Browsing by Subject "Eukaryotic cells"
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Item Characterization of Arabidopsis thaliana translation initiation factor eIF4F(2006-08) Provinzino, Anne Marie Schneider; Browning, Karen S.Eukaryotic translation initiation is a complex process involving many factors. Two vital components of this system are eukaryotic initiation factors (eIF) 4E and 4G, which together form the complex eIF4F. eIF4E is the cap-binding subunit, interacting with the 7-methyl guanosine at the 5'-end of messenger RNA. There are multiple forms of this gene in Arabidopsis thaliana: eIF4E1, eIF4E2, eIF4E3, eIFiso4E, and novel cap-binding protein. The scaffolding component of the eIF4F cap-binding complex is eIF4G. This protein binds not only to eIF4E, but also to eIF3, eIF4A, and poly(A)binding protein. Three versions are found in Arabidopsis: eIF4G, eIFiso4G1, and eIFiso4G2. Plants are unique in that they are the only organisms to contain the isoform versions of these genes. A goal of the research described in this thesis is to determine the mechanisms by which plant eIF4F isoforms specifically select mRNAs for translation. This thesis describes the cloning of dicistronic Arabidopsis eIF4G with some members of the eIF4E cap-binding family. Comparison of the biochemical activities of these complexes with various mRNAs will generate more information about their specific functions. Analysis of Arabidopsis T-DNA insertion mutants is included to elucidate the role(s) of eIF4F, eIFiso4F, and their subunits in plant translation. The phenotype(s) of a mutant may provide clues as to the function of the protein(s) not expressed. The importance of the eIF4F complex to translation initiation makes it a target for regulation. These regulatory mechanisms include inhibition by cleavage, competitive inhibitors, phosphorylation, binding of partner proteins, and exploitation by viruses. Our hypothesis is that the eIF4E and eIF4G genes are also regulated at the level or translation. Part of this thesis describes preparation and sequencing of cDNAs for eIF4G, eIFiso4G1, eIFiso4G2, eIF4E1, eIF4E2, eIF4E3, eIFiso4E, and nCBP to identify full length 5'UTRs and determine if regulatory elements are present.Item Characterization of mitochondrial C₁-tetrahydrofolate synthase transcript and protein expression in adult and embryonic mammalian tissues and the role of the mitochondrial one-carbon pathway in the cytoplasmic methyl cycle(2008-12) Pike, Schuyler Todd, 1966-; Appling, Dean RamsayIn eukaryotes, folate-dependent one-carbon (1-C) metabolism is composed of two parallel pathways compartmentalized to either the cytoplasm or mitochondria. In each, 1-C units, carried on tetrahydrofolate (THF), are interconverted by four catalytic activities. Serine hydroxymethyltransferase transfers the 3-carbon of serine to THF forming 5,10-methylene-THF which is oxidized in 3 successive steps to formate via the intermediates, 5,10-methenyl-THF and 10-formyl-THF. Because of the redox potential in each compartment, 1-C flux is thought by most authors to be from formate to serine in the cytosol and in the opposite direction in mitochondria. Transport of serine, glycine and formate across the mitochondrial membranes creates a 1-C cycle. All eukaryotes characterized to date contain a cytoplasmic trifunctional C1-THF synthase possessing 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities which interconvert the catalytic intermediates between 5,10-methylene-THF and formate. However, despite the observation that adult rat liver mitochondria oxidize serine to formate, no known enzymatic activities correlate with those of cytoplasmic C1-THF synthase. In embryos, a bifunctional protein, containing 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase, accounts for two of these activities. But the 10-formyl-THF synthetase activity has no associated enzyme in mitochondria. Reported here is the discovery of a monofunctional homolog of C1-THF synthase in mammalian mitochondria. Characterization of the protein confirms mitochondrial localization and 10-formyl-THF synthetase activity. Likewise, the adult human transcript is present and differs in size and tissue distribution from cytosolic C1-THF synthase. In mouse embryos, the temporal expression of the mRNA starts out relatively low and increases as the embryos age. The spatial distribution of the transcript is ubiquitous but with areas of elevated expression corresponding to proliferative regions within the embryo. The temporal expression pattern of the protein and transcript correspond well. However, mitochondrial flux studies and immunoblotting data suggest that mitochondrial C1-THF synthase is not the rate-limiting enzyme in mitochondria, at least during the mid to later stages of embryogenesis. Additionally, studies modulating the expression of mitochondria 1-C proteins demonstrate the likelihood that most cytoplasmic 1-C units are mitochondrially derived.Item Defining the late 60S ribosomal subunit maturation pathway from the nucleolus to the cytoplasm(2002-08) Kallstrom, George Harvester; Johnson, Arlen W.Item Development of bacterial delivery systems for the introduction of DNA into eukaryotic cells(2001-08) Seliger, Stefan Siegfriend; Payne, Shelley M.Item Genome-wide mapping of DNA-protein interactions in eukaryotes(2005) Kim, Jonghwan; Iyer, Vishwanath R.The genome of an organism encodes thousands of genes, and their expression needs to be