Extrachromosomal inheritance inSchizosaccharomyces pombe

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Molec. gen. Genet. 164, 309-320 (1978) © by Springer-Verlag 1978

Extrachromosomal Inheritance in

Schizosaccharomycespombe

VII. Studies by Zygote Clone Analysis on Transmission, Segregation, Recombination, and Uniparental Inheritance of Mitochondrial Markers Conferring Resistance to Antimycin, Chloramphenicol, and Erythromycin G. Seitz-Mayr, K. Wolf*, and F. Kaudewitz Genetisches Institut der Universitfit Mfinchen, Maria-Ward-Str. 1a, D-8000 Miinchen 19, Federal Republic of Germany

Summary. Crosses involving mitochondrial markers conferring resistance to antimycin (ana r, AR), chloramphenicol (cap r, C~), and erythromycin (eryr, E R) in cis- and trans-configuration were studied by zygote clone analysis. Mutant and-& from which all other drug - resistant isolates were derived, exhibits a highly biased transmission (6.8% ana r) in an analysis of 100 individual zygote clones: Important results of zygote clone analyses were: - Zygote clones may contain one, two, three, or four mitochondrial genotypes. - The proportion of the two parental and the two recombinant genotypes in individual zygote clones can vary almost over the entire range of percentages. - Proportions of the two corresponding recombinant types in individual clones are usually unequal. - Transmission rates of markers are higher in transthan in cis-crosses, indicating additivity of bias by two mutated alleles in coupling, - Transmission rates are different for the three markers both in cis- and trans-crosses, being lowest for C R and highest for E R. - Up to more than 80% uniform clones, expressing only one genotype, can be produced in cis- and transcrosses. In cis-crosses always the double-sensitive parental type becomes uniform, in trans-crosses this may be the case for parental and/or recombinant genotypes. A tentative map is presented using data from cisand trans-crosses, including a correction by omission of uniform clones. Phenomena of transmission, segregation, and formation of uniform clones are discussed with special regard to the difference brought about by fission versus budding. A comparison with relevant data from Saccharomyces cerevisiae and other organisms is presented. *

To whom reprint requests should be sent

Introduction Analysis of transmission, segregation, and recombination of mitochondrial markers in yeast in the past by several workers was carried out mainly by two types of experiments: the first based essentially on the progeny of a population of cells, the second on the progeny of an individual cell heterozygous for mitochondrial genetic markers. The first approach has led Dujon et al. (1974) to a quantitative application of the Visconti-Delbrtick model for phage crosses (Visconti and Delbrtick, 1953) to mitochondrial inheritance in Saccharomyces cerevisiae (S. c.). Since the model of Dujon et al. fits nicely with many data obtained by random progeny analyses, it is of interest to see, i f it is also applicable to data obtained by zygote clone analysis. Extensive studies of individual clones suggest, that panmixis (an important prerequisite for the phage analogy model), is established very slowly and therefore only to a limited extent in zygotes. This delay in cytoplasmic mixing gives rise to a non-random segregation during the first zygote generations. Indeed, the systematic zygote clone analysis reported by Birky (1975) and Birky etal. (1978) clearly show, that most crosses contain classes of zygotes not predicted by the phage analogy model. Birky et al. have also demonstrated that in some crosses large numbers of zygote clones are pure (uniparental) for one genotype or another. This indicates that there is a mechanism active in at least some zygotes which effectively eliminates one genotype or the other. The model of Dujon et al., does not deal with this phenomenon, and may therefore overlook at least one important feature in yeast mitochondrial genetics. Therefore, a more comprehensive view especially including initial events in transmission and segregation may be obtained by zygote clone analysis. In recent papers (Wolf et al., 1976a, b, c, d; Del Giudice etal., 1977; Seitz etal., 1977; Wolf and

0026-8925/78/0164/0309/$02.40

310

G. Seitz-Mayr et al. : Extrachromosomal Inheritance in Schizosaccharomyces pombe. VII

Kaudewitz, 1977) we have initiated a study of mitochondrial inheritance in the fission yeast Sehizosaccharomyces pombe (S. p. ) for two main reasons. Firstly, we wanted to investigate the genetic properties of the mitochondrial genome of a petite-negative yeast. Secondly, we intended to study consequences of cell division by regular fission (in comparison to budding) on transmission, segregation, and recombination of mitochondrial markers. The study of mitochondrial inheritance by the method of zygote clone analysis in this simple eukaryote may give insights in general features of mitochondrial inheritance in higher eukaryotes. Isolation and preliminary characterization of mutants used in this study already have been described by Seitz et al. (1977).

Materials and Methods

Table 2. Terminology of possible mitochondrial phenotypes of sec-

ondary zygote clones P1

P2

R1

R2

Abbreviation

Terminology

4. + + 4.

+ 4, + +

+ + -

+ 4.

PPRR PPRO PPO0 PPO0

biparental

birecombinant unirecombinant 1 unirecombinant 2 nullirecombinant

44+ 4,

-

4, 4,

+ +

PORR PORO POOR PO00

uniparental 1

birecombinant unireeombinant 1 unireeombinant 2 nullirecombinant

+ + + 4.

