Mary E. Woodworth
Professor Emerita of Microbiology
Before I retired from Miami University, my lab was interested in the molecular biology of tumor viruses, specifically simian virus-40 (SV40), a small DNA virus. Naturally arising variants of SV40 which have evolved because of their selective advantage over wild type and other defectives were being utilized to elucidate the mechanisms of initiation of replication and transcription in eukaryotic cells. By a comparative analysis of the biological activities of cloned variant sequences that during evolution have replaced wild type sequences in the non-coding regulatory region of SV40, we sought to identify cis-acting sequence elements and trans-acting factors which positively and/or negatively regulate replication and gene expression. We also investigated the interdependence of the overlapping functions for replication and transcription in the SV40 genome.
A 69-bp monkey DNA sequence, present in some naturally arising variants of SV40, can in either orientation enhance both replication and transcription through multiple motifs. The analysis of these cis-acting motifs that enhance replication and transcription has implicated certain cellular transcription factors to be involved. The next step would be to investigate these trans-acting factors to determine the mechanism(s) by which they function to stimulate replication and transcription. Protein-binding experiments performed with crude extracts and also with purified proteins could be used to identify factors and define the nucleotides within the monkey sequence with which they interact. Gel retardation assays, DNAse and/or DMS footprinting experiments could be used to define the binding sites of novel replication/transcription factors, and also to ask if the factors facilitate each other's binding, or the binding of viral-encoded large T antigen or the specific cellular transcription factor Sp1. The ability of factors to facilitate T antigen-mediated DNA unwinding could be tested directly by probing with a chemical that can detect alterations in DNA conformation, such as potassium permanganate.
In parallel with the binding experiments, further cis-acting studies should be done. Point mutations in binding sites could be made by site-directed mutagenesis so that the individual motifs can be assayed without altering the remainder of the monkey sequence. Another line of experiments could assay the replication activity of reiterated binding sites to determine if the stimulation of replication conforms to the enhanson model of transcriptional activation.
Once the binding sites for novel replication/transcription factors are defined, they could be used to screen a cDNA expression library to isolate the genes for these factors. The cloned genes could then be used in transfection experiments, or used to produce purified protein for in vitro replication studies. In addition, nuclease treatment and electron microscopy could be performed to determine if the monkey sequence produces a nuclease-sensitive region or possibly a nucleosome-free gap.
- Okuley, S., M. Call, T. Mitchell, B. Hu, and M.E. Woodworth. 2003. Relationship among location of T-antigen-induced DNA distortion, auxiliary sequences, and DNA replication efficiency. J. Virol. 77:10651-10657.
- Turner, W. J. and M. E. Woodworth. 2001. DNA replication efficiency depends on transcription factor-binding sites. J. Virol. 75:5638-5645.
- Wilderman, P.J., Hu, B., and M.E. Woodworth. 1999. Conformational changes in simian virus 40 rearranged regulatory regions: effects of the 21-bp promoters and their location. J. Virol. 73:10254-10263.
- Adiletta, D.C., R.W. Elliott, and M.E. Woodworth. 1993. Characterization of Murine Middle Repetitive DNA. DNA and Cell Biol. 12:319-327.