GENETIC ANALYSIS OF PETITE MUTANTS OF SACCHAROMYCES CEREVZSZAE: TRANSMISSIONAL TYPES PHILIP S. PERLMAN Department of Genetics, The Ohio State University, Columbus, Ohio 43210 Manuscript received June 24, 1975 Revised copy received December 10, 1975 ABSTRACT
We have studied a number of petite [rho-] mutants of Saccharomyces cerevisiae induced in a wild-type strain of mitochondrial genotype [omeCHLR ERYS OLP,,,,, PARS] by Berenil and ethidium bromide, all of which have retained two mitochondrial genetic markers, [CHLE] and [ERYE], but have lost all other known markers. Though stable in their ability to retain these markers in their genome, these mutants vary widely among themselves in suppressiveness and in the extent to which the markers are transmitted on crossing to a common wild-type tested strain. In appropriate crosses all of the strains examined in this study demonstrate mitochondrial polarity, and thus have also retained the [ome-] locus in a functional form; however, five different transmissional types were obtained, several of them quite unusual, particularly among the strains originally induced by Berenil. One of the most interesting types is the one that appears to reverse the parental genotypes with [CHLR ERYs] predominating over [CHLS ERYE] in the diploid [rho+] progeny, rather than the reverse, which is characteristic of analogous crosses with [rho+] or other petites. Mutants in this class also exhibited low o r no suppressiveness. Since all of the petites reported here are derived from the same wild-type parent, and 53 have the same nuclear background, we have interpreted the transmissional differences as being due to different intramolecular arrangements of largely common retained sequences.
HE cytoplasmic petite [rho-] mutation was first discovered and characterand his colleagues in the late Tized in Saccharomyces cerevisiae by EPHRUSSI 3 940's (EPHRUSSI, HOTTINGUER and CHIMENES 1949; EPHRUSSI, HOTTINGUER and TAVLITZKI (1949). These early studies revealed that the mutation behaves as a "-Mendelian character in genetic crosses. Since the irreversible genetic change produced a permanent impairment of mitochondrial respiratory function, it was tentatively concluded that it was due to the loss of a cytoplasmic genetic element, probably localized within the mitochondria. Shortly thereafter EPHRUSSIreported that the cytoplasmic factors of some petite mutant strains interfere with the expression or replication of the normal factor in the diploid state in contrast to the first-reported mutants that did not HOTTINGUER and ROMAN1955). Those in the former affect normal (EPHRUSSI, class were termed suppressive and those in the latter, neutral petites. Suppressive petites were found to vary widely in the extent of their suppressiveness, an Genetics 82 : 645-663 April, 1976.
646
P.
S. PERLMAN
observation that suggested that there exist many, genetically different, petite lesions (EPHRUSSI and GRANDCHAMP 1965; EPHRUSSI, JAKOB and GRANDCHAMP 1966). However, all these petites were indistinguishable in their phenotype which is deficient in cytochrome-linked respiratory and phosphorylating functions (SLONIMSKI 1949; SLONIMSKI and EPEIRUSSI 1949; MAHLER1973) and-as discovered more recently-in mitochondrial protein synthesis (KUZELAand GRECNA 1969; SCHATZ and SALTZGABER 1969). The discovery of mitochondrial DNA (mtDNA) in the early 1960’s was soon €ollowed by the discovery that petite mutants often contained mtDNA with novel physical and chemical characteristics (MOUNOLOU, JAKOB and SLONIMSKI 1966; MEHROTRA and MAHLER 1968). It has since been demonstrated that most, and occasionally all, of the base sequences in these mutant mtDNAs are also found in wild-type [rho+] mtDNA, but that all o i them lack, in variable amounts, the sequences and the genetic information characteristic of this wild1975); thus they can be considered formally type DNA (reviewed by PERLMAN as deletion mutants. Since a number of segments located in different regions of mtDNA code for the RNA components of the mitochondrial translation machinery and since this machinery is essential for the expression of full mitochondrial function, it is not surprising that, at the level of mitochondrial respiratory competence, petites are phenotypically identical. Recently, several laboratories