The 5'-end of eucaryotic mRNAs has a unique structure that is required for mRNA processing, binding to the ribosome, and increased stability. This terminus consists of a 7-methyl guanosine linked by a 5'-5' triphosphate bridge to the penultimate nucleotide (7mGpppNpN- - - - N) which is called a "cap" structure. In our work on cellular methylation, we have isolated the guanine-7-methyltransferase from nuclei of tumor cells and have characterized this enzyme. While this enzyme is elevated about 10-fold in cancer cells, the mRNA is hypomethylated. We would like to understand this paradox and determine how cells regulate the methylation of RNA.

Using recombinant DNA techniques we have synthesized in vitro transcripts that are capped but not methylated which serve as excellent substrates for the guanine-7-methyltransferase. Using these in vitro transcripts as substrates we are characterizing various substrate analogs and inhibitors. Nucleoside analogs and amino acid analogs are being evaluated as potential anti-tumor or anti-viral agents because of their impact on cellular methylation.

We are currently trying to clone the gene for this interesting enzyme in order to understand the regulation of mRNA methylation which is essential for gene expression. Many modern techniques in molecular biology are being used to understand this important post-transcriptional modification.

Kilel, B. and Sitz, T. O. (2003) Comparative Analysis of mRNA Guanine-N7-Methyltransferase in Eucaryotic Organisms Using Informatics Tools. Va. J. Sci. 54:80.

Linkous, T. A. and Sitz, T. O. (2003) Hypomethylation of mRNA in NRK Cells Treated with Cycloleucine. Va. J. Sci. 54:81.

MacCarthy, C. M. and Sitz, T. O. (2003) The Subunit Structure of the Cloned Human Guanine-7-Methyltransferase. Va. J. Sci. 54:81.

Bonham, A. R. and Sitz, T. O. (2002) Active Site Labeling with RNA and DNA. Va. J. Sci. 53:84.

Miller, H. L. and Sitz, T. O. (2002) A Comparison of Subunit Structure of the Guanine-7-Methyltransferase in Mouse and Human. Va. J. Sci. 53:84.

Bonham, A. R. and T. O. Sitz (2001) Active site labeling of guanine-7-methyltransferase. Va. J. Sci. 52(2):103.

Newbrough, K. E. and Sitz, T. O. (2001) Characterization of Mouse Guanine-7-Methyltransferase mRNA. Va. J. Sci. 52:103.

Bautz, D. J., Zulfiger, S., Newbrough, K., and Sitz, T. O. (2000) Characterization of Mouse and Human Guanine-7-Methyltransferase. Va. J. Sci. 51:106.

Bautz, D. J., Simms, B. L., Huggins, J. D., Raulfs, E. C., and Sitz, T. O. (1999) Inactivation of the guanine-7-methyltransferase with N-ethylmaleimide (NEM): essential sulfhydryls? Va. J. Sci. 50.

Prickett, T. D., Tayal, R., DelaCour, J., and Sitz, T. O. (1998) Biochemical characterization of the guanine-7-methyltransferase. Va. J. Sci. 49:104.