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Biogenesis of mitochondria 44

Biogenesis of mitochondria 44
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  Molec. gen. Genet. 145, 43-52 (1976) © by Springer-Verlag 1976 Biogenesis of Mitoehondria 44 Comparative Studies and Mapping of Mitochondrial Oligomycin Resistance Mutations in Yeast Based on Gene Recombination and Petite Deletion Analysis M.K. Trembath, P.L. Molloy*, K.S. Sriprakash, G.J. Cutting, A.W. Linnane, and H.B. Lukins** Department of Biochemistry, Monash University, Clayton, Victoria 3168, Australia . Summary. A comparative study of eight indepen- dently isolated mitochondrial oligomycin resistant mutants obtained from three laboratories show a vari- ety of phenotypes based on cross resistance to venturi- cidin and sensitivity to low temperature. Analysis of recombination between pairs of markers indicate the existence of at least three genetic classes; class A, cross resistant to venturicidin and including the mutations Oil b [olil-r], [OLG1-R], [tso-r]; class B, mutations O~, [olil7-r], [OLG2-R]; and class C, the mutation On. The recombination data is consistent with mutations of each class residing in three separate genes, although mutations of class A and B show very close linkage. Recombination in non-polar crosses has demon- strated that markers of all three classes are linked to the mikl locus in the configuration (AB)-mikl-C. The mapping of this segment with respect to other markers of the mitochondrial genome and the order of classes A and B was established by analysis of co-retention frequencies of markers in primary petite isolates as well as by analysis of marker overlap of genetically and physically defined petite genomes. The unambiguous order eryl-A-B-mikl-C-par was ob- tained. DNA-DNA hybridization studies using mtDNA isolated from selected petites confirms this map and estimates the physical separation of markers. A reasonable correlation exists in this region of the genome between distances estimated physically by hybridization and genetically by frequency of recom- bination in non-polar crosses. It is postulated that the oligomycin-mikamycin linkage group represents a cluster of genes involved in determining a number of mitochondrial membrane proteins associated with the mitochondrial ATPase and respiratory complex III. * Present Address: Department of Human Genetics, Yale Univer- sity, New Haven, Connecticut 06510, USA ** This work was supported by the Australian Research Grants Committee, Project D65/15930 Introduction One aspect of current interest in the study of mito- chondrial genetics in Saccharomyces cerevisiae is the establishment of procedures for the mapping of mutations on the mitochondrial genome. The initial data on the linkage relationships of some mitochon- drial antibiotic resistance markers were obtained from the ordered transmission of markers and the polarity of recombination in polar crosses (Thomas and Wil- kie, 1968, Coen etal., 1970; Bolotin etal., 1971; Howell et al., 1973). However, significant polarities are only observed for markers situated in a relatively small part of the genome close to the co +/o)- alleles (Howell et al., 1973, 1974a; Avner et al., 1973; Wolf et al., 1973; Linnane et al., 1974; Netter et al., 1974): markers distributed elsewhere on the genome cannot be readily mapped by transmission or recombination. The difficulty of mapping by recombination has been essentially overcome by the combined applica- tion of DNA-DNA hybridization using mitochon- drial DNA from stable petite strains and the analysis of co-retention of markers in spontaneous petite mutants derived from multiply marked strains. The principle of this latter procedure is that markers being commonly co-retained are mapped close together whereas those which are often separated following petite mutation are considered to be far apart. From the relative co-retention frequencies observed for pairs of markers a marker order can be established (Molloy et al., 1975). DNA-DNA hybridization stud- ies using mitochondrial DNA from stable petite strains carrying various combinations of markers may then be employed to establish the physical location of the markers on the genome (Sriprakash et al., 1976). Furthermore, physically and genetically character- ised petite