Metallothionein Gene Family Research Articles

Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles

MT-III, a brain-specific member of the metallothionein gene family, binds zinc and may facilitate the storage of zinc in neurons. The distribution of MT-III mRNA within the adult brain was determined by solution and in situ hybridization and compared to that of MT-I mRNA. MT-III mRNA is particularly abundant within the cerebral cortex, hippocampus, amygdala, and nuclei at base of the cerebellum. Transgenic mice generated using 11.5 kb of the mouse MT-III 5' flanking region fused to the E. coli lacZ gene express beta-galactosidase in many of the same regions identified by in situ hybridization. MT-III mRNA was present in readily identifiable neurons within the olfactory bulb, hippocampus, and cerebellum, and beta-galactosidase activity was localized to neurons throughout the brain, but not to glia, as determined by costaining with X-Gal and neural- and glia-specific antibodies. There is marked correspondence between the neurons that are rich in MT-III mRNA and those neurons that store zinc in their terminal vesicles. MT-III is found complexed with zinc in vivo and its expression in cultured cells leads to the intracellular accumulation of zinc and enhanced histochemical detection of zinc. These results are discussed in light of the possibility that MT-III may participate in the utilization of zinc as a neuromodulator.

Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia.

A new member of the metallothionein (MT) gene family was discovered that lies about 20 kb 5' of the MT-III gene in both mouse and human. The MT-IV proteins are highly conserved in both species and have a glutamate insertion at position 5 relative to the classical MT-I and MT-II proteins. Murine MT-IV mRNA appears to be expressed exclusively in stratified squamous epithelia associated with oral epithelia, esophagus, upper stomach, tail, footpads, and neonatal skin. The MT derived from tongue epithelium contains both zinc and copper. Many of these epithelia develop parakeratosis during zinc deficiency in the rat. In situ hybridization reveals intense labeling of MT-IV mRNA in the differentiating spinous layer of cornified epithelia, whereas MT-I is expressed predominantly in the basal, proliferative layer; thus, there is a switch in MT isoform synthesis during differentiation of these epithelia. We suggest that MT-IV plays a special role in regulating zinc metabolism during the differentiation of stratified epithelia.

Enhanced neurotrophic activity in Alzheimer's disease cortex is not associated with down-regulation of metallothionein-III (GIF)

Alzheimer's disease (AD) is a chronic neurodegenerative disorder for which the pathogenic mechanisms are not well understood. Previous studies demonstrated that extracts prepared from AD brains could increase the survival of rat cortical neurons in vitro. Additional studies indicated that this enhanced neurotrophic activity of AD brain was due to a reduction of a growth inhibitory factor (GIF) that was subsequently shown to be a new member of the metallothionein (MT) gene family, and designated MT-III. The study presented here examined the association between neurotrophic activity and MT-III expression in frontal cortices from eight AD and five control brains, and further characterized the inhibitory activity of MT-III. On average, AD extracts stimulated the survival of approximately 2-fold more rat cortical neurons than control extracts, demonstrating that AD brain possesses elevated neurotrophic activity. When recombinant MTs were added to cultures grown in the presence of brain extract, MT-III but not MT-I had an inhibitory effect on neuron survival, confirming that MT-III is a specific inhibitory factor in this assay. However, in contrast to previous reports, neither MT-III mRNA nor MT-III protein levels were significantly decreased in the AD group. Therefore, the difference in neurotrophic activity between the AD and control brain samples examined in this study is probably not directly mediated by MT-III. These results suggest that MT-III down-regulation is not an important pathogenic event in some cases of AD.

Rapid transcriptional autoregulation of a yeast metalloregulatory transcription factor is essential for high-level copper detoxification.

Copper detoxification in the yeast Candida glabrata is carried out in large part by a family of metallothionein (MT) genes: a unique MT-I gene, a tandemly amplified MT-IIa gene, and a single unlinked MT-IIb gene. In response to elevated environmental copper levels, members of this MT gene family are transcriptionally activated by a copper-dependent, sequence-specific DNA-binding transcription factor, AMT1. AMT1 shares several structural and functional features with the Saccharomyces cerevisiae copper metalloregulatory transcription factor ACE1, which is constitutively expressed and poised for rapid transcriptional responses to the toxic metal copper. In this paper, we demonstrate that AMT1 is subject to positive transcriptional autoregulation, which is exerted through binding of copper-activated AMT1 to a single copper responsive element in the AMT1 promoter. A nonautoregulatory amt1 mutant displayed a marked decrease in both copper tolerance and expression of the MT-II genes, which are critical for high-level copper detoxification in Candida glabrata. Kinetic analysis demonstrated the remarkably rapid AMT1 mRNA accumulation in the presence of copper, which is followed by increased expression of the metallothionein gene products. These results demonstrate that AMT1-positive autoregulation plays a critical role in metal detoxification and suggest that the rapid autoactivation of the AMT1 metalloregulatory transcription factor biosynthesis is essential for C. glabrata to quickly build up a cellular defense line to protect cells upon exposure to high environmental copper levels.

Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor.

The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.

Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor.

The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.

Selective and tandem amplification of a member of the metallothionein gene family in Candida glabrata.

Metallothioneins constitute a multigene family in the yeast Candida glabrata. Two genes, designated metallothionein-I (MT-I) and one member of the metallothionein-II family (MT-II), were cloned and sequenced previously (Mehra, R. K., Garey, J. R., Butt, T. R., Gray, W. R., and Winge, D. R. (1989) J. Biol. Chem. 264, 19747-19753). Southern analysis of the genomic DNA samples from different wild-type isolates indicated that the MT-I gene was always present as a single copy but multiple (3-9) and tandemly arranged copies of one MT-II gene were present in different strains. Strains of C. glabrata highly resistant to copper salts were obtained by repeated culturing of wild-type isolates in medium containing increasing concentrations of copper sulfate. These strains showed further stable chromosomal amplification (greater than 30 copies) of the MT-II gene. The MT-I gene remained as a single copy. Amplified copies of the MT-II gene were always arranged tandemly. One of the copper-resistant strains acquired more copies of the MT-II gene by apparent duplication of the chromosome carrying this gene. The size of the amplification unit was 1.25 kilobases. The principal MT-I and -II genes of C. glabrata were shown to map to different chromosomes by electrophoretic karyotypic analysis. The length of chromosome carrying MT-II gene increased appreciably in strains exhibiting the highest amplification of this gene. Northern analysis showed increased basal levels of MT-II mRNA in strains having highly amplified MT-II locus.

Structure of an ectodermally expressed sea urchin metallothionein gene and characterization of its metal-responsive region.

The metallothionein-A gene in the metallothionein gene family of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) was sequenced and found to contain three coding exons plus a 3' entirely noncoding exon. Putative alpha and beta MT domains were encoded, by its exons 2 and 3, respectively, in reverse of the order in vertebrate metallothionein genes. The SpMTA promoter was characterized through the expression of recombinant constructs containing various portions of the proximal 678-base-pair (bp) 5'-flanking region of the SpMTA gene. Zygotes injected with constructs were cultured to the blastula stage in the presence of a heavy-metal chelator and then incubated in the presence or absence of cadmium. The longest constructs were expressed only when heavy-metal ion was present. Two putative metal-responsive elements (MREs a and b) within 240 bp of the transcription start site resembled mammalian MREs in their critical 8-bp cores (TGCRCNCS) and in their locations relative to each other and to the TATA box. Elimination of activity by site-specific mutations in MREs a and b, separately or in both, identified them as metal regulatory elements. Thus, MRE recognition in this invertebrate resembles that in vertebrates. Upstream sites with single-mismatched MREs neither acted as MREs nor amplified the activity of MREs a and b. The SpMTA, Spec1, and CyIIIa actin genes, which have the same ectodermal specificity, have common DNA elements at relatively similar locations in their promoter regions; however, these elements are insufficient in themselves to promote gene expression.

Differential repair of DNA damage in the human metallothionein gene family.

We studied the repair of UV- and aflatoxin B1 (AFB1)-induced damage in the human metallothionein (hMT) gene family. After exposure to either UV or AFB1, DNA damage was initially repaired faster in the DNA fragments containing the transcribed hMT-IA, hMT-IE, and hMT-IIA genes than in the genome overall. By 6 h posttreatment, there was at least twice as much repair in these genes as in the rest of the genome. Repair of UV damage in the hMT-IB gene, which shows cell-type specific expression, and in the hMT-IIB gene, which is a nontranscribed processed pseudogene, was about the same as in the rest of the genome, whereas repair of AFB1-induced damage was deficient in these two genes. Inducing transcription of the three expressed hMT genes with CdCl2 or of only the hMT-IIA gene with dexamethasone increased the initial rate of repair in the induced genes another twofold over the rate observed when they were transcribed at a basal level. The rates of repair in the hMT-IB and hMT-IIB genes were not altered by these inducing treatments. Transcription of the hMT genes was transiently inhibited after UV irradiation. Inducing transcription of the genes did not shorten this UV-induced delay. Thus, the efficiency of repair of damage in a DNA sequence is dependent on the level of transcriptional activity associated with that sequence. However, an increased efficiency in repair of a gene itself is not necessarily coupled to recovery of its transcription after DNA damage.

Differential repair of DNA damage in the human metallothionein gene family.

