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Sleiman, SamaStudy of the role of pax transcription factors and SP-related factors in C. Elegans organ development
Doctor of Philosophy, The Ohio State University, 2008, Molecular, Cellular, and Developmental Biology

Organogenesis requires cells to coordinate their development. The similarities and differences between cells can be explained by looking at their gene expression profiles. At the center of these profiles, there are often transcription factors such as Pax and Sp-related factors that are critical for organ development.

To understand the specific role of Pax factors in organogenesis, it is important to determine how they function with other transcription factors to regulate target genes.

To better understand the different roles of EGL-38, I used an overexpression approach. I tested the effect of ectopic expression of EGL-38 and its mutants in animals. My results indicate that ectopic expression of EGL-38 and its male tail specific alleles (sy287 and gu22) in embryos induce embryonic lethality, whereas ectopic expression of its egg-laying defective allele (n578) does not have any effect. I hypothesized that these defects could be due to misexpression of different EGL-38 targets in response to the different alleles. Using gfp reporter transgenes, I have shown that indeed ectopic expression of EGL-38, induces ectopic expression of lin-48 (a known direct target).

Pax factors have different targets in different cells because they are known to act in a combinatorial manner with other transcription factors. For example, lin-48 is a direct target of EGL-38 in the hindgut, but it is not expressed in other organs where EGL-38 is known to function. To identify EGL-38 cofactors important for lin-48 hindgut expression, I conducted a genetic screen. I have isolated two mutations (gu84 and gu85). I have shown that gu85 is a mutation in sptf-3, a Sp-related factor.

I also characterized the role of sptf-3 in organogenesis. sptf-3(gu85) animals show developmental defects that are similar to these seen in Wnt mutants. Specifically, mutants exhibit the Bivulva phenotype similar to lin-17/frizzled and lin-18/Ryk mutants. I show that sptf-3(gu85) mutants are sensitive to the dose of lin-17. In addition, sptf-3(gu85) animals exhibit P12 to P11 cell fate transformations, and defects in B cell asymmetric division. These results provide the first description for the role of sptf-3 in C. elegans development. They also suggest that SPTF-3 functions in Wnt-dependent decisions.

Committee:

Helen Chamberlin (Advisor)

Subjects:

Biology, Genetics; Biology, Genetics

Keywords:

Sp1; Pax; Organ Development; c. elegans

Nayak, TaniaInvestigations of the Functions of gamma-Tubulin in Cell Cycle Regulation in Aspergillus nidulans
Doctor of Philosophy, The Ohio State University, 2008, Molecular Genetics
Published data have revealed that γ-tubulin has essential, but incompletely understood functions in addition to its established role in microtubule nucleation. Our lab has isolated several conditional γ-tubulin alleles in which γ-tubulin localization is normal, microtubules are abundant and mitotic spindle assembly is not inhibited, but growth is strongly inhibited at restrictive temperatures. Additionally, there were defects in the coordination of late mitotic events (Prigozhina et al., 2004), indicating that γ-tubulin plays a role in the regulation of mitosis. Most mitotic regulatory proteins localize at the spindle pole body during mitosis, as does γ-tubulin, and it is possible that interactions between γ-tubulin and mitotic regulatory proteins are important for coordinating the events of mitosis. One focus of my research has, consequently, been to study the distribution of mitotic regulatory proteins in vivo by fusing them to green fluorescent protein. In order to facilitate GFP tagging, I have generated a strain in which the nkuA (A. nidulans homolog of Ku70) gene is deleted. We have found that this deletion, along with the use of heterologous markers from A. fumigatus, increases the correct gene targeting frequency to >90% (Nayak et al., 2006). Using this system, I have made GFP fusions of many mitotic regulatory proteins and studied their distribution over time by time-lapse live cell imaging in control cells and in cells carrying a γ-tubulin mutant, mipAD159. Three critical mitotic regulatory proteins that are mislocalized in mipAD159: Cyclin B, Cdk1 and MspA. Studies on cyclin B localization in mipAD159 indicate that the mitotic defects of γ-tubulin are at least partially caused by misregulation of an important mitotic regulator, the APC/C. In particular, γ-tubulin plays an essential role in the inactivation of the APC/C at the end of mitosis or in G1. I have determined that this role of γ-tubulin in APC/C inactivation is independent of its role in microtubule nucleation. In addition, I have also characterized the septation initiation network in A. nidulans. This has revealed novel functions of some genes and may be useful in understanding septum initiation, timing and positioning in coenocytic fungi like A. nidulans.

Committee:

Berl Oakley, PhD (Advisor); Stephen Osmani, PhD (Committee Member); Harold Fisk, PhD (Committee Member); Hay-Oak Park, PhD (Committee Member)

Subjects:

Cellular Biology; Genetics; Molecular Biology

Keywords:

&947;-tubulin; APC/C; cyclin B; cell cycle; mitotic exit; spindle pole body

Chinwalla, VandanaCharacterization of trithorax, a protein required for long-term maintenance of homeotic gene expression
Doctor of Philosophy, Case Western Reserve University, 1995, Genetics
Homeotic genes of Drosophila determine the unique development of each segment and must be precisely regulated to ensure normal development. Long-term maintenance of homeotic gene expression requires the presence of positive (trithorax-Group) and negative (Polycomb-Group) regulators. Trithorax (trx) is the prototypic member of the trithorax-Group and is required for the positive maintenance of homeotic gene expression patterns. We developed two different polyclonal antibodies against parts of trx proteins. Using these antibodies we showed that the trx proteins bind to 63 specific sites on the polytene chromosomes of larval salivary glands. These sites include sites of their known targets, the Bithorax and Antennapedia complexes. Binding of trx to the cytological location of the Bithorax complex, which is not expressed in salivary glands, suggests that the trx binding is constitutive. We localized one trx binding site within a 670 bp fragment of the 5′ regulatory region of Ultrabithorax (Ubx). These results suggest that the trx proteins exert their effect by binding directly or indirectly to specific DNA sequences in their target genes. We showed that the trx proteins co-localize with Polycomb-Group proteins at many sites on the polytene chromosomes. The same DNA fragment from the Ubx regulatory region also contains binding sites for Polycomb-Group proteins. Our results also indicate that the trx proteins are co-localized at almost all sites with another member of the trithorax-Group proteins, the absent, small, homeotic 1 protein. These results suggest that the interaction between these positive and negative regulators of the homeotic genes may be important for their mode of action. The trx proteins contain a DNA-binding motif which is a novel variant of the DNA-binding domains of the nuclear receptor superfamily. We expressed this domain as a fusion protein and used an electrophoretic mobility shift assay to investigate and characterize its DNA-binding activity

