CYB rDNA rDNA. 0. 8 6. RESULTS ... probes for yeast CytOx I, yeast Cyt b (CYB), wheat mitochondrial. 18S/5S rRNA ..... MT M H K L P L F V W. F IT A1m, L I. L.
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3340-3344, May 1985 Genetics
Genes for respiratory chain proteins and ribosomal RNAs are present on a 16-kilobase-pair DNA species from Chlamydomonas reinhardtii mitochondria (niitochondrial DNA/cytochrome oxidase subunit I gene/rRNA genes)
Poppo H. BOER*, LINDA BONEN*, ROBERT W. LEEt, AND MICHAEL W. GRAY* Departments of *Biochemistry and tBiology, Dalhousie University, Halifax, NS B3H 4H7, Canada
Communicated by Hewson Swift, December 31, 1984
We have used heterologous hybridization ABSTRACT and DNA sequence analysis to determine whether the 16-kilobase-pair (kbp) DNA from Chlamydomonas reinhardtii mitochondria is the functional equivalent of mtDNA in other eukaryotes. Restriction fragments corresponding to a continuous internal stretch spanning 75% of the 16-kbp DNA have been cloned and mapped, and regions hybridizing with probes specific for the cytochrome oxidase subunit I [CytOx I (acronym COI)] and apocytochrome b (Cyt b) genes of yeast and the mitochondrial 26S and 18S rRNA genes of wheat have been identified. Sequence analysis has verified the presence of CytOx I and the large and small subunit rRNA genes in the C. reinhardtii 16-kbp DNA. In the region of the 16-kbp DNA corresponding to exon 4 in the yeast CytOx I gene, the derived amino acid sequence is 61% and 63% identical with the CytOx I amino acid sequences of yeast and human mitochondria, respectively. Notably, tryptophan is specified by TGG rather than by TGA in this section of the C. reinhardt& CytOx I gene. A probe from the CytOx I region of the 16-kbp DNA hybridizes only with this 16-kbp DNA in Southern blots of total cellular DNA from C. reinhardtii but with a larger DNA species in the total cellular DNA of C. moewusii and C. eugametos-two species that lack a 16-kbp DNA. These observations provide evidence that C. reinhardtii 16-kbp DNA comprises at least part of the mitochondrial genome of this organism and that a homologous DNA exists in other species of Chlamydomonas.
piratory chain. Moreover, although the 16-kbp DNA appears to be highly conserved among a number of C. reinhardtii strains (10), DNA molecules of this size are not present in two other Chlamydomonas species, C. eugametos and C. moewusii (11, 12). This has raised the possibility that the 16kbp DNA is in fact a plasmid-like DNA specific to C. reinhardtii mitochondria, rather than the mitochondrial genome itself. This is an important consideration in view of the fact that plasmid-like DNA species, including linear ones, have been identified in plant mitochondria (4). Indeed, although C. moewusii lacks a 16-kbp DNA, it does possess two smaller plasmid-like DNAs that are not present in either C. reinhardtii or C. eugametos (11, 12). In this report, we describe the cloning of a large part of the 16-kbp DNA and the identification of regions hybridizing with probes specific for the cytochrome oxidase subunit I [CytOx I (acronym COI)] and apocytochrome b (Cyt b) genes of yeast mtDNA and the 26S and 18S rRNA genes of wheat mtDNA. The identities of the CytOx I and rRNA genes have been verified by sequence analysis. Our data constitute direct evidence that the 16-kbp DNA is at least part of the mitochondrial genome of C. reinhardtii. In addition, we demonstrate that a DNA species related to the 16kbp DNA is present in both C. eugametos and C. moewusii.
