Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN 968-878-105-3
MOLECULAR EVOLUTION OF MITOCHONDRIAL SEQUENCES
ENCODING A FAMILY B DNA POLYMERASE AND THE SSU-rRNA, AMONG EDIBLE
MUSHROOMS (AGROCYBE SPP, AGARICUS BISPORUS, LENTINULA EDODES)
G. Barroso and J. Labarère
Laboratory of Molecular Genetics and Breeding of Cultivated Mushrooms
University Victor Segalen Bordeaux 2 – INRA, I. B. V. M., CRA de Bordeaux,
BP 81, 33883 Villenave d'Ornon Cedex, France.
<labarère@bordeaux.inra.fr>
ABSTRACT
In this study, we compared two mitochondrial genes (SSU-rDNA and DNA pol B), sequenced in
three edible mushrooms: Agrocybe spp, Agaricus bisporus and Lentinula edodes. The SSU-rRNA
gene is conserved on all mtDNA described to date and possesses highly conserved regions and
variable domains (V1 to V9). Among Basidiomycota, most of the variable domains evolve by
insertion/deletion of long nucleotide motifs and three small domains (V3, V5 and V7) seem to be
affected only by point mutations. The second gene which encodes a family B DNA polymerase, has
been described in the mtDNA of the mushroom A. aegerita and of only three other eukaryots: two
algae and one plant. In Basidiomycota, this gene seems to evolve by duplication events followed by
elimination (truncation and disruption) from the mitochondrial genome. In this case, the transfer of
a functional copy of the gene to a mitochondrial plasmid or to the nucleus compartment is
discussed.
INTRODUCTION
Mitochondrial genomes (mtDNA), because of their small size, content of highly conserved genes
and rapid rate of sequence divergence, are appealing molecules to use in the study of eukaryotic
population and evolutionary biology. Hence, comparison of mitochondrial genomes can lead to the
estimation of genetic variability, the establishment of genetic relationships between species or
higher taxa, and to the molecular characterization of strains, species or genus. However, in higher
fungi, studies on the organization and evolution of mitochondrial genome and genes have been
largely limited to a few yeasts and filamentous ascomycetes (for a review, see Gray et al. 1999).
In 1990 we began a study of the mitochondrial genome of Basidiomycota, its natural variability and
gene organization, in a biological model: the edible and industrially cultivated mushroom Agrocybe
aegerita. In this species, the first complete sequences for the Basidiomycota division of some
mitochondrial genes were obtained, such as cox1 encoding the first sub-unit of the cytochrome
oxydase (Gonzalez et al. 1998), or the two structural genes encoding the small (SSU-rDNA) and
large (LSU-rDNA) RNA of the mitoribosome (Gonzalez et al. 1997, 1999).
In this paper we report on two mitochondrial genes of edible mushrooms, previously described in
the A. aegerita genome:
- the first one, the SSU-rRNA gene, is present and conserved on all mitochondrial genomes
described to date.
- the second one, encoding a family B DNA polymerase, is present on the A. aegerita
mitochondrial genome of all the 36 European wild-type strains studied. In eukaryotic organisms,
such a gene has been reported on the mtDNA of only two algae and one plant.
The analysis of the complete sequence of the A. aegerita SSU-rDNA (Gonzalez et al. 1997)
revealed the presence of a large group-IC2 intron. When compared to the prokaryotic model
applicable to SSU-rRNAs from Archae, Bacteria, plastids and mitochondria (Neefs et al. 1993), the
61
A. aegerita SSU-rRNA secondary structure showed the presence of large nucleotidic extensions
mainly located in three variable domains of the SSU-rRNA (V4, V6 and V9 domains). Comparison
of the length and sequence variations of these three variable domains (V4, V6 and V9) within
Agrocybe (Gonzalez and Labarère 1999) and Pleurotus genera (Gonzalez and Labarère 2000)
suggested that their variations were species-specific and represented efficient molecular markers for
the taxonomy of Basidiomycota and for the investigation of phylogenetic relationships between
related species. It is to be noted that, besides the A.aegerita SSU-rRNA, numerous sequences from
Basdiomycota species are now available in the Genbank, for a 5' region of the SSU-rRNA (around
500 nt in length) overlapping the V4 domain. These sequences extended from the helix 18 of the V3
domain to downstream the V5 domain and the helix 31. These sequences have been used to study
the phylogenetic relationships between various basidiomycete species (Hibbett et al. 1997,
Moncalvo et al. 2000).
