Molec. gen. Genetics 139, 189--202 (1975) © by Springer-Verlag 1975

Genetic Analysis of Unequal Transmission of the Mitochondrial Markers in Saccharomyces cerevisiae N o r i o Gunge Central Research Laboratory, Mitsubishi Chemical Industries Limited, Yokohama, Japan Received February 12, 1975

Summary. The presence of mitochondrial sex factor, co, was demonstrated in haploid strains of yeast Saccharomyces cerevisiae which came from our laboratory. Transmission and recombination of the mitochondrial genes (C~/C s, E~/E s and OR/O s), conferring the resistance] sensitivity to ehloramphenieol, erythromycin and oligomyein, respectively, were non-polar in homosexual crosses and highly polar in heterosexual crosses. Different results were obtained in crosses involving an erythromyein resistant mutant G706E11 (CSERO s) which was found to contain cellular DNA of diploid level This strain was co- and showed no recombination polarity when crossed to co- haploid strains having the genotype CRE soil, but there was a highly polar transmission, that is, the alleles from G706E11 (Cs, E ~ and 0 s) were transmitted tothe zygote progeny in preference to the CR, Es and OR alleles. When crossed to co+ haploid strains, there was a highly polar recombination, but no transmission was seen for the E and 0 alleles. Polar transmission of markers from co+ haploid parental strain, characteristic of heterosexual crosses, was noticed only for the C allele. The crosses of G706Ell to co+ haploids featured an increase in the recombination frequency. The values of % suppressiveness of ~- petite mutants were relatively low when determined by crossing to G706Ell or to e+ diploid strain M2-8C rather than by crossing to ~+ haploid strains, indicating that there is a positive correlation between the polar transmission of drug resistance markers and the suppressiveness degrees. Genetic mechanism of the anomalous behaviors if mitochondrial genes in crosses involving G706Ell was discussed and interpreted as due to an unbalanced supply of mitochondrial genomes from parental strains. Introduction S t u d i e s on t h e t r a n s m i s s i o n a n d r e c o m b i n a t i o n of m i t o c h o n d r i a l genes in y e a s t were r e c e n t l y a d v a n c e d b y genetic analysis of m u l t i f a e t o r i a l crosses inv o l v i n g t h e c y t o p l a s m i c r e s i s t a n t m u t a n t s to a n t i b i o t i c s such as chloramphenicol, e r y t h r o m y c i n , oligomycin, s p i r a m y c i n a n d p a r o m y c i n (Thomas a n d Wilkie, 1968; Coen et al., 1970; B o l o t i n et al., 1971 ; Kleese et al., 1972; R a n k a n d B e c h - H a n s e n , 1972 ; A v n e r et al., 1973 ; Howell et al., 1973 ; L u k i n s et al., 1973 ; Michaelis et al., 1973 ; T r e m b a t h et al., 1973 ; W i l k i e arid T h o m a s , 1973 ; W o l f et al., 1973 ; D e u t s e h et al., 1974; S u d a a n d U c h i d a , 1974; W a k a b a y a s h i , 1974). I m p o r t a n t f e a t u r e s in t h e m i t o c h o n d r i a l genetics a r e t h e t r a n s m i s s i o n p o l a r i t y (unequal t r a n s m i s s i o n f r o m p a r e n t a l strains of allelic forms of m i t o c h o n d r i a l genes controlling t h e d r u g r e s i s t a n c e / s e n s i t i v i t y ) a n d t h e r e c o m b i n a t i o n p o l a r i t y ( a s y m m e t r i c r a t i o of recipr o c a l - t y p e r e c o m b i n a n t s ) . B o l o t i n ~t al. (1971) h a v e p r o p o s e d t h a t t h e m i t o c h o n d r i a l r e c o m b i n a t i o n is controlled b y t h e m i t o c h o n d r i a l sex factor to a n d h a v e d e s c r i b e d t h a t h e t e r o s e x u a l crosses (~+ xco-) show a h i g h l y p o l a r r e c o m b i n a t i o n , because genomes of to+ m i t o c h o n d r i a are u n i d i r e c t i o n a l l y t r a n s f e r r e d Go ~o- m i t o -

190

N. Gunge

chondria to be led to the r e c o m b i n a t i o n . The transfer of w + m i t o c h o n d r i a l genomes starts at t h e w + locus a n d markers linked to the w + are preferentially t r a n s m i t t e d , causing t h e t r a n s m i s s i o n polarity. I n homosexual crosses (w-×~o- a n d ¢o+ ×~o+), on the contrary, there is no polar t r a n s m i s s i o n a n d r e c o m b i n a t i o n . On the other h a n d , nuclear gene(s) a n d some u n d e f i n i t e factor(s) were reported to affect the p o l a r i t y p h e n o m e n o n of m i t o c h o n d r i a l genes ( B u n n et al., 1970; l~ank a n d B e c h - H a n s e n , 1972; A v n e r et al., 1973; B e c h - t I a n s e n a n d l~ank, 1973 ; Howell et al., 1973 ; W a x m a n et al., 1973 ; Callen, 1974; Gouhier-Monnerot, 1974; W a x m a n a n d E a t o n , 1974). I n t h e present c o m m u n i c a t i o n , we will describe a modification of the t r a n s m i s s i o n p o l a r i t y b y a n a l t e r a t i o n of the nuclear ploidylevel of one of p a r e n t strains used i n m i t o c h o n d r i a l genetic crosses. The ploidy effect is also observed i n d e t e r m i n i n g the percent of suppressiveness of @- m u t a n t s .

