Proc. Nati. Acad. Sci. USA Vol. 85, pp. 7288-7292, October 1988 Genetics Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA (Saccharomyces cerevisiae/oxil/rhol-/high-velocity microprojectile bombardment) THOMAS D. FOX*t, JOHN C. SANFORD*, AND THOMAS W. MCMULLIN* *Section of Genetics and Development, Cornell University, Ithaca, NY 14853; and tDepartment of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456 Communicated by Gerald R. Fink, June 10, 1988

ABSTRACT The mitochondrial gene oxil, carried on a properties outlined above for a rho- strain carrying reiterated bacterial plasmid, has been used to transform the mitochondria copies ofthe plasmid. The ability to generate such "synthetic of a yeast strain lacking mtDNA (rhoo). The plasmid DNA rho-" strains by transformation will now allow transfer of behaved in a manner entirely consistent with the known mutations generated in vitro to wild-type rho+ mtDNA as properties of normal yeast rho- mtDNA after its introduction well as examination of the function of altered genes in trans. by high-velocity microprojectile bombardment. Like the mtDNA sequences retained in natural rho- strains, the plas- mid DNA in the transformants was reiterated into concatemers MATERIALS AND METHODS whose size was indistinguishable from that of wild-type Media, Yeast Strains, and Genetic Methods. Minimal me- mtDNA. The oxil sequences in the transformants were sur- dium (SD), complete medium containing glucose (YPD), rounded by restriction sites derived from the plasmid that were complete medium containing the nonfermentable carbon not present in wild-type mtDNA. oxil genetic information in sources ethanol and glycerol (YPEG), and standard genetic these "synthetic rho"- strains could be expressed in diploids as described (8, 11). Key strains and their either after "marker rescue" by recombination with rho+ manipulations were mtDNA carrying an appropriate oxil point mutation or in trans genotypes are listed in Table 1. TD28rho° was generated by during the growth of diploids heteroplasmic for both the treatment of TD28 (12) with ethidium bromide (13). TF138 plasmid-derived oxil sequences and rho' mtDNA with oxil and TF140 were progeny of crosses between DAU2 (MATa, deleted. The ability to generate such "synthetic rho-" strains ade2, ura3, [rho+]) and the oxil mutants M8-171 and M11-30, by transformation will allow transfer ofmutations generated in respectively (8, 14, 15). TF145 was similarly derived from a vitro to wild-type rho' mtDNA as well as examination of the spontaneous oxil deletion isolated in this laboratory. All function of altered genes in trans. other strains have been described previously (8, 16, 17). Plasmids and Transformations. The plasmid pMT36 (18) An unusual and very useful feature ofmitochondrial genetics carries the wild-type oxil gene inserted at the Pst I site of in the yeast Saccharomyces cerevisiae is the occurrence of pBR322 (19) after GC-tailing such that the mtDNA insert mutations, termed rho-, that block respiration by deleting could be cleaved out by using either the Pst I sites derived large portions of wild-type (rho+) mtDNA (extensively from the plasmid or the Msp I (Hpa II) sites derived from reviewed in ref. 1). rho - mutations may delete any segment mtDNA (8) (see Fig. 1). pCGE137 carries the wild-type URA3 of mtDNA, and in the limit case such mutations produce gene and a 1.56-kb fragment containing the origin of repli- strains, termed rho', whose mitochondria contain no mtDNA cation of the 2-,um plasmid of yeast in pBR322 and has been at all (reviewed in ref. 1). DNA sequences retained in rho- used previously (20). Equal amounts of these plasmid DNAs strains are present as concatemers whose size roughly equals were mixed and precipitated onto 1-,um tungsten particles as that of wild-type mtDNA. Genetic information encoded in described (5), with the following modifications. A 2.5-,ul rho - mtDNA can be expressed by crosses to appropriate aliquot of DNA (1 ,ug/,u) was added to 25 ,ul of a suspension rho + strains, either after recombination with rho + mtDNA of 1-,um tungsten particles (50 mg/ml) in 50% glycerol. Then (marker rescue) or in trans by selective maintenance of a 25,ul of 2.5 M CaCI2 and 5 ,ul of 1 M spermidine were added. heteroplasmic state. Because of these properties, the ability The solution was allowed to sit for 10 min. The particles were to manipulate the content of rho - mtDNA