Spelling suggestions: "subject:"andfunctional"" "subject:"fosfunctional""
1 |
Genomic and Molecular Analyses of the Core DNA Replication Machinery in PlantsShultz, Randall William 04 April 2007 (has links)
Accurate and complete DNA replication is essential for maintaining the integrity of the genome. In eukaryotes, this process requires the coordinated action of numerous molecular machines. Based on yeast and animal model systems, we defined a set of fifty-one ?core DNA replication proteins? that are integral to the initiation, DNA synthesis, and Okazaki fragment maturation functions of DNA replication. We used computational analyses to identify putative homologs in the genomes of two plants, Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice), providing the first comprehensive view of the core DNA replication machinery in plants. Our results indicated that the overall composition of this apparatus is conserved, but plants are unique in that multiple DNA replication genes exist as small gene families. Fourteen of the genes we annotated in this study have not been previously reported in the literature, and we have provided revised gene models for seventeen plant proteins. To better understand how the DNA replication machinery functions in plants, we cloned multiple subunits of the pre-replication complex (pre-RC) from Arabidopsis and generated antibodies against four key components of this complex ? AtORC1, AtORC2, AtMCM5, and AtMCM7. We demonstrated that the pre-RC is developmentally regulated in Arabidopsis and, consistent with a role in DNA replication, is abundant in proliferating tissues. We used immunocytochemical and biochemical methods to characterize MCM7 in plants. We observed two distinct localization patterns for plant MCM7 proteins. In most cells, MCM7 was nuclear and colocalized with DNA. In a small fraction of cells, MCM7 was dispersed throughout the cytoplasmic compartment. Biochemical analysis confirmed that MCM7 binds to chromatin and that it is present in the nucleus at least during the G1, S and G2 cell cycle stages. Together, these analyses support a model where the MCM complex is loaded onto DNA in late M and early G1, released into the nucleoplasm during S phase followed by a brief dispersion into the cytoplasmic compartment concurrent with nuclear envelope breakdown in mitosis.
|
2 |
Molecular and Structural Characterization of Proteins Involved in Bacterial Adaptive ResponsesSullivan, Daniel Michael 22 April 2008 (has links)
Bacteria are remarkable in their ability to adapt to environmental conditions that are continually in flux between growth-promoting and growth-limiting. Responses to a host of environmental situations are equally varied, ranging from the secretion of antimicrobial compounds and polymer degrading enzymes, to the up-regulation of alternative cellular developmental pathways leading to complete physiological transformation. In endospore forming bacteria this results in a metabolically inert, yet highly resistant endospore. The first study presented here deals with the NMR structural and dynamic characterization of a class of proteins in Bacillus subtilis known as transition-state regulators, responsible for global gene regulation during the transition from the vegetative mode of growth to the semi-quiescent stationary phase. The utilization of protein-DNA docking protocols further allows for the first description of a structural model for the interaction between these DNA-binding proteins and a cognate DNA promoter sequence. The later portions of this dissertation deal with the characterization of proteins involved in the ubiquitous bacterial signal transduction system known as the two-component signal transduction pathway. In the basic two-component signal transduction paradigm, an environmental signal is detected by a multi-domain sensor kinase that, via phosphorylation, activates a response regulator protein for its cellular role (be it DNA-binding, RNA-binding, enzymatic, etc). In the second study, a comparative modeling analysis of the predicted receiver domains the response regulators from Vibrio vulnificus YJ016 was performed, using the hydrophobic characteristics of the response regulator surface known to interact with the four-helix bundle of the cognate sensor kinase as the basis for sub-classification. In the final study, a new mass spectrometric technique to detail the structural changes in proteins resulting from oxidative damage was applied to the single domain response regulator Spo0F from B. subtilis.