strains could be employed as a means of mapping new markers (or markers not previously assigned an unambiguous map location). This would be achieved by the analysis of diploid progenies de-  44 M.K. Trembath et al. : Mapping of Mitochondrial Oligomycin Resistance Mutations in Yeast rived from crosses of a [rho ] strain carrying the unmapped marker with a series of stable petite strains carrying various different physically defined segments of the mitochondrial genome. Two alleles of the marker in question would be present amongst the diploid progeny of a cross only when the petite parent retained the segment of the genome overlapping the marker. In this communication this latter procedure is applied, together with an analysis of recombination, to the mapping of several mutations to resistance to oligomycin and a mikamycin resistant mutation. These markers all occur in a region of the genome in which there is little or no polarity of recombination and hence uncertainty as to the relative marker order. Recent articles by Avner et al. 1973) and Lancashire and Griffiths 1975), have described three mitochon- drial loci for resistance to oligomycin based on recom- bination and cross resistance behaviour of their mutants. Several laboratories employ a number of indepen- dently isolated oligomycin resistant mutants Avner and Griffiths, 1970; Stuart, 1970; Wakabayashi and Gunge, 1970; Griffiths et al., 1972; Rank and Martin, 1972; Mitchell etal., 1973; Shannon etal., 1973; Callen, 1974; Trembath et al., 1975). A comparative study of the mutants from three laboratories Lin- nane, Griffiths and Callen) is made in this paper and the map location of the oligomycin resistance loci and the locus for mikamycin resistance is established. Materials and Methods Strains. The strains used and their genotypes are listed in Table 1. The srcin of strains carrying mutations to oligomycin resistance are also listed in Table 1. Strains D22/A15, D22/A19 and D22/A21 were generously provided by D.E. Griffiths and D8 and El5 by D. Callen. Other mutant strains were derived in this laboratory Linnane et al., 1968; Bunn et al., 1971 ; Mitchell et al., 1973; Mol- loy et al., 1973; Howell et al., 1974b; Trembath et al., 1975) or constructed by selection of recombinant diploids from appropriate crosses followed by sporulation and tetrad dissection. The nomen- clature used is that given by the authors srcinally describing the genetic identity of the markers. Table 1. Strains used Strain Name Oligomycin Other markers Source of strain resistance marker aL4100 [olil-r] co+ ~ ura his Bunn L4200 ,, co + a ura his L4400 ,, co+ a adel trpl lys2 863-2C(Y) ,, co + aura [capl-r eryl-r mikl-r par-r/ 770-7B (U) ,, co+ aura arg4-17 lys2 [capl-r eryl-r mikl-r] bD22/A19 Om CO + a ade2 gal Griffiths T247-1C ,, co + ~ trpl lys2 T247-2B ,, co+- ~ ade lys2 c D8 [OLG1-R] co + ~ leu Callen D8' ,, co+ a trp dL748 [tso-r] co + c~ ura his Trembath T131-3D ,, co+ a adel trpl ura T 108-6B [olil 7-r] co + c~ ura Trembath T108-2A ,, co + a trpl lys2 e D22/A21 Ol co + a ade2 gal Griffiths T248-1D ,, co + ~ adel E 15 [OLG2-R] co + ~ leu Callen ElY co+ a leu e D22/A15 01i co + a ade2 gal Griffiths T245-3A co + ~ trpl lys2 9003-2D ,, co+ c~ ade2 [capl eryl-r mikl-r par-r/ 690/13 - co+ c~ trpl lys2 [mikl-r] D515-1B - co+ ~ his2 Sherman D311-3A - co + a trp2 lys2 him Sherman a Mitchell et al. (1973) b Strain reported by Avner and Griffiths (1973b), O m notation Lancashire and Griffiths (1975) ° Callen (1974) d Trembath et al. (1975) ° Strains reported by Avner and Griffiths (1973a), O I and OH notation Avner et al. (1973)  M.K. Trembath et al. : Mapping of Mitochondrial Oligomycin Resistance Mutations in Yeast 45 Media. The complex media used and the concentrations of antibio- tic supplements used were as described by Molloy et al. (1975). Venturicidin, added to media at 5 gg/ml, was a generous gift from D.E. Griffiths. Strains were termed temperature sensitive when there was no evidence of growth on YEPE medium after 4 days at i9 ° wild-type clones at this temperature are fully developed after 4 days. Analysis of Recombination in Crosses between [rho+] strains. Crosses to assess the capability of various oligomycin resistance markers to genetically recombine, were conducted as described by Trembath et al. (1973) for similar