Current Genetics
Current Genetics(1983) 7:151-157
© Springer-Verlag 1983
Mitochondrial DNA from Podospora anserina IV. The Large R i b o s o m a l R N A Gene Contains T w o L o n g Intervening Sequences
Richard M. Wright and Donald J. Cummings Department of Microbiologyand Immunology, Campus Box B-175, School of Medicine, University of Colorado, Health Sciences Center, Denver, Colorado 80262, USA
Summary. We have examined the structure of the rRNA genes from the mitochondrial genome of Podospora anserina. Using R-loop analysis, nuclease protection experiments, and Southern blot hybridization analysis we have observed two intervening sequences (IVS) in the large rRNA gene, and none in the small rRNA gene. the IVS sequences are 1.65 kbp and 2.73 kbp long, and the larger of the two is in the position of the conserved IVS found in the mitochondrial genomes of other fungi. We have detected precursor transcripts for the large rRNA, and these data support the observation of two IVS in this gene. We also note that the large and small rRNA genes are separated by approximately 6 kbp of DNA. Key words: Podospora anserma - Mitochondrial DNA Ribosomal Genes - Intervening Sequences - Transcripts
Introduction
The structure and evolution of mitochondrial genomes has been the subject of three recent reviews (Grant and Lambowitz 1982; Gray and Doolittle 1982; Wallace 1982). Within the fungi, there appears to be little conservation of genetic organization. In general, both the position and relative spacing between protein coding genes varies between different genera. For example, genes for cytochrome c oxidase subunits 1, 2, and 3; cytochrome b; subunit 6 of the oligomycin sensitive ATPase; the large and small ribosomal RNA (rRNA) genes, and 22-24 transfer RNA (tRNA) genes are all present in fungal mitochondrial genomes. However, they
Offprint requests to: D. J. Cummings
are not located in the same relative position and are separated by variable amounts of "spacer" DNA. Genes for subunit 9 of the oligomycin sensitive ATPase and the mitochondrial ribosomal protein Var-1 are present in some but not all fungal mitochondrial DNAs. Some structural features of the rRNA genes are conserved within the fungi. The small rRNA gene has not been found to contain any coding discontinuities in any of the organisms examined. A single, but variably sized, intervening sequence (IVS) has been found in the large rRNA gene in Saccharomyces cerevisiae (Dujon 1980), Neurospora crassa (Hahn et al. 1979; Heckman and Raj Bhandary 1979), and Aspergillus nidulans (Lazarus et al. 1980). The IVS is located 400 to 600 bp from the 3' end of the gene (Grant and Lambowitz 1982) and is 1.1 to 2.3 kb long. Some concern has focused upon the distance of separation between rRNA coding loci and the genetic content of the DNA separating the two genes. In the fungi, this region varies from 1 kbp to 25 kbp and is known to code for both tRNAs and mRNAs (Wallace 1982). The inference is that the rRNA genes are most likely independently transcribed. Transcript biogenesis and processing modes of the large rRNA have been explored in an effort to understand transcript splicing mechanisms in mitochondria. Much of this work has centered uponNeurospora crassa (Mannella et al. 1979; Grimm and Lambowitz 1979; Green et al. 1981, Bertrand et al. 1982)or Saccharomyces cerevisiae (Boset al. 1980; Merten et al. 1980; Boerner et al. 1981). In general, precursor transcripts can accumulate in certain nuclear mutants, suggesting that rRNA processing enzymes are nuclear coded. Exact splicing pathways have yet to be determined, but multiple nuclear genes are likely involved (Bertrand et al. 1982) in catalyzing the single excision event. 5' end processing almost certainly occurs in Neurospora crassa (Grant and Lambowitz 1982), but not in Saccharornyces cerevisiae.
152
R.M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina
In the present paper, we demonstrate that the ascomycete Podospora anserina possesses two long intervening sequences in the large rRNA gene, 2.73 and 1.65 k b p in length. We present some details o f the organization o f the rRNA genetic loci. Based upon contrast to the other fungi, we infer that the extra IVS in Podospora is the shorter o f the two and begins 1.3 k b p from the 5' end o f the gene.
Sl Nuclease and Exonuclease VII Mapping. DNA-RNA duplexes were formed at tss-1 °C as described previously (Wright et al. 1982b). Strand separation temperatures (tss) were determined electrophoretically as described by Rosbash et al. (1979). For the large rRNA gene tss was 56 °C for E5, 54 °C for E7; and for the small rRNA gene tss was 53.5 °C for El. $1 nuclease analysis was performed by the Weaver and Weissmann (1979) modification of the Berk and Sharp (1977) procedure, and details of the cleavage reaction will be published elsewhere (Wright and Cummings, manuscript submitted). Exonuclease VII analysis was performed as described by Villarreal et al. (1979) with the modifications described fully by Wright and Cummings (manuscript submitted).