have undertaken detailed analyses of mtDNA from a small number of petite mutants (FAYE et al. 1973; LOCKER, RABINOWITZ and GETZ19741a,b; MOLet al. 1974; reviewed in PERLMAN 1975). These elegant studies show clearly that the mtDNA in these mutants retains wild-type sequences in reiterated form, as well as rearrangements of them, such as inversions. The size of the basic repeating units can be as small as 70 base pairs (Mor, et al. 1974) or as large as 30-50% of the wild-type genome (LAZOWSKA et al. 1974; MICHEL et al. 1974). I n contrast to this extensive information on the molecular level, the transmission genetics of these mutants has been largely neglected. It is known that it is possible to obtain petites that retain any particular mitochondrial marker; two or more markers may also be retained, but not in all conceivable combinations et al. 1969; RANK1970; F-~YE et al. 1973; UCHIDAand SUDA1973; (GINGOLD DEUTSCH et al. 1974; MOLLOY, LINNANE and LUKINS1975; PERLMAN 1975). Some quantitative information of the ability of petites to transmit particular and LINNANE 1972; RANK markers has been reported ( COENet al. 1970; NAGLEY et al. 1971; FAYE et al. 1970), including some multifactorial crosses (BOLOTIN 1973; MOLLOY,LINNANEand LUKINS.1975); published data indicate that mutants of apparently identical genotype may differ in their degree of transmission and in their suppressiveness. Suppressiveness does not appear to be correlated with any particular markers but, according to LINNANE and RANK,all those strains that did retain any markers at all were found to be suppressive; at least some marker-retaining petites in the collection at Gif are neutral when crossed [rho+] testers (P. P. SLONIMSKI, personal communication; P. PERLMAN, unpublished data), but this point is not apparent from published data with these strains.
647
P E T I T E TRANSMISSIONAL TYPES
We have isolated and characterized a number of mutants induced by Berenil and PERLMAN 1973; PERLMAN and MAHLER 1973) and ethidium bromide (EtdBr) (SLONIMSKI. PERRODIN and CROFT 1968), all of which have , have retained two mitochondrial genetic markers[ CHLR321] and [ E R Y E ]but lost others, i.e. [OLZs,,,,,] and [ P A R S ] .Though stable in their ability to retain these markers in their genome, these strains vary widely in suppressiveness and in the extent to which the markers are transmitted on crossing to [rho+] tester strains. Each strain examined in this study demonstrates mitochondrial polarity (in appropriate crosses) and thus has also retained the locus [ome] in a functional form [OW-] ; however, several unusual transmissional types were obtained, particularly among the strains induced by Berenil. One of the most interesting types is one that appears to reverse the parental genotype with [CHLRERYE] predominating over [CHLSE R Y R ] in the diploid [rho+] progeny, rather lhan the reverse which is characteristic o€ analogous crosses with [rho+] or other petites. Mutants of this type also exhibited low or no suppressiveness. Since all of the petites reported here are derived from the same wild-type parent and, therefore, possess the same nuclear background, we have interpreted the transmissional differences as being due to different intramolecular arrangements of largely common retained sequences. (MAHmR
MATERIALS A N D METHODS
Yeast strains and culture conditions-Yeast strains used in these studies are listed in Table 1; all petite strains used are derived from IL16-1OB. The symbols for mitochondrial loci reflect the suggestions of SHERMAN and LAWRENCE (1974); [CHL], [ E R Y ] , [OLZl,s,3] and [ P A R ] are 1 x i influencing sensitivity o r resistance to chloramphenicol, erythromycin, oligomycin (three separate loci) and paromomycin, respectively; [ome] is the mitochondrial locus, omega (BOLOTIN et al. 1971; DUJON,SLONIMSKI and WEIL 1971.; PERLMAN and BIRKY1974; HOWELL et al. 1974); the superscripts R, S, and 0 denote resistance. sensitivity or gene absence, respectively. Allele numbers are those assigned by the laboratory that provided each strain. Media (minimal medium [MD], semi-synthetic medium with dextrose [RD] or glycerol [RG] and RG plus antibiotics) and BIRKY(1974). are described i n PERLMAN TABLE 1 Yeast strains used Strain
IL16-1OB 55R5-3C/221 D22 D22-ER2 D22-Al5 D22-A21 DPl-IB/517 D243-4A-OR 4810 AMFP-9 (32)l-2/3
