Biochemistry, Biophysics, and Structural Biology Commons^™

Genomic Analysis Of Homotypic Vacuole Fusion, E Scott Seeley, Masashi Kato, Nathan Margolis, William Wickner, Gary Eitzen

Dartmouth Scholarship

Yeast vacuoles undergo fission and homotypic fusion, yielding one to three vacuoles per cell at steady state. Defects in vacuole fusion result in vacuole fragmentation. We have screened 4828 yeast strains, each with a deletion of a nonessential gene, for vacuole morphology defects. Fragmented vacuoles were found in strains deleted for genes encoding known fusion catalysts as well as 19 enzymes of lipid metabolism, 4 SNAREs, 12 GTPases and GTPase effectors, 9 additional known vacuole protein-sorting genes, 16 protein kinases, 2 phosphatases, 11 cytoskeletal proteins, and 28 genes of unknown function. Vacuole fusion and vacuole protein sorting are catalyzed by …

Erv14p Directs A Transmembrane Secretory Protein Into Copii-Coated Transport Vesicles, Jacqueline Powers, Charles Barlowe

Dartmouth Scholarship

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are …

Nuclear Export Of 60s Ribosomal Subunits Depends On Xpo1p And Requires A Nuclear Export Sequence-Containing Factor, Nmd3p, That Associates With The Large Subunit Protein Rpl10p, Olivier Gadal, Daniela Strau, Jacques Kessl, Bernard Trumpower

Dartmouth Scholarship

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We …

Structure And Function Analysis Of Lin-14, A Temporal Regulator Of Postembryonic Developmental Events In Caenorhabditis Elegans, Yang Hong, Rosalind C. Lee, Victor Ambros

Dartmouth Scholarship

During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternativelin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternativelin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. …

Sec34p, A Protein Required For Vesicle Tethering To The Yeast Golgi Apparatus, Is In A Complex With Sec35p, Susan M. Vanrheenen, Xiaochun Cao, Stephanie K. Sapperstein, Elbert C. Chiang, Vladimir V. Lupashin, Charles Barlowe, M. Gerard Waters

Dartmouth Scholarship

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE …

Cbfa2 Is Required For The Formation Of Intra-Aortic Hematopoietic Clusters, Trista North, Ting-Lei Gu, Stacy Terryl, Qing Wang, Louisa Howard, Michael Binder, Miguel Marín-Padilla, Nancy A. Speck

Dartmouth Scholarship

Cbfa2 (AML1) encodes the DNA-binding subunit of a transcription factor in the small family of core-binding factors (CBFs). Cbfa2 is required for the differentiation of all definitive hematopoietic cells, but not for primitive erythropoiesis. Here we show that Cbfa2 is expressed in definitive hematopoietic progenitor cells, and in endothelial cells in sites from which these hematopoietic cells are thought to emerge. Endothelial cells expressing Cbfa2 are in the yolk sac, the vitelline and umbilical arteries, and in the ventral aspect of the dorsal aorta in the aorta/genital ridge/mesonephros (AGM) region. Endothelial cells lining the dorsal aspect of the aorta, and …

A Role For The Light-Dependent Phosphorylation Of Visual Arrestin, Paul G. Alloway, Patrick J. Dolph

Dartmouth Scholarship

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in the Drosophila visual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a Ca2+/calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascade in vivo. Here, we report the isolation of nine alleles of the Drosophila photoreceptor cell-specific arr2 gene. Flies carrying each of these alleles underwent light-dependent retinal degeneration and displayed electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca2+/calmodulin-dependent protein …

Ets-Core Binding Factor: A Common Composite Motif In Antigen Receptor Gene Enhancers, Batu Erman, Marta Cortes, Barbara S. Nikolajczyk, Nancy A. Speck, Ranjan Sen

Dartmouth Scholarship

A tripartite domain of the murine immunoglobulin μ heavy-chain enhancer contains the μA and μB elements that bind ETS proteins and the μE3 element that binds leucine zipper-containing basic helix-loop-helix (bHLH-zip) factors. Analysis of the corresponding region of the human μ enhancer revealed high conservation of the μA and μB motifs but a striking absence of the μE3 element. Instead of bHLH-zip proteins, we found that the human enhancer bound core binding factor (CBF) between the μA and μB elements; CBF binding was shown to be a common feature of both murine and human enhancers. Furthermore, mutant enhancers that bound …

