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Item Inexpensive alternative to M&S medium for selection of Arabidopsis plants in culture(Elsevier, 1999-02-01) Pollock, Mary A.; Oppenheimer, David G.Item Differentially Expressed Forms of 1-L-myo-Inositol-1-Phosphate Synthase (EC 5.5.1.4) in Phaseolus vulgaris(American Society for Biochemistry and Molecular Biology, 1996-07-19) Johnson, Margaret Dean; Wang, XiaohongWe have characterized two distinct polypeptides with 1-L-myo-inositol-1-phosphate synthase (MI-1-P synthase) activity that are differentially expressed during development in Phaseolus vulgaris. Western analyses, enzyme assays, and partial purification of MI-1-P synthase during embryonic and postembryonic development show that its expression is temporally and spatially regulated. Developmental Western analyses of soluble proteins detect a small protein, approximately 33 kDa, with MI-1-P synthase activity during the globular stage (stage II) of embryogenesis and in mature roots. Expression of this small protein is also enriched in thylakoidal membranes of fractionated leaf chloroplasts, although Western analyses of total soluble leaf proteins show no cross-reacting material. In contrast, a larger protein, approximately 56 kDa, with MI-1-P synthase activity is present during the cotyledonary phase (stage IV) of embryogenesis in green cotyledons of seedlings and in young roots.Item Tryptophan-containing α-Subunits of the Escherichia coli Tryptophan Synthase: ENZYMATIC AND UREA STABILITY PROPERTIES(American Society for Biochemistry and Molecular Biology, 1995-07-28) Choi, Shin-Geon; O'Donnel, Stephen E.; Sarken, Krishna D.; Hardman, John K.Early studies suggested that the Escherichia coli tryptophan synthase α-subunit unfolded in a two-step process in which there was a stable intermediate composed of a native α-1 folding unit (residues 1-188) and a completely unfolded α-2 folding unit (residues 189-268). More recent evidence has indicated that such a structure for the intermediate seems unlikely. In this report, single Trp residues (absent in the wild-type α-subunit) are substituted separately for Phe residues at positions 139 (in α-1) and 258 (in α-2) to produce the F139W, F258W, and F139W/F258W mutant α-subunits. The UV absorbance and fluorescence properties of the F139W/F258W double mutant are identical with those of equimolar mixtures of the single mutants, suggesting that the Trp residue at each position can independently report the behavior of its respective folding unit. Each mutant α-subunit is wild-type enzymatically, and when UV absorbance is monitored, the urea-induced unfolding of the three tryptophan-containing α-subunits is virtually identical to the wild-type protein. These wild-type properties make these proteins attractive candidates for a fluorescence examination of the behavior of the individual folding units and the structure of potential intermediate(s) and as host proteins for the insertion of our existing destabilizing and/or stabilizing mutational alterations.Item Unfolding Properties of Tryptophan-containing a-Subunits of the Escherichia coli Tryptophan Synthase(American Society for Biochemistry and Molecular Biology, 1995-11-24) Choi, Shin-Geon; Hardman, John K.The urea-induced unfolding of the Escherichia coli tryptophan synthase α-subunit is examined via fluorescence measurements with tryptophan-containing α-subunit mutants, constructed by in vitro mutagenesis. Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable intermediate composed of a native α-1 folding unit (residues 1-188) and a completely unfolded α-2 folding unit (residues 189-268). Recently, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediates seems unlikely. Previously, we described the introduction of Trp residues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically and in their stability to urea. The unfolding behavior of these α-subunits, monitored by fluorescence intensity changes at the discrete emission λmax for each, in both equilibrium and kinetic experiments, suggest that: (a) both folding units commence unfolding simultaneously (near 2 M urea); (b) the larger α-1 unit unfolds in a multistep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller α-2 unit unfolds in a single step event. These results are also clearly incompatible with the early proposals on the structure of the intermediate. It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of α-1 attached to either a completely folded or completely unfolded form of α-2. These results are consistent with and provide an added dimension to the recent description of the proposed structure of the intermediate.Item Multiple mRNAs from the Punch locus of Drosophila melanogaster encode isoforms of GTP cyclohydrolase I with distinct N-terminal domains(American Society for Biochemistry and Molecular Biology, 1993-12-25) McLean, Janna R.; Krishnakumar, Sujatha; O'Donnell, Janis M.The GTP cyclohydrolase I gene of Drosophila melanogaster, the Punch locus, encodes alternative transcripts of 1.7 and 1.75 kilobases (kb). These transcripts are differentially expressed throughout Drosophila development. cDNA