+ + -

+ +

OPRR OPRO OPOR OP O 0

uniparental 2

birecombinant unirecombinant l unireeombinant 2 nullirecomb inant

+ +

+ OORR - " OORO + O00R

nulliparental

birecombinant unirecombinant 1 unirecombinant 2

1. Yeast Strains and their origins are listed in Table 1 2. Nomenclature is described in detail by Del Giudice et al. (1977) 3. M e d i a and Chemicals are described by Wolf et al. (1976a) and

Seitz et al. (1977) 4. S t a n d a r d Crosses (Type 2 - s e g r e g a t i o n analysis) are described

by Del Giudice et al. (1977)

P1 =parental type 1 P 2 = parental type 2 R l = r e c o m b i n a n t type 1

R2=recombinant type 2 + / - =presence or absence of this particular phenotype (P1, P2, R1, R2) among secondary zygote clones issued from a single primary zygote clone P P R O = P1, P2, and R 1 present, R 2 absent in a sample of secondary zygote clones

5. Z y g o t e Clone Analysis Haploid strains of the mating types h and m e i 1-102 were grown in glucose-complete-medium (GLU) to

late logarithmic phase, washed twice with 0.85% NaC1 and resuspended in the same volume. 0.1 ml of each strain was pipetted onto the surface of synthetic sporulation agar (SPA) and incubated at 25 ° C for 18~4 h to allow zygote formation. The mating mixture containing approximately 1% zygotes was suspended in 1 ml NaC1solution and plated 10- and 100fold diluted onto minimal me-

dium +Phloxin (MMP) to select prototrophic diploid clones. Each diploid colony issued from a single zygote is defined as primary z y g o t e clone. After incubation at 30 ° C (approximately 20 generations after zygote formation) these primary zygote clones were picked off and suspended separately in NaC1. Several replica master plates (MMP) were then prepared and incubated to allow colony formation. These colonies, issued from primary zygote clone cells, are called secondary z y g o t e clones. These secondary clones are then replicated onto the respective drug plates.

Table 1, List of strains 6. Terminology in Z y g o t e - C l o n e Analysis. The terminology used ade 7-50 h + cap'-101-3 C ade 7-50 m e i 1-102 cap~-lO1 arg 1-1 m e i 1-102

U. Leupold (Berne) spore from a cross anal-8 cap'-lOl-23 A ade 7-50 h + x ura 1-171 arg 1-1 mei 1-102

spore from a cross capr-lO1-23A ade 7-50 h + x ura 1-171 arg-l-1 m e i 1-102

e r y ~ - l l 6 - 9 B his 3-237 mei 1-102 capr-lOl e r y r - l l 6 ade 7-50 h -

spore from a cross ana'-8 e r y r - l l 6 ade 7-50 h + x ura 1-171 his 3-237 mei 1-102

spore from a cross eapr-lO1-23A ade 7-50 h + x e r y r - l l 6 - 1 4 B ura 1-171 h -

anal-8 cap'-lO1 e r J - 1 1 6 ade 7-50 h

spontaneous m u t a n t derived f r o m anal-8 e r y ' - l l 6 ade 7-50 h

All other strains are described by Seitz et al. (1977)

in zygote-clone analysis will be illustrated by an example: The following cross was set up and a sample of 200 500 secondary zygotic clones was analyzed: anar-8 cry s ade 7-50 h (parent 1 = P 1 ) x ana s eryr-l l 6 - 9 B his 3-277 mei 1-102 (parent 2 = P 2 ) (ana r and e r J are in trans-configuration or repulsion). Secondary zygote clones issued from a single primary zygote clone exhibited the following genotypic composition: ana r eryS=parental type 1 = P1 ana s ery ~=parental type 2 = P 2 ana r ery r = recombinant type 1 = R 1

A primary zygote clone is, by analysis of secondary zygote clones, shown to be biparental unirecombinant 1 (abbreviation P P R O ) . Primary clones showing a single phenotype among their secondary progeny (parental or recombinant) are called uniform zygote clones.

All possible configurations of primary zygote clones are given in Table 2, the experimental details are summarized in Figure 1.

G. Seitz-Mayr et al. : Extrachromosomal Inheritance in Schizosaccharomyces pombe. VII

anar-8 eryr-1161~I ade 7-50 h-(P1}U precultures

HanaSeryS Uura1-171arg1-1mei1--102(P2~

~

1

zygotes

zygote formationon SPA

i]

20 generat ions

diluteland plate~onMNP

primaryzygote) L clones

20 generations

selection of diploid prototrophic danes

~[U

/;\

I~l harvest individuoUy and dilute

~

secondary

311

tive), and 81% sensitive diploid clones. Since a primary clone comprises the whole population from the zygote up to the twentieth generation, the high percentage of pure sensitive clones is very striking. A velveteen replica of a n o r m a l - s i z e d colony (3 mm) contains approximately 2-4× 105 cells, and consequently 1 resistant among 2 4 x 105 cells would be detectable as papilla on drug medium. In view of the following data it is of importance to stress that there is no significant difference in growth rates in drug free glucose medium between anal-8 and its sensitive wild-type. The same holds true for all other haploid and diploid mutant strains harbouring one or more mutated alleles. Therefore a bias of the resistant strains on the cellular level can be ruled out. On the other hand we observed zygote clones which were still heteroplasmic after at least 50 generations after zygote formation (Del Giudice et al., 1977). This demonstrates another, very slow process of segregation, overlapping with the fast one described above. In various standard crosses (see Methods 4), where diploids were allowed to segregate for another 20 generations, transmission of ana r decreased to 5-14%.

replicate on GLY,GLY,E,GLY+A,GLY+A+E Fig. 1. Diagramatic presentation of the methodology of zygote clone analysis (SPA, sporulation agar; M M P , minimal medium +phloxin; GL Y, glycerol complete medium; E, erythromycin; A, antimycin)

70

60 7. Analysis fo Uniform Clones by Aliquot Plating.To estimate transmission rates less than 1%, zygote clones were suspended and 0,1 ml of the undiluted suspension (approximately 2 x 106 cells/ml) are plated on drug medium. In parallel, titers were determined by plating an appropriate dilution on drugfree medium.

