Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

A biochemical and structural characterization of Drosophila neuroglian

A number of cell-cell interactions in the nervous system are mediated by immunoglobulin gene superfamily members. For example, neuroglian, a homophilic neural cell adhesion molecule in Drosophila, has an extracellular portion comprising six C- 2 type immunoglobulin-like domains followed by five fibronectin type III (FnIII) repeats. Neuroglian shares this domain organization and significant sequence identity with Ll, a murine neural adhesion molecule that could be a functional homologue. Here I report the crystal structure of a proteolytic fragment containing the first two FnIII repeats of neuroglian (NgFn 1,2) at 2.0Å. The interpretation of photomicrographs of rotary shadowed Ng, the entire extracellular portion of neuroglian, and NgFnl-5, the five neuroglian Fn III domains, is also discussed.

The structure of NgFn 1,2 consists of two roughly cylindrical β-barrel structural motifs arranged in a head-to-tail fashion with the domains meeting at an angle of ~120, as defined by the cylinder axes. The folding topology of each domain is identical to that previously observed for single FnIII domains from tenascin and fibronectin. The domains of NgFn1,2 are related by an approximate two fold screw axis that is nearly parallel to the longest dimension of the fragment. Assuming this relative orientation is a general property of tandem FnIII repeats, the multiple tandem FnIII domains in neuroglian and other proteins are modeled as thin straight rods with two domain zig-zag repeats. When combined with the dimensions of pairs of tandem immunoglobulin-like domains from CD4 and CD2, this model suggests that neuroglian is a long narrow molecule (20 - 30 Å in diameter) that extends up to 370Å from the cell surface.

In photomicrographs, rotary shadowed Ng and NgFn1-5 appear to be highly flexible rod-like molecules. NgFn 1-5 is observed to bend in at least two positions and has a mean total length consistent with models generated from the NgFn1,2 structure. Ng molecules have up to four bends and a mean total length of 392 Å, consistent with a head-to-tail packing of neuroglian's C2-type domains.

The short-timescale behavior of glacial ice

Glaciers are often assumed to deform only at slow (i.e., glacial) rates. However, with the advent of high rate geodetic observations of ice motion, many of the intricacies of glacial deformation on hourly and daily timescales have been observed and quantified. This thesis explores two such short timescale processes: the tidal perturbation of ice stream motion and the catastrophic drainage of supraglacial meltwater lakes. Our investigation into the transmission length-scale of a tidal load represents the first study to explore the daily tidal influence on ice stream motion using three-dimensional models. Our results demonstrate both that the implicit assumptions made in the standard two-dimensional flow-line models are inherently incorrect for many ice streams, and that the anomalously large spatial extent of the tidal influence seen on the motion of some glaciers cannot be explained, as previously thought, through the elastic or viscoelastic transmission of tidal loads through the bulk of the ice stream. We then discuss how the phase delay between a tidal forcing and the ice stream’s displacement response can be used to constrain in situ viscoelastic properties of glacial ice. Lastly, for the problem of supraglacial lake drainage, we present a methodology for implementing linear viscoelasticity into an existing model for lake drainage. Our work finds that viscoelasticity is a second-order effect when trying to model the deformation of ice in response to a meltwater lake draining to a glacier’s bed. The research in this thesis demonstrates that the first-order understanding of the short-timescale behavior of naturally occurring ice is incomplete, and works towards improving our fundamental understanding of ice behavior over the range of hours to days.

Radiation from a short electric dipole antenna in a hot uniaxial plasma

The effects of electron temperature on the radiation fields and the resistance of a short dipole antenna embedded in a uniaxial plasma have been studied. It is found that for ω < ω_p the antenna excites two waves, a slow wave and a fast wave. These waves propagate only within a cone whose axis is parallel to the biasing magnetostatic field B_o and whose semicone angle is slightly less than sin ^(-1) (ω/ω_p). In the case of ω > ω_p the antenna excites two separate modes of radiation. One of the modes is the electromagnetic mode, while the other mode is of hot plasma origin. A characteristic interference structure is noted in the angular distribution of the field. The far fields are evaluated by asymptotic methods, while the near fields are calculated numerically. The effects of antenna length ℓ, electron thermal speed, collisional and Landau damping on the near field patterns have been studied.

The input and the radiation resistances are calculated and are shown to remain finite for nonzero electron thermal velocities. The effect of Landau damping and the antenna length on the input and radiation resistances has been considered.

The radiation condition for solving Maxwell's equations is discussed and the phase and group velocities for propagation given. It is found that for ω < ω_p in the radial direction (cylindrical coordinates) the power flow is in the opposite direction to that of the phase propagation. For ω > ω_p the hot plasma mode has similar characteristics.

A short thesis on arc-welding

During the past few years the attention of architects, engineers and others encased in or connected with the building industry has been attracted to the possibilities of the application of welding processes to the joining of structural members. As oxy-acetylene welding was developed before electric arc welding became perfected it was only natural that the gas torch should be first considered. It developed however on examination of the two processes that while acetylene welding gave better results in most cases it was only in the hands of experts that it could consistently outscore the arc as a welding medium. Arc-welding has the advantage over the acetylene process, where each individual operator must use his own judgement as to the proper flame, in that a squad of arc-welders can work under the direction of a single expert supervisor who accepts the responsibility of fixing the current value and of determining the proper size of welding rod to be used on any given type of work.

Dielectric recovery of short A-C arcs between low-boiling-point electrodes

The re-ignition characteristics (variation of re-ignition voltage with time after current zero) of short alternating current arcs between plane brass electrodes in air were studied by observing the average re-ignition voltages on the screen of a cathode-ray oscilloscope and controlling the rates of rise of voltage by varying the shunting capacitance and hence the natural period of oscillation of the reactors used to limit the current. The shape of these characteristics and the effects on them of varying the electrode separation, air pressure, and current strength were determined.

The results show that short arc spaces recover dielectric strength in two distinct stages. The first stage agrees in shape and magnitude with a previously developed theory that all voltage is concentrated across a partially deionized space charge layer which increases its breakdown voltage with diminishing density of ionization in the field-tree space. The second stage appears to follow complete deionization by the electric field due to displacement of the field-free region by the space charge layer, its magnitude and shape appearing to be due simply to increase in gas density due to cooling. Temperatures calculated from this second stage and ion densities determined from the first stage by means of the space charge equation and an extrapolation of the temperature curve are consistent with recent measurements of arc value by other methods. Analysis or the decrease with time of the apparent ion density shows that diffusion alone is adequate to explain the results and that volume recombination is not. The effects on the characteristics of variations in the parameters investigated are found to be in accord with previous results and with the theory if deionization mainly by diffusion be assumed.