In order to extend the knowledge on the evolution of the mitochondrial SSU-rRNA sequences in the
Basidiomycota division, we determined the complete sequences and secondary structure of this
gene in the two most widely cultivated mushrooms in the world: A. bisporus (family Agaricaceae)
and L. edodes (family tricholomataceae). The following are conclusions drawn from the comparison
between a large region (1800 nt in size) of the A. aegerita, L. edodes and A. bisporus SSU-rRNAs.
The compared sequences represent 94 % of the A. aegerita complete sequence and include all the
variable domains V1 to V9.
The region located upstream of the 5' end of the A. aegerita SSU-rDNA carries, on the opposite
strand, a gene, named Aa-polB, encoding a family B DNA polymerase (Bois et al. 1999). This gene
encodes a putatively functional 571 amino-acid (aa) protein possessing all the conserved domains
and residues involved in 3'-5' exonucleolytic and polymerisation activities. The family B DNA
polymerases are a group of enzymes, related to E. coli Pol II and generally involved in the
replication of linear DNA genomes. Based on the high conservation of the  DNA polymerases,
from yeast to man (Lecrenier et al. 1997), a group of nucleus-encoded enzymes involved in the
replication of the mtDNA, Aa-polB could constitute a second DNA polymerase activity in the A.
aegerita mitochondria. To date, such a gene was described in only three other eukaryots: the two
algae Ochrommonas danica (Coleman et al. 1991) and Porphyra purpurea (Burger et al. 1999) and
the plant Beta vulgaris (EMBL Accession N° Q39432).
Moreover, the A. aegerita mitochondrial genome contains in a distant region (about 20 kpb apart
from Aa-polB) a second family B DNA polymerase gene (paralog) named Aa-polB P1 (Barroso et
al. 2001). The nucleotide sequence of this paralog gene is 86% identical to Aa-polB but it appears
truncated in its 3' portion leading to a non-functional protein that lacks the Pol III, Pol IV and Pol V
polymerization domains. Two naturally occurring alleles of Aa-polB P1 carry one or two inverted
copies of the disrupted sequence. Hence, the A. aegerita family B DNA polymerase mitochondrial
gene appears to evolve by duplication events followed by elimination (truncation and disruption) of
one copy. Such disrupted sequences homologous to family B DNA polymerase genes have been
reported on the mtDNA of an other eukaryot: the rye Secale cereale (Dohmen and Tudzynski
1994). To understand the evolution modalities of such genes, we decided to investigate the presence
of mitochondrial sequences homologous to Aa-polB in Basidiomycota species phylogenetically
closely related or not to A. aegerita, that is to say Agrocybe chaxingu and A. bisporus.
MATERIALS AND METHODS
Strains, media and culture condition
The dikaryotic A. aegerita strain SM47 and A. chaxingu strain SM960903 were obtained by subculture on CYM medium (Raper and Hoffman, 1974) of a small fragment of basidiocarp collected
in nature, in Agen (France) and Thaïland, respectively. The A. bisporus strain SC 930503 (ATCC
62
62489) used in this study has a snow-white phenotype. All these strains, as well as the L. edodes
strain SM 74 are conserved in the International Culture Collection of the Laboratory of Molecular
Genetics and Breeding of Cultivated Mushrooms. The E. coli strain used for the molecular cloning
of the A. bisporus and A. chaxingu mitochondrial DNA fragments was E. coli JM83 or E. coli XL1blue.
DNA extraction and molecular cloning
The "miniprep" extraction method of fungal total DNA from mycelia collected on a single petri dish
was previously described (Barroso et al. 1995). The A. chaxingu and A. bisporus mtDNA were
purified on a CsCl gradient in the presence of bisbenzimide, according to Fukumasa-Nakai et al.
(1992). The plasmid used as cloning vector was pGEM7Zf(+) from Promega Corp. (Madison, WI).