Committee:

Peter Harte (Advisor)

Subjects:

Biology, Genetics

Keywords:

Trithorax; Homeotic genes

Niswander, Lee AnnThe genetic control of early postimplantation development: An embryological and molecular analysis
Doctor of Philosophy, Case Western Reserve University, 1990, Genetics
The albino-deletion complex located on chromosome 7 of the mouse represents a model system for the study of mammalian embryonic, neonatal and adult development. This series of 37 overlapping deficiencies that surround and include the albino coat color locus, when homozygous, are associated with specific developmental abnormalities. Nine genetic units have been defined by complementation analyses. These studies reveal the location of gene products needed during preimplantation, early postimplantation, perinatal and juvenile stages of development, formation of the ear labyrinth, and growth and differentiation of the embryonic ectoderm and extraembryonic ectoderm. Also included within this area of chromosome 7 is the albino locus and the structural gene for mitochondrial malic enzyme. Embryological examination of the lethal phenotype associated with the deletion chromosomes of the original Bi complementation group defined two new complementation groups, Bex and Bem. The microclones were localized within the deletion complex by Southern analysis of the DNA hybridization pattern produced by Mus spretus/ M. musculus interspecies cross and homozygous and double heterozygous deletion mice. Two genomic clones distinguish proximal deletion chromosome breakpoint differences within the Bem and Bex complementation groups, a distinction which was not possible genetically. These molecular markers define the limits for the Bem and Bex proximal breakpoints, establish the distal boundary associated with the perinatal survival functional region and provide a molecular starting point to enter the region of the genome needed for development of the embryonic ectoderm and extraembryonic ectoderm

Committee:

Terry Magnuson (Advisor)

Subjects:

Biology, Genetics

Keywords:

Genetic control; Postimplantation; Embryological, molecular analysis

Sinha, RitwikEFFICIENT CONFIDENCE SETS FOR DISEASE GENE LOCATIONS
Doctor of Philosophy, Case Western Reserve University, 2007, Epidemiology and Biostatistics
In positional cloning of disease susceptibility genes, identification of a linked chromosomal region via linkage studies is often followed by fine mapping with association studies. Efficiency can be gained via an intermediate step where confidence regions for the locations of disease genes are constructed. We proposed and explored the properties of two novel practical approaches, one frequentist and one Bayesian, to constructing such intervals using affected sibling pair data. The first approach draws upon a promising paradigm, Confidence Set Inference (CSI) [Papachristou and Lin, 2006a], that converts a sequence of tests to obtain the interval. CSI replaces the traditional null hypotheses of no linkage with a new set of null hypotheses where the chromosomal position under consideration is in tight linkage with a trait locus, and was proposed for the Mean test statistics (CSI-Mean). We postulate that a more efficient test statistic, the Maximum LOD Score (MLS), will lead to more efficient confidence sets when used in the CSI framework. We propose a procedure that tests the CSI null hypotheses using the MLS statistic (CSI-MLS). Compared to CSI-Mean, CSI-MLS provides tighter confidence regions over a range of single- and two-locus disease models. In addition, the MLS test is more powerful than the Mean test in testing the CSI null over a wide range of disease models. Furthermore, CSI-MLS is computationally much more efficient than CSI-Mean. The CSI framework requires knowledge of some disease model related parameters. In practice, such knowledge is often absent and a two-step procedure may be employed. The advantages of CSI-MLS over CSI-Mean is preserved in this practical setting as well. Though the CSI framework compares favorably with other competitors, and CSI-MLS has further improved its statistical and computational efficiency, the two-step procedure is often conservative. This motivates us to explore a Bayesian approach, that formulates the disease gene location as a parameter, to seek possible improvements. In this case, credible intervals with a uniform prior on the location are confidence regions. A Metropolis-Hastings algorithm is implemented to sample from the posterior distribution and Highest Posterior Density Intervals of the disease gene location are constructed. The proposed Bayesian method is shown to provide precise confidence sets with correct coverage probabilities when compared to competing methods. The two novel methods are applied to a Rheumatoid Arthritis data example.

Committee:

Yuqun Luo (Advisor)

Subjects:

Biology, Genetics; Statistics

Keywords:

Confidence Set Inference; disease gene localization; model-free linkage; maximum lod score; effect of disease models; CSI-MLS; Bayesian methods in linkage

Kurzhals, Rebeccah LynnGENETIC AND BIOCHEMICAL ANALYSIS OF THE ROLE OF EXTRA SEX COMBS-LIKE IN POLYCOMB SILENCING IN DROSOPHILA MELANOGASTER
Doctor of Philosophy, Case Western Reserve University, 2006, Genetics
Maintenance of gene expression states is important for cellular differentiation during development. Chromatin mediates the faithful propagation of expression states, but the exact mechanism of how this occurs is unknown. Drosophila development provides an excellent model to study the maintenance of silenced transcription states. Homeotic gene expression domains are established early in development by transiently expressed repressors. The products of the Polycomb group (PcG) genes maintain gene silencing of the homeotic genes outside of their normal expression domains for the rest of development. The Polycomb group proteins ESC and E(Z) are required for Polycomb silencing. ESC has the distinction of being required only in early development in contrast to other PcG gene products. More recently E(Z) was shown to be a histone methyltransferase (HMTase) and its activity is required continuously throughout development. E(Z) activity is dependent on its association with ESC. This implies that either E(Z) HMTase activity in vivo is not as dependent on ESC or another protein replaces the function of ESC later in development. Here I describe a new PcG gene, extra sex combs-like (escl), a paralog of esc, that encodes the ESCL protein. ESCL is expressed throughout development in contrast to ESC. I demonstrate that ESCL interacts with E(Z) like ESC and that ESCL is associated with components of the embryonic ESC-E(Z) complex in embryogenesis and in larvae when ESC is no longer detectable. E(Z) dependent methylation of histones is absent when both ESC and ESCL are removed demonstrating that either ESC or ESCL is required for histone H3 K27 methylation. A mutation in escl enhances the phenotypes of esc mutants. I demonstrate that over-expression of ESCL rescues esc mutant phenotypes and over-expression of ESCL can functionally substitute for ESC. The findings described in this thesis resolve the paradox that ESC is only required and expressed early in embryogenesis, but ESC is continuously required for the HMTase activity of E(Z). I propose that ESCL replaces ESC later in development in E(Z) complexes required for the continued maintenance of Polycomb silencing.