50 c

Results

1. Properties of the Antimycin-Resistant Mutant aria*-8

u

z,0

o o~ >, U

*6

30

.Q

Genetic and physiological properties of mutant and-8 were described in previous papers (Lang et al., 1975; Wolf et al., 1976a) and will be summarized here only briefly. A n d - 8 is a nitrosoguanidine-induced mutant which differs from its wild-type by a "mutator activity" which can be separated from the antimycin-resistant phenotype (Del Giudice et al., 1977; Seitz et al., 1977). Strains carrying the "mutator", in contrast to the wild-type, produce both mitochondrially inherited spontaneous antibiotic-resistant- and respiratory-deficient mutants of the mit--type (Wolf et al., 1976b; Seitz et al., 1977). Replica-plating of primary zygote clones (see Methods 6.) on antimycin medium revealed 9.5% resistant, 9.5% sectored (resistant/sensi-

E ¢-

20

10

I

0

10 20 30 40 50 60 70 % ono r transmission

80

90 100

Fig. 2. Transmissional pattern of 100 individual zygote clones from the cross anar-8 ade 7-50 h- x ana "~ura 1-171 arg 1-1 mei 1-102, analyzed by replica-plating

312

G. Seitz-Mayr et al. : ExtrachromosomalInheritance in Schizosaccharomyces pombe. VII

Table 3. Analysis of 100 zygote clones of the cross: ana'-8 ade 7-50 h x anas ura 1-171 arg 1-1 mei 1-102 (300-500 secondary clones were scored per primary zygote clone) ariar transmission (%)

Number or primary zygote clones

alia r

transmission (%)

Number of primary zygote clones

0.0 0.001- 0.01 0.011 0.1 0.11 - 0.5 0.51 - 5.0 5.1 10.0 10.1 15.0 15.1 ~0.0 20.1 -25.0 25.1 -30.0 30.1 -35.0 35.1 -40.0 40.1 -45.0

38" 18" 8~ 73 1 7 4 0 4 3 1 3 2

45.1 50.1 55.1 60.1 65.1 70.1 75.1 80.1 85.1 90.1 95.1 100.0

0 1 0 0 2 0 0 0 0 0 0 1

-50.0 -55.0 ~50.0 ~65.0 -70.0 -75.0 -80.0 -85.0 -90.0 -95.0 -99.9

Analyzedby aliquot plating (approximately 10s cells plated on antimycin medium) Other values determined by replica plating. Mean transmission of and in 100 zygote clones: 6.8%

(markers in coupling or repulsion). In addition, a three-factor cross with all three markers in cis-configuration was performed. The general features for all crosses are: a) Individual zygotes may give rise to clones containing one, two, three, or four mitochondrial genotypes. b) The proportion in individual zygote clones of either of the parental genotypes und also of either of the recombinant genotypes, can vary almost over the entire range of percentages. c) The proportions of the two recombinant genotypes in individual clones are not equal. d) In all crosses uniform clones can be found, containing either one parental or one recombinant type. 3. Crosses in cis-Configuration ( M a r k e r s in Coupling)

a

To study transmission of the anar-phenotype in more detail, we analyzed the progeny of 100 individual zygote clones from a cross between anar-8 and the sporulation-deficient, antimycin sensitive strain mei 1-102, scoring 200-1000 secondary clones per primary zygote clone. Results obtained both by replicaplating and by aliquot-plating revealed the highly biased frequency distribution for anar-transmission. The transmission values are presented in 5% intervals in Table 3 (including results from aliquot plating) and in the diagram on Figure 2 (without aliquot plating). This pattern of distribution is characteristic for mutant anar-8 and not shared by other mitochondrial mutants like diu~-301 (resistant to diuron, Wolf and Kaudewitz, 1977), analyzed in the same way. We were interested, how other mitochondrial markers like chloramphenicol-(C n) or erythromycin-resistance (E n) behave in transmission and segregation in absence or presence of the antimycin-marker anar-8, and in the interaction of the three markers in cis- and trans-crosses. It should be pointed out that the percentage distributions reported for zygote clones represent the final genotype composition after about 20 generations from the zygote to the primary zygote clone, plus another 20 generations from a diploid cell of a primary clone to a secondary clone. 2. General Features in Z y g o t e Clone Analysis Involving Crosses in cis- and trans-Configuration Two factor crosses involving the markers A n, C R, and E n were performed in cis- and trans-configuration

a) Transmission. Since we do not know the genetic basis of unequal distribution among parental- and recombinant types, we shall use the term " b i a s ' throughout, following the suggestion of Avner et al. (1973). A s y m m e t r y and polarity will be reserved for unequal distributions caused by nuclear or mitochondrial determinants. Tables 4A, B, C show the results of zygote clone analyses of the crosses ARC R × Ascs(4A), A R E n × ASES(4B), and CnE R × CSES(4C). Table 4D presents the results of a threefactor cross ( A R C n E n x ASCSE s) which is included as two-factor crosses in Tables 4A, B, C. The last column in each table specifies the class of the primary zygote clone as explained in Table 2. In all crosses transmission of the double (or triple) sensitive parental type was much higher than that of the double (or triple) resistant one. In the cross A n C n × A s C s 3 out of 61 clones transmit A R C n, in the cross A n E n × A S E s 4 among 23 clones transmit A n E n, and in the cross CnE n × CSE s none of the 48 clones is C n E R. Transmission values for individual markers (sum of p a r e n t a l - a n d recombinant types) is different for the three markers involved. E n shows the highest transmission (average of 4 crosses: 6.1%), A n slightly lower (4.4%), and C n the lowest transmission value. Transmission values are also dependent from the other marker in c i s - c o n f i g u r a t i o n . As an example, transmission of A n is 1.1%, when it is coupled with C R, but 7.5% with E n. If one omits uniform zygote clones (the double sensitive ones) from the calculation, transmission is increased by a factor 3 5, but still C n has the lowest, E R the highest transmission rate (Table 6A). c) Uniform Z y g o t e Clones. All four crosses yielded a considerable percentage of uniform clones, i.e.