Both An N-Terminal 65-Kda Domain And A C-Terminal 30-Kda Domain Of Seca Cycle Into The Membrane At Secyeg During Translocation, Jerry Eichler, William Wickner

Dartmouth Scholarship

SecA, a 102-kDa hydrophilic protein, couples the energy of ATP binding to the translocation of preprotein across the bacterial inner membrane. SecA function and topology were studied with metabolically labeled [35S]SecA and with inner membrane vesicles from cells that overex- pressed SecYEGDFyajC, the integral domain of preprotein translocase. During translocation in the presence of ATP and preprotein, a 65-kDa N-terminal domain of SecA is protected from proteolytic digestion through insertion into the mem- brane, as previously reported for a 30-kDa C-terminal domain [Economou, A. & Wickner, W. (1994) Cell 78, 835–843]. Insertion of both domains occurs at saturable SecYEGDFyajC sites …

I2b Is A Small Cytosolic Protein That Participates In Vacuole Fusion, Paul Slusarewicz, Zuoyu Xu, Kimberly Seefeld, Albert Haas, William T. Wickner

Dartmouth Scholarship

Saccharomyces cerevisiae vacuole inheritance requires two low molecular weight activities, LMA1 and LMA2. LMA1 is a heterodimer of thioredoxin and protease B inhibitor 2 (I^B2). Here we show that the second low molecular weight activity (LMA2) is monomeric I^B2. Though LMA2 / I^B2 was initially identified as a protease B inhibitor, this protease inhibitor activity is not related to its ability to promote vacuole fusion: ( i ) Low M r protease B inhibitors cannot substitute for LMA1 or LMA2, ( ii ) LMA1 and LMA2 promote the fusionof vacuoles from a strain that …

Docking Of Yeast Vacuoles Is Catalyzed By The Ras-Like Gtpase Ypt7p After Symmetric Priming By Sec18p (Nsf), Andreas Mayer, William Wickner

Dartmouth Scholarship

Vacuole inheritance in yeast involves the for- mation of tubular and vesicular “segregation struc- tures” which migrate into the bud and fuse there to es- tablish the daughter cell vacuole. Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion. We have developed biochemical and micro- scopic assays for the docking step of in vitro vacuole fusion and characterized its requirements. The vacu- oles must be primed for docking by the action of Sec17p ( a -SNAP) and Sec18p (NSF). Priming is neces- sary for both fusion …

A Heterodimer Of Thioredoxin And Ib2 Cooperates With Sec18p (Nsf) To Promote Yeast Vacuole Inheritance, Zuoyu Xu, Andreas Mayer, Eric Muller, William Wickner

Dartmouth Scholarship

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between saltwashed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as IB2, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo …

Numa Assembles Into An Extensive Filamentous Structure When Expressed In The Cell Cytoplasm, Alejandro Saredi, Louisa Howard, Duane A. Compton

Dartmouth Scholarship

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-DeltaNLS) in BHK-21 cells. During interphase, NuMA-DeltaNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-DeltaNLS associates normally with the mitotic spindle without causing any apparent deleterious effects …

Circadian Clock-Controlled Genes Isolated From Neurospora Crassa Are Late Night- To Early Morning-Specific, Deborah Bell-Pedersen, Mari L. Shinohara, Jennifer J. Loros, Jay C. Dunlap

Dartmouth Scholarship

An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs …

Cloning Of Human Acetyl-Coa Carboxylase-Beta And Its Unique Features., Joohun Ha, Jung-Kee Lee, Kyung-Sup Kim, Lee A. Witters, Ki-Han Kim

Dartmouth Scholarship

Acetyl-CoA carboxylase, which has a molecular mass of 265 kDa (ACC-alpha), catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. In this study we report the complete amino acid sequence and unique features of an isoform of ACC with a molecular mass of 275 kDa (ACC-beta), which is primarily expressed in heart and skeletal muscles. In these tissues, ACC-beta may be involved in the regulation of fatty acid oxidation, rather than fatty acid biosynthesis. ACC-beta contains an amino acid sequence at the N terminus which is about 200 amino acids long and may be uniquely related to the …

A Truncated Form Of The Pho80 Cyclin Of Saccharomyces Cerevisiae Induces Expression Of A Small Cytosolic Factor Which Inhibits Vacuole Inheritance., Teresa Nicolson, Barbara Conradt, William Wickner

