Thesis (M.S.)--Youngstown State University, 1997. / Includes bibliographical references (leaves 82-88).

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains v, 125 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 71-87).

Homologous recombination is a highly regulated and complex biological process required for sexual reproduction in humans and many other species. The mechanisms of initiation and control of this process, however, are only partially understood. The aim of this work is to tease apart and utilize the differences in recombination localization between human populations to understand the underlying biological processes.

In order to study and compare spontaneous and radiation induced recombination frequencies of the euchromatic and heterochromatic regions of chromosome 2 Drosophila melanogaster females were exposed to 0, 2, 3, 4, or 5 krads of gamma radiation. Exchange was measured in the b-Bl, Bl-It, lt-rl, rl-pk, and pk-cn intervals. The lt-rl interval defines a wholly heterochromatic segment. Analysis of the recombination data demonstrates that spontaneous recombination occurs in the heterochromatic interval at a frequency of approximately 0.1% and the frequency of induced recombination is primarily increased in the heterochromatic interval. Moreover, the frequency of recombination in the heterochromatic interval is correlated with the dose of radiation. There are slight or no increases in the recombination frequencies of the euchromatic segments and the regions containing the heterochromatic-euchromatic boundaries show responses intermediate to the heterochromatic and euchromatic regions. Testing of the recombinant chromosomes indicates that 22% of them are associated with recessive lethals. The association is greater in eggs laid the first four days after radiation treatment than in thoselaid five-eleven days after the radiation treatment. It is postulated that induced recombination can occur via symmetrical as well as asymmetrical interchange. The interchromosomal effect of chromosome 3_, heterozygous for In(3LR)DcxF, on recombination in chromosome 1_ has been studied. The results show that its effect is not significant in the heterochromatic region. Thus, the alterations in the recombination frequencies owing to radiation treatment appear to be independent of those owing to the interchromosomal effect. Recombination was also measured in the presence of the heterochromatic deficiency - Df(2R)MS2¹º. The results indicate that the frequency of recombination is decreased in the chromosome arm containing the deficiency and in the heterochromatic interval of the left arm. The euchromatic regions of the opposite arm show a slight increase in recombination. A higher number of multiple crossover progeny are recovered than would be expected according to map distances in the presence of the heterochromatic deficiency, Df(2R)MS2¹º, the heterozygous inversion, In(3LR)DcxF, and for double crossovers involving the heterochromatic region, but only at high doses of radiation. / Science, Faculty of / Zoology, Department of / Graduate

The spectral phase of high harmonic and attosecond pulses is typically shaped by the interaction of the recollision electron with the strong field in the continuum. However, the phase of the transition moment coupling bound and continuum states can be significant in shaping the emitted radiation. The measurement of transition moment phase shifts can reveal information about attosecond electron dynamics and structure. Here, I demonstrate that all-optical approaches to attosecond measurement, based on perturbing recollision with a weak infrared field, are sensitive to transition moment phase shifts arising from electronic structure and multielectron interaction using analytical theory, ab initio simulation, and experiment. The insensitivity of all-optical approaches to transition moment phase shifts arising from ionic structure is found to be a result of a first-order cancellation of the effect of the perturbing field on the recollision electron wave packet and the transition moment. Prior to these findings, it was widely believed that all-optical methods were insensitive to the transition moment phase. The insensitivity of all-optical measurement to both ionic structure and propagation effects will permit for the unambiguous isolation of electron structure and multielectron interaction in attosecond measurement. These results will allow any laboratory capable of generating attosecond pulses to perform measurements of the transition moment phase without an additional experimental apparatus, even at wavelengths where the single photoionization cross-section becomes small.

No description available.

An experimental method (the two-dose-rate method) for the correction of general recombination losses in liquid ionization chambers has been developed and employed in experiments with different liquids and radiation qualities. The method is based on a disassociation of initial and general recombination, since an ionized liquid is simultaneously affected by both of these processes. The two-dose-rate method has been compared to an existing method for general recombination correction for liquid ionization chambers, and has been found to be the most robust method presently available. The soundness of modelling general recombination in liquids on existing theory for gases has been evaluated, and experiments indicate that the process of general recombination is similar in a gas and a liquid. It is thus reasonable to employ theory for gases in the two-dose-rate method to achieve experimental corrections for general recombination in liquids. There are uncertainties in the disassociation of initial and general recombination in the two-dose-rate method for low applied voltages, where initial recombination has been found to cause deviating results for different liquids and radiation qualities. Sensitivity to ambient electric fields has been identified in the microLion liquid ionization chamber (PTW, Germany). Experimental data may thus be perturbed if measurements are conducted in the presence of ambient electric fields, and the sensitivity has been found to increase with an increase in the applied voltage. This can prove to be experimentally limiting since general recombination may be too severe for accurate corrections if the applied voltage is low.

