Biology Commons^™

The A326s Mutant Of G(Iα1) As An Approximation Of The Receptor-Bound State, Bruce A. Posner, Mark B. Mixon, Mark A. Wall, Stephen R. Sprang, Alfred G. Gilman

Biological Sciences Faculty Publications

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their α subunits. In search of an approximation of the receptor-α subunit complex, we have considered the properties of A326S G(iα1), a mutation discovered originally in G(sα) (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the α(i1)β₁γ₂ heterotrimer by 250-fold. Nevertheless, affinity of mutant G(iα1) for GTPγS is high in the presence of Mg²⁺, …

Multiple Duplications Of Yeast Hexose Transport Genes In Response To Selection In A Glucose-Limited Environment, Celeste J. Brown, Kristy M. Todd, R. Frank Rosenzweig

Biological Sciences Faculty Publications

When microbes evolve in a nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the …

Molecular Analysis Of Bacterial Communities In A Three-Compartment Granular Activated Sludge System Indicates Community-Level Control By Incompatible Nitrification Processes, William Holben, Kazuhiko Noto, Tatsuo Sumino, Yuichi Suwa

Biological Sciences Faculty Publications

Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined, Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge(-1) day(-1). After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K, Note, T. Ogasawara, Y, Suwa, and T. Sumino, Water Res. 32:769-773, 1998), presumably mediated by different bacterial …

Rad52 Associates With Rpa And Functions With Rad55 And Rad57 To Assemble Meiotic Recombination Complexes, Stephen L. Gasior

Biological Sciences Faculty Publications

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for …

Exchange Of Substrate And Inhibitor Specificities Between Adenylyl And Guanylyl Cyclases, R. K. Sunahara, A. Beuve, J. J.G. Tesmer, S. R. Sprang, D. L. Garbers, A. G. Gilman

Biological Sciences Faculty Publications

The active sites of guanylyl and adenylyl cyclases are closely related. The crystal structure of adenylyl cyclase and modeling studies suggest that specificity for ATP or GTP is dictated in part by a few amino acid residues, invariant in each family, that interact with the purine ring of the substrate. By exchanging these residues between guanylyl cyclase and adenylyl cyclase, we can completely change the nucleotide specificity of guanylyl cyclase and convert adenylyl cyclase into a nonselective purine nucleotide cyclase. The activities of these mutant enzymes remain fully responsive to their respective stimulators, sodium nitroprusside and G(s)α. The specificity of …

Insect Herbivory Above- And Belowground: Individual And Joint Effects On Plant Fitness, John L. Maron

Biological Sciences Faculty Publications

Understanding of the selective effects of insect herbivory on plants comes primarily from studies of herbivory aboveground. The impact of belowground herbivory, either in isolation or in concert with herbivory aboveground, on plant fitness is only beginning to be understood.

I reduced the densities of root-boring ghost moth (Hepialus californicus) larvae and/or flower- and seed-feeding insects of bush lupine (Lupinus arboreus), a woody shrub, and followed fecundity and survival of plants for three years. In year one, suppression of aboveground herbivores increased mean seed output by 31%, but suppression of belowground herbivores had no significant effect …

Coreceptor Utilization By Human Immunodeficiency Virus Type 1 Is Not A Primary Determinant Of Neutralization Sensitivity, Rachel A. Lacasse, Kathryn E. Follis, Tarsem Moudgil, Meg Trahey, James M. Binley, Vicente Planelles, Susan Zolla-Pazner, Jack H. Nunberg

Biological Sciences Faculty Publications

We have examined the relationship between coreceptor utilization and sensitivity to neutralization in a primary isolate of human immunodeficiency virus type I and its T-cell line-adapted (TCLA) derivative. We determined that adaptation of the primary-isolate (PI) virus 168P results in the loss of the unique capacity of PI viruses to utilize the CCR5 coreceptor and in the acquisition by the TCLA 168C virus of sensitivity to neutralization by V3-directed monoclonal antibodies (MAbs). In experiments wherein infection by 168P is directed via either the CCR5 or the CXCR4 pathway, we demonstrate that the virus, as well as pseudotyped virions bearing a …

