INTRODUCTION

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The genes in the fungal mitochondrial genome generally belong to a small set of highly conserved genes that probably originated from a prokaryotic ancestral endosymbiont (for a review, see reference 12). They encode components of complexes I to V of the electron transport chain, as well as the RNA portion of the translation system, mitochondrial rRNAs, and mitochondrial tRNAs. Most other genes encoding mitochondrial proteins are located on the nuclear chromosomes. In particular, the highly conserved  DNA polymerases (17) responsible for the replication of the mitochondrial DNA (mtDNA) are nucleus encoded.

Agrocybe aegerita is a cultivated basidiomycete whose mitochondrial genome has been cloned and mapped (21) and from which three mitochondrial genes have been sequenced: cox1 (10) and the small-subunit (SSU) and large-subunit rRNA genes (rDNAs) (9, 11). A potentially functional family B DNA polymerase gene named Aa-polB is near the SSU rDNA, on the opposite strand, in the 20 A. aegerita wild strains that have been studied (4). This gene putatively encodes a 571-amino-acid (aa) protein possessing all the conserved domains and residues involved in 3'-5' exonucleolytic and polymerization activities (4). Other sequences similar to Aa-polB are present in the mitochondrial genome about 20 kb from the Aa-polB gene. This region, named H4, has two alleles, H4-1 and H4-2, that differ in length and that are present in 28 and 8 strains, respectively, of 36 A. aegerita field strains (3).

Our objectives in this study were to determine the relationship between Aa-polB and a putative copy of this gene some 20 kb distant and to determine if the distal copy of this sequence affects allelic variability. The duplicated copies of this family B DNA polymerase gene may have arisen by reverse transcription of mRNAs primed by a mitochondrial tRNA.

	MATERIALS AND METHODS

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Strains, media, and culture conditions. We sequenced the HindIII restriction fragments H4 and H4a, corresponding respectively to the allelic forms H4-2 and H4-1. The 4.2-kb H4 fragment from the H4-2 allele was previously isolated from an mtDNA library from A. aegerita strain WT-3 (= SM47) (21); the 3.5-kb H4a fragment from the H4-1 allele was isolated from a library of mtDNA from strain WT-11 (= SM751002). Both strains are preserved on CYM medium (23) in the International Culture Collection of the Laboratory of Molecular Genetics and Breeding of Cultivated Mushrooms (collection number GMACC WDCM 786) and were previously described (3). Escherichia coli JM83 (29) was used for cloning and propagation of plasmids in Luria-Bertani medium (20).

DNA manipulations. HindIII or HaeIII mitochondrial fragments were cloned into the HindIII or SmaI sites of pUC18, respectively, by using conventional cloning procedures (8, 20). The H4a restriction fragment was isolated by colony hybridization, using H4 as a probe. Probes were digested with the appropriate restriction endonucleases, separated by electrophoresis in a 0.8% (wt/vol) Nusieve GTG agarose gel (FMC Bioproducts, Rockland, Maine), recovered by using a Geneclean kit (Bio 101 Inc., Vista, Calif.), and labeled with [-³²P]dCTP (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom) by using a Promega (Madison, Wis.) Random Primer DNA labeling kit.

DNA sequencing and sequence analyses. Mitochondrial sequences were subcloned in both orientations in pUC18 (29), then processed to generate nested deletions by using the Erase-a-Base system according to the manufacturer's (Promega) recommendations. Recombinant plasmids were purified from the E. coli JM83 clones by a conventional miniprep method (20). Both strands were sequenced in reactions using the M13-40 primer, the M13 reverse primer, or specific 18-mer oligonucleotides (Eurogentec, Seraing, Belgium). The sequencing reactions were performed by the dideoxy-chain termination method (26) with a Sequenase II kit (United States Biochemical Corp., Cleveland, Ohio). Labeled fragments were denatured, separated by electrophoresis on 6% (wt/vol) polyacrylamide gels, and identified by autoradiography. Sequence analyses were performed with the DNA Strider 1.2 software (Commissariat, à l'Energie Atomique, Gif-sur-Yvette, France). Comparisons with sequences in the GenBank and EMBL databases were performed by using the BLAST search algorithm (1), and nucleotide and protein sequences were aligned with the Clustal W package (13, 14).

