1 Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada z D~partement de ... using a Sau3AI partial digest of a C. moewusii DNA.
Mol Gen Genet (•99•) 23•:53-58
MG(3
© Springer-Vertag 1991
Cloning and characterization of the Chlamydomonas moewusii mitochondrial genome Robert W. Lee 1, Carole Dumas 2, Claude Lemieux 2, and Monique Turmei 2 1 Department of Biology,Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada z D~partement de biochimie, Facult6 des scienceset de g6nie, Universit~ Laval, Qu6bec, Quebec G1K 7P4, Canada ReceivedJune 18, 1991 / August 7, 1991
Summary. We report that the mitochondrial genome of Chlamydomonas moewusii has a 22 kb circular map and thus contrasts with the mitochondrial genome of Chlamydomonas reinhardtii, which is linear and about 6 kb shorter. Overlapping restriction fragments spanning over 90% of the C. moewusii mitochondrial DNA (mtDNA) were identified in a clone bank constructed using a Sau3AI partial digest of a C. moewusii DNA fraction enriched for mtDNA by preparative CsC1 density gradient centrifugation. Overlapping Sau3AI clones were identified by a chromosome walk initiated with a clone of C. moewusii mtDNA. The mtDNA map was completed by Southern blot analysis of the C. moewusii mtDNA fraction using isolated mtDNA clones. Regions that hybridized to C. reinhardtii or wheat mitochondrial gene probes for subunit I of cytochrome oxidase (coxl), apocytochrome b (cob), three subunits of N A D H dehydrogenase (nadl, nad2 and had5) and the small and the large ribosomal RNAs (rrnS and rrnL, respectively) were localized on the C. moewusii mtDNA map by Southern blot analysis. The results show that the order of genes in the mitochondrial genome of C. moewusii is completely rearranged relative to that of C. reinhardtii. Key words: Chlamydomonas moewusii - Mitochondrial DNA - Map - Gene localization - Evolution
Introduction The most extensively studied green algal mitochondrial genome is that of Chlamydomonas reinhardtii. Coding regions in this completely sequenced 15.8 kb linear DNA have been identified for five different subunits of respiratory NADH dehydrogenase (nadl, nad2, had4, had5 and nad6), apocytochrome b (cob), subunit I of cytochrome oxidase (coxl), a reverse transcriptase-like protein (rtl), only three tRNAs and the small subunit (rrnS) and large
Offprint requests to: Robert W. Lee
subunit (rrnL) mitochondrial ribosomal RNAs (see review by Gray and Boer 1988 and recent summaries by Michaelis et al. 1990; Boer and Gray 1991). An unusual feature of the C. reinhardtii mitochondrial genome is the division of its rrnS and rrnL genes into a number of separate coding regions that are interspersed with one another and with genes specifying proteins and tRNAs (Boer and Gray /988a). Another distinctive feature of this DNA is the absence of genes specifying subunits II and III of cytochrome oxidase (cox2 and cox3) or any subunits of the ATPase complex (Michaelis et al. 1990). The mtDNA of Chlamydomonas smithii, an interfertile relative of C. reinhardtii, is colinear with that of C. reinhardtii, except for a mobile intron in the cob gene (Boynton et al. /987; Matagne et al. 1988; Colleaux et al./990). Information about mitochondrial genomes in other members of the Chlorophyta is scant. A circular 76 kb mtDNA has been identified in an exsymbiotic Chlorellalike alga (Waddle et al. 1990) and based on heterologous hybridization experiments with mitochondrial gene probes, eight coding regions have been assigned to this genome, including coxl, cox2, cob, rrnS, rrnL and genes for subunits 6, 9 and e of the ATPase complex (atp6, atp9 and atpc~, respectively). Wolff and Kfick (1990) have identified and sequenced the mitochondrial