G. Seitz-Mayr et al. : Extrachromosomal Inheritance in Schizosaccharomyces pombe. VII Table 4. Zygote clone analysis of crosses in cis-configuration (markers in coupling) (values in percent, about 300 500 secondary clones scored per primary clone) A, Cross anal-8 capr-101 ade 7-50 h

(Pl)

x a n a ~ caps ura 1-171 arg 1-1 mei 1-102 (P2)

S and R refer to sensitive and resistant alleles for the loci A and C, in that order Clone number

R2 SR

Class

0.0 0.0 0.0

0.0 0.0 0.0

PPO0

38.6 99.4

61.4 0.6

0.0 0.0

OPRO

97.8 97.0 98.5 93.5 94.6 93.6

0.0 0.0 0.0 0.0 0.0 0.0

0.2 3.0 1.0 6.5 5.4 6.4

OPOR

P1 RR

P2 SS

l 2 3

4.1 3.0 1.4

95.9 97.7 98.6

4 5

0.0 0.0

6 7 8 9 10 11

0.0 0.0 0.0 0.0 0.0 0.0

R1 RS

12-61

0.0

100.0

0.0

0.0

mean mean - UC

0.1 0.8

98.4 91.3

1.0 5.6

0.4 2.0

mean 4D

0.07

95.27

4.67

0.0

mean 4 D - UC

0.2

87.4

12.4

0.0

mean 4A + 4 D + UC

0.09

96.8

2.8

0.2

m e a n 4A + 4 D - UC

0.5

89.4

9.0

1.0

OPO0

B. Cross anal-8 ery~-ll6 ade 7-50 h - (P1) x a n a s ery s ura 1-171 arg 1-1 mei 1-102 (P2) (order A E) R2 SR

Class

3.8 9.8 0.4

0.5 37.9 42.4

PPRR

64.2

0.0

0.0

PPO0

P2 SS

1 2 3

11.2 9.2 3.5

84.5 43.1 53.7

4

35.8

R1 RS

5

0.0

82.3

2.1

15.6

OPRR

6 7

0.0 0.0

99.7 3.2

0.3 96.8

0.0 0.0

OPRO

8 9 10

0.0 0.0 0.0

84.9 49.5 84.5

0.0 0.0 0.0

15.1 50.5 15.5

OPOR

11 23

0.0

100.0

0.0

0.0

mean

2.6

84.5

4.9

7.7

11.3

17.8

mean -- UC

6.0

64.9

mean 4D

3.46

93.01

1.28

2.26

mean 4 D - - UC

9.2

81.4

3.4

6.0

mean4B+4D+UC

3.1

88.8

3.1

5.0

mean4B+4D--UC

7.6

73.2

7.4

11.9

(Pl)

x ana s cap s ery ~ ura 1-171 arg I-1 mei 1-102 (P2) (order C-E)

Clone number

P1 RR

P2 SS

R1 RS

R2 SR

Class

1

0.0

78.2

1.4

20.4

OPRR

2 3 4 5 6 7 8

0.0 0.0 0.0 0.0 0.0 0.0 0.0

68.2 96.9 93.4 81.8 97.4 93.8 73.6

0.0 0.0 0.0 0.0 0.0 0.0 0.0

31.8 3.1 6.6 18.2 2.6 6.2 26.4

OPOR

948

0.0

100.0

0.0

0.0

OPO0

mean

0.0

0.01

2.4

97.57

mean - UC

0.0

85.4

0.18

14.4

mean 4D

0.07

94.3

0.0

5.7

mean 4 D - UC

0.2

84.8

0.0

15.0

mean 4 C + 4 D + UC

0.035

95.9

0.005

4.1

mean 4 C + 4 D -

0.1

85.1

0.09

14.7

Clone P1 number RRR

= c r o s s 4A = c r o s s 4D (three-factor cross) - U C = w i t h o u t uniform clones + U C = w i t h uniform clones

P1 RR

C. Cross ana s capr-lO1 eryr-ll6 ade 7-50 h

UC

D. Cross anar-8 cap~-lO1 ery~-ll6 ade 7-50 h - (PI) x anas cap s ery s ura 1-171 arg 1-1 mei 1-102 (P2) (order A - C - E )

4A 4D

Clone number

313

P2 SSS

R1 RSS

R2 SSR

R3 SRS

R4 RSR

R5 RRS

R6 SRR

1

1.7

98.3

0.0

0.0

0.0

0.0

0.0

0.0

2

0.0

50.5

0.0

44.6

0.0

4,9

0.0

0.0

3 4

0.0 0.0

76.5 92.8

23.5 7.2

0.0 0.0

0.0 0.0

0.0 0.0

0.0 0.0

0.0 0.0

5 6

0.0 0.0

98.8 91.8

0.0 0.0

1.2 8.4

0.0 0.0

0.0 0.0

0.0 0.0

0.0 0.0

7 8 9

0.0 0.0 0.0

81.3 44.9 97.4

0.0 0.0 0.0

0.0 0.0 0.0

0.0 0.0 0.0

18.7 55.1 2.6

0.0 0.0 0.0

0.0 0.0 0.0

100.0

10~4

0.0

0.0

0.0

0.0

0.0

0.0

0.0

mean

0.07

93.01

1.28

2.26

0.0

3.39

0.0

0.0

mean

0.2

81.4

3.4

6.0

0.0

9.0

0.0

0.0

-UC

clones that express only a single mitochondrial t y p e . I n c i s - c r o s s e s it is a l w a y s

the double

sensitive parent (P2), which becomes OPO0

ental), The percentage of uniform is 5 7 % f o r t h e c r o s s A R E R x A s E for the crosses ARcR × Asc s and

geno-

(or triple)

uniform

(unipar-

clones (Table 6A) s, 8 2 % a n d 8 3 % C R E R × C S E S, a n d

63 % f o r t h e t h r e e - f a c t o r c r o s s . A g a i n c r o s s e s i n v o l v ing the C-marker show a higher proportion of uniform clones, suggesting a correlation between input bias of C R and formation of uniform clones. Possible mechanisms

involved

will be discussed.