|
3 |
Identification and characterization of the protein disulfide isomerase multigene family in plants.Houston, Norma L 15 June 2007 (has links)
Protein disulfide isomerases (PDIs) contain thioredoxin domains and aid in the formation of proper disulfide bonds during protein folding. Iterative BLAST searches of sequence databases were used to identify 22 PDI-like (PDIL) genes in Arabidopsis thaliana and maize (Zea mays) and 19 in rice (Oryza sativa). The PDIL genes were resolved into 10 phylogenetic groups. Genes in groups I-V had two active thioredoxin domains while members of groups VI-X had one active thioredoxin domain. One single domain PDIL, maize PDIL5-1, showed increased accumulation in the endosperm mutants that produced defective storage proteins, but PDIL5-1 was not localized to endomembrane fractions. Expression analysis was done in eighteen PDIL genes in maize endosperm, a storage tissue, and two vegetative organs, embryo and leaves. Eight PDIL genes were expressed mainly in endosperm, and two of these genes (PDIL1-2 and 2-3) had increased expression levels only in the endosperm that produced defective storage proteins. There were three PDIL genes (1-3, 1-4, 1-5) that showed the highest expression levels in the embryo, while two other genes, adenosine 5?-phosphosulfate reductase-like (APRL) 2 and APRL8, had elevated expression levels in leaves. To further characterize members of the gene family, isomerase and reductase assays were conducted to test for recombinant maize PDIL1-1, 1-3, 2-3 and 5-1 enzymatic activity in vitro. Recombinant and endogenous maize PDIL1-1 showed both isomerase and reductase activity while recombinant PDIL1-3 showed anti-chaperone activity. Recombinant PDIL2-3 and PDIL5-1 showed no activity under the assay conditions tested.
|
4 |
Functional Genomic Characterization of the Anti-Adipogenic Effects of trans 10, cis 12-Conjugated Linoleic Acid (t10c12-CLA) in a Polygenic Obese Line of MiceHouse, Ralph Lee 29 July 2004 (has links)
We analyzed gene expression during t10c12-CLA-induced body fat reduction in a polygenic obese line of mice. Adult mice (N=185) were allotted to a 2x2 factorial experiment consisting of a non-obese (ICR-control) and an obese (M16-selected) line of mice fed a 7% fat, purified diet containing either 1% linoleic acid (LA) or 1% t10c12-CLA. Body weight (BW) gain by day 14 was 12% lower in CLA compared to LA fed mice (P<0.0001). By day 14, t10c12-CLA reduced weights of epididymal, mesenteric and brown adipose tissues as a percentage of BW in both lines by 30, 27 and 58%, respectively, and increased liver weight/BW by 34% (P<0.0001). Total RNA was isolated and pooled (4-5 mice per composite) from epididymal adipose (day 5 & 14) and liver (day 14) of the obese mice to analyze gene expression profiles using Agilent mouse oligo microarray slides (4 per tissue?day) representing >20,000 genes. Numbers of genes differentially expressed by ≥ two fold in epididymal adipose (day 5 & 14) and liver (day 14) were 29, 125, and 80, respectively. Of particular interest in adipose, CLA putatively increased expression of the uncoupling proteins (1 and 2), carnitine palmitoyltransferase (L and M), and carnitine translocase, but decreased expression of PPAR-gamma, GLUT-4, perilipin, caveolin-1, adiponectin and resistin (P<0.01). In conclusion, this experiment has revealed candidate genes that will be useful in elucidating mechanisms underlying the potent anti-adipogenic effects of t10c12-CLA.
