Recombination is an essential component in the scheme of events that maintain telomeric homeostasis. I have been interested in understanding the recombination events that lead to telomere tract maintenance and survival in the budding yeast Saccharomyces cerevisiae. One of the foci of my dissertation has been an investigation of recombination events in cells carrying a novel allele of the multifaceted MRE11 gene. This allele, mre11A470T, confers a variety of phenotypes influencing nuclear telomeres, the most interesting of which is an altered survival in telomerase negative cells. Through an investigation of the genetic dependencies of this phenotype, I found that senescence bypass is mediated by homologous recombination. The analysis of telomere profiles from this allele reveals a unique extended telomere tract phenotype accompanying the amplification of subtelomeric elements. Homeologous recombination and sister chromatid exchange (SCE) between repeats are increased in the absence of Mre11 (19) and Mre11-associated proteins, (144). Our data therefore suggest a model in which mre11A470T generates extended wildtype length telomere tracts through increased homeologous recombination and subsequent break induced replication (BIR) to the end of the chromsosome. (Figure 1.12) BIR is likely to increase viability by increasing the range of telomere tract sizes in telomerase null yeast cells. mre11A470 is invariant among organisms and falls into a highly conserved 13 amino-acid stretch. hsMre11 is associated with alternate lengthening of telomeres (ALT) cancers that use recombination for survival. Our studies may help uncover the role of Mre11 in the regulation of human telomeric recombination in cancers. Elongated telomere tracts in vegetative cells of budding yeast are known to shorten to the size of the majority of telomeres through a one-step recombination process called telomere rapid deletion (TRD) (106) (21). TRD dramatically differs from the slow attrition of telomere tracts observed in the absence of telomerase and has now been found in a multiplicity of organisms suggesting that TRD is part of a common scheme of telomere maintenance among organisms. The second focus of my dissertation is a study of the occurrence and significance of TRD in meiosis. To this end, I calculated the rate of deletions of elongated telomeres in meiosis and investigated its dependency on a key protein regulator of the bouquet assembly, Ndj1. Our results show that meiotic deletions occur at elevated frequencies during meiotic recombination, raising the possibility that TRD may be responsible for the resetting of telomere size during meiosis / acase@tulane.edu

No description available.

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2005. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0033. Adviser: Lynda F. Delph. "Title from dissertation home page (viewed Feb. 9, 2007)."

<p>The Idiopathic Interstitial Pneumonias (IIPs) are complex conditions, with limited treatment options and unknown etiology. Thus, given the complex nature of the disease and the likelihood of genetic heterogeneity, phenotypic and environmental factors must be taken into consideration when searching for genetic components involved in IIP. Families with 2 or more cases of IIP (classified as familial interstitial pneumonia, FIP) provide a unique opportunity to study IIP genetics. Therefore, in order to better define the FIP phenotype, families with a homogeneous pattern of disease diagnosis (IPF only, with all individuals diagnosed with IPF) were compared to families with a heterogeneous phenotype (mixed, with multiple different IIP diagnoses within a single family, including at least one case of IPF). Survival was decreased in the mixed (46%) compared to the IPF only (60%) families (p=0.006) along with the mean age of death (69 IPF only, 64 mixed, p=0.007). Surprisingly, the same results were found when only individual diagnosed with IPF from both types of families were compared (survival 40% vs. 60%, p=0.0003 and age of death 65 vs. 69, p=0.03). Using this same phenotypic classification scheme a whole genome microsatellite screen for FIP was conducted. Two peaks suggestive of linkage to chromosome 11 (LOD=3.3) and chromosome 10 (LOD=2.1) were identified in all 82 families, along with a third peak on chromosome 12 only seen in homogeneous families (LOD=2.5). In order to determine if the two linkage peaks seen in all 82 families were the result of genetic heterogeneity, ordered subset analysis (OSA) was conducted. Applying OSA, which uses family level covariate data to define a more homogeneous subset of families that maximize linkage, low linkage to chromosome 11 maximized linkage to chromosome 10 within a subset of 63 of the 83 families (LOD=3.4) and 27 of the mixed families (LOD=5.1). Furthermore, OSA revealed that families with a lower proportion of smokers among affected individuals contributed significantly to evidence in favor of linkage on chromosome 11 (LOD=4.9). It therefore appears that chromosomes 10 and 11 represent distinct susceptibility factors for FIP. Conducting further fine-mapping of the chromosome 11 region also identified 2 potential candidate genes, MUC2 and MUC5AC. Re-sequencing of both genes followed by selective genotyping of the 10 most interesting SNPs revealed 7 SNPs significantly associated with FIP and 7 SNPs significantly associated with IPF, 6 of which were significant in both FIP and IPF cases as compared to spouse controls. A haplotype consisting of 4 SNPs (1 in MUC2 and 3 in MUC5AC) was also found to be significant in both FIP (p=0.002) and IPF cases (p=0.001). While the SNP in MUC2 is intronic, all 3 MUC5AC SNPs produce amino acid changes. Thus, non-synonymous polymorphisms in MUC5AC are associated with both FIP and IPF.</p> / Dissertation