in formation

of uniform

clones

314

G. Seitz-Mayr et al. : Extrachromosomal Inheritance in Schizosaccharomyces pombe. VII

Table 5. Zygote clone analysis of crosses in trans-configuration (markers in coupling) (values in percent, about 300-500 secondary clones scored per primary clone; for further legend see Table 4) A. Cross anar-8 caps ade 7-50 h- (P1) x ana~ eapr-lO1-3 C arg 1-1 mei 1-102 (P2) (order A-C) Clone number

P1 RS

P2 SR

R1 RR

R2 SS

class

1 2 3 4 5

59.0 48.3 73.2 23.4 40.8

2.6 1.4 3.7 0.7 6.8

0.0 0.0 0.0 0.0 0.0

38.4 50.3 23.1 75.9 52.4

PPOR

6 7

93.9 98.2

6.1 1.8

0.0 0.0

0.0 0.0

PPO0

8 9 10 11 12 13 14

98.0 83.6 91.5 80.5 10.8 81.0 73.1

0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0

2.0 16.4 8.5 19.5 89.2 19.0 26.9

POOR

100.0

0.0

0.0

0.0

PO00

27

0.0

80.0

0.0

20.0

OPOR

28-32

0.0

0.0

0.0

100.0

O00R

mean

67.4

3.2

0.0

29.4

mean - UC

63.7

6.9

0.0

29.4

15 26

B. Cross anar-8 ery s ade 7-50 h- (P1) x anas eryr-ll6-9B his 3-237 mei a-102 (P2) (order A-E) Clone number

P1 RS

P2 SR

R1 RR

R2 SS

Class

1 2 3 4

13.4 3.8 10.4 0.9

36.3 93.2 65.1 77.6

7.6 0.4 14.5 0.8

42.7 2.6 10.0 20.7

PPRR

5 6 7 8 9

33.8 25.9 12.0 24.4 56.4

43.9 40.3 84.9 75.0 31.9

0.0 0.0 0.0 0.0 0.0

22.3 33.8 3.1 0.6 11.7

PPOR

10

0.0

58.6

7.6

33.8

OPRR

11 12 13

0.0 0.0 0.0

0.8 27.9 35.3

0.0 0.0 0.0

99.2 72.1 64.7

OPOR

14 32

0.0

0.0

0.0

100.0

O00R

mean

5.7

21.0

1.0

72.3

13.9

51.6

2.4

32.1

mean - U C

c) R e c o m b i n a t i o n . A s a l r e a d y m e n t i o n e d , r e c i p r o c a l recombinant genotypes are unequally distributed in the whole population and in individual zygote clones. The differences at the level of zygote clones, however, e x c e e d t h a t a t t h e p o p u l a t i o n level. I n o r d e r t o d e t e r -

C. Cross cap~-lO1 ery s ade 7-50 mei 1-102 (P1) x caps eryr-ll6-14B ura 1-171 h (P2) (order C-E) Clone number

P1 RS

P2 SR

R1 RR

R2 SS

Class

1 2 3 4 5 6 7 8 9 10 11 12

1. l 18.2 0.5 1.7 5.6 6.6 4.6 1.1 1.7 8.7 0.9 8.7

94.3 81.5 47.9 64.7 89.8 88.5 85.4 84.8 91.7 82.2 97.7 88.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

4.6 0.3 51.6 33.6 4.6 4.9 10.0 14,1 6.6 9.1 1.4 3.3

PPOR

13 14 15 16 17 18

2.2 3.9 1.9 9.3 9.5 11.0

97.8 96.1 98.l 90.7 90.5 89.0

0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0

PPO0

19 20 21 22 23 24 25 26

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

99.6 82.0 72.2 85.9 98.5 93.9 60.8 75.4

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.4 18.0 27.8 14.1 1.5 6.1 39.2 24.6

OPOR

27 35

0.0

100.0

0.0

0.0

OPO0

mean

2.8

89.3

0.0

7.9

mean - UC

3.7

85.7

0.0

10.6

mine relative distances between the three markers, recombination frequencies were calculated both on t h e b a s i s o f all z y g o t e c l o n e s a n a l y z e d , a n d a f t e r o m i s sion of uniform zygote clones. This lead to a prelimin a r y m a p , w h e r e A E is t h e l o n g e s t , A C t h e s h o r t e s t d i s t a n c e . V a l u e s g i v e n i n F i g u r e 3 A r e p r e s e n t recombination values of the two-factor crosses, without and with the values of the three-factor corss. Inclusion o r o m i s s i o n o f u n i f o r m c l o n e s l e a d t o c o n s i s t e n t results, s u g g e s t i n g t h e o r d e r A C E . T h e s i g n i f i c a n c e o f t h e s e d a t a is d i s c u s s e d t o g e t h e r w i t h t h e r e s u l t s o f trans-crosses i n a l a t e r p a r a g r a p h .

4. Crosses in t r a n s - C o n f i g u r a t i o n ( M a r k e r s in R e p u l s i o n ) ( T a b l e s 5 A , B, (2) a) Transmission. T r a n s m i s s i o n a l bias c a u s e d by the C R - m a r k e r is a l s o o b s e r v e d i n trans-crosses. T h e r a t i o o f t h e p a r e n t a l - t y p e s i n t h e c r o s s A R E s x A s E R is 1 : 3 . 7 . I n t h e c r o s s A S C R x A R C s it is 1 : 2 1 . 1 a n d 1 : 3 1 . 9 f o r c r o s s E s C R x E R C s. T h e o v e r a l l t r a n s m i s -

sion rates of individual markers in different crosses ( T a b l e 6 B ) a r e r o u g h l y t e n t i m e s h i g h e r t h a n in cis-

315

G. Seitz-Mayr et al. : Extrachromosomal Inheritance in Schizosaccharomyces pombe. VII Table 6. Marker transmission and formation of uniform zygote clones in cis- and trans crosses A. Crosses in cis-configuration (data from Table 4A, B, C, D) Crosses