Materials and Methods Strains. The origin and genotype of strains, heterothallic Saccharomyces cerevisiae, employed in this study are listed in Table 1. They are all haploids, except for G706Ell, M1-7C and M2-8C. G706Ell was derived from a presumably autopolyploidized cell in the haploid culture, G706E. M1-7C and M2-8C were diploid segregants from an aac~o~tetraploid yeast (Gunge and Nakatomi, 1972). The drug resistance mutations in G706 and G102D were judged as mutations in mitoehondrial genes on the following bases: (1) the resistances were frequently eliminated by @- mutation with the acriflavine or ethidium bromide treatment and (2) there was mitotic segregation, but no meiotic segregation for the resistance phenotype in crosses between the drug resistant and sensitive strains. When resistant mutants to the same drug were crossed, no sensitive clone appeared, which indicated that they were allelic. The CR, E R and 01~ mutations in the above strains were also allelic to their corresponding mutations in the a~ tester strains which were kindly sent from Dr. P. P. Slonimski. Resistant mutants to a given drug did not show any cross resistance to other drugs. Nomenclature. Genetic symbols a and ~ indicate the mating types, his4, leu2, thrg, ado1 ilv, ural, trpl, lys, phe, and arg refer to auxotrophic requirements for histidine, lencine, threonine, adenine, isoleucine, uracil, tryptophan, lysine, phenylalanine and arginine, respectively. The subscript numbers denote the genetic loci. petA, petB and petC are non-aUelic nuclear peptite genes which were formerly designated as rl, r2 and r3, respectively (Gtmge, 1966). @represents the mitochondrial gone responsible for the respiration, m is the mitochondrial sex factor. CR/Cs, ER/E s and OR/Os denote the mitochondrial genes conferring the resistance/sensitivity to chloramphenicol, erythromycin and oligomycin, respectively. In the evaluation of the polarity of transmission and recombination of the drug resistance markers, the C, E and O alleles derived from the mating type a or ~ parental strains were designated, for convenience, Ca or C% E a or E a and 0 a or 0 ~ (in homosexual crosses) and the alleles from the m+ or ~o- mitochondria, C+ or C-, E+ or E- and O+ or O- (in heterosexual crosses), respectively, irrespective of whether the markers indicated the drug resistance or sensitivity. Media. YEPD (complete) medium contained 1% yeast extract, 1% peptone and 2% glucose. Lactate medium contained 0.3% yeast extract, 0.35% peptone, 2% sodium lactate, 0.2% KH2PO4, 0.1% MgSO4.7H20 and 0.1% (Ntt4)2SO~. Drug resistance test media, LC, LE, and LO are lactate medium supplemented with chloramphenicol (3 mg/ml), erythromycin-glucoheptonate (3 mg/ml) and oligomycin (50?/surface of a plate), respectively. Lactate media containing two or three kinds of drugs simultaneously at the above concentrations, LCE, LCO, LEO and LCEO, were used to check the double or triple drug resistance mutation. NBG (minimal) medium contained 0.67% Difco's yeast nitrogen base without amino acids and 2% glucose. NBLG medium which was used for the determination of the percent of suppressiveness of ~- mutants consisted of 0.67% Difco's yeast nitrogen base without amino acids, 2% sodium lactate and 0.1% glucose. For solid media, 2.3% agar was added.

Transmission Polarity of Mitochondrial Genes in Yeast

191

Table 1. The origin and genotype of strains employed in mitoehondrial cross Strain

Genotype

Origin

Nuclear

Mitoehondrial

G706 G102D

a hisg leu2 thr4 ~ adel ilv

co- C s E s o s Q+ co- CSESO s ~+

From our laboratory

G706C G706E G706R

a hisg leu2 thrd a his4 leu2 thrd a his4 leu2 thr4

co- CREsO s Q+ co- CSERO s ~+ co- C s E s o R q+

Spontaneous mutant of G706

G706Ell

Phenotypically the same as G706E

co- CsEROS ~+

Autopolyploidization of G706E

G102E G102R

a adel ilv ~ adel ilv

co- CsE~O s ~+ co- C s E s o ~ ~+

Spontaneous mutant of G102D

O596R-1A O596R-3A

a his4 leu2 thrd a adel

w- CsERO R q+ co- CSERO R Q+

Segregant from a cross, G706R × G102E

0763-61-4C 0763-61-5C

a adel ~ adel

co- CREsO R ~+ co- C~EsO ~ Q+

Segregant from a cross, G706C × G102R

55R5-3C/1 55R5-3C/321 IL126-1C IL836-2A ILS-SD DPI-IB DPl-1B/517

aural aural aural aural aural c~his1 trpl c~his1 trpl

e0- CSESO R ~+ co- CREsO s Q+ co- C~ERO s ~+ co+ CSESO ~ ~+ co+ CREDO s q+ ¢o+ CSES0S ~+ co+ CRES0 s ~+

|

0708-11-8A O708-11-16A

c¢adel ural a adel ural

co+ Cr~E~O R ~+ co+ CREI~O R Q+

t Segregant from a cross, IL8-8D × G102R

M1-7C M2-8C O748-1-6D O747-121-2B 0749-2-11B

~/c¢ trpl/trpl a d e l / A D E a/a trpl/trpl a d e l / A D E ~ ilv lys petA ~ adel phe petB c¢adel arg petC

CSE s o s Q+ C SE so s ~+ C ~ E s 0 s qCRER0 R ~CRERO ~ ~-

I From Dr. Slonimski

Form our laboratory

Clonal Analysis o/the Drug Resistance. Ceils from drug sensitive and resistant strains or ceils from resistant mutants to different drugs having the opposite mating types and complementary auxotrophie markers were mixed in Y E P D medium for 4.5 hrs at 30 ° and a portion of the mating mixture was transferred into N B G medium to allow only the zygote cells to proliferate. After more than 30 generations of growth, the cells of the zygotic progeny which became wholly homoplasmie were spread on N B G medium and a number of resultant colonies were replica-plated on the drug(s) supplemented media: LC, LE, LO, LCE, LCO, LEO and LCEO. The resistance/sensitivity to the drug(s) was diagnosed by the presence/absence of growth of cells after incubation at 30 ° for 3 to 5 days. The Determination o/the Degree o/Suppressiveness. Cells from petite (~-) and wild-type (~+) strains having different auxotrophic markers were mixed in Y E P D medium at 30 ° for 4.5 hrs. After the dilution, the resultant zygote cells were spread on N B L G medium and incubated at 30 ° for 4 days. Since peptite mutants form small colonies on this medium while wild-type cells produce large ones, the percent suppressiveness (% S) of the petite mutant can be claeulated according to the formula, % S -~ (X--Y)/(I.00--Y) × 100 where X is the proportion of small colonies from zygotes and Y is that of spontaneous petites in the wild-

192

~N. Gunge

type parental strain (Sherman and Ephrussi, 1962). When necessary, small colonies were isolated and checked for their inability to grow on lactate medium ~o confirm the petite phenotype. Tetrad Analysis and the Determination o/DNA Content. These were carried out as described previously (Gunge and Nakatomi, 1972).

