Autonomously Replicating RNA in Mitochondria of Maize Plants with S

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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 5175-5179, July 1986 Genetics Autonomously replicating RNA in mitochondria of maize plants with S-type cytoplasm (cytoplasmnic male sterility/genetic RNA/RNA plasmids/double-stranded RNA/in organello RNA synthesis) PATRICK M. FINNEGAN AND GREGORY G. BROWN Centre for Plant Molecular Biology, Department of Biology, McGill University, Montreal, PQ, Canada H3A iB1 Communicated by Hewson Swift, March 17, 1986

ABSTRACT Mitochondria isolated from maize plants with in organello RNA synthesis system to search for differences S-type male-sterile cytoplasms are capable of synthesizing four in mitochondrial gene expression among the various maize species of RNA at concentrations of actinomycin D that cytoplasmic types. We have made the surprising discovery eliminate all DNA-directed RNA synthesis. No RNA synthesis that several R$As unique to the S cytoplasm are synthesized occurs under the same conditions with mitochondria from in a DNA-independent manner. Our observations indicate plants possessing normal (N) cytoplasm or with other that these RNAs represent an RNA-based genetic system subcellular fractions from plants with S cytoplasm. The that is endogenous to the mitochondnron. No other such actinomycin D-resistant RNA synthesis occurs within the system has been found in an organelle. Because these RNAs mitochondria since the labeling ofthese species is unaffected by replicate independently of cellular chromosomes, we have inclusion of RNase in the incubation medium and since they termed them RNA plasmids. become completely sensitive to RNase upon lysis of the mitochondria with low concentrations of Triton'X-100. Two of the actinomycin D-resistant products are double stranded. MATERIALS AND METHODS These are 2850 and 900 base pairs in length, whereas the remaining two are 2150 and 850 bases. The synthesis ofall four Materials. Inbred WF9 seeds were from Marc Albertsen RNAs occurs in at least five different accessions ofS cytoplasm, (Pioneer Hi-Bred International, Johnston, IA); inbred B73 suggesting it is a general feature of S qitochondria. The seeds were from Mike Brayton Seeds (Ames, IA); Vg double-stranded RNAs show homology to single-stranded S cytoplasm seed (open-pollinated) was from C. S. Levings III mitochondrial RNA but not to N niutochondrial RNA. Our (North Carolina State University, Raleigh, NC). Miracloth is observations indicate that the replication of these RNAs occurs a product of Calbiochem. [a-32P]UTP (410 or 3000 Ci/mmol, independently of mtDNA and that they thus represent a novel 1 Ci = 37 GBq) was obtained from Amersham. Percoll was type of inheritable element in organelles, an RNA plasmid. from Pharmacia, CF-li cellulose was from Whatman, and GeneScreen transfer membrane is a product of New England Many viruses store genetic information as RNA, but such Nuclear. genetic RNA is rarely encountered in uninfected cells. In the Mitochondrial Isolation and Cell Fractionation. Mitochon- only well-documented examples in which genetic RNA is dria were prepared from 5- or 6-day-old etiolated maize found as a normal cellular constituent, it is associated with shoots and roots essentially as described (10). Extramito- virus-like capsid structures in the cytosol, as in the cases of chondrial RNA was removed by treating the mitochondria, the killer plasmid ofyeast (1), the virus-like particles ofother suspended in homogenization buffer, with 10 pug of RNase A fungi (2), and the cryptic viruses ofplants (3). In each case the per g of starting material for 60 min. The mitochondria were capsid-associated RNA is usually double stranded, and collected from the reaction mixture by centrifugation through replicative and messenger RNA synthesis takes place using a cushion of 2 vol of 0.6 M sucrose/10 mM N-[tris(hydroxy- an RNA