Human metallothionein genes: molecular cloning and sequence analysis of the mRNA.

From a cDNA clone bank prepared from cadmium-treated HeLa cells, we isolated clones representing mRNAs whose concentration is increased after cadmium induction. Several metallothionein cDNA clones were isolated by cross-hybridization to mouse metallothionein-I cDNA. The nucleotide sequence of one of these clones, containing a nearly full-length cDNA copy of human metallothionein-II mRNA, was determined. The homology between the human and mouse metallothionein sequences is strictly limited to the coding region of the mRNA. Codon usage in metallothionein mRNA is not random. Seventy-nine percent of the codons have G or C residues at the third position, resulting in a GC-rich sequence.     

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.     

Wheat Ec metallothionein genes. Like mammalian Zn2+ metallothionein genes, wheat Zn2+ metallothionein genes are conspicuously expressed during embryogenesis.

A cDNA library was prepared from the bulk mRNA of mature wheat embryos and screened with mixed 32P-labeled oligonucleotide probes that encoded parts of the partial amino-acid sequence for the Zn-containing Ec protein. Each DNA insert in 11 positives from a screen of 10(5) plaques encoded a 5' untranslated and a 3' untranslated region, in addition to an open reading frame (of 81 amino acids) which, in every case, corresponded to at least 56 of the 59 amino acids in the partial polypeptide sequence previously determined for the Ec protein. The three different mRNA sequences encoded in the cDNA probably correspond to single-copy genes in the A, B and D genomes of hexaploid wheat. A wheat genomic library was screened with 32P-labeled cDNA and gave a single positive in a screen of 5 x 10(5) plaques. A 3.1-kb genomic fragment (gf-3.1) was sequenced and a cap site for the encoded mRNA was determined by primer extension. The gf-3.1 sequence encodes an intronless mRNA for the Ec protein and contains appreciable amounts of 5' and 3' flanking sequences. In addition to a putative TATA box, two inverted-repeat sequences and one direct-repeat sequence, the 5' flank in gf-3.1 contains a sequence similar to the abscisic-acid-responsive element in other higher-plant genes but does not contain sequences similar to the metal-responsive elements in animal metallothionein genes. Consistent with these findings, RNA blotting shows that accumulation of Ec mRNA is abundant in immature embryos, undetectable in germinated embryos and can be induced by adding abscisic acid, but not by adding Zn2+ to the medium in which mature wheat embryos are germinated. The findings suggest that the wheat Ec metallothionein genes, like mammalian liver metallothionein genes, are conspicuously expressed during embryogenesis.     

Isolation and characterization of Mytilus edulis metallothionein genes

Metallothioneins (MTs) are crucial proteins in all organisms for the regulation of essential metals and the detoxification of heavy metals. Many studies have estimated MT levels in mussel tissues to detect marine metal pollution. In this study, we investigated the MT gene structures of the forms present in Mytilus edulis (blue mussel). One MT-10 (2413 bp) gene and one MT-20 (1906 bp) gene were obtained. These MT genes contain three exons and two long introns. The splicing signals for MT-10 and MT-20 were GTA(T/A)GT-(C/T)AG. The structural organization (length of intron, splicing signals, AT content) of MT-10 and MT-20 is compared with other MT genes.     

Differential expression of four linked sheep metallothionein genes

Regulation of the closely linked endogenous sheep MT-Ia, MT-Ib, MT-Ic and MT-II genes by heavy metals and dexamethasone was studied in cultured sheep fibroblasts. Only MT-II mRNA was detectable before addition of any inducer. Addition of copper, zinc or cadmium salts to the culture medium increased the level of each mRNA; however, the magnitude of this response varied greatly between the four metallothionein genes. Following induction, levels of MT-Ia mRNA were the highest, followed by MT-II and MT-Ic mRNAs. The MT-Ib mRNA was only present at low levels. Zinc and cadmium were found to be the most effective inducers of each gene. The maximal response of the sMT-Ia, Ib and II genes to copper was only 30% of zinc and cadmium. The sMT-Ic gene responded very weakly to copper, less than 5% of the levels achieved with zinc. Only the MT-II mRNA increased in response to dexamethasone. In the liver of sheep on normal diets, the levels of MT-Ia, Ic and II mRNAs were found to be unexpectedly high and comparable to induced levels in fibroblasts.     

Cloning, nucleotide sequence and molecular evolution of a rabbit processed metallothionein MT-2 pseudogene

A rabbit metallothionein-2 pseudogene (MT-2 psi) has been isolated from a partial rabbit genomic library. Its unusual sequence shows evidence of complex rearrangements involving recombination and deletion events. There are no intervening sequences, 3' poly A tract or 5' regulatory DNA sequences. The pseudogene is flanked by two sets of direct repeats (CT)3 GT(CT)4 and CTGG(G)CTC. They are most probably the sites of insertion of MT-2 psi in the rabbit genome. In addition, a number of repetitive DNA sequences are observed flanking the MT-2 psi gene. These are features of a processed retrogene.     