Cloning of the HinP1I or HaeIII mitochondrial fragments at the Cla I and SmaI site of pGEM7Zf
(+), respectively, was achieved by using conventional cloning procedures (Maniatis et al. 1982)
from purified mtDNA. From the HinP1I and HaeIII library of mtDNA from A. chaxingu and A.
bisporus, respectively, restriction fragments were isolated by hybridization on colonies, using as a
probe the previously cloned Ha-pol fragment of the A. aegerita strain WT-3 carrying the Aa-polB
gene (Bois et al. 1999). This cloned mitochondrial fragment used as probe was recovered after
digestion of the appropriate recombinant plasmids and electrophoresis in a 0.8% (wt/v) agarose gel,
by using a "Geneclean Kit" (Bio 101 Inc., CA, USA), then labelled with 925 kBq of [32P] dCTP
(110 TBq/mmol, Amersham, UK) using the Promega Corp. (Madison, WI, USA) "Random Primers
DNA Labelling Kit". The probes had a specific radioactivity higher than 108 cpm/g DNA.
DNA Labelling and Hybridizations
For Southern (1975) hybridizations, A. chaxingu or A. bisporus total DNA was extracted from 5 g
of vegetative dikaryotic mycelium cultivated in Roux flasks containing 100 ml of CYM liquid
medium, by using the N-cetyl-N,N,N-trimethylammonium bromide (CTAB) extraction method
adapted to Basidiomycetes by Noël and Labarère (1987). The digested total DNA (10 g) was
transferred after agarose (0.8% wt/v) gel electrophoresis in TBE buffer to Hybond N+ (Amersham,
UK) membrane by the Southern method (1975), with the help of a vacuum transfer system
(Appligène, France), in the presence of NaOH 0.4N. Prehybridizations, hybridizations and high (0.2
x SSC) or low (2 x SSC) stringency washings were carried out as previously described (Barroso et
al. 1995).
PCR amplifications
PCR amplifications of the mitochondrial SSU-rDNA were conducted using the Extensor Hi-fidelity
PCR
kit
(ABgene,
Epsom,
Surrey,
UK)
and
primers
SSU1
(5’
TTCTGATTGAACGTTTTTCAGTAG 3’) and SSU2 (5’ TACAAGCTACCTTGCTATGACTT 3’)
deduced from the A. aegerita SSU-rDNA (Genbank Accession N° U54637) and synthezised by
Eurobio (Les Ullis, France). PCR was performed in a Programmable Thermal Cycler PTC 100 (MJ
Research Inc., Watertown, Mass). Each reaction contained 10 to 30 ng of genomic DNA, 4 M of
the primer, 200 M of each dNTP, 1 unit of Taq DNA polymerase, 50 mM KCl, 10 mM Tris
(pH=8.3), 2 mM MgCl2, 0.1 % (vol/vol) triton X-100 in a final volume of 25 l. Reactions were
run for 40 cycles of 94 ° C for 1 min, 62° C (i. e. two degrees below the Tm of both
oligonucleotides) for 1 min, 72° C for 2 min, and one final cycle of 72° C for 5 min. The reaction
products were then analysed by agarose (1.5 %, wt/vol) gel electrophoresis for 3 hours at 90 V in 1
x TBE and visualized by ethidium bromide staining (Maniatis et al. 1982). The PCR products were
purified on agarose gels, eluted using the Qiaquick gel extraction kit (Qiagen, Santa Clarita,
California).
63
Sequencing
Before sequencing, the A. chaxingu and A. bisporus mitochondrial inserts were sub-cloned in their
two orientations in pGEM7ZF (+) vector, then processed to generate nested deletions using the
"Erase-a-base system", according to the manufacturer's recommendations (Promega Corp., WI,
USA). Recombinant plasmids were purified from the E. coli clones by a conventional miniprep
method (Maniatis et al. 1982).
Purified PCR products or cloned mitochondrial inserts were manually sequenced by the Sanger's
method (Sanger et al. 1977), on both strands using the ThermoSequenase sequencing kit and
33P-labeled dideoxynucleosides triphosphates (United Stades Biochemicals, Cleveland, Ohio).