by genetic then sedimented by brief centrifugation, 25 ul of supernatant transformation would be an enormous asset in the study of was removed, and the particles were resuspended in the function and expression of yeast mitochondrial genes. Standard procedures for genetic transformation of yeast remaining supernatant prior to bombardment. Cells were nuclei (2, 3) have so far failed to introduce DNA into spread on Petri plates containing minimal medium plus mitochondria. However, high-velocity microprojectile bom- sorbitol (1.0 and 0.5 M) at a density of 108 cells per plate and bardment (4, 5) was recently found to deliver DNA into both bombarded with the particles (J.C.S., T. M. Klein, K. B. chloroplasts of Chlamydomonas (6) and mitochondria of Shark, and S. A. Johnston, unpublished procedure). Approx- yeast (7), where it became incorporated into preexisting imately 1 u.g of DNA on 0.5 mg of particles was used per organellar DNA (rho+ in the case ofyeast) by recombination. plate. Since TD28rho° proved to be somewhat osmotically We report here that yeast mitochondria lacking mtDNA sensitive, after 4 days the bombarded plates were replicated can be transformed by bombardment of a rho' strain with to SD medium plates without sorbitol to allow more rapid microprojectiles carrying a mitochondrial gene, oxil (8-10), growth of transformed clones. on a plasmid. Genetic and molecular analysis of the trans- Nucleic Acid Analysis. Total yeast DNA was prepared as formants demonstrates that they exhibit all the expected described (11). Agarose gel electrophoresis, blotting, hybrid- ization, and radioactive labeling of probe DNAs by nick- translation were as described (21). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 7288 Downloaded by guest on September 25, 2021 Genetics: Fox et al. Proc. Natl. Acad. Sci. USA 85 (1988) 7289 Table 1. Key strains subclones that retained oxil but differed slightly in their Mitochondrial genetic behavior (see below) were picked from one initial Strain Nuclear genotype genotype transformant for further study and termed TF182 and TF183. DA1 MA Ta, ade2 rho+ (wild-type) The Transformants Contain Reiterated Copies of Plasmid TD28rho' MATa, ura3-52, inol-13 rho' DNA. The genetic behavior of the transformants described TF182 MATa, ura3-52, inol-13 rho-, oxil + (pMT36*) above strongly indicated that DNA had been transferred to TF183 MATa, ura3-52, inol-13 rho-, oxil+ (pMT36) the mitochondrial compartment. The oxil coding sequence 8-12 MATa, his4-580 rho-, oxil + can be translated only in mitochondria due to the presence of AB-4D/V25 MATa, opi, adel, met3 rho', oxil-V25 five UGA codons in the structural gene (9), and we have (UAA) experimentally verified that oxil sequences located in the TF138 MA Ta, ade2, ura3 rho', oxil-M8-171 nucleus are genetically inert (P. P. Muller and T.D.F., (deletion) unpublished results). The fact that these sequences were TF140 MATa, ade2, ura3 rho', oxil-Mii-30 replicated is not surprising, since any segment ofmtDNA can (insertion) be replicated in rho - strains (1), including subsets of the TF145 MATa, ade2, ura3 rho, oxil-17 mtDNA sequence carried in pMT36 (8). (deletion) To examine the mtDNA ofthe transformants, total cellular Items in parentheses describe relevant features of the mitochon- DNA was isolated and subjected to DNA-gel-blot hybridiza- drial genotype. pMT36* refers to the altered form ofpMT36 revealed tion analysis, using as a probe mtDNA from the oxil-specific by the experiment of Fig. 2. rho - strain 8-6 (8). DNA from both TF182 and TF183 carried oxil on the same 2.4-kb Msp I (Hpa II) fragment as that RESULTS previously described (8) for the wild-type, DA1, and a Conversion of rho° to rho- by Transformation. The strain previously characterized (8) rho - strain, 8-12 (Fig. 2). DNA TD28rho° contains no mtDNA. Thus when TD28rho° is from TD28rho° lacked this fragment. crossed to other strains containing mutations in the mito- If the oxil DNA in the transformants TF182 and TF183 chondrial gene oxil the resulting diploids carry the mutant were derived from pMT36 it should be distinguishable from mtDNA and fail to grow on nonfermentable carbon sources. wild-type and standard rho - strain DNAs by the Pst I sites [oxil encodes cytochrome c oxidase subunit II (22).] In surrounding oxil on the plasmid. Pst I sites do not surround contrast, standard rho- strains of yeast that carry the oxil in mtDNA of any