rrnS gene of Prototheca wickerhamii, a colourless heterotrophic alga sharing several characteristics with certain members of the genus ChlorelIa. The small subunit rRNA specified by this gene was shown to have more features in common with the corresponding mitochondrial RNA of land plants than with that of C. reinhardtii. Finally, a small linear DNA has been identified in Pandorina morum, a green alga in the same order (Volvocales) as Chlamydomonas (Moore and Coleman 1989). This DNA, which varies in size from ca. 20 to 38 kb in different syngens (sexually isolated populations) of P. morum, is likely to be mtDNA by virtue of its cross-hybridization with C. reinhardtii mtDNA and with probes containing the maize mitochondrial rrnS gene. A cross-hybridizing component has been identified in preparations of total cellular DNA from Chlamydo-
54 Table 1. Heterologous mitochondrial gene probes
Gene
Product
Source a
Fragment b
Location c
rrnS rrnL atp6 cob coxl cox2 nadl nad2 nad5 nad6 rtl
18S rRNA 26S rRNA Subunit 6 of ATP synthetase Apocytochrome b Subunit I ofcytochrome oxidase Subunit II of cytochrome oxidase Subunit 1 of NADH dehydrogenase Subunit 2 ofNADH dehydrogenase Subunit 5 ofNADH dehydrogenase Subunit 6 of NADH dehydrogenase Reverse transcriptase-like protein
Wheat Wheat Wheat Wheat Cr Wheat Cr Cr Cr Cr Cr
1.7 kb 3.1 kb 1.9 kb 0.7 kb 1.0 kb 0.9 kb 0.8 kb 0.9 kb 1.3 kb 0.5 kb 0.7 kb
- 27/+1755 + 40/+ 3043 --519/+ +426 + 462/+ 1183 +456/+ 1440 - 180/+ 760 - 25/+ 826 - 6/+914 +239/+1533 + 12/+480 +274/+992
PstI-SmaI SmaI-SalI MluI-EcoRI HindIII-BamHI SstI-XbaI MluI-BamHI BamHI-HindIII EcoRI-HindIII TaqI-TaqI EcoRI-HpaII NruI-PvuII
Referenced 1 2 3 4 5, 6, 7 8 9 6, 10 5, 7, 11 12, 13 9
Cr = C. reinhardtii b Sizes are approximations bases on electrophoretic mobility of the fragments c The extents of the various probes are given relative to the first base of the coding region; + + indicates that the probe ends beyond the termination codon
a 1, Falconet et al. 1984; 2, Falconet et al. 1985; 3, Bonen and Bird 1988; 4, Boer et al. 1985b; 5, Vahrenholz et al. 1985; 6, Boer and Gray 1986a; 7, Kfick and Neuhaus 1986; 8, Bonen et al. 1984; 9, Boer and Gray 1988b; 10, Pratje et al. 1984; ll, Boer and Gray 1986b; 12, Ma et al. 1988; 13, Boer and Gray 1989
monas moewusii by Southern blot analysis using a C. reinhardtii coxl probe (Boer et al. 1985 a). Because these
form E. coli strain DH5c~ (Life Technologies, Inc.) using standard techniques (Maniatis et al. 1982). Over 1000 ampicillin-resistant recombinant clones were individually patched onto LB ampicillin plates and replica plated onto H y b o n d N (Amersham) nylon filters for colony hybridization (Maniatis et al. 1982). Overlapping clones were identified in this bank by a chromosome walk initiated with a cloned 1.8 kb EcoRI fragment of C. moewusii m t D N A (Turmel et al. 1987). Recombinant plasmids were isolated by the alkaline extraction procedure of Birnboim and Doly (1979) and characterized by restriction analysis and by hybridization with various restriction digests of the C. moewusii fl-DNA fraction. D N A inserts used as hybridization probes were isolated from Sea Plaque (FMC Corporation, Marine Colloids Division) low-melting agarose gels (Maniatis et al. 1982).
two algae represent evolutionarily distant lineages of this polyphyletic genus (Buchheim et al. 1990; Turmel et al. 1991), it was of particular interest to compare the structure and gene organization of their mtDNAs. We have therefore undertaken the cloning and molecular analysis of the C. moewusii m t D N A and report here that this D N A has a 22 kb circular map and a gene organization very different from that described for C. reinhardtii.