crosses between spiramycin- resistant strains, except that recombinant clones of the type [olix-s oliy-s] were detected by their ability to grow on YEPE medium but not on oligomycin supplemented YEPE medium. The diploids of crosses between strains carrying [mikl-r] and strains carrying a particular oligomycin resistance marker were analysed on YEPE, YEPE +oligomycin, YEPE +mikamycin and YEPE + oligomycin + mikamycin media. In such crosses the replica plating involved suspending each individual colony in sterile water and transferring samples with a metal inoculator to the series of test plates. This technique, which has the advantage of transferring fewer cells and producing a more even patch than velveteen replica plating, was used for all determinations of resistance to mikamycin. The concentration of mikamycin necessary for clear discrimination between resistance and sensitivity is criticai, and where a heavy patch of cells is transferred, as often occurs using the velveteen method, growth of sensitive cells not in close contact with the antibiotic may be observed. Isolation of Petites. The isolation of spontaneous petites and the determination of loss or retention of antibiotic resistance marker in these petites were as described by Molloy et al. (1975). Results Classification of Oligomycin Resistance Markers on the Bases of Phenotype and Genetic Recombination Table 1 lists the strains which carry the eight mito- chondrial oligomycin resistance markers used in this study. These eight markers can be divided into two groups on the basis of resistance or sensitivity they confer to the antibiotic venturicidin; the markers [olil- F] Oli D [OLG1-R] and [tso-r] confer resistance to ven- turicidin simultaneously with the resistance to oligo- mycin, whereas [oli17-r], 0~, [OLG2-R] and On confer resistance only to oligomycin Table 2). Results for markers O~, On and O m are consistent with those reported by Avner and Griffiths 1973a) and Lanca- shire and Griffiths 1975). Amongst the mutants show- ing cross resistance to venturicidin [tso-r] strains showed a markedly reduced capacity for growth on nonfermentable substrates at 19 °, a characteristic not shared with [olil-r], Om or [OLG1-R] strains. In order to determine whether the members of a single phenotypic group represented mutations at the same or different loci, and to estimate genetic distances between different loci, a series of crosses Table 2. Phenotypes for several mitochondrial markers for oligo- mycin resistance Marker Phenotype Oligomycin Venturicidin Low temperature [olil-d R R R 0 m R R R [O/~G1-R] R R R [tso-r] R R S [olilT=r] R S R Oi R S R [OLG2-R] R S R 0 H R S R R and S denote resistance and sensitivity to an antibiotic or low temperature as defined in Materials and Methods Table 3. Frequencies of recombination between oligomycin resistance markers in non-polar crosses [olil-r] <0.04 2,375 1.8 1,802 0.07 4,552 [OLG1-R] < 0.04 2,494 <0.07 1,486 Om <0.05 0.2 3.1 2,389 1,442 [olil7-r] 0.2 2,577 1.1 2,039 <0.06 1,577 [OLG2-R] 0.2 0.3 <0.05 <0.07 1,380 1,512 1,782 1,462 5.8 822 1,928 [tso-r] 0.4 1.9 1,132 2,311 0.3 1.5 1,416 1,422 1.3 0.9 1,836 2,134 6.9 8.0 1,214 1,837 6.7 866 7.4 1,060 6.4 920 Pairs of strains carrying different oligomycin resistance markers as shown in Table 2 were crossed and the percentage ofoligomycin sensitive diploids colonies was scored. Crosses involving each pair of markers were performed in the a x ct and c~ x a configurations and since the crosses were non-polar the data was pooled. The upper figure in each square is me perccnta~ of diploid colonies which were oligomycin sensitive; tl frequency of recombination be- tween markers is estimal by multiplying this number by 2. The lower figure is the total number of diploids analysed. Each was performed at least twice. Ol 6.3 1,308 O n  46 M.K. Trembath et al. : Mapping of Mitochondrial Oligomycin Resistance Mutations in Yeast was performed involving pairs of oligomycin resis- tance markers. The crosses were scored for the percent- age of oligomycin sensitive diploids generated by a recombination event in the region of the genome be- tween the two markers. As all crosses performed were non-polar (co + x co+), the total frequency of recom- bination could be estimated as twice the frequency of oligomycin sensitive diploids. The