Life Sciences Commons^™

The Relationship Between Frq-Protein Stability And Temperature Compensation In The Neurospora Circadian Clock, Peter Ruoff, Jennifer J. Loros, Jay C. Dunlap

Dartmouth Scholarship

Temperature compensation is an important property of all biological clocks. In Neurospora crassa, negative-feedback regulation on the frequency (frq) gene's transcription by the FRQ protein plays a central role in the organism's circadian pacemaker. Earlier model calculations predicted that the stability of FRQ should determine the period length of Neurospora's circadian rhythm as well as the rhythm's temperature compensation. Here, we report experimental FRQ protein stabilities in frq mutants at 20 degrees C and 25 degrees C, and estimates of overall activation energies for mutant FRQ protein degradation. The results are consistent with earlier model predictions, i.e., temperature compensation of …

Genetic And Molecular Analysis Of Phytochromes From The Filamentous Fungus Neurospora Crassa, Allan C. Froehlich, Bosl Noh, Richard D. Vierstra, Jennifer Loros, Jay C. Dunlap

Dartmouth Scholarship

Phytochromes (Phys) comprise a superfamily of red-/far-red-light-sensing proteins. Whereas higher-plant Phys that control numerous growth and developmental processes have been well described, the biochemical characteristics and functions of the microbial forms are largely unknown. Here, we describe analyses of the expression, regulation, and activities of two Phys in the filamentous fungus Neurospora crassa. In addition to containing the signature N-terminal domain predicted to covalently associate with a bilin chromophore, PHY-1 and PHY-2 contain C-terminal histidine kinase and response regulator motifs, implying that they function as hybrid two-component sensor kinases activated by light. A bacterially expressed N-terminal fragment of PHY-2 covalently …

Growth Factor–Induced Shedding Of Syndecan-1 Confers Glypican-1 Dependence On Mitogenic Responses Of Cancer Cells, Kan Ding, Martha Lopez-Burks, José A. Sánchez-Duran, Murray Korc, Arthur D. Lander

Dartmouth Scholarship

The cell surface heparan sulfate proteoglycan (HSPG) glypican-1 is up-regulated by pancreatic and breast cancer cells, and its removal renders such cells insensitive to many growth factors. We sought to explain why the cell surface HSPG syndecan-1, which is also up-regulated by these cells and is a known growth factor coreceptor, does not compensate for glypican-1 loss. We show that the initial responses of these cells to the growth factor FGF2 are not glypican dependent, but they become so over time as FGF2 induces shedding of syndecan-1. Manipulations that retain syndecan-1 on the cell surface make long-term FGF2 responses glypican …

Principal Component Analysis For Predicting Transcription-Factor Binding Motifs From Array-Derived Data, Yunlong Liu, Matthew P Vincenti, Hiroki Yokota

Dartmouth Scholarship

The responses to interleukin 1 (IL-1) in human chondrocytes constitute a complex regulatory mechanism, where multiple transcription factors interact combinatorially to transcription-factor binding motifs (TFBMs). In order to select a critical set of TFBMs from genomic DNA information and an array-derived data, an efficient algorithm to solve a combinatorial optimization problem is required. Although computational approaches based on evolutionary algorithms are commonly employed, an analytical algorithm would be useful to predict TFBMs at nearly no computational cost and evaluate varying modelling conditions. Singular value decomposition (SVD) is a powerful method to derive primary components of a given matrix. Applying SVD …

Sara Positively Controls Bap-Dependent Biofilm Formation In Staphylococcus Aureus, María P. Trotonda, Adhar C. Manna, Ambrose L. Cheung, Iñigo Lasa, José R. Penadés