The primers used for the PCR amplifications as well as internal primers were used for the
sequencing reactions. The sequencing products were separated on 6% polyacrylamide gels and the
dried gels were exposed for 48 h with Kodak X-Omat LS films.
Sequence analyses were performed with the DNA Strider 1.2 software (Commissariat à l'Energie
Atomique, Gif-sur-Yvette, France). Comparisons with sequences of the GenBank and EMBL
databases were performed using the search algorithm BLAST (Altshul et al. 1990). Alignments of
nucleotide and protein sequences were carried out with Clustal W (1.8) software (Higgins and
Sharp 1989).
RESULTS AND DISCUSSION
Evolution of the mitochondrial SSU-rDNA sequences from A. bisporus, L. edodes and A.
aegerita.
The complete sequence and secondary structure of the mitochondrial SSU-rRNA of A. aegerita was
published in 1977 (Gonzalez et al. 1997) and constitutes, along with that of Schizophyllum
commune (Genbank Accession number N° AF402141), the only two complete sequences to date
available for mitochondrial SSU-rRNAs in Basidiomycota. In order to extend our knowledge within
Basidiomycota, of molecules with important evolutionary dimensions and structural complexity,
the mitochondrial SSU-rDNA was sequenced in two other mushrooms which represent the most
industrially cultivated mushrooms in the world: A. bisporus and L. edodes.
For that purpose, a 24-mer oligonucleotides named SSU1 (nt 20- nt 44) located 20 nt downstream
the 5' end of the gene and a primer SSU2, located on the opposite strand at 36 nt of the SSU-rRNA
3' end (nt 3241-nt 3277) on the A. aegerita sequence (Gonzalez et al. 1997, Genbank Accession N°
U54637) were defined. When used in PCR experiments with the A. aegerita total DNA, the two
primers lead to an amplification product of 3221 nt in size, corresponding to 1850 nt of the SSUrRNA structural gene interrupted by its 1371 bp group-IC2 intron. The 1802 nt of the sequence
between the two oligonucleotides SSU1 and SSU2 represent 94 % of the complete sequence (1906
nt) and lack 44 nt in the 5' region and 60 nt in the 3' region. The two oligonucleotides were used as
primers in PCR experiments using the total DNA of A. bisporus and L. edodes as template. The size
of the amplification product was 1875 nt for A. bisporus and 2080 nt for L. edodes. These
amplification products were sequenced on both strands by using the SSU1 and SSU2 primers and
internal primers deduced from the sequences. The resulting sequences located between the two
primers had a size of 1827 nt (A. bisporus) and 2033 nt (L. edodes), i. e. a longer size than with A.
aegerita (1802 nt). The three sequences were aligned by using Clustal W software.
Alignments have shown that the group I intron localized at the foot of the helix 34 in the A. aegerita
SSU-rDNA is absent in the Agaricus bisporus and Lentinula edodes SSU-rDNAs, despite the fact
that the sequence surrounding the intron insertion site is highly conserved between the three
species. This result confirms the hypothesis of a recent acquisition of this intron by a lateral transfer
between the Ascomycota Scerotinia sclerotiorum and the Basidiomycota Agrocybe aegerita
(Gonzalez et al. 1997).
64
A comparison of more than 500 SSU-rRNAs from Archae, Bacteria, plastids and mitochondria has
shown that these molecules possessed conserved and variable domains and a model was proposed to
represent a consensus secondary structure of the prokaryotic SSU-rRNA (Neefs et al. 1993). Nine
variable domains, named V1 to V9, were defined from the 5' end to the 3' end of the rRNA.
Comparisons of the three mushrooms SSU-rRNAs have shown that the sequences can be classified
in two groups:
- (i) the highly conserved regions constituted by long stretches of nucleotides with a little
number of point mutations in the three basidiomycetes studied, and
- (ii) the regions with high variations between each of the three species; these variations were
mainly constituted by length variations due to the insertion/deletion of large nucleotidic motifs.