known yeast strain (there is only a wild-type oxil gene yield respiring diploids after mating with single Pst I site in wild-type mtDNA; see ref. 23). Indeed, the oxil mutants (1, 8). pMT36-derived oxil-carrying 2.5-kb Pst I fragment could be We sought to introduce into the mitochondria ofTD28rho° cleaved from the DNA of TF182 and TF183 (Fig. 2). As a plasmid, pMT36 (Fig. 1; ref. 18), that carries the wild-type oxil gene. The scheme for isolating such transformants was MspI Pst I Uncut to first bombard the rho° host strain with microprojectiles ABC D E F A BC E F A BC D E F carrying a mixture of pMT36 and a replicating yeast vector that carries the selectable nuclear marker URA3. Uracil- synthesizing (Ura+) transformants were selected and then -"C tested for their ability to yield respiring diploids when mated, 2.4 -- * _w _ W - by replica plating, to lawns of a tester strain carrying an oxil mutation. In two separate experiments 1850 Ura+ transform- ants were obtained on 50 Petri plates after bombardment of TD28rho° with the mixture of plasmids. Two of the Ura+ transformants (one from each experiment) yielded respiring diploids when crossed to the strain AB-4D/V25, which carries a UAA mutation in oxil (16). Upon subcloning, the Pst I Uncut Ura+ phenotype, determined by the plasmid replicating in A E F B CA E F the nucleus, segregated mitotically as expected. The ability to yield respiring diploids when crossed to the oxil UAA mutant s0 was quite stable (retained by greater than 90% of the sub- g 4.3No -.f -"C clones). There was no linkage between the Ura+ phenotype and retention of oxil during mitotic segregation. Two Ura-

M oxi1 M mtDNA MML-- T

FIG. 2. Molecular analysis of plasmid sequences in DNA of P M M P mitochondrial transformant and control strains. The plasmid pMT36 (lanes A) and total yeast DNA from the wild-type DA1 (lanes B), the pMT36 oxii-carrying rho- 8-12 (lanes C), the recipient TD28rho° (lanes D), the transformant TF182 (lanes E), and the transformant TF183 (lanes F) were subjected to agarose gel electrophoresis after digestion with 1 kb either Msp I (an isoschizomer of Hpa II) or Pst I as indicated. Undigested DNAs were electrophoresed in lanes marked "Uncut." FIG. 1. Restriction map of mtDNA and the plasmid pMT36 in the The gels were blotted to nitrocellulose filters, which were hybridized region surrounding oxii. The location of oxil relative to cleavage either to radioactively labeled mtDNA from the oxil-specific rho- sites for Msp I (M) and Pst I (P) is indicated by the solid bars. The 8-6 (8) (Upper) or to radioactively labeled pBR322 (Lower). The wavy lines indicate sequences derived from pBR322 and the open positions ofbands corresponding to the Msp I fragment carrying oxil boxes represent poly(dG)poly(dC) tracts formed during the con- (2.4), open circular (0) and closed circular (C) pMT36, and linear struction of pMT36 (18). kb, Kilobase pairs. pBR322 (4.3) are marked. Downloaded by guest on September 25, 2021 7290 Genetics: Fox et al. Proc. Natl. Acad. Sci. USA 85 (1988) expected, oxil sequences in Pst I cleaved DNA from DA1 having TF182 as a parent. The explanation for this property and 8-12 ran with the mobility of uncut DNA (Fig. 2). remains to be determined. Uncut DNA from TF182 and TF183 contained oxil se- Maintenance of Plasmid-Derived mtDNA Heteroplasmically quences that ran with the mobility ofuncut wild-type mtDNA with rho' mtDNA. A second major difference in growth (Fig. 2), demonstrating that, after entry into the mitochon- phenotype of the diploids generated in the experiment of dria, plasmid sequences became reiterated in a manner Table 2, which depended on the nature of the rho', oxil similar to that of retained mtDNA sequences in standard mutant parent, was in the mitotic stability of respiratory rho- strains. In addition, bands corresponding to monomer competence. Diploids formed by mating TF182 with strains circles of pMT36 were visible in the uncut DNA of TF183 carrying either the UAA mutation (AB-4D/V25) or the (Fig. 2), and indeed we have recovered apparently intact insertion mutation (TF140) were mitotically stable. How- pMT36 from this DNA by transformation ofEscherichia coli. ever, diploids having either oxil deletion mutant (TF138 or Whether or not these monomers are located in mitochondria TF145) as a parent exhibited marked mitotic instability: is at present an open question [the oxil DNA fragment colonies