Proc. Natl. Acad. Sci. USA Vol. 81, pp. 7156-7160, November 1984 Genetics Yeast mitochondrial genomes consisting of only AT base pairs replicate and exhibit suppressiveness (Saccharomyces cerevisiae/replication origins/DNA excision) WALTON L. FANGMAN* AND BERNARD DUJON Centre de Gdndtique Moldculaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France Communicated by Herschel L. Roman, July 16, 1984 ABSTRACT Mutants, called p-, that result from exten- genome in the zygote. The p- genome, in a fraction of the sive deletions of the 75-kilobase Saccharomyces cerevisiae zygotes, monopolizes the mitochondrial replication or segre- mitochondrial genome arise at high frequency. The remaining gation apparatus, excluding the p+ genome, which is ulti- mitochondrial DNA is amplified in the p- cells, often as head- mately destroyed or diluted out. [Recombination between to-tail multimers, producing a cell with the normal amount of the p- and the p+ genomes resulting in destruction of respi- mitochondrial DNA. In matings, some of these p- mutants ex- ratory competence has also been postulated as a mechanism hibit zygotic hypersuppressiveness, excluding the wild-type for suppressiveness. This mechanism is ruled out because mitochondrial genome (p+) from all the diploids that are pro- the p- mitochondrial DNA of the zygotic progeny is the duced. From a hypersuppressive p- strain, we isolated two same as the p- parent (7, 8).] p- mutants that exhibit >95% mutants with reduced suppressiveness. These mutants, one zygotic suppressiveness have been called hypersuppressives moderately suppressive and one nonsuppressive, retain only (9). Hypersuppressive p- mutants retain a short [usually 89 and 70 base pairs, respectively, of the wild-type mitochon- s2500 base pairs (bp)] tandemly repeated segment that drial genome. Their sequences consist of 100% A-T base arises from only three different regions of the p+ genome of pairs. Replication of DNA in the mitochondrion, formation the strain KL14-4A. Each hypersuppressive p- contains a and amplification of new deletion genomes, and exhibition of 300-bp stretch that is almost identical (8-11). These 300-bp suppressiveness do not require a single G C base pair. sequences in the three different regions have been called rep1, rep2, and rep3. While the rep sequences may corre- The dispensable nature of the Saccharomyces cerevisiae spond to three major origins of replication used by the p+ mitochondrial genome has allowed an extensive genetic genome, a role in another process, such as mitochondrial analysis of mitochondrial DNA gene organization and genome segregation, has not been excluded. expression (reviewed in ref. 1). However, because the mito- The p- genome HS3324, which contains rep2, exhibits au- chondrial genome cannot be isolated as a discrete DNA mol- tonomous replication sequence (ARS) activity when trans- ecule but only as fragments, the molecular details of its repli- formed into yeast cells as part of an Escherichia coli-yeast cation have not been determined. The wild-type 75-kilobase chimeric plasmid (8). The transformed plasmid appears to (kb) circular mitochondrial genome (p+) gives rise, by mas- reside in the yeast nucleus. Deletion analysis of the cloned sive deletion of p+ DNA, to respiratory-deficient mutants, 963-bp HS3324 DNA has shown that ARS activity requires p-s, at a high frequency (reviewed in ref. 1). Two observa- both a G+C-rich segment (Fig. 1, I) in the rep2 sequence, tions about p- mutants are clearly related to the mechanism and a short sequence from one of two A+T-rich segments (B of mitochondrial DNA replication. First, p- strains contain or B') in flanking unique DNA (11). Thus, while hyper- the same amount of mitochondrial DNA per cell as does the suppressiveness in the mitochondria may require only rep p+ wild type. That is, the DNA segment retained in a dele- sequences, ARS activity in the nucleus requires a second tion mutant is amplified during or immediately after the dele- element. tion event, and it is often present as a series of head-to-tail We began an analysis to determine more directly which circular multimers (although inverted repeats and more com- particular sequences in the mitochondrial DNA of a hyper- plex arrangements also exist). Second, p- mutants can arise suppressive p- mutant are required for suppressiveness in from many different regions of the p+ genome. Therefore, vivo. Using the HS3324 hypersuppressive p- strain, we iso- many different segments of the p+ genome are capable of lated mutants with reduced suppressiveness. The rationale promoting their own replication in the mitochondrion. was to determine whether such mutants had altered or de- When two genetically marked p+ strains are mated, the leted sequences in the rep region, the secondary elements resulting diploid segregates the different parental and recom- required for ARS activity, or other regions of the hyper- binant p+ genomes within a few cell divisions (2-4). Matings suppressive p- DNA. We find that