The size of the domains V2 to V9 were deduced from the alignments of the SSU-rDNAs sequences
for the three studied mushrooms. Our results show that, in the three Basidiomycota, five variable
domains are affected by insertion/deletion events involving long nucleotide motifs: the V2, V4, V6,
V8 and V9 domains (Table 1). It is to be noted that the domains V4, V6 and V9 were previously
shown to be highly variable in size between closely related species belonging to the genus Agrocybe
or Pleurotus (Gonzalez and Labarère 1998, 2000) and that these domains constituted efficient
molecular markers for the taxonomy of Basidiomycota. From our results, it appears that two
additional domains are highly variable in length (V2 and V8).
Table 1. Comparison of the size of the variable domains V2 to V9 of the mithochondrial
SSU-rRNAs from Agrocybe aegerita, Agaricus bisporus and Lentinula edodes.
Domains
Size (nt) of the domain in the SSU-rRNA of
A. aegerita
L. edodes
A. bisporus
V2
82
138
144
V3
35
35
35
V4
153
64
118
V5
29
29
29
V6
158
160
104
V7
20
20
20
V8
31
220
52
V9
168
139
130
On the contrary, the small variable domains V3, V5 and V7 appeared not subject to length
variations, but affected by point mutations, as suggested by the percentage of nucleotide identity
between these three variable domains in the three studied species (Table 2), which varied from 66
% (V5: A. bisporus and L. edodes) to 100 % (V7: A. aegerita and A. bisporus). From a comparison
of these three small domains without length variation, A. aegerita appears more closely related to A.
bisporus than to L. edodes, which is equally distant from both species.
65
Table 2. Percentage of nucleotide identity between the SSU-rDNA variable domains V3, V5 and
V7, in Agrocybe aegerita, Agaricus bisporus and Lentinula edodes.
A. bisporus
L. Edodes
V3 A. aegerita
94%
89%
V5 A. aegerita
76%
69%
V7 A. aegerita
100%
75%
V3 A. bisporus
100%
86%
V5 A. bisporus
100%
66%
V7 A. bisporus
100%
75%
Molecular cloning and sequencing of an ortholog of the Aa-polB gene in Agrocybe chaxingu
In 1999, we reported the presence, on the mitochondrial genome of 36 A. aegerita wild-type strains,
of a gene named Aa-polB, encoding a potentially functional family B DNA polymerase (Bois et al.
1999). The presence of a homologous gene (ortholog) was searched in Agrocybe chaxingu, which is
the phylogenetically nearest species of Agrocybe aegerita (Gonzalez and Labarère 1998), by
Southern hybridization with a cloned Aa-polB probe. This probe was constituted by a 0.9 kbp XbaI
restriction fragment carrying the part of the gene encoding the Exo III, Pol I, Pol IIa and Pol IIb
domains, isolated from the cloned Ha-pol mitochondrial fragment (4143 nt).
This radioactively labelled probe was used in Southern hybridization of total DNA and purified
mitochondrial DNA digested by 12 different restriction endonucleases. For ten endonucleases, the
hybridization patterns obtained after high stringency washings (0.2 x SSC, 50°C) were constituted
by one band of high molecular weight (> 10 kbp). Only the endonucleases Hind III and HaeIII
revealed two homologous fragments with a size below 10 kbp: 3 + 12 kbp for Hind III, 3 + 1.4 kbp
for HaeIII. These results suggested the presence in the A. chaxingu DNA of sequences homologous
to Aa-polB. The presence of two hybridizing bands can be explained by: (i) a site for the restriction
enzyme in the target sequence of the probe, or (ii) by two copies (paralogs) of the DNA polymerase
gene in the DNA of the strain, as described in the A. aegerita mitochondrial DNA (Barroso et al.
2001). For each endonuclease, the hybridization patterns obtained with the purified mitochondrial
DNA were strictly identical to the patterns obtained with the total DNA, suggesting that the A.
chaxingu sequences homologous to Aa-polB were located on the mitochondrial DNA of the strain.