Dartmouth Scholarship

The biofilm-associated protein Bap is a staphylococcal surface protein involved in biofilm formation. We investigated the influence of the global regulatory locus sarA on bap expression and Bap-dependent biofilm formation in three unrelated Staphylococcus aureus strains. The results showed that Bap-dependent biofilm formation was diminished in the sarA mutants by an agr-independent mechanism. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation. As expected, the diminished capacity to form biofilms in the sarA mutants correlated with the decreased presence of Bap in the bacterial surface. Using transcriptional fusion and …

Calmodulin And Pf6 Are Components Of A Complex That Localizes To The C1 Microtubule Of The Flagellar Central Apparatus, Matthew J. Wargo, Erin E. Dymek, Elizabeth F. Smith

Dartmouth Scholarship

Studies of flagellar motility in Chlamydomonas mutants lacking specific central apparatus components have supported the hypothesis that the inherent asymmetry of this structure provides important spatial cues for asymmetric regulation of dynein activity. These studies have also suggested that specific projections associated with the C1 and C2 central tubules make unique contributions to modulating motility; yet, we still do not know the identities of most polypeptides associated with the central tubules. To identify components of the C1a projection, we took an immunoprecipitation approach using antibodies generated against PF6. The pf6 mutant lacks the C1a projection and possesses flagella that only …

Cellulose Utilization By Clostridium Thermocellum: Bioenergetics And Hydrolysis Product Assimilation, Yi-Heng P. Zhang, Lee R. Lynd

Dartmouth Scholarship

The bioenergetics of cellulose utilization by Clostridium thermocellum was investigated. Cell yield and maintenance parameters, Y(X/ATP)True = 16.44 g cell/mol ATP and m = 3.27 mmol ATP/g cell per hour, were obtained from cellobiose-grown chemostats, and it was shown that one ATP is required per glucan transported. Experimentally determined values for G(ATP)P-T (ATP from phosphorolytic beta-glucan cleavage minus ATP for substrate transport, mol ATP/mol hexose) from chemostats fed beta-glucans with degree of polymerization (DP) 2-6 agreed well with the predicted value of (n-2)/n [corrected] (n = mean cellodextrin DP assimilated). A mean G(ATP)(P-T) value of 0.52 +/- 0.06 was calculated …

Integrated Recombinant Protein Expression And Purification Platform Based On Ralstonia Eutropha, Gavin C. Barnard, Jesse D. Mccool, David W. Wood, Tillman U. Gerngross

Dartmouth Scholarship

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer poly

A Gene Expression Fingerprint Of C. Elegans Embryonic Motor Neurons, Rebecca M. Fox, Stephen E. Von Stetina, Susan J. Barlow, Christian Shaffer, Kellen L. Olszewski, Jason H. Moore

Dartmouth Scholarship

Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo.

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From The Cover: Assignment Of An Essential Role For The Neurospora Frequency Gene In Circadian Entrainment To Temperature Cycles, Antonio M. Pregueiro, Nathan Price-Lloyd, Deborah Bell-Pedersen, Christian Heintzen, Jennifer J. Loros, Jay C. Dunlap

Dartmouth Scholarship

Circadian systems include slave oscillators and central pacemakers, and the cores of eukaryotic circadian clocks described to date are composed of transcription and translation feedback loops (TTFLs). In the model system Neurospora, normal circadian rhythmicity requires a TTFL in which a White Collar complex (WCC) activates expression of the frequency (frq) gene, and the FRQ protein feeds back to attenuate that activation. To further test the centrality of this TTFL to the circadian mechanism in Neurospora, we used low-amplitude temperature cycles to compare WT and frq-null strains under conditions in which a banding rhythm was elicited. WT cultures were entrained …

A Rab Requirement Is Not Bypassed In Sly1-20 Suppression, Nicole Ballew, Yiting Liu, Charles Barlowe