Population Genetics And Pattern Of Larval Dispersal Of The Endemic Hawaiian Freshwater Amphidromous Gastropod Neritina Granosa (Prosobranchia: Neritidae), Marc H. Hodges, Fred W. Allendorf

Biological Sciences Faculty Publications

Protein electrophoresis was used to study the population genetics of the endemic Hawaiian freshwater amphidromous gastropod Neritina granosa Sowerby. The genetic information was used to infer the pattern and degree of planktonic larval dispersal. Samples were taken from 12 streams located throughout the Hawaiian Archipelago during July, August, and September 1991. Overall mean heterozygosity was 0.052. Heterozygote deficiency was comparable with that found in other mollusks and marine invertebrates. Gene flow was substantial and was generally sufficient to maintain similar allele frequencies among stream populations. An island model of migration was indicated. However, significant heterogeneity among populations was observed and …

Systematic Studies And Conservation Status Of Claytonia Lanceolata Var. Flava (Portulacaceae), J. Stephen Shelly, Peter Lesica, Paul G. Wolf, Pamela S. Soltis, Douglas E. Soltis

Biological Sciences Faculty Publications

A biosystematic study of Claytonia lanceolata and related taxa in the Rocky Mountains was undertaken to evaluate the taxonomic status of C. lanceolata var. flava. This study was part of a broader assessment to determine the need for protection of the latter taxon under the federal Endangered Species Act. Electrophoretic and morphological studies revealed that C. lanceolata var. flava in Southwestern Montana and northwestern Wyoming represents a distinct diploid species (n=8) whose populations consist of yellow and/or white-flowered plants. Morphological, allozyme, and cytological data all indicate that this taxon does not belong in the C. lanceolata complex, but is …

Crystal Structure Of The Adenylyl Cyclase Activator G(Sα), Roger K. Sunahara, John J.G. Tesmer, Alfred G. Gilman, Stephen R. Sprang

Biological Sciences Faculty Publications

The crystal structure of G(sα), the heterotrimeric G protein α subunit that stimulates adenylyl cyclase, was determined at 2.5 Å in a complex with guanosine 5'-O-(3-thio-triphosphate) (GTPγS). G(sα) is the prototypic member of a of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of G(sα)·GTPγS with that of G(iα)·GTPγS suggest that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the α3-β5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of …

Crystal Structure Of The Catalytic Domains Of Adenylyl Cyclase In A Complex With G(Sα)·Gtpγς, John J.G. Tesmer, Roger K. Sunahara, Alfred G. Gilman, Stephen R. Sprang

Biological Sciences Faculty Publications

The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein α subunit (G(sα)) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substrate recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by G(sα).

Ecophysiological Responses Of Three Riparian Graminoids To Changes In The Soil Water Table, Anna Sala, Robert S. Nowak

Biological Sciences Faculty Publications

We examined the effect of changes in soil water table on the water relations, leaf gas exchange, and aboveground biomass of three riparian graminoids native to the semiarid western United States: Carex lanuginosa, Juncus balticus, and Carex nebrascensis. All three species co-occur at the wettest microhabitats within riparian corridors, but J. balticus and C. nebrascensis extend into drier areas. Lowering the water table to 1 m had little effect on the leaf gas exchange characteristics of the three graminoids. In the greenhouse, experimental reductions of the water table when plants had three fully mature leaves did not affect gas exchange …

Competition And Facilitation: A Synthetic Approach To Interactions In Plant Communities, Ragan M. Callaway, Lawrence R. Walker

Biological Sciences Faculty Publications

Interactions among organisms take place within a complex milieu of abiotic and biotic processes, but we generally study them as solitary phenomena. Complex combinations of negative and positive interactions have been identified in a number of plant communities. The importance of these two processes in structuring plant communities can best be understood by comparing them along gradients of abiotic stress, consumer pressure, and among different life stages, sizes, and densities of the interacting species. Here, we discuss the roles of life stage, physiology, indirect interactions, and the physical environment on the balance of competition and facilitation in plant communities.