template. methyl)methyl]aminoethanesulfonic acid (Tes)/20 mM The mitochondrial genomes of higher plants possess a EDTA (sucrose buffer) at 10,000 x g for 20 min before further number of features that are not found in nuclear, viral, or purification by centrifugation through 20-60% sucrose gra- other organelle genomes. They exist as a set of large dients (11). The purified mitochondria were slowly diluted recombining circular molecules in which some circles are over 15 min with sucrose buffer before collection by centrifugation at 12,000 x g for 10 min. The pellet fraction of the more abundant than others, resulting in unequal representa- sucrose-gradient centrifugation consisted mainly of nuclear tion of the various sequences that they comprise (4, 5). contamination and is referred to as the nuclear pellet. The Minicircular plasmid DNAs are also frequently present in pellet resulting from the first centrifugation of the tissue plant mitochondria (6) and linear DNA episomes that recom- homogenate is referred to as the 1000 x g pellet. Microsome bine with specific sequences on the large circular molecules and cytosol fractions are the pellet and supernatant material, have been found as well (7, 8). In these and in other organelles respectively, obtained from centrifugation of the postmito- the only identified molecules with the potential of storing chondrial supernatant at 140,000 x g for 30 min. genetic information are DNAs. Radiolabeling the Products of Mitochondrial RNA (mtRNA) Another interesting feature of plant mitochondrial DNA Synthesis. Mitochondria purified from 30 g of tissue, without (mtDNA) is that variation in it can result in the phenotype of nuclease treatment, were preincubated for 10 min in 250 ,ul of male sterility. In maize there are four distinct cytoplasmic 60 mM mannitol/3 mg of bovine serum albumin per ml/20 and hence mitochondrial types, S, C, T, and N; S, C, and T mM Tris phosphate (pH 7.3)/10 mM potassium phosphate/ can confer male sterility when present in specific nuclear 150 mM KCl/10 mM MgC12/1 mM EGTA/5 mM sodium genetic backgrounds, whereas N cannot (9). We have used an succinate/5 mM phosphoenolpyruvate/30 Ag of pyruvate

The publication costs of this article were defrayed in part by page charge Abbreviations: mtRNA, mitochondrial RNA; bp, base pair(s); Tes, payment. This article must therefore be hereby marked "advertisement" N-[tris(hydroxymethyl)methyl]aminoethanesulfonic acid; ds RNA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. double-stranded RNA. 5175 Downloaded by guest on September 27, 2021 5176 Genetics: Finnegan and Brown Proc. Natl. Acad Sci. USA 83 (1986) kinase per ml/2.5 mM ATP/0.3 mM CTP/0.3 mM GTP/0.1 buffer before eluting the double-stranded RNA (ds RNA) mM UTP. Other additions were made as indicated in the with ethanol-free wash buffer. The aurintricarboxylic acid figure legends. The products of RNA synthesis were labeled was removed by passage of RNA samples through a G-25 by adding [a-32P]UTP to a final specific activity of2 Ci/mmol Sepharose column (12). and incubating at 230C with agitation for 60-90 min. RNA for S1 Nuclease Digestion. RNA dissolved in 50 ,ul of 280 mM use as a hybridization probe was labeled in a reaction mixture NaCl/50 mM NaOAc, pH 4.6/4.5 mM ZnSO4/20 gg of containing 2 ,M UTP at 3000 Ci/mmol. The RNA synthesis heat-denatured salmon sperm DNA per ml was digested with reaction was terminated by collecting the mitochondria in a 2000 units of S1 nuclease per ml at 42°C. The digestion microcentrifuge for 5 min and lysing immediately as de- products were extracted with phenol and precipitated with scribed below. ethanol prior to electrophoretic analysis as above. Purification of mtRNA. Mitochondria were resuspended RNA Transfer and Hybridization. After extensive washing and lysed in 50 mM Tes, pH 7.2/10 mM EDTA/0.2% diethyl of the agarose/urea gels with water, RNA was transferred to pyrocarbonate/2% NaDodSO4. One-half volume of redis- a GeneScreen