Program in Evolutionary Biology, Canadian Institute for Advanced Research, Département de Biochimie, Université de Montréal, Montreal, Quebec, Canada
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Abstract |
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Introduction |
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In most instances, the significance of such repeat sequences and structural elements for mitochondrial function remains unknown. With biochemical experiments being difficult to perform for mitochondria, especially for nonmodel species, valuable hints as to the nature of such motifs have emerged from the comparison of mtDNA sequences from closely related species (e.g., de Zamaroczy and Bernardi 1986
; Lang, O'Kelly, and Burger 1998
; Nedelcu and Lee 1998
). They might serve as control elements, involved in regulation of transcription, translation, or replication, and be conserved during evolution. Alternatively, they might simply result from errors of the mitochondrial DNA replication and DNA repair machinery or transposition of mobile elements. In any case, the accumulation of repeat elements likely favors recombination events that lead to mitochondrial genome reorganization.
Families of tandem repeats of varying complexity occur in the divergent regions of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei, forming arrays of up to 3 kb (Muhich, Neckelmann, and Simpson 1985
; Simpson et al. 1987
). In the mtDNA of the green alga Pedinomonas minor, a 9-kb region contains exclusively repeat elements (13 families of 6389 bp of dispersed repeats forming an elaborate superstructure), occupying more than a third of the genome (Turmel et al. 1999
). The mtDNA of another chlorophyte alga, that of Prototheca wickerhamii, also contains families of repeats consisting of nine sequence motifs of 30200 nt in length. In this case, instead of being organized in large arrays, the repeats are dispersed throughout the genome (Wolff et al. 1994
). The mtDNA of the fungus Podospora anserina contains a large number of short repeats, dispersed mostly in single units throughout the genome (Koll et al. 1996
). Finally, the mtDNA of the red alga Porphyra purpurea contains two inverted copies of a sequence unit of 291 bp, located opposite each other on the 37-kb circular mapping mtDNA (Burger et al. 1999
). None of these repeats has an obvious cellular function; however, some if not all are recombinogenic, favoring genomic rearrangements (Muhich, Neckelmann, and Simpson 1985
; Koll et al. 1996
; Burger et al. 1999
).
Structural sequence elements constitute a second class of repetitive sequences. In the mtDNA of the fungus N. crassa, inverted repeats forming long, highly stable hairpins have been identified (Yin, Heckman, and RajBhandary 1981
). These Pst palindromes (so-called for the presence of PstI restriction sites in their primary sequence) are scattered throughout intergenic spacers and introns of the genome. In the mtDNA of the yeast S. cerevisiae, stretches of G+C-rich sequences are dispersed throughout the genome. These G+C-rich clusters have been subdivided into eight families according to their sequence similarities, and many of them can be folded into a variety of stem-and-loop structures (deZamaroczy and Bernardi 1986). They have been characterized as preferential recombination sites (Dieckmann and Gandy 1987
; Clark-Walker 1989
; Weiller, Schueller, and Schweyen 1989
; Weiller et al. 1991
), and their presence in replication origins may serve regulatory functions (deZamaroczy and Bernardi 1986). In addition, some G+C-rich clusters are unidirectionally transmitted at the DNA level during genetic crosses (Butow, Perlman, and Grossman 1985
; Wenzlau and Perlman 1990
). Interestingly, G+C-rich clusters occur in several Saccharomyces species (deZamaroczy and Bernardi 1986; Wenzlau and Perlman 1990
) but are absent from the mtDNA of the yeast Candida (Torulopsis) glabrata (Clark-Walker, McArthur, and Sriprakash 1985
), hence reinforcing the view that G+C-rich clusters are most likely mobile elements. In plant mitochondria, two different types of structural elements have been identified. Members of one family, termed palindromic repeat sequences (PRSs), are highly conserved at the primary sequence level and are believed to be mobile because of their variable occurrence in closely related plant species (Nakazono et al. 1994
). Elements in the second group are also characterized by stem-and-loop structures but are less conserved at the primary sequence level than are PRS elements and are apparently involved in RNA processing (Schuster et al. 1986
; Dombrowski, Brennicke, and Binder 1997
). Finally, 11 palindromic sequences (up to 65 nt long) displaying conserved primary sequence have been identified in the mtDNA of the green alga Chlamydomonas reinhardtii. The distribution of these sequences within the genome suggests a role in gene expression (Boer and Gray 1991
).
All of the above-mentioned sequence elements are confined to groups of closely related species, and much of their distribution can be explained by direct exchange of genetic material following sexual crosses. We identified a novel type of structural element that is different from all of the previously described elements: it is widespread in fungi and folds into a characteristic double-hairpin structure (also referred to as a double-hairpin element [DHE]). DHEs were first recognized in the 57.5-kb-long mtDNA of the chytridiomycete Allomyces macrogynus (Paquin and Lang 1996
). These elements are spread throughout intergenic spacers and introns (five of the DHEs inserted in introns introduce frameshifts of intronic ORFs), and some are inserted in variable domains of the rRNA-coding regions. DHEs are also found in the mtDNA of the closely related species Allomyces arbusculus, as well as in those of distantly related fungal groups such as Monoblepharidales, Spizellomycetales (Paquin et al. 1997
), Zygomycota (unpublished results), and Ascomycota. In this paper, we present a detailed analysis of the DHE structural features and describe the distribution patterns of DHEs in various mtDNAs. We discuss evidence that DHEs are active mobile elements and that they are transferred across species boundaries.