Materials and methods D N A isolation and restriction analysis. Total cellular
D N A was prepared from the wild type mating-type " p l u s " strain of C. moewusii ( U T E X 97). The major satellite or fi-DNA fraction (enriched in chloroplast and mitochondrial DNA), was collected after equilibrium CsC1 density gradient centrifugation of total cellular DNA. The conditions employed for these procedures as well as for restriction endonuclease digestion and agarose gel electrophoresis have been described previously (Lemieux et al. 1980). Bisbenzimide - CsCI gradient fractionation of total cellular DNAs. Conditions of centrifugation were as de-
scribed by Lemieux et al. (1980) except for the addition of bisbenzimide (Hoechst dye 33258; Polysciences) according to Aldrich and Cattolico (1981). The gradient was fractionated by upward displacement using an Isco fractionator (Model 184) and Fluorinert (Isco) as the displacement fluid. Fractions (170 ~tl) were extracted three times with equal volumes of isopropanol saturated with CsC1 and precipitated with two volumes of ethanol. Isolation and characterization of m t D N A clones. Fragments (4-10 kb) from a Sau3AI partial digest of the C. moewusii fl-DNA fraction were ligated into the dephosphorylated BamHI site of pUC18 and used to trans-
Hybridization. For hybridization with homologous probes, digests o f / % D N A or cloned Sau3AI fragments were transferred from agarose gels to H y b o n d N (Amersham) nylon filters according to Southern (1975) and the hybridization conditions of Lemieux and Lemieux (1985) were used. Hybridizations with heterologous mitochondrial gene probes (Table 1) employed nitrocellulose filters (Schleicher and Schuell BA-85) and the less stringent hybridization conditions of Woessner et al. (1984). These probes were prepared from clones and purified by preparative gel electrophoresis as described above. All probes were radiolabeled to high specific activity by the random hexanucleotide priming method of Feinberg and Vogelstein (1983, 1984) using ~ (32p) dCTP. Results Identification of the m t D N A fraction in bisbenzimide Cs Cl gradients
The absence of cell wall-deficient mutant strains of C. moewusii precluded the preparation of C. moewusii
55 M
1
2
3
4
5
6
7
8
9
101112131415161718
A
M
kb --20
--1.8
B --20
.
.
.
.
.
pattern and sedimented into fractions 5-7. These D N A components have densities of 1.718 and 1.700 g/cm 3, respectively, as determined by CsC1 density gradient centrifugation in the analytical ultracentrifuge (Lemieux et al. 1980). Additional D N A density components with less complex EcoRI restriction patterns and of unknown cellular origin were associated with fractions 7-8 and 9-10. The cloned 1.8 kb EcoRI fragment of C. moewusii m t D N A hybridized maximally to fraction 5 and at a position corresponding to 1.8 kb on the Southern blot of the EcoRI digest. This suggests that the m t D N A of C. moewusii has a density similar to but slightly less than that of the cpDNA. Consistent with the conclusion that fraction 5 was enriched for m t D N A is the detection of a non-hybridizing high molecular weight fragment in the EcoRI digest of that fraction (estimated to be about 20 kb from gel mobility); the C. reinhardtii coxl probe hybridizes exclusively to a similarly sized EcoRI fragment of C. moewusii total cellular D N A (Boer et al. 1985a).
