Division of Biology

I have developed a new experimental preparation of the fruit fly, Drosophila melanogaster. A fly is glued to a steel pin, which is held in the field between two magnets such that the fly is free to rotate about only one axis. Such magnetically tethered flies perform rapid yaw turns, similar to the behaviors termed body saccades in free flight. Saccades can be evoked by visual stimulation, in a manner suggesting that the underlying neural circuitry may be performing an angular threshold calculation. Once a saccade is initiated, however, visual feedback has very little effect on its dynamics, but rotational feedback from the haltere system plays an important role in structuring the time course of saccades. Vision is important, though, in maintaining a stable orientation in both intact flies and flies with asymmetrical wing alterations. The halteres are known to mediate responses to Coriolis forces correlated with the fly's rotations in flight, but flies with modified halteres also exhibit distorted saccade dynamics when they are not free to rotate. This suggests that the halteres may be involved in saccade initiation, although the precise mechanisms are not clear. There is preliminary evidence suggesting that the haltere strokes may be actively modulated during flight.

The structure of the gene regulatory networks that drive animal development is encoded in the genome in cis-regulatory regions. Locating these regions and understanding how they integrate regulatory information to produce specific spatiotemporal patterns of gene expression is a major challenge facing developmental biology. This thesis presents computational and experimental work on finding, dissecting, and understanding regulatory regions. I discuss the use of comparative sequence analysis or phylogenetic footprinting to locate regulatory regions in animals. I then present experimental work on dissecting the information encoded in the cyIIIa cis-regulatory system of the California purple sea urchin, Strongylocentrotus purpuratus. Finally, I present a computational investigation of binding site validation techniques in E.coli.

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.The enzymatic hydrolysis of amide bonds was studied in two systems by site-specific mutagenic techniques. In the first study, I developed an expression system for the serine protease, [alpha]-lytic protease, from Lysobacter enzymogenes 496, using a previously constructed synthetic gene, which contained numerous unique restriction sites. Since the wild type enzyme is expressed as a zymogen, which is self-processed into the mature proteolytic enzyme, a unique chimeric expression system utilizing the concomitant expression of the pro-domain from the wild type enzyme was created. The system expresses the properly-folded, mature protease-domain of the enzyme, thus allowing for production of active-site mutants of [alpha]-lytic protease, that otherwise could not be obtained in the mature form.The ability of the enzymatic machinery to enhance the nucleophilicity of a chemical group other than the active-site serine hydroxyl was investigated through the mutation of serine 195 to an alanine. The removal of the serine hydroxyl was hypothesized to provide sufficient volume in the active site to allow a water molecule to bind and possibly function as the attacking nucleophile in the hydrolysis. The structure of the enzyme would be minimally disturbed by the removal of the single atom. The mutant enzyme was assayed for activity on the serine protease inhibitor diethyl [...]-nitrophenyl phosphate. No enzymatic hydrolysis of the substrate was detected. Analysis of structural constraints of the enzyme suggests that a serine 195 glycine mutation might provide a more hydrophilic environment in the active site for the binding of a water molecule.In the second study, I investigated the mechanism of RTEM-1 [beta]-lactamase, an enzyme which hydrolyzes the amide bond of [beta]-lactam antibiotics, conferring antibiotic resistance to bacterial cells. Serine 130, a conserved active-site residue in the class A [beta]-lactamases, has been proposed to be involved in positioning the conserved lysine 234 through a hydrogen bond interaction (Moews et al. (1990) Proteins 7 156). The function of lysine 234 is known to be one solely of substrate binding (D. M. Long (1991) Ph.D. Thesis, California Institute of Technology). I performed site-saturation mutagenesis of serine 130, and the resulting 20 mutant enzymes were assayed for the ability to confer resistance to E. coli towards several [beta]-lactam antibiotics. Four mutants (Ser 130 Gly, Ser 130 Thr, Ser 130 Asn, and Ser 130 Gln) conferred notable resistance to the penam antibiotics. These four mutants were purified to homogeneity, and the steady-state kinetic parameters for hydrolysis of benzylpenicillin were measured for each. The values of KM for all four mutants were no more than ten-fold more than the wild type value. However, values of kcat for the four mutants were decreased at least 1000-fold from that of the wild type, demonstrating clearly the involvement of serine 130 in catalysis. These results, along with an analogy to structural mutants of Thr 157 in T4 lysozyme, suggest how the four serine 130 mutants might maintain the native hydrogen bonding interactions of the serine with lysine 234, as well as participate in the catalytic mechanism.