Materials and Methods Podospora anserina Strains and Cultivation. Podospora anserina of the s+ race was used throughout. Mycelia were propagated on solid corn-meal agar plates and these were used to inoculate 24 1 of liquid culture as described (Smith and Rubenstein 1973; Cummings, Belcour and Grandchamp 1979). Liquid cultures were the source of mitochondria. Mitochondrial DNA and RNA. Mitochondrial DNA (mtDNA) was prepared from mitochondria purified by differential centrifugation as described by Cummings et al. (1979). MtDNA was purified through two cycles of Dapi-CsC1gradient centrifugation. Mitochondrial RNA (mtRNA) was prepared as previously described (Wright et al. 1982a) and stored as an ethanol precipitate. Specific transcripts were purified from 7.5 mM CH3HgOH1.2% agarose gels as described by Wright et al. (1982a). Cloned EcoRI fragments from our clone bank were utilized as required (pPMT-1, 2, etc., Wright et al. 1982a). Northern and Southern Blot Hybridization Analyses. MtDNA was blotted to nitrocellulose filters (Schleicher and Schuell) by the method of Southern (1975) and hybridized to cloned, nick-translated (Rigby et al, 1977) probes as described by Wright et al. (1982a). Northern analysis of mitochondrial transcripts was performed essentially as described by Thomas (1980) with the modifications previously described (Wright et al. 1982a). MtRNA was displayed on 7.5 mM CH3HgOH-1.2% agarose gels according to Bailey and Davidson (1976). Gels were washed, stained, photographed, and blotted to nitrocellulose (Wright et at. 1982a). Cloning IVS-2 Sequences. Approximately 60% of IVS-2 can be isolated from the genomic fragment E7 as a single BglII fragment. For this reason, the clone pPMT-7 was cleaved with the restriction endonuclease BglII and the fragment corresponding to IVS-2 was purified on agarose gels. This gragment was inserted at the single BglII site of the vector pSVO10 (Learned et al. 1981). Ampicillin resistant colonies were screened for IVS-2 sequences using the colony hybridization procedure of Grunstein and Hogness (1975). The hybridization probe employed was the nick-translated IVS-2 region from pPMT-7. One clone was selected for use in the present study (pSV-I2). R-loop Formation and Electron Microscopy. R-loops were formed to cloned, linearized fragments of the mitochondrial genome as described by Wright et al. (1982b). In each case, the ribosomal transcript used for hybridization was purified on CH3HgOH-agarose gels. Snap-back hybr.ids were formed as described in the legend to Fig. 2 using cloned mtDNA fragments. Electron microscopy, staining, and shadowing were performed as described by Wright et al. (1982b).
Results R-loops o f the Ribosomal Genes Using Southern blot (Southern 1975) hybridization analysis, we had shown previously (Wright et al. 1982a) that the small rRNA gene was contained on the genomic fragment E l . The large rRNA gene was shown to span up to 10 kbp o f DNA contained on the adjacent fragments E5 and E7. We have formed R-loops to the clones o f these fragments as described in the legend to Fig. 1. It can be seen from Fig. 1A that the small rRNA gene is continuous and uninterrupted b y intervening sequences. However, a short segment o f the gene fails to form an obvious R-loop. This region is associated with an inverted snap-back repeat sequence (Fig. 2) which m a y disrupt R-loop formation. R-loops formed to the cloned segments E5 and E7, using purified large rRNA, both exhibit long intervening sequences (IVS-1 and IVS-2). These are illustrated in Fig. 1B and C. Summaries o f R-loops formed between the large and small rRNAs and the respective substrates are illustrated in Figs. 3 and 4, and a map o f this region o f the mitochondrial chromosome is shown in Fig. 5. F r o m these data, we have calculated the size o f the coding region for the small rRNA gene to be 1.9 kbp and 7.9 kbp for the large rRNA gene. The intervening sequences in the large rRNA gene measure 1.65 kbp and 2.73 kbp. To our knowledge, these data constitute the first demonstration o f two large coding discontinuities in mitochondrial ribosomal RNA genes. The separation between rRNA genes is approximately 6 kbp.
Direction o f Transcription and Location o f 5' ends We have used Weaver and Weissmann's (1979)modification o f the Berk and Sharp (1977) mapping procedure to determine transcriptional polarity in the rRNA genes. S1 nuclease digestion and exonulcease VII digestion were used to define the location o f the 5' termini. F o r this analysis, numerous genomic probes were constructed
R. M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina
153
Fig. 1A-C. R-loops to the large and small rRNA genes. The large and small rRNAs were purified on CH3HgOHagarose gels as described (Wright et al. 1982b). R-loops were formed to the purified transcripts, spread for electron microscopy, photographed and calibrated as described (Ibid). A R-loop formed between the small rRNA and the cloned genomic fragment El. B R-loop formed between the large rRNA and the cloned genomic fragment E5. C R-loop formed between the large rRNA and the cloned genomic fragment E7. On the left is the photomicrograph and on the right is the interpretive drawing. Hatched lines correspond to RNA, narrow lines to single strand DNA, thick lines to double strand DNA, intervening sequences are indicated. The bar represents 1 kbp of duplex DNA
Fig. 2A and B. Identification of an inverted snap-back repeat at the locus of the small rRNA gene. The E1 fragment was isolated from the clone pPMT-1 by EcoRI digestion. Samples were suspended in reannealing mix (50% formamide; 0.08 M PIPES buffer [pH 7.9]; 10 mM EDTA; final Na+ ion concentration was made 0.30 M with NaC1), denaturated at 90 °C for 5 min. Single strand snap-back molecules were obtained by cooling samples to 0 °C and immediately spreading for electron microscopy as described in Wright et al. (1982b). Underwound double strand superhelices were obtained by cooling denatured samples to 21 °C and allowing them to incubate for 5 to 60 min (Broker et al. 1977). Resolution of metastable underwound superhelices was usually complete by 60 min and most molecules could be trapped in the above intermediate state with 15 min of incubation. A Single strand snap-back molecule. B Double strand snapback molecule showing an underwound superhelix
from the E5, E7, and E1 regions, and the probes illustrated in Fig. 5 are the only probes which were protected b y mtRNA. For example, a probe made by 5' end labeling the cloned E7 fragment at EcoRI sites (one site o f which lies in exon II) was n o t protected b y mtRNA.