Committee:

Peter Harte (Advisor)

Subjects:

Biology, Genetics

Keywords:

Polycomb; Drosophila; chromatin; histone methylation; ESC; ESCL; E(Z)

Cramer, Todd JamesGenetic Mosaicism Between The Bacteriophage φ80 And Bacteriophage λ
Master of Science (MS), Bowling Green State University, 2008, Biological Sciences
Mosaicism may occur among the genomes of organisms as well as the viruses or bacteriophage that infect them. Some of these changes result from mutations, insertions, deletions, hybridization, generalized or specialized transduction, transformation, and conjugation. These changes to the genome often lead to strains and/or species different from the original. Comparing sequences of these new forms of the genome to their purported origins can help us further understand the evolutionary links between them and how they may have diverged. As bacteriophage lack ribosomal RNA, and no other universal characteristic exists that allows comparison of many different bacteriophage species to each other, it is clear that genomic and proteomic comparisons will play an invaluable role in the future of bacteriophage phylogenetics. The bacteriophage φ80 has an ~45,000 nucleotide linear dsDNA genome and infects several Gram-negative bacteria, including Escherichia coli, exploiting proteins in the bacteria’s outer membrane for host recognition and the bacterial cell itself for reproduction. The bacteriophage φ80 is a lambdoid bacteriophage, genetically and morphologically related to bacteriophage λ, which also infects E. coli. As φ80 and λ utilize different receptors for host recognition, it is likely that their respective genomes will differ in at least those aspects of the genomes that dictate host range specificity. Many other bacteriophage also infect E. coli, and it is possible that co-infection may occur. When this happens, recombination and reorganization between genomes may occur, creating a new version when viable progeny are released. By comparing the approximately forty percent (40%) of the known sequences of the genome of φ80 to already completed sequences of λ and other lambdoid and non-lambdoid bacteriophage, a clearer understanding of how these two bacteriophage are related has emerged and suggests that they are not as similar as previously assumed. The data presented here suggest that although φ80 is related to λ phylogenetically, its genome has diverged from the genome of λ considerably over a relatively short amount of time.

Committee:

Ray Larsen (Advisor); Scott Rogers (Committee Member); Vipa Phuntumart (Committee Member)

Subjects:

Genetics

Keywords:

genetics; mosaicism; bacteriophage; &955;; &966;80

Smith, Christopher T.Characterization of the 5' flanking region of SRY in Rattus norvegicus
Master of Science, University of Akron, 2007, Biology
Hypertension is a growing disease affecting millions of people and is a leading risk factor in heart disease. In the spontaneously hypertensive rat model the Y chromosome raises blood pressure approximately 30 mm HG. Sry is a gene found on the rat Y chromosome that may be responsible for this effect. The 5’ flanking region of Sry1 was cloned, sequenced and characterized to develop a 9.6 kb contig. Analysis of different methods for generating sequencing vectors was completed indicating the insertion of a marker transposon was preferred over traditional cloning procedures. Transcription factor binding sites were identified and characterized to determine which sites may play a role in regulation of Sry1. A CREB site, an intracellular signaling molecule, was identified adjacent to the start site. CREB activation is known to occur in response to a number of factors including hormones and may be a potential activator due to physiological responses. An ETF site known to activate transcription in the absence of a TATA box was also identified. The identification of this factor is significant as the only TATA box identified is distant from the start site suggesting ETF could be the primary activator of transcription. A number of other sites were found near the potential transcriptional start site including WT-1 and EN-1, which are developmental regulators. Furthermore, binding sites for SRY were identified, which could regulate the Sry1 gene by direct activation or a feedback response system. The contig sequence was used to design primers to amplify the coding region plus the 5’ flanking region to allow for comparison of the multiple 5’ flanking regions. A modified PCR procedure with these primers using single stranded binding protein was employed with success to amplify through known secondary structure. The identification of the 5’ flanking region ahs enabled a characterization of the control region promoting future research. Efforts are need to identify a core promoter and other known regulatory regions. Additionally, comparisons need made to determine conservation among the different copies 5’ flanking region. Inexorably this research should focus on whether each copy is functional and how it contributes to hypertension.

Committee:

Monte Turner (Advisor)

Subjects:

Biology, Genetics

Keywords:

Sry; hypertension; shr; wky; 5' flanking region; ETF; SSBP;

Wang, Shu-HueiRegulation of vein, an activating ligand of the drosophila EGF receptor
Doctor of Philosophy, The Ohio State University, 2003, Molecular Genetics
Signaling through the EGF receptor (EGFR) family of receptor tyrosine kinases is important in both normal development and in disease processes. In Drosophila, there is a single EGF receptor and multiple ligands. I show that EGFR signaling stimulated by its activating ligand, Vein, has a fundamental role in regulating two of these cell fate choices: 1) Vn/EGFR signaling directs cells to become notum by antagonizing wing development and by activating notum-specifying genes. 2) Vn/EGFR signaling directs cells to become part of the dorsal compartment by induction of apterous, the dorsal selector gene, and consequently also controls wing development, which depends upon an interaction between dorsal and ventral cells. To understand the control of Vn activity, I investigated transcriptional and post-translational regulation. I analyzed the transcriptional regulation of vn expression in both early and late wing discs using enhancer-reporter study and sequence comparison in Drosophila species. Using the yeast two hybrid system, I identified an interaction between gutfeeling (guf), a homologue of the vertebrate ornithine decarboxylase antizyme (OAZ), and the Ig domain of Vn. This interaction was confirmed in in vitro binding assays and by co-immunoprecipitation. In mammals, OAZ has been shown to bind to Ornithine decarboxylase (ODC) and Smad1, and targets these proteins for degradation by the 26S proteasome in an ubiquitin-independent manner. Whether OAZ has a more general role in protein degradation and has other targets is unknown. In support of a functional relationship between Guf and Vn, the results of genetic tests suggest that guf negatively regulates Vn/EGFR activity. Furthermore, I found genetic evidence that Vn may be degraded by 26S proteasome, and that Guf is involved in 26S mediated protein degradation. These data raise the possibility that the binding of Guf to Vn might be involved in targeting Vn to be degraded by the 26S proteasome. Further experiments to localize the proteins in cells and to investigate the mechanism of Vn degradation will be critical for examining this hypothesis.