<p>Recombination of <em>Tcrb</em> gene segments in DN thymocytes is subject to allelic exclusion, such that only a single functional V_β - DJ_β rearrangement is generated per T cell. For <em>Tcrb</em> to be allelically excluded the two alleles must initiate recombination asynchronously and once a β-protein is selected, feedback signals must suppress further recombination. Earlier studies of antigen-receptor loci implicated directed monoallelic association with pericentromeric heterochromatin in the initiation or maintenance of allelic exclusion. In this study we used three-dimensional fluorescent <em>in situ</em> hybridization to directly visualize the nuclear localization of <em>Tcra</em> and <em>Tcrb</em>, pericentromeric heterochromatin, and the nuclear lamina. Here we provide evidence for a fundamentally different basis for <em>Tcrb</em> allelic exclusion. We demonstrate that <em>Tcrb</em> is highly associated with pericentromeric heterochromatin and the nuclear lamina in pro-B cells and in DN and DP thymocytes. We also find that <em>Tcrb</em> does not associate with peri-centromeric heterochromatin and the nuclear lamina in a strict monoallelic fashion. Rather, <em>Tcrb</em> alleles independently associate with the two compartments, leading to a stochastic distribution of nuclei containing both, one, or neither allele associated. In the subset of DN thymocyte nuclei with monoallelically associated <em>Tcrb</em> alleles, the non-rearranged allele is most often associated with repressive compartments. This suggests that association with these compartments inhibits recombination prior to β-selection. This inhibition occurs without altering the conformation of the locus. Moreover, the introduction of an ectopic enhancer into <em>Tcrb</em>, led to both a repositioning of <em>Tcrb</em> away from these repressive compartments. This repositioning was correlated with an increase in the frequency of recombination and a break in allelic exclusion. These data lead us to propose that stochastic rather than directed interactions of <em>Tcrb</em> alleles with repressive nuclear compartments bias initial <em>Tcrb</em> recombination to be monoallelic in developing thymocytes and that such interactions are essential for <em>Tcrb</em> allelic exclusion.</p> / Dissertation