1,8
Recovery o f clones o f C. moewusii m t D N A
Fig. 1A, B. Identification of mtDNA in a bisbenzimide-CsC1 gradient of C. moewusii total cellular DNA. A Agarose gel (0.75%) electrophoresis of gradient fractions (numbered 1-18) digested with EcoRI. The least dense fractions were collected first. The molecular weight markers in the extreme left and right lanes (labeled M) are HindIII fragments of bacteriophage lambda DNA: 27.5 (23.1 + 4.4), 9.4, 6.7, 2.3, and 2.0 kb. The cohesive ends of the 23.1 and 4.4 kb fragments were not separated by heating. B Southern blot hybridization of the EcoRI-restricted gradient fractions with a 1.8 kb EcoRI clone of C. moewusii mtDNA
m t D N A from DNase-treated mitochondrial pellets as was done with C. reinhardtii (Ryan et al. 1978). However, the inadvertant recovery of a clone of C. moewusii m t D N A made it possible to detect, by homologous hybridization, the m t D N A component of total cellular C. moewusii D N A after fractionation by CsC1 density gradient centrifugation. The clone, a 1.8 kb EcoRI fragment (Turmel et al. 1987), was identified as m t D N A by virtue of its hybridization to C. moewusii restriction fragments that also hybridized to a C. reinhardtii cox1 probe (clone IIe; Boer et al. 1985a); i.e. both the C. moewusii and the C. reinhardtii probes hybridized exclusively to 19 kb and 14 kb fragments in SalI and BstEII digests, respectively, of C. moewusii total cellular D N A (data not shown). The EcoRI digestion patterns and Southern blot hybridization analysis of C. moewusii D N A fractions collected from a bisbenzimide CsC1 gradient are shown in Fig. 1. The bisbenzimide binds preferentially to ATrich D N A sequences and enhances buoyant density differences of cellular D N A (Mfiller and Gautier 1975). The main band or ~-DNA component (predominantly nuclear DNA) was concentrated in fractions 11-13. The major satellite D N A o r / % D N A component (predominantly chloroplast DNA) gave a less complex restriction
The previous experiments demonstrate that the sedimentation patterns of the C. moewusii chloroplast and mitochondrial D N A components in bisbenzimide - CsC1 density gradients are too similar to allow the recovery from such gradients of a m t D N A fraction sufficiently enriched to allow its direct cloning. It was decided, therefore, to use a partial Sau3AI restriction digest to construct a clone bank from t h e / % D N A density fraction, as recovered from CsC1 gradients without bisbenzimide, and to identify m t D N A clones in this bank by a chromosome walk initiated with the 1.8 kb clone of C. moewusii mtDNA. It was anticipated that the concentration of m t D N A molecules in the fl-DNA fraction of C. moewusii D N A (hereafter referred to as the mtDNA-enriched fraction) would be considerably higher than that of unfractionated total cellular DNA. Restriction maps of the cloned Sau3AI fragments identified by the chromosome walk experiment support the idea that they are contiguous (Fig. 2), as do studies of hybridization between the clones using Southern blot analysis (data not shown). Collectively, these putative m t D N A clones span a D N A segment of 20.4 kb. The HETTE
I
AHA
III II III I I
I I I
A
M S V A AH A V
I
II I1 II
I
t
I I
92.1 nl
I i
20.75
I I I
I
M S VVABP1HH T A
III1 I11 II III I
Ill
III
i
G I
I
I
10.96
I I
HH
I
II
13.38 II U
II
C
I
__
= 1 kb
Fig. 2. Restriction maps of overlapping Sau3AI partial digest clones spanning 20.4 kb of C. moewusii mtDNA. The cloned fragments were inserted into the BamHI site of the polytinker cloning region of the pUC18 vector. The XbaI site of the vector polylinker region is adjacent to the left end of all fragments with the exception of clone 92.1, in which it is adjacent to the right end. The restriction sites are as follows: A, AeeI; B, BamHI; E, EeoRI; H, HindIII; M, SmaI; P1, PstI; S, SalI; T, BstEII; V, AvaI
56
rrnS
1.8 kb EcoRI clone used to initiate the c h r o m o s o m e walk maps completely within clone 20.75 and partially within clone 92.1.
~ - - " -
/ The mitochondrial genome o f C. moewusii is 22 kb in size and circular
~ad2 b
/
In order to confirm and extend the m a p of the cloned region described above, each clone (or a portion of a clone in the case of clone 10.96) was hybridized to Southern blots of HindIII, AccI, AvaI and BstEII digests of the C. moewusii mtDNA-enriched fraction. The fragments identified by each probe are listed in Table 2. For each digest, the hybridizing fragments were found to span a total length of 22 kb (Table 3) and the same restriction fragments were recognized by the probes from the left and right ends of the linear cloned region, i.e. clones 20.75 and 13.38 (see Fig. 2). These results support
\ \ \ \~ S / / ' / g L 8 / / /rrnS ~'~'~~Y/~5 U /rrnL rt I~
cox1 nad5
HindIII
AceI
AvaI
BstEII
Fig. 3. Restriction site and gene maps of the C. moewusiimitochondrial genome. The size of this genome is 22 kb. Starting from the outside, the four circles represent the HindIII, AecI, AvaI and BstEII restriction site maps. Restriction sites on the fifth and innermost circle are as follows: B, BamHI; E, EcoRI; M, SmaI; Pl, PstI and S, SalI. Gene localizations represent the maximum extent of hybridization of the gene-specific probes employed (see Table 1). The broken line represents a region showing weak hybridization with the rrnS probe
14.2, 6.8, 1.0 14.2
the conclusion that the linear clondd region is part of a circular m a p 22 kb in size as presented in Fig. 3.
