Thesis advisor: Hugh P. Cam / Meiotic homologous recombination (HR) is not uniform across eukaryotic genomes, creating regions of strong recombination activity dubbed recombination hotspots, and regions of low recombination activity dubbed coldspots. Considerable attention has led to discoveries of a host of factors controlling the formation of hotspots. However, the determinants of coldspots are not as clearly defined. I have previously shown that CENP-B homologs of the fission yeast Schizosaccharomyces pombe have a genome surveillance role in regulating the nuclear organization and expression of Tf2 retrotransposons. Here, I reveal an additional role for CENP-Bs in suppressing meiotic recombination of Tf2s. I describe the development of a random sporulation assay to rapidly screen thousands of meiotic progeny for recombination across a locus in a variety of genetic backgrounds. Loss of any CENP-B family members (Abp1, Cbh1, Cbh2), results in increased HR at Tf2s. I show that Abp1, which acts as the primary determinant of HR suppression at Tf2s, is required to maintain proper recombination exchange of homologous alleles flanking a Tf2. In addition, Abp1-mediated suppression of HR at Tf2s requires all three of its domains with distinct functions in transcriptional repression and higher-order genome organization. I show that this suppression is likely mediated by Abp1 binding to specific motifs near the 3’end of flanking LTRs. I demonstrate that HR suppression of Tf2s can be robustly maintained despite disruption to chromatin factors essential for transcriptional repression and nuclear organization of Tf2s. Intriguingly, I uncover a surprising cooperation between the histone methyltransferase Set1 responsible for histone H3 lysine 4 methylation and the non-homologous end joining pathway in ensuring the suppression of HR at Tf2s. Furthermore, I identify a role for the architectural protein condensin involved in 3D chromatin organization and chromosome condensation in restricting HR at Tf2s. My study identifies a molecular pathway involving functional cooperation between a transcription factor with epigenetic regulators, DNA repair pathway, and chromosome organizers to regulate meiotic recombination at interspersed repeats. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Understanding how genetic modifications, individual or in combination, affect organismal fitness or other phenotypes is a challenge common to several areas of biology, including human health & genetics, metabolic engineering, and evolutionary biology. The importance of a gene can be quantified by measuring the phenotypic impact of its associated genetic perturbations "here and now", e.g. the growth rate of a mutant microbe. However, each gene also maintains a historical record of its cumulative importance maintained throughout millions of years of natural selection in the form of its degree of sequence conservation along phylogenetic branches. This thesis focuses on whether and how the phenotypic and evolutionary importance of genes are related to each other. Towards this goal, I developed a new approach for characterizing the phenotypic consequences of genetic modifications in genome-scale biochemical networks using constraint-based computational models of metabolism. In particular, I investigated the impact of gene loss events on fitness in the model organism Saccharomyces cerevisiae, and found that my new metric for estimating the cost of gene deletion correlates with gene evolutionary rate. I found that previous failures to uncover this correlation using similar techniques may have been the result of an incorrect assumption about how isoenzymes deletions affect the reaction they catalyze. I next hypothesized that the improvement my metric showed in predicting the cost of isoenzyme loss could translate into an improved capacity to predict the impact of pairs of gene deletions involving isoenzymes. Studies of such pair-wise genetic perturbations are important, because the extent to which a genetic perturbation modifies any given phenotype is often dependent on the genetic background upon which it has been performed. This lack of independence within sets of perturbations is termed epistasis. My results showed that, indeed, the new metric displays an increased capacity to predict epistatic interactions between pairs of genes. In addition to shedding light on the relationship between the functional and evolutionary importance of genes, further developments of our approach may lead to better prediction of gene knockout phenotypes, with applications ranging from metabolic engineering to the search for gene targets for therapeutic applications.

