Isolation and Characterization of Copper- and Zinc- Binding Metallothioneins from the Marine Alga Ulva compressa (Chlorophyta)

In this work, transcripts encoding three metallothioneins from Ulva compressa (UcMTs) were amplified: The 5′and 3′ UTRs by RACE-PCR, and the open reading frames (ORFs) by PCR. Transcripts encoding UcMT1.1 (Crassostrea-like), UcMT2 (Mytilus-like), and UcMT3 (Dreissena-like) showed a 5′UTR of 61, 71, and 65 nucleotides and a 3′UTR of 418, 235, and 193 nucleotides, respectively. UcMT1.1 ORF encodes a protein of 81 amino acids (MW 8.2 KDa) with 25 cysteines (29.4%), arranged as three motifs CC and nine motifs CXC; UcMT2 ORF encode a protein of 90 amino acids (9.05 kDa) with 27 cysteines (30%), arranged as three motifs CC, nine motifs CXC, and one motif CXXC; UcMT3 encode a protein of 139 amino acids (13.4 kDa) with 34 cysteines (24%), arranged as seven motifs CC and seven motifs CXC. UcMT1 and UcMT2 were more similar among each other, showing 60% similarity in amino acids; UcMT3 showed only 31% similarity with UcMT1 and UcMT2. In addition, UcMTs displayed structural similarity with MTs of marine invertebrates MTs and the terrestrial invertebrate Caenorhabtidis elegans MTs, but not with MTs from red or brown macroalgae. The ORFs fused with GST were expressed in bacteria allowing copper accumulation, mainly in MT1 and MT2, and zinc, in the case of the three MTs. Thus, the three MTs allowed copper and zinc accumulation in vivo. UcMTs may play a role in copper and zinc accumulation in U. compressa.


Introduction
Metallothioneins (MTs) are low molecular weight proteins, of around 10 kDa, that are rich in cysteine residues allowing the binding of divalent or monovalent metal ions such as Zn 2+ , Cd 2+ , Pb 2+ , Hg 2+ , Cu 1+ , Ag 1+ , among others [1][2][3]. MTs participate in metal accumulation and detoxification in vertebrates, invertebrates, plants, algae, and bacteria [1][2][3]. Cysteine residues in MTs are usually arranged as CC, CXC, and/or CXXC motifs and they correspond to around 30% of amino acids. In vertebrates, cysteine residues in MTs are contained in two domains, α and β, that are separated by a linker of variable sizes. Vertebrate MTs are rich in glycine and alanine amino acids, ranging from 10% to 20% residues [2] and invertebrate and plant MTs can contain histidine and aromatic residues [4,5]. MTs were first discovered in horse kidney, although they have been isolated from kidney and liver

Sequences of Transcripts Encoding UcMTs
Total RNA and mRNAs were isolated from U. compressa cultivated with 10 µM copper for three days. The 5 and 3 untranslated regions (UTR) of transcripts encoding three UcMTs were amplified using RACE-PCR technique, as well as the open reading frame (ORF) using conventional PCR. UcMT1.1 transcript (formerly Crassostrea-like mt) and protein are described in Table 1 (Figure 1). It is important to mention that two other transcripts that were closely related with UcMT1.1 were isolated, corresponding to UcMT1.2 and UcMT1.3; UcMT1.2 showed a deletion of 48 nucleotides after G in position 471 of the 3´UTR region of UcMT1.1, and UcMT1.3 displayed the same deletion mentioned before and a deletion of 58 nucleotides after C in position 607 in the 3´UTR of UcMT1.1 (Figure 1). UcMT2 transcript (formerly Mytilus-like mt) and protein is described in Table 1 ( Figure 2) as well as UcMT3 transcript (formerly Dreissena-like mt) and protein ( Figure 3). The linker region of UcMT1.1, UcMT2, and UcMT2 correspond to, 9,9, and 23 amino acids, respectively (Figures 1-3).

