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Small Metallothionein MT-10 Genes in Coastal and HydrothermalMussels Laboratoire de Biologic et Ge´ne´tique Evolutive, Universite´ du Maine, 72085 Le Mans, France Received: 25 November 2003 / Accepted: 20 August 2004 / Online publication: 5 May 2005 motif: Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys (whereX is an amino acid other than cysteine). Mollusk Metallothioneins (MTs) are important proteins in MTs all belong to class I, as do mammalian and the intracellular regulation of metals. In the Mytil- crustacean MTs (Kagi, 1993; Picinni et al., 1999).
idae family, which includes many economically The spatial arrangement of these repetitive motifs important species, 2 major forms of MTs have been seems to determine the metal-binding properties of reported: MT-10 (10 kDa) and MT-20 (20 kDa). Many the MT (George and Hodgson, 1995). The cysteine different MT-10 proteins have been isolated from the residues constitute 2 domains (a and b) (metal-thio- common species Mytilus edulis, which suggests that late clusters) that bind the metal ions (Kagi et al., distinct MT-10 genes may occur in a single speci- 1984; Braun et al., 1992). If the intracellular con- men. Some MT genes, involving 3 exons and 2 large centrations of essential metals are too high, or toxic introns, have been isolated in Mytilidae. Our aim metals are present within the cell, then synthesis of was to determine whether intron-free forms of the the MT proteins is induced. These proteins bind the MT-10 genes can exist, which could allow rapid metal ions and convey them to the intracellular transcription in response to exposure to metals. Our study focused on 2 species living under very different Many reports have suggested that MT concen- environmental conditions: Mytilus edulis (a coastal trations could constitute a potential indicator of mussel) and Bathymodiolus thermophilus (a hydro- marine pollution by metals (Hennig, 1986; Cajara- thermal mussel). We report here the first description ville et al., 2000; Dallinger et al., 2000). In the My- of small, intron-free MT-10 genes, possessing a cor- tilidae and Ostreidae families, 2 major marine rect open reading frame in these 2 species.
bivalve families, the use of metallothioneins hasbeen investigated in several genera: Mytilus (Am- Key words: metallothionein — MT-10 — gene — iard-Triquet et al., 1998; Petrovic et al., 2001; Geret Mytilus edulis — Bathymodiolus thermophilus and Cosson, 2002), Perumytilus (Riveros et al.,2003), and Crassostrea (Geffard et al., 2002). Manystudies have also attempted to characterize the pro- tein structure and complementary DNA sequencesof MTs, but few publications have focused on Metallothioneins (MTs) constitute a family of ubiq- uitous cysteine-rich, low molecular weight proteins In the Mytilidae, 2 major forms have been re- that are involved in intracellular regulatory mecha- ported: MT-10 and MT-20 (Frazier et al., 1985; nisms (Bauman et al., 1993; Kag, 1993; Roesijadi et Mackay et al., 1993; Barsyte et al., 1999; Ivankovic et al., 1997; Legras et al., 2000; Park et al., 2001; Wu et al., 2002). High Zn and Cd concentrations induce the al., 2002) and play an antioxidant role (Viarengo et synthesis of MT-10 (monomeric form, 10 kDa), al., 1999, 2000; Cavaletto et al., 2002; Sato and whereas the more specific MT-20 (dimeric form, 20 Kondoh, 2002). The MTs have been divided into 3 kDa) is induced by exposure to Cd (Frazier et al., classes according to the distribution of the Cys-Cys 1985; Isani et al., 2000; Lemoine et al., 2000). In theMytilus genus MT-10 and MT-20 contain 73 aminoacids (21 cysteines) and 72 amino acids (23 cyste- Correspondence to: V. Leignel; E-mail: ines), respectively (Mackay et al., 1993). The addi- DOI: 10.1007/s10126-004-0135-8  Volume 7, 236–244 (2005)  Ó Science+Business Media, Inc. 2005 V. LEIGNEL ET AL.: SMALL METALLOTHIONEIN GENES IN MUSSELS