Results The Mitochondrial Sex in Drug Sensitive and Resistant Strains. B i a n d t r i f a c t o r i a l crosses for t h e m i t o c h o n d r i a l d r u g r e s i s t a n c e m a r k e r s (C~/C s, E R / E s a n d 0 ~ / 0 s) w e r e c a r r i e d o u t b e t w e e n v a r i o u s d r u g s e n s i t i v e a n d r e s i s t a n t h a p l o i d s d e r i v e d f r o m o u r l a b o r a t o r y . T h e r e s u l t s of a r a n d o m clonal a n a l y s i s of t h e dist r i b u t i o n of m i t o c h o n d r i a l t y p e s a r e g i v e n i n T a b l e 2 A . T h e d a t a of t r a n s m i s s i o n a n d r e c o m b i n a t i o n of i n v o l v e d m a r k e r s w e r e c a l c u l a t e d as s h o w n i n T a b l e 2 B , i n d i c a t i n g t h a t t h e r e c i p r o c a l r e c o m b i n a t i o n was n o n - p o l a r , t h a t is, t h e r a t i o s of r e c o m b i n a n t s , CaE~/C~E a, c a o ~ / c a 0 a a n d EaO~/EaO a, a v e r a g e d 0.64/1, 0.95/1 a n d 1.07/1, r e s p e c t i v e l y , t h e v a l u e s b e i n g n o t g r e a t l y d e v i a t e d f r o m 1/1. F u r t h e r m o r e , t h e t r a n s m i s s i o n f r e q u e n c i e s f r o m b o t h p a r e n t s of t h e C, E a n d 0 alleles w e r e n e a r l y e q u a l , a v e r a g i n g 4 7 : 5 3 , 4 8 : 5 2 a n d 4 9 : 5 1 r e s p e c t i v e l y . As d e m o n s t r a t e d in T a b l e s 3 A a n d B, crosses of t h e a b o v e h a p l o i d s t o t h e w - t e s t e r s t r a i n s g a v e s i m i l a r r e s u l t s (crosses nos. 12-15). W h e n crossed t o t h e co+ t e s t e r strains, h o w e v e r , t h e r e was a h i g h l y p o l a r t r a n s m i s s i o n a n d r e c o m b i n a t i o n (crosses nos. 18-23). F r o m t h e s e o b s e r v a t i o n s a n d also f r o m c o m p a r i s o n w i t h d a t a f r o m t y p i c a l h o m o a n d h e t e r o - s e x u a l crosses b e t w e e n co t e s t e r s t r a i n s (crosses nos. 16, 17, 24 a n d 25), l i s t e d in T a b l e s 3 A a n d B for r e f e r e n c e , it c a n b e c o n c l u d e d t h a t G706, G 1 0 2 D a n d t h e d r u g r e s i s t a n t m u t a n t s d e r i v e d f r o m t h e m a r e all 0 7

Table 2A. Analysis of zygotic progeny from bi and tri factorial crosses for the mitochondrial drug resistance markers Number of colonies

Cross No.

Bifactorial cross 1 2 3 4 5 6 7 8

G706C G706C G706 G706 G706E GT06R G706 O596R-1A

x G102E ×G102R x 0763-61-4C x O763-61-5C x G102R x G102E x 0596R-3A x G102I)

954 480 300 360 761 620 265 212

306 178 40 27 -----

547 ---293 272 25 28

-192 30 44 270 196 34 20

34 --------

-37 125 145 -----

----66 51 98 61

---------

67 73 105 144 132 101 108 103

300 360 650

25 31 237

129 114 47

11 9 22

1 5 5

86 140 37

30 38 268

8 17 19

10 6 15

Tr~actorial cross 9 10 11

G706E GT06E G706

x 0763-61-4C x 0763-61-5C × O596R-3A

Transmission Polarity of Mitochondrial Genes in Yeast a n d t h e r e f o r e t h a t crosses nos. 1 - 1 5 a r e h o m o s e x u a l nos. 1 8 - 2 3 a r e h e t e r o s e x u a l (co-×co+).

(co-×co-)

193 w h i l e crosses

T h e p o l a r r e c o m b i n a t i o n i n t h e a b o v e h e t e r o s e x u a l crosses (crosses nos. 1 8 - 2 5 ) w a s n o t i c e d t o b e c h a r a c t e r i z e d b y a n a s y m m e t r y t h a t t h e m a j o r classes of rec o m b i n a n t s a l w a y s c o n s i s t e d of clones of C+E -, C+O - a n d E + O - t y p e s w h i l e t h e m i n o r o n e s c o n s i s t e d of c l o n e s of C - E + , C - O + a n d E - O + t y p e s . I n a d d i t i o n , t h e p o l a r i t y w a s t h e m o s t r e m a r k a b l e i n t h e C - E g e n e p a i r , i.e., c l o n e s of t h e C+E t y p e w e r e p r o d u c e d a b o u t 130 t i m e s m o r e f r e q u e n t l y t h a n t h o s e of t h e C - E + t y p e . T h e p o l a r i t y i n t h e C - O a n d E - O g e n e p a i r s w a s l o w e r , b u t still t h e m a j o r classes p r e v a i l e d a b o u t t e n t i m e s o v e r t h e m i n o r ones. A s t o t h e t r a n s m i s s i o n p o l a r i t y , t h e m a r k e r s f r o m co+ p a r e n t a l s t r a i n s p r e d o m i n a t e d a m o n g t h e d i p l o i d p r o g e n y , b u t t h e m a r k e r f r e q u e n c y w a s f o u n d t o v a r y d e p e n d i n g o n t h e alleles c o n c e r n e d as s h o w n b y t h e a v e r a g e v a l u e s ; 9 0 : 1 0 f o r C + : C - , 7 7 : 2 3 f o r E + : E - a n d 6 4 : 3 6 f o r O +: O - . T h e s e r e s u l t s of t h e p o l a r i t y of r e c o m b i n a t i o n a n d t r a n s m i s s i o n a r e e s s e n t i a l l y t h e s a m e as t h o s e r e p o r t e d b y o t h e r w o r k e r s ( C o e n et al., 1 9 7 0 ; B o l o t i n et al., 1971 ; A v n e r et al., 1973 ; W o l f et al., 1973) a n d a r e c o n s i s t e n t w i t h t h e v i e w t h a t t h e m i t o c h o n d r i a l g e n e o r d e r is c o - C - E - O a n d t h a t t h e p o l a r i t y v a l u e s of m a r k e r s closer t o t h e 6o l o c u s a r e h i g h e r . T h e e s t i m a t e d g e n e o r d e r C - E - 0 is c o m p a t i b l e w i t h t h e v a l u e s of r e c o m b i n a t i o n f r e q u e n c y b e t w e e n t h e r e s p e c t i v e m a r k e r s i n t h e h e t e r o s e x u a l c r o s s e s : 13.3 % i n C - E , 2 5 . i % i n C - O a n d 2 2 . 5 % i a E - O w h i c h i n d i c a t e t h a t t h e E l o c u s lies b e t w e e n t h e C a n d 0 loci, a l t h o u g h a n a l t e r n a t i v e g e n e o r d e r E - C - O m a y b e s u g g e s t e d f r o m t h e r e s u l t s i n t h e h o m o s e x u a l crosses (crosses nos. 1 - 1 7 ) w h e r e t h e f r e q u e n c y v a l u e i n E - O w a s s l i g h t l y h i g h e r t h a n that in C-O.