A R c R x AsC s

+ UC UC

--

AR

CR

1.1 6.4 7.5 17.3

0.5 2.8 -

4.74 12.6

0.01 0.18 0.07 0.2

× ASE s

+ UC - UC

CREg x CSEs

+ UC

-

-

-

A R E

g

ARcRE R x ASCSE s

Genotypes of uniform zygote clones

%uniform zygote clones

Ascs(p2)

82

10.3 23.8

ASES(p2)

57

2.4 14.4 5.72

CSES(p2)

83

ASCSES(p2)

63

Genotypes of uniform zygote clones

% uniform zygote clones

ARcS(p1) ; AsCS(R2)

38 ; 16

ASES(R2)

59

CSER (P2)

26

% transmission of marker

U C

+ UC UC

ER --

--

15.2

mean

+ UC

4.4

0.2

6.1

mean

- UC

12.1

1.1

17.8

+ UC=with uniform clones; -UC=without uniform clones B. Crosses in trans-configuration (data from Table 5A, B, C) Crosses

% transmission of marker AR

Ce

ER

22.0 54.0 89.3 85.7

ARE s x ASE g

+ UC -- UC

67.4 63.7 6.7 16.3

CREs x CSE R

+ UC - UC

-

3.2 6.9 -2.8 3.7

mean

+ UC

37.1

3.0

55.7

mean

- UC

40.0

5.3

69.9

ARC s x A S c R

+ UC -- UC

crosses. This can be e x p l a i n e d b y the fact, t h a t then b o t h p a r e n t a l strains are b i a s e d in t r a n s m i s s i o n b y the presence o f one m u t a t e d allele. L i k e in c i s - c r o s s e s , t r a n s m i s s i o n is high for E R ( 5 5 . 7 % ) , l o w e r for AR(37.1%) a n d lowest for C R (3.0%).

U n i f o r m C l o n e s . In the cross A R C s x A S C R b o t h p a r e n t a l type 1 ( A R C s) a n d r e c o m b i n a n t t y p e 2 ( A s C s) are p r e s e n t in u n i f o r m clones. In cross A R E s x A S E g the u n i f o r m class is r e p r e s e n t e d b y the r e c o m b i n a n t type A S E s, in the cross C R E s × C S E R b y the p a r e n t a l type 2 ( C S E R ) . It is r a t h e r p u z z l i n g to see, t h a t a z y g o t e clone b e c o m e s u n i f o r m for a r e c o m b i n a n t gen o t y p e , since r e c o m b i n a n t m o l e c u l e s are c e r t a i n l y o n l y a m i n o r f r a c t i o n a m o n g the t o t a l after z y g o t e f o r m a t i o n . Since there is n o selection on the cellular level, r e c o m b i n a n t m o l e c u l e s o f this t y p e m u s t have a c o n s i d e r a b l e selective (replicative?) a d v a n t a g e over b o t h p a r e n t a l m o l e c u l e s a n d their r e c o m b i n a n t c o u n b)

t e r p a r t . Possible m e c h a n i s m s will be a p o i n t o f discussion.

Like in c&-crosses, there is a s t r o n g bias f o r the d o u b l e r e s i s t a n t versus the d o u b l e sensitive type. O n l y in cross A R E s x A s E g 5 o u t o f 32 clones were able to t r a n s m i t the d o u b l e r e s i s t a n t rec o m b i n a n t type A R E R. In the o t h e r two crosses, these r e c o m b i n a n t types were absent, a g a i n p o i n t i n g o u t the selective d i s a d v a n t a g e b r o u g h t a b o u t b y the C Rm a r k e r . R e c o m b i n a t i o n d a t a o b t a i n e d with a n d witho u t u n i f o r m clones are c o n s i s t e n t with the d a t a f r o m eis-crosses, insofar EA is the greatest d i s t a n c e (Fig. 3 B ) . A c c o r d i n g to these d a t a , the d i s t a n c e A C is g r e a t e r t h a n C E . This d i s c r e p a n c y b e t w e e n results o f eis- a n d t r a n s - c r o s s e s shows, t h a t m a p p i n g is r e n d e r e d quite difficult, p r e s u m a b l y by the high degree o f i n t e r m o l e c u l a r selection. It is evident, t h a t by o m i s s i o n o f u n i f o r m clones o n l y the e x t r e m e cases c) R e c ombination.

316

@

G. Seitz-Mayr et al. : Extrachromosomal Inheritance in A C W 1L(3.0) _W 76(10.0)

E

• 2/.(4.1) 126(81)1z'N14-8)

29~9.31 A •

C •

29.4

E •

79

29.4. 73.3 34.5

10.6

Fig. 3. Relative map positions of the markers A, C, and E from cis @ - a n d trans-crosses @ . Upper values: including uniform clones, Lower values: without uniform clones, Values in brackets: three-factor cross (Table 4D) included

of such a selection process are eliminated from the calculation.

D i s c u s s i o n

It is an early observation, that the mitochondrial genomes present in zygotes of S. c. with mixed (heteroplasmic) mitochondrial genotypes sort out during mitotic divisions of the zygote. Thus in most cases within roughly twenty generations virtually all progeny is pure (homoplasmic) for one mitochondrial genotype or another, parental or recombinant (for review see Perlman et al., 1977). It has been shown that in many cases the progeny of early divisions is already homoplasmic (Lukins etal., 1973; Waxman etal., 1973; Wilkie and Thomas, 1973; Dujon et al., 1974; Callen, 1974; Strausberg and Perlman, 1978). Sorting out of mitochondrial genomes can be considerably accelerated by the following events: - Formation of homoplasmic end buds due to slow cytoplasmic mixing, followed by deviations of the composition of the remaining mitochondrial genomes. The consequences are a more rapid further segregation, as discussed in detail by Birky (1975), Birky et al. (1978), Birky and Skavaril (1976), and Birky et al. (1978). Formation of segregational units containing several mitochondrial DNA molecules (chondriolites: Williamson, 1976; Williamson and Fennell, 1975; Williamson et al., 1977). A mechanism acting in zygotes, preventing replication or initiating destruction of certain mitochondrial DNA (mit DNA molecules (Birky, 1975). -