precisely controlled at the right place and time for normal cellular functioning. Control of gene expression occurs predominantly at the level of transcription and the transcription of a particular gene is determined by the interactions between diverse regulatory proteins and their specific cis-acting binding elements. However, our understanding about how and when transcription factors function in the context of the whole genome is limited. Hundreds to thousands of transcription factors can potentially interact with DNA in the genome and modulate the level of transcription of protein coding genes into mRNAs in response to regulatory signals. Identifying the chromosomal targets of regulatory transcription factors in various conditions is crucial for reconstructing the transcriptional regulatory networks underlying global gene expression programs. Recently-developed chromatin immunoprecipitation (ChIP)-based genome-wide studies such as ChIP-microarrays allow us to identify the genomic loci occupied by transcription factors in vivo. In combination with expression profiling analysis, the ChIP-microarray method is a powerful tool to study DNA-protein interactions in vivo. However, there are some limitations to applying this ChIP-microarray method to every sequenced large genome due to the complexity and size of large genomes such as human or mouse. To gain a better understanding of the functions of transcription factors in mediating eukaryotic gene regulation, we performed ChIP-microarray analysis in yeast and human systems. First, we determined the role of the yeast TATA-box binding protein (TBP) in controlling global gene expression and revealed that PolIII genes are the strongest targets of TBP in yeast. Second, we determined that the human oncogenic transcription factor Myc has more than a thousand target genes in human cells. Finally, to address some of the current limitations in ChIP-microarrays, we developed an alternative method, sequence tag analysis of genomic enrichment (STAGE), to study genome-wide mapping of DNA-protein interactions.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 Nmd3p, the nuclear export adapter for the 60S ribosomal subunit: characterization of its recycling mechanism and novel interaction with the nuclear pore complex in yeast(2005) West, Matthew Blaine; Johnson, Arlen W.Item On the structural response of eukaryotic cells(2003) Ananthakrishnan, Revathi; Käs, Josef A.; Moon, T. J. (Tess J.)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.Item Toward group II intron-based genome targeting in eukaryotic cells(2009-12) Vernon, Jamie Lee; Lambowitz, AlanMobile group II introns consist of a self-splicing RNA molecule and an intron-encoded protein with reverse transcriptase activity that function together in an RNP and catalyze the insertion of the intron into specific DNA target sites by a process known as retrohoming. The mechanism of insertion requires the intron RNA to bind and reverse splice into one strand of the DNA target site, while the intron-associated protein cleaves the opposite DNA strand and reverse transcribes the intron RNA. DNA target site recognition and binding are dependent upon base pairing between the intron RNA and the target DNA molecule. By modifying the recognition sequences in the intron RNA, group II introns can be engineered to insert into virtually any desired target DNA. Based on this technology, a novel class of commercially available group II intron-based gene targeting vectors, called targetrons, has been developed. Targetrons have been used successfully for gene targeting in a broad range of bacteria. Previously, our laboratory demonstrated that group II introns retain controllable retrohoming activity in mammalian cells, albeit with very low targeting efficiency. However, the gene targeting capability of group II introns is not limited to direct insertion of the intron. Group II introns can also create double-strand breaks that stimulate homologous recombination. By virtue of these attributes, mobile group II introns offer great promise for applications in genetic engineering, functional genomics and gene therapy. Here I present the results of experiments in which I tested group II introns for gene targeting activities in eukaryotic cells. First, I demonstrated that group II introns injected into zebrafish (Danio rerio) embryos retain in vivo plasmid targeting activity that is enhanced by the addition of magnesium chloride and deoxynucleotides. I also verified that similar in vivo targeting activity is retained in Drosophila melanogaster embryos. Further, I describe repeated experiments in zebrafish embryos designed to target the zebrafish genome with inconclusive results. Group II introns were also delivered to cultured human cells for genome targeting. Here I present promising evidence for the ability of group II introns to stimulate homologous recombination between an exogenously introduced donor DNA molecule and the chromosome. The donor DNA was delivered either as a linearized double-stranded plasmid by electroporation or as a single stranded genome of a recombinant adeno-associated virus (AAV). In both cases, cells receiving both the group II intron RNP and the donor DNA showed more efficient integration of the donor DNA than introduction of the donor DNA alone. The studies presented here provide insight into the potential of using group II introns for future applications in gene targeting in eukaryotes.