clones representing these transcripts, corresponding genomic regions and polymerase chain reaction-amplified primer extensions of the 5' ends of the RNAs were sequenced. Both RNAs contain five exons and derive from primary transcripts that extend over approximately 8 and 4 kb for the 1.7- and 1.75-kb RNAs, respectively. Their 5'-most exons are unique and spliced onto common 3' exons. The cDNAs each contain a single long open reading frame, which can be translated into a polypeptide of 273 amino acids for the 1.7-kb mRNA and 308 amino acids for the 1.75-kb mRNA. The unique exons confer distinct N-terminal domains to each predicted protein. Sequence comparisons reveal that the Drosophila GTP cyclohydrolase isoforms encoded by the multiple transcripts are highly similar to GTP cyclohydrolases from humans, rodents, and bacteria, with one significant exception. The N-terminal domains encoded by the transcript-specific 5' exons cannot be aligned with the N termini of any other GTP cyclohydrolases. These domains are predicted to confer distinct physical characteristics to the alternate isoforms, and it is hypothesized that they aid in regulating the expression of the enzyme in diverse cellular environments.Item Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits(American Society for Biochemistry and Molecular Biology, 1991-10-25) Lim, Woon Ki; Sarkar, Sanjoy K.; Hardman, John K.Thirty-nine mutant tryptophan synthase alpha subunits have been purified and analyzed (in the presence of the beta 2-subunit) for their enzymatic (kcat, Km) behavior in the reactions catalyzed by the alpha 2.beta 2 complex, the fully constituted form of this enzyme. The mutant alpha subunits, obtained by in vitro random, saturation mutagenesis of the encoding trpA gene, contain single amino acid substitutions at sites within the first 121 residues of the alpha polypeptide. Four categories of altered residues have been tentatively assigned roles in the catalytic functions of this enzyme: 1) catalytic residues (Glu49 and Asp60); 2) residues involved in substrate binding or orientation (Phe22, Thr63, Gln65, Tyr102, and Leu105); 3) residues involved in alpha.beta subunit interactions (Gly51, Pro53, Asp56, Asp60, Pro62, Ala67, Phe72, Thr77, Pro78, Tyr102, Asn104, Leu105, and Asn108); and 4) residues with no apparent catalytic roles. Catalytic residue alterations result in no detectable activity in the alpha-subunit specific reactions. Substrate binding/orientation roles are detected enzymatically primarily as rate defects; alterations only at Tyr102 result in apparent Km effects. alpha.beta interaction roles are detected as rate defects in all tryptophan synthase reactions plus Km increases for the alpha-subunit substrate, indole-3-glycerol phosphate, only when L-serine is present at the beta 2-subunit active site. A substitution at only one site, Asn104, appears to be unique in its potential effect on intersubunit channeling of indole, the product of the alpha-subunit specific reaction, to the beta 2-subunit active site.Item Hydride transfer stereospecificity of rat liver aldehyde dehydrogenases(American Society for Biochemistry and Molecular Biology, 1987-08-15) Jones, Kimberly Harper; Lindahl, Ronald; Baker, David C.; Timkovich, RussellThe stereospecificity of hydride transfer to NAD+ by several forms of rat liver aldehyde dehydrogenase was determined by a nuclear magnetic resonance method. The forms included several mitochondrial and microsomal isozymes from normal liver, as well as isozymes from xenobiotic-treated and tumor cells. The proton added to NAD+ comes exclusively from the aldehyde substrate and in all cases was A (pro-R)-stereospecific.Item Rat liver aldehyde dehydrogenase. II. Isolation and characterization of four inducible isozymes(American Society for Biochemistry and Molecular Biology, 1984-10-10) Lindahl, Ronald; Evces, SusanThe purification and properties of 4 inducible cytosolic rat liver aldehyde dehydrogenase isozymes are described. Based on their behavior during purification and their properties, the activities can be grouped into 2 classes. The isozyme inducible in normal liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the tumor-specific isozyme found in hepatocellular carcinomas have apparent molecular weights of 110,000, prefer NADP+ as coenzyme, and preferentially oxidize benzaldehyde-like aromatic aldehydes, but not phenylacetaldehyde. They also have identical pH profiles and responses to effectors. These isozymes differ slightly in isoelectric point and thermal stability. The normal liver phenobarbital-inducible isozyme and the isozyme appearing during the promotion phase of hepatocarcinogenesis appear to be identical. Both have apparent molecular weights of 165,000, are NAD-specific and prefer aliphatic aldehydes. They can oxidize phenylacetaldehyde, but not benzaldehyde-like aromatic aldehydes. They also have identical pH and thermal stability profiles and responses to effectors. While the 4 inducible isozymes share identical subunit molecular weights (54,000) with the normal liver millimolar Km aldehyde dehydrogenases, they are distinctly different enzymatic species. The interrelationships of the various normal liver and inducible rat liver aldehyde dehydrogenases are discussed.Item Rat liver aldehyde dehydrogenase. I. Isolation and characterization of four high Km normal liver isozymes.