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

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).

Siu Yau Lung, Philip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 99-119). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.iv / Declaration --- p.v / Scientific publication --- p.vi / Abbreviations --- p.vii / Genetic symbols --- p.ix / Table of contents --- p.x / List of tables --- p.xiv / List of figures --- p.xv / Chapter Chapter One --- Introduction / Chapter 1.1 --- Thesis outline --- p.1 / Chapter 1.2 --- Candida albicans --- p.2 / Chapter 1.3 --- Physical characterization of C. albicans --- p.3 / Chapter 1.3.1 --- Strain identification --- p.3 / Chapter 1.3.2 --- Dimorphism --- p.5 / Chapter 1.3.3 --- Genome of C. albicans --- p.10 / Chapter 1.3.4 --- Karyotype --- p.11 / Chapter 1.4 --- Candidiasis --- p.12 / Chapter 1.4.1 --- Superficial candidiasis --- p.15 / Chapter 1.4.2 --- Systemic candidiasis --- p.16 / Chapter 1.4.3 --- Virulence --- p.16 / Chapter 1.4.4 --- Multi-drug resistance --- p.17 / Chapter 1.5 --- Parasexual genetics --- p.20 / Chapter 1.5.1 --- Mutant isolation --- p.20 / Chapter 1.5.2 --- Spheroplasts complementation --- p.21 / Chapter 1.5.3 --- Mitotic complementation --- p.22 / Chapter 1.6 --- Natural heterozygosity in C. albicans --- p.22 / Chapter 1.7 --- Adenine biosynthesis --- p.26 / Chapter 1.7.1 --- de novo pathway --- p.26 / Chapter 1.7.2 --- Salvage pathway --- p.29 / Chapter 1.7.3 --- Importance of C. albicans ADE1 and ADE2 genes --- p.29 / Chapter 1.8 --- Aim of study --- p.30 / Chapter Chapter Two --- Construction of disrupted C. albicans ADE1 gene / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.34 / Chapter 2.2.1 --- Strains --- p.34 / Chapter 2.2.2 --- Construction of plasmid pGEMTE-ADEl --- p.34 / Chapter 2.2.2.1 --- Isolation of Candida genomic DNA --- p.34 / Chapter 2.2.2.2 --- Isolation of C. albicans ADE1 gene from CAM --- p.36 / Chapter 2.2.2.2.1 --- Amplification of C. albicans ADE1 gene --- p.36 / Chapter 2.2.2.2.2 --- Purification of PCR product --- p.37 / Chapter 2.2.2.3 --- Cloning of ADEl gene into pGEMT-Easy vector --- p.38 / Chapter 2.2.2.3.1 --- Cloning vector pGEMT-Easy --- p.38 / Chapter 2.2.2.3.2 --- Ligation --- p.38 / Chapter 2.2.2.4 --- Transformation of E. coli DH5a cells --- p.39 / Chapter 2.2.2.4.1 --- Preparation of competent E. coli DH5a cells --- p.39 / Chapter 2.2.2.4.2 --- Plasmid DNA transformation --- p.40 / Chapter 2.2.2.4.3 --- Isolation ofplasmid DNA from E. coli --- p.40 / Chapter 2.2.3 --- Construction of pGEMTE-ADElA-URA3 --- p.41 / Chapter 2.2.3.1 --- Isolation of C. albicans URA3 gene from plasmid pCUB-6 --- p.41 / Chapter 2.2.3.2 --- Preparation of cloning vector pGEMTE-ADE 1Δ --- p.42 / Chapter 2.2.3.2.1 --- PCR amplification of vector pGEMTE-ADElΔ --- p.42 / Chapter 2.2.3.2.2 --- Modification of PCR vector pGEMTE-ADElΔ --- p.44 / Chapter 2.2.3.2.3 --- Dephosphorylation --- p.45 / Chapter 2.2.3.3 --- Cloning and isolation of plasmid pGEMTE-ADE1Δ-URA3 --- p.46 / Chapter 2.3 --- Results and Discussion --- p.47 / Chapter Chapter Three --- Gene disruption of C. albicans CAI4 by electroporation / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.2.1 --- Strains --- p.54 / Chapter 3.2.2 --- Transforming DNA --- p.54 / Chapter 3.2.3 --- Purification of PCR product --- p.55 / Chapter 3.2.4 --- DNA transformation --- p.55 / Chapter 3.2.5 --- Transformation efficiency --- p.56 / Chapter 3.2.5.1 --- Pulse length --- p.56 / Chapter 3.2.5.2 --- Amount of DNA --- p.57 / Chapter 3.2.6 --- Southern analysis of transformants --- p.57 / Chapter 3.2.6.1 --- Isolation of Candida genomic DNA --- p.57 / Chapter 3.2.6.2 --- Preparation of Candida genomic DNA for Southern analysis --- p.57 / Chapter 3.2.6.3 --- Southern hybridization --- p.58 / Chapter 3.2.6.4 --- Preparation of radioactive probe --- p.60 / Chapter 3.2.6.5 --- Radioactive labelling of the probe --- p.61 / Chapter 3.2.6.6 --- Hybridization of nylon membrane --- p.62 / Chapter 3.2.6.7 --- Stringency washes --- p.62 / Chapter 3.2.6.8 --- Auto-radiography --- p.62 / Chapter 3.3 --- Results and Discussion --- p.64 / Chapter Chapter Four --- UV mutagenesis of disrupted C. albicans / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Materials and Methods --- p.76 / Chapter 4.2.1 --- Strains --- p.76 / Chapter 4.2.2 --- Generation of recombinants by UV irradiation --- p.76 / Chapter 4.2.3 --- Analyses of twin-sectored colonies --- p.77 / Chapter 4.2.3.1 --- Replica analyses of twin-sectored colonies --- p.77 / Chapter 4.2.3.2 --- Southern analysis of segregants --- p.77 / Chapter 4.3 --- Results and Discussion --- p.78 / Chapter Chapter Five --- Concluding remarks and perspectives --- p.96 / Bibliography --- p.99