|
5 |
Quantitative Trait Transcript Mapping for Drug Response in Drosophila melanogasterPassador-Gurgel, Gisele Candia 17 November 2005 (has links)
Here I used microarrays to identify genes that are activated or repressed by nicotine and caffeine in Drosophila melanogaster. I compared genotypes with differential resistance to each drug in order to select genes that may be involved in resistance to the drugs. Comparison of the genes differentially expressed by both drugs leads me to propose that there are common mechanisms of metabolic resistance to caffeine and nicotine, in particular cytochrome P-450-mediated mechanisms. Caffeine seems to have a more dramatic influence on gene expression than nicotine, in particular on expression of genes involved in energy metabolism. Next, I extended the studies on nicotine resistance to ask whether there are differences in response between two populations of Drosophila. The gene expression patterns of both populations were evaluated separately and in a combined analysis. Most of the differentially expressed genes were up-regulated by nicotine in both populations and in the combined analysis. The induced transcripts were mainly related to protein, nucleic acid, amino acid and energy metabolism, and response to stimulus and stress. Those findings suggest that amino acid and energy metabolism may be important biological processes affected by nicotine and be interesting targets for further investigation related to the nicotine response in Drosophila. The two populations displayed considerable differences in gene expression profiling that may be the result of the observed phenotypic variation for nicotine response between the two populations. Most of the differential expression induced by nicotine seems to be specific to the more resistant population. Finally, I focused on genes whose expression showed significant correlation with survival time on nicotine food. Using a regression approach, it was possible to map quantitative trait transcripts associated to nicotine response. Control expression of alkaline phosphatase and ornithine aminotransferase displayed significant correlation to survival time in drug food. They seem to be linked to regulation of GABA/glutamate neurotransmission and detoxification mechanisms, which ultimately counteracts the stimulatory effects of nicotine.
|
6 |
Bacteriophage Defense Systems and Strategies for Streptococcus thermophilusSturino, Joseph Miland 15 December 2003 (has links)
The genomes of six Streptococcus thermophilus bacteriophages were compared to identify genes that could be targeted by engineered phage defense systems with potentially widespread efficacy. The genes associated with the S. thermophilus phage Sfi21-prototype genome replication module, including a putative primase and a putative helicase, were found to be among the best candidates due to their frequency of distribution in industrial phage isolates, striking sequence conservation between independent isolates, and intrinsic strategic importance in early phage development. Fourteen antisense RNA cassettes targeting the phage k3-derived helicase (hel3) or primase (pri3) genes were expressed in S. thermophilus NCK1125. These constructs consistently reduced the efficiency of plaquing (EOP) of phage k3 to between 5 x 10-1 and 2.0 x 10-3 depending on the (i) gene targeted and (ii) region of the gene that was targeted. The largest antisense RNAs were generally found to confer the largest reductions in EOP, however shorter antisense RNAs designed to the 5' region of the gene retained much of the inhibitory function, especially if they contained sequences complementary to the ribosome binding site. Expression of antisense RNAs correlated with decreased levels of phage encoded primase transcripts, likely due to increased degradation of the dsRNA complex. This, in turn, correlated with diminished phage genome replication and aborted phage development. In a separate study, invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutation within the S. thermophilus phage k3-encoded putative primase. PCR products containing the desired mutation(s) were cloned and expressed in S. thermophilus NCK1125. The majority of the examined constructs remained sensitive to phage k3, however four constructs conferred strong phage resistance to the bacterial host. The mutated residues resided within a putative ATPase/helicase domain suspected to be critical for primase function in vivo. The co-expression in trans of the K238(A/T) or RR340-341AA mutant proteins suppressed the function of the native, phage-encoded primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. According to this model, the plasmid-encoded mutant primase subunits are structurally intact and form stable interactions with the native, phage-encoded primase subunits, thus inhibiting their activity. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing more that nine log cycles. Given the magnitude of the resistance conferred, it was concluded that the putative primase is essential for genome replication in S. thermophilus Sfi21-type phages. Further, it was also clear that host-encoded factors were unable to complement the resultant deficiency. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and phage sensitivity of the host, indicating that translation was required to confer phage resistance. Residues within a critical oligomerization domain were also identified through genetic analysis. Introduction of an E437A mutation downstream of the transdominant K238T mutation completely suppressed phage resistance, indicating that the E437A mutation precluded the association of the mutant and native subunits. To our knowledge, this is the first use of subunit poisoning to inhibit phage replication in the lactic acid bacteria.