Nuclear genotype
a his
a ura a ade a ade
ade ade his trp ade lys a trp a arg met a ade a a a a
Mitochondrial genotype
[rho]
[ome] [CHL] [ERY] [OLI,] [OL2,1 [OLI,I [PARI
S R221
S S
S S
S S
s
s
s
s
s
S
Re S
+f -- R 3 2 1S +
+
+ + + +
+ + + + +
+ +
+ + +
+
-
S S S R517 S S S S S R321 R221 R321 S
S S
s
S S S S S R 1 5 S S S S R 2 1 S S S S S R4 S S S S S S R1 S S S S R4 S S S
Source of strain
SLONIMSKI SLONIMSKI WILKIE WILKIE/BIRKY GRIFFITHS GRIFFITHS SLONIMSKI CRIDDLE KLEESE FRAENKEL
PER LM A N
648
P. S. PERLMAN
Isolation of petite mutants-Petites were induced by treatment of a culture of IL16-IOB growing in 1% glucose medium (RD) with EtdBr (25 pM) for 10 min or Berenil (50 pM) f o r 30 min. The mutagenized cells were then washed and plated on RD solid medium to yield primary mutagenized clones; these were suspended in water and subcloned and the subcloning repeated once again to yield the strains which were analyzed in more detail as described in the text. Mating conditions-For matings of petite with wild-type strains the petites were grown to exponential phase in RD, and the wild-type testers in RG. The testers were grown in RG in order to maintain a high level of [rho+] in the cultures used. Cells were mated in RD liquid medium at IO7 cells/ml (equal numbers of each parent) at 30” with shaking for two hours. The mating mixture was then washed and plated at several dilutions on M D to select for prototrophic diploid clones. In all experiments both parents were found to yield fewer than one in 106 prototrophic revertants. Additionally, the level of [rho-] in the wild-type tester culture used was measured; that value is used in calculating the percent suppressiveness of the cross as noted below. Other genetic procedures-I . The mitochondrial genotypes of diploid progeny of crosses were determined as described by PERLMAN and BIRKY(19748) which is a minor modification of the standard cross as defined by COENet al. (1970); the major differences are the use of RG media instead of YEPGly and mating of cells in suspension instead of as a pellet. 2. Diploids were sporulated as described by ROTHand H~LVORSON 1969; random spores were obtained after digestion of the ascus walls with glusulase. 3. Suppressiveness of petites was determined by scoring the zygotic clones for [rho-] progeny using the triphenyl tetrazolium overlay procedure of OGUR, ST. JOHNand NAGAI(1957). The quantity “percent suppressiveness” was calculated using the equation of SHERMAN and EPHRUSSI (1962) and the experimentally determined values for the percent [rho-] zygotic clones and percent [rho-] in the wild-type tester culture. Materials-Chloramphenicol, erythromycin, ethidium bromide, oligomycin and triphenyltetrazolium chloride were obtained from Sigma Chemical Company, St. Louis, Missouri. Paromomycin sulfate was donated by the Parke, Davis Company, Detroit, Michigan. Berenil was a gift of Dr. H. Loewe of Farbwerke Hoechst, Frankfurt (Main) Germany. Glusulase was purchased from Endo Laboratories, Garden City, New Jersey. All other chemicals were of reagent grade. RESULTS
Mitochondrial marker transmission is reduced by petite induction In preliminary studies it was found that the capability of transmitting each of four mitochondrial markers is reduced by treatment of wild-type cells either with EtdBr or Berenil followed by their mating to a common wild-type tester strain. In all these experiments the treated parent was IL16-lOD, which exhibits the mitochondrial genotype, [rho+ ome- CHLR,,, ERYS OLlsl,z, P A R S ] .WildOLZR41 type tester strains containing the markers [om&] or [ome-], [ERYRZzl or [ P A R n l ] ,all auxotrophic for characters different from IL16, were used SO that transmission of all markers could be studied in both homopolar and heteropolar ( [ome-] x [ome-] , [ome-1 x r o m e f ] , respectively) crosses. In Figure 1 we present data on petite induction f o r IL16-1OB treated with EtdBr (A) or Berenil (B) under growing conditions. At each time-point noted, a sample of cells was removed, washed to remove exogenous mutagen and then mated to one or more [rho+] testers in fresh, drug-free medium for 150 min. The ability of the mutagenized cells to transmit the markers was then analyzed by replica-plating of random diploid prototrophic progeny cells. In many ways
