CD-tagging gives us the opportunity to identify and isolate new proteins or protein-containing structures and to identify the genes that encode them. Tagged cells can be identified using automated or semi-automated immunological screens with antibodies directed towards the "guest peptide".  Or as we do most often now, the guest peptide is the Green Fluorescent Protein, EGFP, which can be visualized in the fluorescence microscope.  Cells expressing internal EGFP CD tags can be isolated using the Flurorescence Activated Cell Sorter (FACS).  We are constructing modified transposon and retroviral CD-cassette delivery vectors to be used in both DNA library and cell tagging experiments.

My laboratory research program has also been interested in answering basic questions regarding how biological structures form and how they perform their designated functions. Prokaryotic viral model systems (Salmonella typhimurium bacteriophage P22 and Escherichia coli bacteriophage T4) were used in our research because they are genetically easy to manipulate.

Cartoon of the P22 Virus

One of the many unanswered biological questions is how does a bacterial virus transport its genome across bacterial membranes during infection? This is an interesting macromolecular transport problem because of the large size and poly-anionic nature of the DNA molecule and the hydrophobic nature of the two cellular membranes that it has to cross during infection. In bacteriophage P22, three different phage-encoded proteins are known to be involved in this "DNA-injection" reaction because mutant phage particles missing any one of these three proteins fail to inject their DNA into the cell. My research group has determined that two of these proteins are degraded by the cell after DNA injection so they seem to be "ejected" from the phage particle during the infection process. The third protein is responsible for the Ca⁺⁺ requirement and Mg⁺⁺ inhibition of the DNA injection process. This remains a poorly characterized but important function of the virus particle.

My research program has also tried to determine the elements of protein structure which are involved in a morphogenetic assembly reaction. We studied the terminal assembly reaction in phage P22 morphogenesis, the attachment of the P22 tailspike protein to the completed capsid structure. We have isolated over 100 absolute defective tail protein mutant strains and tried to characterize their defects. Many of the mutations have been sequenced at the DNA level, and from the mutant phenotypes, describe primary sequence changes which prevent the tail protein monomers from oligomerizing into their native trimeric structure. Thus some of these mutations define amino acid residues essential for proper protein folding. Other mutations have identified residues which are required for the assembly of the tailspike protein with the capsid itself.

The P22 Tailspike Protein X-ray Structure

Using the 3-D x-ray crystallographic structure of the P22 tailspike shown above, these types of studies should reveal why certain amino acid residues are important for the proper folding of the tailspike protein during its synthesis and maturation, and which residues are important for the "docking and stabilization" of an assembly protein during its morphogenetic reaction. Because the tailspike is also a specific glycosidase which cleaves the O-antigen of bacteria sensitive to P22, mutational analyses should also reveal details about its enzymatic mechanism.

In addition, my research program has been very active in "Outreach" activities into the educational system which feeds into the university community. We have developed educational projects and supporting multimedia computer education aids for distribution to secondary schools.

One of our "Outreach" projects was developing a traveling "DNA Fingerprinting Footlocker" to be circulated to secondary school biology teachers. This footlocker contained all the equipment and consumable supplies to perform a relatively sophisticated human DNA fingerprinting experiment. The main experiment contained in this "footlocker" used PCR (Polymerase Chain Reaction) to amplify and type various polymorphic markers in the human genome, enabling the students to develop DNA fingerprints from their own cells. The DNA was collected non-intrusively from cheek (bucal) cells, and extracted with a simple procedure. The human loci studied were of the VNTR (Variable Number of Tandem Repeats) markers class. Markers used were D1S80 and ApoB. We also explored using D17S5. We experimented with RB1 and IGHJ, but without much success. We had planned to incorporate extensive multimedia information into the project, in the form of a compact disk, so that the high school classes will be able to "point and click" to get more information on any topic or aspect of the information. In addition, to aid in the instruction of the techniques and concepts, video clips and diagrams (some animated) were going to be included to illustrate such things as the correct way to pipette and load gels, and the way that DNA polymerase associates with the DNA molecule. These latter portions of the project never materialized. We also looked at a dimorphic Alu element insertion into the tissue plasminogen activator (TPA or PLAT) gene that could serve as a straightforward, easy fingerprinting marker.

My laboratory provides a training environment in genetics, molecular biology, molecular genetics and protein biochemistry. Our research uses the classical analytical techniques of genetics and modern methods of biotechnology such as gene cloning, DNA sequencing, genetic engineering of protein-overproducing plasmids and site-directed mutagenesis.
In addition, we provide students with the opportunity to develop skills in communicating effectively using computer tools and networking. We also provide a nurturing environment in which students could develop the sound academic habit of providing service to the scientific community through outreach activities.