Browsing by Subject "Genetic regulation"
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Item Applications of affinity chromatography to isolation of sequence specific nuclear proteins(Texas Tech University, 1981-05) Sherrod, Peter D.Even before Watson and Crick proposed their brilliant model for the structure of DNA, Jacob and Monod were busy piecing together the information that would lead to the first fully defined mechanism for genetic regulation in the bacteria, Escherichia coli. Since then, other mechanisms have become fell defined in prokaryotes, however eukaryotic mechanisms of control have been difficult to establish. This is primarily due to the fact that the genome of euiiaryotic cells is much more complex than in bacteria. Just the immensity of size defies the precise and controlled study that has yielded so much information in prokaryotic systems. Nevertheless, it is not inconceivable that similar mechanisms of regulation exist in the eukaryotic genome.Item Cell cycle protein interaction with telomerase in a breast cancer culture system(Texas Tech University, 2001-08) Cook, T. KevinBreast cancer causes 40,000 deaths a year and is second only to lung cancer in deaths attributed to neoplastic disease. There is a known age and cell lineage dependence related to the development of the disease. It is vitally important to understand the manner in which this disease develops. One approach is to examine proteins involved in cell cycle regulation. Three such proteins are c-Myc, p53, and pRb. These proteins in conjunction with one another and telomerase perform critical functions within a cell. They have an intertwined pattern of activity. If these functions are not regulated, the cells can proliferate unchecked and give rise to cancer. Our study focused on the differences of protein levels and activity overtime between epithelial and stromal cells of the breast. The data appeared to show that stromal cells exhibit a tighter control over their cell cycle than their epithelial counterparts. Further, it would seem that c-Myc levels are inversely related to telomerase activity. This data points to more studies in the future.Item Directed evolution of T7 RNA polymerase variants using an 'autogene'(2004-08) Chelliserrykattil, Jijumon Pavithran, 1974-; Ellington, Andrew D.Item Dynamics of adaptive evolution in two experimental viral systems(2001-12) Holder, Kristina Kichler; Bull, James J.; Hillis, David M., 1958-Item Engineered regulation of an RNA ligase ribozyme(2001-08) Robertson, Michael Paul; Ellington, Andrew D.Item Exploring the global gene expression programs and regulation in the response of quiescent human fibroblasts to distinct proliferative stimuli(2005) Gu, Jian; Iyer, Vishwanath R.Serum treatment of quiescent human dermal fibroblasts induces proliferation coupled with a complex physiological response that is indicative of their normal role in wound-healing. However, it is not known to what extent such complex transcriptional events are specific to a given cell type and signal, and to what extent these changes are innate programmed responses that are activated in a range of related cell types in response to a variety of stimuli. We have profiled the global transcriptional program of human fibroblasts from two distinct tissue sources to four different growth stimuli and identified a striking conservation in their gene expression signatures. However, there were specific differences among different stimuli with regard to signaling pathways that mediate these transcriptional programs. The use of a specific PI3-kinase pathway inhibitor suggested that this pathway is differentially involved in mediating the responses of cells to serum as compared to individual peptide growth factors. By applying siRNA knockdown technique, we demonstrated that putative targets of two important immediate early transcription factors, Myc and SRF, served functions related to cell cycle progression/cell survival and wound healing, indicating that these two transcription factors may serve as master transcription controllers during the transition of fibroblasts from quiescence to proliferation. In addition, different Myc targets were identified either between different cell types (Hela vs. foreskin fibroblasts) or between different cell states (unsynchronized vs. synchronized), while SRF targets included a group of genes only induced at certain time points during cell cycle progression, which was not observed in the Myc data. MicroRNA (miRNA) expression profiling indicated that let7 and other miRNAs with similar expression profiles may be involved in regulating the transcriptional program in response to proliferative signals. Our results indicate that conservation of transcriptional programs and their regulation among different cell types may be much broader than previously appreciated.Item From developing protein-protein interaction strategies to identifying gene functions: case studies for transcription factor complexes and ribosome biogenesis genes(2007-12) Li, Zhihua, doctor of cell and molecular biology; Marcotte, Edward M.Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.Item Functional characterization of m-Bop, a transcriptional repressor essential for heart development(2002) Sims, Robert Joseph; Gottlieb, Paul D.Item Functional characterization of smyd1, a methyltransferase essential for heart and skeletal muscle development(2006) Zhu, Li; Tucker, Philip W.Item Induction and characterization of tyrosine aminotransferase and its mRNA isolated from D. hydei salivary glands(Texas Tech University, 1983-05) Belew, Karla Jean DavidsonA puff at band II-48C on the salivary gland polytene chromosomes of D_. hydei is induced by pyridoxine. Pyridoxine also induces a 40,000. protein and tyrosine aminotransferase activity. Tyrosine aminotransferase (TAT) was purified and used to prepare antibody in rabbits. The anti-TAT immunoprecipitated the newly synthesized 40,000 D. polypeptide. TAT mRNA containing polysomes were immunoprecipitated. The TAT mRNA was translated in an in vitro translation system. The polypeptide translated had a molecular weight of 40,000 D. and was nuncprecipitated by anti-TAT. Tritiated RNA extracted from TAT mRNA containinq polysomes was in situ hybridized to D. hydei salivary gland squashes. Silver grains were found over the nucleolus and band II-48C. These data identify II-48C as the locus coding for TAT.Item Isolation and characterization of DNA sequences bound by a class of nonhistone proteins(Texas Tech University, 1979-08) Jagodzinski, Linda L.All somatic cells of the same organism contain the same complement of genes. During cellular differentiation transcriptional specialization occurs. This process allows the selected expression of genetic information in specialized cells; e.g., only red blood cell precursors synthesize hemoglobin, only hepatocytes synthesize phenylalanine hydroxylase and serum albumin (159), and only estrogen induced oviduct cells synthesize ovalbumin. During differentiation certain genes function only at specific times and in particular tissues. Hence, portions of the eukaryotic genome must be prevented from expressing, in some manner, their genetic information. Evidence indicates that the chromosomal proteins participate in the regulation of gene activity. How this is accomplished and which components are involved are questions which are now being investigated.Item Pax6 and Six1/2 orthologs in leech ectodermal patterning(2008-12) Quigley, Ian Kirk; Shankland, MartyClitellate annelids display conserved mechanisms of segmental ectodermal and mesodermal patterning. These tissues are generated by asymmetric divisions of large stem cells called teloblasts, elongating the ectoderm and mesoderm of the embryo. Each teloblast-derived lineage makes highly stereotyped contributions to the leech: the N, O, P, and Q contribute specific neurons, epidermis, and other ectodermal tissues along the ventral-to-dorsal axis of the embryo, respectively. The N and Q ectodermal lineages appear to be specified autonomously, but specification of the O and P lineages depends upon interactions with other, neighboring teloblast lineages. Until quite recently, there have been precious few teloblast lineage-specific markers, and virtually no molecular candidates for genes influencing the proper differentiation of any of these lineages. Here, I explore the possibility that members of the Pax-Six-Eyes absent-Dachshund network are involved in leech ectodermal patterning. I show that the leech Helobdella sp. Austin has two Pax6 paralogs, and demonstrate that Hau-Pax6A is expressed early in a subset of N-derived cells and O-derived cells. Next, I demonstrate that an ortholog of the six gene family, Hau-six1/2a, is expressed in the P lineage. I show through a series of cell ablations that Hau-six1/2a expression is regulated by neighboring teloblasts in a manner consistent with P fate induction, hinting that this transcription factor may be involved in P specification. The identification of these genes is a first step towards dissecting the molecular mechanisms of ectodermal teloblast differentiation in the leech embryo. The evolutionary context of the deployment of these genes is also discussed. In the appendices, I present two projects on the evolution of pigment patterns in Danio rerio and its relatives. In the first, I show that the larval melanin-containing pigment cells of Danio nigrofasciatus are uniquely redeployed into the adult pigment pattern, in contrast to seven related fishes. In the second, I show that variation in yellow pigment cell populations in different danio species may be dependent on variable signaling through the receptor tyrosine kinase fms pathway.Item The function and subcellular localization of an arabidopsis 14-3-3 protein, GF14 lambda(Texas Tech University, 2002-08) Li, QingtianThe 14-3-3 proteins play important roles in signal transduction pathways and regulating cellular enzyme activities in eukaryotes. GF14^, one of the fourteen 14-3-3 protein isoforms found in Arabidopsis, interacts with several proteins, including AKR2 (ankyrin repeat-containing protein 2) and APX3 (ascorbate peroxidase 3) proteins. AKR2 may function as a regulatory protein of APX3, because it interacts with APX3 specifically and protects APX3's activity in vitro. The APX3 protein is involved in hydrogen peroxide-scavenging in peroxisomal membranes. These protein-protein interactions may be involved in a novel antioxidation regulation mechanism in higher plants. The GF14X protein may serve as a scaffold protein in this mechanism. GF14>. was localized to the nucleus and cytoplasm by fusing it to GFP (green fluorescent protein) and studying their localization using fluorescence microscopy and immunoelectron microscopy. Furthermore, the function of GF14X was studied in anti-GF14X transgenic plants. Our data indicate that GFUX plays an important role in plant cold tolerance, because reduced expression of GF14X leads to sensitive phenotypes under chilling temperature conditions. The implication of our discovery with respect to the general functions of 14-3-3 proteins in eukaryotes is discussed in this dissertation.