tional cysteine residues in MT-20 could enhance its chloroform protocol. Any messenger RNA trace was metal-chelating properties or create intermolecular visualized during the DNA extractions. The metal- linkages between the 2 monomeric subunits (Mac- lothionein genes were amplified by polymerase kay et al., 1993). Several cDNA isoforms of MT-10 chain reaction (PCR) from genomic DNA (100 ng and MT-20 have been reported in a pool of Mytilus template). PCR was performed using different edulis individuals (Mackay et al., 1993; Barsyte et primers, depending on the species being studied. The al., 1999). The technique used did not make it pos- primers specific for the amplification of the MT sible to find out whether these isoforms were allel- genes of Bathymodiolus thermophilus were BathA (5¢-CCTTGTAACTGTGTCG-3¢) and BathB (5¢-GCAGGAA- The identification of several isoforms could be CAGGCAGG-3¢), and those for Mytilus edulis were accounted for by the existence of several genes in Bath01 (5¢-GACCACTGAGACCACTAC-3¢) and Bath06 individuals or by polymorphism within mussel (5¢-GGAATAAGTTCGAGTATATTGTC-3¢). These 2 pairs populations. In Mytilidae recent genomic charac- of oligonucleotide primers were defined from a terizations indicate that MT genes consist of 3 exons multiple alignment of the mRNA MT sequences of and 2 introns (Khoo and Patel, 1999; Ceratto et al., Mytilus edulis (available in GenBank) and the 2002). This typical genomic organization matches mRNA MT-10 sequence of Bathymodiolus thermo- mammalian MT structure. In Mytilus galloprovin- philus (available in Denis et al., 2002). PCR was cialis the MT-20 gene is 1865 bp in length (Ceratto et performed in a 25-ll reaction volume for 35 cycles: 1 al., 2002). Khoo and Patel (1999) suggested that in minute at 94°C (denaturing phase), 1 minute at 49°C Perna viridis 2 distinct MT sequences may corre- and 57°C (for BathA/BathB and Bath01/Bath06, spond to 2 different metallothionein genes: MT-I1 respectively) (annealing phase), and 1 minute at 72°C and MT-I2, 1657 bp and 2045 bp, respectively.
(elongation phase). The short elongation phase was However, these 2 sequences encode the same pro- designed to permit the replication only of fragments tein. A large second intron has been reported in MT- up to 1000 bases. The amplification of MT genes I1 (1093 bp) and MT-I2 (1291 bp). Each individual with the typical structure (3 exons and 2 introns) can mussel could possess different MT genes, charac- require an elongation phase of about 2.5 minutes in terized by distinct regulatory regions and induced by view of the lengths of the MT genes already identi- different factors. These MT genes could have specific fied in Mytilidae: Perna viridis, 1657 and 2045 bp structures that favor their rapid transcription in re- (Khoo and Patel, 1999); Mytilus galloprovincialis, sponse to exposure to a specific metal or metals.
Our aim was to find out whether several different The PCR products were ligated to the pGEM-T mussel MT genes exist. We analyzed the genomic Easy (Promega) plasmid vector and transfected into MT information of 2 Mytilidae species, collected Escherichia coli JM-109 strain (Promega). The colo- from very different environments: Mytilus edulis nies containing MT inserts were confirmed by PCR (the blue mussel) and Bathymodiolus thermophilus using the BathA/BathB or Bath01/Bath06 primers. A (a hydrothermal mussel). We investigated 2 ques- second confirmation was carried out using a B.
tions. Do Mytilidae, like the Ostreidae (A. Tanguy, thermophilus MT-10 probe. The high sequence personal communication), have small MT genes? similarity of this probe means that it could be ex- And if they do, could we identify any in 2 different pected to hybridize with all the metallothionein mussel species living in very different environments? variants. The selected clones were sequenced byGENOME Express Enterprise using the universalprimers SP6 and T7.