Dartmouth Scholarship

Vacuoles project streams of vesicles and membranous tubules into the yeast bud where they fuse, founding the daughter cell organelle, vac5-1, which encodes a truncated form of the Pho80 cyclin, inhibits normal vacuole inheritance. An in vitro inheritance assay which measures the fusion of vacuoles serves as a model for several steps of this process. We find that cytosol isolated from the vac5-1 mutant is unable to promote the fusion of wild-type vacuoles in the in vitro assay. Wild-type vacuoles are irreversibly inactivated in a time- and temperature-dependent manner if preincubated with vac5-1 cytosol and ATP, suggesting the presence of …

A Novel Iron-Regulated Metal Transporter From Plants Identified By Functional Expression In Yeast., David Eide, Margaret Broderius, Jeanette Fett, Mary Lou Guerinot

Dartmouth Scholarship

Iron is an essential nutrient for virtually all organisms. The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Related sequences were also found in the genomes of rice, yeast, nematodes, and humans. In Arabidopsis, …

Lag-2 May Encode A Signaling Ligand For The Glp-1 And Lin-12 Receptors Of C-Elegans, Samuel T. Henderson, Dali Gao, Eric J. Lambie, Judith Kimble

Dartmouth Scholarship

The C. elegans lag-2 gene is required for several cell-cell interactions that rely on the receptors GLP-1 and LIN-12. In this paper, we report that lag-2 encodes a putative membrane protein with sequence similarity to Drosophila Delta, a proposed ligand for the Notch receptor. Furthermore, we show that the lag-2 promoter drives expression of a reporter protein in the signaling distal tip cell (DTC) of the DTC/germline interaction. By in situ hybridization, we have found that endogenous lag-2 mRNA is present in the DTC but not the germ line. One fusion protein, called LAG-2::beta-gal(intra), rescues a lag-2 null mutant and …

The Pha-4 Gene Is Required To Generate The Pharyngeal Primordium Of Caenorhabditis-Elegans, Susan E. Mango, Eric J. Lambie, Judith Kimble

Dartmouth Scholarship

In the 4-cell Caenorhabditis elegans embryo, two blastomeres are destined to generate pharyngeal cells, each by a distinct developmental strategy: one pathway is inductive, while the other is autonomous. Here, we identify the pha-4 locus. In animals lacking pha-4 activity, an early step in pharyngeal organogenesis is blocked: no pharyngeal primordium is formed and differentiated pharyngeal cells are absent. Most other tissues are generated normally in pha-4 mutants, including cells related to pharyngeal cells by cell lineage and position. Thus, pha-4 activity is required to form the pharyngeal primordium. We propose that pha-4 marks a convergence of the inductive and …

Circadian Clock Locus Frequency: Protein Encoded By A Single Open Reading Frame Defines Period Length And Temperature Compensation., Benjamin D. Aronson, Keith A. Johnson, Jay C. Dunlap

Dartmouth Scholarship

The frequency (frq) locus encodes a key component, a state variable, in a cellular oscillator generating circadian rhythmicity. Two transcripts have been mapped to this region, and data presented here are consistent with the existence of a third transcript. Analysis of cDNA clones and clock mutants from this region focuses attention on one transcript encoding a protein. FRQ, which is a central clock component: (i) mutations in all of the semidominant frq alleles are the result of single amino acid substitutions and map to the open reading frame (ORF) encoding FRQ; (ii) deletion of this ORF, or a frameshift mutation …

Binding Of Matrix Attachment Regions To Lamin Polymers Involves Single-Stranded Regions And The Minor Groove., M. E. Eva Ludérus, Jan L. Den Blaauwen, Oncko J. De Smit, Duane A. Compton, Roel Van Driel

Dartmouth Scholarship

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail …

The Primary Structure Of A Fungal Chitin Deacetylase Reveals The Function For Two Bacterial Gene Products., Dimitris Kafetzopoulos, George Thireos, John N. Vournakis, Vassilis Bouriotis