growing on YPEG medium contained approximately contains a weak origin for nuclear DNA replication (ARS) 50% nonrespiring cells, asjudged by restreaking. Sporulation (P. P. Muller and T.D.F., unpublished results)]. of these unstable diploids yielded no respiring haploid prog- In addition to oxil, both TF182 and TF183 contained eny from the cross ofTF145 x TF182 (0/35), while only 22% pBR322 sequences derived from pMT36 in reiterated form ofthe progeny from TF138 x TF182 (17/76) yielded respiring (Fig. 2). While the pBR322-hybridizing Pst I fragment of sectors. These respiring haploid progeny also exhibited TF183 migrated with that of pMT36, the corresponding mitotic instability. The nonrespiring progeny all contained fragment from TF182 was approximately 1.5 kb larger. This the rho +, oxil deletion mtDNAs, asjudged by their ability to difference in size could be explained by homologous recom- yield respiring diploids when crossed to rho - strains carrying bination between the pBR322 sequences of pMT36 and the the wild-type oxil gene. cotransforming These results can be explained as follows. Since the UAA URA3 plasmid (Materials and Methods). and insertion mutations are flanked on both sides by DNA Restriction analysis of TF182 DNA (not shown) indicated homologous to the wild-type mtDNA fragment on pMT36, that its mtDNA had acquired the 2-,um plasmid origin of these oxil lesions can be corrected by double crossovers replication by a double cross-over with the URA3 plasmid. after mating and the resulting functional mtDNA can be The difference between TF182 and TF183 is noteworthy, stably propagated. In contrast, the deletion mutation in strain since they were mitotic segregants from a single initial TF138 completely removed regions homologous with pMT36 transformant. However, it is well documented that different (8), while the deletion in TF145 left only a few hundred "secondary rho- clones" are frequently isolated from the homologous base pairs in the 5' flanking region of oxil same "primary rho- clone" (1). (T.D.F., unpublished results). Thus, homologous double Molecular analysis of DNA from the second initial trans- recombination between the plasmid and the mtDNA of these formant revealed that, like TF183, it contained concatemers deletion mutants is impossible. Apparently the oxil gene of pMT36 and low levels of monomer circles (not shown). derived from the plasmid functions in trans and is maintained, Reiterated Plasmid DNAs in the Transformants Function unstably, in a heteroplasmic state with the mutant rho+ Like Standard rho- mtDNA in Crosses. We examined the mtDNA during mitosis. We and others have previously genetic behavior ofTF182 and TF183 in crosses to wild-type observed such mitotic instability in strains heteroplasmic for and various oxil mutant strains. To test whether the trans- wild-type mtDNA and rho - mtDNAs that suppress nuclear formants were suppressive [i.e., inhibited transmission of mutations (17, 24-26), as well as a strain carrying a natural wild-type mtDNA from a rho' parent to diploid zygotes (see rho - and an oxil deletion mutation that shared no homology ref. 1)], TF182, TF183, TD28rho°, and 8-12 were mated to the (P. P. Muller and T.D.F., unpublished results). rho + wild-type strain DAL. Zygotic clones and their progeny The hypothesis of the previous paragraph can be tested by were selected on minimal medium and examined for growth examining oxil sequences in the diploid strains. The mitoti- on YPEG. In all cases, greater than 99% ofthe zygotic clones cally stable respiring diploids, formed by mating the oxil as well as their progeny could respire, demonstrating that the UAA mutant with TF182, should not contain the plasmid- plasmid DNA in mitochondria was not suppressive. derived Pst I sites surrounding oxil in the transformant, since The ability of TF182 and TF183 to generate respiring these Pst I sites lie outside the region ofhomology. However, diploids when crossed to strains carrying four different oxil the mitotically unstable respiring diploids from the cross of mutations (Table 1 and Materials andMethods) was tested by the deletion mutant TF145 with TF182 should still contain patch mating (Table 2). The diploids from all ofthese crosses oxil on a Pst I fragment derived from the plasmid. These were able to respire to some extent, although marked dif- predictions were confirmed by the hybridization experiment ferences in