MATERIALS AND METHODS Strains, Culture, and DNA Isolation. The 16-kbp DNA was recovered from a DNase I (Worthington)-treated crude mitochondrial pellet of the mating type " + " strain of the C. reinhardtii cell-wall-less mutant CW-15-2 (no. 277 of the Duke Univ. Culture Collection) by procedure B of Ryan et al. (9). Total cellular DNA was prepared from the mating type " + " wild-type strains of C. reinhardtii (137c), C. eugametos (UTEX 9), and C. moewusii (UTEX 97) as described (12). Cloning, Mapping, and Sequencing of C. reinhardtii 16-kbp DNA. Restriction fragments of the 16-kbp DNA were cloned in the pUC9 vector (13). Plasmid minipreparations (14) were purified by polyethylene glycol precipitation (15), and digests were subcloned in M13 vectors for sequencing by the dideoxy chain-termination method of Sanger et al. (16). Southern hybridizations with DNA probes (labeled with 32p either by nick-translation or M13 primer extension) were conducted in 5x NaCl/Cit (lx NaCl/Cit is 0.15 M NaCl/0.015 M sodium citrate, pH 7) containing 0.1% NaDodSO4, 2x Denhardt's solution (17), and 20 pg of sheared denatured salmon sperm DNA per ml at either 65°C overnight (homologous) or 500C for at least 36 hr (heterologous).
There is marked variation in the size, organization, and expression of mitochondrial genomes from different eukaryotes (1-3), and this is particularly exemplified by the mtDNA of higher plants, which is some 15-150 times as large as animal mtDNA and displays a correspondingly more complex physical structure and genomic arrangement (4-7). As yet, little is known about the mtDNA in unicellular green algae, the group of protists that is thought to share a specific evolutionary ancestry with higher plants (8). In Chlamydomonas reinhardtii, a linear DNA species of approximately 16 kbp in length (about the size of the circular animal mitochondrial genome) has been isolated from DNase-treated mitochondria (9). This molecule has unique ends and a homogeneous sequence (10) and is the only discrete DNA species that can be isolated from a C. reinhardtii mitochondrial fraction treated with DNase (ref. 9 and our unpublished results). Although it has been assumed that the 16-kbp DNA represents the mitochondrial genome of C. reinhardtii, until now it had not been established formally that this DNA has the functions ascribed to mtDNA in other eukaryotes-i.e., that it codes for ribosomal and transfer RNA components of a mitochondrial translation system as well as for a limited number of polypeptide components of the mitochondrial res-
Nitrocellulose blots were then washed for 30 min at the temperature and salt conditions of hybridization and then extenAbbreviations: bp, base pair; mtDNA, mitochondrial DNA; Cyt b, cytochrome b; CytOx I (acronym COI), cytochrome oxidase subunit I.
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.
3340
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Proc. NatL. Acad. Sci. USA 82 (1985)
sively at 37TC. In homologous hybridizations, blots were washed with 0.3 x NaCl/Cit at 65TC. Probes specific for the Saccharomyces cerevisiae CytOx I gene (a subfragment of clone D, which includes most of exon 4; ref. 18) and the Kluyveromyces lactis Cyt b gene (an internal 750-bp fragment) were kindly provided by L. A. Grivell (Univ. of Amsterdam), whereas recombinant plasmids carrying wheat mitochondrial 18S + 5S or 26S rRNA genes (contained within Sal I fragments S19 and S14, respectively; ref. 19) were isolated from a pUC9 clone bank. Plasmid inserts were isolated from low-melting-point agarose.
