Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis.

The primary structure of the cDNA and metallothionein (MT) genomic sequences of the tropical green mussel (Perna viridis) was determined. The complete cDNA sequences were obtained using degenerate primers designed from known metallothionein consensus amino acid sequences from the temperate species Mytilus edulis. The amino acid sequences of P. viridis metallothionein deduced from the coding region consisted of 72 amino acids with 21 cysteine residues and 9 Cys-X-Cys motifs corresponding to Type I MT class of other species. Two different genomic sequences coding for the same mRNA were obtained. Each putative gene contained a unique 5'UTR and two unique introns located at the same splice sites. The promoters for both genes were different in length and both contained metal responsive elements and active protein-binding sites. The structures of the genomic clones were compared with those of other species. J. Exp. Zool. 284:445-453, 1999.     

Roles of the conserved serines of metallothionein in cadmium binding

The sequence of six amino acid residues -Ser-Cys-Cys-Ser-Cys-Cys- is present in all mammalian metallothionein sequences and has been highly conserved during evolution, although the metallothioneins have divergent primary sequences. To determine whether two serines in the sequence play a crucial role in metalbinding of metallothioneins, a mutant metallothionein with these two serines replaced by leucines was obtained using anEscherichia coli expression system. The expressed protein was analyzed for its chemical and spectroscopic properties. It was confirmed that the mutant metallothionein (MT) bound cadmium through a metal-thiolate complex and that there was no strong difference between the mutant and the wild-type MTs in retaining the metal-binding cluster. However, the metal-binding cluster of the mutant metallothionein was more unstable than that of the wild-type metallothionein. The two conservative serines could play a role in the stability of metal-binding ligands.     

Multiple isoforms of metallothionein are expressed in the porcine liver

Isogenes are highly homologous to each other and are often difficult to ascertain, as has been the case with metallothionein, a metal-binding protein rich in cysteines. Conventional separation of metallothionein isoforms relied on ion exchange chromatography of the proteins, or screening for the sequences from gene libraries. In this study, a combination of RT–PCR and partial protein sequencing is used in the identification of metallothionein isogenes expressed in porcine liver. By this approach, we have identified expressed coding sequences which constitute 10 new isogenes. Of the four known groups of metallothioneins (MT), phylogenetic analyses place these pig isogenes in the MT-1 group, except two which are identified as being closely related to MT-2, and none in groups 3 and 4. The isogenes are thus named pMT-1a to -1g, and pMT-2a and -2b. While each of the isogene sequences is unique, two isogenes, pMT-1e1 and pMT-1e2, share an identical amino acid sequence, differing only in specific codons. Two others, pMT-1b and pMT-1g, have a cysteine substituted by arginine, the first such sequence ever detected in MT. pMT-2a and pMT-2b are closely aligned with the MT-2 group of vertebrates, in spite of the absence of a characteristic acidic amino acid at position 10 or 11, common in other mammalian metallothioneins.     

A human metallothionein pseudogene containing AG/CT repetitive elements

Summary A lambda phage recombinant clone, 25 S, which contains a 15.5-kb EcoRI human genomic DNA fragment, has been characterized. Restriction mapping and Southern blot hybridization indicated a 3.0-kb HindIII fragment containing metallothionein (MT)-like sequences. Several interesting features were found upon comparison of this nucleotide sequence with that of other human MT genes: (1) sequences representing the 5 regulatory region, the 5 untranslated region, and the first exon are not contained in the 3.0-kb HindIII fragment; (2) the coding sequence of the second exon (amino acids 10–31 encoding a portion of the -domain of the MT protein) has 11 amino acid changes out of a total of 21, whereas, the third exon (amino acids 32–61, representing the complete -domain of the MT protein) has only 4 amino acid substitutions; however, all cysteine residues are conserved; (3) this MT-like gene retains intron sequences and processing signals; (4) Southern blot analysis of human genomic DNA indicated this MT-like gene is located on a 10.5-kb EcoRI genomic DNA fragment; and (5) unusual AG/CT-rich repetitive elements are located within the second intron and upstream of the second exon of this MT-like gene. This gene is not expressed in response to metal induction in two human cell lines, as shown by northern blot analyses. Based on these observations, this MT-like gene represents a unique nonprocessed pseudogene of the human MT multigene family.     

蓝藻的类金属硫蛋白及其研究进展

单细胞蓝藻中的类金属硫蛋白已经得到分离纯化,并在蛋白质水平上与标准的哺乳动物金属硫蛋白做了对比性分析,发现二者氨基酸组成和序列差异很大,前者只形成一个结构域,但二级结构和金属结合性质具有一定的相似性,是进化上功能趋同的表现．同时克隆并分析了它的基因ORF结构,研究了金属诱导和逆向转录抑制对于蛋白质表达的调控及类似于哺乳动物金属硫蛋白基因具有的放大和重排现象机理,提出了近期研究的重点和方向．     

Cloning and sequencing of a novel metallothionein gene in Mytilus galloprovincialis Lam

Metallothionein (MT) is a ubiquitous, metal-inducible protein with an important role in the homeostasis and in the detoxification of heavy metals. This work reports the cloning and sequencing of a MT gene encoding a MT isoform (MT20-IIIa) in the mussel Mytilus galloprovincialis Lam, a lamellibranch mollusc known to accumulate and to detoxify large amounts of metal. The MT gene, lacking the 5' promoter region, is 1865 bp long and has a tripartite structure consisting of three exons and two introns. The putative open reading frame (ORF) encodes a polypeptide of 72 amino acids, which corresponds to the MT-I class, type 2 family (http://www.unizh.ch/~mtpage/classif.html). The structure of the gene and the putative MT20-III protein have been compared with those of other species. The putative biological significance of the differences at the amino acid level among the different MTs is discussed.     