Characterization Of The Sediment Bacterial Community In Groundwater Discharge Zones Of An Alkaline Fen: A Seasonal Study, Timothy C. Gsell, William E. Holben, Roy M. Ventullo

Biological Sciences Faculty Publications

The cell density, activity, and community structure of the bacterial community in wetland sediments were monitored over a 13-month period. The study was performed at Cedar Bog, an alkaline fen. The objective was to characterize the relationship between the sediment bacterial community in groundwater upwelling zones and the physical and chemical factors which might influence the community structure and activity. DNA, protein, and lipid synthesis were measured at three different upwelling zones by using [H-3]thymidine, [C-14]leucine, and [C-14]glucose incorporation, respectively. The physiological status (apparent stress) of the consortium was assessed by comparing [C-14]glucose incorporation into membrane and that into storage …

Structure Of Rgs4 Bound To Alf4--Activated G(Iα1): Stabilization Of The Transition State For Gtp Hydrolysis, John J.G. Tesmer, David M. Berman, Alfred G. Gilman, Stephen R. Sprang

Biological Sciences Faculty Publications

RGS proteins are GTPase activators for heterotrimeric G proteins. We report here the 2.8 Å resolution crystal structure of the RGS protein RGS4 complexed with G(iα1)-Mg²⁺-GDP-AlF₄. Only the core domain of RGS4 is visible in the crystal. The core domain binds to the three switch regions of G(iα1), but does not contribute catalytic residues that directly interact with either GDP or AlF₄. Therefore, RGS4 appears to catalyze rapid hydrolysis of GTP primarily by stabilizing the switch regions of G(iα1), although the conserved Asn-128 from RGS4 could also play a catalytic role by interacting with …

A Carboxy-Terminal Processing Protease Gene Is Located Immediately Upstream Of The Invasion-Associated Locus From Bartonella Bacilliformis, Samuel J. Mitchell, Michael F. Minnick

Biological Sciences Faculty Publications

A gene with homology to those encoding an unusual class of C-terminal processing proteases that flanks the invasion-associated locus ialAB of Bartonella bacilliformis has been identified. The 1302 bp gene, termed ctpA, is located immediately upstream of the ialA gene and encodes a predicted nascent product of 434 amino acids, producing a mature protein of 411 amino acid residues. The Bartonella CtpA appears to undergo autolysis in vitro, producing multiple products of 43-46 kDa, and a second group of products of 36-37 kDa. Production of CtpA in vivo gives a single product of 41.8 kDa. In addition to a computer-predicted …

Reversion Rates In A Leub Auxotroph Of Escherichia Coli K-12 Correlate With Ppgpp Levels During Exponential Growth, Barbara E. Wright, Michael F. Minnick

Biological Sciences Faculty Publications

Two isogenic strains of Escherichia coli K-12 differing only in relA, as well as two spoT transductants of the relA- strain, were examined with respect to ppGpp levels and reversion rates of a leuB- allele under nine different conditions. A positive correlation was established between reversion rates and the steady-state concentration of ppGpp during exponential growth. The leuB genes from two leuB- strains (isogenic except for relA) were cloned and sequenced and found to contain a single mutation, namely, a C-to-T transition at nucleotide 857. This mutation resulted in a serine-to-leucine substitution at amino acid residue 286 of the LeuB …

Do Biochemical Exaptations Link Evolution Of Defense And Pollination Systems? Historical Hypotheses And Experimental Tests With Dalechampia Vines, Jerome J. Howard

Biological Sciences Faculty Publications

Mapping resin secretion and pollination ecology onto the estimated phylogeny of species of the euphorb vine Dalechampia generated two historical hypotheses: resin rewards offered to pollinators by Dalechampia flowers evolved by minor modification of a preexisting, resin-based, floral defense system, and resin defense of leaves in advanced species evolved by modification of the preexisting resin-reward system. From these hypotheses, we derived two predictions: floral reward resins are chemically similar to putative floral defense resins and exhibit antiherbivore activities, and foliar resins are chemically similar to reward resins and also exhibit antiherbivore activities. We tested these predictions by chemical analyses and …