Biological Samples. Two specimens of Mytilus ed- ulis (blue mussel, a coastal species) were examined Processed Pseudogenes? To find out whether there (individuals coded N1, and N2). They were collected were any processed pseudogenes (reverse transcrip- in Normandy (France). One specimen of Bathy- tion events) in the individual mussels, PCRs were modiolus thermophilus (a hydrothermal mussel) carried out using the primers BathA/oligo(dT) (24- was collected from a site 9°N in the Pacific Ocean in mer) for Bathymodiolus thermophilus and Bath01/ 1994 (oceanographic mission ‘‘Hero’’). These mus- oligo (dT) (24-mer) for Mytilus edulis. The optimum sels were conserved at )70°C until required.
melting temperature of oligo (dT) is 42°C. The PCRconditions were 35 cycles consisting of 1 minute at DNA Isolation, Polymerase Chain Reaction, 94°C, 1 minute at between 37°C and 42°C and 1 and Sequencing. Genomic DNA was isolated and minute at 72°C. The low annealing temperature purified from the mussels using the classic phenol- facilitated PCR amplification with the forward V. LEIGNEL ET AL.: SMALL METALLOTHIONEIN GENES IN MUSSELS primers BathA or Bath01. The 1-minute elongation protein sequences available in GenBank revealed the phase allowed the replication of only small frag- similarity between MT-10A (without the extra G) and the MT-10 IV protein found in Mytilus edulis(Figure 2). Two putative alleloforms of MT-10A were Phylogenetic Analysis. The MT sequences ob- observed in the N2 mussel, MT-10A(1) and MT- tained from Mytilus edulis and Bathymodiolus 10A(2) respectively. These 2 probable alleloforms thermophilus were identified using the BLAST pro- displayed 3 nucleotide polymorphism sites (Fig- gram (Altschul et al., 1997). A multiple alignment ure 1), which induced one distinct amino acid in between the MT sequences of Mytilidae was carried position 4 serine (S) instead of proline (P) (Figure 2).
out to identify the nucleotide differences. The Pileup MT-10B (198 bp) was characterized by a large module of the GCG software (Genetics Computer deletion (site 84–107), 2 short insertions (139–144 Group) and the Genedoc program (Nicholas and and 189–191), and several silent mutations (35, 47, Nicholas, 1997) were used for the multiple align- 52, 108, 138, 150, 169, 170, 175, 197, and 219) (Fig- ment. Phylogenetic trees were constructed using the ure 1). This MT-10B gene possessed an ORF, and the Phylip package (Felsentein, 1993) by the neighbor- translation revealed a protein sequence (66 amino joining method (Jukes and Cantor genetic distance).
acids, including 19 cysteines at the expected posi- The bootstrap option was used (1000 replicates).
tions) identified by the BLAST program as the MT-10 Two trees were established: one using the complete protein (Figure 2). The large nucleotide deletion re- sequences, including the insertion and deletion re- sulted in the elimination of a polypeptide core (pro- gions (indels), and the other based on the sequences tein sites 34–41) incorporating 2 cysteines. Two without the indel regions. The MT mRNA sequence putative alleloforms of MT-10B were observed in the of Perna viridis (accession number AF036904) con- stituted the outgroup of the phylogenetic trees.
whereas only MT-10B(1) was observed in the N1mussel.
The EMBL accession numbers for our sequences MT-10A(1), AJ577124, MT-10A(2), AJ577125; MT- Putative Active Intron-free MT-10 Genes of Mytilus 10B (1); AJ577126; and MT-10B(2), AJ577127.
edulis. Amplification of the MTs from M. edulis(coastal blue mussel) DNA revealed 2 distinct MT Putative Active, Intron-free MT-10 Gene in genes with differing nucleotide sequence lengths Bathymodiolus thermophilus. Metallothionein am- (198 and 222 bp, respectively, from the start [ATG] to pli fication of Bathymodiolus thermophilus (hydro- the stop [TGA] codons) in specimen N2, but only thermal mussel) yielded a partial MT sequence one MT gene (198 bp) in N1. The 2 MT genes were corresponding to a portion of the MT-10 mRNA se- characterized by the absence of an intron and the quence (from bases 10 to 233) of M. edulis. Ampli- presence of a correct open reading frame (ORF).