Sequences of Transcripts Encoding UcMTs
Total RNA and mRNAs were isolated from U. compressa cultivated with 10 µM copper for three days. The 5′and 3′untranslated regions (UTR) of transcripts encoding three UcMTs were amplified using RACE-PCR technique, as well as the open reading frame (ORF) using conventional PCR. UcMT1.1 transcript (formerly Crassostrea-like mt) and protein are described in Table 1 (Figure 1). It is important to mention that two other transcripts that were closely related with UcMT1.1 were isolated, corresponding to UcMT1.2 and UcMT1.3; UcMT1.2 showed a deletion of 48 nucleotides after G in position 471 of the 3´UTR region of UcMT1.1, and UcMT1.3 displayed the same deletion mentioned before and a deletion of 58 nucleotides after C in position 607 in the 3´UTR of UcMT1.1 (Figure 1). UcMT2 transcript (formerly Mytilus-like mt) and protein is described in Table 1 ( Figure 2) as well as UcMT3 transcript (formerly Dreissena-like mt) and protein ( Figure 3). The linker region of UcMT1.1, UcMT2, and UcMT2 correspond to, 9,9, and 23 amino acids, respectively (Figures 1-3). UcMT1.2 presents a deletion of 48 nucleotides located after the G in position 471 in the 3′UTR of MT1.1, and the deletion is underlined. UcMT3 present the deletion previously mentioned, and a deletion located after the C in position 607 in the 3′UTR of UcMT1.1, and the deletion is underlined.

Similarities in Amino Acids of UcMTs and Hierarchical Clustering of MTs
UcMT1.1 and UcMT2 were more closely related among each other and shared 52.2% identity and 60% similarity in amino acids; indeed, both sequences contained arrangement of cysteines corresponding to 3 CC and 9 CXC motifs, but UcMT2 also contained a CXXC motif ( Figure 4A). In contrast, UcMT3 shared only 30% similarity in amino acids with MT1.1 and MT2 and contained arrangements of cysteines corresponding to 7 CC and 7 CXC ( Figure 4A). UcMT3 showed an extra N-terminal sequence of 10 amino acids, an internal additional sequence of 19 amino acids, and an extra C-terminal sequence of 8 amino acids, compared with UcMT1.1 and UcMT2 ( Figure 4A). Thus, UcMT1 and UcMT2 may have derived from UcMT3 by deletions of initial, internal, and terminal nucleotide sequences.
The hierarchical clustering of vertebrate, invertebrate, and plants MTs constructed with 237 protein sequences (including the 3 UcMTs) demonstrated that UcMT1.1 and UcMT2 grouped mainly with marine crustacean MTs, such as those of the lobster Homarus americanus and the crabs Carcinus maena and Scylla serrata ( Figure S1). In addition, UcMT3 clustered with the nematode C. elegans MTs, as well as with MTs of marine equinoderms MTs such as those of Sterechinus neumayeri, Strongylocentrus purpuratus, and Paracentrotus livudus ( Figure S1). On the other hand, UcMTs grouped as a different clade with MTs from Rodophyceae and Phaeophyceae ( Figure 4B). Thus, U. compressa

Similarities in Amino Acids of UcMTs and Hierarchical Clustering of MTs
UcMT1.1 and UcMT2 were more closely related among each other and shared 52.2% identity and 60% similarity in amino acids; indeed, both sequences contained arrangement of cysteines corresponding to 3 CC and 9 CXC motifs, but UcMT2 also contained a CXXC motif ( Figure 4A). In contrast, UcMT3 shared only 30% similarity in amino acids with MT1.1 and MT2 and contained arrangements of cysteines corresponding to 7 CC and 7 CXC ( Figure 4A). UcMT3 showed an extra N-terminal sequence of 10 amino acids, an internal additional sequence of 19 amino acids, and an extra C-terminal sequence of 8 amino acids, compared with UcMT1.1 and UcMT2 ( Figure 4A). Thus, UcMT1 and UcMT2 may have derived from UcMT3 by deletions of initial, internal, and terminal nucleotide sequences.
The hierarchical clustering of vertebrate, invertebrate, and plants MTs constructed with 237 protein sequences (including the 3 UcMTs) demonstrated that UcMT1.1 and UcMT2 grouped mainly with marine crustacean MTs, such as those of the lobster Homarus americanus and the crabs Carcinus maena and Scylla serrata ( Figure S1). In addition, UcMT3 clustered with the nematode C. elegans MTs, as well as with MTs of marine equinoderms MTs such as those of Sterechinus neumayeri, Strongylocentrus purpuratus, and Paracentrotus livudus ( Figure S1). On the other hand, UcMTs grouped as a different clade with MTs from Rodophyceae and Phaeophyceae ( Figure 4B). Thus, U. compressa MTs are more closely related with marine invertebrate MTs and the terrestrial invertebrate C. elegans MTs, and not to MTs from other marine macroalgae. MTs are more closely related with marine invertebrate MTs and the terrestrial invertebrate C. elegans MTs, and not to MTs from other marine macroalgae.