membrane by overnight capillary blotting with tilled phenol equilibrated with 0.1 M Tes (pH 7.2) was quickly 0.3 M NaCl/0.03 M sodium citrate, as recommended by the added and the lysate was extracted, with shaking, 20 min at manufacturer. The membranes were prehybridized for 20 hr 230C. One-half volume of chloroform/isoamyl alcohol (24:1) at 42°C in 50% formamide/0.2% polyvinylpyrrolidone/0.2% was added prior to separation of the phases. The aqueous bovine serum albumin/0.2% Ficoll/50 mM Tris-HCl, pH phase was reextracted twice in this manner before precipi- 7.5/1.0 M NaCl/0.1% sodium pyrophosphate/1% NaDod- tation of the RNA with ethanol. S04/10% dextran sulfate/100 ,ug of heat-denatured salmon Electrophoretic Analysis of RNA. RNA was dissolved in sperm DNA per ml. Hybridizations, with heat-denatured electrophoresis buffer (45 mM Tris/45 mM boric acid/2 mM radiolabeled mtRNA added as probe, were done under the EDTA)/80% deionized formamide, heated 2 min at 70°C, same conditions for 24 hr. The membranes were washed quickly chilled, and loaded onto a vertical 1% agarose slab gel twice for 10 min with wash buffer (0.3 M NaCl/0.03 M sodium containing 2x concentrated electrophoresis buffer/5 M urea. citrate) at 23°C, twice for 30 min with wash buffer/1% Electrophoresis was carried out at 7 V/cm for -4 hr. RNAs NaDodSO4 at 65°C, and twice for 30 min with 0.1 x wash were visualized by ethidium bromide staining after removal buffer at 23°C before autoradiography. of urea by extensive washing with water. Gels containing radiolabeled samples were treated with 10% acetic acid and dried by suction prior to autoradiography. Escherichia coli RESULTS 23S [2.9 kilobases (kb)] and 16S (1.5 kb) RNAs and rabbit RNA Synthesis by Isolated Maize Mitochondria. As shown globin mRNA (0.65 kb) were used as markers to estimate the in Fig. 1, when mitochondria from maize plants possessing N sizes of RNA species from their electrophoretic mobilities. cytoplasm were incubated in a mixture that supports the CF-li Cellulose Chromatography. Purified mtRNA dis- incorporation of labeled nucleoside triphosphates into RNA, solved in 200 mM NaCl/100 mM Tris HCl, pH 7.0/2 mM they synthesized products that have the electrophoretic EDTA/15% ethanol/1 mM aurintricarboxylic acid was load- mobilities ofthe major mtRNAs detected in gels stained with ed onto a column of CF-11 cellulose equilibrated with the ethidium bromide. Several lines of evidence indicate that same buffer. The column was washed extensively with this RNA synthesis by isolated maize mitochondria is an accurate A B C 123 4 56 7 8 1 2 34 56 71B 1 2 3 4 5

23S ---2850 b to1#* -- -2150 b. _ 16S

- ....-..9OO b 9. 9--850 b-'*'* 9 2 C/) FIG. 1. Effects ofactinomycin D and ethidium bromide on RNA synthesis by isolated maize mitochondria. (A and B) RNA synthesis products from inbred WF9 N (lanes 2-4) or S (lanes 5-7) shoot mitochondria or from inbred WF9 S root mitochondria (lane 8). The standard reaction mixture (lanes 2 and 5) was supplemented to 50 Mg of actinomycin D per ml (lanes 3, 6, and 8) or 50 ,ug of ethidium bromide per ml (lanes 4 and 7). RNA was separated on a 1% agarose/5 M urea gel .and visualized by ethidium bromide staining (A) and autoradiography (B). 32P-end-labeled partial Hinfl digestion products of pBR322 were run in lanes 1 to facilitate comparison of the ethidium-stained gel and the autoradiograph. (C) Products ofactinomycin D-resistant RNA synthesis by shoot mitochondria isolated from B73 nucleargenotype, M cytoplasm (lane 1), B73 nuclear genotype, M4 cytoplasm (lane 2), WF9 nuclear genotype, S cytoplasm (lane 3), B73 nuclear genotype, SD cytoplasm (lane 4), and unspecified nuclear genotype, Vg cytoplasm (lane 5). b, Bases. Downloaded by guest on September 27, 2021 Genetics: Finnegan and Brown Proc. Natl. Acad. Sci. USA 83 (1986) 5177 reflection of in vivo mitochondrial events (to be published ence of actinomycin D was applied to a column of CF-11 elsewhere). When