Lai-Han Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 120-129). / Abstracts in English and Chinese. / Members of Thesis Advisory Committee --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / Abbreviations --- p.viii / Table of contents --- p.ix / Chapter Ch 1 --- General Introduction / Chapter 1.1 --- Growth hormone --- p.1 / Chapter 1.1.1 --- Physiology of GH --- p.1 / Chapter 1.1.2 --- Biological Functions of growth hormone --- p.2 / Chapter 1.1.3 --- Structure of growth hormone --- p.3 / Chapter 1.1.4 --- The physiological activities of growth hormone --- p.5 / Chapter 1.1.5 --- The importance of increasing grass carp production yield --- p.6 / Chapter 1.1.6 --- Application of recombinant growth hormone --- p.7 / Chapter 1.1.7 --- Application of recombinant grass carp GH --- p.8 / Chapter 1.2 --- Administration of GH to fish --- p.9 / Chapter 1.2.1 --- Methods in the administration of growth hormone to fish --- p.9 / Chapter 1.3 --- Different approaches in the expression of GH --- p.12 / Chapter 1.3.1 --- Bacterial expression system --- p.12 / Chapter 1.3.2 --- Baculovirus and yeast expression system --- p.14 / Chapter 1.4 --- Expression of grass carp growth hormone in yeast (Saccharomyces cerevisiae & Pichia Pastoris) --- p.14 / Chapter 1.4.1 --- Saccharomyces cerevisiae is widely used in the past --- p.14 / Chapter 1.4.2 --- Pichia pastoris is an alternative approach to overcome drawbacks of Saccharomyces cerevisiae --- p.16 / Chapter 1.5 --- Methods for increasing expression level of a cloned gene --- p.20 / Chapter 1.6 --- Purpose of present study --- p.22 / Chapter Ch 2 --- Materials and Method / Chapter 2.1 --- Bacterial strains --- p.23 / Chapter 2.2 --- Yeast strains --- p.23 / Chapter 2.3 --- Plasmids --- p.23 / Chapter 2.4 --- Bacterial culture Media and Solutions --- p.23 / Chapter 2.5 --- Antibiotic Solutions --- p.24 / Chapter 2.6 --- Restriction endonucleases and other enzymes --- p.24 / Chapter 2.7 --- Culture stock solutions --- p.24 / Chapter 2.8 --- SDS-PAGE and Western blot reagents --- p.28 / Chapter 2.9 --- DNA agarose gel --- p.30 / Chapter 2.10 --- General Techniques --- p.32 / Chapter 2.11 --- Construction of recombinant yeast strain --- p.40 / Chapter 2.12 --- Expression of the recombinant protein --- p.42 / Chapter 2.13 --- Purification of the protein --- p.44 / Chapter 2.14 --- Characterization of the protein --- p.46 / Chapter Ch3 --- Results / Chapter 3.1 --- Construction of Recombinant Plasmids --- p.50 / Chapter 3.2 --- Sequencing of the recombinants --- p.61 / Chapter 3.3 --- Linearization and electroporation of recombinant plasmids --- p.69 / Chapter 3.4 --- Optimization of electroporation condition of producing recombinant yeast cells --- p.70 / Chapter 3.5 --- Screening and selection of AOX characteristics (By MM plates) --- p.71 / Chapter 3.6 --- Screening and selection of AOX characteristics (By PCR) --- p.72 / Chapter 3.7 --- Selection of high expression clones --- p.77 / Chapter 3.8 --- Time course expression studies --- p.82 / Chapter 3.9 --- Batch fermentation --- p.89 / Chapter 3.10 --- Purification of r-gcGH --- p.92 / Chapter 3.11 --- Characterization of r-gcGH --- p.100 / Chapter 3.11.1 --- Immunological property of r-gcGH --- p.100 / Chapter 3.11.2 --- Biological activity of r-gcGH --- p.101 / Chapter Ch4 --- Discussion / Chapter 4.1 --- Evaluation of expression profile --- p.108 / Chapter 4.1.1 --- Expression yield --- p.108 / Chapter 4.1.2 --- Efficiency in the cleavage of signal peptide --- p.111 / Chapter 4.2 --- Evaluation of the biological activity of r-gcGH --- p.113 / Chapter 4.3 --- Evaluation of the post-translational modification of r-gcGH --- p.115 / Chapter 4.4 --- Further studies --- p.116 / Reference --- p.120

The ATP binding cassette (ABC) transporters represent the largest family of transmembrane proteins and play important roles in human inherited disease such as the multi-organ disease cystic fibrosis and cholesterol transport disorder Tangier’s disease. These proteins are also implicated in conferring multidrug resistance, rendering many cancer therapies ineffective, as well as contributing to the pathogenicity of some organisms. The yeast ABC proteins, Pdr12p, a weak acid efflux pump, and Ste6p, the a-factor exporter, were screened for interacting partners using the integrated membrane yeast two-hybrid (iMYTH) system to gain further insight into their biological function. Two interactors were identified for Ste6p, however, the Pdr12p screen identified 13 novel interactions, most notable of which are three other ABC transporters, Pdr5p, Pdr10p and Pdr11p. Subsequent functional analysis of double deletion mutants supports a genetic interaction between Pdr12p and Pdr10p as the pdr12Δ pdr10Δ strain showed resistance to increasing concentrations of weak organic acids.

The ATP binding cassette (ABC) transporters represent the largest family of transmembrane proteins and play important roles in human inherited disease such as the multi-organ disease cystic fibrosis and cholesterol transport disorder Tangier’s disease. These proteins are also implicated in conferring multidrug resistance, rendering many cancer therapies ineffective, as well as contributing to the pathogenicity of some organisms. The yeast ABC proteins, Pdr12p, a weak acid efflux pump, and Ste6p, the a-factor exporter, were screened for interacting partners using the integrated membrane yeast two-hybrid (iMYTH) system to gain further insight into their biological function. Two interactors were identified for Ste6p, however, the Pdr12p screen identified 13 novel interactions, most notable of which are three other ABC transporters, Pdr5p, Pdr10p and Pdr11p. Subsequent functional analysis of double deletion mutants supports a genetic interaction between Pdr12p and Pdr10p as the pdr12Δ pdr10Δ strain showed resistance to increasing concentrations of weak organic acids.