A 5' end labeled probe composed of E5 labeled at its EcoRI sites was protected by mtRNA. Thus, the direction o f transcription is that shown in Fig. 5. Comparable experiments were conducted on the small rRNA locus, and the only probe which was protected by m t R N A is
R.M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina
154
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Fig. 3. R-loops formed at the small rRNA locus. R-loops and snap-back molecules were measured along the distances A - E as shown above. Means and standard deviations are given for each distance. The distance "A" on the R-loops was calculated by subtracting 1,300 bp from the total length of the distance from the start of the molecule to the start of the R-loop. This corresponds to the 1,300 bp separating the EcoRI and BamHI sites in pBR325 (Bolivar 1978). The snap-back molecules have been aligned as shown because cleavage with the restriction endonuclease XhoI alters the distance from one end of the molecule to the start of the snap-back loop. This distance is 6,400 bp. Summary data are listed in kilobase pairs
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the one shown in Fig. 5. These data are illustrated in Fig. 6 where it can be seen that S1 nuclease analysis located the 5' end o f the small rRNA gene to a point 1,000 bp from the internal BglII site. We observe that both the large and small rRNA genes are transcribed in the same direction, and, in fact, all genes so far examined on Podospora m t D N A are also transcribed in this direction. Because the large rRNA gene is complex in structure, we have confirmed our determination o f the 5' end b y using exonuclease VII digestion. A 5 . 5 kbp probe, end labeled at the EcoRI site in exon II, was used in the protection experiments. These data are shown in Fig. 6B where titration from probe excess to RNA excess is also shown. The major site o f S1 nuclease resistance is 1.0 k b p from the labeled EcoRI site. This identifies the position o f the acceptor site o f IVS-1. A t high RNA
Fig. 4. R-loops formed at the large rRNA locus. Rloops formed between the clones pPMT-5 and pPMT-7 to purified large rRNA were calibrated as indicated in the legend to Fig. 3. The EeoRI site located at the junction between A and B segments marks the disruption of exon 2 which occurred by cloning the EcoRl fragments
concentration, precursor molecules are detected from which, presumably, IVS-1 has not been spliced. The major exonuclease VII resistant fragment occurs at 3.9 kbp, although some cleavage also occurs at the major site o f S1 trimming. The size o f exonuclease VII resistant fragments is in excellent a g r e e m e n t with R-loop data and allows us to def'me the 5' end o f the gene as shown in Fig. 5, 3.9 kbp from the EcoRI cleavage site in exon II. The resolution o f these experiments does not allow us to eliminate the possibility o f 5' end processing.
Precursor Transcripts from the Large rRNA Locus In a previous publication (Wright et al. 1982a), we used Northern blot hybridization analysis to confirm the
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Fig. 5. The ribosomal RNA genetic locus of Podospora anserina mitochondrial DNA. Data from Figs. 1, 3, 4 and 6 have been summarized. The restriction endonuctease cleavage map has been published previously (Wright et al. 1982). Line A: Restriction endonuclease cleavage map. Line B: organization of the coding region. Line C: Transcriptional polarity. Line D: Synopsis of $1 nuclease protection experiments. Narrow lines indicate probes, wide lines are protected fragments. Line E; Synopsis of exonuclease VII protection experiments
Fig. 6. A Transcriptional polarity and location of 5' ends of ribosomal RNA genes. $1 nuelease analysis was conducted as described in Materials and Methods. Probes of the large ribosomal RNA locus were constructed by 5' end labeling the EcoRI sites of the fragments E5 and E7 from clones pPMT-5 and pPMT-7. The E5 probe was cleaved with PstI to produce the single headed probe shown. E7 was used as a double headed probe. Probes of the small ribosomal RNA locus were produced from pPMT-1 by 5' end labeling the BgllI cleavage products. These correspond to the genome fragments Bg12/13 and Bg4. Lane A: Mobility standards. Lane B: E5 probe. Lane C: S1 nuclease protection experiment using E5 probe. Lane D: E7 probe. Lane E: S1 nuclease protection experiment using E7 probe. Lane F: Bg4 probe. Lane G: S1 nuclease protection experiment using Bg4 probe. Lane H: Bg12/13 probe. Lane I: $1 nuclease protection experiment using Bg 12/13 probe; B S1 nuclease and exonuclease VII resistant fragments from the large ribosomal RNA gene were produced as described in Materials and Methods. Lane A: Mobility standards. Lane B: S1 nuclease treated probe from Lane C. Lane C: Large ribosomal 5' end labeled probe. Lane D: S1 nuclease protection experiment for probe Lane C. Lane E: Exonuclease VII protection experiment for probe in Lane C. Lane F: Blank. Lanes G - K : Titration of large ribosomal RNA probe from probe excess to RNA excess. Lane G: 100,000 cpm probe, 2/~g RNA; Lane H: 20,000 cpm probe, 5 ~tg RNA: Lane I: 20,000 epm probe, 10 t~g RNA: Lane J: 20,000 cpm probe, 15 t~g RNA, Lane K: 10,000 epm probe, 15 ~g RNA
R.M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina
156
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Fig. 7. A Northern blot hybridization analysis of the large rRNAs. Probes were constructed, RNA was blotted, hybridization was done as described in Materials and Methods. The first lane shows mtRNA stained with ethidium brorriide. Lane A: hybridization to IVS-1 enriched probe. Lane B: hybridization to the IVS-2 specific probe. The bands marked have been observed in repetitions of these experiments. The unmarked bands in Lane B may represent various degradation products; they do not arise consistently in repetitions of these experiments. B summarizes the structure and transcripts obtained form the large rRNA locus. The probes used in 7A are illustrated along with the transcripts hybridized
coding sequence identification for large and small rRNA loci. We showed that these transcripts could be isolated from Podospora mitochondria with a minimum of degradation. At that time, we employed large probes of each ribosomal region to identify the major ribosomal transcripts. Here, we have used the cloned IVS-2 sequence as a specific nick-translated probe of Northern blotted mtRNA, as well as a probe from IVS-1 which overlaps exons 1 and 2. These results are illustrated in Fig. 7A where precursor transcripts can be defined for the large rRNA at 7.9 kb, 6.3 kb, and 5.2 kb. The IVS-1 enriched probe hybridizes to three transcripts larger than the mature rRNA. Three transcripts are hybridized by the IVS-2 specific probe. The two largest transcripts (7.9 kb and 6.3 kb) are hybridized by both IVS-1 enriched and IVS-2 probes. IVS-1 enriched probe hybridized to a large transcript (5.2 kb) not detected by the IVS-2 probe, and it hybridizes to the mature rRNA. IVS-2 hybridizes to an excision product not hybridized by the IVS-1 enriched probe. These observations are summarized in Fig. 7B. We have not detected an excision product for IVS-1, and perhaps it is very unstable in the mitochondria. We also observe that the largest transcript detected occurs in very low concentration and is more readily revealed with longer probes. The occurrence of intermediate transcripts in approximately equivalent concentration suggests that splicing of either IVS may be random.