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Materials and Methods |
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Sequence Analysis
The complete sequence of the A. macrogynus mtDNA (Paquin and Lang 1996
; accession number U41288) and part of the A. arbusculus mtDNA (accession number AF281324) were analyzed using programs supplied in the FASTA package (Pearson and Lipman 1988
) and our collection of programs (http://megasun.bch.umontreal.ca/ogmp/ogmpid.html). DHEs were identified by visual examination following the criteria defined in the text.
cDNA Amplification
Total RNA was extracted according to Deeley et al. (1977)
, except that a 6 M guanidine-HCl solution was employed for cell lysis. One microgram of RNA was incubated with FPLC pure DNAse I (Pharmacia) at 37°C for 15 min. to eliminate DNA contaminants. The RNA was then extracted with phenol/chloroform and precipitated with ethanol. First-strand DNA synthesis was carried out with AMV reverse transcriptase (Boehringer Mannheim), as detailed by the manufacturer using the oligonucleotide 5'-AGATGGTAACCTAACTCTAC-3', a primer hybridizing downstream of DHE10 within the ORF of cobi6 (intron 6 of the apocytochrome b gene). PCR amplification was carried out after adding the primer 5'-AGCTCCTCCGAGACTACATG-3' and Vent DNA polymerase (New England Biolabs), to yield a fragment of about 800 bp covering the region in which DHE9 and DHE10 are inserted. Control experiments without first-strand cDNA synthesis were performed, showing that the amplified DNA had been amplified from reverse-transcribed RNA and not from contaminating genomic DNA.
Southern Hybridization
The PCR-amplified DNAs were separated by electrophoresis on a 0.8% agarose gel. Only one major band of the same size was observed, both for the cDNA and the genomic DNA. To ensure the detection of weaker bands indicative of partial DHE removal, the DNA was transferred on a nylon membrane (Hybond-N, Amersham) according to the manufacturer's instructions and hybridized with the gel-purified PCR fragment.
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Results and Discussion |
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Are DHEs Spliced from Precursor RNAs?
Eleven DHEs are inserted in coding regions: six in the rRNA genes and five in intronic ORFs. The latter introduce frameshift in four ORFs, potentially inhibiting their expression. Apart from these DHE insertions, there are no further nonsense mutations in the intronic ORFs, suggesting either that insertions occurred only recently or that they are spliced out from precursor RNAs and do not interfere with the expression of the intronic ORFs. We experimentally tested the latter possibility.
We chose the ORF of cob intron 6 (cobi6), which contains two DHEs (DHE9 and DHE10), and as detailed in the methods section, we generated the cDNA encoding a portion of the intronic ORF and amplified it by PCR. After electrophoretic separation of the products in a 0.8% agarose gel, we observed a single cDNA band of the same size as PCR-amplified genomic DNA (data not shown). To further test the possibility that some spliced RNA was present at a low concentration, we transferred the amplified DNAs to a nylon membrane and hybridized with the radio-labeled PCR fragment. Again, only a single band was detected (data not shown). Finally, the PCR fragment obtained from the cDNA was cloned, and 10 independent clones were sequenced. The resulting sequences were all identical to the genomic sequence. This result implies that DHE9 and DHE10 are not spliced from this precursor RNA, at least not within the detection limits of the experiment performed.
The finding that DHE insertions are not removed from the cobi6 transcript is consistent with the observation that they never occur in structurally important portions of genes, e.g., protein-coding genes, tRNAs, highly conserved domains of rRNA structures, or the conserved core structures of group I and group II introns. Our results also parallel conclusions for G+C-rich clusters in yeast mitochondria, which are not removed or edited at the RNA level either in the var1 ORF or in rRNAs (Sor and Fukuhara 1982, 1983
; Zinn et al. 1988
).
DHEs Are Widespread in mtDNAs of Lower Fungi
In order to test the presence and distribution of DHEs in other Allomyces species, we sequenced a 4-kb fragment of the A. arbusculus mtDNA comprising the 3' region of nad5, atp6, and the 5' portion of cox1. Within this and other fragments (unpublished results), the sequence of A. arbusculus mtDNA is highly similar to that of A. macrogynus (>95%), both in coding and in intergenic regions. Nine DHEs were detected in A. arbusculus, and eight of these were 90%100% identical to DHEs in A. macrogynus mtDNA. Five of these eight DHEs were inserted at the same position as in A. macrogynus, while three others appeared in different genome locations in the two species (table 1
). For example, DHE16 is located in the intergenic spacer between nad5 and atp6 of A. macrogynus. The corresponding region of A. arbusculus contains an identical DHE insertion (DHEa1; fig. 6a
). However, it has an insertion of a twin DHE of 62 nt (DHEa2; fig. 6b
), whereas DHE16 is uninterrupted. An almost identical copy of the DHEa2 sequence was also found in intron 1 of cox1 (DHEa3) of A. arbusculus. In addition, DHE14 (inserted in the nad5 intron 3 of A. macrogynus) is identical in sequence to DHEa2 (fig. 6b
). These results indicate that not only are DHEs present in A. arbusculus mtDNA, but they also seem to show similar patterns of distribution and organization within both Allomyces mtDNAs.
