Research Summary

We are interested in molecular genetic processes and their application to new techniques for biological studies. We are using DNA transposition, non-homologous recombination, and gene fusions with reporter genes. We begin our studies and applications with the well-developed bacterium E. coli and then extend them to other prokaryotic and eukaryotic organisms. We have used the high frequency bacteriophage Mu transposon to fuse reporter genes and regulated promoters to other genes for studies of gene expression and regulation and also to clone genes in vivo without in vitro recombinant DNA. Genetic applications of Mu at least provide futuristic model systems for higher organisms. Other experiments with Mu involve targeting it's transposition to specific DNA regions with gene fusions of the B targeting gene to specific DNA binding proteins. This is part of our quest for a universal, high frequency transposon, which can be used in all organisms. Our work with reporter genes involves the development of new, more sensitive reporter genes and their application to new processes including protein-protein interactions. We have focused on genes for hydrolytic enzymes, which can use a wide spectrum of substrates for chromogenic assays and growth selections. The tbg gene from Thermus aquaticus encodes a thermostable -galactosidase which can not only function at high temperatures, where most proteins from eukaryotes and mesophilic bacteria would denature, but also in adverse conditions such as with detergents on polyacrylamide gels. Potentially these hybrid proteins may have new applications in studies of protein structure and interaction.

Selected Publications

Weber, J. M., Johnson, S. P., Vonstein, V., Casadaban, M. J. and Demirjian, D. C. (1995). "A chromosome integration system for stable gene transfer into Thermus flavus." Biotechnology (N Y) 13: 271-5. (PubMed)

Vonstein, V., M. J. Casadaban, and D. C. Demirjian (1995). Molecular cloning of the pyrE gene from the extreme thermophile Thermus flavus. J. Bacteriol. 177:4540-4543. (PubMed)

Demirjian, D., G. Stanfield, and M. Casadaban (1993). Altering transposition of bacteriophage Mu with a chimeric transposition protein. Protein Eng. 6 suppl., 60.

Roncero, C., K. Sanderson, and M. Casadaban (1992). Genetic analysis of the genes involved in the synthesis of the lipopolysaccharide core in Escherichia coli: three operons in the rfa locus. J. Bacteriol. 174:3250-3260. (PubMed)