	RESULTS

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Hybridization of the Aa-polB gene with a sequence located in a polymorphic region of the A. aegerita mtDNA. The Aa-polB gene is carried on a 4.2-kb HaeIII and a 7.2-kb HindIII restriction fragment (4). Four fragments were identified in Southern hybridizations of the HindIII- or HaeIII-digested mtDNA from WT-3, using the 4.2-kb HaeIII fragment or the H4 fragment (21) as a probe: 7.2- and 4.2-kb (H4) HindIII fragments and 4.2- and 11-kb HaeIII fragments. If mtDNA from WT-11 was probed in a similar manner, then the DNA fragments typical of the H4-2 allele (4.2-kb HindIII and 11-kb HaeIII) were not seen and those typical of the H4-1 allele (3.5-kb HindIII and 5.3-kb HaeIII) were evident instead. From these results, we concluded that a sequence hybridizing with the Aa-polB gene was present in two locations in both the WT-3 and WT-11 mtDNAs.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Comparison of the restriction maps and molecular organizations of the A. aegerita mtDNA H4 regions carrying the Aa-polB P1 genes of strain WT-11 (allelic form H4-1) and WT-3 (allelic form H4-2). The sizes of the restriction fragments are indicated in kilobase pairs when established by agarose gel electrophoresis or in nucleotides when deduced from the sequence. The location of the mitochondrial tRNAMet gene is indicated by an asterisk. Duplicated sequences are indicated by hatched bars; the sequence specific to the H4a fragment is indicated by a black bar.

Sequence analysis. We identified open reading frames (ORFs) in the H4 and H4a sequences by following coding rules for Neurospora crassa mtDNA (9). In H4a, we found a single large ORF of 1,857 nt, beginning at the HindIII site. In H4, this ORF was found in both inverted copies; no additional ORF was present in the 317-nt central nonrepeated sequence. The ORF was interrupted at its 5' end by the HindIII site. We sequenced the region surrounding the H4a fragment on the overlapping 5.3-kb HaeIII fragment (Fig. 1) and found a TAA stop codon in the same reading frame immediately before the HindIII site. Thus, the ORF is entirely contained in the H4a sequence. The putatively encoded protein had a size of 500 aa from the first ATG (nt 355 to 357) codon or of 617 aa from the termination codon just before the HindIII site.

Comparison of the large ORF of the polymorphic region with Aa-polB and other related family B DNA polymerase genes. In the 2,282-nt region from the HindIII site (nt 1) to the end of the tRNA^Met (nt 2071) in both H4 and H4a, the ORF region (nt 1 to 1955) can be aligned with a part (nt 644 to 2602) of the A. aegerita mitochondrial sequence (GenBank accession no. AF061244) containing the Aa-polB gene preceded by the intergenic region between the 5' end of the SSU rDNA and Aa-polB (86% sequence identity). The remaining 116 nt of H4 or H4a, from nt 1956 to the 5' end of the tRNA^Met (nt 2071), had no significant sequence identity with the Aa-polB gene sequence, suggesting that the 3' end of this gene was missing from the H4 or H4a sequence. If the two protein sequences are aligned (Fig. 2), then there is 96% aa similarity and 78% aa identity from the methionine (aa 1) constituting the Aa-POLB protein NH₂ terminus to the KLF motif (aa 515 to 517) located in the DNA polymerase III (Pol III) domain. Thus, the Aa-polB P1 paralog is a truncated copy that should produce a nonfunctional protein that lacks the Pol III, Pol IV, and Pol V polymerization domains. The Aa-POLB P1 protein also should contain 16 aa before the methionine residue aligning with the NH₂ terminus of Aa-POLB, since there is another Met amino acid codon in frame there.

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Partial aa alignment of the family B DNA polymerases encoded by the Aa-polB (B) and Aa-polB P1 (P) genes (GenBank accession no. AF061244 and AF269233, respectively), carried out with the Clustal W package (13, 14). The conserved aa are indicated by stars, and the similar and equivalent aa residues are indicated by periods and colons, respectively. The conserved domains involved in the exonuclease (Exo) and polymerase activities previously defined on the Aa-POLB protein (4) and the numbers of amino acids between conserved domains are indicated. For each protein, the first Met aa (M) and the Z, corresponding to the first stop codon TAA, are framed. The COOH-terminal region of the Aa-POLB protein truncated in Aa-POLB P1 is in italics.