Expression of UcMTs-GST in Bacteria and Detection of GST-Tag
The ORFs of UcMTs were cloned in an E. coli expression vector, which allows the expression of MTs fused with the enzyme glutathione-S-transferase (GST) from the platyhelminthe Schistosoma japonicum (26 kDa), an enzyme that contain a single cysteine in the N-terminal domain, and do not bind metals. After 1 to 12 h of culture, the induction of protein expression with IPTG allows the visualization of increasing levels of UcMT1.1-GST (34.2 kDa. Figure 5A), UcMT2-GST (35.05 KDa, Figure 5B), and UcMT3-GST (39.4 KDa, Figure 5C). These proteins were detected by Western blot using an antibody prepared against S. japonicum GST indicating that the overexpressed proteins correspond to UcMTs fused with GST ( Figure 5C).

Expression of UcMTs-GST in Bacteria and Detection of GST-Tag
The ORFs of UcMTs were cloned in an E. coli expression vector, which allows the expression of MTs fused with the enzyme glutathione-S-transferase (GST) from the platyhelminthe Schistosoma japonicum (26 kDa), an enzyme that contain a single cysteine in the N-terminal domain, and do not bind metals. After 1 to 12 h of culture, the induction of protein expression with IPTG allows the visualization of increasing levels of UcMT1.1-GST (34.2 kDa. Figure 5A), UcMT2-GST (35.05 KDa, Figure 5B), and UcMT3-GST (39.4 KDa, Figure 5C). These proteins were detected by Western blot using an antibody prepared against S. japonicum GST indicating that the overexpressed proteins correspond to UcMTs fused with GST ( Figure 5C).

UcMTs-GST-Mediated Accumulation of Copper or Zinc In Vivo
Transformed bacteria expressing MTs-GST were cultivated with 0.5 mM IPTG for 30 min, and with 1 mM copper and IPTG for 6 h. Control bacteria expressing only GST accumulate 0.35 µg of copper mg -1 of dry weight (DW), whereas those expressing UcMT1.1-GST, UcMT2-GST, and UcMT3-GST accumulate 1.8, 1.7, and 1.4 times more copper than the control, respectively ( Figure 6A). On the other hand, transformed bacteria were cultivated with 0.5 mM IPTG for 30 min and, with 1 mM zinc and IPTG for 6 h. Control bacteria accumulate 0.49 µg mg -1 of zinc mg -1 of DW whereas those expressing UcMT1.1-GST, UcMT2-GST, and UcMT3-GST accumulate 4,1, 3.8, and 3.4 times more zinc than the control, respectively; although these increases were not significantly different among each other ( Figure 6A). Thus, expression of UcMTs-GST mediates accumulation of copper and zinc in vivo.

UcMTs-GST-Mediated Accumulation of Copper or Zinc In Vivo
Transformed bacteria expressing MTs-GST were cultivated with 0.5 mM IPTG for 30 min, and with 1 mM copper and IPTG for 6 h. Control bacteria expressing only GST accumulate 0.35 µg of copper mg −1 of dry weight (DW), whereas those expressing UcMT1.1-GST, UcMT2-GST, and UcMT3-GST accumulate 1.8, 1.7, and 1.4 times more copper than the control, respectively ( Figure 6A). On the other hand, transformed bacteria were cultivated with 0.5 mM IPTG for 30 min and, with 1 mM zinc and IPTG for 6 h. Control bacteria accumulate 0.49 µg mg −1 of zinc mg −1 of DW whereas those expressing UcMT1.1-GST, UcMT2-GST, and UcMT3-GST accumulate 4,1, 3.8, and 3.4 times more zinc than the control, respectively; although these increases were not significantly different among each other ( Figure 6A). Thus, expression of UcMTs-GST mediates accumulation of copper and zinc in vivo.

UcMTs-GST-Mediated Accumulation of Copper or Zinc In Vivo
Transformed bacteria expressing MTs-GST were cultivated with 0.5 mM IPTG for 30 min, and with 1 mM copper and IPTG for 6 h. Control bacteria expressing only GST accumulate 0.35 µg of copper mg -1 of dry weight (DW), whereas those expressing UcMT1.1-GST, UcMT2-GST, and UcMT3-GST accumulate 1.8, 1.7, and 1.4 times more copper than the control, respectively ( Figure 6A). On the other hand, transformed bacteria were cultivated with 0.5 mM IPTG for 30 min and, with 1 mM zinc and IPTG for 6 h. Control bacteria accumulate 0.49 µg mg -1 of zinc mg -1 of DW whereas those expressing UcMT1.1-GST, UcMT2-GST, and UcMT3-GST accumulate 4,1, 3.8, and 3.4 times more zinc than the control, respectively; although these increases were not significantly different among each other ( Figure 6A). Thus, expression of UcMTs-GST mediates accumulation of copper and zinc in vivo.