Discussion In this paper we have presented evidence for the existence and transcription of two long intervening sequences in the mitochondrial large rRNA gene of Podospora anserina. For this analysis, we have drawn upon data from R-loop experiments, Northern blot hybridization analysis, $1 nuclease and exonuclease VII protection experiments. The structures of the large and small rRNA genes have been characterized, and precursor transcripts for the large rRNA have been detected in the mitochondria. As far as we can determine, the situation described here for Podospora anserina is unique among mitochondrial genomes. All of the Ascomycete fungi which have been examined exhibit one IVS close to the 3' terminus of the large rRNA gene (Grant and Lambowitz 1982). This IVS is preserved in Podospora, although it is larger (2.73 kbp) than the other IVS reported. The "extra" IVS in Podospora, located near the 5' end of the gene, is the unique feature of this situation. In yeast, the large rRNA gene in the co+ allelic state contains a single large IVS, while in the co- allelic state this IVS is missing (Dujon 1980). In Neurospora crassa and Aspergillus nidulans a single IVS is found at this locus. Thus, large rRNA genes from the fungi can be found containing none, one, or two long discontinuities in the coding sequences. A high degree of variability in IVS occurrence has also been observed in the cob locus of
R. M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina yeast (Lazowska et al. 1980) where two IVS are found in the short form of the gene and five in the long form. In Aspergillus nidulans only one is found (Davies et al. 1982). This is comparable to the situation of the oxi3 locus which is uninterrupted in Podospora (Wright et al. 1982b); twice interrupted in Aspergillus nidulans (Kuntzel et al. 1982); and extensively (seven IVS) interrupted in Saccharomyces cerevisiae (Bonitz et al. 1980).
Acknowledgements. This work was supported in part by a grant from the National Institues of Health, AGO1367.
References Bailey JM, Davidson N (1976) Anal Biochem 70:75-85 Berk AJ, Sharp P (1977) Cell 12:721-732 Bertrand H, Bridge P, Collins RA, Garriga G, Lambowitz AM (1982) Cell 29:517-526 Boerner P, Mason TL, Fox TD (1981) Nucleic Acids Res 9: 6379-6390 Bolivar F (1978) Gene 4:121-136 Bonitz SG, Coruzzi G, Thalenfeld BE, Tzagoloff A, Macino G (1980) J Biol Cliem 255:11927-11941 Bos JL, Osinga KA, Von Der Horst G, Hecht NB, Tabak HF, Van Ommen G-JB, Borst P (1980) Cell 20:207-214 Broker TR, Soil L, Chow LT (1977) J Mol Biol 113:579-589 Cummings DJ, Belcour L, Grandchamp C (1979) Mol Gen Genet 171:229-238 Davies RW, Scazzocchio C, Waring RB, Lee S, Grisi E, Berks MM, Brown TA (1982) In: Slonimski P, Borst P, Attardi G (eds) Mitochondrial Genes: Cold Spring Harbor Monographs 12 Dujon B (1980) Cell 20:185-197 Grant DM, Lambowitz AM (1982) In the Cell Nucleus, Busch H, Rothblum L (eds), vol X. Academic Press, New York, NY Gray MW, Doolittle WF (1982) Microbiol Rev 46:1-42 Grimm MF, Lambowitz AM (1979) J Mol Biol 134:667-672
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Green MR, Grimm MF, Goewert RR, Collins RA, Cole MD, Lambowitz AM, Heckmart JE, Yin S, Raj Bhandary AL (1981) J Biol Chem 256:2027-2034 Grunstein M, Hogness DS (1975) Proc Natl Acad Sci USA 72:3961-3965 Hahn U, Lazarus CM, Lunsdorf H, Kuntzel H (1979) Cell 17: 191-200 Heckman JE, Raj Bhandary UL (1979) Cell 17:583-595 Kuntzel H, Kochel HG, Lazarus CM, Lunsdorf H (1982) In: Slonimski P, Borst P, Attardi G (eds) Mitochondfial Genes. Cold Spring Harbor Monographs 12 Lazarus CM, Lunsdorf H, Han U, Stepien PP, Kuntzel H (1980) Mol Gen Genet 177:389-397 Lazowska J, Jacq C, Slonimski PP (1980) Cell 22:333-348 Learned RM, Meyers RM, Tjian R (1981) In: Ray DS (ed) The Initiation of DNA Replication. Academic Press, New York, pp 555-566 Mannella CA, Collins RA, Green MR, Lambowitz AM (1979) Proc Natl Acad Sci USA 76:2635-2639 Merten S, Synenki RM, Locker J, Christianson T, Rabinowitz M (1980) Proc Natl Acad Sci USA 77:1417-1421 Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) J Mol Biol 113:239-251 Rosbash M, Blank D, Fahrner K, Hereford L, Riccardi R, Roberts B, Ruby S, Woolford T (1979) Meth Enzymol 68:454-469 Smith JR, Rubenstein I (1973) J Gen Microbiol 76:297-304 Southern EM (1975) J Mol Biol 98:503-517 Thomas P (1980) Proc Natl Acad Sci USA 77:5201-5205 Villarreal LP, White RI, Berg P (1979) J Virol 29:209-219 Wallace DC (1982) Microbiol Rev 46:208-240 Weaver RF, Weissman C (1979) Nucleic Acids Res 7:11751193 Wright RM, Laping JL, Horrum MA, Cummings DJ (1982a) Mol Gen Genet 185:56-64 Wright RM, Horrum MA, Cummings DJ (1982b) Cell 29:505515
Communicated by C. W. Birky Jr. Received November 5 / December 20, 1982
Current Genetics(1983) 7:151-157
© Springer-Verlag 1983
Mitochondrial DNA from Podospora anserina IV. The Large R i b o s o m a l R N A Gene Contains T w o L o n g Intervening Sequences