mitochondrial fractions were subjected to cellulose, under conditions that allow the selective binding of isopycnic centrifugation in linear sucrose gradients, RNA ds RNA (22), the 2850- and 900-base species were retained, synthetic activity coincided with the mitochondrial marker whereas the bulk of the 2150- and 850-base species was not enzyme succinate:cytochrome c reductase. Bacterial and (Fig. 2). All of the unbound material was sensitive to the plastid RNA synthesis did not appear to contribute signifi- action of S1 nuclease, confirming its single-stranded charac- cantly; a prominent radioactive 16S RNA band was not ter (Fig. 2, lane 3), whereas the bound material was resistant observed among the products, synthesis was inhibited to to digestion by this enzyme (Fig. 2, lane 5). Thus, the 2850- >85% by rifampicin at concentrations (175 ,ug/ml) that have and 900-base RNAs are double stranded, whereas the 2150- been shown to have no effect on RNA synthesis in isolated and 850-base RNAs appear to be single stranded. The 2850- maize chloroplasts and etioplasts (13), and synthesis was and 900-base-pair (bp) species are highly abundant in inhibited only slightly by rifampicin at concentrations (20 unlabeled S mtRNA preparations that have been enriched for jug/ml) that completely block bacterial RNA synthesis (14). double-stranded molecules, indicating that their formation is Actinomycin D and ethidium bromide completely prevented not an artifact of the in organello labeling system. RNA synthesis by isolated N mitochondria (Fig. 1B, lanes 3 Actinomycin D-Resistant RNA Synthesis Occurs in and 4). Although the labeled products showed a preponder- Mitochondria. Most of the RNA synthesized from RNA virus ance of high molecular weight RNA relative to total mtRNA, and encapsidated RNA plasmid templates is mRNA that must this probably reflects an increased abundance of labeled be released into the cytoplasm for translation and is thus precursors, as it does in other isolated mitochondrial systems susceptible to exogenous RNase. When mitochondria isolat- (15-18). ed from the shoots ofplants with S cytoplasm were incubated Actinomycin D-Resistant RNA Synthesis by S Mitochondria. in an RNA synthesis mixture that included RNase at con- Two major products of RNA synthesis by S mitochondria centrations preliminary experiments established to be suffi- (Fig. 1B, lane 5) were not found in the N mitochondrial ciently high to digest ds RNA, however, the profile of products. These species have sizes of approximately 900 and S-specific mitochondrial RNA products was unaffected (Fig. 850 bases, the smaller corresponding to a major ethidium- 3A, lane 1). This result indicates that the RNAs are synthe- staining RNA specific to S cytoplasm that Schuster et al. (19) sized and remain within an RNase-impermeable compart- termed S/Rw-RNA-b (Fig. lA, lanes 5-8). Since the nuclear ment. Since virus capsids, in general, remain genotypes were the same for the strains used in this exper- structurally iment (inbred WF9), nuclear gene effects cannot account for intact in the presence of low concentrations of nonionic these differences. Surprisingly, these and two other S- detergents (23), we would not expect mild detergent treat- specific species, of 2850 and 2150 bases, continued to be ment to cause RNAs enclosed in such structures to become synthesized at concentrations of actinomycin D that com- susceptible to RNase. Addition of 1% or 0.1% Triton X-100 pletely block all other RNA synthesis in N and S cytoplasms to S mitochondria that had synthesized RNA in the presence (Fig. 1B, lane 6). All four RNAs were synthesized by 1 2 3 4 5 mitochondria isolated from the roots as well as the shoots of S-cytoplasm seedlings and the synthesis of all four was prevented by ethidium bromide (Fig. 1B, lanes 7 and 8). Results of several experiments showed that the actinomycin D-resistant labeling is due to incorporation of 5' UMP into the internal portion of RNA