G Protein Mechanisms: Insights From Structural Analysis, Stephen R. Sprang

Biological Sciences Faculty Publications

This review is concerned with the structures and mechanisms of a superfamily of regulatory GTP hydrolases (G proteins). G proteins include Ras and its close homologs, translation elongation factors, and heterotrimeric G proteins. These proteins share a common structural core, exemplified by that of p21(ras) (Ras), and significant sequence identity, suggesting a common evolutionary origin. Three-dimensional structures of members of the G protein superfamily are considered in light of other biochemical findings about the function of these proteins. Relationships among G protein structures are discussed, and factors contributing to their low intrinsic rate of GTP hydrolysis are considered. Comparison of …

Conservation Implications Of A Multi-Scale Study Of Flammulated Owl (Otus Flammeolus) Habitat Use In The Northern Rocky Mountains, Usa, Vita Wright, Sallie J. Hejl, Richard L. Hutto

Biological Sciences Faculty Publications

Our multi-scale analysis of Flammulated Owl (Otus flammeolus) habitat use in the northern Rocky Mountains indicates some landscapes may be unsuitable for this species. As a result, there may be less habitat available for Flammulated Owls than thought based on the results of microhabitat studies. Thus, we suggest Flammulated Owl habitat conservation measures be based on the results of landscape-level, as well as microhabitat studies. Habitat conservation and restoration efforts in the ponderosa pine ecosystem should retain large trees, large snags, understory tree thickets, and grassland openings within landscapes that contain an abundance of suitable forest types.

Recovery Of Crenarchaeotal Ribosomal Dna Sequences From Freshwater-Lake Sediments, Christa Schleper, William E. Holben, Hans-Peter Klenk

Biological Sciences Faculty Publications

We report several novel environmental sequences of archaea from the kingdom Crenarchaeota, recovered from anaerobic freshwater-lake sediments in Michigan. A nested PCR approach with Archaea- and Crenarchaeota-specific primers was used to amplify partial Small-subunit ribosomal DNAs. Phylogenetic analysis of seven sequences shows that these DNAs represent a monophyletic lineage diverging prior to all recently identified crenarchaeotal phylotypes isolated from temperate environments. Including our lineage, all uncultured crenarchaeotal sequences recovered from moderate or told environments form a distinct, monophyletic group separate from the ''genuine'' thermophilic crenarchaeota. Our finding extends the emerging picture that crenarchaeota, thought until recently to be solely extreme …

Seasonal Abundance Of Autotrophic Picoplankton In The Pagan River, A Nutrient Enriched Subestuary Of The James River, Virginia, Lillian N. Davis, Karen A. Phillips, Harold G. Marshall

Biological Sciences Faculty Publications

Autotrophic picoplankton had average monthly concentrations of 73.5 X 10⁶ cells/L in the Pagan River, with summer-early fall maxima of 10⁸ cells/L. The abundance peaks increased with rising water temperatures, declining to their least abundance in mid-winter (10⁵ cells/L).

Problems With The Interpretation Of Mark-Release-Recapture Data In Subterranean Termites (Isoptera: Rhinotermitidae), Anthony D. Curtis, Deborah A. Waller

Biological Sciences Faculty Publications

The Lincoln index (Peterson method) is frequently used to estimate animal population size in mark-release-recapture studies. We tested the accuracy of this method to estimate termite colony size using logs infested with termites that were maintained in the laboratory. Termites were fed paper towels dyed either with 0.05% or 0.1% (w/w) of the dye marker Nile blue and released into their host logs in the laboratory. Following recapture a week later, estimates of termite population size for termites dyed with 0.05% Nile blue, and were approximately 3 times greater for termites dyed with 0.1% Nile blue. Concentrations of 0.1% Nile …