Richard M. Wright and Donald J. Cummings Department of Microbiologyand Immunology, Campus Box B-175, School of Medicine, University of Colorado, Health Sciences Center, Denver, Colorado 80262, USA
Summary. We have examined the structure of the rRNA genes from the mitochondrial genome of Podospora anserina. Using R-loop analysis, nuclease protection experiments, and Southern blot hybridization analysis we have observed two intervening sequences (IVS) in the large rRNA gene, and none in the small rRNA gene. the IVS sequences are 1.65 kbp and 2.73 kbp long, and the larger of the two is in the position of the conserved IVS found in the mitochondrial genomes of other fungi. We have detected precursor transcripts for the large rRNA, and these data support the observation of two IVS in this gene. We also note that the large and small rRNA genes are separated by approximately 6 kbp of DNA. Key words: Podospora anserma - Mitochondrial DNA Ribosomal Genes - Intervening Sequences - Transcripts
Introduction
The structure and evolution of mitochondrial genomes has been the subject of three recent reviews (Grant and Lambowitz 1982; Gray and Doolittle 1982; Wallace 1982). Within the fungi, there appears to be little conservation of genetic organization. In general, both the position and relative spacing between protein coding genes varies between different genera. For example, genes for cytochrome c oxidase subunits 1, 2, and 3; cytochrome b; subunit 6 of the oligomycin sensitive ATPase; the large and small ribosomal RNA (rRNA) genes, and 22-24 transfer RNA (tRNA) genes are all present in fungal mitochondrial genomes. However, they
Offprint requests to: D. J. Cummings
are not located in the same relative position and are separated by variable amounts of "spacer" DNA. Genes for subunit 9 of the oligomycin sensitive ATPase and the mitochondrial ribosomal protein Var-1 are present in some but not all fungal mitochondrial DNAs. Some structural features of the rRNA genes are conserved within the fungi. The small rRNA gene has not been found to contain any coding discontinuities in any of the organisms examined. A single, but variably sized, intervening sequence (IVS) has been found in the large rRNA gene in Saccharomyces cerevisiae (Dujon 1980), Neurospora crassa (Hahn et al. 1979; Heckman and Raj Bhandary 1979), and Aspergillus nidulans (Lazarus et al. 1980). The IVS is located 400 to 600 bp from the 3' end of the gene (Grant and Lambowitz 1982) and is 1.1 to 2.3 kb long. Some concern has focused upon the distance of separation between rRNA coding loci and the genetic content of the DNA separating the two genes. In the fungi, this region varies from 1 kbp to 25 kbp and is known to code for both tRNAs and mRNAs (Wallace 1982). The inference is that the rRNA genes are most likely independently transcribed. Transcript biogenesis and processing modes of the large rRNA have been explored in an effort to understand transcript splicing mechanisms in mitochondria. Much of this work has centered uponNeurospora crassa (Mannella et al. 1979; Grimm and Lambowitz 1979; Green et al. 1981, Bertrand et al. 1982)or Saccharomyces cerevisiae (Boset al. 1980; Merten et al. 1980; Boerner et al. 1981). In general, precursor transcripts can accumulate in certain nuclear mutants, suggesting that rRNA processing enzymes are nuclear coded. Exact splicing pathways have yet to be determined, but multiple nuclear genes are likely involved (Bertrand et al. 1982) in catalyzing the single excision event. 5' end processing almost certainly occurs in Neurospora crassa (Grant and Lambowitz 1982), but not in Saccharornyces cerevisiae.
152
R.M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina
In the present paper, we demonstrate that the ascomycete Podospora anserina possesses two long intervening sequences in the large rRNA gene, 2.73 and 1.65 k b p in length. We present some details o f the organization o f the rRNA genetic loci. Based upon contrast to the other fungi, we infer that the extra IVS in Podospora is the shorter o f the two and begins 1.3 k b p from the 5' end o f the gene.
Sl Nuclease and Exonuclease VII Mapping. DNA-RNA duplexes were formed at tss-1 °C as described previously (Wright et al. 1982b). Strand separation temperatures (tss) were determined electrophoretically as described by Rosbash et al. (1979). For the large rRNA gene tss was 56 °C for E5, 54 °C for E7; and for the small rRNA gene tss was 53.5 °C for El. $1 nuclease analysis was performed by the Weaver and Weissmann (1979) modification of the Berk and Sharp (1977) procedure, and details of the cleavage reaction will be published elsewhere (Wright and Cummings, manuscript submitted). Exonuclease VII analysis was performed as described by Villarreal et al. (1979) with the modifications described fully by Wright and Cummings (manuscript submitted).