Discussion
In this work, we isolated the complete sequence of transcripts encoding three MTs from the marine alga U. compressa. These transcripts encode UcMT1.1, UcMT2, and UcMT3 with a MW of 8.3, 9.05, and 13.4 kDa, respectively, and containing 24-30% of cysteines. It is important to mention that UcMT1.1 and UcMT2 seemed to be structurally more related among each other than to UcMT3. UcMT1.1 and UcMT2 showed deletions in amino acids sequences compared with MT3 at the initial, internal, and terminal part of the protein compared with UcMT3. Thus, UcMT2 and UcMT3 genes may have derived from UcMT3 gene. In the case of UcMT1, it seems that more than a single copy exists in U. compressa genome since three different transcripts of UcMT1 were isolated corresponding to UcMT1.1, UcMT1.2 and UcMT1.3. In this sense, it has been shown that the increase in number of copies of Cup1 MT gene in the yeast S. cereviciae allowed an increased tolerance to copper [30]. In addition, several copies of domains α and β of MTs exist in the mollusk C. gigas [12]. Thus, it is not surprising that UcMT1 may be present in multiple copies in U. compressa genome, but the latter remained to be confirmed. The sequencing of U. compressa genome is already in course.
Interestingly, UcMTs resemble marine invertebrate MTs. Mollusk MTs are constituted by 73-75 amino acids and contain 20-21 cysteine residues (28%), arranged mainly as CXC and CC motifs [5]. UcMTs are longer than mollusk MTs since they are constituted by 81, 90, and 139 amino acids; although the content of cysteines is similar to mollusk MTs (28-30%). However, mollusk MTs reported until now do not contain histidine or aromatic residues [5]. In addition, MT from marine crustacean and equinoderms showed cysteines (29-30%) arranged as CC and CXC and they do not contain histidine or aromatic aminoacids [25,26]. It is important to mention that we showed that UcMT1.1 contains a tyrosine, UcMT2 a histidine, and UcMT3 a tryptophan. In this sense, the two MTs found in the worm C. elegans are constituted by 75 amino acids and contain 19 cysteines (25%), arranged as CXC and CC, and contain a tyrosine and histidine residues [8]. Thus, UcMTs are more similar to C. elegans MTs but longer and contain a higher percentage of cysteines (28-30%). This indicates that UcMTs are unique MTs, and their sequences are more closely related with MTs of marine invertebrates and C. elegans MTs. It is not surprising that UcMTs showed similarities to marine invertebrate MTs since F. vesiculosus MT is also more related to mollusk MTs than to plant MTs [20]. F. vesiculosus MT showed a linker of 14 amino acids, which is longer than the linker of vertebrates MTs (4 aa) and the linkers of plant MTs (4 and 40 aa) [20]. The linker of UcMTs has a size of 9-13 amino acids that is more closely related with F. vesiculosus and invertebrate MTs linker size.
In addition, the sequence of UcMTs is different from the MT cloned from F. vesiculosus, which is constituted by 67 amino acids (6.9 kDa) and contains 16 cysteine residues (24%) arranged as CXC, but not as CC [3]. The only structural similarity of Fucus mt is that this transcript showed a short 5 UTR of 64 nucleotides and a long 3 UTR of 960 nucleotides [8]. Furthermore, MTs identified in the genomes of Rodophyceae C. cripus and E. denticulatum are constituted by 69 to 71 amino acids and contain 12 to 14 cysteines (20%) arranged mainly as CXC, but not as CC [2]. Furthermore, UcMTs grouped in a different clade compared with MTs from other marine macroalgae. Thus, UcMTs of the green macroalga U. compressa are distantly related with those found in red and brown macroalgae.
On the other hand, we showed that UcMTs-GST mediates the accumulation of copper and zinc in bacteria. In particular, UcMT1.1 and UcMT2, which are structurally more closely related, allowed higher accumulation of copper compared with UcMT3. Moreover, the three MTs allowed accumulation of zinc with similar efficiencies among them. Thus, the three UcMTs differentially bind copper and similarly bind zinc in vivo but their affinity for copper and zinc need to be further investigated. In this sense, it has been demonstrated that MTs are either more Zn-or Cu-binding thioneins, preference associated with cysteine arrangements and the nature of the other amino acids that constitute the MT [4,31]. Likewise, mouse MT1, MT2, and MT3 are more Zn-thionein and, in contrast, yeast cup-1, Drosophila MntA and MntB, and mouse MT4, are more Cu-thioneins [4,31]. Thus, the nature of UcMTs regarding their affinity for copper or zinc need to be further investigated.
It is now clearly established that green and red macroalgae are more closely related among each other, and with terrestrial plants, than with brown macroalgae [32][33][34]. Green and red macroalgae belong to the kingdom Plantae, as terrestrial plants [35] whereas brown macroalgae belong to the kingdom Chromalveolta [34]. The latter is based on the observations that green and red algae contain a plastid that derive from a single event of endosymbiosis by a cyanobacteria, whereas brown algae plastids derive from a secondary or tertiary endosymbiosis event of green or red microalgae [32,33]. Thus, it is unexpected that the three UcMTs of the green macroalga U. compressa are not closely related with other marine alga MTs, in particular to red macroalgae MTs. In contrast, the major similarity is with MTs of marine invertebrate and C. elegans MTs. Considering that marine algae appeared on earth around a billion years ago, and marine invertebrates emerged around 500 million years ago [34,36] and, moreover, that UcMTs are longer than MTs of marine invertebrates, it is then possible that marine invertebrates have acquired MTs genes from marine green macroalgae by horizontal gene transfer; however, this hypothesis need to be further investigated. Furthermore, it is possible that U. compressa contain additional MTs that differentially bind copper, zinc, and other heavy metals, as it has been predicted in [23], but the latter need to be further analyzed. In this sense, it has been shown that the equinoderm Paracentrotus livudus exhibits 7 MTs [35] suggesting that the four other potential UcMTs can be functional MTs. Thus, additional UcMTs may exist in the genome of U. compressa and these MTs will be cloned and characterized in the future.
In conclusion, we showed that transcripts encoding three MTs were cloned and sequenced; they encode unique MTs with homology with marine invertebrate and C. elegans MTs. UcMTs expressed in bacteria allowed copper and zinc accumulation in vivo. Thus, it is likely that these UcMTs may participate in copper and zinc accumulation in the marine alga U. compressa.