(American Society for Biochemistry and Molecular Biology, 1984-10-10) Lindahl, Ronald; Evces, SusanFrom normal rat liver mitochondrial and microsomal fractions, 4 distinct aldehyde dehydrogenase isozymes with millimolar substrate Km values have been purified and characterized. Two isozymes were isolated from mitochondria and 2 from microsomes. A mitochondrial aldehyde dehydrogenase with a substrate Km in the micromolar range was also identified. Subunit molecular weights for all millimolar Km isozymes is 54,000. The mitochondrial and microsomal millimolar Km isozymes are clearly distinguishable from each other by substrate and coenzyme specificity, pH velocity profiles, and thermal stability. By these same properties, the 2 isozymes from each organelle are virtually identical. The 2 mitochondrial isozymes can be distinguished by apparent molecular weight (I, 170,000; II, approximately 250,000), Km for NADP+, effect of inhibitors, and pI. The 2 microsomal isozymes are of the same apparent molecular weight (approximately 250,000), but are distinguishable by their Km values for benzaldehyde and NADP+, response to inhibitors, and pI.Item Movement of Cichla species (Cichlidae) in a Venezuelan floodplain river(Sociedade Brasileira de Ictiologia, 2003) Hoeinghaus, David J.; Layman, Craig A.; Arrington, D. Albrey; Winemiller, Kirk O.In their native habitats of black- and clear-water Neotropical rivers, large predatory cichlids of the genus Cichla are an important food and recreational resource. Understanding patterns of movement for these species is necessary for effective management and conservation; however, no information is available on movement in natural fluvial populations. Therefore, we initiated a cooperative mark-recapture program with local sport-fishing groups to evaluate movement of Cichla in the Cinaruco River, Venezuela and to promote conservation awareness. Between January 1999 and May 2003, we tagged 2,224 individuals of three species of Cichla (C. temensis, C. orinocensis, and C. intermedia) with uniquely numbered floy tags. Over 52 months, 2.8% of the tagged fishes were recaptured. Most recaptures occurred within 1 km of the tagging location; however, a few large C. temensis moved up to 21 km, demonstrating the potential for longer distance movement in this species. The distance between tagging and recapture locations was not significantly correlated with the time interval between tagging and recapture. Distance moved was significantly related to fish size and period of the annual hydrological cycle. Implications for management and conservation of these species and future research directions are discussed.Item Microbiological Assay for Estimating Salivary Concentrations of the Cariostatic Antibiotic Actinobolin(American Society for Microbiology, 1970-10-01) Hunt, D.E.; Bradley, E.L.; Bachmann, Joan W.A logarithmic-ratio microbiological assay has been developed for the estimation of concentrations of actinobolin (or inhibitory equivalents) in human saliva. The utility of this assay has been demonstrated by presenting data that show concentrations of this antibiotic (or inhibitory equivalents) detected in saliva or saline after incubation at 37 C for periods as long as 6 hr. The results of some statistical analyses of the assay data have been included.Item Survey of the Sensitivity of Microorganisms to Rubratoxin B(American Society for Microbiology, 1970-06-30) Hayes, A. Wallace; Wyatt, Elwanda P.Of the 133 microorganisms tested, Tetrahymena pyriformis and Volvox aureus were the most sensitive to rubratoxin B, being inhibited at 25 and 50 mug/ml, respectively.Item Environmental and Nutritional Factors Affecting the Production of Rubratoxin B by Penicillium rubrum Stoll(American Society for Microbiology, 1970-09-01) Hayes, A. Wallace; Wyatt, Elwanda P.; King, Patricia A.Rubratoxin B can be produced in a semisynthetic medium by Penicillium rubrum under varying environmental and nutritional conditions. Maximum production (552.0 mg/500 ml) was obtained with P. rubrum NRRL A-11785 grown in stationary cultures of Mosseray's simplified Raulin solution supplemented with 2.5% malt extract broth at ambient temperature. Zinc is required at levels of at least 0.4 mg per liter. In the absence of iron sulfate, there was a 50-fold reduction in rubratoxin B production but not in growth. No toxin was produced by this isolate in 5- or 7-liter fermentors.Item A Terminal Inversion in Drosophila Ananassae(National Academy of Sciences, 1936-10-15) Kaufmann, B.P.Item Mitotic Behavior of Induced Chromosomal Fragments Lacking Spindle Attachments in the Neuroblasts of the Grasshopper(National Academy of Sciences, 1938-11-15) Carlson, J. GordonItem Morphology of the Chromosomes of Drosophila ananassae(The Japan Mendel Society, 1937-08-31) Kaufmann, Berwind P.1) The chromosome complement of D. ananassae, studied in the neurocytes, consists of three pairs of V-shaped autosom.es and the sex chromosomes. These are a pair of V-shaped X-chromosomes in the female, an X and a J-shaped Y-chromosome in the male. The chromosomes may be distinguished by relative, sizes and characteristic constrictions. 