Materials and Methods Podospora anserina Strains and Cultivation. Podospora anserina of the s+ race was used throughout. Mycelia were propagated on solid corn-meal agar plates and these were used to inoculate 24 1 of liquid culture as described (Smith and Rubenstein 1973; Cummings, Belcour and Grandchamp 1979). Liquid cultures were the source of mitochondria. Mitochondrial DNA and RNA. Mitochondrial DNA (mtDNA) was prepared from mitochondria purified by differential centrifugation as described by Cummings et al. (1979). MtDNA was purified through two cycles of Dapi-CsC1gradient centrifugation. Mitochondrial RNA (mtRNA) was prepared as previously described (Wright et al. 1982a) and stored as an ethanol precipitate. Specific transcripts were purified from 7.5 mM CH3HgOH1.2% agarose gels as described by Wright et al. (1982a). Cloned EcoRI fragments from our clone bank were utilized as required (pPMT-1, 2, etc., Wright et al. 1982a). Northern and Southern Blot Hybridization Analyses. MtDNA was blotted to nitrocellulose filters (Schleicher and Schuell) by the method of Southern (1975) and hybridized to cloned, nick-translated (Rigby et al, 1977) probes as described by Wright et al. (1982a). Northern analysis of mitochondrial transcripts was performed essentially as described by Thomas (1980) with the modifications previously described (Wright et al. 1982a). MtRNA was displayed on 7.5 mM CH3HgOH-1.2% agarose gels according to Bailey and Davidson (1976). Gels were washed, stained, photographed, and blotted to nitrocellulose (Wright et at. 1982a). Cloning IVS-2 Sequences. Approximately 60% of IVS-2 can be isolated from the genomic fragment E7 as a single BglII fragment. For this reason, the clone pPMT-7 was cleaved with the restriction endonuclease BglII and the fragment corresponding to IVS-2 was purified on agarose gels. This gragment was inserted at the single BglII site of the vector pSVO10 (Learned et al. 1981). Ampicillin resistant colonies were screened for IVS-2 sequences using the colony hybridization procedure of Grunstein and Hogness (1975). The hybridization probe employed was the nick-translated IVS-2 region from pPMT-7. One clone was selected for use in the present study (pSV-I2). R-loop Formation and Electron Microscopy. R-loops were formed to cloned, linearized fragments of the mitochondrial genome as described by Wright et al. (1982b). In each case, the ribosomal transcript used for hybridization was purified on CH3HgOH-agarose gels. Snap-back hybr.ids were formed as described in the legend to Fig. 2 using cloned mtDNA fragments. Electron microscopy, staining, and shadowing were performed as described by Wright et al. (1982b).
Results R-loops o f the Ribosomal Genes Using Southern blot (Southern 1975) hybridization analysis, we had shown previously (Wright et al. 1982a) that the small rRNA gene was contained on the genomic fragment E l . The large rRNA gene was shown to span up to 10 kbp o f DNA contained on the adjacent fragments E5 and E7. We have formed R-loops to the clones o f these fragments as described in the legend to Fig. 1. It can be seen from Fig. 1A that the small rRNA gene is continuous and uninterrupted b y intervening sequences. However, a short segment o f the gene fails to form an obvious R-loop. This region is associated with an inverted snap-back repeat sequence (Fig. 2) which m a y disrupt R-loop formation. R-loops formed to the cloned segments E5 and E7, using purified large rRNA, both exhibit long intervening sequences (IVS-1 and IVS-2). These are illustrated in Fig. 1B and C. Summaries o f R-loops formed between the large and small rRNAs and the respective substrates are illustrated in Figs. 3 and 4, and a map o f this region o f the mitochondrial chromosome is shown in Fig. 5. F r o m these data, we have calculated the size o f the coding region for the small rRNA gene to be 1.9 kbp and 7.9 kbp for the large rRNA gene. The intervening sequences in the large rRNA gene measure 1.65 kbp and 2.73 kbp. To our knowledge, these data constitute the first demonstration o f two large coding discontinuities in mitochondrial ribosomal RNA genes. The separation between rRNA genes is approximately 6 kbp.
Direction o f Transcription and Location o f 5' ends We have used Weaver and Weissmann's (1979)modification o f the Berk and Sharp (1977) mapping procedure to determine transcriptional polarity in the rRNA genes. S1 nuclease digestion and exonulcease VII digestion were used to define the location o f the 5' termini. F o r this analysis, numerous genomic probes were constructed
R. M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina
153
Fig. 1A-C. R-loops to the large and small rRNA genes. The large and small rRNAs were purified on CH3HgOHagarose gels as described (Wright et al. 1982b). R-loops were formed to the purified transcripts, spread for electron microscopy, photographed and calibrated as described (Ibid). A R-loop formed between the small rRNA and the cloned genomic fragment El. B R-loop formed between the large rRNA and the cloned genomic fragment E5. C R-loop formed between the large rRNA and the cloned genomic fragment E7. On the left is the photomicrograph and on the right is the interpretive drawing. Hatched lines correspond to RNA, narrow lines to single strand DNA, thick lines to double strand DNA, intervening sequences are indicated. The bar represents 1 kbp of duplex DNA
Fig. 2A and B. Identification of an inverted snap-back repeat at the locus of the small rRNA gene. The E1 fragment was isolated from the clone pPMT-1 by EcoRI digestion. Samples were suspended in reannealing mix (50% formamide; 0.08 M PIPES buffer [pH 7.9]; 10 mM EDTA; final Na+ ion concentration was made 0.30 M with NaC1), denaturated at 90 °C for 5 min. Single strand snap-back molecules were obtained by cooling samples to 0 °C and immediately spreading for electron microscopy as described in Wright et al. (1982b). Underwound double strand superhelices were obtained by cooling denatured samples to 21 °C and allowing them to incubate for 5 to 60 min (Broker et al. 1977). Resolution of metastable underwound superhelices was usually complete by 60 min and most molecules could be trapped in the above intermediate state with 15 min of incubation. A Single strand snap-back molecule. B Double strand snapback molecule showing an underwound superhelix
from the E5, E7, and E1 regions, and the probes illustrated in Fig. 5 are the only probes which were protected b y mtRNA. For example, a probe made by 5' end labeling the cloned E7 fragment at EcoRI sites (one site o f which lies in exon II) was n o t protected b y mtRNA.