Table 2 ]3. Analysis of transmission and recombination. (On the basis of the data in Table 2A) Cross No.

Transmission polarity

Recombination polarity

Recombination frequency %

c~:c~

~

c-E

c-o

s-o

----66/132 101/51 108/98 28/20 38/35 55/37 59/52

10.6 -------10.0 10.3 9.4

-22.9 23.7 19.8 ----22.3 23.0 18.8

----26.0 24.6 22.2 22.6 24.3 25.5 17.2

1.07

10.1

21.8

23.2

~:E~

o~:o~

6

~

o

o

5

6

1 2 3 4 5 6 7 8 9 10 11

36:64 45:55 45:55 52:48 ----60:40 46:54 46:54

39:61 ---47:53 48:52 54:46 42:58 56:44 48:52 48:52

-48:52 48:52 47:53 56:44 60:40 50:50 38:62 55:45 43:57 47:53

34/67 -------21/9 24/37

-37/73 30/40 44/27 ----41/26 47/36 56/66

Average

47:53

48:52

49:51

0.64

0.95

15/22

194

N. Gunge

Table3A. Analysis of zygotic progeny from ~he mitochondrial homo and heterosexual crosses

Cross ~o.

Number of colonies

o

B

o

o

o

B

~-X~-

12 13 14 15

G102E G102D G102E G1021~

16 17

IL8-8D IL836-2A

x 551~5-3C/321 × IL126-1C X 55P~5-3C/1 × IL126-1C

832 539 475 360

478 4 -2

324 11 199 0

I -182 120

19 316 -177

---3

--25 3

---33

11 208 69 22

DPI-IB DPI-IB/517

300 417

16 198

20 --

-142

124 --

-31

---

---

140 46

× IL8-8D × DPI-IB/517 X 0708-11-8A X DPI-IB/517 x IL836-2A x ILS-8D × DPI-IB x DPl-1B/517

599 358 540 536 607 358 688 717

83 263 -294 -5 0 493

0 47 42 -176 0 92 --

---108 263 11 -24

467 46 103 --244 34 --

---102 -41

--6 -13 0

199

--

--389 --56 ---

49 2 -32 155 1 562 1

o,)+ X o9+

~-X

18 19 20 21 22 23 24 25

X X

0) +

G102D G706E G706E G706R GL02E G102R IL126-1C 55R5-3C/1

Tetrad Analysis o] Mitochondrial Recombinants.

W h e n cells of m i t o c h o n d r i a l

recoml)iuants were spornlated and subjected to tetrad analysis, there was no meiotic segregation for the r e c o m b i n a n t p h e n o t y p e , while nuclear a u x o t r o p h i c m a r k e r s a n d m a t i n g t y p e s s h o w e d t h e 2 : 2 s e g r e g a t i o n i n all asci d i s s e c t e d . T h e s e g r e g a t i o n d a t a of a C ~ E ~ O ~ r e c o m b i n a n t f r o m a cross, G102t~ × I L S - 8 D , a r e g i v e n i n T a b l e 4 as a n e x a m p l e . U n e x p e c t e d l y , a C ~ E ~ O ~ r e c o m b i n a n t f r o m a cross, G706C × 0 5 9 6 R - 3 A , p r o d u c e d t h e i r r e g u l a r a s c i ; o u t of 27 asci a n a l y z e d , t h r e e g a v e t h e p h e n o t y p i c s e g r e g a t i o n of 3 C ~ E a O R: 1 CSERO ~ w h i l e t h e i n v o l v e d n u c l e a r m a r k e r s w e r e all s e g r e g a t e d i n a 2 : 2 r a t i o . W h e n c r o s s e d t o CSESO s h a p l o i d s t r a i n s , h o w e v e r , all of t h e s e C S E ~ O ~ s e g r e g a n t s w e r e f o u n d t o p r o d u c e C ~ E ~ O ~ d i p l o i d c l o n e s c a p a b l e of g r o w i n g o n L C E O m e d i u m c o n t a i n i n g t h e t h r e e d r u g s s i m u l t a n e o u s l y , so i t is h i g h l y l i k e l y t h a t t h e y c a r r i e d t h e C ~ g e n e b u t the p h e n o t y p i c expression was m a s k e d for some reasons. Thus, the results of t e t r a d a n a l y s i s c o u l d b e t a k e n t o i n d i c a t e t h a t clones of r e c o m b i n a n t - t y p e w e r e all d u e t o t r u e g e n e t i c r e c o m b i n a t i o n .

Analysis o/ Crosses Involving G706E11. A n e r y ~ h r o m y c i n r e s i s t a n t m u t a n t G 7 0 6 E 1 1 h a v i n g t h e cell size l a r g e r t h a n h a p l o i d w a s i s o l a t e d f r o m t h e G 7 0 6 E c u l t u r e w i t h t h e a i d of a m i c r o m a n i p u l a t o r . A s l i s t e d i n T a b l e 5, t h e c o m p a r a t i v e d e t e r m i n a t i o n of D N A c o n t e n t of cells f r o m v a r i o u s c r o s s e s i n d i c a t e d t h a t a

T r a n s m i s s i o n P o l a r i t y ol M i t o c h o n d r i a l Genes in Y e a s t

195

T a b l e 3 B . A n a l y s i s of t r a n s m i s s i o n a n d r e c o m b i n a t i o n . (On t h e b a s i s of t h e d a t a i n T a b l e 3A ) H o m o s e x u a l cross Cross

Transmission polarity

Recombination polarity

Recombination frequency %

~o.