RESULTS Characterization of the 16-kbp DNA. Grant and Chiang (10) have demonstrated that C. reinhardtii 16-kbp DNA is linear, with unique ends that can be 5'-labeled by phosphate exchange with polynucleotide kinase and [r- 2P]ATP. We found that the 3' ends also can be labeled specifically by using the Klenow fragment of Escherichia coli DNA polymerase I and [a-32P]dATP in the presence of the remaining three dNTPs. Label is incorporated into a single discrete DNA species with the size of 16-kbp, which upon digestion with various restriction endonucleases yields the predicted end fragments (10). For example, Sal I cleaves the 16-kbp DNA into two fragments of 11.0 and 4.8 kbp (Fig. 1). Digestion with EcoRI produces two end fragments of 6.9 and 0.6 kbp (the latter not shown in Fig. 1), although the two internal fragments of 7.1 and 1.25 kbp are also labeled to a minor extent as a result of incorporation at single-strand breaks in isolated 16-kbp DNA. These data establish that the DNA species characterized here is the same as that previously isolated from C. reinhardtii mitochondria (9, 10). The simple Sal I restriction pattern of 16-kbp DNA presents a marked contrast to the complex pattern (>50 fragments) produced by Sal I digestion of wheat mtDNA (Fig. 2A). Hybridization of Heterologous Probes to the 16-kbp DNA. Probes specific for mitochondrial respiratory chain protein (D
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FIG. 2. Hybridization of the 16-kbp DNA with mitochondrial gene probes. (A) UV fluorescence patterns of Sal I-digested wheat mtDNA (lane a) and C. reinhardtii 16-kbp DNA (lane b). Arrowheads indicate the wheat Sal I fragments S19 (6.3 kbp, containing 18S + 5S rRNA genes) and S14 (8.6 kbp, containing a 26S rRNA gene) (19). (B) Southern blots of 16-kbp DNA hybridized with probes for yeast CytOx I, yeast Cyt b (CYB), wheat mitochondrial 18S/5S rRNA genes, and wheat mitochondrial 26S rRNA gene.
and rRNA genes were used in hybridization experiments to search for homologous regions in Sal I-digested 16-kbp DNA. Probes derived from the CytOx I region of yeast mtDNA and the 18S + 5S and 26S rRNA gene regions of wheat mtDNA hybridized to the larger Sal I fragment of 16kbp DNA, whereas a yeast Cyt b gene probe hybridized to the smaller Sal I fragment (Fig. 2B). These results suggested the presence of the analogous genes on 16-kbp DNA, prompting us to undertake more definitive studies with cloned 16-kbp DNA. Localization of Genes on Cloned 16-kbp DNA. Recombinant clones with inserts covering a continuous internal stretch of about 75% of the 16-kbp DNA were used together with end-labeling data to construct the restriction map shown in Fig. 3 Upper. This map confirms and extends that of Grant and Chiang (10). Only internal restriction fragments but not the ends of the 16-kbp DNA were obtained from cloning attempts that included pretreatment of 16-kbp DNA with ligase and/or Klenow polymerase to fill in recessed 3' ends, whose existence is suggested by labeling experiments (Fig. 1). These observations suggest that the ends of C. reinhardtii 16-kbp DNA possess structural features that preclude their cloning by the usual approaches. In heterologous hybridization experiments with cloned 16kbp DNA, we used isolated gene-specific fragments from wheat and yeast mitochondrial clones to minimize vector hybridization. Screening of recombinant clones indicated that pEl contained the regions hybridizing with rRNA-specific probes, whereas pHi contained the CytOx I hybridizing region. As noted above, in preliminary experiments. Cyt b hybridization had been localized to the smaller Sal I fragment (Fig. 2B). We subsequently determined that pH1 did not hybridize with the Cyt b probe, so that the putative Cyt b gene must lie within the leftward uncloned terminal region of 16kbp DNA. In a HindIII/Sal I/Sst I digest of pH1, hybridization with the yeast CytOx I-specific insert was centered around the Sst I site. Strongest hybridization was to the 0.8-kbp Sst ISal I subfragment, while the 1.15-kbp HindIII-Sst I subfragment hybridized more weakly (Fig. 3 Lower, lanes a). Hybridization of the probe to pUC9 reflects vector contamination of the isolated fragment. The apparent hybridization of the yeast probe to the 2.0-kbp Sal 1-HindIII fragment is like-
Genetics: Boer et aL
3342
Proc. Natl. Acad. Sci. USA 82 (1985)
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FIG. 3. (Upper) Restriction map of the 16-kbp DNA and regions of homology with yeast and wheat mitochondrial probes (dashed lines). The cloned EcoRI, HindIII, and EcoRI-Sal I fragments are indicated above the map. The terminal regions (x x x x x) have not yet been cloned. The DNA sequence in the CytOx I gene region, indicated by a stippled block, is shown in Fig. 5. The solid triangle indicates a cluster of three HindIII sites revealed by sequence analysis. Restriction enzyme sites: B, BamHI; C, Cla I; E, EcoRI; H, HindIII; HI, Hpa I; K, Kpn I; S, SalI; SI, Sst I; and X, Xba I; C and SI are only within cloned regions. (Lower) Hybridization of mitochondrial gene probes to cloned 16-kbp DNA fragments. (a-c) Left lanes are UV fluorescence patterns, and right lanes are Southern blots. Clone pHi was digested with HindIII, Sal I, and Sst I (a), and clone pEl was digested with EcoRI, BamHI, Cla I, Hpa I, and Kpn I (b and c). Hybridization with yeast CytOx I (a), wheat mitochondrial 18S (SSU)/55 (b), and wheat mitochondrial 26S (LSU) rRNA gene probes is indicated by solid arrowheads, and pUC9 cloning vector, by open arrowheads. Sizes are in kbp. CYB, Cyt b.