A 5' end labeled probe composed of E5 labeled at its EcoRI sites was protected by mtRNA. Thus, the direction o f transcription is that shown in Fig. 5. Comparable experiments were conducted on the small rRNA locus, and the only probe which was protected by m t R N A is
R.M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina
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the one shown in Fig. 5. These data are illustrated in Fig. 6 where it can be seen that S1 nuclease analysis located the 5' end o f the small rRNA gene to a point 1,000 bp from the internal BglII site. We observe that both the large and small rRNA genes are transcribed in the same direction, and, in fact, all genes so far examined on Podospora m t D N A are also transcribed in this direction. Because the large rRNA gene is complex in structure, we have confirmed our determination o f the 5' end b y using exonuclease VII digestion. A 5 . 5 kbp probe, end labeled at the EcoRI site in exon II, was used in the protection experiments. These data are shown in Fig. 6B where titration from probe excess to RNA excess is also shown. The major site o f S1 nuclease resistance is 1.0 k b p from the labeled EcoRI site. This identifies the position o f the acceptor site o f IVS-1. A t high RNA
Fig. 4. R-loops formed at the large rRNA locus. Rloops formed between the clones pPMT-5 and pPMT-7 to purified large rRNA were calibrated as indicated in the legend to Fig. 3. The EeoRI site located at the junction between A and B segments marks the disruption of exon 2 which occurred by cloning the EcoRl fragments
concentration, precursor molecules are detected from which, presumably, IVS-1 has not been spliced. The major exonuclease VII resistant fragment occurs at 3.9 kbp, although some cleavage also occurs at the major site o f S1 trimming. The size o f exonuclease VII resistant fragments is in excellent a g r e e m e n t with R-loop data and allows us to def'me the 5' end o f the gene as shown in Fig. 5, 3.9 kbp from the EcoRI cleavage site in exon II. The resolution o f these experiments does not allow us to eliminate the possibility o f 5' end processing.
Precursor Transcripts from the Large rRNA Locus In a previous publication (Wright et al. 1982a), we used Northern blot hybridization analysis to confirm the
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Fig. 5. The ribosomal RNA genetic locus of Podospora anserina mitochondrial DNA. Data from Figs. 1, 3, 4 and 6 have been summarized. The restriction endonuctease cleavage map has been published previously (Wright et al. 1982). Line A: Restriction endonuclease cleavage map. Line B: organization of the coding region. Line C: Transcriptional polarity. Line D: Synopsis of $1 nuclease protection experiments. Narrow lines indicate probes, wide lines are protected fragments. Line E; Synopsis of exonuclease VII protection experiments
Fig. 6. A Transcriptional polarity and location of 5' ends of ribosomal RNA genes. $1 nuelease analysis was conducted as described in Materials and Methods. Probes of the large ribosomal RNA locus were constructed by 5' end labeling the EcoRI sites of the fragments E5 and E7 from clones pPMT-5 and pPMT-7. The E5 probe was cleaved with PstI to produce the single headed probe shown. E7 was used as a double headed probe. Probes of the small ribosomal RNA locus were produced from pPMT-1 by 5' end labeling the BgllI cleavage products. These correspond to the genome fragments Bg12/13 and Bg4. Lane A: Mobility standards. Lane B: E5 probe. Lane C: S1 nuclease protection experiment using E5 probe. Lane D: E7 probe. Lane E: S1 nuclease protection experiment using E7 probe. Lane F: Bg4 probe. Lane G: S1 nuclease protection experiment using Bg4 probe. Lane H: Bg12/13 probe. Lane I: $1 nuclease protection experiment using Bg 12/13 probe; B S1 nuclease and exonuclease VII resistant fragments from the large ribosomal RNA gene were produced as described in Materials and Methods. Lane A: Mobility standards. Lane B: S1 nuclease treated probe from Lane C. Lane C: Large ribosomal 5' end labeled probe. Lane D: S1 nuclease protection experiment for probe Lane C. Lane E: Exonuclease VII protection experiment for probe in Lane C. Lane F: Blank. Lanes G - K : Titration of large ribosomal RNA probe from probe excess to RNA excess. Lane G: 100,000 cpm probe, 2/~g RNA; Lane H: 20,000 cpm probe, 5 ~tg RNA: Lane I: 20,000 epm probe, 10 t~g RNA: Lane J: 20,000 cpm probe, 15 t~g RNA, Lane K: 10,000 epm probe, 15 ~g RNA
R.M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina
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Fig. 7. A Northern blot hybridization analysis of the large rRNAs. Probes were constructed, RNA was blotted, hybridization was done as described in Materials and Methods. The first lane shows mtRNA stained with ethidium brorriide. Lane A: hybridization to IVS-1 enriched probe. Lane B: hybridization to the IVS-2 specific probe. The bands marked have been observed in repetitions of these experiments. The unmarked bands in Lane B may represent various degradation products; they do not arise consistently in repetitions of these experiments. B summarizes the structure and transcripts obtained form the large rRNA locus. The probes used in 7A are illustrated along with the transcripts hybridized
coding sequence identification for large and small rRNA loci. We showed that these transcripts could be isolated from Podospora mitochondria with a minimum of degradation. At that time, we employed large probes of each ribosomal region to identify the major ribosomal transcripts. Here, we have used the cloned IVS-2 sequence as a specific nick-translated probe of Northern blotted mtRNA, as well as a probe from IVS-1 which overlaps exons 1 and 2. These results are illustrated in Fig. 7A where precursor transcripts can be defined for the large rRNA at 7.9 kb, 6.3 kb, and 5.2 kb. The IVS-1 enriched probe hybridizes to three transcripts larger than the mature rRNA. Three transcripts are hybridized by the IVS-2 specific probe. The two largest transcripts (7.9 kb and 6.3 kb) are hybridized by both IVS-1 enriched and IVS-2 probes. IVS-1 enriched probe hybridized to a large transcript (5.2 kb) not detected by the IVS-2 probe, and it hybridizes to the mature rRNA. IVS-2 hybridizes to an excision product not hybridized by the IVS-1 enriched probe. These observations are summarized in Fig. 7B. We have not detected an excision product for IVS-1, and perhaps it is very unstable in the mitochondria. We also observe that the largest transcript detected occurs in very low concentration and is more readily revealed with longer probes. The occurrence of intermediate transcripts in approximately equivalent concentration suggests that splicing of either IVS may be random.