fication of these MT genes revealed a duplication Multiple alignment of these sequences (with or (CAG) at position 155, a correct ORF, and no an in- without the indel positions) and the MT-10 and MT- tron. It appeared to be closer to MT-10 than to MT- 20 cDNA available in GenBank was performed using 20 (Mytilus edulis), and was designated the MT-10Bt the BLAST program. This analysis revealed that gene. Two sequences were obtained, MT-10Bt(1) and small MT genes were closer to MT-10 than to MT-20 MT-10Bt(2). These probable alleles displayed 3 sequences of M edulis. These 2 small MTs were polymorphic sites (positions 39, 197, and 218) (Fig- named MT-10A (222 bp in length) and MT-10B (198 ure 1). The level of similarity between the mRNA bp in length), respectively. The most obvious dif- MT-10 sequence of B. thermophilus (Denis et al, ference between these 2 small MT-10 genes is the 2002) and MT-10Bt (1) or MT-10Bt(2) was 99% or large deletion in the region corresponding to exon 2 99.5%. The nucleotide substitutions observed in the of the gene. However, many other polymorphic sites 2 alleles do not change the protein information are observable in the regions corresponding to exon 2 (Figure 2). The EMBL accession numbers for our se- quences are as follows: MT-10Bt(1), AJ577128; and MT-10 A (222 bp) also had 3 CGG nucleotides (positions 119–121), that were not observed in otherMT-10 isoforms. These additional nucleotides cor- Phylogenetic Analysis. The 2 trees produced respond to a duplication of the neighboring codon using the neighbor-joining method (Jukes and Cantor (Figure 1). If correctly incorporated in the ORF this genetic distance) with or without the indel regions insertion induces one additional amino acid glycine observed in genomic sequences (MT-10A, MT-10B, (G) in the protein (at site 41). Comparison with the and MT-10Bt), displayed the same typology (Fig- V. LEIGNEL ET AL.: SMALL METALLOTHIONEIN GENES IN MUSSELS Fig. 1. Multiple alignments of MT-10 cDNA metallothionein available in GenBank with small MT-10 genes isolated fromMytilus edulis (MT-10A and MT-10B) and Bathymodiolus thermophilus (MT-10Bt). The GenBank accession numbers ofthe cDNA sequences are as follows: MT-10III (AJ005454), MT-10II (AJ005453), MT-10Ib (AJ005452), MT-10Ia (AJ005451),MT-10IV (AJ007506). Dots indicate deletions. The motif corresponding to MT-10I and MT-10II is shown in bold faceletters. The specific insertion and deletion sites for MT-10A and MT-10B (the MT-10 genes of Mytilus edulis) are enclosedwith dashed and solid boxes, respectively. The specific sites of the MT-10Bt sequences in Bathymodiolus thermophilusare indicated by italics and underlining.
V. LEIGNEL ET AL.: SMALL METALLOTHIONEIN GENES IN MUSSELS Fig. 2. Multiple alignments of a protein sequence obtained in silico (bioinformatic approach) from the MT-10 nucleicsequence (showed in the Figure 1) obtained from Mytitus edulis and Bathymodiolus thermophilus. The GenBankaccession numbers of the cDNA sequences are as follows: MT-10III (AJ005454), MT-10II (AJ005453), MT-10Ib (AJ005452),MT-10Ia (AJ005451), and MT-10IV (AJ007506). The cysteine residues characteristic of metallothioneins are underlined.