Dartmouth Scholarship

The Rab GTPase Ypt1p and the large homodimer Uso1p are both required for tethering endoplasmic reticulum-derived vesicles to early Golgi compartments in yeast. Loss-of-function ypt1 and uso1 mutations are suppressed by SLY1-20, a dominant allele that encodes the Sed5p-associated protein, Sly1p. Here, we investigate the mechanism of SLY1-20 suppression. In wild-type strains, Ypt1p can be coimmunoprecipitated with Uso1p; however, in a ypt1Δ/SLY1-20 strain, which lacks this complex, membrane binding of Uso1p was reduced. In spite of Ypt1p depletion, Uso1p-dependent vesicle tethering was not bypassed under the ypt1Δ/SLY1-20 condition. Moreover, tethering and fusion assays with ypt1 …

A Three-Component Regulatory System Regulates Biofilm Maturation And Type Iii Secretion In Pseudomonas Aeruginosa, Sherry L. Kuchma, John P. Connolly, George A. O'Toole

Dartmouth Scholarship

Biofilms are structured communities found associated with a wide range of surfaces. Here we report the identification of a three-component regulatory system required for biofilm maturation by Pseudomonas aeruginosa strain PA14. A transposon mutation that altered biofilm formation in a 96-well dish assay originally defined this locus, which is comprised of genes for a putative sensor histidine kinase and two response regulators and has been designated sadARS. Nonpolar mutations in any of the sadARS genes result in biofilms with an altered mature structure but do not confer defects in growth or early biofilm formation, swimming, or twitching motility. After …

Yos1p Is A Novel Subunit Of The Yip1p–Yif1p Complex And Is Required For Transport Between The Endoplasmic Reticulum And The Golgi Complex, Matthew Heidtman, Catherine Z. Chen, Ruth N. Collins, Charles Barlowe

Dartmouth Scholarship

Yeast Yip1p is a member of a conserved family of transmembrane proteins that interact with Rab GTPases. Previous studies also have indicated a role for Yip1p in the biogenesis of endoplasmic reticulum (ER)-derived COPII transport vesicles. In this report, we describe the identification and characterization of the uncharacterized open reading frame YER074W-A as a novel multicopy suppressor of the thermosensitive yip1-4 strain. We have termed this gene Yip One Suppressor 1 (YOS1). Yos1p is essential for growth and for function of the secretory pathway; depletion or inactivation of Yos1p blocks transport between the ER and …

Effects Of Food Deprivation On Blood Lipid Concentration And Composition In Steller Sea Lions (Eumetopias Jubatus), Michelle Lea Berman

Electronic Theses and Dissertations

Steller sea lions, the largest Otariid, fast during their breeding season; during this time they refrain from ingesting food for a period of 12-43 days. Fasting, while undertaking an extremely energetically demanding activity (breeding and pupping), requires specific physiological adaptations. This study examined the physiological response to fasting of two age classes, juveniles and sub-adults, during the breeding and non-breeding seasons to determine how these animals utilize lipids and the pattern of fatty acid mobilization from lipid stores during fasting. Four juveniles and 5 sub-adults were fasted for one and two weeks, respectively, and blood samples were collected approximately every …

Does Intracellular Metabolite Diffusion Limit Post-Contractile Recovery In Burst Locomotor Muscle?, Stephen T. Kinsey, Pragyansri Pathi, Kristin M. Hardy, Amanda Jordan, Bruce R. Locke

Biological Sciences

Post-metamorphic growth in the blue crab entails an increase in body mass that spans several orders of magnitude. The muscles that power burst swimming in these animals grow hypertrophically, such that small crabs have fiber diameters that are typical of most cells (600μm). Thus, as the animals grow, their muscle fibers cross and greatly exceed the surface area to volume ratio (SA:V) and intracellular diffusion distance threshold that is adhered to by most cells. Large fiber size should not impact burst contractile function, but post-contractile recovery may be limited by low SA:V and excessive intracellular diffusion distances. A number of …

