 1977), diuron (Colson et al., 1977) and antimycin A resistance (Pratje and Michaelis, 1977) respectively. A genetic analysis is required to allocate the various resistance and mit- mutations to the mitochondrial genetic loci involved in the specification of components of the cytochrome bcl complex. Funiculosin resistance has previously been reported to map at the second antimycin A resistance locus (Pratje and Michaelis, 1977). Preliminary experiments indicate that the two loci for antimycin A resistance are allelic to the two diuron resistance loci (Colson et al., personal communication). In the present work
A~I Fun5
% Rec. total % Rec. total % Rec. total colonies colonies colonies M10-152 M21-71 M17-162 M33-119 M18-68 M8-181 M6-200 M17-231 M15-207 M8-53 M13-101 M24-241 M7-40
0 1.9 0.07 2.4 3.3 2.5 3.4 8.1 3.5 6.9 6.9 1.8 4.7
967 2079 1495 2145 2702 2011 3044 2564 1287 976 1701 883 784
0 2.8 0.07 3.2 3.1 3.2 2.7 6.3 3.9 5 2.4 1.5 1.8
1886 1759 1430 1847 2709 1020 2344 1709 2184 1862 2286 1629 2607
1.3 0.8 0.7 0 0 1.2 3.4 4.8 2.2 2.5 2.1 0.3 2.6
1052 857 1210 1568 930 1065 872 806 679 791 900 1196 574
The c o b - mutants were crossed to D243-4A/A32,/D732,/Fun5 as described in Materials and Methods. After about 10 generations the diploids were spread on WO plates. These WO masters were replicated on N3 and N3 antibiotic media (50 ng antimycin A/ml, 25 pg D C M U / m l or 5 pg funiculosin/ml). Growth on glycerol and the antibiotic media was scored after 2-3 days incubation at 28 ° C
AI
83
All I 1
I I|
I
I
1
I 1
¢xt
C~
!
!
~
I
1
I
C~
C~
1
1
I
I
I
I
I
/
cob 2
cob 1
/
J
J
7 /" f f J
J
Fig. 1. Arrangement of the two antimycin A resistance loci A 1 and A~I on the mitochondrial genome of Saccharomyces cerevisiae. The order of the cob markers follows Tzagoloff et al. (1976). The two antimycin A resistance loci are placed on the map according to the results presented in Tables 2, 3 and 4
84
G. Michaelis and E. Pratje : Mapping of the Two Mitochondrial Antimycin A Resistance Loci
we have mapped the two antimycin A resistance loci AI and A~I in the O i - O n region of mitochondrial DNA, and more precisely in the cob2 locus defined by Tzagoloff et al. (1976). Two independent series of petite clones were used for the deletion mapping. The petite mutants of the first series, isolated and described by Tzagoloff et al. (1976), were characterized by their ability to restore glycerol growth to various cob- mutants. These petite testers were crossed to the antimycin A and funiculosin resistant p ÷ strains, and the diploids were tested for the occurrence of antibiotic sensitive p + cells. Such sensitive p+ colonies are only produced with those petite testers retaining the respective sensitive allele. We have isolated a second series of petite testers, retaining one or both antimycin A resistance loci. These petite strains were crossed to the cob mutants o f T z a g o l o f f e t al. (1976), and the diploids were tested for growth on glycerol. The result of this restoration test is in agreement with the arrangement of cobmutations as determined by Tzagoloff et al. (1976). The two series of petite testers yielded coherent mapping results and allowed the two A g loci to be placed in the cob region. It should be mentioned that the cob2 locus has already been subdivided in four loci, designated box 1, 2, 3 and 4, by Kotylak and Slonimski (1976). Two factor crosses between cob- and antibiotic resistant mutants produced ambiguous results. The cob mutants could not be separated into two groups based on the frequency of recombinants. Never; theless, the very close association of A~ with cob mutation M 10-152 and A R with cob mutation M 18-68 is confirmed since no recombinants could be detected in these crosses. The results of our mapping studies are summarized in Figure 1. In this article we present evidence that the cob region specifies two sites involved in the binding of antimycin A and funiculosin. Several important questions remain to be answered. The number of mitochondrial structural and regulatory genes for the cytochrome bca complex, the number of cytochrome b polypeptide species, and the gene-product relationship for the two A R loci and cytochrome b remain to be established. A biochemical comparison of proteins from antimycin A resistant and sensitive strains, now under investigation in our laboratory, should contribute to the solution of these problems. Acknowledgements. We wish to thank Dr. A. Tzagoloff for kindly providing us with his petite and cob- mutants. Careful reading of the manuscript by Dr. D. Miller is greatly acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft.