Functional homologs of fungal metallothionein genes from Arabidopsis.

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Two cDNAs encoding proteins with homology to animal and fungal MTs have been isolated from Arabidopsis. The genes represented by these cDNAs are referred to as MT1 and MT2. When expressed in an MT-deficient (cup1 delta) mutant of yeast, both MT1 and MT2 complemented the cup1 delta mutation, providing a high level of resistance to CuSO4 and moderate resistance to CdSO4. Although the MT-deficient yeast was not viable in the presence of either 300 microM CuSO4 or 5 microM CdSO4, cells expressing MT1 were able to grow in medium supplemented with 3 mM CuSO4 and 10 microM CdSO4, and those expressing MT2 grew in the presence of 3 mM CuSO4 and 100 microM CdSO4. In plants, MT1 mRNA was more abundant in roots and dark-grown seedlings than in leaves. In contrast, MT2 mRNA accumulated more in leaves than in either roots or darkgrown seedlings. MT2 mRNA was strongly induced in seedlings by CuSO4, but only slightly by CdSO4 or ZnSO4. However, MT1 mRNA was induced by CuSO4 in excised leaves that were submerged in medium. These results indicated that Arabidopsis MT genes are involved in copper tolerance. Plants also synthesized metal binding phytochelatins (poly[gamma-glutamylcysteine]glycine) when exposed to heavy metals. The results presented here argue against the hypothesis that phytochelatins are the sole molecules involved in heavy metal tolerance in plants. We conclude that Arabidopsis MT1 and MT2 are functional homologs of yeast MT.     

Expression of the Drosophila melanogaster metallothionein genes in yeast

The metallothionein system in Drosophila melanogaster is composed of two genes, Mto and Mtn, that code for distinctly different proteins. In order to compare the properties of Mto and Mtn, we transformed yeast with several fusion plasmids. The Mto and Mtn cDNAs, when placed under the control of CUP1 or PGK promoters, can confer a copper-resistance phenotype to copper-hypersensitive cells. Both Mto and Mtn proteins can be characterized in extracts from transformed yeast cells.     

Tissue-specific regulation of zinc metabolism and metallothionein genes by interleukin 1

Interleukin 1 (IL 1) production is stimulated by infection, cellular injury, and inflammation. This cytokine directs a wide spectrum of host responses. Human interleukin 1 alpha (IL 1 alpha) was used to examine the time course of effects on zinc metabolism as part of the acute phase response. IL 1 produced a transient depression in the serum zinc concentration and increased serum ceruloplasmin. Metallothionein levels were increased in liver 14-fold after IL 1. Increased expression of metallothionein-1 and -2 genes following IL 1 were observed in liver, bone marrow, and thymus. Pulse-labeling experiments with i.v.-administered 65Zn showed that IL 1 drastically altered zinc distribution kinetics among tissues. More 65Zn was taken up (and/or retained) by the liver, bone marrow, and thymus 6 h after IL 1, whereas correspondingly less 65Zn was found in bone, skin, and intestine. Uptake by other tissues was not affected by IL 1. Chromatography of cytosol from tissues with increased 65Zn uptake suggests the IL 1-induced redistribution may be driven by enhanced metallothionein synthesis. Collectively, the results show that IL 1 regulates zinc metabolism and may direct its preferential, tissue-specific distribution via elevated metallothionein-1 and -2 gene expression.     

Molecular cloning and sequencing of metallothionein in squamates: New insights into the evolution of the metallothionein genes in vertebrates

Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian metallothioneins, we have cloned and sequenced the cDNAs encoding for metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of metallothioneins in reptiles. Phylogenetic analysis shows that reptilian metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of metallothionein genes along the major vertebrate lineages.     

Human height genes and cancer

Body development requires the ability to control cell proliferation and metabolism, together with selective ‘invasive’ cell migration for organogenesis. These requirements are shared with cancer. Human height-associated loci have been recently identified by genome-wide SNP-association studies. Strikingly, most of the more than 100 genes found associated to height appear linked to neoplastic growth, and impose a higher risk for cancer. Height-associated genes drive the HH/PTCH and BMP/TGFβ pathways, with p53, c-Myc, ERα, HNF4A and SMADs as central network nodes. Genetic analysis of body-size-affecting diseases and evidence from genetically-modified animals support this model. The finding that cancer is deeply linked to normal, body-plan master genes may profoundly affect current paradigms on tumor development.