Committee:

Amanda Simcox (Advisor)

Subjects:

Biology, Genetics

Keywords:

vein; Drosophila

Smith, Nathan CharlesIdentification and Validation of Quantitative Trait Loci Affecting the Milling and Baking Quality of Soft Red Winter Wheat
Master of Science, The Ohio State University, 2008, Horticulture and Crop Science
The quality of flour derived from wheat is an important breeding objective. Wheat products require flours of specific composition and rheological functionality. The baking quality of wheat is governed by two general characteristics: gluten strength and water absorption. The milling quality of wheat is also governed by two characteristics: flour yield, and grain texture. A better understanding of the genes that control the milling and baking quality of wheat will enable breeders to use marker assisted selection to more efficiently develop cultivars that meet the varied demands placed upon. Two bi-parental populations of RILs and one population composed of full- and half-sib RILs were used to identify and validate QTL affecting milling and baking quality of wheat. We identified and validated important QTL for flour yield and gluten strength on chromosome 1B, pentosan and partially hydrated gliadin content on chromosome 2B, and water absorption on chromosome 3B.

Committee:

Clay Sneller, PhD (Committee Chair); David Francis, PhD (Committee Member); Richard Pratt, PhD (Committee Member); Ed Souza, PhD (Advisor)

Subjects:

Agriculture; Genetics

Keywords:

wheat; quality; QTL

Jantasuriyarat, ChatchawanIdentification and characterization of genes involved in the interaction between rice and rice blas fungus, Magnaporthe grisea
Doctor of Philosophy, The Ohio State University, 2006, Plant Pathology
To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. Ubiquitination-regulated protein degradation is a common regulatory mechanism that controls a range of cellular processes in eukaryotes. We have recently identified 43 rice ubiquitination-related E2 and E3 genes in our microarray analysis. These genes were induced or suppressed in Pi2 and Pi9 resistant plants after rice blast inoculation. The expression patterns of these E2 and E3 genes were confirmed using northern blot and RT-PCR methods. Among these E2 and E3 genes, we chose the homologue (OsSINAT5) of the Arabidopsis SINAT5 for a detailed functional analysis. Northern blot hybridization and RT-PCR confirmed that OsSINAT5 was induced by rice blast infection as early as 6 hr after inoculation. Functional analysis of this gene in resistance to rice pathogens was conducted using reverse genetics and biochemical approaches.

Committee:

Guo-Liang Wang (Advisor)

Subjects:

Biology, Genetics

Keywords:

Rice blast; Resistant gene; ubiquitination

Sprong, Amy NicoleX Chromosome Aneuploidy: A Look at the Effects of X Inactivation
Bachelor of Science, Miami University, 2008, College of Arts and Sciences - Zoology
The creation of the Barr body has been understood to account for dosage compensation of the X chromosome between males and females. Various X chromosomal aneuploidies such as Klinefelter syndrome (XXY), Turner syndrome(X0), and triple X syndrome (XXX) do not exhibit phenotypic qualities that would indicate that the extra X chromosomes were successfully condensed into Barr bodies. After reviewing current research, it was found that problems with the X inactivation process due to CTCF proteins and CpG islands can lead to some genes on the inactivated X chromosome remaining active. In addition, the X inactivation process does not occur at the beginning of the embryo development, and some genes on the X chromosome always remain expressed. The incomplete and late X inactivation results in the phenotypical differences between XXY, X0, XXX, and normal XX females and XY males.

Committee:

David Pennock, PhD (Advisor); Susan Hoffman, PhD (Committee Member); Robert Balfour, Mr. (Committee Member)

Subjects:

Genetics

Keywords:

Genetics; X Chromosome; Aneuploidy; Turner; Klinefelter; triple X

Keddache, MehdiRelationship between triplet repeat polymorphisms and HapMap tagSNPs
PhD, University of Cincinnati, 2011, Engineering and Applied Science: Biomedical Engineering

Single Amino Acid Repeat Proteins (SARPs) are a class of peptides that contain extended stretches of the same amino acid. At the DNA level they are represented by a repetition of the same triplet of bases. Their influence can be on gene regulation, transcription and protein function depending on the number of repeats. Mutations that add or subtract repeat units are both frequent and reversible.

Several human diseases have been identified that are caused by variations in the size of repeated DNA sequences. Animal and plant genetic studies showed that variations in repeat sequences can lead to complex phenotypes and that variations within a normal (i.e. non-pathological) range of repeat number commonly yields small, quantitative functional effects.

Even though triplet repeats present an intriguing disease mechanism for other complex human diseases, methods for a fast, inexpensive and systematic search have never been utilized. We have devised an assay that can be used for a high throughput, genome wide detection of SARPs in those diseases. We have optimized our method for low cost and validated our results through direct sequencing.

We also showed that inferring SARP alleles from SNP data derived from genome wide association studies (GWAS) is often not possible and therefore a SNP based GWAS approach will be underpowered or may even fail if the disease studied is caused by a triplet repeat allele. In addition, we found that current methods of next generation sequencing may miss the detection of triplet repeat variations, probably due to technical limitations of the technology.

Committee:

Bruce Aronow, PhD (Committee Chair); William S. Ball, MD (Committee Member); Jeffrey Johnson, PhD (Committee Member)

Subjects:

Genetics

Keywords:

triplet repeats

Benson, JenniferEffect of the DNA Methylation Inhibitor 5-aza-2'-Deoxycytidine on the Virulence of the Soybean Pathogen Phytophthora Sojae
Master of Science (MS), Bowling Green State University, 2012, Biological Sciences
Phytophthora sojae is an oomycete plant pathogen classified in the Stramenopile kingdom. It specifically targets the soybean crop plant. Infection by P. sojae causes ‘damping off' in seedlings and root rot in older plants. The main form of dispersal is a motile zoospore released when the oomycete is under nutrient or water stress. Diverse gene regulation mechanisms control growth, development, virulence and dispersal of zoospores. Due to the pathogen's ability to rapidly adapt, it is thought that not only genetic but also epigenetic signaling plays a role in P. sojae growth and virulence. The focus of this study is to investigate epigenetic gene regulation mechanisms employed by P. sojae via DNA methylation and its effect on pathogenicity. Treatment with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, altered hyphal growth rates, zoospore morphology, gene expression, and infection pattern, possibly due to a loss of methylation.