ly due to a comigrating 1.95-kbp HindIII-Sal I fragment resulting from incomplete Sst I digestion, because the former fragment is in the region of the 16-kbp DNA, which does not show hybridization in Sal I digests (see Fig. 2). Digestion of pEl, as shown in Fig. 3 Lower, lanes b and c, generated six insert fragments. Only one fragment hybridized strongly with the wheat mitochondrial 18S + 5S probe, namely the 2.2-kbp Cla I-EcoRI fragment at the rightward end of fragment El (covering fragment H5). The wheat mitochondrial 26S rRNA gene hybridized with three different fragments, corresponding to EcoRI-Kpn 1 (1.3 kbp), Kpn IBamHI (950 bp), and BamHI-Kpn I (1.75 kbp). These three fragments are contiguous in the restriction map of 16-kbp DNA (Fig. 3 Upper); they span the 4-kbp region encompassing fragments H4, H8, and H6. Characterization of the CytOx I Gene in 16-kbp DNA. In view of the relatively strong hybridization of the yeast CytOx I probe to the Sst I-Sal I subfragment of Hl (Fig. 3 Lower, lanes a), the sequence surrounding the Sst I site was
determined by using the strategy shown in Fig. 4. This analysis revealed strong identity at the level of both nucleotide sequence and derived amino acid sequence between the yeast CytOx I probe and this region of 16-kbp DNA. The sequence of the 468 nucleotides of 16-kbp DNA, shown in Fig. 5, is equivalent to exon 4 of the yeast CytOx I gene. When the CytOx I region that is represented in the yeast probe is aligned with the C. reinhardtii sequence, allowing for a six-nucleotide deletion, nucleotide identity between the two is 56%. Another six-nucleotide deletion in the algal sequence is required to align the entire exon 4 region that encompasses residues 80-239 in the human CytOx I protein. The corresponding C. reinhardtii DNA sequence and derived amino acid sequence is shown aligned with those of yeast and human. Overall identity is 61% between C. reinhardtii and yeast (20) and 63% between C. reinhardtii and human (21), compared to 68% between the yeast and human sequences. At 84 out of 160 of the positions, the same amino acid is present in all three sequences. Although a meaningful analysis of codon usage will have to await determination of the complete sequence, it is noteworthy that the C. reinhardtii gene follows the universal genetic code by using TGG to specify tryptophan at five positions (residues 81, 103, 126, 186, and 236) where tryptophan is encoded by TGA in the human and yeast CytOx I genes. Sequence Analysis of the rRNA Regions in 16-kbp DNA. In view of the strong hybridization of wheat mitochondrial rDNA probes to the El region of 16-kbp DNA and the pronounced structural similarities between the wheat mitochondrial and E. coli rRNA genes (ref. 22 and unpublished results), we would anticipate finding stretches of high homology to these latter genes within fragment El. Although sequence analysis is at a preliminary stage, we have identified regions that correspond in primary sequence and potential secondary structure to both small- and large-subunit rRNA genes. One such stretch of 202 nucleotides within fragment H5 has a potential secondary structure that precisely matches that proposed for positions 766-993 of E. coli 16S rRNA (cf. ref. 23), encompassing universal regions U4 and U5 in small-subunit rRNAs (22). Excluding the variable hairpin