chains (not shown): limited 5' or 3' exonuclease digestion of the products yielded labeled mate- 2850 b- rial of significantly smaller size, the products were completely sensitive to digestion with DNase-free RNase, and the only radioactive nucleotide obtained from them in a complete nuclease P1 digest was 5' UMP. When the products of actinomycin D-resistant RNA synthesis by mitochondria from different accessions of S cyto- 2150 b- plasm, in different nuclear genotypes, were analyzed, all were found to synthesize the RNAs (Fig. 1C). This suggests that their synthesis, like the synthesis of S-specific polypep- tides (11) and the presence of the linear DNA episomes S-1 and S-2 (7, 8, 10), is a general molecular characteristic of S-type maize cytoplasms. Their synthesis also occurs in plants that are male fertile, either as a result of nuclear fertility restoration (9) (Fig. 1C, lanes 1 and 2) or cytoplasmic reversion (9) (not shown), indicating that it is unlikely to be _ 0 -900 b related to the male-sterile phenotype. 850 b- A Double-Stranded S-Specific Products. The synthesis of the S-specific RNAs takes place at actinomycin D concentrations that block all DNA-directed RNA synthesis (20). RNA synthesis arising from the replication or transcription of RNA-based genetic systems, however, is usually unaffected by this compound (20, 21). It seemed possible, therefore, that FIG. 2. Products of actinomycin D-resistant, mtRNA synthesis. the S-specific RNA synthesis was due to an RNA virus or mtRNA from inbred B73 (M4 cytoplasm) was labeled in the standard plasmid within or tightly associated with the mitochondrion. reaction mixture supplemented with 50 jug of actinomycin D per ml Since either the genomic or the (lane 1) and separated by CF-11 cellulose chromatography into an replicative forms of such unbound, presumably single-stranded, fraction (lane 2) and a bound, RNA-based systems, with the exception of retroviruses, are presumably double-stranded, fraction (lane 4). The unbound and ds RNA, it was of interest to determine if any products of the bound fractions were then treated with S1 nuclease (lanes 3 and 5, actinomycin D-resistant RNA synthesis were double strand- respectively). The RNA in lanes 4 and 5 was derived from -50 times ed. When RNA synthesized by S mitochondria in the pres- the amount of tissue as the RNA in lanes 1-3. b, Bases. Downloaded by guest on September 27, 2021 5178 Genetics: Finnegan and Brown Proc. Natl. Acad. Sci. USA 83 (1986)

A B A B C 1 2 3 1 2 3 4 5 6 7 8 9 10 11 1 2 3 1 2 3 1 2 3

FIG. 3. Intracellular location of actinomycin D-resistant RNA synthesis. (A) Mitochondria from inbred B73 (SD cytoplasm) were labeled in the standard reaction mixture supplemented with 0.5 mg of RNase A per ml. After 60 min, either mitochondria were left 850 b untreated (lane 1) or Triton X-100 was added to a final concentration of 1% (lane 2) or 0.1% (lane 3), and the incubation was continued for an additional 15 min. (B) RNA synthesis products from inbred B73 (SD cytoplasm) were labeled in the standard reaction mixture in the presence (lanes 2, 4, 6, 8, and 10) or absence (lanes 3, 5, 7, 9, and 11) FIG. 4. Hybridization characteristics of the mtRNA synthesized of 50 ,ug of actinomycin D per ml. Lanes 2 and 3, sucrose in the presence of actinomycin D. WF9 N mtRNA (lanes 1), WF9 S gradient-purified mitochondria; lanes 4 and 5, 1000 x g pellet; lanes mtRNA (lanes 2), and WF9 S CF-11 unbound mtRNA (lanes 3) were 6 and 7, microsomes; lanes 8 and 9, sucrose gradient-purified nuclei; subjected to agarose/urea gel electrophoresis, stained with ethidium Hinfl lanes 10 and 11, cytosol; lane 1, 32P-end-labeled partial bromide (A), transferred to a hybridization membrane, and probed same digestion products of pBR322 as in Fig. 1. Approximately the with CF-11 bound (B) or CF-11 unbound (C) WF9 S mtRNA labeled amount of RNA was loaded in each lane of the gel. in the presence of 50 ,ug of actinomycin D per ml. b, Bases.