Committee:

Vipaporn Phuntumart, PhD (Committee Chair); Paul Morris, PhD (Committee Member); Raymond Larsen, PhD (Committee Member)

Subjects:

Agriculture; Biochemistry; Biology; Botany; Genetics; Plant Biology; Plant Pathology; Plant Sciences

Keywords:

epigenetics; oomycete; soybean; DNA methylation; methyltransferase; 5-aza-2'-deoxycytidine; phytophthora; P. sojae

Riedl, Ann ElizabethIdentification destabilizing sequences the fushi tarazu messenger RNA
Doctor of Philosophy, Case Western Reserve University, 1994, Genetics

The instability of fushi tarazu (ftz) mRNA is important for its correct spatial distribution in the embryo. We used deletion constructs in transgenic flies to identify sequences involved in the regulation of this instability. The constructs are derived from the r5f3 hybrid gene, which consists of the 5' half of the rpA1 gene fused to the 3' half of the ftz gene.

We have developed a method of measuring the stability of hybrid deletion constructs in early embryos without the use of drugs. The r5f3 hybrid message and its derivatives are driven by the rpA1 promoter and are therefore expressed maternally during oogenesis. Since this promoter is not active in the early embryo, the half-life can be estimated from the decrease in steady-state levels of the maternal message over time.

Using this method, we have delineated a 201 nucleotide region which contains the destabilization signal of the 3' half of the ftz message. This region is sufficient to destabilize a heterologous mRNA. Further deletions provide evidence that there are at least two interacting elements within this 201 nucleotide region. A model which attempts to interpret these interactions is proposed.

We are also using the maternal expression of the hybrid messages to analyze changes in mRNA stability as a functi on of development. The r5f3 hybrid transcript was found to be stable during oogenesis and then becomes destabilized upon fertilization.

Committee:

Marcelo Jacobs-Lorena (Advisor)

Subjects:

Biology, Genetics

Keywords:

Identification destabilizing sequences thefushi tarazu messenger RNA

Zentner, Gabriel EtienneGenomic analysis of ribosomal DNA and its application to the investigation of disease pathogenesis
Doctor of Philosophy, Case Western Reserve University, 2011, Genetics
The synthesis of rRNA is critical to all growing organisms, accounting for well over half of all cellular transcription. Highlighting its central function in cellular life, dysregulation of rRNA biogenesis and subsequent ribosome assembly has been implicated in human genetic diseases and cancer. While the transcription of rRNA is highly regulated at the chromatin level, it has not been analyzed by genomic methods due to the exclusion of rDNA from current genome assemblies. This work describes a novel method of analysis that enables the alignment of high-throughput sequencing data to a single copy of rDNA in the context of the full human genome. Integrated analysis of genomic datasets reveals that the coding region of rDNA is contained within nucleosome-poor open chromatin with high transcriptional activity. We find that histone modifications are enriched not only at the rDNA promoter but also at novel sites within the noncoding intergenic spacer. The distribution of active modifications is more similar within and between cell types than that of repressive modifications. Using ChIP-seq, we show that the nucleolar protein UBF is bound to sites throughout the genome and may play a role in regulating the transcription of nucleoplasmic genes. Lastly, the insulator-binding protein CTCF is bound to a site proximal to the junction between adjacent rDNA repeats, potentially indicating a role for transcriptional insulation in the regulation of rRNA transcription. We apply this method to a disease-relevant protein, CHD7, a chromatin-remodeling enzyme mutated in the developmental disorder CHARGE syndrome. CHARGE syndrome shares several clinical features with known disorders of ribosome biogenesis. ChIP-seq analysis reveals robust association of CHD7 across the transcribed region of rDNA. Immunofluorescence and subcellular fractionation confirm the nucleolar localization of a substantial fraction of CHD7. Knockdown experiments show that CHD7 functions to positively regulate rRNA biogenesis by counteracting DNA methylation at the rDNA promoter. Lastly, CHARGE-relevant tissues from Chd7-mutant mouse embryos display reduced levels of precursor rRNA. Taken together, these studies provide a novel means for assessing protein occupancy at rDNA that can be applied to disease-relevant chromatin-associated proteins in order gain novel insights into disease pathogenesis. Additionally, the work presented herein defines a novel role for CHD7 as a positive regulator of rRNA synthesis and suggests that dysregulated rRNA synthesis is involved in the pathogenesis of CHARGE syndrome.

Committee:

Peter Scacheri, PhD (Advisor); Guangbin Luo, PhD (Committee Chair); Helen Salz, PhD (Committee Member); Derek Abbott, MD/PhD (Committee Member)

Subjects:

Genetics

Keywords:

rDNA; ribosomal DNA; rRNA; ribosomal RNA; CHD7; CHARGE syndrome; ChIP-seq; UBF; upstream binding factor; CTCF; histone modifications; haploinsufficiency; TCOF1; treacle

Incardona, John PatrickGenetic analysis of glycolipid anchor function using Drosophila acetylcholinesterase as a model protein
Doctor of Philosophy, Case Western Reserve University, 1995, Genetics
Glycoinositol phospholipid (GPI)-anchored proteins in many mammalian polarized epithelial cell lines are found on the apical surface, incorporated into cold Triton-insoluble complexes at the level of the trans-Golgi, and frequently found sequestered in cholesterol-dependent plasma membrane invaginations called caveolae. To assess the role of GPI anchoring in vivo in Drosophila, we have focused on acetylcholinesterase (AChE) encoded by the Ace locus on the third chromosome. We constructed secreted (SEC) and chimeric transmembrane (TM) forms of AChE and substituted these for the wild type CPI-anchored form in transgenic flies. Expression in a Drosophila cultured cell line (S2) showed that the biochemical properties of the mutants were not altered except as predicted. Control experiments with a variety of GPI-AChE transgene inserts expressing a range of AChE activities demonstrated a threshold of only about 10% of normal activity for adult viability; AChE levels of <5% of normal resulted in late embryonic lethality. ce mutant flies were rescued by GPI-AChE transgenes that expressed 12-40% of normal activity and were essentially unchanged from wild type flies in behavior. In contrast, no SEC1 transgene insert could rescue Ace null alleles, indicating that AChE must be me mbrane-bound to function appropriately in the fly nervous system. Our data suggested that secreted AChE is either degraded within neurons or is unstable in the extracellular space of neuropil. TM-AChE transgenes were able to rescue Ace null alleles, although with a slightly higher threshold than that for GPI-AChE. Animals expressing 15% of normal activity from a TM-AChE transgene showed an abnormal phenotype not observed in rescued flies expressing 12% of normal from a GPI-AChE transgene: most animals died shortly after eclosion and many were unable even to eclose. However, flies expressing TM-AChE at about 30% of normal levels were essentially unchanged from wild type flies in behavior and fertility but had a reduced lifespan secondary to subtle coordination defects and tended to be entrapped in the food. Light level and electron microscopic immunocytochemistry showed no differences in the localization of GPI- and TM-AChE. Furthermore, expression of both AChEs in epithelial tissues of the adult and embryo, respectively, showed that they were sorted identically, although the various tissues differed in their sorting behavior. Immunocytochemistry on primary cultures of CNS neurons with antibodies to GPI and TM AChE, GPI-anchored fasciclin I, and insect glyscosphingolipids showed discrete clusters of all antigens at both light and electron microscopic levels. Overlap of the clusters appeared to be random, however, and the clusters were not sensitive to sterol-binding drugs. Our data suggest that rather than having a primary role in protein sorting, the GPI anchor of AChE plays some other more subtle cellular role