loop (E. coli positions 818-879), we have compared the 16-kbp sequence with the homologous regions in other small-subunit rRNA genes. The sequence identity values calculated in this way are 69o with E. coli 16S rRNA (23), 70% with C. reinhardtii chloroplast 16S rRNA (24), 72% with wheat mitochondrial 18S rRNA (22), 53% with human mitochondrial 12S rRNA (21), and 63% with yeast mitochondrial 15S rRNA (25). An Analog of 16-kbp DNA Is Present in Two Other Species of Chlamydomonas. A clone derived from the CytOx I gene of C. reinhardtii 16-kbp DNA (M13 clone lie, Fig. 4) was used to probe the total DNA of C. reinhardtii, C. eugametos, and C. moewusii under hybridization conditions of high stringency (Fig. 6). In C. reinhardtii, the probe detected a single discrete DNA species, corresponding to linear 16-kbp DNA, ES2
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Genetics: Boer et aL
Proc. Natl. Acad. Sci. USA 82 (1985)
3343
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before restriction endonuclease digestion, and the expected smaller fragments after digestion with either Sal I or EcoRI. In contrast, with undigested total DNA from C. moewusfi or C. eugametos, the CytOx I probe hybridized to DNA of heterogeneous mobility and apparent molecular weight greater than that of 16-kbp DNA. However, after digestion with either Sal I or EcoRI, the probe hybridized to a discrete band, having an apparent size of 18.5 (C. moewusit) or 20.2 (C. eugametos) kbp. This suggests that a DNA species analogous to C. reinhardtii 16-kbp DNA but with a minimum size
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FIG. 6. Autoradiograph showing that sequences homologous to the C. reinhardtii CytOx I gene are present in other Chlamydomonas species. Southern blots of total cellular DNA (2 Pg) from C. reinhardtii (lanes r), C. moewusii (lanes m), and C. eugametos (lanes e), digested as indicated, were hybridized with the C. reinhardtii CytOx I gene region contained in clone Ile (Fig. 4). The EcoRJ digest of C. reinhardtii DNA was partial. Homologous hybridization conditions were used followed by stringent washes. The lane at right contained 2 ng of HindIl-digested clone pHi, and the 2.7- and 3.8kbp bands represent vector and insert, respectively. Open arrowheads indicate the position of the C. moewusii plasmid-like DNAs discussed in the text. Sizes are in kbp.
of 18.5 and 20.2 kbp is present in C. moewusii and C. eugametos, respectively. We emphasize that the same-sized hybridizing fragments were detected in C. moewusii DNA digested with either Sal I or EcoRI; in C. eugametos digests, the probe again hybridized to Sal I and EcoRI fragments of the same size. The simplest interpretation of these results is that the 16-kbp analog in these two species is a circular molecule containing single Sal I and EcoRI sites, so that cleavage at either of these sites produces a homogeneous linear fragment representing the entire genome. The possibilities remain, however, that the hybridizing Sal I and EcoRJ fragments do not represent the entire 16-kbp DNA analog of these species and that, by chance, both enzymes produced fragments of the same size. Note that the CytOx I probe does not hybridize to either of the two small plasmid-like DNAs (indicated by open arrowheads in Fig. 6) present in the C. moewusii total cellular DNA.