Increasing gene dosage provides a powerful means of probing gene function, as it tends to cause a gain-of-function effect due to increased gene activity. In the budding yeast, Saccharomyces cerevisiae, systematic gene overexpression studies have shown that in wild-type cells, overexpression of a small subset of genes results in an overt phenotype. However, examining the effects of gene overexpression in sensitized cells containing mutations in known genes is a powerful means for identifying functionally relevant genetic interactions. When a query mutant phenotype is rescued by gene overexpression, the genetic interaction is termed dosage suppression. I comprehensively investigated dosage suppression genetic interactions in yeast using three approaches. First, using one of two novel plasmid libraries cloned by two colleagues and myself, I systematically performed dosage suppression screens and identified over 130 novel dosage suppression genetic interactions for more than 25 essential yeast genes. The plasmid libraries, called the molecular barcoded yeast ORF (MoBY-ORF) 1.0 and 2.0, are designed to streamline dosage analysis by being compatible with high-throughput genomics technologies that can monitor plasmid representation, including barcode microarrays and next-generation sequencing methods. Second, I describe a detailed analysis of the novel dosage suppression interactions, as well as of literature-curated interactions, and show that the gene pairs exhibiting dosage suppression are often functionally related and can overlap with physical as well as negative genetic interactions. Third, I performed a systematic categorization of dosage suppression genetic interactions in yeast and show that the majority of the dosage suppression interactions can be assigned to one of four general mechanistic classifications. With this comprehensive analysis, I conclude that systematically identifying dosage suppression genetic interactions will allow for their integration into other genetic and physical interaction networks and should provide new insight into the global wiring diagram of the cell.

Increasing gene dosage provides a powerful means of probing gene function, as it tends to cause a gain-of-function effect due to increased gene activity. In the budding yeast, Saccharomyces cerevisiae, systematic gene overexpression studies have shown that in wild-type cells, overexpression of a small subset of genes results in an overt phenotype. However, examining the effects of gene overexpression in sensitized cells containing mutations in known genes is a powerful means for identifying functionally relevant genetic interactions. When a query mutant phenotype is rescued by gene overexpression, the genetic interaction is termed dosage suppression. I comprehensively investigated dosage suppression genetic interactions in yeast using three approaches. First, using one of two novel plasmid libraries cloned by two colleagues and myself, I systematically performed dosage suppression screens and identified over 130 novel dosage suppression genetic interactions for more than 25 essential yeast genes. The plasmid libraries, called the molecular barcoded yeast ORF (MoBY-ORF) 1.0 and 2.0, are designed to streamline dosage analysis by being compatible with high-throughput genomics technologies that can monitor plasmid representation, including barcode microarrays and next-generation sequencing methods. Second, I describe a detailed analysis of the novel dosage suppression interactions, as well as of literature-curated interactions, and show that the gene pairs exhibiting dosage suppression are often functionally related and can overlap with physical as well as negative genetic interactions. Third, I performed a systematic categorization of dosage suppression genetic interactions in yeast and show that the majority of the dosage suppression interactions can be assigned to one of four general mechanistic classifications. With this comprehensive analysis, I conclude that systematically identifying dosage suppression genetic interactions will allow for their integration into other genetic and physical interaction networks and should provide new insight into the global wiring diagram of the cell.

Tumor Susceptibility Gene, TSG101, has been identified as a tumor susceptibility gene with multiple functions. TSG101 encodes a 46 kDa protein composed of 390 amino acids. As previous studies reported, TSG101 participates in cell-cycle control, membrane proteins¡¦ trafficking, and transcriptional regulation. To identify the proteins that mediated function involved TSG101, we perform yeast two-hybrid cDNA library screening to search for TSG101-interacting proteins. A construct pAS2-1-TSG101 was used as a bait to screen a human testis cDNA library. This screening selected 68 TSG101 interacting clones, including 17 known proteins. These proteins were functionally classified as participating in cell-cycle alteration, protein sorting, transcriptional regulation, modification, signal transduction and other functions. Our results provide the evidences which not only confirm the results of previous studies, but also provide further information related to TSG101 biological functions worth intensive study. Among these clones, we choose KLIP1 gene, which encodes a transcription factor, for further study to elucidate the functional role of TSG101 in nucleus. In vitro GST pull-down assay and in vivo co-immunoprecipitation assay were performed using GST-KLIP1 and HA-tagged KLIP1, respectively, have demonstrated that TSG101 and KLIP1 indeed interact with each other within mammalian cells. Detailed biological function mediated through this TSG101 and KLIP1 interaction awaits further investigation.

Dissertation (PhD)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 130-138). Also available in electronic version. Access restricted to campus users.