two reduced suppressive between a p+ and different p- mutants can give very skewed mutants represent large deletions of the original hyper- results (5). Some p-s give rise to zygotic diploid clones, all suppressive p- genome with loss of the entire rep sequence of which contain p+ cells. Such a p- is said to be neutral with and all the other sequences required for ARS activity. The regard to zygotic suppressiveness. Other p-s produce some mutants, one neutral and one moderately suppressive, retain zygotic diploid clones that are entirely composed of p- cells. only 70 and 89 bp, respectively, from the p+ genome. These The fraction of zygotic diploid clones that are entirely p- is a DNAs contain only A T base pairs. characteristic of the particular p- mutant (6). A mutant that produces, for example, 20% p- diploid clones is said to be MATERIALS AND METHODS 20% suppressive. Strain KL14-4A/I21/HS3324 (a, hisi, trp2, leu2, p-), here Suppressiveness can be thought of as a consequence of called HS3324, and the growth media have been described competition between the amplified p- segment and the p' Abbreviations: kb, kilobase(s); bp, base pair(s); ARS, autonomous The publication costs of this article were defrayed in part by page charge replication sequence. payment. This article must therefore be hereby marked "advertisement" *Permanent address: Department of Genetics, SK-50, University of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Washington, Seattle, WA 98195. 7156 Downloaded by guest on September 28, 2021 Genetics: Fangman and Dujon Proc. Natl. Acad. Sci. USA 81 (1984) 7157 Mbo Ava1i Nde I Ava 11 Mbo I bp (11) containing the rep2 sequence. Individual colonies of ijct Ois HS3324 were tested for reduced suppressiveness by crossing ,.......... .....rp~ ~ " them on plates with a lawn of p+ cells. The zygotic diploids, selected by complementation of nutritional markers, form B I B patches that are replica-plated to glycerol plates to distin- 11110111i1111111111ilii S5 guish p+ and p- growth. Most patches, which contain many independent zygotes, give rise to only small papillae of growth, expected for the -98% suppressiveness of HS3324; FIG. 1. Map of HS3324 DNA. Map is arbitrarily linearized at the that is, -2% of the zygotes give rise to p+ clones, which can single Mbo I site. The map and the positions of the regions essential grow aerobically. An occasional patch produced confluent for ARS activity (I and B', or I and B) are taken from ref. 11. Arrow or nearly confluent growth on glycerol (Fig. 2a). In these labeledjct indicatesjunction between two excision points of HS3324 was and a subclone was mitochondrial DNA from the p+ genome. S4 and S5 are described in cases, the original colony purified, kesults. retested as large patches on a Petri dish (Fig. 2b). Approxi- mately 1% of the original HS3324 colonies were mutants by (11). A p0 derivative of HS3324 was obtained by growing these criteria. HS3324 in complete glucose medium with ethidium bromide Two of the spontaneous mutants, HS3324-S4 and HS3324- (20 ,g/ml). Suppressiveness tests used strain 777-3A/6 (a, S5 (here called S4 and S5), were analyzed further. Quantita- adel, pet9, p'). Media (2) and quantitative suppressiveness tive zygotic suppressiveness test results are shown in Table tests (12) have been described, except that zygotic colonies 1. The p+ tester culture contained 2.7% p- cells, as shown were scored for p' or p- phenotypes directly on unsupple- by the control mating to a p0 strain (a strain lacking mito- mented glucose plates after 10-14 days incubation at 28TC. chondrial DNA). These p- cells represent spontaneous p- p colonies remain small and white, while p+ colonies are mutants that arose during the cross or immediately before large and yellowish. Mitochondrial DNA was isolated by the mating, because the pet9 p+ tester culture cannot accumu- procedure described in ref. 13. late p- cells. Therefore, the results of each of the other mat- For yeast colony hybridization, portions of colonies were ings were adjusted for this background (31). Although S4 and picked from Petri dishes by laying a nitrocellulose sheet on S5 were isolated from the same culture, they appear to be the them for a few minutes. The inverted nitrocellulose sheet result of different mutations. S4 is essentially neutral (1.5% was incubated consecutively, at room temperature, on paper suppressive), whereas S5 is still moderately suppressive at filter sheets saturated with the following: (i) 50 mM 35%. Tris HCl, pH 7.5/25 mM sodium EDTA/1% 2-mercapto- A clone with reduced suppressiveness could actually be a ethanol/500 mM NaCl (30 min); (ii) solution i containing Zy- heterogeneous population of cells in which two kinds of molyase 60,000 (Seikagaku Kogyo, Tokyo) (65 min); (iii) 500 mitochondrial genomes are segregating (1), one the original mM NaOH (8 min); (iv) 1 M Tris HCl, pH 7.8 (2 min); (v) 500 hypersuppressive and the other a neutral variant or even a mM Tris HCl, pH 7.8/1.5 M NaCl (2 min, and then repeated p°.