2) The fourth chromosomes and the Y-chromosome appear totally heteropyknotic in resting and early prophase stages of mitotic cells. Short heteropyknotic regions lie adjacent to the spindle attachment regions of the X-chromosomes and the four longer autosomes. 3) In salivary gland nuclei there are but six chromosome arms with euchromatic sections. Four of these represent the paired arms of the longer autosomes, the other two the X-chromosome arms. The fourth chromosomes are represented in salivary gland nuclei by a small bipartite mass of heterochromatin which forms part of the chromocenter. 4) In mitotic prophases of female larvae the fourth chromosomes are associated with the nucleolus, which separates a small satellite-like portion from the remainder of the long arm of these chromosomes. In the male the Y-chromosome forms a third member of the group associated with the nucleolus. 5) The fourth chromosome-nucleolus-satellite relationship is. also evident, in salivary gland nuclei, the satellite appearing as a banded body associated with the nucleolus, and also connected with the fourth chromosomes by chromatic strands. 6) The chromocenter of mitotic cells and of the salivary gland cells is not amorphous, but discloses, under suitable conditions, the limits of the component chromosomes.Item A New Leptothorax From Alabama (Hymenoptera: Formicidae)(Cambridge Entomological Club, 1950-01-01) Wilson, Edward OsborneItem Suboxic Deposition of Ferric Iron by Bacteria in Opposing Gradients of Fe(II) and Oxygen at Circumneutral pH(American Society for Microbiology, 2001) Sobolev, Dmitri; Roden, Eric E.The influence of lithotrophic Fe(II)-oxidizing bacteria on patterns of ferric oxide deposition in opposing gradients of Fe(II) and O(2) was examined at submillimeter resolution by use of an O(2) microelectrode and diffusion microprobes for iron. In cultures inoculated with lithotrophic Fe(II)-oxidizing bacteria, the majority of Fe(III) deposition occurred below the depth of O(2) penetration. In contrast, Fe(III) deposition in abiotic control cultures occurred entirely within the aerobic zone. The diffusion microprobes revealed the formation of soluble or colloidal Fe(III) compounds during biological Fe(II) oxidation. The presence of mobile Fe(III) in diffusion probes from live cultures was verified by washing the probes in anoxic water, which removed ca. 70% of the Fe(III) content of probes from live cultures but did not alter the Fe(III) content of probes from abiotic controls. Measurements of the amount of Fe(III) oxide deposited in the medium versus the probes indicated that ca. 90% of the Fe(III) deposited in live cultures was formed biologically. Our findings show that bacterial Fe(II) oxidation is likely to generate reactive Fe(III) compounds that can be immediately available for use as electron acceptors for anaerobic respiration and that biological Fe(II) oxidation may thereby promote rapid microscale Fe redox cycling at aerobic-anaerobic interfaces.Item Microbially Catalyzed Nitrate-Dependent Oxidation of Biogenic Solid-Phase Fe(II) Compounds(American Chemical Society, 2001) Weber, Karrie A.; Picardal, Flynn W.; Roden, Eric E.The potential for microbially catalyzed NO3—-dependent oxidation of solid-phase Fe(II) compounds was examined using a previously described autotrophic, denitrifying, Fe(II)-oxidizing enrichment culture. The following solid-phase Fe(II)-bearing minerals were considered: microbially reduced synthetic goethite, two differ ent end products of microbially hydrous ferric oxide (HFO) reduc tion (biogenic Fe3O4 and biogenic FeCO3), chemically precipitated FeCO3, and two microbially reduced iron(III) oxide-rich subsoils. The microbially reduced goethite, subsoils, and chemically precip itated FeCO3 were subject to rapid NO3 —-dependent Fe(II) oxida tion. Significant oxidation of biogenic Fe3O4 was observed. Very little biogenic FeCO3 was oxidized. No reduction of NO3 — or ox idation of Fe(II) occurred in pasteurized cultures. The molar ratio of NO3 — reduced to Fe(II) oxidized in cultures containing chemi cally precipitated FeCO3, and one of the microbially reduced sub soils approximated the theoretical stoichiometry of 0.2:1. However, molar ratios obtained for oxidation of microbially reduced goe thite, the other subsoil, and the HFO reduction end products did not agree with this theoretical value. These discrepancies may be related to heterotrophic NO3 — reduction coupled to oxidation of dead Fe(III)-reducing bacterial biomass. Our findings demonstrate that microbally catalyzed NO3 —-dependent Fe(II) oxidation has the potential to significantly accelerate the oxidation of solid-phase Fe(II) compounds by oxidized N species. This process could have an important influence on the migration of contaminant metals and radionuclides in subsurface environments.Item Book Review of Strictly for the Chickens by Frances Hamerstrom(1981-03-01) Sandy, John H.