Discussion In this paper we have presented evidence for the existence and transcription of two long intervening sequences in the mitochondrial large rRNA gene of Podospora anserina. For this analysis, we have drawn upon data from R-loop experiments, Northern blot hybridization analysis, $1 nuclease and exonuclease VII protection experiments. The structures of the large and small rRNA genes have been characterized, and precursor transcripts for the large rRNA have been detected in the mitochondria. As far as we can determine, the situation described here for Podospora anserina is unique among mitochondrial genomes. All of the Ascomycete fungi which have been examined exhibit one IVS close to the 3' terminus of the large rRNA gene (Grant and Lambowitz 1982). This IVS is preserved in Podospora, although it is larger (2.73 kbp) than the other IVS reported. The "extra" IVS in Podospora, located near the 5' end of the gene, is the unique feature of this situation. In yeast, the large rRNA gene in the co+ allelic state contains a single large IVS, while in the co- allelic state this IVS is missing (Dujon 1980). In Neurospora crassa and Aspergillus nidulans a single IVS is found at this locus. Thus, large rRNA genes from the fungi can be found containing none, one, or two long discontinuities in the coding sequences. A high degree of variability in IVS occurrence has also been observed in the cob locus of
R. M. Wright and D. J. Cummings: Mitochondrial DNA from P. anserina yeast (Lazowska et al. 1980) where two IVS are found in the short form of the gene and five in the long form. In Aspergillus nidulans only one is found (Davies et al. 1982). This is comparable to the situation of the oxi3 locus which is uninterrupted in Podospora (Wright et al. 1982b); twice interrupted in Aspergillus nidulans (Kuntzel et al. 1982); and extensively (seven IVS) interrupted in Saccharomyces cerevisiae (Bonitz et al. 1980).
Acknowledgements. This work was supported in part by a grant from the National Institues of Health, AGO1367.
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Green MR, Grimm MF, Goewert RR, Collins RA, Cole MD, Lambowitz AM, Heckmart JE, Yin S, Raj Bhandary AL (1981) J Biol Chem 256:2027-2034 Grunstein M, Hogness DS (1975) Proc Natl Acad Sci USA 72:3961-3965 Hahn U, Lazarus CM, Lunsdorf H, Kuntzel H (1979) Cell 17: 191-200 Heckman JE, Raj Bhandary UL (1979) Cell 17:583-595 Kuntzel H, Kochel HG, Lazarus CM, Lunsdorf H (1982) In: Slonimski P, Borst P, Attardi G (eds) Mitochondfial Genes. Cold Spring Harbor Monographs 12 Lazarus CM, Lunsdorf H, Han U, Stepien PP, Kuntzel H (1980) Mol Gen Genet 177:389-397 Lazowska J, Jacq C, Slonimski PP (1980) Cell 22:333-348 Learned RM, Meyers RM, Tjian R (1981) In: Ray DS (ed) The Initiation of DNA Replication. Academic Press, New York, pp 555-566 Mannella CA, Collins RA, Green MR, Lambowitz AM (1979) Proc Natl Acad Sci USA 76:2635-2639 Merten S, Synenki RM, Locker J, Christianson T, Rabinowitz M (1980) Proc Natl Acad Sci USA 77:1417-1421 Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) J Mol Biol 113:239-251 Rosbash M, Blank D, Fahrner K, Hereford L, Riccardi R, Roberts B, Ruby S, Woolford T (1979) Meth Enzymol 68:454-469 Smith JR, Rubenstein I (1973) J Gen Microbiol 76:297-304 Southern EM (1975) J Mol Biol 98:503-517 Thomas P (1980) Proc Natl Acad Sci USA 77:5201-5205 Villarreal LP, White RI, Berg P (1979) J Virol 29:209-219 Wallace DC (1982) Microbiol Rev 46:208-240 Weaver RF, Weissman C (1979) Nucleic Acids Res 7:11751193 Wright RM, Laping JL, Horrum MA, Cummings DJ (1982a) Mol Gen Genet 185:56-64 Wright RM, Horrum MA, Cummings DJ (1982b) Cell 29:505515
Communicated by C. W. Birky Jr. Received November 5 / December 20, 1982