phenotype were observed. Diploids from crosses of Fig. 3. Interestingly, the oxil-carrying Pst I fragment from between TF183 and either the insertion mutant (TF140) or the the TF145 x TF182 diploid (Fig. 3, right lane E) was slightly oxil deletion mutants (TF145 and TF138) grew markedly larger than the corresponding fragment from TF182, and the slower on YPEG medium than did the corresponding diploids same fragment hybridized weakly with a pBR322 probe (not shown). Thus it appears that during propagation of this Table 2. Respiratory ability of diploids formed by mating diploid the plasmid-derived sequences suffered a deletion transformants with oxil mutant strains that removed one of the Pst I sites flanking oxil and most of Respiratory ability of diploid the pBR322 DNA. Diploids from the cross of the other AB-4D/V25 TF138 TF145 TF140 deletion mutant, TF138, with TF182 contained the predicted oxil-V25 oxil-M8-171 oxii-17 oxil-M11-30 oxil Pst I fragment, but they also had concatemeric oxil Transformant (UAA) (deletion) (deletion) (insertion) sequences lacking Pst I sites (not shown). TF182 + + + + + TF183 + + +/- +/- +/- DISCUSSION 8-12 + + + + + + + The experiments described in this paper demonstrate that TD28rhol - - - - plasmid DNA can be introduced into mitochondria of a yeast Respiratory ability was scored by the ability to grow on nonfer- strain that lacks mtDNA. The plasmid DNA behaved in a mentable YPEG medium. + +, Strong growth; -, no growth. manner entirely consistent with the known properties of Downloaded by guest on September 25, 2021 Genetics: Fox et al. Proc. Natl. Acad. Sci. USA 85 (1988) 7291 can then be based on a high-frequency event by mating the Msp I Pst I synthetic rho- strains (transformants) to appropriate rho+ testers. A B C DE A B C D E An example of the potential utility of this approach is provided by our work on nuclear gene products that act in the 5' untranslated leaders of specific mitochondrial mRNAs to U- promote their translation. Mitochondrial gene rearrange- ments that suppress these nuclear mutations are carried on 2. 4 _ of>* rho - mtDNAs that exist heteroplasmically with rho + mtDNA (17, 18, 26, 27). To date we have only been able to study rearrangements that could be selected genetically on the basis oftheir ability to function. While it has been possible FIG. 3. Molecular analysis ofoxil sequences from diploid strains to some extent to manipulate 5' untranslated leaders in this formed by crossing the transformant TF182 with oxil mutants. Total fashion (28, 29), we have heretofore not been able to study yeast DNA from the transformant TF182 (lane A), the oxil UAA rearrangements that destroyed a site of action. We can now mutant AB-4D/V25 (lane B), a diploid formed by mating TF182 with construct mutant genes encoding mitochondrial mRNAs with AB-4D/V25 (lane C), the oxil deletion mutant TF145 (lane D), and altered regulatory sites in their 5' leaders. These genes can a diploid formed by mating TF182 with TF145 (lane E) was digested then be used to construct synthetic rho- strains, identifiable with either Msp I or Pst I, as indicated, and analyzed by electro- by their ability to "marker rescue" structural gene muta- phoresis and hybridization to the oxil-specific probe as described in the legend to Fig. 2. The positions of the oxil Msp I fragment (2.4) tions. The synthetic rho - strains can then be mated to and uncut mtDNA (U) are marked. appropriate partners to test whether the altered regulatory sites still function. normal yeast rho- mtDNAs (1) after its introduction by A second advantage of the ability to generate synthetic high-velocity microprojectile bombardment (5). Like the rho - strains carrying defective mitochondrial genes is that it mtDNA sequences retained in natural rho- strains, the will make possible the transfer of in vitro-generated muta- plasmid DNA in our transformants, including pBR322 se- tions to wild-type rho+ mtDNA. It is well established that quences, was reiterated into concatemers whose size was mutations carried on rho - mtDNA are transferred in crosses indistinguishable from that of wild-type mtDNA. This "syn- to rho+ mtDNA by recombination (1). Moreover, our ex- thetic rho- mtDNA" was replicated and efficiently trans- periments demonstrate the feasibility of introducing foreign mitted to mitotic progeny despite the absence of any preex- DNA, in this case pBR322 sequences, into yeast mitochon- isting