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

[摘要] This thesis is a compilation of inquiries into the molecular biology of two disparateorganisms, each using site-specific recombination to generate diversity in and regulate theproduction of a protein. Coincidentally, each of these proteins functions in the context of thevertebrate immune system: one is the major defensive weapon of a eubacterial pathogen, andthe other the sine qua non of the system designed to recognize and destroy infectious agents, theantibody. The first section describes a series of experiments designed to explore the molecularbasis for antigenic variation in Borrelia hermsii, the eubacterial agent responsible for relapsingfever. A serotype 7 vmp gene fragment was cloned using mixed sequence oligonucleotideprobes derived from the sequencing of CNBr peptides from VMP 7. Use of this fragment innorthern and southern blot experiments demonstrated that B. hermsii DNA sequences duplicateand rearrange, and that these duplications correlate with differential expression of VMPs (in apattern remarkably reminiscent of the trypanosomes). These striking results formed the basisfor several subsequent studies, which are also discussed. The final section details two separateprojects involving V(D)J recombination. Inital effort was directed at producing a non-lymphoidcell line capable of performing V(D)J recombination. Our strategy was based upon the ability ofretroviruses to transcriptionally activate genes distant from the site of integration. Due toreports of the cloning of RAG-1 and -2, the project was discontinued, but not before producingone line with an interesting phenotype. Following largely anecdotal reports of a previouslyunnoticed pattern of base addition during V(D)J recombination, we decided to perform arigorous examination of the hypothesis, using both experiment and a detailed examination ofpublished data. While we were able to confirm the existence of palindromic, non-templatedbases, our results contradicted other reports with regard to the origins and characteristics ofthese inserts. Some surprises arose, most notably in the influence primary DNA sequence hason the spectrum of product molecules; this adds a new dimension to a process previouslythought to be well understood. 1his work represents the most thorough study of P nucleotideaddition to date.

[发布机构] University:California Institute of Technology;Department:Biology

[关键词] Biology