Ca:C ~

12 13 14 15

Ea:E ~

Oa:O ~

~

~

5

5

~

C-E

C -O

E-O

60:40 40:60 -60:40

59:41 3 9:61 53:47 5 9:41

--44:56 56:44

19/11 4/11 -5/3

---36/22

--69/25 36/24

3.6 2.8 -2.3

---16.1

--19.7 16.7

16 17

47:53 45:55

48:52 --

-41:59

16/20 ---

-46/31

---

12.0 --

-18.4

---

Average

50:50

52:48

47:53

0.98

1.55

2.14

5.2

17.3

18.2

CO+ X O) ÷

H e t e r o s e x u a l cross Cross No.

Transmission polarity

Recombination polarity

Recombination frequency %

C+:C -

~

© +

+ ©

C-E

C -O

E-0

I

I

I

+

+

+

83/0 46/2 ---46/0 92/0 --

--103/6 102/32 -97/1 -85/1

----155/13 56/6 ---

13.8 12.9 ---12.9 13.4 --

--20.2 25.0 -27.3 -28.0

----27.7 17.3 ---

133.5

9.68

11.1

13.3

25.1

22.5

E+:E-

0+:0 -

~- X ~+

18 19 20 21 22 23 24 25

92:8 86:14 91:9 74:26 -97:3 95:5 96:4

78:22 74:26 --69:31 84:16 82:18 --

--73:27 61:39 45:55 70:30 -69:31

Average

90:10

77:23

64:36

cross of G706Ell

to a haploid

0763-61-5C

of t r i p l o i d c e l l l e v e l a n d t h e r e f o r e t h a t In addition, with

respect

quirements.

G706Ell to

autopolyploidization cross,

showed the same phenotypes

nuclear

Thus,

consists of cells containing

one

markers could

in the

G706E11 ×0763-61-5C,

such

assume

G706E were

the DNA

G706E11 is in fact diploid or near diploid. as the original culture G706E

as the mating that

G706E11

type

and

emerged

auxotrophic as the

culture. To check this genetically, sporulated

and

a tetrad

analysis

re-

result

of

cells of a was

con-

196

N. Gunge Table 4. Meiotic segregation data from mitochondrial recombinants

Strain

No. of asci tested

Phenotypic segregation in ascus (CRERO~:CSE~OR)

C~E~0 ~ type recombinant from a cross, G1021~ × ILS-SD

25

4:0 (in all asci)

C~E~O R type recombinant from a cross, G706C × 0596P~-3A

27

4:0 (in 24 asei) 3 : 1 (in 3 asci)

All of the nuclear markers involved were segregated in a 2: 2 ratio in all asei.

Table 5. Determination of DNA content/cell Strain G706C (a) G706C (a) M1-7C (as) G706E (a) G706Ell (a')

× × × × ×

G1021~ (~) 05961~3A (=) 0708-11-16A (a) 0763-61-5C (=) 0763-61-5C (~)

Diploid Diploid Triploid Diploid

DNA (~g/10s cells)

DNA/Ploidy

3.96 4.39 6.00 4.28 6.40

1.98 2.20 2.00 2.14 2.13 a

a G706Ell was estimated as diploid.

ducted. Because of poor g e r m i n a t i o n of spores, complete t e t r a d s were n o t o b t a i n e d , b u t t h e 60 s u r v i v e d r a n d o m spores showed t h e p h e n o t y p i c segregation of 10 a ' : 5 ~ ' : 4 5 n o n - m a t e r s for t h e m a t i n g types. The r a t i o d e v i a t e s from 30 a ' : 1 0 ~ ' : 2 0 n o n - m a t e r s which is e x p e c t e d of a n a a : c t r i p l o i d meiosis (Emerson, 1956) a n d hence, it m a y he i n a d e q u a t e to r e g a r d G 7 0 6 E l l s i m p l y as a n a a diploid. H o w e v e r , t h e frequent segregation of n o n - m a t e r s a n d t h e u n e q u a l r a t i o of a ' : ~ ' still f a v o r t h e view t h a t G T 0 6 E l l is a p o l y p l o i d h a v i n g t h e m a t i n g p h e n o t y p e a (Gunge a n d N a k a t o m i , 1972). Thereupon, it a p p e a r e d interesting to analyze how t h e a u t o p o l y p l o i d i z a t i o n would affect t h e genetic t r a i t s of m i t o c h o n d r i a l genes. F o r t h i s purpose, G706E11 was crossed t o h a p l o i d strains which were p r e v i o u s l y i n v o l v e d in t h e crosses b y GT06E (crosses nos. 5, 9, 10, 19 a n d 20), in order to c o m p a r e t h e results of elonal analysis. The daYa are given in Tables 6 A a n d B. F r o m these it is clear t h a t G T 0 6 E l l showed no p o l a r i t y of r e c o m b i n a t i o n when crossed t o t h e ~o- strains (crosses nos. 26-28), b u t gave p o l a r r e c o m b i n a t i o n when crossed to t h e w + strains (crosses nos. 29 a n d 30), i n d i c a t i n g t h a t t h e m i t o e h o n d r i a l sex is ~o- like G706E. As to t h e t r a n s m i s s i o n p o l a r i t y , however, t h e results of crosses b y G 7 0 6 E l l were different from those of crosses b y G706E. F i r s t l y , in t h e crosses t o t h e costrains, which were homosexual, alleles from G T 0 6 E l l (C a, Ea a n d O a) were p r e f e r e n t i a l l y c o - t r a n s m i t t e d into t h e zygote p r o g e n y w i t h a f r e q u e n c y of a b o u t 70 to 80%. Secondly, in t h e crosses to t h e o~+ strains, which were heterosexual, frequent t r a n s m i s s i o n of m a r k e r s from t h e co+ strains was seen only for t h e C allele a n d t h e r a t i o of C+ to C- was significantly lower t h a n t h a t of crosses b y G706E, while t h e t r a n s m i s s i o n of t h e E a n d O alleles was n o n - p o l a r as if t h e y were h o m o s e x u a l crosses. I n addition, t h e h e t e r o s e x u a l crosses involving G706E11

T r a n s m i s s i o n P o l a r i t y of M i t o c h o n d r i a l G e n e s in Y e a s t

197

T a b l e 6 A . A n a l y s i s of z y g o t i c p r o g e n y f r o m crosses of G 7 0 6 E l l to t h e w - a n d w + h a p l o i d s N u m b e r of colonies

Cross No.

