Faculty Bibliography

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-IB 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-IB 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.

The human metallothionein-IIA (hMT-IIA) gene contains an enhancer element within its 5' regulatory region. This enhancer element can compete with the SV40 enhancer for one or more cellular factors in vivo. The competition between the two elements is modulated by cadmium, an inducer of hMT-IIA transcription. The data presented are consistent with a model in which heavy metal ions control the ability of the hMT-IIA enhancer to bind a positive factor, leading to increased transcription. The same factor is required for maximal activity of the SV40 enhancer, which suggests that viruses utilize factors that have a normal role in cellular gene expression to control their own genes.

Incubation of cultured human cells with interleukin 1 leads to increased expression of the human metallothionein-IIA gene. Recently, metallothionein has been shown to be an efficient free radical scavenger, and induction by interleukin 1 may be part of a protective response to minimize damage by hydroxyl radicals

We describe a region of human DNA containing four metallothionein (hMT) genes. One of these genes, hMT-IA, was found to encode a functional protein that confers heavy metal resistance to NIH 3T3 cells after transfer on a bovine papilloma virus-derived vector. This gene is expressed in cultured human cell lines, but at a lower basal level than the hMT-IIA gene; it shows a different induction response to heavy metals and glucocorticoids than the hMT-IIA gene. Induction of the human MT family therefore does not represent an equivalent elevation in the level of expression of individual genes, but is the sum of the differential responses of active members. The differential response is due to functional differences of the respective promoter/regulatory regions of the genes as shown by gene-fusion experiments. While the hMT-IIA promoter is responsive to Cd++, Zn++, and glucocorticoids, the hMT-IA promoter mediates response only to Cd++.

Published information on marsupials DNA is limited to a group of species belonging to only one genus. No previous reports have been written on South American species. In this paper we characterize the DNA of three out of the four marsupials found in Uruguay. Analytical and preparative ultracentrifugations in neutral CsCl gradients, including four intercalating agents and in Cs2SO4 gradients in presence of increasing amounts of Hg++ ion did not allow us to separate any satellite fraction. The buoyant density of the unique peak measured in CsCl gradients was in every case 1.697 g/cc with a G-C content of 37.7%. Digestion of total DNA with 11 restriction endonucleases produced a different pattern of bands for the three species, although some possible homologies could be established. Hybridization with 32P-rRNA of Southern blots of the gels containing digested DNAs demonstrated that the repeated sequences evidenced do not correspond to the ribosomal cistrons.