Committee:

Terrone Rosenberry (Advisor)

Subjects:

Biology, Genetics

Keywords:

Glycolipid; Drosophila acetylcholinesterase

Crough, Elizabeth MarieThe distribution of myosin heavy-chain protein isoforms during Drosophila development
Doctor of Philosophy, Case Western Reserve University, 1995, Biology
The sarcomeric myosin heavy-chain (sMHC) gene of Drosophila is single-copy; and RNA transcription from this gene is developmentally regulated. Numerous sMHC mRNAs that differ in exon composition can be formed by alternate RNA processing. These transcriptional events result in the presence of multiple sMHC isoforms in the developing organism. I have characterized two antibodies which are specific for different types of sMHC protein isoforms in Drosophila; and investigated the tissue distribution and function of various classes of protein isoforms which they recognize. In addition, I have generated an antibody against cytoplasmic myosin heavy-chain (cMHC); and have used this antibody to compare the expression of cMHC with sMHC during embryonic development. I have used the two sMHC-specific antibodies to show that sMHC protein isoforms are distributed in a tissue-specific manner in the adult fly. In addition, sMHC protein isoforms were localized to the mitotic spindles of preblastoderm embryos, indicating the possibility that a muscle myosin may play a role in a non-muscle environment. Genetic experiments were performed to investigate the function of sMHC proteins in embryonic development prior to muscle formation, resulting in the conclusion that the protein is either not essential at that stage of development, or its function can be complemented by another protein. A comparison of the expression of sMHC and cMHC identified a novel localization of cMHC protein to the developing larval tracheal system. The work presented here has provided new information about the distribution of MHC protein isoforms during Drosophila development, and will provide a good foundation for future work investigating the function of these proteins in the Drosophila embryo

Committee:

Charles Rozek (Advisor)

Subjects:

Biology, Genetics

Keywords:

Drosophilia; Myosin heavy chains

Lam, Man-Yee JosephineGenetic Control of Susceptibility to Testicular Cancer
Doctor of Philosophy, Case Western Reserve University, 2005, Genetics

Testicular germ cell tumors (TGCTs) are the most common solid tumors affecting young to middle aged men. It is the fifth most rapidly increasing type of cancer. Therefore, a better understanding is urgently needed of risk factors, early diagnosis, and treatment before metastasis. To date, there has not been a susceptibility gene identified in humans. We study a mouse model, the 129/Sv inbred mouse strain, in which TGCTs arise spontaneously by 3-weeks of age. Linkage studies with the 129/Sv strain, which has a low rate (1-5%) of TGCTs, reveal exceptional complexity in the genetic control of susceptibility. Various mutations that are inherited as Mendelian traits in laboratory mice affect susceptibility to spontaneous TGCTs on the 129/Sv genetic background. These genes, alone or in combination, provide important clues to the control of susceptibility and resistance to tumorigenesis. We undertook a systematic survey to test pairwise interactions of all known TGCT susceptibility loci, by comparing the frequency of TGCTs in double-mutant mice to identify combinations that show evidence of enhancer or suppressor effects. We identified three pairs of genes that enhanced the susceptibility of TGCTs and two pairs of genes that suppress the development of TGCTs. These mouse models can help simplify the complexity of TGCTs.

We can extrapolate from the findings in the mouse model to help identify candidate susceptibility genes in human. Recently, we identified the Ter mutation to be a premature stop codon in the Dnd1 gene that causes significantly increased susceptibility to TGCTs. The human ortholog of Dnd1 maps to Chr5q31. We performed an association study to test whether the human ortholog of Dnd1 contributes to susceptibility of TGCTs in human. We did not identify a mutation within the coding region in the cases, which indicates that Dnd1 may be a low frequency variant in human population.

Committee:

Joseph Nadeau (Advisor)

Subjects:

Biology, Genetics

Keywords:

TGCTs; SlJ; 129/Sv; P53; Ter; TUMOR; TGCT susceptibility

Kara, Jacqueline M.A child's genotype predicted CYP2D6 phenotype correlates with parent initiated contact following tonsillectomy
MS, University of Cincinnati, 2011, Medicine: Genetic Counseling
Children are routinely prescribed codeine for pain management following tonsillectomy; however, the analgesic effects of codeine depend on its enzymatic conversion by cytochrome P450 2D6 (CYP2D6) to the active drug morphine. There are 4 classic phenotypes associated with the enzyme activity of CYP2D6; ultra-rapid metabolizer, extensive metabolizer, intermediate metabolizer, and poor metabolizer. The purpose of this study was to determine whether a CYP2D6 genotype-predicted phenotype was associated with parent initiated contact with a healthcare provider during a 3-week post-operative period when children were prescribed codeine for home pain management following tonsillectomy. This was a retrospective cohort study of 201 children between the ages of 6 and 15, who were scheduled for an elective tonsillectomy. At the time of surgery, blood was drawn and DNA was isolated and frozen. Cohort DNA was analyzed in batch for 23 CYP2D6 alleles after the three week post-operative period for all children. The association of parent initiated contact and the child’s CYP2D6 predicted phenotype was tested using logistic regression. A different CYP2D6 predicted phenotype effect was detected in different races. In white children, increasing CYP2D6 predicted metabolic capacity was significantly associated with higher probability of parent-initiated contact with a healthcare professional (p=0.0051), while in black children, no association was detected. Our study supports the need for future research investigating the clinical utility of pharmacogenetics testing to select and dose codeine and other narcotics for pain management.