<p>Recently, the study of invertebrate innate immunity has garnered considerable attention after the discovery that mammalian homologues of the <italic>Drosophila melanogaster</italic> </p><p>Toll pathway play a role in mammalian innate immunity. One invertebrate model system that has begun to be intensely studied is the nematode <italic>Caenorhabditis elegans</italic>. Immunity in <italic>C. elegans</italic> has been shown to be inducible in that it responds uniquely to different pathogens. These changes in gene expression require transcription factors in order for certain genes to be transcribed. We utilized an RNA interference screen of potential transcription factors to identify the GATA transcription factor ELT-2 as a possible transcription factor involved in immunity. We then demonstrated that ELT-2 was required for resistance to a wide range of pathogens and was responsible for regulating expression of the C-type lectin <italic>clec-67</italic>, a marker of immunity. </p><p>We also studied another transcription factor known to play a role in C. elegans immune function, the FOXO transcription factor DAF-16. We specifically focused in on the role of DAF-16 in germline-deficient mutants, and we demonstrated that such mutants are resistant to many different pathogens. This led to further investigation of the germline-deficient mutant glp-4, which should also show broad range resistance to pathogens but fails to do so. Through whole genome sequencing, we identified mutations that may be responsible for the glp-4 phenotype. We also demonstrated that DAF-16 was active in glp-4 mutants, leading to us proposing a model where glp-4 plays a role in influencing <italic>C. elegans</italic> immunity besides its involvement in germline development.</p> / Dissertation

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Kristi A. Wharton. Includes bibliographical references.

<p> There are two important features that characterize the canonical genetic code. First, the genetic code is redundant. There are 64 three-letter words that can be assembled from four letters (4³), but only 20 types of amino acids are translated. Combinations of only 20 chemistries have been more than sufficient to generate the amazing diversity of life. However, if we could minimize this redundancy to encode additional nonstandard amino acids (NSAAs), this could permit the evolution of entirely new biological functions and possibly, new organisms.</p><p> Second, the genetic code is conserved. All species utilize the same biochemical foundation to sustain life. This conservation permits organisms to share beneficial traits via horizontal gene transfer (<i>4</i>) and enables accurate expression of heterologous genes in non-native organisms (<i>10</i>). However, the conservation of the genetic code also allows viruses to hijack host translation machinery (<i>15</i>) and compromise cell viability. It further allows unwanted sharing of information between genetically modified organisms (GMOs) and their environment. Virus resistance and intrinsic biocontainment &ndash; biological barriers limiting the spread and survival of microorganisms in natural environments &ndash; are among today's major unsolved problems in biotechnology.</p><p> Changing the genetic code to create genomically recoded organisms (GROs, whose codons have been reassigned to create an alternate genetic code) could solve these challenges. The focus of my graduate work was to construct and characterize GROs that: 1) provide dedicated codons to enable sustained incorporation of more than 20 amino acids as part of an expanded genetic code, and 2) depend on synthetic biochemical building blocks for viability, to advance orthogonal barriers between organisms and their environment.</p><p> <b>Chapter 2.</b> Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of an orthogonal translation system (OTS) consisting of an engineered elongation factor and archaeal aminoacyl-tRNA synthetase (aaRS)/tRNA pair to the normal <i>E. coli</i> translation machinery (<i>16</i>). However, protein yield suffered horn expression of the OTS and competition with release factor 1 (RF1). In a strain lacking RF1, phosphoserine phosphatase, and where only seven essential TAG codons were converted to TAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability. </p><p> <b>Chapter 3.</b> To create a GRO we replaced all known TAG stop codons in <i>E. coli</i> with synonymous TAA codons, which permitted the deletion of RF1 and reassignment of UAG translation function. This GRO exhibited improved cellular fitness and improved properties for incorporation of NSAAs that expand the chemical diversity of proteins <i>in vivo.</i> The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.</p><p> <b>Chapter 4.</b> GMOs are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels, and chemicals (<i> 17</i>). Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems (<i>18, 19</i>), which include bioremediation (<i>20</i>) and probiotics (<i>21</i>). Here, we describe the construction of a series of GROs (<i>22</i>) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced in-frame TAG codons into 22 essential genes of a GRO containing an OTS, linking their expression to the incorporation of sAAs. Of the 60 variants isolated, notable strains were not rescued by environmental cross-feeding, maintained robust growth, and exhibited undetectable escape frequencies upon culturing &sim;10¹¹ cells in liquid media for 20 days, a significant improvement over existing biocontainment approaches (<i>18, 19, 23&ndash;27</i>). </p>