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Do DHEs Have a Function in Mitochondrial Recombination?
Because of their sequence similarity and their structure, DHEs could be used as preferential recombination sites, similar to GC clusters in yeast mitochondria (Dieckmann and Gandy 1987
; Clark-Walker 1989
; Weiller, Schueller, and Schweyen 1989
; Weiller et al. 1991
). When comparing A. macrogynus and A. arbusculus sequences, we found three macrodeletions within three genomic fragments of approximately 4 kb each (this study and unpublished data). One, a 300-bp deletion in A. macrogynus mtDNA, occurred in the intergenic region between nad4l and atp9 (coding for subunits of the NADH-ubiquinone dehydrogenase and of the ATP synthetase complex, respectively). This region contains three DHEs in A. macrogynus (table 1
), and, most interestingly, the deletion is mapped within one of them (DHE59). Instead of the 300-bp fragment present in A. arbusculus, DHE59 in A. macrogynus contains a twin DHE (DHE81) not present in the former. In the other two cases (a 1-kb deletion that includes an unidentified ORF in A. arbusculus and a 600-bp deletion that includes an intronic ORF in A. macrogynus), the deletions were not mapped within DHEs, although we identified several of them near the deletion boundaries. These results provide evidence that DHEs actively contribute to the plasticity of mtDNAs.
Are DHEs Mobile?
The distribution pattern of DHEs is reminiscent of mobile elements. They are widely dispersed throughout A. macrogynus and A. arbusculus mtDNAs. Five of the nine DHEs identified in A. arbusculus are highly similar to and located at the same positions as their A. macrogynus counterparts, suggesting that DHEs were present in the mtDNA of the common ancestor of the two species. The difference in location of three other similar DHEs most likely reflects transposition events that occurred only recently, after the divergence of the two closely related Allomyces species, suggesting that DHEs are still active. Their scattered distribution in mtDNAs of lower fungi also favors the hypothesis that DHEs are mobile. Some mtDNAs, like that of Monoblepharella sp., contain numerous DHEs, whereas that of close relatives like Harpochytrium sp. contain relatively few. Similarly, the mtDNA of S. octosporus contains few DHEs, whereas that of its close relative Schizosaccharomyces pombe contains none (unpublished data). We believe that such a sporadic, uneven distribution is due to lateral acquisition of DHEs and their ongoing propagation.
This is not the first claim that small palindromic sequences could be mobile. PRSs are variably inserted into the mtDNA of rice species and are believed to be mobile (Nakazono et al. 1994
). In some yeast strains, two optional G+C-rich clusters called a and cc are inserted into the coding region of the mitochondrial var1 gene. During genetic crosses between a-cc- and a+cc+, nonparental genotypes were observed at a high frequency. Since there was no coconversion of flanking markers, these results were explained by G+C cluster mobility rather than by regular, homologous recombination (Butow, Perlman, and Grossman 1985
; Wenzlau and Perlman 1990
). There is compelling evidence that the transposition of the yeast mitochondrial elements occurs at the DNA level (Wenzlau and Perlman 1990
), probably following a "cut and paste" model (reviewed in Grindley and Reed 1985
). This is supported by the observation of in vivo double-strand cuts near G+C-rich clusters (Zinn et al. 1988
) and the duplication of two nucleotides flanking the mobile G+C-rich clusters.
The existence of twin DHEs in which the insertion was limited to the double-hairpin structure without disrupting that of the surrounding DHE (figs. 3 and 6 ) indicates that mobility of DHEs does not result in the coconversion of flanking markers. However, the mechanism of transposition of DHEs is likely to be different from that of G+C-rich clusters, since duplications of nucleotides are not consistently observed in the flanking sequences of DHEs. In addition, the evolutionary pressure to maintain the double-hairpin structure of DHEs includes the option that they transpose via an RNA intermediate and that the secondary structure is essential for mobility.
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Acknowledgements |
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Footnotes |
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1 Present address: Galileo Genomics Inc., Montreal, Quebec, Canada.
1 Keywords: double hairpins
repetitive sequences
mobile elements
fungi
comparative RNA modeling
2 Address for correspondence and reprints: B. Franz Lang, Département de Biochimie, Université de Montréal, CP 6128 succ. Centre-Ville, Montreal, Quebec, Canada H3C 3J7. E-mail: franz.lang{at}umontreal.ca
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