Dots indicate deletions. The specific mutation sites for MT-10A and MT-10B are enclosed by dashed and solid boxes,respectively. The specific sites for the MT-10Bt(1) and MT-10Bt(2) sequences in Bathymodiolus thermophilus are indi-cated in bold face letters.
ure 3). These trees presented 2 groups with robust Patchun et al., 2004). We tried to determine whether branches: the first consisted of the MT-10 sequences, the cosmopolitan coastal family Mytilidae (bivalves and the second of the MT-20 sequences. Within the commonly chosen as sentinel organisms for detec- MT-10 group, the Bathymodiolus MT-10Bt se- tion of metal pollution) has others MT-10 genes that quences were similar to the MT-10 cDNA of B.
can be rapidly transcribed when the mussels are thermophilus and similar to the MT-10I and MT- exposed to various metals. We confirmed the exis- 10II isoforms of M. edulis. The small MT-10A se- tence of intron-free MT-10 genes in Mytilus edulis quences were similar to MT-10IV and MT-10III, and Bathymodiolus thermophilus. In Mytilus edulis whereas the MT-10B isoform was quite distinct.
we identified 2 distinct MT-10 genes (MT-10A andMT-10B, respectively, with putative alleles), char-acterized by an ORF and any intron sequence. Our investigations in Bathymodiolus thermophilus (a Are the Small MT-10 Genes Processed Pseudogenes hydrothermal mussel) also revealed a distinctive or Putative Active Genes? The few MT genes that MT-10 gene possessing an ORF and no intron. The have been isolated from bivalves all contain 3 exons MT-10Bt gene (2 putative alleles isolated) is very and 2 introns (Crassostrea gigas, Tanguy et al, 2001; similar to the mRNA MT-10 of B. thermophilus.
Mytilus galloprovmcialis, Ceratto et al., 2002; Perna We suggest 2 hypotheses that could account for viridis, Khoo and Patel, 1999), and this structure is the existence of these distinctive intron-free MT typical of the MTs found in other organisms such as genes: they could be active gene; or they could be birds, fish, mammals, and rice (Fernando and An- recent pseudogenes, resulting from reverse tran- drews, 1989; Hung et al., 1991; Wenming et al., 1998; Guo et al., 2003). The expression studies demon- On the one hand, the small MT genes found in strated of high induction metallothionein genes of M. edulis (MT-10A) and B. thermophilus (MT-10Bt) following metal exposure (Cousins, 1979; Cherian display a high degree of similarity to the MT-10 and Nordberg, 1983; Karin and Richards, 1984; cDNA sequences. Thus these small MT-10 genes Lemoine et al., 2000; Tanguy and Moraga, 2001; possess an ORF, and their cysteines, which confer V. LEIGNEL ET AL.: SMALL METALLOTHIONEIN GENES IN MUSSELS Wegnez, 1995). In Crassostrea gigas, another atypi-cal MT gene, CgMT2, which has an organizationcharacterized by an exon duplication, has been iso-lated and this also seems to be an active gene (Tan-guy and Moraga, 2001; Tanguy et al., 2001).
Metallic intoxication induced the expression of many genes other than metallothioneins, such ascytosolic O-acetylserine (thiol) 1yase, decarboxylase,OSISPAP1, Phytochelatin synthases, and proline-rich-protein (PvSR1to PvSR7) genes in plants or thecysteine-rich protein (CRP) gene in yeast (Tuan-Yaoet al., 1998; Clemens et al., 1999; Dominguez-Soliset al., 2001; Tschuschke et al., 2002; Fujimori andOhta, 2003; Mukhopadhyay et al., 2004). PvSR2,PvSR5, and OSISPAP1 genes confer metal resistancein plants, and they do not possess any introns in thecoding region (Zhang et al., 2001; Mukhopadhyay etal., 2004). PvSR2 is a specific heavy-metal-respon-sive gene that might play an important role inresistance to the damage caused by several metalssuch as mercury, arsenic, and cadmium (Zhang etal., 2001). In addition, the PvSR2 protein cannot be Fig. 3. Phylogenetic tree based on metallothionein cDNA metallothionein, as no cysteine domains were ob- and genomic sequences isolated from Mytilus edulis and served in the sequence. The other PvSR genes encode Bathymodiolus thermophilus, using the neighbor-joining proteins that interact in different metabolic re- method with 1000 replicates (bootstrap) (top value, analy- sponses. Indeed, the PvSRT1 gene encodes a proline- sis including insertions and deletions (indels); bottom va- rich protein; PvSR3, a dehydrin protein; PvSR4, a lue, analysis without indel sites). (Outgroup: mRNA MTsof Perna viridis; AF036904). GenBank accession numbers: pathogenesis-related protein; PvSR5, a polyubiqu- itin; PvSR6, a DNA-like protein; and PvSR7, a new AJ005453; MT-10III, AJ005454; MT-10IV, AJ007506; MT- Hg-C12 protein in the plant Phaseolus vulgaris 10A(1), AJ577124; MT-10A(2), AJ577125; MT-10B(1), (Tuan-Yao et al., 1998). The OSISPAP1 gene, also AJ577126; MT-10B(2), AJ577127; MT-10Bt(1), (AJ577128); displaying an intronless structure, encodes a zinc- finger protein, which is essential to the intracellularregulation of metal. Thus the identification of in- their metal-binding capacity, are correctly posi- tronless MTs genes could be another example of tioned relative to the M. edulis cDNA MT sequence.