Cross to ~ 26 27 28

G706Ell G706Ell G706E11

Cross

29 30

× 0763-61-4C × O763-61-5C ×G102R

600 359 955

31 24 --

391 235 611

12 8 175

3 3

79 37

47 37 81

15 5 --

22 10 88

× DPI-1B/517 × 0708-11-8A

830 297

402 --

235 52

---

188 101

---

-3

-141

5 --

t o ~o+

G706Ell G706Ell

T a b l e 6 B. A n a l y s i s of ~ r a n s m i s s i o n a n d r e c o m b i n a t i o n . (On t h e basis of t h e d a t a in T a b l e 6 A) Cross :No.

Transmission polarity

Recombination polarity

Recombination frequency %

¢d

Ca:C ~

Ea:E ~

Oa:O ~

~

•©

~©

C-E

C-O

E-O

26 27 28

79:21 81 : 19 --

76:24 78: 22 72:28

74:26 76: 24 73:27

34/18 18/8 --

59/34 45/27 --

62/53 42/34 81/88

8.7 7.2 --

15.2 20.0 --

19.2 21.0 17.7

Average

80:20

75:25

74:26

2.00

1.68

1.07

8.0

17.6

19.3

Cross No.

Transmission polarity C +: C -

E +: E -

Recombination polarity

0 +: O -

+

+

+

+

+

+

Recombination frequency% C-E

C-O

E-O

29 30

71:29 82:18

49:51 --

-48:52

188/5 --

-100/3

---

23.2 --

-34.7

---

Average

77:23

49:51

48:52

37.6

33.3

--

23.2

34.7

--

featured an increase in the recombination frequency as compared with the corr e s p o n d i n g c r o s s e s b y G 7 0 6 E , i.e. f r o m 1 2 . 9 t o 2 3 . 2 % i n t h e C - E g e n e p a i r a n d from 20.2 to 34.7%

in the C-O gene pair.

The E//ect o~ Ploidy-elevation on the Degree o~ Suppressiveness. ~)- m u t a n t s total,

to wild-type

(Q+) s t r a i n s , t h e p r o p o r t i o n

( ~ - / ~ - @ Q+), v a r i e s b e t w e e n

0 and near

100%,

o f ~)- z y g o t i c depending

111 c r o s s e s o f clones to the

o a Q- m u t a n t s

198

N. Gunge Table 7. Suppressiveness of @- mutants when crossed to various @+strains

@- mutant

% suppressiveness when crossed to the following @+ strains +

¢ I

G1021~-SP, Spontaneously arisen

50

42

54

49

51

9

13

G102R-EB, Ethidium bromide induced

31

27

35

33

41

21

14

G102D-AF, Acriflavine induced

10

11

13

12

11

5

7

0748-1-6D, Nuclear petite a gene (petA) associated

72

70

81

71

66

51

43

O747-121-2B, Nuclear petite a gene (petB) associated

47

58

62

55

60

33

23

0749-2-11B, Nuclear petite a gene (petC) associated

55

51

46

56

56

29

19

a These were isolated as spontaneous mutants, respectively, from the nuclear petites (TetA,

petB and petC) which are associated with the highly unstable @factor (Gunge, 1966).

and the values are defined as the degree of suppressiveness after the correction for the frequency of spontaneous @- m u t a t i o n in the wild-type mating partners (Ephrussi et al., 1955). Considering the fact t h a t the @- factor is m u t a t e d mitochondrial D N A (Mounolou et al., 1966; Bernardi et al., 1968 ; Hollenberg et al., 1972), it seemed interesting to know whether the degree of suppressiveuess would be affected b y alterations in genetic conditions responsible for the recombination and/or transmission of the drug resistance genes. For this purpose, various @m u t a n t s (sponteneously arisen, chemically induced and nuclear petite geae associated) were examined for their suppressiveness-degrees b y crossing to wild-type straius differing in the mitochondrial sex or in the level of nuclear ploidy. As listed in Table 7, the values of suppressiveness of petite m u t a n t s tested were found to v a r y considerably from cross to cross ranging from 5 to 81%. However, when the comparison was made amongst a series of crosses of a given petite to various wild-type strains, the effect of ploidy-elevatioa was clearly noticed, t h a t is, the crosses involving G 7 0 6 E l l or diploid M2-8C as a @+parental strain resulted in relatively low degrees of suppressiveness. No significant difference was observed a m o n g the crosses where haploid strains were employed as a @+ parent, irrespective of whether t h e y were ~o+ or ~o-.

Discussion Mitochondrial resistant m u t a n t s to chloramphenicol, e r y t h r o m y c i n or oligom y c i u were isolated from G706 and G102D, haploids of Saccharomyces cerevisiae. The recombination s t u d y of the resistance markers indicated t h a t all of the drug

Transmission Polarity of Mitochondrial Genes in Yeast

199

resistant and sensitive strains were co- with respect to the mitochondrial sex. A polyploid erythromycin resistant m u t a n t G706Ell derived from a haploid G706E probably as the result of an autopolyploidization was also co-. Thus it appears that the nature of mitochondrial sex factor ~o is stable and is not affected either by the resistance mutations to drugs or b y an alteration of nuclear ploidy. Contrary to the recombination polarity, on the basis of which the nature of the o factor was determined, the transmission polarity of the mitoehondrial markers was greatly affected b y the elevation of nuclear ploidy-level of parental strains employed in the mitoehondrial genetic crosses. I n other words, in homosexual crosses, G706E11 showed a highly polar transmission of its markers. However, the degrees of transmission polarity of the C, E and 0 alleles were almost equal, in contrast to typical haploid by haploid heterosexual crosses where the marker frequertey varied according to whether the alleles concerned were close to or distant from the co locus, indicating that the mechanisms responsible for the transmission polarity were different from each other. The phenomenon of polar transmission in homosexual crosses was also observed when diploid mater such as M1-7C or M2-8C was used as one of parent strains (Gnnge in preparation). Thus it seems certain t h a t the above effect of G706Ell is ascribable to the elevation of nuelear-ploidy up to the diploid or near diploid level and m a y be accounted for in the light of the fact that diploid cells contain twice as much mitoehondrial DNA as haploid cells (Marmur, 1972; Grimes et al., 1974), because, under such circumstances, one could expect that when a cross is made between c)- haploid and co- diploid cells, the resulting zygotes receive twice as m a n y mitoe.hondrial genomes from the diploid parent as from the haploid parent and therefore preferentially co-transmit the diploid derived markers into triploid progeny. I n heterosexual crosses between co- diploid and co+ haploid cells, on the other hand, the patterns of marker transmission would be rather complex on account of interactions between both polarity phenomena, one due to an excess supply of co- genomes from the diploid parent, as discussed above, and the other due to the selective transfer of co+ genomes from the haploid parent to co- mitochondria fr0m the diploid parent. I n such crosses, it could Joe expected that polar recombination betweer~ the resistance markers would first occur between pairs of coand co+ genomes derived from both parents and then that the resulting recombinant-type genomes which should be co+ according to the mechanism proposed b y Bolotin et al. (1971), as well as parental-type co+ genomes, would be led to further polar recombination with the rest of co- genomes derived from the diploid parent. Such being the ease, the transmission frequency of the C, E and O alleles from the co+ haploid parent could he roughly estimated as 81, 59 and 41%, respectively, b y squaring the corresponding values obtained in haploid b y haploid heterosexual crosses which amounted to 90, 77 and 64%, respectively, according to the data given in Table 3B (crosses nos. 18-25). The estimated values are in good accordance with the experimental results, i.e. the decreased polar transmission of the C allele from an co+ haploid parent and the non-polar transmission of the E and O alleles. An assumption of repeated occurrences of polar recombination is also compatible with the observation that the recombination frequency was increased in crosses of G706Efl to co+ haploids.