-

1. Budding versus Fission A study, by zygote clone analysis, of transmission and segregation of mitochondrial markers in an or-

Schizosaccharomycespombe. VII

ganism dividing by regular fission may provide a more general view of mechanisms involved in mitochondrial inheritance, than the mere analysis of these processes in budding yeast. In a budding yeast, the zygote serves as source for continuous production of up to 20-50 buds until the zygote dies. In a fission yeast the zygote divides, producing two daughter cells, comprising the whole cytoplasm of the zygote. After this division, the zygote itself does not exist any more. This fundamental difference in cellular multiplication has some important bearings on extrachromsomal heredity. In view of the facts, that cytoplasm is not well mixed, when the first buds emerge, and that half of the cells of a zygote clone are daughters of the first bud, bud position is of critical importance for the genotype composition of a zygote clone and for the further segregation processes. These effects of acceleration of segregation by homoplasmic first buds have been discussed in great detail by Strausberg and Perlman (1978). The study of mitochondrial inheritance in fission yeast reduces the number of parameters influencing the segregation process, since all primary and secondary influences of budding are eliminated.

2. Possible Modes of Segregation Some ways how to explain segregation data presented in this paper and known from literature are schematically depicted in Figure 4 and discussed in the following paragraph. One possibility is, that fission occurs in the zygote before fusion of mitochondria, thus preventing genetic exchanges between mitDNA molecules (Fig. 4A). This does not seem unlikely in view of the slow cytoplasmic mixing reported for S.c., and could account for those clones in Sch. p., where only the two parental genotypes were detected (see Table 4A: clones 1-3; Table 4B: clone 4; Table 5A: clones 6 and 7; Table 5C: clones 13-18). This mechanism would produce primary zygote clones containing two types of homoplasmic diploids, which then give rise to pure secondary clones. The other alternative possibility is that cytoplasm is well mixed before the first fission (Fig. 4B). This would be the case for all clones containing parental plus recombinant genotypes. Provided no selective advantage of one cellular type over the other and absence of any bias on mitDNA level this would lead to a continuously segregating clone. It has been observed by Del Giudice et al. (1977) in crosses with paromomycin-resistant strains of Sch. p., that segregation may persist over more than 50 diploid generations. Rank and Bech-Hansen (1972) reported that the heteroplasmic state is fairly common at the 19-20 cell division stage in S. c., and can even persist

G. Seitz-Mayr et al. : Extrachromosomal Inheritance in

Schizosaccharomycespombe. VII

317

Fig. 4. Schematic presentation of possible models of segregation. © mitochondrial D N A molecule conferring sensitivity. • mitochondrial D N A molecule conferring resistance to a certain drug. For explanations see Discussion

beyond two passages through selective medium. Forster and Kleese (1975) have demonstrated by analysis of zygote pedigrees that in each cross they analyzed, a small proportion of zygotes (usually less than 10%) did not purify for one or both loci (C R or E R) involved in these crosses. They also describe an exceptional strain which produces a much higher proportion (approximately 30%) of zygotes that never purify for one or both loci. This indicates that segregation of mitochondrial alleles is locus-specific as well as strainspecific, which would explain the differences to other authors. An interesting case of slow segregation has also been reported by Butow et al. (1977). In the case of the mitochondrial locus var 1 rapid segregation, as observed for other mitochondrial markers in these crosses, seems to occur only when the two parental var 1 species are present in a cell. On the other hand, in crosses where a high proportio n of an intermediate form of var 1 is produced, segregation to pure mitochondrial genotypes requires, in some cases, more than 60 generations. Persistent heteroplasmons, socalled "persistent cytohets" have also been described by Sager and Ramanis (1971) in chloroplast genetics of Chlamydomonas. Such extreme slow segregation, however, seem to be exceptions in Sch. p., S. c., and Chlamydomonas. Which mechanisms could then speed up segregation in zygotes where cytoplasmic mixing has occurred, as shown by the presence of recombinant genotypes in a zygote clone? It is evident from data of S. c. and from our data in Sch. 1). that segregation cannot be a random sorting out or diluting out of m i t D N A molecules. Michaelis (1976) considered the case where cytoplasmic segregating units are partitioned at random at cell division and showed that the number of generations required for complete sorting out is a function of the number of segregating units (N); roughly 10 N generations are required where Nis not a small number. In S. c., where diploid cells have about 100 m i t D N A molecules (Grimes et al., 1974) and zygotes may have roughly twice as many (Sena et al., 1976) segregation should require more than 1000 generations if each m i t D N A molecule is an independent segregating unit. It has been reported by Williamson et al. (1977) that most S. c. strains fall in the range of 10-15% m i t D N A per total D N A in logarithmic phase and perhaps 15-20% in

stationary phase. For Sch. p. m i t D N A amounts are roughly half: 6% in log-phase and up to 14% in stationary phase (Bostock, 1969). One can estimate the number of m i t D N A molecules in Sch. 1). according to published data for both yeast species. Total D N A amount in S. c. is about 0.045 pg/cell (Moustacchi and Williamson, 1966), that of Sch. 1). between 0.0399 pg/cell (Bostock, 1970) and 0.0405-0.0503 pg/ cell (Sipiczki and Ferenczy, 1977). Assuming that the molecular weight of m i t D N A of Sch. 1). is 17 x 106 daltons (Tabak and Weijers, 1976) and that this roughly corresponds to a length of 8.5 ~tm of a m i t D N A molecule, the same amount of m i t D N A would contain approximately three times more m i t D N A molecules in Sch. 1). compared to S.c.. Since, however, the amount of m i t D N A in Sch. 1). is about half of S. c., a haploid cell of Sch. 1). should then contain about 75 m i t D N A molecules. F r o m this calculation it is evident that also for Sch. p. the individual m i t D N A molecule cannot be the unit of segregation. A considerable acceleration of segregation could be achieved, when several m i t D N A molecules form a segregational unit. Williamson and Fennel (1975) showed that m i t D N A is distributed in aggregates in S. c., termed "chondriolites" (Williamson, 1976; Williamson etal., 1977), which c o n t a i n 2 - 8 m i t D N A molecules on an average. This possibility is illustrated in Fig. 4C). The assumption of chondriolites, however, does not explain the rapid segregation processes leading to uniform zygote clones. In these clones it appears that the mitochondrial D N A of one parent (in Sch. 1). usually the sensitive one) is replicated and transmitted to the diploid progeny, while the mitochondrial D N A of the other (resistant) parent is not replicated and transmitted, and perhaps is destroyed outright. This possibility is illustrated in Fig. 4D and discussed in the following paragraph. 3. Formation o f Uniform Clones

