Structure and tissue-specific expression of the human metallothionein IB gene.

Abstract

The human metallothionein (MT) IB gene (hMT-IB) is located in a region of human DNA containing at least four tandemly arranged MT genes. As deduced from its sequence, hMT-I] is likely to encode a functional protein. However, the predicted amino acid sequence differed from the hMT-I amino acid sequence in four positions. Most remarkable was the presence of an additional cysteine. Like other MT genes, hMT-IB has at least two copies of the metal-responsive element upstream from the transcription initiation site. These elements probably are responsible for the metal responsiveness of the hMT-IB promoter, leading to inducible expression of fused heterologous genes. Unlike the hMT-IIA and hMT-IA genes described previously, which are expressed in many different cell types, a high level of expression of the endogenous hMT-In gene could be detected only in human hepatoma and renal carcinoma cell lines. Therefore, this is the first MT gene described which exhibits tissue specificity of expression. This specificity is controlled by a cis-acting mechanism involving methylation, since incubation of nonexpressing cells with an inhibitor of DNA methylation led to activation of the hMT-IB gene. In support of this notion, we found that the 5' flanking region of the hMT-IB gene was highly methylated in HeLa cells, a nonexpressing cell type, but it was not methylated in a hepatoma (expressing) cell line.

Downregulation of metallothionein-IIA expression occurs at immortalization

“…MT-IIA expression changes have previously been reported in tumor-derived human cells. Tumor cell lines generally have a lower MT-IIA expression level than normal human cells (Heguy et al, 1986;Schmidt and Hamer, 1986;Zhang et al, 1997). For example, MT-IIA is expressed 27-fold less in colorectal tumors and tumor cell lines compared with normal colorectal tissue (Zhang et al, 1997).…”

Molecular Cloning of Human Growth Inhibitory Factor CDNA and Its Down-Regulation in Alzheimer’s Disease

“…As expected from chemically determined amino acid sequences of human GIF, nucleotide sequence analysis of the human GIF cDNA has shown striking homology to those of metallothioneins (Karin and Richards, 1982;Schmidt et al, 1985;Heguy et al, 1986;Varshney et al, 1986;Foster et al, 1988). Metallothioneins are ubiquitous, low molecular weight proteins that are characterized by an unusually high cysteine content (23-33 mol%) and a selective capacity to bind heavy metal ions such as zinc, copper and cadmium (Hamer, 1986).…”

“…The sense primer (488) was designed from the amino-terminus of human GIF (amino acid residues 1-7), and the antisense primer (487) was designed from the carboxy-terminus of GIF (amino acid residues 61 -68). Taking account of the codon usage preference of human metallothionein genes (Karin and Richards, 1982;Schmidt et al, 1985;Heguy et al, 1986;Varshney et al, 1986;Foster et al, 1988), we selected a unique codon for each amino acid. Oligonucleotides were synthesized using an automated DNA synthesizer (Applied Biosystems Inc., Foster City, CA, USA) according to the manufacturer's instructions.…”

“…(i) The GIF cDNA shows a striking homology to metallothioneins (Karin and Richards, 1982;Schmidt et al, 1985;Heguy et al, 1986;Varshney et al, 1986;Foster et al, 1988) (54% identity in nucleotide sequence and 63% identity in amino acid sequence). (ii) All cysteine residues are conserved between GIF and the mammalian metallothioneins, and the cysteine content of the GIF molecule is as high (29 mol%) as that of mammalian metallothioneins.…”

“…The other primer (# 487; 5'-CTG-GCAGCAGCTGCACTTCTC) was synthesized on the basis of the amino acid sequence at the carboxy-terminus of the human GIF. As the high homology of human GIF to metallothionein suggested that both genes may have been derived from a common ancestor, we assumed that the codon usage preference of GIF cDNA would be similar to that of metallothionein cDNAs (Karin and Richards, 1982;Schmidt et al, 1985;Heguy et al, 1986;Varshney et al, 1986;Foster et al, 1988 Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu Ala TGC AAA GGC GGA GAG GCA GCT GAG GCA GAA GCA 235 for each amino acid rather than producing oligonucleotides with a high degeneracy for each amino acid, as is the usual method for creating oligonucleotide probes for cDNA cloning.…”