649
PETITE TRANSMISSIONAL TYPES 100 0
100
-
10
m c I
E W
E
m
02
U L
m n
1000
10 0
I
10
0
40
I
I 80
i
We have studied a number of petite [rho-] mutants of Saccharomyces cerevisiae induced in a wild-type strain of mitochondrial genotype [omeCHLR ERYS OLP,,,,, PARS] by Berenil and ethidium bromide, all of which have retained two mitochondrial genetic markers, [CHLE] and [ERYE], but have lost all other known markers. Though stable in their ability to retain these markers in their genome, these mutants vary widely among themselves in suppressiveness and in the extent to which the markers are transmitted on crossing to a common wild-type tested strain. In appropriate crosses all of the strains examined in this study demonstrate mitochondrial polarity, and thus have also retained the [ome-] locus in a functional form; however, five different transmissional types were obtained, several of them quite unusual, particularly among the strains originally induced by Berenil. One of the most interesting types is the one that appears to reverse the parental genotypes with [CHLR ERYs] predominating over [CHLS ERYE] in the diploid [rho+] progeny, rather than the reverse, which is characteristic of analogous crosses with [rho+] or other petites. Mutants in this class also exhibited low o r no suppressiveness. Since all of the petites reported here are derived from the same wild-type parent, and 53 have the same nuclear background, we have interpreted the transmissional differences as being due to different intramolecular arrangements of largely common retained sequences.
HE cytoplasmic petite [rho-] mutation was first discovered and characterand his colleagues in the late Tized in Saccharomyces cerevisiae by EPHRUSSI 3 940's (EPHRUSSI, HOTTINGUER and CHIMENES 1949; EPHRUSSI, HOTTINGUER and TAVLITZKI (1949). These early studies revealed that the mutation behaves as a "-Mendelian character in genetic crosses. Since the irreversible genetic change produced a permanent impairment of mitochondrial respiratory function, it was tentatively concluded that it was due to the loss of a cytoplasmic genetic element, probably localized within the mitochondria. Shortly thereafter EPHRUSSIreported that the cytoplasmic factors of some petite mutant strains interfere with the expression or replication of the normal factor in the diploid state in contrast to the first-reported mutants that did not HOTTINGUER and ROMAN1955). Those in the former affect normal (EPHRUSSI, class were termed suppressive and those in the latter, neutral petites. Suppressive petites were found to vary widely in the extent of their suppressiveness, an Genetics 82 : 645-663 April, 1976.
646
P.
S. PERLMAN
observation that suggested that there exist many, genetically different, petite lesions (EPHRUSSI and GRANDCHAMP 1965; EPHRUSSI, JAKOB and GRANDCHAMP 1966). However, all these petites were indistinguishable in their phenotype which is deficient in cytochrome-linked respiratory and phosphorylating functions (SLONIMSKI 1949; SLONIMSKI and EPEIRUSSI 1949; MAHLER1973) and-as discovered more recently-in mitochondrial protein synthesis (KUZELAand GRECNA 1969; SCHATZ and SALTZGABER 1969). The discovery of mitochondrial DNA (mtDNA) in the early 1960’s was soon €ollowed by the discovery that petite mutants often contained mtDNA with novel physical and chemical characteristics (MOUNOLOU, JAKOB and SLONIMSKI 1966; MEHROTRA and MAHLER 1968). It has since been demonstrated that most, and occasionally all, of the base sequences in these mutant mtDNAs are also found in wild-type [rho+] mtDNA, but that all o i them lack, in variable amounts, the sequences and the genetic information characteristic of this wild1975); thus they can be considered formally type DNA (reviewed by PERLMAN as deletion mutants. Since a number of segments located in different regions of mtDNA code for the RNA components of the mitochondrial translation machinery and since this machinery is essential for the expression of full mitochondrial function, it is not surprising that, at the level of mitochondrial respiratory competence, petites are phenotypically identical. Recently, several laboratories have undertaken detailed analyses of mtDNA from a small number of petite mutants (FAYE et al. 1973; LOCKER, RABINOWITZ and