mtDNA in the host strain. dria. It may be possible to use plasmids such as pMT36 as The oxil sequences of the transformants were surrounded vectors to bring appropriately modified foreign genes into by restriction sites derived from the plasmid that were not these organelles. present in wild-type mtDNA. oxil genetic information in In summary, the results reported here demonstrate that a these transformants could be expressed in diploids either plasmid DNA molecule can initiate DNA replication de novo after "marker rescue" recombination with rho' mtDNA after introduction into mitochondria lacking preexisting by mtDNA. In the process, the plasmid becomes reiterated in a an oxil or in trans carrying appropriate point mutation, fashion resembling natural rho - mtDNA, although the mech- during the growth of diploids heteroplasmic for both the anism by which this reiteration occurs is unknown for both plasmid-derived oxil sequences and rho+ mtDNA with oxil deleted. The genetic activity of oxil in these transformants natural and synthetic rho- strains. provided the strongest evidence for mitochondrial localiza- We thank K. B. Shark and M. K. Reiffor technical assistance and tion ofthe DNA, since the oxil coding sequence contains five M. C. Costanzo for helpful discussions and a critical reading of the UGA codons and can be properly translated only in mito- manuscript. T.W.M. is a Postdoctoral Associate of the Cornell chondria (9). Furthermore, we previously found that oxil Biotechnology Program. Biolistics, Inc. (Geneva, NY) provided the sequences located on plasmids in the nucleus were geneti- apparatus used for microprojectile bombardment. This work was cally inert in crosses (P. P. Muller and T.D.F., unpublished supported by Research Grant GM29362 and Research Career De- results). velopment Award HDO0515 to T.D.F. from the National Institutes of Microprojectile bombardment has previously been used to Health. introduce functional genes into both chloroplasts of Chlam- 1. Dujon, B. (1981) in The Molecular Biology of the Yeast ydomonas (6) and mitochondria ofyeast (7). In those studies, Saccharomyces: Life Cycle and Inheritance, eds. Strathern, plasmid-derived wild-type genes were incorporated into mu- J. N., Jones, E. W. & Broach, J. R. (Cold Spring Harbor Lab., tant organellar chromosomes by double recombination. In Cold Spring Harbor, NY), pp. 505-635. cotransformation experiments with yeast similar to those 2. Hinnen, A., Hicks, J. B. & Fink, G. R. (1978) Proc. Natl. described here, approximately one mitochondrial transform- Acad. Sci. USA 75, 1929-1933. ant was found for every 1000 uracil-synthesizing colonies (7). 3. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983) J. Bacteriol. 153, 163-168. In both cases the rare organellar transformants were detected 4. Sanford, J. C., Klein, T. M., Wolf, E. D. & Allen, N. (1987) by virtue of the phenotypic change in the host due to the Part. Sci. Technol. 5, 27-37. introduction of functional genes. 5. Klein, T. M., Wolf, E. D., Wu, R. & Sanford, J. C. (1987) A key advantage ofthe approach described in this paper to Nature (London) 327, 70-73. mitochondrial transformation is that the initial identification 6. Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., of low-frequency transformants does not depend upon func- Johnson, A. M., Jones, A. R., Randolph-Anderson, B. L., tion of the mtDNA being introduced. With respect to mito- Robertson, D., Klein, T. M., Shark, K. B. & Sanford, J. C. chondrial functions, the transformants were phenotypically (1988) Science 240, 1534-1537. identical to the host strain. Successful 7. Johnston, S. A., Anziano, P., Shark, K., Sanford, J. C. & rho° transformation Butow,.R. A. (1988) Science 240, 1538-1541. was detected by a "marker rescue" assay dependent only 8. Fox, T. D. (1979) J. Mol. Biol. 130, 63-82. upon double recombination between the introduced DNA 9. Fox, T. D. (1979) Proc. Natl. Acad. Sci. USA 76, 6534-6538. and known point mutations in a rho' partner. Thus, this 10. Coruzzi, G. & Tzagoloff, A. (1979) J. Biol. Chem. 254, 9324- procedure will readily allow introduction and detection of 9330. defective mitochondrial genes. Studies of mutant phenotypes 11. Sherman, F., Fink, G. R. & Hicks, J. B. (1986) Methods in Downloaded by guest on September 25, 2021 7292 Genetics: Fox et al. Proc. Natl. Acad. Sci. USA 85 (1988)

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