http://hdl.handle.net/1721.1/7761 http://hdl.handle.net/1721.1/33751 Investigations of tetraspanin functions using large extracellular loops Liu, Christopher C This thesis describes our characterization of a specific tetraspanin domain: the large extracellular loop (LEL). Tetraspanins are involved in cellular migration, adhesion, and metastasis, sperm-egg fusion, and viral infectivity. The large extracellular loop domain is the major extracellular domain of tetraspanins and the binding of a monoclonal antibody against the tetraspanin CD9 serves to inhibit fertilization, consistent with the CD9-null mouse model. The first area of focus in this thesis is the characterization of the murine CD9-LEL domain. We present a methodology to express and purify the mCD9-LEL to homogeneity. Biophysical characterization of the mCD9-LEL protein reveals that it is an autonomously folding, [alpha]-helical dimer. Mutagenesis over much of the mCD9-LEL protein reveals that it is composed of two subdomains: a dimerization subdomain and a variable subdomain proposed to mediate heterotypic interactions. These results suggest both a means for exploring endogenous tetraspanins functions and a mechanism by which tetraspanins may oligomerize. Surprisingly, we were not able to detect oligomerization of the intact CD9 molecule, in discordance with our biophysical data on the mCD9-LEL. (cont.) In the latter part of this thesis, we expand our methodology to purify and characterize three different tetraspanins-LELs, the hCD9-LEL, the hCD63-LEL, and the hCD8 1-LEL. These tetraspanins-LELs all exhibit similar characteristics to the mCD9-LEL, consistent with a published crystal structure of the hCD81-LEL. Lastly, we demonstrate the ability of our tetraspanin-LEL proteins to bind integrins, to inhibit sperm-egg fusion, and to inhibit hepatitis C viral infectivity. Taken as a whole, these studies present novel, biophysically validated tetraspanins-LELs that lend insight into endogenous tetraspanins functions. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005. Includes bibliographical references. http://hdl.handle.net/1721.1/31193 [RNA polymerase ribozymes] Lawrence, Michael S. (Michael Scott), 1975- The RNA World is a hypothetical ancient evolutionary era during which RNA was both genome and catalyst. During that time, RNA was the only kind of enzyme yet in existence, and one of its chief duties was the replication of RNA. This scenario presupposes that among all possible RNA sequences, there exist RNA replicase ribozymes, capable of synthesizing RNA using the information in an RNA template. The goal of the present work is to provide experimental evidence in support of this conjecture, by isolating such ribozymes in the laboratory. We created a large pool of RNA molecules each containing a previously isolated RNA ligase ribozyme and a large stretch of random RNA. Applying in vitro evolution to select for molecules that could extend a tethered RNA primer using nucleoside triphosphates, we isolated nine distinct classes of polymerase ribozymes. Two of these rudimentary polymerases were further evolved to the point that they each could add 14 nucleotides to an untethered primer-template. One of them was subjected to a detailed further characterization. The polymerization it catalyzes was shown to be accurate, with an average fidelity of nearly 97%. It was shown to be general, with primer-templates of all sequences and lengths being accepted as substrates. Finally, it was shown to be partially processive, with the polymerase achieving processivity as high as 90% in a few instances. The polymerase is currently limited by its low affinity for the primer-template. Future work will focus on improving primer- template binding, in order to produce a polymerase that can synthesize longer RNA. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005. Title supplied by cataloger from abstract. Includes bibliographical references. http://hdl.handle.net/1721.1/10692 A tannery waste disposal problem Canter, Bernard Heifetz, Arthur Thesis (B.S.)--Massachusetts Institute of Technology, Dept. of Biology and Public Health, 1930. Includes bibliographical references (leaves 72-83). http://hdl.handle.net/1721.1/41424 Isolation of GCN5 and ADA5 in a selection for transcriptional adaptors Marcus, Gregory A Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1997. Includes bibliographical references.