In order to characterize the A. chaxingu sequences homologous to Aa-polB, a representative library
of the Hae III digested-mtDNA was achieved in E. coli. The Aa-polB homologous sequences were
searched by colony hybridization with the probe used in the Southern hybridizations described
above. One clone giving a strong hybridization signal with the Aa-polB probe was selected; the
recombinant plasmid of this clone possessed a 3 kbp mitochondrial insert, corresponding to the size
of one of the two fragments detected by Southern. Unfortunately, we failed to recover the second
(1.4 kbp) small fragment detected by Southern. A sequence of 1993 nt from one Hae III cloning site
was determined and analysed. This sequence possessed a 76 % (A+T) content, according to its
mitochondrial origin. Comparison of the sequence with those of the Genbank, has shown that it
66
possessed high homologies (> 85%) with the Aa-polB and its flanking regions, with the exception of
a small region of 78 nt (nt 697 to 774 in the A. chaxingu sequence), which, surprisingly, did not
reveal any sequence homology with the sequences from databases.
ORFs have been searched in the six reading frames by using the Neurospora crassa genetic code
and has shown the presence of a large ORF of 1653 nt (551 aa) from the initiating codon ATG (nt
136-138) to the termination codon TAA (nt 1789-1791). It is to be noticed that this large ORF is
interrupted by two stop codons TAA (nt 727-729 and 739-741), located in the small 78 nt region (nt
697 - 774) without homology with Aa-polB. In order to precise the sequence homology between AapolB and its ortholog in A. chaxingu and to precise the rearrangement leading to the 78 nt sequence
which interrupts the ORF, the two sequences were aligned, at the nucleotidic and proteic levels
using Clustal W software (Higgins and Sharp 1989).
The alignment of nt 532-812 of the Aa-polB sequence with the nt 670-902 of the A. chaxingu
ortholog (Figure 1 A) clearly shows two rearrangement events: (i) the insertion in A. chaxingu of a
78 nt sequence not recovered in Aa-polB, and (ii) 30 nt upstream this insertion, a 126 nt deletion. It
will be noticed that the nucleotides upstream the inserted sequence downstream the deleted one and
the nt between these two sequences are highly homologous between Aa-polB and the A. chaxingu
ortholog.
At the proteic level (Figure 1 B), the alignment confirms the observed insertion and deletion events.
The 78 nt inserted in the A. chaxingu sequence do not lead to a frame shift but this small sequence
contains two stop codons TAA. The deletion leads to the lack in A. chaxingu of a block of 42 aa
between the nine last aa of the Exo III domains and the 33 following aa. The two stop codons and
the lack of a part of the coding region lead to a non-functional protein encoded by the A. chaxingu
ortholog.
These two events located in a small region of the gene contrast with the high conservation of the
remaining part of the sequence. We have verified by PCR amplification that these events do not
constitute a particularity of the cloned insert but were present in the mitochondrial genome of A.
chaxingu.
In conclusion, our results have shown the presence in A. chaxingu of an ortholog of the A. aegerita
Aa-polB gene. Both genes have 85% identity at the nucleotidic level. At the proteic level, the
deduced proteins possess 81 % aa identity and 95% aa similarity. It will be noticed that such high
percentages have been described between Aa-polB and its paralog Aa-polB P1 P1 (86 % nucleotide
identity, 96 % aa similarity), both present on the A. aegerita mtDNA (Barroso et al. 2001).
In contrast with Aa-polB and as reported for Aa-polB P1, the A. chaxingu ortholog appears nonfunctional. Indeed, we have shown that the ORF is interrupted by the insertion of a 78 nt sequence
of mitochondrial origin and a 126 nt deletion. Both events are located in the 5' part of the gene, in
the Exo III motif. In Aa-polB P1, the ORF is disrupted in its 3' part, leading to the loss of the Pol III,
Pol IV and PolV domains. From this, it can be deduced that the events leading to the non-functional
proteins in A. aegerita Aa-polB P1 and in A. chaxingu, have appeared independently in intact copies
of the gene. In both cases, the maintenance of a long homologous sequence with Aa-polB suggests
that the rearrangements would be recent, i. e. after the speciation event between the two Agrocybe
species.
Finally, the isolation and characterization of the second copy of the gene detected by hybridization
with the Aa-polB probe in the A. chaxingu mtDNA (fragment Hae III of 1.4 kbp), will allow us to
determine if, as described in A. aegerita, a potentially functional copy of a family B DNA
polymerase gene is maintained on the A. chaxingu mitochondrial genome.