of RNase, followed by an additional 15 min of incubation, one another. For example, the single-stranded species might however, resulted in the complete disappearance of all result from transcription of the ds RNAs. This predicts that labeled RNA (Fig. 3A, lanes 2 and 3). This indicates that the homology should exist between the double- and single- RNA had been protected from RNase by a Triton X-i00- stranded forms. To test for this, S mtRNA was labeled in the sensitive, presumably membranous, compartment. This sug- presence ofactinomycin D to high specific activity, separated gests that the mitochondrion and not a virus or virus-like by CF-li cellulose chromatography into single- and double- particle is the site of synthesis of the RNAs. stranded fractions, and hybridized to total N mtRNA and Cell-fractionation experiments provide additional evidence total and single-stranded S mtRNA. The labeled double- that the synthesis of these RNAs takes place in the stranded fraction hybridized to the 850-base RNA oftotal and mitochondrion. Only weak synthesis of products lacking single-stranded S mtRNA but not to N mtRNA (Fig. 4B). This discrete sizes was observed when the nuclear and cytosolic indicates that complementarity exists between at least one of fractions were incubated in the mitochondrial incorporation the ds RNAs and the 850-base single-stranded RNA. No mixture. This synthesis was uniformly inhibited by hybridization was observed between the labeled single- actinomycin D (Fig. 3B). No incorporation was detected with stranded fraction and single-stranded S mtRNA, total S the microsomal fraction. Although the crude 1000 x g pellet mtRNA, or N mtRNA (Fig. 4C). Interestingly, no hybrid- fraction possesses some RNA synthetic activity, the prod- ization was observed between either of the labeled RNA ucts resembled those made by mitochondria. The only fractions and mtDNA or between nick-translated, 32P-labeled detectable product of actinomycin D-resistant RNA synthe- S mtDNA and unlabeled ds RNA under conditions in which sis by the 1000 x g pellet was the 850-base mitochondrial an equivalent amount of mitochondrial rRNA hybridized species. This suggests that most of the RNA synthesis in this strongly (not shown). Thus, the RNAs are not homologous to fraction is due to the small amount of mitochondria present mtDNA, a further indication that their synthesis is DNA in it. In addition, when a crude mitochondrial pellet was independent. subjected to centrifugation in sucrose or Percoll (24) density gradients, the actinomycin D-resistant RNA synthesis DISCUSSION cosedimented with the mitochondrial marker enzyme succinate:cytochrome c reductase and with the mitochondrial- The actinomycin D-resistant mode of synthesis of the RNAs specific 26S and 18S rRNAs. described here indicates that their replication proceeds in- Sequence Homology Between Single- and Double-Stranded dependently of mtDNA and other cellular chromosomes. S-Specific RNAs. It seemed likely that the four RNAs syn- Autonomous replication is also suggested by the double- thesized in the presence of actinomycin D might be related to stranded character of the 2850- and 900-bp species. It is Downloaded by guest on September 27, 2021 Genetics: Finnegan and Brown Proc. Natl. Acad. Sci. USA 83 (1986) 5179 unlikely that these RNAs arise from infection with a hori- Formation de Chercheurs et Action Concertee of the Province of zontally transmitted agent, such as a virus or viroid, since Quebec (EQ2177). plants in which they are found show no disease symptoms and since they occur in a wide variety of maize plants of 1. Tipper, D. J. & Bostian, K. A. (1984) Microbiol. Rev. 48, plants 125-156. different origins. Moreover, their occurrence only in 2. Lemke, P. A. (1976) Annu. Rev. Microbiol. 30, 105-145. with S-type cytogenes strongly indicates a vertical, cytoplas- 3. Boccardo, G., Lisa, V. & Milne, R. G. (1983) in Double- mic mode of transmission. They are thus most properly Stranded RNA Viruses, eds. Compans, R. W. & Bishop, classified as plasmids, analogous to the cytoplasmically D. H. L. (Elsevier, New York), pp. 425-430. transmitted RNA plasmids of killer strains of yeast (1) and 4. Palmer, J. D. & Shields, C. R. (1984) Nature (London) 307, other cellular virus-like particles (2, 3). They differ from other 437-440. RNA genomes in that they are associated with the 5. Lonsdale, D. M., Hodge, T. P. & Fauron, C. M.-R. (1984) mitochondrion. Since we have not observed any virus-like Nucleic Acids Res. 12, 9249-9261. structures during electron microscopic examination of our 6. Sederoff, R. R. (1984) Adv. Genet. 