GETZ19741a,b; MOLet al. 1974; reviewed in PERLMAN 1975). These elegant studies show clearly that the mtDNA in these mutants retains wild-type sequences in reiterated form, as well as rearrangements of them, such as inversions. The size of the basic repeating units can be as small as 70 base pairs (Mor, et al. 1974) or as large as 30-50% of the wild-type genome (LAZOWSKA et al. 1974; MICHEL et al. 1974). I n contrast to this extensive information on the molecular level, the transmission genetics of these mutants has been largely neglected. It is known that it is possible to obtain petites that retain any particular mitochondrial marker; two or more markers may also be retained, but not in all conceivable combinations et al. 1969; RANK1970; F-~YE et al. 1973; UCHIDAand SUDA1973; (GINGOLD DEUTSCH et al. 1974; MOLLOY, LINNANE and LUKINS1975; PERLMAN 1975). Some quantitative information of the ability of petites to transmit particular and LINNANE 1972; RANK markers has been reported ( COENet al. 1970; NAGLEY et al. 1971; FAYE et al. 1970), including some multifactorial crosses (BOLOTIN 1973; MOLLOY,LINNANEand LUKINS.1975); published data indicate that mutants of apparently identical genotype may differ in their degree of transmission and in their suppressiveness. Suppressiveness does not appear to be correlated with any particular markers but, according to LINNANE and RANK,all those strains that did retain any markers at all were found to be suppressive; at least some marker-retaining petites in the collection at Gif are neutral when crossed [rho+] testers (P. P. SLONIMSKI, personal communication; P. PERLMAN, unpublished data), but this point is not apparent from published data with these strains.
647
P E T I T E TRANSMISSIONAL TYPES
We have isolated and characterized a number of mutants induced by Berenil and PERLMAN 1973; PERLMAN and MAHLER 1973) and ethidium bromide (EtdBr) (SLONIMSKI. PERRODIN and CROFT 1968), all of which have , have retained two mitochondrial genetic markers[ CHLR321] and [ E R Y E ]but lost others, i.e. [OLZs,,,,,] and [ P A R S ] .Though stable in their ability to retain these markers in their genome, these strains vary widely in suppressiveness and in the extent to which the markers are transmitted on crossing to [rho+] tester strains. Each strain examined in this study demonstrates mitochondrial polarity (in appropriate crosses) and thus has also retained the locus [ome] in a functional form [OW-] ; however, several unusual transmissional types were obtained, particularly among the strains induced by Berenil. One of the most interesting types is one that appears to reverse the parental genotype with [CHLRERYE] predominating over [CHLSE R Y R ] in the diploid [rho+] progeny, rather lhan the reverse which is characteristic o€ analogous crosses with [rho+] or other petites. Mutants of this type also exhibited low or no suppressiveness. Since all of the petites reported here are derived from the same wild-type parent and, therefore, possess the same nuclear background, we have interpreted the transmissional differences as being due to different intramolecular arrangements of largely common retained sequences. (MAHmR
MATERIALS A N D METHODS
Yeast strains and culture conditions-Yeast strains used in these studies are listed in Table 1; all petite strains used are derived from IL16-1OB. The symbols for mitochondrial loci reflect the suggestions of SHERMAN and LAWRENCE (1974); [CHL], [ E R Y ] , [OLZl,s,3] and [ P A R ] are 1 x i influencing sensitivity o r resistance to chloramphenicol, erythromycin, oligomycin (three separate loci) and paromomycin, respectively; [ome] is the mitochondrial locus, omega (BOLOTIN et al. 1971; DUJON,SLONIMSKI and WEIL 1971.; PERLMAN and BIRKY1974; HOWELL et al. 1974); the superscripts R, S, and 0 denote resistance. sensitivity or gene absence, respectively. Allele numbers are those assigned by the laboratory that provided each strain. Media (minimal medium [MD], semi-synthetic medium with dextrose [RD] or glycerol [RG] and RG plus antibiotics) and BIRKY(1974). are described i n PERLMAN TABLE 1 Yeast strains used Strain
IL16-1OB 55R5-3C/221 D22 D22-ER2 D22-Al5 D22-A21 DPl-IB/517 D243-4A-OR 4810 AMFP-9 (32)l-2/3
Nuclear genotype
a his
a ura a ade a ade
ade ade his trp ade lys a trp a arg met a ade a a a a