Sampling of Algae and Water Collection
The green macroalga U. compressa was collected during spring 2017 from the high intertidal zone at Cachagüa Beach (32 • 34 S), a site in central Chile with no history of metal pollution [23]. Algal samples were transported to the laboratory in sealed plastic bags inside a cool-box. In the laboratory, material was rinsed three times with filtered seawater, cleaned manually, and sonicated three times for 2 min using an ultrasound bath (HiLab Innovation Systems, model SK221OHP) to remove epiphytes. The algae were maintained in aerated seawater under an irradiance of 50 µmoles m −2 s −1 on a photoperiod of 12 h:12 h light:dark cycle, at 14 • C for 4 days, prior to experimentation. Seawater was obtained from Quintay (33 • 12 S), a pristine site in central Chile, filtered through 0.45 and 0.2 µm pore size membranes and stored in darkness at 4 • C.

Algal Culture and RNA Extraction
U. compressa (1 g of FT) was cultivated in 100 mL of filtrated seawater containing 10 µM CuCl 2 (Merck, Darmstat, Germany) for 3 days. The alga was washed with 10 mM Tris-50 mM EDTA pH 7.0, in order to eliminate copper and other metals bound to cell walls [37].

Purification of Total RNAs and mRNAs for RACE-PCR
Total RNAs were extracted as described in [38]. U. compressa (150 mg of FT) was frozen in liquid nitrogen and pulverized in a mortar. One mL Trizol reagent (Invitrogen, Carlsbad, CA, USA) was added and the alga was homogenized with a pestle until thawing. The mixture was centrifuged at 12,000× g for 10 min at 4 • C, and the supernatant was recovered. Chloroform (200 µL) was added and the mixture was vortexed for 10 s and left at room temperature for 3 min. The mixture was centrifuged at 12,000× g for 15 min at 4 • C, and the aqueous phase was recovered. Isopropanol (500 µL) was added and the solution incubated for 10 min at room temperature. The solution was centrifuged at 12,000× g for 10 min at 4 • C, and the supernatant removed. The pellet was washed twice with 1 mL of 75% ethanol, gently vortexed, and centrifuged at 7000× g for 5 min at 4 • C. The ethanol phase was removed, the pellet dried for 15 min at room temperature, dissolved in 50 µL of ultrapure water treated with DEPC (water-DEPC), and incubated for 10 min at 60 • C.
Total RNAs were quantified using Nanodrop spectrophotometer (Tecan, Zürich, Switzerland); the integrity was verified by agarose gel electrophoresis and stored at −80 • C. Messenger RNAs were purified from 100 µg of total RNA using NucleoTrap mRNA minikit (Macherey-Nagel, Düren, Germany), mRNAs were eluted in 25 µL of water-DEPC, and normally 1 µg of mRNAs was obtained from 100 µg of total RNAs.
The first round of amplification was performed using PCR kit (Favorgene, London, UK) and 1 µL of C-tailed cDNAs mixed with 10 µL of PCR mixture, 0.6 µL of 5 RACE adapter primer (5 GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG3 ), and with 0.6 µL of primer 4 for MT1 (5 GGCATACGCACGTCTCGGG3 ), primer 5 for MT2 (5 CTGCGTAACGACATAGCCGA3 ), or primer 6 for MT3 (5 GCAGCCAGAATCGCAACTAC3 ), at 10 µM, and 6.8 µL of water-DEPC to complete a final volume of 20 µL. The mixture was incubated at 95 • C for 3 min and subjected to 40 cycles of denaturation at 95 • C for 5 s, hybridization at 63 • C for 10 s and amplification at 72 • C for 15 s, using a real-time thermocycler RotorGene 6000. The second round of amplification was performed with the mixture diluted 100 times in distilled water-DEPC, and 14.4 µL of the cDNAs were mixed with 10 µL of PCR mixture, 0.6 µL of abridged universal adaptor primer (AUAP) (5 GGCCACGCGTCGACTAGTAC3 ) and primer 7 for MT1, (5 GCAACCATCTTCGGTTTGGC3 ), primer 8 for MT2, (5 ATCCTTCGCGGGTGAGCAAG3 ), and primer 9 for MT3 (5 CACAGTTGCATTCTGCGGTT3 ) at 10 µM and with 6.8 µL of water-DEPC to complete a final volume of 20 µL. The amplification was performed using 40 cycles of amplification mentioned before. The amplified fragments were analyzed in a 2% agarose gel, stained with SYBR green (Invitrogen, Carlsbad, CA, USA), and visualized on a UV trans-illuminator.

Amplification of 3 RACE cDNAs
Initial cDNAs were obtained using MMLV reverse transcriptase kit (Promega, Madison, WI, USA). To this end, 6.8 µL of mRNAs (250 ng) were mixed with 5 µL of 3 RACE adapter primer with an oligo-dT tail (5 GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT3 ) at 10 µM and with 3.2 µL of water-DEPC to complete a final volume of 15 µL. The mixture was denatured at 65 • C for 5 min and then cooled on ice for 1 min. Then, 0.7 µL of RNAse inhibitor (10 U µL −1 ), 5 µL of MMLV buffer 5×, 1.5 µL of dNTPs mixture (10 mM of each dNTP), 1 µL de MMLV reverse transcriptase (200 U µL −1 ), and 1.8 µL of water-DEPC were added to complete a final volume of 25 µL. The mixture was incubated at 42 • C for 1 h and at 70 • C for 10 min to inactivate MMLV reverse transcriptase and diluted to 50 µL with water-DEPC. cDNAs were treated with RNAse H, purified using GEL/PCR purification mini kit (Favorgen Biotech Corp., Changzhi, Taiwan), and eluted in 40 µL of water-DEPC.
The first round of amplification was performed using PCR kit (Favorgene, London, UK) and 1 µL of cDNAs were mixed with 0.6 µL of AUAP (5 GGCCACGCGTCGACTAGTAC3 ), 0.6 µL of primer 10 for MT1 (5 CAGTGCCAAACCGAAGATGG3 ), primer 11 for MT2 (5 GATGAGGGCTGTCCTTGCTC3 ), or primer 12 for MT3 (5 AGTGTGATGCTGAGTGCTGT3 ) at 10 µM and 6.8 µL of water-DEPC to complete a final volume of 20 µL. The mixture was incubated at 95 • C for 3 min and subjected to 40 cycles of denaturation at 95 • C for 5 s, hybridization at 63 • C for 10 s, and amplification at 72 • C for 15 s, using a real-time thermocycler RotorGene 6000. The second round of amplification was performed with the mixture diluted 100 times in distilled water-DEPC, and 14.4 µL of the cDNAs were mixed with 10 µL of PCR mixture, 0.6 µL of AUAP forward (5 GGCCACGCGTCGACTAGTAC3 ) and with primer 13 for MT1 (5 GGTTGCAAGTGCTAGCTGAC3 ), primer 14 for MT2 (5 GCTTGTTAGGCCTCAGTGGT3 ), or primer 15 for MT3 (5 TGTCAGTGCGACAGCCTAA3 ) and with 6.8 µL of water-DEPC to complete a final volume of 20 µL. The amplification was performed using 40 cycles of amplification mentioned before. The amplified fragments were analyzed in a 2% agarose gel, stained with SYBR green (Invitrogen, Carlsbad, CA, USA) and visualized on a UV trans-illuminator.