22, 1-108. a further 7. Pring, D. R., Levings, C. S., Hu, W. W. L. & Timothy, D. H. mitochondrial preparations, it is possible that (1977) Proc. Natl. Acad. Sci. USA 74, 2904-2908. difference from other RNA genomes may be their lack of 8. Schardl, C. L., Lonsdale, D. M., Pring, D. R. & Rose, K. R. encapsidation. (1984) Nature (London) 310, 293-2%. These RNA plasmids represent a novel type of inheritable 9. Laughnan, J. R. & Gabay-Laughnan, S. (1983) Annu. Rev. element in organelles. The extent to which these elements Genet. 17, 27-48. occur within and outside the plant kingdom is at present 10. Kemble, R. J., Gunn, R. E. & Flavell, R. B. (1980) Genetics unknown. They may occur in at least some other species, 95, 451-458. since ds RNA has also been found to be associated with sugar 11. Forde, B. G. & Leaver, C. J. (1980) Proc. Natl. Acad. Sci. beet mitochondria (25). In addition, ds RNAs have been USA 77, 418-422. 12. Hallick, R. B., Chelm, B. K., Gray, P. W. & Orozco, E. M., found in the mitochondrial fraction in yeast (26) and in maize Jr. (1977) Nucleic Acids Res. 4, 3055-3064. plants of a specific nuclear genotype (inbred W182BN) 13. Bottomley, W., Spencer, D., Wheeler, A. M. & Whitfield, carrying the L or LBN accession of S cytoplasm (27). The P. R. (1971) Arch. Biochem. Biophys. 143, 269-275. yeast mitochondrial ds RNAs differ from the ones described 14. Chamberlin, J. J. (1974) in The Enzymes, ed. Boyer, P. D. here in that they hybridize to mtDNA and thus probably (Academic, New York), Vol. 10, pp. 333-374. result from symmetrical DNA transcription. In the maize 15. Groot, G. S. P., van Horten-Loosbroek, N., van Ommen, example, the two ds RNAs, LBN-1 and LBN-2, were not G.-J. B. & Pijst, H. L. A. (1981) Nucleic Acids Res. 9, found in the same inbred with other forms ofS cytoplasm (28) 6369-6377. or in plants with L cytoplasm in different nuclear genotypes. 16. Boerner, P., Mason, T. L. & Fox, T. D. (1981) Nucleic Acids Res. 9, 6379-6390. Because oftheir similar size and hybridization characteristics 17. Newman, D. & Martin, N. (1982) Plasmid 7, 66-76. (19), these RNAs are probably related to the ds RNAs we 18. Gaines, G. & Attardi, G. (1984) J. Mol. Biol. 172, 451-466. show here to be synthesized in a DNA-independent manner. 19. Schuster, A. M., Sisco, P. H. & Levings, C. S., III (1983) in It is possible that the copy number of a variant form of these Plant Molecular Biology, UCLA Symposia on Molecular and RNAs specific to L cytoplasm is under strong nuclear gene Cellular Biology, New Series, ed. Goldberg, R. (Liss, New control, resulting in an increased abundance in inbred York), Vol. 12, pp. 437-444. W182BN. 20. Gale, E. F., Cundliffe, E., Reynolds, P. E., Richmond, M. H. & Waring, M. J. (1981) The Molecular Basis of Antibiotic Since the RNA plasmids are found only in the S cytotype, Action (Wiley, New York), pp. 258-401. it is likely that they arose in this maternal lineage within the 21. Nassuth, A., Alblas, F., van der Geest, A. J. M. & Bol, J. F. relatively recent evolutionary past, subsequent to the diver- (1983) Virology 126, 517-524. gence of S from the other maize cytoplasms. Although we 22. Franklin, R. M. (1966) Proc. Natl. Acad. Sci. USA 55, consider it premature to speculate on the origin of these 1504-1511. RNAs, it is interesting to note that some evolutionary relation- 23. Welling, G. W., Groen, G. & Welling-Wester, S. (1983) J. Chromatogr. 266, 629-632. ship between genetic RNA and mitochondria is suggested by 24. Neuberger, M., Journet, E.-P., Bligny, R., Carde, J.-P. & the low level of sequence homology found between proteins Douce, R. (1982) Arch. Biochem. Biophys. 217, 312-323. encoded by a group ofplant RNA viruses and those encoded by 25. Powling, A. (1981) Mol. Gen. Genet. 183, 82-84. certain yeast mitochondrial introns (29). 26. Beilharz, M. W., Cobon, G. S. & Nagley, P. (1982) Nucleic Acids Res. 10, 1051-1070. 27. Sisco, P. H., Garcia-Arenal, F., Zaitlin, M., Earle, E. D. & We thank Howard Bussey, Sarah Gibbs, and Desh-Pal Verma for Gracen, V. E. (1984) Plant Sci. Lett. 34, 127-134. critical comments on the manuscript, Annette Nassuth for helpful 28. Sisco, P. H., Gracen, V. E., Everett, H. L., Earle, E. D., discussions, and C. S. Levings and Marc Albertsen for gifts of seed. Pring, D. R., McNay, J. W. & Levings, C. S., III (1985) This research was supported by grants from National Sciences and Theor. Appl. Genet. 71, 5-15. Engineering Research Council of Canada (A7624) and the Fonds 29. Zimmern, D. (1983) J. Mol. Biol. 171, 345-352. Downloaded by guest on September 27, 2021