Mitochondrial genotype
[rho]
[ome] [CHL] [ERY] [OLI,] [OL2,1 [OLI,I [PARI
S R221
S S
S S
S S
s
s
s
s
s
S
Re S
+f -- R 3 2 1S +
+
+ + + +
+ + + + +
+ +
+ + +
+
-
S S S R517 S S S S S R321 R221 R321 S
S S
s
S S S S S R 1 5 S S S S R 2 1 S S S S S R4 S S S S S S R1 S S S S R4 S S S
Source of strain
SLONIMSKI SLONIMSKI WILKIE WILKIE/BIRKY GRIFFITHS GRIFFITHS SLONIMSKI CRIDDLE KLEESE FRAENKEL
PER LM A N
648
P. S. PERLMAN
Isolation of petite mutants-Petites were induced by treatment of a culture of IL16-IOB growing in 1% glucose medium (RD) with EtdBr (25 pM) for 10 min or Berenil (50 pM) f o r 30 min. The mutagenized cells were then washed and plated on RD solid medium to yield primary mutagenized clones; these were suspended in water and subcloned and the subcloning repeated once again to yield the strains which were analyzed in more detail as described in the text. Mating conditions-For matings of petite with wild-type strains the petites were grown to exponential phase in RD, and the wild-type testers in RG. The testers were grown in RG in order to maintain a high level of [rho+] in the cultures used. Cells were mated in RD liquid medium at IO7 cells/ml (equal numbers of each parent) at 30” with shaking for two hours. The mating mixture was then washed and plated at several dilutions on M D to select for prototrophic diploid clones. In all experiments both parents were found to yield fewer than one in 106 prototrophic revertants. Additionally, the level of [rho-] in the wild-type tester culture used was measured; that value is used in calculating the percent suppressiveness of the cross as noted below. Other genetic procedures-I . The mitochondrial genotypes of diploid progeny of crosses were determined as described by PERLMAN and BIRKY(19748) which is a minor modification of the standard cross as defined by COENet al. (1970); the major differences are the use of RG media instead of YEPGly and mating of cells in suspension instead of as a pellet. 2. Diploids were sporulated as described by ROTHand H~LVORSON 1969; random spores were obtained after digestion of the ascus walls with glusulase. 3. Suppressiveness of petites was determined by scoring the zygotic clones for [rho-] progeny using the triphenyl tetrazolium overlay procedure of OGUR, ST. JOHNand NAGAI(1957). The quantity “percent suppressiveness” was calculated using the equation of SHERMAN and EPHRUSSI (1962) and the experimentally determined values for the percent [rho-] zygotic clones and percent [rho-] in the wild-type tester culture. Materials-Chloramphenicol, erythromycin, ethidium bromide, oligomycin and triphenyltetrazolium chloride were obtained from Sigma Chemical Company, St. Louis, Missouri. Paromomycin sulfate was donated by the Parke, Davis Company, Detroit, Michigan. Berenil was a gift of Dr. H. Loewe of Farbwerke Hoechst, Frankfurt (Main) Germany. Glusulase was purchased from Endo Laboratories, Garden City, New Jersey. All other chemicals were of reagent grade. RESULTS
Mitochondrial marker transmission is reduced by petite induction In preliminary studies it was found that the capability of transmitting each of four mitochondrial markers is reduced by treatment of wild-type cells either with EtdBr or Berenil followed by their mating to a common wild-type tester strain. In all these experiments the treated parent was IL16-lOD, which exhibits the mitochondrial genotype, [rho+ ome- CHLR,,, ERYS OLlsl,z, P A R S ] .WildOLZR41 type tester strains containing the markers [om&] or [ome-], [ERYRZzl or [ P A R n l ] ,all auxotrophic for characters different from IL16, were used SO that transmission of all markers could be studied in both homopolar and heteropolar ( [ome-] x [ome-] , [ome-1 x r o m e f ] , respectively) crosses. In Figure 1 we present data on petite induction f o r IL16-1OB treated with EtdBr (A) or Berenil (B) under growing conditions. At each time-point noted, a sample of cells was removed, washed to remove exogenous mutagen and then mated to one or more [rho+] testers in fresh, drug-free medium for 150 min. The ability of the mutagenized cells to transmit the markers was then analyzed by replica-plating of random diploid prototrophic progeny cells. In many ways
649
PETITE TRANSMISSIONAL TYPES 100 0
100
-
10
m c I
E W
E
m
02
U L
m n
1000
10 0
I
10
0
40
I
I 80
i