10.96 °
5.7
G
5.7, 5.3
C
5.3, 2.5, 0.6 5.3, 4.4, 2.5, 0.6, 0.4
8.8, 1.2, 1.0 8.8, 2.5, 1.2, 1.0 2.5, 1.35, 0.8 2.9, 1.35, 0.8 3.6, 2.9
16.8
92.1
5.7, 4.4, 3.4 5.7, 3.4
Table 2. Hybridization patterns of cloned Sau3AI fragments of C. moewusii mtDNA to restriction endonuclease digests of the C. moewusii mtDNA-enriched fraction Probe"
20.75
13.38
Hybridizing filter-bound DNA (sizes in kb) b
8.8, 3.6
16.8 3.5
14.2
3.5, 1.7 16.8, 1.7 16.8
14.2
Localization o f genes on the C. moewusii m t D N A map
14.2 14.2, 6.8
a The relative positions of the probes on the C. moewusii mtDNA are shown in Fig. 2 b Underlined hybridizing fragments join the left (20.75) and right (13.38) ends of the cloned linear map (Fig. 2) into a circular map Probe 10.96 was a 2.4 kb subfragment of clone 10.96, which extends from the AvaI (SmaI) site of the insert to the right end of the insert as depicted in Fig. 2 Table 3. C. moewusii mtDNA fragments generated by HindIII, AccI, AvaI and BstEII Fragment number
Size (kb) HindlII
1
2 3 4 5 6 7 8
AccI
AvaI
BstEII
5.7 5.3 4.4 3.4 2.5 0.6 0.4
8.8 3.6 2.9 2.5 1.35 1.2 1.0 0.8
16.8 3.5
14.2 6.8
1.7
1.0
22.3
22.15
22.15
0.15
22.0
Mitochondrial gene-specific probes derived from C. reinhardtii and wheat (Table 1) were used to identify hybridizing regions on the 22 kb circular m a p of the C. moewusii m t D N A . Such experiments were performed in order to confirm the identity of this D N A and also to establish whether its gene organization differs significantly from that reported for C. reinhardtii. The gene probes were hybridized to filter-bound HindIII and AccI fragments of the m t D N A - e n r i c h e d preparation, to AccI fragments of clones 92.1 and G, to AvaI fragments of clones 10.96 and C and also to the HindIII-EcoRI double digestion products of clones 20.75 and 13.38. The coxl, nadI, nad5 and rtl gene probes identified hybridizing fragments in digests of the Sau3AI clones and the m t D N A - e n r i c h e d preparation. Examples of coxl, nadl and nad5 probe hybridizations with genomic D N A digests are shown in Fig. 4. The rrnS and rrnL hybridizing regions of the C. moewusii m t D N A were identified on blots prepared with the Sau3AI clones only; the hybridization patterns on the genomic blots were complex and uninformative because of hybridization with chloroplast rrnS and rrnL genes (data not shown). The cob- and nad2-hybridizing regions were detected with the Sau3AI clones but not with the m t D N A enriched fraction (data not shown), presumably because of low sequence similarity between these gene probes and the corresponding C. moewusii mitochondrial genes. Overall, these results led to the localization of mitochondrial gene-hybridizing regions shown in Fig. 3. Finally,
57 cox1 H
A
nadl H A
nad5 H A
kb
--5.3 --3.6 --2.9
--1.35
Fig. 4. Mapping of nadl, nad5 and coxl on the C. moewusii mitochondrial genome. Southern blots of HindIII (H) and AccI (A) digests of the C. moewusii mtDNA-enriched fraction were hybridized with the corresponding 32P-labeled nadl, nad5 and coxl genespecific probes described in Table 1
the atp6, cox2 and nad6 gene probes showed no hybridization to either the cloned C. moewusii mtDNA or the C. moewusii mtDNA-enriched fraction. Discussion
Although the size and conformation of mitochondrial genomes vary considerably among different organisms, all such genomes appear to contain coxl, cob, and coding regions for the small and the large mitochondrial RNAs (Gray 1988). Because C. reinhardtii or wheat mtDNAs specific for these four genes were found to hybridize to the circular 22 kb D N A described here, we conclude that this D N A is the mitochondrial genome of C. moewusii. This conclusion is further strengthened by the identification of nadl-, nad2-, nad5- and rtl-hybridizing regions in this D N A using C. reinhardtii mitochondrial gene probes. Although cox2 and atp6 genes are typically associated