	DISCUSSION

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The A. aegerita mitochondrial genome contains the previously described Aa-polB gene, which encodes a putatively functional family B DNA polymerase (4), and a truncated, probably nonfunctional paralog, Aa-polB P1. Moreover, two widely distributed mitochondrial alleles carry one or two copies of the truncated Aa-polB P1 sequence. These DNA polymerase genes may have arisen by duplications occurring in two steps (Fig. 3). A first duplication of Aa-polB, occurring before A. aegerita speciation, led to the divergent (14%) paralog Aa-polB P1 present in all strains (Fig. 3A and B); then a duplication of Aa-polB P1, occurring after A. aegerita speciation, led to the copies accounting for the allelic variability of the H4 region (Fig. 3C).

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Hypothetical model for the duplications of the family B DNA polymerase genes in the A. aegerita mitochondrial genome. (A) Distal (20-kb) duplication involving reverse transcription (RT) of the Aa-polB mRNA primed by the tRNAMet followed by integration of the cDNA (Aa-polB-tRNAMet) by recombination at the tRNAMet locus. (B) Sequence divergence (up to 14%) between Aa-polB and its paralog, leading to the disruption of the Aa-polB P1 copy. (C) Proximal (317-nt) duplication generating two inverted copies of Aa-polB P1 by recombinational integration at the tRNAMet locus of a cDNA (Aa-polB P1-tRNA) obtained after RT of the Aa-polB P1 mRNA. This duplication is accompanied by a large (>0.6-kb) deletion of neighboring mitochondrial sequences.

The putative origin of both duplications could be due to an illegitimate recombination between two mitochondrial genomes or, more probably, to the integration of a cDNA at a tRNA mitochondrial locus (Fig. 3). The first hypothesis is supported only by the fact that recombinant mtDNA molecules in A. aegerita heteroplasmons have been described (2). Because the Aa-polB P1 gene is followed by a tRNA^Met, it seems more probable that an mRNA of the Aa-polB gene captured a tRNA^Met whose 3'-OH end was used as a primer for a mitochondrial reverse transcriptase activity (Fig. 3A) (see, e.g., references 5 and 18). The resulting cDNA (Aa-polB-tRNA^Met) was integrated into the mtDNA by recombination at the tRNA^Met locus (Fig. 3A and B). We have no information about the other recombination site on the mtDNA; this region of recombination could be nonhomologous (illegitimate recombination) or, more probably, consist of a short homologous sequence present both on the cDNA and on the mtDNA near the tRNA^Met locus. The high percentage of A+T in the recombining molecules favors the presence of such a homologous small sequence.

The second duplication event leading to the H4-2 allele appears to require a complex mechanism involving the duplication of Aa-polB P1 and the deletion of a large sequence of size greater than 0.6 kb (Fig. 3C). Since this duplication could have affected the regions preceding the sequenced fragment, our results do not allow the determination of the size of the deleted sequence or the size of the duplicated one.

The mechanism(s) leading to the formation of the Aa-polB P1 truncated gene is unknown; in particular, we failed to find in the short sequence separating the truncation site and the tRNA^Met any sequence or secondary structure reminiscent of a group I or group II mitochondrial intron.

The  DNA polymerases represent a highly conserved family of nucleus-encoded DNA polymerases responsible for the replication of circular mitochondrial genomes of all eukaryotic organisms. However, recent reports on plant or fungal mitochondria have described the presence of additional DNA polymerase activities. For example, a nucleus-encoded  DNA polymerase activity in yeast mitochondria has been recently reported (19), and family B DNA polymerase genes in the chrysophyte alga Ochromonas danica (6) and in the plant Beta vulgaris (GenBank accession no. Z34298) have been described. The products of these family B DNA polymerase genes could be involved in the replication of linear genomes, and it has been recently reported that most of the plant and fungal genomes are present as linear multimeric molecules (16).

Comparison of Aa-POLB and Aa-POLB P1 sequences with other family B DNA polymerases shows that they are distantly related to the polymerases of Sulfolobus solfataricus (7). If mitochondria originated from a fusion between a Clostridium-like eubacterium and a Sulfolobus-like archaebacterium (15), the A. aegerita family B DNA polymerase genes could be remnants of the archaebacterial genes.

The presence of Aa-polB and its paralog Aa-polB P1 in the mtDNA of the basidiomycete A. aegerita suggests that a family B DNA polymerase activity could exist in a fungal mitochondrion; the lack of such a gene in all the ascomycete mitochondrial genomes sequenced to date leads us to hypothesize that in these fungi this gene was eliminated after transfer of a duplicated copy to the nucleus. The search for sequences homologous to Aa-polB in other basidiomycetes and the determination of their genomic location(s) (nucleus and/or mitochondria) will allow us to assess this hypothesis and to reconstruct the evolutionary histories of such mitochondrial family B DNA polymerase genes.