Dartmouth Scholarship

Chitin deacetylase (EC 3.5.1.41) hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin. A cDNA to the Mucor rouxii mRNA encoding chitin deacetylase was isolated, characterized, and sequenced. Protein sequence comparisons revealed significant similarities of the fungal chitin deacetylase to rhizobial nodB proteins and to an uncharacterized protein encoded by a Bacillus stearothermophilus open reading frame. These data suggest the functional homology of these evolutionarily distant proteins. NodB is a chitooligosaccharide deacetylase essential for the biosynthesis of the bacterial nodulation signals, termed Nod factors. The observed similarity of chitin deacetylase to the B. stearothermophilus gene product suggests that this gene …

The Interplay Of Light And The Circadian Clock. Independent Dual Regulation Of Clock-Controlled Gene Ccg-2(Eas), Guiseppina Arpaia, Jennifer J. Loros, Jay C. Dunlap, Giorgio Morelli, Guiseppe Macino

Dartmouth Scholarship

Ambient light is the major agent mediating entrainment of circadian rhythms and is also a major factor influencing development and morphogenesis. We show that in Neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene under the control of the circadian clock and allelic to the developmental gene easy wettable (eas), is regulated by light in wild-type strains. Light elicits a direct and important physiological effect on ccg-2(eas) expression as demonstrated using several mutant Neurospora strains. In white collar mutants (wc-1 and wc-2) that are “blind” to blue light, ccg-2(eas) mRNA shows no variation following illumination with saturating light. …

Differential Regulation Of Collagenase Gene Expression By Retinoic Acid Receptors--Alpha, Beta And Gamma, Luying Pan, Stephen H. Chamberlain, David T. Auble, Constance E. Brinckerhoff

Dartmouth Scholarship

The mechanisms involved in retinoic acid (RA)-mediated regulation of the collagenase gene in a rabbit synovial fibroblast cell line (HIG82) were investigated. When HIG82 cells are cotransfected with expression vectors containing cDNAs for retinoic acid receptor (RAR) α1, β2, or γ1 and collagenase promoter-driven CAT reporter constructs, only RAR-γ1 represses basal CAT expression upon RA treatment, while RAR-α1, β2, and γ1 all suppress phorbol-induced CAT expression. Thus, transcriptional regulation of collagenase by RA is mediated by RARs in an RAR-type specific manner. Using mutatlonal and deletional analysis, we find that interaction between elements within 182 bp collagenase promoter plays an …

Evidence For Helical Structures In Poly(1-Olefin Sulfones) By Transmission Electron Microscopy, George C. Ruben, W H. Stockmayer

Dartmouth Scholarship

Transmission electron microscope images were obtained of fractions of poly(1-tetradecene sulfone) and poly(cyclohexene sulfone) cast from very dilute solutions (0.007%, wt/vol) and rapidly freeze-dried on a mica surface. The samples were then vertically platinum-carbon (Pt-C) replicated with 9 +/- 0.3-A Pt-C and held together with 128 A of electron-transparent evaporated carbon. The Pt-C coating enlarges the molecular chain diameters by approximately 5 A, so that a single polysulfone chain has an apparent diameter of 9-12 A in the transmission electron microscope. Poly(1-tetradecene sulfone) forms short helical regions that show irregular helical turns of pitch 7-18 A, two to eight turns …

Interleukin-1 Or Phorbol Induction Of The Stromelysin Promoter Requires An Element That Cooperates With Ap-1., Karen Sirum-Connolly, Constance E. Brinckerhoff

Dartmouth Scholarship

Interieukin-1, a mediator of inflammation, or tumor promoting phorbol esters induce transcription of stromelysin, a metalloproteinase that degrades extracellular matrix molecules and that is overexpressed in diseases such as rheumatoid arthritis. Sequences required for induction of transcription of the human stromelysin promoter are contained on a 46 base pair fragment. This fragment contains a sequence with a high degree of similarity to the binding site for the transcription factor activator protein-1 (AP-1) and indeed, the AP-1 sequence of this fragment is necessary but not sufficient for the maximal response to phorbol 12-myristate 13-acetate (phorbol) or interleukin-1. Maximal induction requires functional …

Fermentation Of Cellulosic Substrates In Batch And Continuous Culture By Clostridium Thermocellum, Lee R. Lynd, Hans E. Grethlein, Richard H. Wolkin