Cloning of UcMTs 5 and 3 UTRs, and UcMT ORFs in pGEM-T Vector
The 5 RACE and 3 RACE amplification fragments obtained in the second PCR (see above) and those of UcMTs ORFs were subjected to electrophoresis in 2% agarose gel. The piece of agarose gel containing the stained fragments was removed from the gel and placed in Eppendorf tubes. The amplified fragments were eluted from agarose using Gel/PCR purification kit (Favorgen, London, UK), recovered in 50 µL of water-DEPC and stored at 4 • C. Amplified fragments were ligated with the cloning vector pGEM-T easy (Promega, Madison, WI, USA) and transformed in E. coli competent cells One Shot TOP 10 (Invitrogen, Carlsbad, CA, USA). Transformed E. coli cells were cultivated in 10 mL of LB medium (10 g tryptone, 5 g yeast extract, and 100 g NaCl in 1 L of distilled water) supplemented with 100 µg mL −1 of ampicillin. The culture was centrifuged at 3,000 x g for 5 min in a centrifuge model Nuwind (Nuaire, Plymouth, MN, USA). Transformed pGEM-T vectors were purified from the bacterial pellet using Wizard Plus SV Miniprep DNA Purification System (Promega, Madison, WI, USA). To check cloning of 5 UTR fragments in pGEM-T, primers AUAP and primers 7, 8, and 9 were used. To check cloning of 3 UTR in pGEM-T, primer AUAP and primers 13, 14, and 15 were used. PCR conditions were identical to those used for the amplification of 5 and 3 RACE ends (mentioned above). To amplify the ORFs, primers 16 and 17 for MT1, primers 18 and 19 for MT2, and primers 20 and 21 for MT3 were used and to verify the insertion PCR conditions were those used to amplify ORFs (mentioned above). Cloned fragments were sequenced using an ABI3730XL (Macrogen, Seoul, Korea).

Cloning of UcMTs ORFS in pGEX Expression Vector
UcMTs were synthesized and subjected to codon optimization for expression in E. coli by Genscript (Piscataway, NJ, USA) and then ligated to the expression vector pGEX-5X-1 (Genscript) which allowed fusion of UcMTs ORFS with the enzyme glutahione-S-transferase (GST) from the platyhelminthes Schistosoma japonicum (26 Kda), an enzyme containing a single cysteine that does not bind metals; the fusion proteins were named UcMT1.1-GST, UcMT2-GST, and UcMT3-GST. The recombinant vectors were sequenced by Genscript to verify the correct insertion of complete ORFs.

Transformation of Expression Vectors in Bacteria
The recombinant expression vectors were transformed in competent E. coli strain BL21 (DE3) (Sigma-Aldrich, Saint Louis, MO, USA). To this end, 200 µL of competent cells BL21 (DE3) were incubated with 50 ng of recombinant expression vector containing UcMTs-GST. Then, 800 µL of SOC medium (2% (w/v) tryptone, 0.5% (w/v) of yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mm MgSO 4 , and 20 mM glucose) were added, and cultivated at 37 • C for 45 min. An aliquot of 200 µL was cultured on solid LB medium containing 100 µg L −1 of carbenicillin in a Petri dish overnight. Individual colonies were selected for each UcMT, cultivated with LB medium, and stored at −80 • C in LB medium containing 15% glycerol.