|
7 |
Functional Deficits in Motor Terminals and their Mitochondria in Mouse Models of Amyotrophic Lateral SclerosisNguyen, Khanh Tu 06 December 2009 (has links)
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which the upper and lower motor neurons die. Most studies aimed at elucidating the cause of this disease have focused on the motor neuron cell body. However, recent work has suggested that the disease may begin in motor nerve terminals. The experiments described in Chapters 2-4 of this dissertation studied functional defects in motor nerve terminals of mice expressing mutant human superoxide dismutase 1 (SOD1-G93A, SOD1-G85R), models of familial ALS. In Chapter 2, the proximal hind limbs of SOD1-G93A mice were subjected to varying durations of a tourniquet-induced ischemia/reperfusion injury to determine whether these motor terminals were more vulnerable to this stress than wild-type terminals. Confocal imaging of yellow fluorescent protein (YFP expressed in neurons) and alpha-bungarotoxin (labels acetylcholine receptors on muscle) was used to determine endplate occupancy. In the distal hind limb of SOD1-G93A/YFP terminals innervating fast type muscles (extensor digitorum longus (EDL) and plantaris) were more vulnerable to ischemia/reperfusion injury than those occupying the slow type muscle (soleus). Increased vulnerability to endplate denervation was evident in presymptomatic mice as early as 31 days old. Experiments in Chapters 3 tested whether mitochondrial handling of Ca2+ loads is altered at presymptomatic stages. These experiements used rhodamine-123 to measure depolarization of the mitochondrial membrane potential (Ψm) evoked by trains of action potentials delivered to the motor nerve in levator auris longus motor terminals. These Ψm depolarizations depended on Ca2+ entry into motor terminals and were relatively small (~1-2 mV) in wild-type terminals. Consistent with the hypothesis that reduced ability to accelerate the electron transport chain (ETC) activity results in larger stimulation-induced Ψm depolarizations, presymptomatic SOD1-G93A (maintains dismutase activity) and SOD1-G85R (lacks dismutase activity) terminals displayed ~5 times greater depolarizations than wild-type terminals. Expression of normal human SOD1 or knockout of SOD1 did not significantly alter Ψm depolarizations. In the presence of a low concentration of rotenone (inhibits complex 1 of the ETC) wild-type terminals also displayed larger Ψm depolarizations. Experiments in Chapter 4 studied stimulation-induced Ψm depolarizations in terminals of older, symptomatic SOD1-G93A and SOD1-G85R mice. These depolarizations decayed more slowly than those in wild-type terminals and incremented with successive trains. Asynchronous depolarizations that were not time linked to the stimulus train were also noted. These behaviors were attenuated when opening of the mitochondrial permeability transition pore (mPTP) was inhibited with cyclosporin A or by replacing bath Ca2+ with Sr2+. Incrementing Ψm depolarizations could be elicited in wild-type terminals when subjected to an oxidative stress (diamide-induced depletion of glutathione). These findings indicate that motor terminals in mutant SOD1 mice display functional deficits even at presymptomatic ages, and that deficits associated with mitochondrial handling of stimulation-induced Ca2+ loads increase with age and may contribute to motor terminal degeneration in mutant SOD1 mice.
|
8 |
Functions of positive type and related topics in general analysis ...Dines, Charles Ross, January 1900 (has links)
Thesis (Ph. D.)--University of Chicago, 1915. / Vita. "A Private Edition Distributed by the University of Chicago Libraries." "Extracted from the Proceedings of the London mathematical society, series 2, vol. 15, part 4." Includes bibliographical references. Also available on the Internet.
|
9 |
Étude sur les équations fonctionelles à une ou à plusieurs variables /Léau, Léopold, January 1897 (has links)
Thèse--Université de Paris.
|
10 |
Minima of functions of lines ... /Le Stourgeon, Elizabeth. January 1900 (has links)
Thesis (Ph. D.)--University of Chicago, 1917. / "Private edition, distributed by the University of Chicago Libraries, Chicago, Illinois, 1920." "Reprinted from Transactions of the American mathematical society, Vol. 21, No. 4, October, 1920." Includes bibliographical references. Also available on the Internet.
|
Page generated in 0.0905 seconds