Committee:

John Greinwald Jr, MD (Committee Chair); Cynthia Prows, , (Committee Member); Daniel Prows, PhD (Committee Member); Xue Zhang, PhD (Committee Member)

Subjects:

Genetics

Keywords:

tonsillectomy;codeine;CYP2D6;poor metabolizer;adverse drug reaction;parent contact

Pack, Jessica K.The Impact of the Myriad Direct-to-Consumer Advertising Campaign for BRCA1/2 Genetic Testing in the Greater Cincinnati Area
MS, University of Cincinnati, 2011, Medicine: Genetic Counseling
Purpose: To describe the impact of the Myriad Genetics direct-to-consumer advertising (DTCA) campaign for BRCA genetic testing on a Hereditary Cancer Program (HCP) in Cincinnati, Ohio. Methods: Patient data was extracted from the HCP Database from two time periods, August 2008-March 2009 (DTCA campaign not in effect) and August 2009-March 2010 (DTCA campaign in effect). Patient demographics, risk assessment, and genetic testing uptake between these time periods were compared for patients referred for BRCA-related genetic services and non-BRCA-related genetic services. Results: Demographics (age, race, religion, education, marital status, and personal cancer diagnosis) did not differ between the time periods for either the BRCA or non-BRCA populations. There was an increased proportion of patients receiving a low risk (<7%) assessment for a BRCA mutation during the DTCA campaign (28%) versus pre-DTCA campaign (17%), a decreased proportion receiving a moderate risk (7-20%) assessment for a BRCA mutation (49% during and 57% pre-DTCA) and no differences in the proportion receiving a high risk (> 20%) assessment for a BRCA mutation. The proportion of individuals declining BRCA testing was higher during the DTCA campaign (20%), compared to the pre-DTCA time period (9%) We did not observe significant differences in mutation based risk-assessment or testing uptake in the non-BRCA related patient population. Conclusion: Patients seen in the HCP during the DTCA campaign for indications related to BRCA testing were at an overall lower risk for carrying a genetic mutation for a hereditary cancer syndrome and were less likely to pursue genetic testing. These results were not seen in the non-BRCA related patient population suggesting they may be a result of the Myriad DTCA campaign. This information may help predict trends in patient populations seen by similar programs in areas targeted by future Myriad DTCA campaigns.

Committee:

Melanie Myers, PhD (Committee Chair); Erin Acra Mundt, MS (Committee Member); Sara Rankin Knapke, MS (Committee Member); Lisa Martin, PhD (Committee Member)

Subjects:

Genetics

Keywords:

DTC;Myriad;BRCA;direct-to-consumer

Snyder, Justine A.Tools and Strategies That a BRCA Positive Population Considers to be Useful in the Result Disclosure Process to Family Members
MS, University of Cincinnati, 2012, Medicine: Genetic Counseling

BACKGROUND: Communicating positive test results to at-risk family members can be difficult for individuals with BRCA mutations. Previous studies have identified barriers to disclosure but have not addressed the utility of supportive materials in the disclosure process.

PURPOSE: The purpose of this study was to identify tools and strategies useful to patients with BRCA mutations in disclosing result information to family members.

METHODS: A questionnaire was mailed to 482 patients with BRCA mutations assessing their experience with disclosing test result information to their families.

RESULTS: Of the 177 completed questionnaires received, 90 participants reported that they told every at-risk biological relative about their BRCA positive test results. These 90 questionnaires were analyzed to assess the utility of specific tools and strategies provided by genetic counselors to assist patients with results disclosure. Participants felt that printed materials, such as a sample letter explaining that a BRCA mutation had been identified in the family was the tool expected to be most helpful in disclosing result information. The strategy expected to be most helpful in disclosing BRCA positive test result information was a step by step plan for informing family members. Eighty-seven participants, those who reported they did not tell one or more family members their results, reported reasons for non-disclosure including those that may be considered to be valid reasons, such as at-risk relatives who were under the age of 18 at the time the results were in active discussion, some family members did not wish to be informed of results, and relatives who previously received results by someone other than the participant.

CONCLUSIONS: Results from this study suggest that patients perceive a benefit from receiving a sample letter to distribute, as well as a clear plan to effectively communicate positive results to family members. It is recommended that both be provided by genetic counselors to all patients with BRCA mutations to increase the likelihood that results will be shared with family members. However, this study found evidence that there are valid reasons some patients may choose not to disclose results to every at-risk relative. This finding requires additional investigation to be completely understood.

Committee:

Robert Hopkin, MD (Committee Chair); Erin Acra Mundt, MS (Committee Member); Valentina Pilipenko, PhD (Committee Member)

Subjects:

Genetics

Keywords:

BRCA disclosure;BRCA mutations and family;Cancer genetic counseling;Disclosure of genetic risk;Tools for communicating genetic risk;;

WILLE, MARTA CECILIAREPRODUCTIVE CONCERNS OF ADULT SURVIVORS OF PEDIATRIC CANCER
MS, University of Cincinnati, 2001, Allied Health Sciences : Genetic Counseling
Purpose: It seems quite feasible that adult long term survivors of childhood cancer can have numerous medical concerns when considering a pregnancy. The aims of this study were to determine whether survivors of childhood cancer, who attend a long-term survivors pediatric oncology clinic, (1) recall their diagnosis accurately, and (2) have reproductive concerns that differ from reproductive concerns of their friends. Patients and Methods: Subjects were all individuals over the age of 18 years who attended a local long term survivors oncology clinic. They were interviewed about reproductive concerns and were asked to contact a friend of similar age and gender to serve as a control. Controls were also interviewed and asked similar questions. Results: Of the 25 long-term survivors of childhood cancer interviewed (mean age 26.3 years; 17 males, 8 females), 24 accurately recalled their diagnoses. The comparison of cancer survivors with their friends (study controls) showed no statistically significant differences in worries regarding reproductive issues. Furthermore, there was no statistically significant difference in perceived risk of future cancer in themselves or of cancer in their offspring when responses from the two groups were compared. Discussion: The results of this study suggest that cancer survivors who attend annual multidisciplinary follow-up clinics have concerns and worries about reproductive issues similar to those of their peers. These findings highlight the need to include an appropriate comparison group when examining these issues for long term survivors.