genetic adaptations, to control rapidly rising metal Despite these deletions and insertions, the MT-10B small genes (in Mytilus edulis) also display a correct On the other hand, many of the small, intron- ORF and correct putative sites for the cysteines.
free MT genes have been shown to be pseudogenes These characteristics led us to think that they could inactivated as a result of mutations. These pseud- be active genes. The observation of putative active, ogenes are characterized by alterations of the coding small MT genes is not exclusive to the Mytilus and sequence with one or several stop codons or repeated Bathymodiolus genera. An unusual intron-free MT sequences (Karin and Richards, 1984; Schmidt et al., gene (MT-3) has been characterized in Crassostrea 1985; Andersen et al., 1986; Peterson et al., 1988; gigas (Mollusca, Ostreidae) (A. Tanguy, personal Tam et al., 1988). Walker and Gedamu (1990) have communication). This particular MT gene has an shown that there is a human MT pseudogene that ORF, a polyadenylation signal, and a correct 3¢ displays numerous substitutions and repetitive ele- untranslated region. In other species several minor ments upstream from the second exon, even though forms of MT genes have been isolated that deviate it conserves the cysteine residues in the protein from the habitual tripartite structure. In the Dro- information. If the small MT-10 genes present in M.
sophila genus (Insecta), 2 active genes, MTn and edulis and B. thermophilus are recent pseudogenes, MTo, have been identified. They are characterized then one could expect to observe several mutations by their distinctive structure, including only one randomly located in the genomic sequence, whereas small intron (265 bp and 61 bp, respectively) (Maroni in fact we observed that these 3 MT genes all have an et al., 1986; Stephan et al., 1994; Bonneton and ORF and correctly positioned cysteines. Our PCR V. LEIGNEL ET AL.: SMALL METALLOTHIONEIN GENES IN MUSSELS test to amplify any processed pseudogene present of the Iberian Peninsula: a practical approach. Sci Total using Bath01/oligodT and BathA/oligodT for Mytilus edulis and Bathymodiolus thermophilus, respec- 9. Cavaletto M, Ghezzi A, Burlando B, Ceratto N, Evan- tively, revealed no amplification. This result could gelisti V, Viarengo A (2002) Effect of hydrogen peroxide suggest that any poly (A) is localized at the 3¢ on antioxidant enzymes and metallothionein level inthe digestive gland of Mytilus galloprovincialis. Comp extremity of these small MT-10 genes (MT-10 A, 10. Ceratto N, Dondero F, Van de Loo JW, Burlando B, MT-10 genes characterized by this tripartite Viarengo A (2002) Cloning and sequencing of a novel organization or having a shorter structure could metallothionein gene in Mytilus galloprovincialis coexist in the same individual. Our data constitute Lam. Comp Biochem Physiol C 131, 217–222 the first description of putative active, intron-free 11. Cherian MG, Nordberg M (1983) Cellular adaptation MT genes in mussel. We are currently attempting to in metal toxicology and metallothionein. Toxicology determine the flanking regions of these 3 genes in order to find out whether they have the regulatory 12. Clemens S, Kim EJ, Neumann D, Shroeder JI (1999) regions characteristic of an active gene.
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