The overall goal of this project is to understand the molecular mechanisms by which heavy metal ions affect gene expression in human cells. Trace metal ions like Zn and Cu are necessary for proper function, growth and development of all living organisms, yet when present in high concentration they can lead to adverse effects. On the other hand, metals like Cd and Hg have no obvious biological role and are significant environmental toxins, teratogens and possibly carcinogens. The long term effects of imbalanced Zn and Cu metabolism and exposure to Cd and Hg are likely to be mediated by the interactions of these metal ions with the gentic apparatus. To understand how these metal ions affect the human transcriptional apparatus we have been studying the structure and expression of the human metallothionein (MT) gene family, used as a model. The transcription of these genes is induced by heavy metal responsive elements via a putative metal responsive factor (MRF) that binds to the metal responsive elements (MRE's), four of which are present in the hMT-IIA promoter. We plan to isolate the MRF, determine a partial amino-acid sequence, raise specific antisera and isolate a cDNA clone encoding this factor. The complete structure of the factor will be determined by sequencing the cDNA. Site-specific mutagenesis will be used to study its mechanism of action. Similarly, we will isolate other factors that are responsible for the basal activity of the hMT-IIA promoter and for its induction by growth factors and tumor promoters. Their corresponding cDNAs will be isolated as well, and used to determine the structure of the factors. The interactions between the various factors which serve to regulate the expression of the hMT-IIA gene will be studied in a reconstituted in-vitro transcription system. In addition, we will examine the effect of heavy metal ions on the activity of regulatory proteins that use the """"""""Zn binding finger motif"""""""" for recognition of specific DNA sequence. For these experiments two well characterized proteins, TFIIIA and the glucocorticoid receptor, will be used as a model. The effect of Cd and Hg on their specific and non-specific DNA binding activities will be determined in order to reach a better understanding of the mechanisms by which these toxic ions can interfere with the function of the transcriptional apparatus. Completion of these studies will provide us with the required biochemcial basis for understanding the effects of beneficial and toxic heavy metal ions on the human transcriptional apparatus.
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