References Burger, G., Lang, B., Bandlow, W., Schweyen, R.J., Backhaus, B., Kaudewitz, F. : Antimycin resistance in Saccharomyces ¢ere-
visiae. a new mutation on the mitDNA conferring antimycin resistance on the mitochondrial respiratory chain. Biochem. biophys. Res. Commun. 72, 1201.-1208 (1976) Colson, A.-M., The Van, L., Convent, B., Briquet, M., Goffeau, A.: Mitochondrial heredity of resistance to 3-(3,4-dichlorphenyl)-l,l-dimethylurea, an inhibitor of cytochrome b oxidation, in Saccharomyces cerevisiae. Europ. J. Biochem. 74, 521-526 (1977) Foury, F., Tzagoloff, A.: Assembly of the mitochondrial membrane system XIX. Genetic characterization of mit- mutants with deficiencies in cytochrome oxidase and coenzyme QHzcytochrome c reductase. Molec. gen. Genet. 149, 43 50 (1976) Groot Obbink, D.J., Hall, R.M., Linnane, A.W., Lukins, H.B., Monk, B.C., Spithill, T.W., Trembath, M.K.: Mitochondrial genes involved in the determination of mitochondrial membrane proteins. In: (C. Saccone, A.M. Kroon, eds.), The genetic function of mitochondrial DNA, pp. 163-173. Amsterdam: Elsevier/ North-Holland Biomedical Press 1976 Groot Obbink, D.J., Spithill, T.W., Maxwell, R.J., Linnane, A.W. : Biogenesis of mitochondria 48. Mikamycin resistance in Saccharomyces cerevisiae- A mitochondrial mutation conferring resistance to an antimycin A-like contaminant in mikamycin. Molec. gen. Genet. 151, 127 136 (1977) Jakob, H.: Technique de synchronisation de la formation des zygotes chez la levure Saccharomyces cerebisiae. C.R. Acad. Sci. (Paris) 254, 3909 (1962) Katan, M.B., Van Harten-Loosbroek, N., Groot, G.S.P.: The cytochrome bcl complex of yeast mitochondria. Site of translation of the polypeptides in vivo. Europ. J. Biochem. 70, 409-417 (1976) Kotylak, Z., Slonimski, P.P.: Joint control of cytochromes a and b by a unique mitochondrial DNA region comprising four genetic loci. In: (C. Saccone, A.M. Kroon, eds.), The genetic function of mitochondriai DNA, pp. 143 154. Amsterdam: Elsevier/North-Holland Biomedical Press 1976 Michaelis, G.: Cytoplasmic inheritance of antimycin A resistance in Saccharomyces cerevisiae. Molec. gen. Genet. 146, 133 137 (1976) Michaelis, G., Douglass, S., Tsai, M.-J., Criddle, R.S.: Mitochondrial DNA and suppressiveness of petite mutants in Saccharomyces cerevisiae. Biochem. Genet. 5, 487495 (1971) Pajot, P., Wambier-Kluppel, M.L., Kotylak, Z., Slonimski, P.P.: Regulation of cytochrome oxidase formation by mutations in a mitochondrial gene for cytochrome b. In: (Th. Bticher, W. Neupert, W. Sebald, S. Werner, eds.), Genetics and biogenesis of chloroplasts and mitochondria, pp. 443-451. Amsterdam: Elsevier/North-Holland Biomedical Press 1976 Pratje, E., Michaelis, G.: Two mitochondrial antimycin A resistance loci in Saccharomyces cerevisiae. In: (Th. Bficher, W. Neupert, W. Sebald, S. Werner, eds.), Genetics and biogenesis of chloroplasts and mitochondria, pp. 443 451. Amsterdam: Elsevier/North-Holland Biomedical Press 1976 Pratje, E., Michaelis, G. : Allelism studies of mitochondrial mutants resistant to antimycin A or funiculosin in Saecharomyces cerevisiae. Molec. gen. Genet. 152, 167-174 (1977) Rieske, J.S.: Composition, structure, and function of complex III of the respiratory chain. Biochim. biophys. Acta (Amst.) 456, 195:-247 (1976) Slonimski, P.P., Tzagoloff, A. : Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QHz-cytochrome c reductase. Europ. J. Biochem. 61, 27-41 (1976) Subik, J.: Mucidin-resistant antimycin A-sensitive mitochondrial mutant of Saccharomyces cerevisiae. FEBS Letters 59, 273 276 (I975) SuNk, J., Kovacova, V., Takacsova, G.: Mucidin resistance in yeast. Isolation, characterization and genetic analysis of nuclear
G. Michaelis and E. Pratje : Mapping of the Two Mkochondrial Antimycin A Resistance Loci and mitochondrial mucidin-resistant mutants of Saccharomyces cerevisiae. Europ. J. Biochem. 73, 275 286 (1977) Tzagoloff, A., Akai, A., Needleman, R.B. : Properties of cytoplasmic mutants of Saccharomyces cerevisiae with specific lesions in cytochrome oxidase. Proc. nat. Acad. Sci. (Wash.) 72, 2054-2057 (1975) Tzagoloff, A., Foury, F., Akai, A. : Assembly of the mitochondrial membrane system XVIII. Genetic loci on mitochondrial DNA involved in cytochrome b biosynthesis. Molec. gen. Genet. 149, 33-42 (1976) Weiss, H.: Subunit composition and biogenesis of mitochondrial cytochrome b. Biochim. biophys. Acta (Amst.) 456, 291 313 (1976)
Note Added in Proof
The classification of cob mutations into cobl and cob2 follows Tzagoloff et al. (1976). Slonimski and coworkers have recently presented evidence that the cob- mutations corresponding to the AH locus should rather be regrouped at the terminal part of the cobl region (Colloquium on Genetics and Biogenesis of Mitochondria, Schliersee, August 1977).
85
Weiss, H., Ziganke, B. : Cytochrome b in Neurospora crassa mitochondria. Site of translation of the heme protein. Europ. J. Biochem. 41, 63-71 (1974)
Communicated
by W. Gajewski
Received July 19, 1977
