A Biological Arms Race: Site Specific DNA Recombination in Competing Immunofunctional Proteins

Citation

Meier, Joseph Thomas (1993) A Biological Arms Race: Site Specific DNA Recombination in Competing Immunofunctional Proteins. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/fd8q-cb34. https://resolver.caltech.edu/CaltechTHESIS:12262012-130125197

Abstract

This thesis is a compilation of inquiries into the molecular biology of two disparate organisms, each using site-specific recombination to generate diversity in and regulate the production of a protein. Coincidentally, each of these proteins functions in the context of the vertebrate immune system: one is the major defensive weapon of a eubacterial pathogen, and the other the sine qua non of the system designed to recognize and destroy infectious agents, the antibody. The first section describes a series of experiments designed to explore the molecular basis for antigenic variation in Borrelia hermsii, the eubacterial agent responsible for relapsing fever. A serotype 7 vmp gene fragment was cloned using mixed sequence oligonucleotide probes derived from the sequencing of CNBr peptides from VMP 7. Use of this fragment in northern and southern blot experiments demonstrated that B. hermsii DNA sequences duplicate and rearrange, and that these duplications correlate with differential expression of VMPs (in a pattern remarkably reminiscent of the trypanosomes). These striking results formed the basis for several subsequent studies, which are also discussed. The final section details two separate projects involving V(D)J recombination. Inital effort was directed at producing a non-lymphoid cell line capable of performing V(D)J recombination. Our strategy was based upon the ability of retroviruses to transcriptionally activate genes distant from the site of integration. Due to reports of the cloning of RAG-1 and -2, the project was discontinued, but not before producing one line with an interesting phenotype. Following largely anecdotal reports of a previously unnoticed pattern of base addition during V(D)J recombination, we decided to perform a rigorous examination of the hypothesis, using both experiment and a detailed examination of published data. While we were able to confirm the existence of palindromic, non-templated bases, our results contradicted other reports with regard to the origins and characteristics of these inserts. Some surprises arose, most notably in the influence primary DNA sequence has on the spectrum of product molecules; this adds a new dimension to a process previously thought to be well understood. 1his work represents the most thorough study of P nucleotide addition to date.

Thesis Files