Human metallothionein genes: structure of the functional locus at 16q13

The functional human metallothionein (MT) genes are located on chromosome 16q13. We have physically mapped the functional human MT locus by isolation and restriction digest mapping of cloned DNA. The mapped region contains all sequences on chromosome 16 that hybridize to metallothionein gene probes and comprises 14 tightly linked MT genes, 6 of which have not been previously described. This analysis defines the genetic limits of metallothionein functional diversity in the human genome.     

Pigeon metallothionein consists of two species

Two isospecies of metallothionein, a cysteine-rich protein that binds metals, exist in all mammals examined, but only one in some invertebrates and lower animals. Lower vertebrates such as fish and birds have one or two metallothionein genes depending upon the organism. In this study, we show by amino acid sequence determinations that two isospecies of metallothionein, 75% homologous to each other, can be induced by zinc to accumulate in pigeon livers. This is in contrast to single isospecies found in chicken and duck. Each of these two sequences consists of 63 amino acids, with all 20 cysteines in positions held invariant in most if not all class I mammalian metallothioneins. One of these two pigeon isometallothioneins is terminated with histidine at the carboxyl end, which is apparently unique to avians. Its sequence differs from that of duck and chicken by only four substitutions and is the predominant isospecies that accumulates upon induction. The other pigeon metallothionein has lysine at its carboxyl terminus and is devoid of arginine. None of these isospecies carries any aromatic amino acid, which is also characteristic of all higher metallothioneins. As this is the first demonstration with sequence data that two isospecies of metallothionein indeed exist in birds, these results suggest that pigeon metallothionein genes evolved from an ancestral form through duplication and mutation upon specification.     

Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality

Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.     

Different types of hypersensitive sites in the mouse metallothionein gene region

We have examined the chromatin structure of the metallothionein (MT) gene region in MT- S49 mouse lymphoma cells and in derivatives which express MT-I alone, MT-II alone, or both genes. In all lines, these genes are contained in a 16-kilobase pair region between two DNase I sensitive sites: one site located 5.3 kilobase pairs 5' of MT-II (the 5' gene) is present in naked DNA and retained in the chromatin of all lines; the other site located 3.1 kilobase pairs 3' of MT-I is hypersensitive. Hypersensitivity at three other sites is dependent on the expression of MT genes. Two sites 5' of MT-II disappear, and a site 3' of MT-I appears regardless of which gene is activated. The fact that these sites respond when either gene is activated suggests that the regulation of the two genes is interdependent and that the region undergoes a general change in conformation with MT activation. In addition, a single site in the 5' region of MT-II becomes hypersensitive with activation of the gene and may be related directly to expression.     

Metallothionein genes from hydrothermal crabs (Bythograeidae, Decapoda): characterization, sequence analysis, gene expression and comparison with coastal crabs

Hydrothermal vent conditions can alter DNA and hydrothermal organisms may develop detoxification mechanisms and/or genetic adaptations. Hydrothermal vent animals notably synthesize a high quantity of metallothioneins (MT). Recent studies have revealed that the levels of MT within hydrothermal crustacean tissues are higher than those found in other vent animals. To improve our understanding of the environmental impacts exerted on the vent organisms, we characterized the metallothioneins (cDNA and Mt genes) of several members of the Bythograeidae (Bythograea thermydron, Cyanagraea praedator and Segonzacia mesatlantica) which is the only endemic hydrothermal crab family. In comparison, the isolation of metallothionein cDNA was also carried out in several coastal crab families. The results showed that the hydrothermal crabs possess Mt composed of three exons and two introns presenting conserved splicing signals. The cDNA sequences isolated from distinct crabs showed multiple substitutions. In spite of the unique environmental conditions, the protein sequence analysis revealed no specific amino acid residue for the MT of the three hydrothermal crabs. However, gene expression analysis performed by real-time PCR based on S. mesatlantica (hydrothermal crab) compared to Pachygrapsus marmoratus (coastal crab) confirmed the higher metallothionein induction in hydrothermal crabs suggested by others authors.     

Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium.

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.     

Spectroscopic characterization of Cicer arietinum metallothionein 1

The plant metallothioneins differ distinctively from other metallothionein families with respect to the cysteine distribution patterns, the presence of aromatic amino acids in most and histidine in some forms, as well as long cysteine-free amino acid stretches between cysteine-rich regions. Although known for more than 25 years, research activity on plant metallothioneins has been low increasing only in the past few years. In the following, we will present the first characterization of Cicer arietinum (chickpea) MT1. In this root-specific protein two cysteine-rich regions with six cysteine residues each are separated by a 42 amino acids long linker region. A synthetic gene encoding MT1 was designed, cloned into a suitable vector, and the protein was over-expressed in Escherichia coli. We could show, that MT1 has the ability to coordinate up to five Zn²⁺ or Cd²⁺ ions and even higher amounts of Hg²⁺. According to titration experiments pH-dependent zinc- and cadmium-thiolate cluster stability in MT1 is considerably lower than in vertebrate metallothioneins. The approximate contribution of secondary structural elements to the overall structure was assessed with circular dichroism and infrared spectroscopy. Hypothetical metal-thiolate cluster structures will be presented.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.