DISCUSSION Although there is pronounced variability in the size, organization, and expression of mitochondrial genomes among and within the major eukaryotic groups, mtDNA does appear to have a universal coding function, in that the genes for certain components of the respiratory chain and the mitochondrial translation system are always encoded by mtDNA. By this criterion, the results presented here establish that the 16-kbp DNA of Chlamydomonas reinhardtii is the functional equivalent of mtDNA in other organisms because (i) defined regions in the 16-kbp DNA hybridize selectively with probes specific for the CytOx I and Cyt b genes of yeast and the mitochondrial 26S and 18S rRNA genes of wheat, and (it) in the case of the CytOx I and rRNA-hybridizing regions of the 16-kbp DNA, sequence analysis has verified that these regions are homologous to portions of the CytOx I, small-subunit, and large-subunit rRNA genes of other mitochondrial genomes. Hybridization experiments, performed under stringent conditions, have not detected CytOx I sequences other than on the 16-kbp DNA. RNA blot-hybridization experiments, again under stringent conditions, indicate that the sequenced CytOx I region is actively transcribed (unpublished data); we conclude, therefore, that this is the function-
3344
Genetics: Boer et aL
al CytOx I gene in C. reinhardtii. Although sequence analysis of the regions hybridizing with wheat mitochondrial rRNA is at a preliminary stage, we do have enough information to suggest that these regions in the 16-kbp DNA are indeed the structural equivalents of the rRNA genes in other organisms and that they are not derived from either the chloroplast or the nuclear DNA of C. reinhardtii. It seems likely that the 16-kbp DNA constitutes the entire mitochondrial genome of C. reinhardtii because 16-kbp DNA, which is present in multiple copies per cell (9), is the only discrete DNA species that can be recovered from crude mitochondrial pellets after treatment with DNase (ref. 9 and our unpublished data). Despite the close correspondence in size between the 16-kbp DNA of C. reinhardtii (15.8 kbp) and the mtDNA of animals (16.5 kbp), the arrangement and expression of genes may well be different in the two. In animals, the small- and large-subunit rRNA genes are separated by only a tRNA gene, whereas they appear to be at least 0.9 kbp apart in the 16-kbp DNA. In view of the marked difference in size between the mitochondrial genomes of C. reinhardtii (15.8 kbp) and higher plants (220-1200 kbp), it should be of considerable interest to determine how much of the former genome is contained in the latter. In the context of an endosymbiotic origin of mitochondria (26), analysis of conserved sequences in small-subunit rRNA has suggested a separate branching within the eubacterial lineage of plant mitochondria and fungal/animal mitochondria (27). It is, therefore, intriguing that the rRNA data we have to date suggest a closer evolutionary link between a green algal and a higher plant mtDNA than between a green algal and an animal (or fungal) mtDNA. Although this link can only be considered tentative at the moment, it does draw additional support from our finding that the 16-kbp DNA, in concert with plant mtDNA, apparently uses TGG to encode tryptophan at positions where TGA specifies tryptophan in animal and fungal mitochondrial genes. It also should be noted that the C. reinhardtii 16-kbp DNA has the same buoyant density (1.706 g.cm-3; refs. 9 and 12) as the mtDNA of almost all higher plants (4). These indications of a specific phylogenetic relationship between the mitochondrial genomes of higher plants and green algae are of particular interest because the same conclusion [based on SS rRNA sequence comparisons (28, 29)] has emerged in the case of the nuclear genomes of these two groups of organisms. If the 16-kbp DNA is indeed the functional equivalent of mtDNA in other organisms, then homologs should exist in other Chlamydomonas species. Our identification of sequence homologs of 16-kbp DNA in C. moewusii and C. eugametos that are larger than 16-kbp provides an explanation for the absence of a discrete 16-kbp DNA species in these two cases and removes an important objection to the view that the 16-kbp DNA is mtDNA. Although the physical form of mtDNA in C. eugametos and C. moewusii has not been directly determined, the Southern hybridization patterns of native and restriction enzyme-digested DNA are most consistent with a circular conformation. In view of the close evolutionary relationship between C. eugametos and C. moewusfi and their greater evolutionary distance from C. reinhardtii (30), it should be of interest to compare in detail the mitochondrial genomes in the three species. Moreover, the ability to recover viable progeny from crosses between C. eugametos and C. moewusfi opens up the possibility of searching for restriction site polymorphisms that could be used to evaluate the mode of inheritance of Chlamydomonas mtDNA.
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