We studied the repair of UV- and aflatoxin B1 (AFB1)-induced damage in the human metallothionein (hMT) gene family. After exposure to either UV or AFB1, DNA damage was initially repaired faster in the DNA fragments containing the transcribed hMT-IA, hMT-IE, and hMT-IIA genes than in the genome overall. By 6 h posttreatment, there was at least twice as much repair in these genes as in the rest of the genome. Repair of UV damage in the hMT-IB gene, which shows cell-type specific expression, and in the hMT-IIB gene, which is a nontranscribed processed pseudogene, was about the same as in the rest of the genome, whereas repair of AFB1-induced damage was deficient in these two genes. Inducing transcription of the three expressed hMT genes with CdCl2 or of only the hMT-IIA gene with dexamethasone increased the initial rate of repair in the induced genes another twofold over the rate observed when they were transcribed at a basal level. The rates of repair in the hMT-IB and hMT-IIB genes were not altered by these inducing treatments. Transcription of the hMT genes was transiently inhibited after UV irradiation. Inducing transcription of the genes did not shorten this UV-induced delay. Thus, the efficiency of repair of damage in a DNA sequence is dependent on the level of transcriptional activity associated with that sequence. However, an increased efficiency in repair of a gene itself is not necessarily coupled to recovery of its transcription after DNA damage.

The sheep metallothionein gene family. Structure, sequence and evolutionary relationship of five linked genes.

Southern blot analysis of the sheep genome revealed a metallothionein gene family with at least nine members. Two overlapping cosmid clones spanning approximately 67 kb and containing five metallothionein genes have been isolated. DNA sequence analysis reveals that one of these is a metallothionein II variant, three are metallothionein I variants and one is a truncated metallothionein pseudogene containing only the first exon. The predicted amino acid sequence was compared with previously reported amino acid composition data of sheep metallothioneins [Whanger, P. D., Oh, S.-H. & Deagen, J. T. (1981) J. Nutr. 111, 1207-1215], and this suggests that we have isolated the genes encoding the major protein isoforms found in the sheep liver. The promoter regions of these genes contain many conserved elements, among them metal-regulatory elements and putative glucocorticoid-responsive elements. However, there are a number of differences between these genes in the arrangement of these elements. Sequence comparisons indicate that the multiple metallothionein I genes and the pseudogene appear to have resulted from sequential duplication events, and a larger cluster of metallothionein I genes may have been disrupted leading to the formation of the pseudogene.

Regulation of human metallothionein (MT) genes. Differential expression of MTI-F, MTI-G, and MTII-A genes in the hepatoblastoma cell line (HepG2).

We have analyzed the pattern of expression of three human metallothionein (MT) genes (MTI-F, MTI-G, and MTII-A) in the hepatoblastoma cell line, HepG2, in response to the metal ion inducers cadmium, copper, and zinc. The absolute number of transcripts of each of the three genes were measured, and the data clearly suggest differential regulation of these members of the MT gene family by the different inducers both in terms of the rate and the extent of transcript accumulation. The lowest levels of transcript accumulation was observed for MTI-F gene (maximum 4,000 molecules of mRNA per cell) and copper was shown to be its poorest inducer (up to 2,000 molecules per cell). Cadmium is the poorest inducer of MTI-G gene even though transcripts of this gene accumulated at comparatively higher levels than those of MTI-F. Copper- and zinc-induced MTI-G transcript accumulation was up to 12,000 molecules per cell whereas the corresponding value for cadmium was only 4,500. MTII-A was the only gene expressed in the absence of any externally added inducer. Also, in contrast to the MTI genes, the MTII-A gene was equally responsive to all the metal ions tested and the induced levels of accumulation were much higher (up to 75,000 molecules of MTII-A mRNA per cell).

The human metallothionein gene family: structure and expression.

Metallothioneins (MTs) are encoded by a multigene family in man. We have isolated those genes and analyzed the structure of some of them. The MT-II variant is encoded by a single functional gene: MT-IIA. The MT-IIB gene is a processed pseudogene derived from a reverse transcript of MT-II mRNA. On the other hand, the MT-I class of variants are encoded by a large number of genes, arranged in tandem. The MT-IIA and the MT-IA genes show a differential response to glucocorticoid hormones and heavy metals, yet they are both expressed in primary human fibroblasts and in HeLa cells. Expression of both of those genes, in high level after transfer on bovine papilloma virus vectors, leads to increased resistance of the host cells to cadmium-induced toxicity.

Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene.

The complete nucleotide sequence of two of the human metallothionein gene family has been compared. One is a functional metallothionein-II gene, the other a pseudogene, lacking introns, terminating in a poly(A) tail and flanked by two direct repeats. In addition, we have detected a size polymorphism associated with the processed gene in the population, examined, and we have observed a region of apparent secondary structure homology between of 5' flanking region of the functional metallothionein-II gene and that of a mouse metallothionein-I gene.