results of all pairwise crosses are presented in Table 3. Within the first phenotypic group showing venturicidin resistance ([olil-r], Oli D [OLG1-R] and [tso-r]) no recombination was detected between the pairs [olil-r]/[OLG1-R] and [OLG1-R]/Oln. Similarly, crosses involving the pairs of markers [tso-r]/Om and [tso-r]/[OLG1-R] showed no oligomycin sensitive dip- loids amongst their progeny, and only a very low level of recombination (0.14% total) was observed to take place between the [tso-r] and [olil-r] mutations. However, a significant level of recombina- tion was apparent between the pair [olil-r]/Oni. Although the spatial arrangement of these mutations cannot be defined on the basis of these data, it appears likely that Oni and [olil-r] are the distal mutations within this group, and one possible interpretation of the data is that [OLG1-R] is a small deletion which overlaps both the olil and Onl loci. The lack or very low frequency of recombination observed between these four markers is probably consistent with them being mutations of a single gene. On the bases of the phenotypic and genetic behaviour of these four mutations it is concluded they belong to the same class, designated class A, as illustrated in Table 4. Crosses between pairs of markers of the second phenotypic group [olil7-r], [OLG2-R], O1 and On clearly indicated that the Oil mutation was located a considerable distance from the other three, the recombination frequencies being 14.8%, 12.8% and 12.6% between Oil and [olil7-r], [OLG2-R] and O 1 respectively. Hence, O n clearly constitutes a separate class from the other three markers, to be referred to as class C (Table 4). The remaining three markers [o#17-r], [OLG2-R] and Ol were indistinguishable from each other, yielding no recombinants in almost Table4. Classification of mitochondrial oligomycin resistance markers based on genetic behaviour Class A Class B Class C [olil-r] [olil7- d 0 n [OLG-1-R] [OLG2-R] Om Oi [tso-r] Probably I gene 1 gene 1 gene 5,000 diploids. The phenotypes and genetic behaviour associated with these markers could not eliminate the possibility that they represent similar mutations iso- lated independently in three laboratories. Thus [oli17- r], [OLG2-R] and O1 constitute class B of oligomycin resistance markers (Table 4). From the recombination data of pairwise crosses between markers of class A and class B (Table 3) it was apparent that the two classes were closely linked with an average recombinant frequency of 2.5%. A consideration of the recombination frequen- cies between individual markers of class A and class B does not reveal any consistent pattern to allow the determination of marker order within class A. Neither is it possible to resolve whether class A or class B is closer to On (class C), the average total recombination frequencies being 13.7% and 13.4% respectively. Ordering of Classes A and C with Respect to the mikl and eryl Loci by Petite Deletion Analysis The location of oligomycin resistance markers can be approximated in relation to other known loci of the mitochondrial genome by the measurement of the co-retention or co-deletion of markers resulting from petite mutations (Molloy et al., 1975). For these analyses two strains each carrying five markers, but differing in the oligomycin resistance marker they contained, were employed; strains 863-2C [par-r mik l-r olil-r ery i-r cap l-r] and 9003-2D [par-r mik l-r eryl-r capl-r]On. Table 5a presents the frequencies of primary petite clones of different genotypes found among spontaneous petites isolated from these strains. As has been previously noted (Molloy et al., 1975) there was marked variation in the overall fre- quency of marker retention between different strains; 60% and 19% of the petites isolated from 863-2C and 9003-2D respectively retained no markers, while 9.8% and 6.6% respectively retained all markers. The significance of this observation is not immediately apparent, but is not related to the relative petite mutation fi'equencies of the two strains which are both about 1%. An analysis of the significant results relating to the patterns of co-retention of markers in petites retaining one or more markers is presented in Table 5b. In the case of strain 863-2C, petites which retained both [mikl-r] and [eryl-r], also retained [olil-r] in all cases. Further, 95% of petites which had been deleted in both of these loci had also lost [olil-r]. These data are consistent with the previously deter- mined physical and genetic location of the olil locus between mikl and eryl (Molloy et al., 1975 ; Sriprak-  M.K. Trembath et al. : Mapping of Mitochondrial Oligomycin Resistance Mutations in Yeast 47 Table 5a-e. Spontaneous petites were isolated from two strains each carrying five markers: 863-2C [capl-r ervl-r olil-r mikl-r par-r] and 9003-2D [capl-r eryl-r mikl-r par-riO n. The retention or deletion of markers in petites was established by the method described in Materials and Methods. The [rho+] tester strains to which the petites were crossed were D515-1B for petites from 863-2C and D311-3A for petites from 9003-2D. a) Lists the frequencies of all the different genotypes observed in petites of strains 863-2C and 9003-2D. r represents the retention of the resistance marker, O represents its deletion. b) Lists the frequency with which oligomycin resistance was retained or deleted in petites of strains 863-2C and 9003-2D known to have retained or lost concomitantly the pairs of loci mikl/eryl and par/mikl. The table was derived from data presented in part A. c) Lists the frequency with which oligomycin resistance was retained or deleted concomitantly with either of the loci of the mikl/eryl or par/mikl pairs in petites where only one of each pair of loci had been retained. The table was derived from data presented in part A ash et al., 1976). However, no such co-retention or co-deletion was observed between On and the mikl/ eryl pair of loci; 72% of [mikl-r eryl-r] petites from strain 9003-2D had lost O n and 48% of [mikl-o eryl- o] petites still retained O11. Thus it appears that, unlike olil, 0~ is not located between mikl and eryl. This conclusion was confirmed by analysis of co-retention and co-deletion of O u with the par/mikl pair of loci. As seen in Table 5b On was retained whenever [par-r] and [mikl-r] were retained together and deleted whenever these markers were both lost, strongly sug- gesting that O n lies between the mikl and par loci. When the par and mikl loci were separated by petite deletion (as shown in Table 5c) the OH locus was more frequently co-retained or co-deleted with the rnikl locus than par suggesting a closer linkage with the mikl locus. From similar analyses it is appar- ent that the olil locus is located approximately mid- way between mikl and eryl. Recombination between [mikl-r] and O1 Oil and Om The recombination data obtained from crosses be- tween the oligomycin resistance markers presented in Table 3, though based on the analysis of only one recombinant type, indicated a separation of O11 from the other oligomycin resistance markers of approxi- mately 13-14%. The availability of [mikl-r], which apparently is linked to each of the classes, introduces a marker of different phenotype which enables com- plete recombination data to be obtained from crosses involving [mikl-r] and oligomycin resistance markers of each class in turn. Thus a more precise location may be defined for the various classes of oligomycin resistance markers and the order of classes A and B may be more clearly resolved. Table 5a. Frequencies of different genotypes among spontaneous petite isolates from strains carrying five markers Petite genotype Frequency of genotype (%) Strain par mikl olil eryl capl 863-2C 9003-2D or 0~ r 1- r O O 3.0 14.1 O O O r r 1.6 12.8 r r O O O 1.2 0 O O r 1 r 4.2 0 r O O O O 7.3 6.6 O r r r r 2.8 4.3 r O r r r 3.7 0.7 O r O O O 0 3.0 1 O O t r 4.2 13.2 O r r O O 1.2 4.1 1 r O r r 0 2.7 O O r O O 0.2 2.5 r O r O O 0.2 2.5 O r O r r 0 6.8 O O O O r 0 0.5 O O O r O 0 0.5 r O r O r 0 0.5 r r r r r 9.8 6.6 O O O O O 60 18.7 Total petites analysed (500) (439) Table 5b Petite genotype Frequency of retention or deletion of oligomycin resistance (%) r 0 olil 100 0 [mikl-r eryl-r] OH 28 72 olil 5 95 [mikl-o eryl-o] O11 48 52 olil 71 29 [par-r mikl-r] Oil 100 0 olil 72 28 Lvar-o mik l-o] 0tl 0 100 Table 5 c Petite genotype Frequency of co-retention and co-deletion with particular markers (%) par mikl eryl [mikl-r eryl-o] + [mikl-o eryl-r] [par-r mik -o] + [par-o mik l-r] ofil 52 48 O n -- 92 8 o~1 24 76 -- Oil 32 68 --
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