Purification of UcMTs-GST by Affinity Column
A sample of 2 mL of E. coli transformed with expression vectors containing a UcMT-GST were added to 100 mL LB medium containing 100 µg mL −1 ampicillin and cultured at 37 • C overnight. A sample of 10 mL was added to 1 L of LB medium containing 100 µg mL −1 ampicillin and 0.5 mM IPTG, in quadruplicate, until OD 600 = 0.6 (aprox. 2.5 h). The 4 L of culture were centrifuged at 6000× g for 10 min. The pellet was washed twice with 300 mL of PBS and bacteria were suspended in 10 mL of PBS containing 5 mM dithiotreitol (DTT) and 1 tablet of protease inhibitor cocktail (Roche, Manheim, Germany). The bacterial suspension was sonicated for 20 s, with 20 s of pause, for 5 min. The suspension was centrifuged at 6000 rpm for 10 min and supernatant was recovered. Protein concentration was determined using Bradford method [39] and adjusted with PBS-5 mM DTT (PBS-DTT) to 1 mg mL −1 . UcMTs-GST were purified by HPLC using GSTrap HP (General Electric, Uppsala, Sweden) at 5 bars of pressure, washed with PBS-DTT, and eluted with 3 mL of buffer 50 mL Tris-HCl-10 mM GSH. Normally, 1-2 mg of purified UcMT-GST was obtained from 4 L of bacterial culture and proteins were quantified using Bradford method [40].

Detection of UcMTs-GST with Anti-GST Antibody
Transformed bacteria were cultured in 100 mL of LB medium until OD 600 = 0.6, 0.5 mM IPTG was added and the mixture incubated for 6 h. The culture was centrifuged at 6000× g for 5 min, the pellet suspended in 5 mL of buffer PBS, and sonicated for 20 s, with 20 s of pause, for 5 min. Proteins (20 µg) were separated by electrophoresis in a denaturant 12% polyacrylamide gel and transferred to a nitrocellulose membrane for 10 min using Trans Blot Turbo apparatus (BioRad, Hercules, CA, USA). The membrane was stained with Ponceau Red dye and washed with 10 mL of distilled water. The membrane was incubated in 10 mL TTBS (20 mM Tris-HCl pH 7.5, 0.1 mM NaCl, 0.05% Tween-20) containing 5% skim milk for 1 h, washed twice with 10 mL TTBS for 15 min, incubated with 10 mL TTBS containing 3% skim milk and the antibody anti-GST (Sigma-Aldrich, St Louis, MO, USA) diluted 5000 times, and washed four times in TTBS for 15 min. The membrane was incubated in TTBS containing 3% skim milk and the secondary antibody prepared against rabbit IgG coupled to hoseradish peroxidase (Agrisera, Vännas, Sweden) diluted 2000 times, for 1 h, and washed four times with TTBS for 15 min. Proteins were detected using ECL Western Blotting System kit (Amersham, Buckinghamshire, UK) and revealed using a C-Digits chemiluminiscence Western blot scanner Li-Cor (Lincoln, NE, USA) and Image Studio Digits software version 4.0 Li-Cor.

Quantification of Copper and Zinc in Bacteria Expressing UcMTs-GST
Recombinant bacteria were cultured in 100 mL of LB medium containing 100 mg mL −1 of carbenecillin until DO 600 = 0.6, with 0.5 mM IPTG for 30 min, and with 1 mM CuSO 4 or 1 mM ZnCl 2 and IPTG for 6 h. Bacterial pellets showed a weight of 26-42 mg for copper cultures and 46-62 mg for zinc cultures. Pellets were dried at 60 • C for 48 h, suspended in 5 mL of 60% (v/v) HNO 3 , and incubated at 85 • C for 2 h. The solutions were filtered through 0.22 µm MCE filters (TCL, Santiago, Chile) and analyzed by flame atomic emission spectrophotometry using an atomic emission spectrophotometer ThermoFisher (Waltham, MA, USA).

Hierarchical Clustering of UcMTs
Amino acid sequences corresponding to MTs of different animal and plant species (234 in total) were selected from revised SwissProt repository of the UniprotKB database (www.uniprot.org). Alignment of these sequences was performed with Clustal W software with default setting. This alignment was used to generate the phylogenetic reconstruction to represent a hierarchical clustering using UPGMA algorithm based on distance. Phylogenetic and hierarchical clustering analyses were conducted using MEGA software version X [39].

Statistical Analyses
Statistical analyses were performed with the Prism 6 statistical package (Graph Pad software Inc., San Diego, CA, USA). Following confirmation of normality and homogeneity of variance, significant differences among treatments were determined by two-way ANOVA and Tukey's multiple comparison post-hoc test, at a 95% confidence interval.