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 114-125).

Dysgenic male recombination (MR) induced by the P strains T-007 and OKI rarely, if ever, occurs in the heterochromatin of chromosome two. One possible explanation is that the lack of heterochromatic exchange is due to the highly condensed chromatin in this region. Butyrate (a suspected modifier of chromatin structure) induced significant levels of heterochromatic MR in dysgenic hybrids derived from crosses involving two different P strains. This finding is consistent with the hypothesis that chromatin structure can influence the insertion and excision of P elements and hence MR. Analogous experiments were performed using third chromosome suppressor of variegation (Su(var)) mutations. Neither suppressor mutation induced any heterochromatic MR, suggesting that the mode of action of these Su(var) genes is different from, and more specific than, that of butyrate. One of the mutations (325) which is thought to influence meiotic recombination frequencies, causes some alterations in euchromatic MR in crosses involving the OKI strain. The other mutation, 318, affects neither meiotic nor dysgenic recombination. Su(var) 325 is the first known "factor" to influence meiotic and dysgenic recombination similarly. / Science, Faculty of / Zoology, Department of / Graduate

Meiotic recombination promotes genetic variation by reciprocal exchange of genetic material producing novel allelic combinations that influence important agronomic traits in crop plants. Therefore, harnessing meiotic recombination has the potential to accelerate crop improvement via classical breeding. Numerous genes involved in crossover formation have been identified in model systems. For example, SPO11 mediates generation of meiotic DNA double-strand breaks (DSBs) across all eukaryotes, which may be repaired as crossovers. However, downstream regulators of recombination remain to be identified, including those with species-specific roles. To isolate crossover frequency modifiers I performed a high-throughput forward genetic screen using EMS mutagenesis of Arabidopsis carrying a fluorescent crossover reporter line called 420. The primary screen isolated nine mutants from ~3,000 scored individuals that showed significantly higher (high crossover rate, hcr) or lower (low crossover rate, lcr) crossover frequency, including a new fancm allele. Four mutants (hcr1, hcr2, hcr3 and lcr1) were mapped by sequencing and candidate genes identified. The hcr1 mutation was confirmed as being located within the PROTEIN PHOSPHATASE X-1 (PPX-1) gene, using isolation of an independent allele and complementation studies. Similarly, the lcr1 mutation was confirmed to be within the gene TBP-ASSOCIATED FACTOR 4B (TAF4B). Using immunocytological staining I observed that hcr1 did not show changes in DSB-associated foci (RAD51), but it did show a significant increase in crossover-associated MLH1 foci. The hcr1 mutation increases crossovers mainly in the sub-telomeric chromosome regions, which remain sensitive to crossover interference. Also the genetic interaction between the hcr1 and fancm mutations is additive. These results support a model where PPX- 1 acts to limit recombination via the Class I interfering CO pathway, downstream of DSB formation. In summary, this genetic screen has led to discovery of novel genes that regulate meiotic recombination and their functional characterization may find utility in crop breeding programs.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 109-140).

Thesis (Ph. D.)--Stanford University, 1997. / Submitted to the Department of Biochemistry and the Committee on Graduate Studies of Stanford University. Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 130-133).