200

N. Gunge

I n the present study, we have reported t h a t no polarity of transmission was seen in haploid by haploid homosexual crosses, however, it must be noted that this was not always true and that polar transmission of markers occurred in some haploid by haploid homosexual crosses in other lines of experiments (Gunge, unpublished data). Similar observation was made b y other workers and designated as the D6 effect (Avner et al., 1973). The effects of the mating types or other nuclear genes on the transmission polarity were also suggested (Coen et al., 1970; Bunn et al., 1970 ; Bech-ttansen and Rank, 1973 ; W a x m a n et al., 1973 ; W a x m a n and Eaton, 1974 ; Callen, 1974). Thus, m a n y different mechanisms m a y be considered as reasons for the modifications of the transmission polarity, however, it appears necessary to add that an unbalanced supply of mitochondrial genomes from haploid parents is one of the possible mechanisms. The proportion of mitochondrial DNA to nuclear or total DNA in yeast cells was reported to v a r y among strains from different sources (Fuknhara, 1969 ; Wiliamson, 1970) or under different conditions of growth (Moustacchi and Williamson, 1966; Smith et al., 1968; Bleeg et al., 1972). The degrees of suppressiveness of a given @- petite mutant were about the same when crossed to a group of @+ haploid strains, irrespective of the nature of their mitochondrial sex factor, but were relatively decreased when crossed to GT06Ell or to diploid mater M2-8C. The results m a y also be accounted for b y assuming the increased amounts of mitochondrial DNA per cell in yeast having the elevated ploidy, since the suppressiveness phenomenon was interpreted as due to the difference in replicative rates between the wild-type (@+) and mutated (@-) mitochondrial DNA in zygote or zygotic progeny (Carnevali et al., 1969; Rank, 1970a; Rank, 1970b); in such a case, it can be expected that an excess supply of the @+ DNA from a diploid parent counteracts the rapid replication of the @- DNA and consequently gives rise to a decrease in the values of suppressiveness. The above conception would be well supported b y the t~ank's observation that the ploidy-elevation of a @- petite mutant was characterized by an increase in the suppressiveness value (Rank, 1970b). Recently, an alternative mechanism was proposed to suggest that the variation in values of suppressiveness is ascribable to the different frequency of recombination between the @+ and @- mitochondrial D ~ A (Coen et al., 1970; Deutsch et al., 1974). I n this connection, it seems interesting to remember that the frequency of recombination between the resistance markers was affected by the change in nnclear ploidy-level of parental strains. Whatever the mechanism of the suppressiveness phenomenon, i t should be noted that a positive correlation was observed be~ tween the polar transmission of the drug resistance markers and the degrees of suppressiveness.

References Avner, P. t~., Coen D., Dujon, B., Slonimski, P. P. : Mitochondrial genetics. IV. Allelism and mapping studies of oligomycin resistant mutants in S. cerevisiae. Molec. gen. Genet. 12~, 9-52 (1973) Bech~H~nsen, N.T., Rank, G.H.: The bivious suppressiveness of cytoplasmic petites of S. cerevisiae lacking in mitochondrial DNA. Molec. gem Genet. 120, 115-124 (1973)