Committee:

Dr. Robert Noll (Advisor)

Subjects:

Biology, Genetics

Keywords:

reproductive concerns; long term survivors of cancer; adult pediatric cancer survivors; genetic counseling

Jian, ZhengwenGenetic Diversity and Expression Variation in Human Cytochrome P450 Genes
PhD, University of Cincinnati, 2008, Medicine : Environmental Health
Variation in activity of cytochrome P450s is one of the major factors responsible for interindividual difference in drug clearance rate which may cause serious toxicity or inefficacy of clinically used drugs. Such variation was reported to be determined at mRNA level for major P450 genes albeit it to a various degree. Little is known about mechanisms underlying the huge expression variation of most P450 genes among individuals. Identification of genetic variants affecting expression levels of P450 genes can lead to a cost-effective personalized medicine. This dissertation aims to 1) determine the genetic diversity and genetic structure of the CYP1A1_1A2 region; 2) search for determinants of interindividual variation in expression level of seven cytochrome P450 genes; 3) identify cis-acting genetic variants affecting the expression level of CYP1A1 and CYP1A2. Single nucleotide polymorphisms in a 39.6kb segment encompassing the whole CYP1A1 and CYP1A2 genes and the spacer region were identified. The linkage disequilibrium structure, haplotypes and tagSNPs in this region were determined. A humanized BAC-transgenic mouse line was established as a in vivo platform to study activity of different haplotypes of the human CYP1A1_1A2 region. Four factors were identified as major determinants of interindividual variability in expression level of P450 genes in human liver with various contribution for each P450 gene. These four factors include (1) the functional efficacy of global regulators affecting expression level of most active genes; (2) the expression level of specific P450 regulators; (3) exposure to environmental factors responsible for activation or modulation of activity of specific P450 regulators; (4) the presence of cis-acting genetic variants in P450 genes. At least four major cis-acting regulatory SNPs (rSNP) were demonstrated to exist in regulation of CYP1A1 or CYP1A2 expression. One of the four rSNPs was CYP1A1-940C>T (rs4646418), which is located in a G-rich domain about 1kb upstream of the CYP1A1 transcription initiation site, but affects both CYP1A1 and CYP1A2 expression. Another three SNPs – CYP1A1+2573C>T (rs4646422); CYP1A2-2217G>A (rs2069521); CYP1A2+734A>C (rs762551) – were identified to be top candidates as cis-acting genetic variants affecting the expression level of either CYP1A1 (by +2573C>T) or CYP1A2 (by -2217G>A; +734A>C).

Committee:

Alvaro Puga (Committee Chair); Li Jin (Advisor); Daniel Nebert (Committee Member); Timothy Dalton (Committee Member); Ranjan Deka (Committee Member); Anil Menon (Committee Member); Ying Xia (Committee Member)

Subjects:

Biology; Genetics; Toxicology

Keywords:

single nucleotide polymorphism (SNP); regulatory SNP (rSNP); linkage disequilibrium (LD); allelic expression imbalance(AEI); CYP1A1; CYP1A2; expression variation

Xiong, YiCharacterization of γ-tubulin complex proteins and investigation of the regulation of nuclear proteasome localization in Aspergillus nidulans
Doctor of Philosophy, The Ohio State University, 2011, Molecular Genetics
γ-Tubulin is conserved and essential for microtubule nucleation from lower eukaryotes like unicellular fungi to higher eukaryotes like humans. γ-Tubulin together with other proteins forms complexes and functions in microtubule nucleation and organization. Although these γ-tubulin complex proteins (GCPs) have been identified in several model organisms, the functions of individual GCPs remain unclear and contradictory data from studies using different model organisms have prevented the establishment of a coherent model. Although the existence of GCPs in the filamentous fungus, Aspergillus nidulans, was suggested by early biochemical experiments (Akashi et al., 1997), these proteins remain unidentified and their functions remain undetermined. In my dissertation research, I have identified and deleted each of the genes that encode these proteins, created GFP and mCherry fusions of each of them, and observed them in vivo using spinning disk confocal microscopy. Briefly, I have found that different GCPs play different roles: two are required for the existence of the γ-tubulin complex in cells and are thus essential for formation of the mitotic apparatus and for cell reproduction. Three others are not essential, but play a role in the assembly of large γ-tubulin complexes and have minor functions in the fidelity of chromosomal segregation. We were also able to establish a hierarchy of binding of GCPs to the spindle pole body and, based on these findings, we were able to propose a model for the structure of the γ-tubulin complexes. To understand the microtubule-nucleation and non-nucleation functions of γ-tubulin, our lab created a series of conditionally lethal γ-tubulin mutants. Extensive analysis of one mutant, mipAD159, revealed that the coordination of late mitotic events is disrupted even though mitotic spindles assemble with normal kinetics (Prigozhina et al., 2004). Further investigations revealed that this allele caused a nuclear autonomous failure of inactivation of the anaphase promoting complex/cyclosome (APC/C), which caused constitutive destruction of mitotic regulatory proteins (Nayak et al., 2010). As degradation of proteins targeted by the APC/C is carried out by the proteasome, we were interested in determining if γ-tubulin mutations affect the proteasome. Surprisingly, time-lapse imaging of a γ-tubulin mutant and γ-tubulin deletants as well as deletants of two essential GCPs revealed that the proteasome, which is highly enriched in the nucleus, separates physically from the chromosomes in mitosis. This resulted in the formation of nucleus-like structures containing little or no chromatin but often having a nucleolus. This implies that the proteasome binds to, or itself forms, a nuclear matrix and our high resolution multi-dimensional optical reconstructions of the nuclei support this notion. In an attempt to determine the nature of the nuclear matrix by using a candidate approach, I found that the nuclear localization of the proteasome does not rely on the Mlp1 (the orthologue of Megator) scaffold but does depend on the presence of the A. nidulans homolog of Schizosaccharomyces pombe Cut8 (An-Cut8). The lack of proteosomes in mitotic nuclei of An-cut8 deletants, however, does not cause a delay in cyclin B degradation and obvious defects in mitotic progression. My data do not seem to support the hypothesis that Cut8 as an anchor for the nuclear proteasome as has been proposed in S. pombe, instead, they appear more consistent with the notion that Cut8 is involved in the nuclear transport of the proteasome or proteasomal subunits.

Committee:

Stephen A. Osmani, PhD (Advisor); Berl R. Oakley, PhD (Committee Member); Anita K. Hopper, PhD (Committee Member); Harold A. Fisk, PhD (Committee Member); Jian-Qiu Wu, PhD (Committee Member)

Subjects:

Cellular Biology; Genetics

Keywords:

&947;-Tubulin; &947;-Tubulin Complex protein; microtubule; mitosis; proteasome; Cut8; nuclear pores

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