with mitochondrial genomes, they are absent form C. reinhardtii mtDNA (Michaelis et al. 1990; Boer and Gray 1991). Wheat probes derived from these genes as well as probes for nad6 of C. reinhardtii failed to show hybridization with the C. moewusii mtDNA. A definitive answer to the question of whether or not these genes are missing from C. moewusii mtDNA will have to await the complete sequencing of this gehome. Such a study may also help determine the function, if any, of the extra 6 kb of D N A present in the mtDNA of this alga but not in the mitochondrial gehome of C. reinhardtii. The order of mitochondrial genes mapped here on the C. moewusii mtDNA is completely rearranged relative to their order on the C. reinhardtii mtDNA (Michaelis et al. 1990; Boer and Gray 1991). A similarly extensive rearrangement of genes has been observed for the
chloroplast DNA of C. moewusii relative to C. reinhardtii (Lemieux et al. 1985). These differences, in both mitochondrial and chloroplast DNAs, are not surprising in the light of recent primary sequence comparisons of Chlamydomonas chloroplast and nuclear genes suggesting that C. reinhardtii and C. moewusii may be quite distantly related (summarized by Buchheim et al. 1990; Turmel et al. 1991). In C. reinhardtii, the mitochondrial rrnS and rrnL regions comprise several coding modules that are interspersed with each other and with protein-coding regions (Boer and Gray 1988a). Our hybridization results are consistent with there being a similarly unorthodox rRNA gene structure in the C. moewusii mitochondrial genome, because rrnS- and rrnL-hybridizing regions of this mtDNA are widely separated and are interrupted by putative protein-coding regions. Moreover, one 0.8 kb AccI fragment of C. moewusii mtDNA showed hybridization with both the rrnS and the rrnL gene probes. Clarification of the C. moewusii mitochondrial rRNA gene structure will require D N A sequence analysis. The circular structure of the C. moewusii mitochondrial genome contrasts with the linear structure of its homologue from C. reinhardtii and the putative mitochondrial genomes of different syngens of P. morum (Moore and Coleman 1989). As more studies or volvocalean algal mtDNAs become available, it will be interesting to see whether the distribution of circular versus linear molecules is consistent with the phylogenetic relationships between these algae as deduced from gene sequence comparisons. In this connection, it should be pointed out that although the map of the C. moewusii mtDNA is circular, gel electrophoresis studies have established that the vast majority of the mtDNA molecules migrate as 22 kb linear molecules (T. Locke, B. Langille and R.W. Lee, unpublished results). We do not know whether this preponderance of linear molecules exists in vivo or is the result of random breaks introduced during DNA isolation. Similarly, most of the mtDNA molecules recovered from Physarum polycephalum strain M3 are linear fragments (Bohnert 1977) and this DNA also has a circular restriction map (Jones et al. 1990). Acknowledgements. We thank Poppo Boer, Linda Bonen, Michael Gray and David Spencer for providing C. reinhardtii and wheat mtDNA clones and for information, prior to publication, about the sequence and/or restriction maps of these clones. We thank Eileen Denovan-Wright for confirming some of the results reported here and Eileen Denovan-Wright and Michael Gray for critically reading this manuscript. Most of the work reported here was performed in the laboratories of M.T. and C.L. while R.W.L. was on sabbatical leave. The salary of R.W.L. was provided in part by the Universit6 Laval. This research was supported by grants from NSERC of Canada. R.W.L. and M.T. are Associates, and C.L. a Scholar in the Evolutionary Biology Program of the Canadian Institute for Advanced Research.
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