Cytokine Expression By T. Helper Cells Responding To Stress, Dina Zanetti

Master's Theses

Stress causes alterations to the immune system. These alterations have lead to increased susceptibility to antigens. The present study explores shifts in T helper (TH) cell balance (TH1 and TH2), through their cytokine expression. This study was performed in three phases, and focused mainly on animals subjected to a 10 day a chronic unpredictable stress paradigm. Phase one used ELISAs to confirm the cytokine profile produced from TH 1 and TH2 murine cell-lines. Phase two used SDS PAGE and Western Immunoblotting to show that splenocytes from Long Evan's Hooded rats subjected to the stress paradigm, had decreased in intercellular in …

Silencing Of Retrotransposons In Dictyostelium By Dna Methylation And Rnai, Markus Kuhlmann, Branimira E. Borisova, Markus Kaller, Pontus Larsson, Dirk Stach, Jianbo Na, Ludwig Eichinger, Frank Lyko, Victor Ambros, Fredrik Soderbom, Christian Hammann, Wolfgang Nellen

Dartmouth Scholarship

We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is ∼0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding …

Crystal Structure Of The Gtpase Domain Of Rat Dynamin 1, Thomas F. Reubold, Susanne Eschenburg, Andreas Becker, Marilyn Leonard, Sandra L. Schmid, Richard B. Vallee, F. Jon Kull, Dietmar J. Manstein

Dartmouth Scholarship

Here, we present the 1.9-A crystal structure of the nucleotide-free GTPase domain of dynamin 1 from Rattus norvegicus. The structure corresponds to an extended form of the canonical GTPase fold observed in Ras proteins. Both nucleotide-binding switch motifs are well resolved, adopting conformations that closely resemble a GTP-bound state not previously observed for nucleotide-free GTPases. Two highly conserved arginines, Arg-66 and Arg-67, greatly restrict the mobility of switch I and are ideally positioned to relay information about the nucleotide state to other parts of the protein. Our results support a model in which switch I residue Arg-59 gates GTP binding …

Dietary Fats And Membrane Function: Implications For Metabolism And Disease, Anthony J. Hulbert, Nigel Turner, Leonard H. Storlein, Paul Else

Faculty of Health and Behavioural Sciences - Papers (Archive)

Lipids play varied and critical roles in metabolism, with function dramatically modulated by the individual fatty acid moities in complex lipid entities. In particular, the fatty acid composition of membrane lipids greatly influences membrane function. Here we consider the role of dietary fatty acid profile on membrane composition and, in turn, its impact on prevalent disease clusters of the metabolic syndrome and mental illness. Applying the classical physiological conformer-regulator paradigm to quantify the influence of dietary fats on membrane lipid composition (i.e. where the membrane variable is plotted against the same variable in the environment - in this case dietary …

Health And Lifestyle Profiles Across The Lifespan: Results From The Louisiana Healthy Aging Study, Madlyn Irene Frisard

LSU Doctoral Dissertations

Aging and age related disease affects individuals differently. One possible explanation could be free radical production varies among individuals and this variation determines the aging process and the progression of disease. The purpose of this study was to test whether nonagenarians have a relatively low metabolic rate when compared to younger individuals and whether this low metabolic rate is associated with lower levels of oxidative stress and less incidence of disease. Resting metabolic rate (RMR), markers of oxidative stress to lipids, proteins, and DNA, components of the metabolic syndrome, and physical activity level were measured in 3 groups of individuals …

Use Of Bovine Somatotropin In The Management Of Transition Dairy Cows, Mehmet Şükrü Gülay, Fatma Şefi̇ka Hati̇poğlu

Turkish Journal of Veterinary & Animal Sciences

The major objectives of an efficient dairy farm operation include a successful lactation period, high milk yield relative to the feed costs, reproductive competence, and finally the returning of the cow to the body condition score (BCS) that existed before lactation so she will be prepared for another lactation. In cows a milk yield response to bovine somatotropin (bST) treatments at 60 d after parturition has been well studied and fully documented. Milk production response to bST occurs because of its known effects on partitioning of nutrients and because a greater proportion of the nutrient intake is used for milk …