67
A
Aa-polB
A.chaxingu
GAATATAATAATTATAAATCAAATTTT--------------------------------GAATATAATAATTATCTAAAGACTTTTGTGGCTTCGCAACTTTTTATTTATATTTTATAA
*************** *
* ****
Insertion of 78 nt
697
Aa-polB
A.chaxingu
AAAGTATGGAATTTT
--------------------------------------------ATAAAAATATAAACCGAGGGAAAAAACCAGCACTTTGTGGAAAATAAAATATGGAATTTC
*** **********
774
Exo II
AGGGAAGAAGCTATAAAATATTGTAATCTAGATTGTATATCTCTATATGAAATTTTATA
AAAGAAGAAGCTATA-------------------------------------------* ************
Aa-polB
A.chaxingu
[
]
[
Aa-polB
A.chaxingu
TAAATTTAATACCCTAGTTTTTAATAAGTTTGAGTTAAATATAAATAAATATCCTACTTT
------------------------------------------------------------
Aa-polB
A.chaxingu
ACCTAGTTTATCATTTGCCTTATTTAAAACAAAGTATCTTAAAGAAAATGAAGTTCATAT
---TTTAAAACAAAATATCTTAAAAATGACACTGTGCATAT
------------------*********** ********* * *
** *****
Aa-polB
A.chaxingu
Pol I
GTTATCAGGTTCAATAGCTACAAATATTAGAAAATCTTATACTGGAGGATCTGTAGATAT
GCTATCAGGTCAAATAGCAAAAGATATAAGAAAAGGTTATACAGGAGGATCCGTAGATAT
* ******** ****** * * **** ****** ****** ******** ********
Deletion of 126 nt
]
B
A.chax:
NYSITFRDSYLLLPASLRKLCKSFNNETHKDIFPYLFSDINYVGEVPDFKYFNSISLEEY
YSITF+DSYLLLP+SLRKLCKSFN +T KDIFPYL DINY+GEVPD+KYF ++ +EEY
Aa-polB: KYSITFKDSYLLLPSSLRKLCKSFNTQTQKDIFPYLLDDINYIGEVPDYKYFCNLEMEEY
A.chax:
Aa-polB:
A.chax:
Aa-polB:
Exo III
NNYLKTFVASQLFIYIL*IKI*TEGKNQHFVENKIWNFKEEAI----------------NNY
F
K+WNF+EEAI
NNYKSNF--------------------------KVWNFREEAIKYCNLDCISLYEILYKF
Pol I
-------------------------FKTKYLKNDTVHMLSGQIAKDIRKGYTGGSVDMYI
FKTKYLK + VHMLSG IA +IRK YTGGSVDMYI
NTLVFNKFELNINKYPTLPSLSFALFKTKYLKENEVHMLSGSIATNIRKSYTGGSVDMYI
Figure 1. Alignment of homologous regions of Aa-polB (A. aegerita) and of the A. chaxingu
ortholog sequences, at the nucleotide level (A) and at the amino acids level for the corresponding
proteins (B).
Molecular characterization of vestigial sequences of a family B DNA polymerase in the
Agaricus bisporus mitochondrial genome
The search of Aa-polB homologous sequence in A. chaxingu has led to the characterization of a
non-functional ortholog gene on the mtDNA of this species. This study has been extended to a
phylogenetically more distant species: A. bisporus. Southern hybridizations using the Aa-polB
68
probe have shown hybridizing bands in the total DNA of A. bisporus digested by Hind III (1.6 + 6
kbp), Hae III (3 kbp) or HinP1 I (1.8 + 3.2 kbp). In contrast to the results obtained with A.
chaxingu, hybrids were denatured by a high stringency washing (0.2 x SSS, 50° C), but not by a
lower stringency washing (2 x SSC, 50° C), suggesting the presence in A. bisporus of Aa-polB
homologous sequence with a percentage of nucleotide identity lower than that of the A. chaxingu
ortholog (85%).