Transmission Polarity of Mitoehondrial Genes in Yeast

201

Bernardi, G., Carnevali, F., Nieolaieff, A., Piperno, G., Tecee, G. : Separation and charaeter~ ization of a satellite DNA from a yeast cytoplasmic petite mutant. J. tool. Biol. 37, 493505 (1968) Bleeg, H. S., Leth Bak, A., Christiansen, C., Smith, K . E . , Stenderup, A. : MitoehondriM DNA and glucose repression in yeast. Biochem. biophys. Res. Commun. 47, 524-530 (1972) Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo, E., Slonimski, P. P.: La recombinaison des mitochondries chez Saccharomyces cerevisiae. Bull. Inst. Pasteur (Paris) 69, 215-239 (1971) Bunn, C.L., Mitchell, C.H., Lukins, H.B., Linnane, A. W. : Biogenesis of mitochondria, XVIII. A new class of cytoplasmieally determined antibiotic resistant mutants in Saccharomyces cerevisiae. Proc. nat. Aead. Sci. (Wash.) 67, 1233-1240 (1970) Callen, D. : The effect of mating type on the polarity of mitochondrial gene transmission in Saceharomyces cerevisiae. Molec. gem Genet. 128, 321-329 (1974) Carnevali, F., Morpurgo, G., Tecee, G. : Cytoplasmic DNA from petite colonies of Saecharomyees cerevisiae: A hypothesis on the nature of the mutation. Science 163, 1331-1333 (1969) Coen, D., Deutsch, J., Netter, P., Petroehilo, E., Slonimski, P. P. : Mitochoudrial genetics. I. Methodology and phenomenology. In: Control of organelle development. Symp. Soe. Exp. Biol. (P. L. Miller, ed.), vol. 24, p. 449-496. Cambridge: Cambridge University Press 1970 Deutseh, J., Dujon, B., Netter, P., Petroehilo, E., Slonimski, P. P., Bolotin-Fukuhara, M., Coen, D. : Mitoehondrial genetics. VI. The petite mutation in Saccharomyces cerevisiae: Interrelations between the loss of the @+factor and the loss of the drug resistance mitochondrial genetic markers. Genetics 76, 195-219 (1974) Emerson, S. : Notes on the identification of different cases of aberrant tetrad ratios in Saccharomyces. C. R. Lab. Carlsberg, S6r. Physiol. 26, 71-86 (1956) Ephrussi, B., Margerie-Hottinguer, H., Roman, H.: Suppressivene_ss: A new factor in the genetic determinism of the synthesis of respiratory enzymes in yeast. Proc. nat. Acad. Sci. (Wash.) 41, 1065-1071 (1955) Fukuhara, It. : P~elative proportions of mitochondrial and nuclear DNA in yeast under various conditions of growth. Europ. J. Biochem. 11, 135-139 (1969) Gouhier-Monnerot, M. : Ethidium bromide resistance and enhancement of mitoehondrial recombination. Molec. gen. Genet. 130, 65-79 (1974) Grimes, G.W., Mahler, It., Perlman, P. S.: Nuclear gene dosage effects on mitochondrial mass and DNA. J. Cell Biol. 61, 565-574 (1974) Gunge, N. : Complementary genes controlling respiration of yeast and their relation to the stability of the cytoplasmic factor. Jap. J. Genet. 41, 215-223 (1966) Gunge, N., Nakatomi, Y. : Genetic mechanisms of rare matings of the yeast Saceharomyces eerevisiae heterozygous for mating type. Genetics 70, 41-58 (1972) ltollengerg, C.P., Borst, P., van Bruggen, E. F. J. : Nitochondrial DNA from cytoplasmic mutants of yeast. Bioehim. biophys. Acta (Amst.) 277, 35-43 (1972) Howell, N., Trembath, M. K., Linnane, A. W., Lukins, It. B. : Biogenesis of mitochondria, 30. An analysis of polarity of mitochondrial gene recombination and transmission. Molee. gen. Genet. 122, 37-51 (1973) Kleese, R.A., Grotbeek, t~. C., Snyder, J . R . : Recombination among three mitoehondrial genes in yeast (Saecharomyees cerevisiae). J. Bact. 122, 1023-1025 (1972) Lukins, H. B., Tate, J. R., Saunders, G. W., Linnane, A. W. : The biogenesis of mitochondria, 26. Mitochondrial recombination: the segregation of parental and recombinant mitochondrial genotypes during vegetative division of yeast. Molec. gem Genet. 129, 17-25 (1973) )~:[armur, J. : Personal eommun. (1972) )~ichaelis, G., Petrochilo, E., Slonimski, P. P. : Mitoehondrial genetics. II1. Recombined molecules of mitochondrial DNA obtained from crosses between cytoplasmic petite mutants of S. cerevisiae: physical and genetic characterization. Molec. gen. Genet. 123, 51-65 (1973) ~[ounolou, J. C., Jakob, H., Slonimski, P. P. : Mitochondrial DNA from yeast petite mutants. Specific changes of buoyant densities corresponding to different cytoplasmic mutations. Bioehem. biophys. Res. Commun. 24, 218-224 (1966)

202

N. Gunge

Moustacchi, E., Williamson, D. H. : Physiological variations in satelite components of yeast DNA detected by density gradient centrifugation. Biochem. biophys. Res. Commun. 23, 56-61 (1966) Rank, G. It. : Genetic evidence for 'Darwinian' selection at the molecular level. I. The effect of the suppressive factor on cytoplasmically-inherited erythromycin-resistance in Saccharomyces cerevisiae. Canad. J. Genet. Cytol. 12, 129-136 (1970a) Rank, G. H. : Genetic evidence for 'Darwinian' selection at the molecular level. II. Genetic analysis of cytoplasmieally-inherited high and low suppressivity in Saccharomyees cerevisiae. Canad. J. Genet. Cytol. 12, 340-346 (1970b) Rank, G.H., Bech-Hansen, N.T.: Somatic segregation, recombination, asymmetrical distribution and complementation tests of eytoplasmically-inherited antibiotic-resistance mitochondrial markers in S. eerevisiae. Genetics 72, 1-15 (1972) Sherman, F., Ephrussi, B. : The relationship between respiratory deficiency and suppressiveheSS in yeast as determined with segregational mutants. Genetics 47, 695-700 (1962) Smith, D., Tauro, P., Schweizer, E., Halvorson, H. O. : The replication of mitochondrial DNA during the cell cycle in Saccharomyces laetis. Proc. nat. Acad. Sci. (Wash.) 60, 936-942 (1968) Suda, K., Uchida, A. : The linkage relationship of the cytoplasmic drug-resistance factors in Saccharomyces cerevisiae. Molec. gen. Genet. 128, 331-339 (1974) Thomas, D. Y., Wilkie, D. : Recombination of mitoehondrial drug-resistance factors in Saceharomyces cerevisiae. Biochem. biophys. Res. Commun. 80, 368-372 (1968) Trembath, M. K., Bunn, C. L., Lukins, H. B., Linnane, A. W. : Biogenesis of mitochondria, 27. Genetic and biochemical characterization of cytoplasmic and nuclear mutations to spiramyein resistance in Saccharomyces cerevisiae. Molec. gen. Genet. 121, 35-48 (1973) Wakabayashi, K. : Studies of the mitochondrial gene. 1. The recombination of mitoehondrial drug resistances. J. Antibiot. (Tokyo) 27, 373-378 (1974) Waxman, M. F., Eaton, N. R. : Nuclear factors and the control of suppressiveness in petite mutants of Saecharomyees cerevisiae. Molec. gen. Genct. 188, 37-45 (1974) Waxman, M. F., Eaton, N. R., Wilkie, D. : Effect of antibiotics on the transmission of mitochondrial factors in Saceharomyces cerevisiae. Molec. gen. Genet. 127, 277-284 (1973) Wilkie, D., Thomas, D.Y.: Mitochondrial genetic analysis by zygote cell lineages in Saccharomyces cerevisiae. Genetics 78, 367-377 (1973) Williamson, D.H.: The effect of environmental and genetic factors on the replication of mitochondrial DNA in yeast. In: "Control of organelle development" Syrup. Soc. Exp. Biol. (P. L. Miller, ed.), vol. 24, p. 247-276. Cambridge: Cambridge University Press 1970 Wolf, K., Dujon, B., Slonimski, P. P. : Mitochondrial genetics. V. Multifactorial mitochondrial crosses involving a mutation conferring paromyein-resistance in S. cerevisiae. Molec. gen. Genet. 125, 53-90 (1973) C o m m u n i c a t e d b y F. K a u d e w i t z Dr. Norio Gunge Central Research Laboratory Mitsubishi Chemical Industries Ltd. 1000 Kamoshida-cho Yokohama 227

Japan