Dartmouth Scholarship

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was comparedinbatchandcontinuouscultures.Maximumspecificgrowthratesper hourobtainedon cellulosic substrateswere 0.1inbatchcultureand>0.13incontinuousculture.Cellyields(gramsofcellsper gram of substrate)inbatchculturewere 0.17forpretreatedwoodand0.15forAvicel.Ethanolandacetatewere the mainproductsobservedunderalconditions.Ethanol:acetateratios(ingrams)were approximately1.8:1in batchcultureand generallyslightlylessthan 1:1incontinuousculture.Utilizationofcellulosicsubstrateswas essentially complete in batch culture. A prolonged lag phase was initialy observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuousculturewith-5g ofglucoseequivalentper literinthefeed,substrateconversionrelativeto theoreticalrangedfrom0.86ata dilutionrate(D)of0.05/hto0.48ata D of0.167/hforAvicelandfrom0.75 ata D of0.05/hto0.43ata D of0.11/hforpretreatedwood.Atfeedconcentrationsof<4.5g ofglucose equivalentperliter,conversionofpretreatedwoodwas80to90%atD= 0.083/h.Lowerconversionwas obtainedathigherfeedsubstrateconcentrations,consistentwitha limitingfactorotherthancellulose.Free Avicelaseactivitiesof12to84mU/mlwere observed,withactivityincreasinginthisorder:batchceliobiose, batchpretreatedwood< batchAvicel,continuouspretreatedwood< continuousAvicel.Freecellulaseactivity was higheratincreasingextentsofsubstrateutilizationforbothpretreatedwoodandAvicelunderal conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity,are essentiallythesame forpretreatedmixedhardwoodandAvicelundera varietyofconditions. HydrolysisyieldsobtainedwithC.thermocellumcellulaseactingeitherinvitroor invivowere comparableto thosepreviouslyreportedforTrichodermareeseion thesame substrates.

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was comparedinbatchandcontinuouscultures.Maximumspecificgrowthratesper hourobtainedon cellulosic substrateswere 0.1inbatchcultureand>0.13incontinuousculture.Cellyields(gramsofcellsper gram of substrate)inbatchculturewere 0.17forpretreatedwoodand0.15forAvicel.Ethanolandacetatewere the mainproductsobservedunderalconditions.Ethanol:acetateratios(ingrams)were approximately1.8:1in batchcultureand generallyslightlylessthan 1:1incontinuousculture.Utilizationofcellulosicsubstrateswas …

Modification Of Chromium(Vi)-Induced Dna Damage By Glutathione And Cytochromes P-450 In Chicken Embryo Hepatocytes., Doreen Y. Cupo, Karen E. Wetterhahn

Dartmouth Scholarship

The role of glutathione and cytochrome P-450 in the production of DNA damage by chromium(VI) was examined in chicken embryo hepatocytes by the alkaline elution technique. Cellular levels of glutathione and cytochrome P-450 were altered by treating the hepatocytes with N-acetyl-L-cysteine, buthionine sulfoximine, isopentanol, or beta-naphthoflavone. A dramatic increase in chromium(VI)-induced DNA strand breaks was observed after increasing glutathione levels in the cells. Chromium(VI)-induced DNA strand breaks were even more numerous when the level of cytochrome P-450 was also increased. Upon depletion of glutathione levels and induction of cytochrome P-450 or cytochrome P-448, little or no DNA strand breaks or …

Autoregulation Of Collagenase Production By A Protein Synthesized And Secreted By Synovial Fibroblasts: Cellular Mechanism For Control Of Collagen Degradation, Constance Brinckerhoff, Mary Benoit, William Culp

Dartmouth Scholarship

No abstract provided.

Spermidine-Condensed Phi X174 Dna Cleavage By Micrococcal Nuclease: Torus Cleavage Model And Evidence For Unidirectional Circumferential Dna Wrapping., Kenneth A. Marx, Thomas C. Reynolds

Dartmouth Scholarship

Spermidine-condensed phi X174 replicative form (RF) II DNA was digested with micrococcal nuclease to yield seven identifiable DNA bands forming an arithmetic fragment-length series. The DNA monomer unit length was found to be 780 +/- 80 base pairs. This result is most consistent with a proposed model for micrococcal nuclease cleavage of a DNA torus organized by the unidirectional, circumferential wrapping of B-geometry DNA. By a topological consideration, the blunt-end-rod-fusion model for torus formation [Eickbush, T. H. & Moudrianakis, E. N. (1978) Cell 13, 295-306] is shown to be inconsistent with our empirical solution results. We propose a continuous, circumferential …