The hybridization patterns obtained with the total DNA are identical with those obtained with the
purified mtDNA, suggesting that the Aa-polB homologous sequences are carried by the A. bisporus
mtDNA. This mtDNA has been digested by HinP1 I and cloned at the Cla I site of pGEM7ZF(+). A
representative library of 560 recombinant clones has been obtained in E. coli. Two clones
possessing a recombinant plasmid between PGEM7ZF(+) and a mitochondrial insert of size 1.8 kbp
and 3.2 kbp respectively, have been selected by colony hybridizations. The size of these two inserts
corresponded to those revealed by the Aa-polB probe on the A. bisporus mtDNA.
Both fragments have been sequenced and display short sequences possessing homologies with AapolB. For example, the analysis of the 1795 nt of the small 1.8 kbp insert has shown, at the
nucleotide level, the presence of two typical mitochondrial tRNA genes and of a short sequence (nt
1206 – 1237) homologous to Aa-polB. The analysis at the proteic level extended the homology with
Aa-POLB protein to the nt 1124-1237 region.
The small ORF (nt 1138-1237) of this region has been aligned, at the nucleotide and aa levels with
the corresponding region of the Aa-polB gene upstream the Pol IIa motif (Figure 2).
AapolB
ORF H
AAATTCTATGGGG---ATATACCTTTGAATCTAAAAATATATTTTCAGAAATAATTAGTGATTTATATAAAATGAGACTAGA
TCTTACTATACCTTATATAAAGCGAT-AATCTTTAAAGATTCTGT---AAATCAATTAT-ATCAACTTAGACTTAGACTTGA
* ****
*** * * * ***** *** ** * *
**** * * * ** * ** * * ***** **
1118
1124
AapolB
ORF H
ATACCAGAAAAGTGATCCAATGAATTATATTGCTAAAATTTTAATGAATTCATTATACGGAAGGTTCGGAATGGACGATAATTT
TTTTCTTAAGTGTGATCCAATTAATTATATTGCTAAGATTTTATT---TTTATTAAATAG-----TCTTAATGGGGTTTTATAA
* * ** ********** ************** ****** *
** **** * *
** *****
* **
1268
Pol IIa
1237
1206
Aa-polB
ORF-H
WGYTFESKNIFSEIISDLYKMR--LEYQKSDPMNYIAKIL--MNSLYGRFGMDD
SYYTLYKAIIFKDSVNQLYQLRLRLDFLKCDPINYIAKILFLLNSLNGVLZ
**: . **.: :.:**::* *:: *.**:******* :*** * :
Figure 2. Alignments, at the nucleotide and proteic levels of Aa-polB and the A. bisporus ORF
possessing sequence homology with the A. aegerita family B DNA polymerase gene.
The compared sequences possessed 67 % of identity at the nucleotide level, 44 % of aa identity and
74 % of aa similarity. These results suggest that the A. bisporus sequence homologous to Aa-polB
are short and highly disrupted sequences which can be considered as remnants of a family B DNA
polymerase gene ancestrally present on the A. bisporus mtDNA.
In the same way, three small ORFs have been considered as remnants of a family B DNA
polymerase gene on the mtDNA of Marchantia polymorpha (Weber et al. 1995). The small size of
the conserved sequences between Aa-polB and the A. bisporus sequence confirms the phylogenetic
distance between the two species and the evolution of this gene to the elimination. In this
hypothesis, a functional copy could exist on a mitochondrial plasmid or could have been transferred
to the nuclear compartment. Indeed, it has been shown recently by PCR (Robison and Horgen 1999)
that, except A. bisporus, ten studied Agaricus species possessed a linear mitochondrial plasmid,
encoding a family B DNA polymerase and a RNA polymerase. More generally, those plasmids (for
a review, see Meinhardt et al. 1990), whose functions are still unknown are frequently found in
mitochondria of Ascomycota and Basidiomycota fungi or in plants.
69
ACKNOWLEDGMENTS
This work was supported by grants from the European Community (Fond Européen de
Développement Régional), the Conseil Scientifique de l'Université Victor Segalen Bordeaux 2,
Monsieur le Préfet de la région Aquitaine, Préfet de la Gironde (Fond National d'Aménagement et
de Développement du Territoire), the Conseil Régional d'Aquitaine, and the Institut National de la
Recherche Agronomique.
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