Murine embryonic stem (ES) cells have provided researchers with a useful tool to investigate genome function and the consequences of genome mutation. One mutational approach is gene-targeting, this involves the introduction of DNA sequences of choice, precisely, to almost any location in the target genome by homologous recombination. At present, most gene-targeting strategies introduce DNA constructs that derive from plasmids. Plasmids can stably propagate up to approximately 30 kb of DNA. Therefore, this size limit may place a restriction on the range of mutations that may be made to a genome using a single plasmid-derived gene-targeting construct alone. To overcome this limitation, multiple rounds of sequential gene-targeting experiments may be performed, however such an approach may be too lengthy to be practicable. In order to address this current limitation with gene-targeting a novel strategy was tested, implementing Cre-lox site-specific recombination (SSR) technology and the bacterial artificial chromosome (BAC) vector system. Two sequential gene-targeting events in murine E14Tg2a ES cells (HPRT) were performed at separate locations to chromosome 11. The aim of gene-targeting was to create an interval on chromosome 11 that included a single copy of the murine alpha-globin locus, between the hetero-specific lox sites, loxP and lox511, an interval of approximately 64 kb. To this end the first targeting event delivered lox511 /hygromycin/I See Illox51 J sequences and the second event frt/I See I/5'hprt//oxP/neomycin sequences. ES cells that were confirmed to have correctly undergone the two desired targeting events (double-targeted) were then assessed to determine whether these events had occurred to the same chromosome 11 (in eis ), as desired, or to the alternate copies of chromosome 11 (in trans). This assessment involved restricting DNA from the double-targeted ES cell lines with the rare-cutting restriction endonuclease I See I and resolving the products of this restriction by pulsed field gel electrophoresis. This analysis identified two in cis lines (CAT-A3 and CAT-B3) and an in trans line (CATCIO). The double-targeted ES cell lines were then further characterised to determine whether the hetero-specific lox sites they harboured would participate in ere-mediated SSR. The positive result of this analysis was the generation of ES cell clones that were hemizygous for the alpha-globin locus, a deletion of 64 kb. Hemizygous ES cell clones were obtained from the CAT-A3 and CAT-B3 ES cell lines, as predicted, but not from the CAT-C 10 line, although all the lines tested showed evidence of SSR occurring. In parallel to achieving the interval between loxP and lox51 l in ES cells, a BAC, harbouring the alpha-globin locus, was similarly modified with lox sites using recombination-mediated cloning. The aim of the BAC modification was to create an interval between lox sites in the BAC identical to that achieved in the ES cells. The BAC was targeted sequentially with two separate constructs, lox511/k.anamycin/lox511/HSVtk and then blasticidin/loxP/3'hprt/I See 11.frt. The correct targeting of the BAC was verified by restricting its DNA with a panel restriction endonucleases. The lox sites were then tested in an in vitro analysis with purified Cre recombinase and found to be competent to participate in SSR reactions. The modified BAC was co-electroporated with a Cre expression plasmid into the CAT-A3 and CAT-B3 ES cell lines, previously characterised as targeted in eis, with the aim of exchanging the interval sequences in the ES cell with those of the BAC. The ultimate aim of such an exchange would be to deliver any combination of mutations that would be previously engineered to the BAC interval, to that of the ES cell, by a single SSR event. This experimental approach should expedite and facilitate the mutational analysis of gene loci. To generate comparative data the result of SSR between the modified BAC and an in trans targeted ES cell line (CAT-CI 0) was also assessed. The selection for the desired exchange involved reconstruction of an Hprt minigene and exclusion of a thymidine kinase gene, cells which haboured these events could therefore be selected for in HAT and ganciclovir supplemented media respectively. ES cell clones generated from both of the in cis lines tested (CAT-A3 and CAT-B3) had the correct selection resistance profiles, thus indicating that the desired exchange had been achieved in these clones. Additionally, Southern blot analysis from the DNA from these clones was consistent with the achievement of the desired exchange. However, the results obtained from clones generated from the in trans line (CAT-CI 0) were not consistent with their predicted genetic arrangement following SSR with the modified BAC. Thus far similar experimental approaches have been implemented to exchange smaller intervals of I to 5 kb and have been termed recombinase mediated cassette exchange (RMCE). However the experiments described within this thesis are the first test whether the same rationale may be applied to larger intervals. The strategy described and tested in this thesis has therefore been termed large interval RMCE (liRMCE).

No description available.

No description available.