In this paper we showed that any cross involving the mitochondrial markers A R, C R, or E R can produce substantial amounts of clones transmitting only a single mitochondrial genotype. This phenomenon is called uniparental or maternal inheritance, since in all cases reported so far, it is a parental type, in

318

G. Seitz-Mayr et al. : Extrachromosomal Inheritance in

higher organism usually the female one, which becomes uniform. This mechanism is widespread among organisms and analyzed and reviewed in great detail by Birky (1975a, b), Birky et al. (1978), Birky and Skavaril (1976), and Birky et al. (1977). Despite of various data suggesting uniparental inheritance in yeast (Waxman et al., 1975: Tables 3 and 4; Coen et al., 1970: Table 4; Lukins et al., 1973: Table 2; Callen, 1974: Table 2; Waxman, 1975: Tables 3 and 4) this phenomenon was not explicitly dealt with up to 1975 by Birky. He found that all crosses can produce uniparental zygotes, provided that one parent contributes substantially more m i t D N A molecules to the zygote than does the other parent. He suggests a mechanism of differential marking of molecules in the two parents, followed by a mechanism which "counts" molecules and replicates only the majority type. There is a correlation between transmissional bias and formation of uniparental clones. Our results with anar-8 are compatible with this finding, since anal-8 has a low transmission rate (6.8%). and produced 38 pure sensitive and one pure resistant among 100 zygote clones (Table 3). F r o m a one-factor cross it cannot yet be decided if a zygote or its progeny becomes uniparental for individual loci (e.g. for ana) or for whole m i t D N A molecules. In two- and threefactor crosses of S. c. loci behave coordinately, rather than independently: minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire m i t D N A molecules rather than on individual loci. Rare exceptions to the coordinate behaviour of different loci can be interpreted as marker rescue via recombination. All ciscrosses in Sch. p. follow this rule, since the uniparental class in always the double sensitive, while the double resistant class is lost. In these cases one may assume that the uniparental mechanism acts before recombination takes place. The reverse sequence of events may be suggested for the trans-crosses ARC s x A S c R and ARU x ASE R, where the double sensitive recombinant types A S c s and ASE s become uniform. Here it is necessary that recombination takes place first, and the clone then becomes uniform for the recombinant type. These recombinant m i t D N A molecules must have a strong selective advantage, since at the initial state they are in the minority, compared to parental m i t D N A molecules. This represents an interesting type of uniform clones, which has not been found so far in S. c. 4. Parallels to Organisms Other Than S. c.

The strong bias of certain marker combinations like CRER or ARC a found in the crosses described here, is also found in mitochondrial genetics of other organisms. CRER strains in Paramecium aurelia are slow

Schizosaccharomycespumbe. VII

growing and their mitochondria are very quickly eliminated in mixed cells (Adoutte and Beisson, 1972). The authors assume that the accumulation of mutations concerning mitochondrial ribosomes leads to a less efficient mitochondrial protein synthesis (Adoutte, 1974). In the petite-negative budding yeast Kluyveromyces lactis crosses ORCs x o S c R (O R =resistance to oligomycin) fail to produce ORO R recombinants, while OsC s clones appear (Brunner et al., 1977). No attempts have been reported, however, to isolate an ORCR double mutant and to perform cis-crosses. Another case is reported by Belcour and Begel (1977) for crosses of the mutants 64 or 561 (Belcour, 1975), which affect precise stages of development, with the chloramphenicol-resistant mutant capr-1 in Podospora anserina. It was not possible to detect the recombinant classes (64,capr-1) or (561,cap~-1). The failure could be due to an altered phenotype of the double mutants of to lethality of this type. 5. Mapping o f Mitochondrial Markers

Deletion mapping is not possible in petite-negative yeasts, since the rho -mutation is lethal (Bulder, 1964). Therefore recombination analysis is the only method applicable so far for mapping of mitochondrial markers in Sch. p.. According to the predictions of the phage analogy model of Dujon et al., there should be no transmissional bias of any mitochondrial genotype on the population level. As a consequence, cis- and trans-crosses should yield identical results concerning transmission- and recombination-frequencies. In spite of the fact that this condition is not fulfilled, we have tried to determine relative distances between the three markers. In both cis- and trans-crosses recombination frequencies are highest for AE, and lower for A C and CE, suggesting the order A C E (Figure 3). In trans-crosses, however, distance A C is greater than CE, while the reverse is true for cis-crosses. Omission of uniform clones does not alter these features. Mapping studies with mutant strains exhibiting higher transmission rates than ana r8 will reveal, if these bias effects are specific for a certain set or combination of markers or if they are general effects in mitochondrial inheritance of Sch. p. Acknowledgements. The authors are indebted to Mrs. B. Sydow, Miss R. Stets, and Miss M. Schropp for excellent technical assistence. We are grateful to Dr. R. J. Schweyen for valuable discussions. This work was supported by the Deutsche Forschungsgemeinschaft.

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by W. Gajewski

Received May 16, 1978

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