Magnaporthe grisea is a filamentous ascomycete fungus that causes blast disease on rice and other grasses. Blast is a serious deterrent to rice production and negatively affects production of other cereals, forage crops and economically important grasses. The primary means of blast disease management involves the development and implementation of genetically resistant plants. Understanding the molecular basis of plant resistance is the foundation for the development of unique and durable plant protection. The results presented here focus on genes in the rice blast fungus called avirulence genes that encode molecules acting as effectors of host resistance. Until recently, two avirulence loci had been shown to induce resistance in rice cultivar Maratelli. This study gives an update on the current status of one, the AVR1-MARA locus, and describes a new Maratelli avirulence locus that is not allelic to AVR1-MARA or AVR2-MARA. Additionally, evidence is given that indicates a genome rearrangement is responsible for generation of the newly described avirulence locus. Genetic data, hybridization results and DNA sequence analysis demonstrate the translocation of a large AT-rich fragment from one chromosomal location to another. Molecular detection of the translocation is demonstrated by hybridization of certain AVR1-MARA markers that only follow the avirulent phenotype in strains after the rearrangement. The rearrangement is detectable genetically, as the avirulent phenotype controlled at this locus segregates independently from progenitor strains that also contain a single Maratelli-specific avirulence gene. CHEF electrophoretic separation of chromosome-sized DNA shows that the AT rich sequences are located on one of the larger M. grisea chromosomes both before and after cross 4134. Hybridization of CHEF blots indicates that two chromosomes may have been involved in a translocation, however a reorganization of chromosome 2 cannot be ruled out. A homing enzyme strategy for determining the size of the translocated fragment is described. These results demonstrate an example of genomic plasticity leading to a translocation and creation of a new avirulence locus in the rice blast fungus M. grisea.

Many plants produce toxic compounds, called phytoalexins, in response to infection by microorganisms. Some fungal pathogens of these plants can detoxify their host's phytoalexins and genetic studies of the ascomycete, Nectria haematococca Mating Population VI have established an association between detoxification of the pea phytoalexin, pisatin (Pda), and pathogenicity. Previous studies of one of the six genes (PDA) that confer this trait (PDA6-1) was on a dispensable chromosome. In the current study, a technique was developed that uses the pea plant to select for highly virulent recombinant progeny from crosses in which such progeny were relatively rare. It was demonstrated that when pea plants are inoculated with a mixture of ascospores that isolates recovered from pea lesions showed a strong bias for Pda and for being more virulent on pea, compared with ascospore progeny which did not undergo selection on plant. Additionally, all highly virulent isolates had PDA1-1, one of the three PDA genes present in the cross parents, showing that PDA1-1, or a linked gene, was necessary for virulence on pea. In the current study, highly virulent isolates were also identified by screening progeny from crosses that involved a highly virulent parent, 34-18. Analysis of 34-18 and its progeny showed that this isolate contains three PDA genes, PDA5 and PDA9, which were characterized in this study, and an allele of a previously characterized PDA gene. All three genes were associated with virulence on pea and could be lost during genetic crosses. Electrophoretic karyotype (CHEF) analysis showed that this was due to loss of a 1.5 Megabase chromosome carrying PDA1-2 and at least a portion of a 4.9 Mb chromosome carrying PDA5 and PDA9. CHEF analysis also showed that the other previously characterized PDA genes (PDA1-1, PDA2, PDA3, PDA4, PDA6-1 and PDA6-2) were on dispensable chromosomes. These dispensable chromosomes were not required for pathogenicity on carrot and ripe tomato. The results from this work provide evidence to support the hypothesis that the PDA chromosomes are dispensable, that some of them contain genes conferring virulence specifically on pea and genes for pathogenicity on other hosts were on non-PDA chromosomes.