Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.

Optimal guidance of low-thrust interplanetary space vehicles

The low-thrust guidance problem is defined as the minimum terminal variance (MTV) control of a space vehicle subjected to random perturbations of its trajectory. To accomplish this control task, only bounded thrust level and thrust angle deviations are allowed, and these must be calculated based solely on the information gained from noisy, partial observations of the state. In order to establish the validity of various approximations, the problem is first investigated under the idealized conditions of perfect state information and negligible dynamic errors. To check each approximate model, an algorithm is developed to facilitate the computation of the open loop trajectories for the nonlinear bang-bang system. Using the results of this phase in conjunction with the Ornstein-Uhlenbeck process as a model for the random inputs to the system, the MTV guidance problem is reformulated as a stochastic, bang-bang, optimal control problem. Since a complete analytic solution seems to be unattainable, asymptotic solutions are developed by numerical methods. However, it is shown analytically that a Kalman filter in cascade with an appropriate nonlinear MTV controller is an optimal configuration. The resulting system is simulated using the Monte Carlo technique and is compared to other guidance schemes of current interest.

An economic air pollution control model-application : photochemical smog in Los Angeles County in 1975

An economic air pollution control model, which determines the least cost of reaching various air quality levels, is formulated. The model takes the form of a general, nonlinear, mathematical programming problem. Primary contaminant emission levels are the independent variables. The objective function is the cost of attaining various emission levels and is to be minimized subject to constraints that given air quality levels be attained.

The model is applied to a simplified statement of the photochemical smog problem in Los Angeles County in 1975 with emissions specified by a two-dimensional vector, total reactive hydrocarbon, (RHC), and nitrogen oxide, (NO_x), emissions. Air quality, also two-dimensional, is measured by the expected number of days per year that nitrogen dioxide, (NO₂), and mid-day ozone, (O₃), exceed standards in Central Los Angeles.

The minimum cost of reaching various emission levels is found by a linear programming model. The base or "uncontrolled" emission levels are those that will exist in 1975 with the present new car control program and with the degree of stationary source control existing in 1971. Controls, basically "add-on devices", are considered here for used cars, aircraft, and existing stationary sources. It is found that with these added controls, Los Angeles County emission levels [(1300 tons/day RHC, 1000 tons /day NO_x) in 1969] and [(670 tons/day RHC, 790 tons/day NO_x) at the base 1975 level], can be reduced to 260 tons/day RHC (minimum RHC program) and 460 tons/day NO_x (minimum NO_x program).

"Phenomenological" or statistical air quality models provide the relationship between air quality and emissions. These models estimate the relationship by using atmospheric monitoring data taken at one (yearly) emission level and by using certain simple physical assumptions, (e. g., that emissions are reduced proportionately at all points in space and time). For NO₂, (concentrations assumed proportional to NO_x emissions), it is found that standard violations in Central Los Angeles, (55 in 1969), can be reduced to 25, 5, and 0 days per year by controlling emissions to 800, 550, and 300 tons /day, respectively. A probabilistic model reveals that RHC control is much more effective than NO_x control in reducing Central Los Angeles ozone. The 150 days per year ozone violations in 1969 can be reduced to 75, 30, 10, and 0 days per year by abating RHC emissions to 700, 450, 300, and 150 tons/day, respectively, (at the 1969 NO_x emission level).

The control cost-emission level and air quality-emission level relationships are combined in a graphical solution of the complete model to find the cost of various air quality levels. Best possible air quality levels with the controls considered here are 8 O₃ and 10 NO₂ violations per year (minimum ozone program) or 25 O₃ and 3 NO₂ violations per year (minimum NO₂ program) with an annualized cost of $230,000,000 (above the estimated $150,000,000 per year for the new car control program for Los Angeles County motor vehicles in 1975).

High frequency wave propagation in the earth: theory and observation

The wave-theoretical analysis of acoustic and elastic waves refracted by a spherical boundary across which both velocity and density increase abruptly and thence either increase or decrease continuously with depth is formulated in terms of the general problem of waves generated at a steady point source and scattered by a radially heterogeneous spherical body. A displacement potential representation is used for the elastic problem that results in high frequency decoupling of P-SV motion in a spherically symmetric, radially heterogeneous medium. Through the application of an earth-flattening transformation on the radial solution and the Watson transform on the sum over eigenfunctions, the solution to the spherical problem for high frequencies is expressed as a Weyl integral for the corresponding half-space problem in which the effect of boundary curvature maps into an effective positive velocity gradient. The results of both analytical and numerical evaluation of this integral can be summarized as follows for body waves in the crust and upper mantle:

1) In the special case of a critical velocity gradient (a gradient equal and opposite to the effective curvature gradient), the critically refracted wave reduces to the classical head wave for flat, homogeneous layers.

2) For gradients more negative than critical, the amplitude of the critically refracted wave decays more rapidly with distance than the classical head wave.

3) For positive, null, and gradients less negative than critical, the amplitude of the critically refracted wave decays less rapidly with distance than the classical head wave, and at sufficiently large distances, the refracted wave can be adequately described in terms of ray-theoretical diving waves. At intermediate distances from the critical point, the spectral amplitude of the refracted wave is scalloped due to multiple diving wave interference.

These theoretical results applied to published amplitude data for P-waves refracted by the major crustal and upper mantle horizons (the Pg, P*, and Pn travel-time branches) suggest that the 'granitic' upper crust, the 'basaltic' lower crust, and the mantle lid all have negative or near-critical velocity gradients in the tectonically active western United States. On the other hand, the corresponding horizons in the stable eastern United States appear to have null or slightly positive velocity gradients. The distribution of negative and positive velocity gradients correlates closely with high heat flow in tectonic regions and normal heat flow in stable regions. The velocity gradients inferred from the amplitude data are generally consistent with those inferred from ultrasonic measurements of the effects of temperature and pressure on crustal and mantle rocks and probable geothermal gradients. A notable exception is the strong positive velocity gradient in the mantle lid beneath the eastern United States (2 x 10^-3 sec^-1), which appears to require a compositional gradient to counter the effect of even a small geothermal gradient.

New seismic-refraction data were recorded along a 800 km profile extending due south from the Canadian border across the Columbia Plateau into eastern Oregon. The source for the seismic waves was a series of 20 high-energy chemical explosions detonated by the Canadian government in Greenbush Lake, British Columbia. The first arrivals recorded along this profile are on the Pn travel-time branch. In northern Washington and central Oregon their travel time is described by T = Δ/8.0 + 7.7 sec, but in the Columbia Plateau the Pn arrivals are as much as 0.9 sec early with respect to this line. An interpretation of these Pn arrivals together with later crustal arrivals suggest that the crust under the Columbia Plateau is thinner by about 10 km and has a higher average P-wave velocity than the 35-km-thick, 62-km/sec crust under the granitic-metamorphic terrain of northern Washington. A tentative interpretation of later arrivals recorded beyond 500 km from the shots suggests that a thin 8.4-km/sec horizon may be present in the upper mantle beneath the Columbia Plateau and that this horizon may form the lid to a pronounced low-velocity zone extending to a depth of about 140 km.