Structures Of The Extracellular Domain Of The Type I Tumor Necrosis Factor Receptor, James H. Naismith, Tracey Q. Devine, Tadahiko Kohno, Stephen R. Sprang

Biological Sciences Faculty Publications

Background: Tumor necrosis factor (TNF) is a powerful cytokine that is involved in immune and pro-inflammatory responses. Two TNF receptors that belong to the cysteine-rich low affinity nerve growth factor receptor family (TNF-R1 and TNF-R2) are the sole mediators of TNF signalling. Signalling is thought to occur when a trimer of TNF binds to the extracellular domains of two or three receptor molecules, which permits aggregation and activation of the cytoplasmic domains. The complex is then internalized within an endocytic vesicle, whereupon it dissociates at low pH. Structure of the soluble extracellular domain of the receptor (sTNF-R1) both in the …

Structure Of The Gdp-Pi Complex Of Gly203→Ala G(Iα1): A Mimic Of The Ternary Product Complex Of Galpha-Catalyzed Gtp Hydrolysis, Albert M. Berghuis, Ethan Lee, André S. Raw, Alfred G. Gilman, Stephen R. Sprang

Biological Sciences Faculty Publications

Background: G proteins play a vital role in transmembrane signalling events. In their inactive form G proteins exist as heterotrimers consisting of an α subunit, complexed with GDP and a dimer of βγ subunits. Upon stimulation by receptors, G protein α subunits exchange GDP for GTP and dissociate from βγ. Thus activated, α subunits stimulate or inhibit downstream effectors. The duration of the activated state corresponds to the single turnover rate of GTP hydrolysis, which is typically in the range of seconds. In G(iα1), the Gly203→Ala mutation reduces the affinity of the substrate for Mg²⁺, inhibits a key …

Directed Insertion Of A Selectable Marker Into A Circular Plasmid Of Borrelia Burgdorferi, Patricia Rosa, D. Scott Samuels, Daniel Hogan, Brian Stevenson, Sherwood Casjens, Kit Tilly

Biological Sciences Faculty Publications

Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A(1) as a selectable marker. We have transformed B. burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid. We have identified coumermycin A(1)-resistant transformants in which gyrB had …

Competition And Facilitation: Contrasting Effects Of Artemisia Tridentata On Desert Vs Montane Pines, Ragan M. Callaway, Eh Delucia, Darrin Moore, R Nowak, W. H. Schlesinger, Brenda J. Moor

Biological Sciences Faculty Publications

Circumstantial evidence suggests that Artemisia tridentata may out-compete Pinus ponderosa and P. jefferyi for water at ecotones between shrub steppe and montane forest vegetation in the Great Basin. Other studies indicate that within the shrub steppe Artemisia may act as a nurse plant for a third species of pine, P. monophylla. We used field experiments to study these contrasting effects of Artemisia on P, ponderosa and P. monophylla within the contest of the distributional patterns in western Nevada of all three species on andesite, and on sites where hydrothermal activity has altered the andesite. At intermediate elevations in the Great …

A Protocol For Ecosystem Management, Jack A. Stanford, G. C. Poole

Biological Sciences Faculty Publications

No abstract provided.

Abiotic Stress And The Relative Importance Of Interference And Facilitation In Montane Bunchgrass Communities In Western Montana, John T. Greenlee, Ragan M. Callaway

Biological Sciences Faculty Publications

No abstract provided.

Evidence For Acquisition In Nature Of A Chromosomal 2,4-Dichlorophenoxyacetic Acid/(Alpha)-Ketoglutarate Dioxygenase Gene By Different Burkholderia Spp., V. Grace Matheson, Larry J. Forney, Yuichi Suwa, Cindy H. Nakahatsu, Alan J. Sexstone, William E. Holben

Biological Sciences Faculty Publications

We characterized the gene required to initiate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2,4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage of the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an (alpha)-ketoglutarate-dependent reaction,characteristic of TfdA (F. Fukumori and R. P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tfdA gene was identified in a recombinant plasmid which complemented a tfdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli …