DOCSLIB.ORG

Mapping Three Classical Isozyme Loci in Tetrahymena: Meiotic Linkage of Esta to the Chxa Linkage Group

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mapping Three Classical Isozyme Loci in Tetrahymena: Meiotic Linkage of Esta to the Chxa Linkage Group Copyright 0 1996 by the Genetics Society of America Mapping Three Classical Isozyme Loci in Tetrahymena: Meiotic Linkage of EstA to the ChxA Linkage Group Sally Lyman Allen,* Dawn Zeilingert and Eduardo Oriast *Department of Biology, University of Michigan, Ann Arbor, Mirhigan 48109-1048 and ?Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California 9?106 Manuscript received July 1, 1996 Accepted for publication September 5, 1996 ABSTRACT We demonstrate a reliable method for mappingconventional loci and obtainingmeiotic linkage data for theciliated protozoan Tetrahymena thermophila.By coupling nullisomic deletion mapping with meiotic linkage mapping, loci known to be located on a particular chromosome or chromosome arm can be tested for recombination. This approach has been used to map three isozyme loci, EstA (Esterase A), EstB (Esterase B), and AcpA (Acid Phosphatase A), with respect to the ChxA locus (cycloheximide resistance) and 11 RAPDs (randomly amplified polymorphic DNAs). To assign isozyme loci to chromo- somes, clones of inbred strains C3 or C2 were crossed to inbred strain B nullisomics. EstA, EstB and AcpA were mapped to chromosomes IR, ?L and X,respectively. To test EstA and AcpA for linkage to known RAPD loci on theirrespective chromosomes, a panel of Round I1 (genomic exclusion) segregants from a B/C3 heterozygote was used. Using the "MAKER program, EstA was assigned to the ChxA linkage group on chromosome IR,and a detailed map was constructed that includes 10 RAF'Ds. AcpA (on X),while unlinked to all the RAPDs assigned to chromosome ? by nullisomic mapping, does show linkage to a RAF'D not yet assignable to chromosomes by nullisomic mapping. SOZYMES were among the first loci investigated ge- meiosis, with respect to ploidy or chromosomal mor- I netically in Tetrahymenathermophila (ALLEN 1960, phology (McCoy 1977). McCoy (1977) also detected 1961, 1965, 1968; ALLEN et al. 1963a,b; BORDENet ul. inbred strain differences with regard to a possible link- 1973). Seven loci were identified, each with two alleles: age of EstA and ChxA (cycloheximide resistance). He Esterase A (EstA),Esterase B (EstB), Acid Phosphatase observed linkage when inbred strains B and C2 were A (AcpA),Tyrosine Aminotransferase A ( TutA), NADP- crossed but not with crosses of inbred strains B and D. Isocitrate dehydrogenase A (IdhA),NADP-Malate dehy- In crosses involving the 40 loci known then, a total drogenase (MdhA), and Tetrazolium Oxidase A ( TzoA). ofsix potential linkage groups was found (DOERDER Genetically determined variation was observed among 1973; McCoy 1977). At least three of these linkage the inbred strains (Table 1) . groups have not stood the test of time: when mapping There arefive pairs of chromosomes in the micronu- with nullisomic strains, those supposedly linked mark- cleus of T. thermophila. With at least 40 loci identified ers mappedto different chromosomes (BRUNSand CAS by the early 1970's, linkage between some of these loci SIDY-HANLEY1993). might have been expected. No obvious meiotic linkages The nullisomic strains are a uniqueset of strains that were observed among six of the seven isozyme loci nor have proved very useful in genetic studies. They are with any of the other 30-35 loci, except for one case: characterized by having micronuclei (germ-line nuclei) EstA and mat (mating type locus) (ALLEN 1964; ALLEN that have lost both copies of one or more chromosomes and GIBSON1973; DOERDER1973). However, even this or chromosome arms (BRUNSet al. 1982, 1983). They linkage was disputed (MCCOY1977) since crosses involv- survive vegetativelybecause they are heterokaryons hav- ing different combinations of inbred strains did not ing normal somatic macronuclei. The macronucleus is behave uniformly with regard to recombination fre- the only nucleus active in gene expression and vegeta- quency between mat and EstA. It was suggested that tive reproduction, and therefore a population of cells crosses between certain inbred strains showing linkage can be propagated indefinitely while missing one or involved structural heterozygosity while crosses ofother more chromosomes in theirmicronuclei. During sexual strains not showing linkage for thesame markers lacked reproduction the micronuclei of conjugating cells un- this structural heterozygosity. However, no gross chro- dergo meiosis. When a nullisomic strain is crossed to a mosomal rearrangementscould be detectedduring normal strain, progeny with monosomic micronuclei are produced. Using this type of cross and a battery of nullisomic strains missing different chromosomes, Curresponding author Sally Lyman Allen, Department of Biology, University of Michigan, Ann Arbor, MI 48109-1048. genes can be assigned to their respective chromosomal E-mail: [email protected] location. To date over 100 different loci, >50 cloned Genetics 144: 1489-1496 (December, 1996) 1490 S. L. Allen, D. Zeilinger and E. Orias TABLE 1 Allelic constitution of inbred strains of T. thennophila Isozyme locus Inbred strain EstA EstB AcpA TatA 7ioAIdhA MdhA A B B A s s S F B B B B S S S F C3 C B A S S s F C2 C C B F F F F D B B A s S s S EstA, Esterase A, EstB, Esterase B; AcpA, Acid Phosphatase A TutA, Tyrosine Aminotransferase A IdhA, NADP-Isocitrate Dehydrogenase A; MdhA, NADP-Malate Dehydrogenase A TzoA, Tetrazolium Oxidase A. Alleles A, B and C refer to theparticular inbred strain in which the allele was first observed. Alleles Fand S refer to the relative migration after electrophoresis: S, slow; F, fast. sequences and 47 RAPDs (randomly amplified polymor- chromosome(s), is used to indicate the micronuclear compc- phic DNAs) have beenmapped to specific chromo- sition of each nullisomic line. Recently thawed stocks were used in all crosses. Clone C2-2671(C) was a gift from Dr. somes or chromosome arms using this technique THOMASNERAD at the American Type Culture Collection (BRUNSand CASSIDY-HANLEY 1993). (ATCC) and has the ATCC number 30389. The other clones Combining nullisomic deletion mappingwith recom- listed in Table 2 have been kept frozen in the ORIA~lab (as bination data allowed thedetermination of meiotic described by FLACKS1979) since shortly after their isolation linkage between the mat locus and the ribosomal RNA and initial characterization in the BRUNS'lab. Culture media: PP210 (nutritionallyrich, proteose-pep- gene (BLEYMANet al. 1992). Nullisomic mapping re- tone-based growth medium), 2% BP (bacterizedproteose vealed that both mat and the ribosomal RNA gene are peptone used for sequentially growing and starving cells) and located on the left arm of chromosome 2. Appropriate Dryl's buffered salts solution (used for starving cells) have crosses revealed meiotic linkage. The distance between previously been described (Omand BAUM1984). Toselect the two loci is estimated to be 34.3 cM (LYNCHet al. for progeny, cycloheximide (cycl) (Sigma) was added to PP210 medium at a final concentration of 15 pg/ml. 1995). Genetic procedures: Routine procedures for the mainte- This approach was used here to map three of the nance of strains and growth of cells have been previously isozyme loci, EstA,EstB and AcpA. They were first as- described (Owand BRUNS1975; ORLASand HAMII.TON signed to a particular chromosome, or chromosome 1979). For long-term maintenance, stocks obtained in this arm, by deletion mapping using crosses to 10 different work were frozen in liquid nitrogen byJuDrTH DODGEORUS. Unless otherwise specified all the work was done at 30". nullisomic strains. Linkage to known RAPD loci was Crosses: Cultures were sequentially grown, starved, mixed, then tested by appropriate crosses and, when found, refed, and cycloheximide-selected in Petri dishes (Oms and maps were constructed. EstA was assigned to the ChxA BRUNS1975). The progeny were then grown 5-6 days in linkage group previously located to 1R and built by Erlenmeyer flasks (100 ml in 250-ml flasks) before extracts mapping RAPD loci (BRICKNERet al. 1996).Here, a were prepared forisozyme phenotyping. Twoday-old cultures were used for preparing DNA when required for PCR and linkage group is defined as a group of loci such that RAPD testing. every member shows statistically significant linkage to Isozymephenotyping: Whole-cell extracts were made by at least one other member of the group. As more loci concentrating the cells by centrifugation and rinsing them are mapped, the ChxA linkage group should become with Dryl's salts solution. The cells were then frozen-thawed coextensive with the entire chromosome 1. No linkage six times in a dry-ice ethanol bath and stored at -20". Gelswere madein 12% hydrolyzed potatostarch was found between AcpA, located to chromosome X, (STARCHART,Smithville, TX: lots W560-2 and W562-2). The and six knownRAPD loci on chromosome 3, but it does starch buffer was 30 mM boric acid-Tris, pH 7.7 for the ester- show linkage to a RAPD not yet assigned to chromo- ases and pH 7.15 for the acid phosphatases. We had to lower somes by nullisomic mapping. EstB, known from this the pH to 7.15 to reveal the B allele of AcpA, which is very work to be located on chromosome X, has yet to be sensitive to pH compared to the A (C3) allele. The end-tray buffer was 0.3 M boric acid-Tris, pH 7.34 for both theesterases tested for linkage to these RAPD loci because inbred and acid phosphatases. Except for the pH of the buffers, strains B and C3 are not polymorphic for E&. details of the technique used for starch-gel electrophoresis followed published procedures (ALLEN 1960, 1964; ALI.EN et MATERIALSAND METHODS al. 1963a,b). Gels were run for 4 3/4 hours at 245V for the esterases and 225V for the acid phosphatases. Strains The isozyme genotypes for each of theinbred The gels were then sliced into pieces and marked; the strains of T. thennophila are listed in Table 1. pieces were placed in a tray (for the esterases) or test tubes The clones used (Table 2) were derived from various inbred (for theacid phosphatases), and anenzyme-specific substrate strains of T.
Load more

Recommended publications
  • Isozymes: Methods and Applications*
    Chapter 9 Isozymes: Methods and Applications* J. A. Micales and M. R. Bonde TABLE OF CONTENTS I. Introduction . II. Principles A. Multiple Alleles at Single Locus B. Single or Multiple Alleles at Multiple Loci C. Secondary Isozymes III. Methodology A. Sample selection and Preparation B. Electrophoretic Techniques C. Staining D. Genetic Interpretation IV. Applications of Isozyme Analysis A. Taxonomy B. Identification of Unknown Organisms C. Genetics D. Epidemiology E. Pathogenicity and Virulence V. Advantages and Disadvantages VI. Conclusions References Recommended Reading Keywords – Isozymes, plant pathology, genetics, taxonomy I. INTRODUCTION Isozyme analysis is a powerful biochemical technique with numerous applications in plant pathology. It has long been used by geneticists to study the population genetics of fish, mammals, insects, nematodes, and higher plants. Mycologists and plant pathologists more recently adopted the procedure, and it is now being used routinely to settle taxonomic disputes, identify “unknown” cultures, “fingerprint” patentable fungal lines and plant cultivars, analyze genetic variability, trace pathogen spread, follow the segregation of genetic loci, and determine ploidy levels of fungi and other plant pathogens. These topics have been recently reviewed.1,2 The large number of publications in this field each year indicates the widespread interest in isozyme analysis. In this paper, we discuss some major applications of isozyme analysis in basic and applied plant pathology. The technique is particularly useful with fungi; the greatest advances have been mostly with fungal pathogens. Isozyme banding patterns obtained from fungi are usually relatively uncomplicated and easy to interpret. Isozyme analysis can be readily performed in most laboratories with relatively little expense. With the development of computer programs that enable large numbers of comparisons at the gene level, much information cart be obtained about the population genetics and life cycle of the organism.
    [Show full text]
  • Human Glucokinase Gene
    Proc. Nati. Acad. Sci. USA Vol. 89, pp. 7698-7702, August 1992 Genetics Human glucokinase gene: Isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus (glucose/metabolism/phosphorylation/structure4unctlon/chromosome 7) M. STOFFEL*, PH. FROGUELt, J. TAKEDA*, H. ZOUALItt, N. VIONNET*, S. NISHI*§, I. T. WEBER¶, R. W. HARRISON¶, S. J. PILKISII, S. LESAGEtt, M. VAXILLAIREtt, G. VELHOtt, F. SUNtt, F. lIRSt, PH. PASSAt, D. COHENt, AND G. I. BELL*"** *Howard Hughes Medical Institute, and Departments of Biochemistry and Molecular Biology, and of Medicine, The University of Chicago, 5841 South Maryland Avenue, MC1028, Chicago, IL 60637; §Second Division of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-32, Japan; IDepartment of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107; IlDepartment of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794; tCentre d'Etude du Polymorphisme Humain, 27 rue Juliette Dodu, and Service d'Endocrinologie, H6pital Saint-Louis, 75010 Paris, France; and tG6ndthon, 1 rue de l'Internationale, 91000 Evry, France Communicated by Jean Dausset, May 28, 1992 ABSTRACT DNA polymorphisms in the glucokinase gene by maintaining a gradient for glucose transport into these cells have recently been shown to be tightly linked to early-onset thereby regulating hepatic glucose disposal. In (3 cells, glu- non-insulin-dependent diabetes mellitus in "80% of French cokinase is believed to be part of the glucose-sensing mech- families with this form of diabetes. We previously identified a anism and to be involved in the regulation ofinsulin secretion.
    [Show full text]
  • Practical Isozyme Genetics by N. Pasteur, G. Pasteur, F. Bonhomme
    Book reviews 244 throughout its development, based on a combination and sexual selection, which also reports many interes- of cell lineage studies and reconstructions of electron ting discoveries. The primary sex-determining signal is micrographs of serial sections, has made possible a the X/A ratio, which appears to set the states of a genetic analysis of the control of cell lineage by studies small number of sex-determining genes, which then of a large number of mutants which distort particular direct development along a particular sexual pathway. cell lineages. Many types of such mutants remain to be Of the seven major genes so far identified, the three tra identified by new types of selection procedure, but this genes appear to be required in the hermaphrodite but work is at the stage where molecular analysis of not in the male, the her-1 gene is required in the male lineage mutants is possible - obviously a very im- only, and the three fern genes are required for male portant area for experimental embryology. somatic development and also for spermatogenesis in Muscle anatomy, assembly and function have been both males and hermaphrodites. Dosage compen- studied mainly on the body-wall musculature re- sation is thought to be achieved by increasing sponsible for movement of the animal forwards and transcription from the single X chromosome of the backwards. The major muscle components are similar XO male or decreasing transcription from the two X to those of other animals: muscle extracts contain chromosomes of the XX hermaphrodite. Thus there myosin with both heavy- and light-chain subunits, are similarities between the sex determination and actin, paramyosin, tropomyosin and troponin-like dosage compensation systems of C.
    [Show full text]
  • Immunoelectron Microscopic Localization of 3Β-Hydroxysteroid Dehydrogenase and Type 5 17Β-Hydroxysteroid Dehydrogenase In
    11 Immunoelectron microscopic localization of 3-hydroxysteroid dehydrogenase and type 5 17-hydroxysteroid dehydrogenase in the human prostate and mammary gland G Pelletier, V Luu-The, M El-Alfy, S Li and F Labrie Oncology and Molecular Endocrinology Research Center, Laval University Hospital (CHUL), Quebec, G1V 4G2, Canada (Requests for offprints should be addressed to G Pelletier, Oncology and Molecular Endocrinology Research Center, Laval University Hospital (CHUL), 2705, Laurier Boulevard, Quebec, Quebec, G1V 4G2, Canada; Email: [email protected]) ABSTRACT The subcellular distribution of steroidogenic organelle could be observed, although some concen- enzymes has so far been studied mostly in classical tration of gold particles was occasionally found over endocrine glands and in the placenta. In the bundles of microfilaments. In mammary gland peripheral intracrine organs which synthesize sex sections immunolabelled for 3-HSD or type 5 steroids there is no indication about the organelles 17-HSD localization, labelling was observed in the which contain the enzymes involved in steroid cytoplasm of the secretory epithelial cells in both biosynthesis. We have thus investigated the sub- the acini and terminal ducts. Immunolabelling was cellular localization of two enzymes involved in also found in the endothelial cells as well as in the production of sex steroids, namely 3- fibroblasts in stroma and blood vessels. The gold hydroxysteroid dehydrogenase (3-HSD) and type particles were not detected over any organelles, 517-hydroxysteroid dehydrogenase (17-HSD). except with the occasional accumulation of gold Using specific antibodies to these enzymes, we particles over microfilaments. The present data on conducted immunoelectron microscopic studies in the localization of two steroidogenic enzymes two peripheral tissues, namely the human prostate leading to the synthesis of testosterone indicate that and mammary gland.
    [Show full text]
  • Isozyme Studies on the Origin and Evolution of Puccinia Graminis F
    Aust. J. Bioi. Sci., 1982,35, 231-8 Isozyme Studies on the Origin and Evolution of Puccinia graminis f. sp. tritici in Australia J. J. Burdon/ D. R. Marshall,A.c N. H. Lui~ and D. J. S. GoW» A Division of Plant Industry, CSIRO, P.O. Box 1600, Canberra City, A.C.T. 2601. B Plant Breeding Institute, University of Sydney, P.O. Box 180, Castle Hill, N.S.W. 2154. C Present address: I. A Watson Wheat Research Centre, University of Sydney, P.O. Box 219, Narrabri, N.S.W. 2390. Abstract Isozyme phenotypes for eight enzyme systems were used to assess the origins and evolution of P. graminis f. sp. tritici (the wheat stem rust pathogen) in Australia. The results obtained by this approach agreed with pathways postulated on the basis of virulence studies, confirming the sugges­ tion that most of the major changes in the wheat stem rust pathogen flora of Australia have resulted from overseas introductions. Moreover, they suggest that, although the more recent of these were from Africa, the first major change detected occurred as a result of an introduction from elsewhere. Introduction Australia offers unique opportunities for studies in the variation and evolution of the wheat stem rust pathogen, Pucdnia graminis Pers. f. sp. tritid Eriks. & Henn.. Barberry bushes (Berberis vulgaris L.), on which the sexual stage occurs, are extremely rare and the region is isolated from other wheat-growing regions of the world. On the other hand, the occasional arrival of new rust types provides variability additional to that occurring through mutation.
    [Show full text]
  • Neurosteroid Metabolism in the Human Brain
    European Journal of Endocrinology (2001) 145 669±679 ISSN 0804-4643 REVIEW Neurosteroid metabolism in the human brain Birgit Stoffel-Wagner Department of Clinical Biochemistry, University of Bonn, 53127 Bonn, Germany (Correspondence should be addressed to Birgit Stoffel-Wagner, Institut fuÈr Klinische Biochemie, Universitaet Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany; Email: [email protected]) Abstract This review summarizes the current knowledge of the biosynthesis of neurosteroids in the human brain, the enzymes mediating these reactions, their localization and the putative effects of neurosteroids. Molecular biological and biochemical studies have now ®rmly established the presence of the steroidogenic enzymes cytochrome P450 cholesterol side-chain cleavage (P450SCC), aromatase, 5a-reductase, 3a-hydroxysteroid dehydrogenase and 17b-hydroxysteroid dehydrogenase in human brain. The functions attributed to speci®c neurosteroids include modulation of g-aminobutyric acid A (GABAA), N-methyl-d-aspartate (NMDA), nicotinic, muscarinic, serotonin (5-HT3), kainate, glycine and sigma receptors, neuroprotection and induction of neurite outgrowth, dendritic spines and synaptogenesis. The ®rst clinical investigations in humans produced evidence for an involvement of neuroactive steroids in conditions such as fatigue during pregnancy, premenstrual syndrome, post partum depression, catamenial epilepsy, depressive disorders and dementia disorders. Better knowledge of the biochemical pathways of neurosteroidogenesis and
    [Show full text]
  • Inheritance of Isozyme Variation and Heterozygosity in Pinus Ponderosa
    University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 1980 Inheritance of isozyme variation and heterozygosity in Pinus ponderosa David M. O'Malley The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation O'Malley, David M., "Inheritance of isozyme variation and heterozygosity in Pinus ponderosa" (1980). Graduate Student Theses, Dissertations, & Professional Papers. 2706. https://scholarworks.umt.edu/etd/2706 This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. COPYRIGHT ACT OF 1976 THIS IS AN UNPUBLISHED MANUSCRIPT IN WHICH COPYRIGHT SUB­ SISTS. ANY FURTHER REPRINTING OF ITS CONTENTS MUST BE APPROVED BY THE AUTHOR. MANSFIELD LIBRARY UNIVERSITY OF MONTANA DATE: JAN S 1981 INHERITANCE OF ISOZYME VARIATION AND HETEROZYGOSITY IN PINUS PONDEROSA By David M. O'Malley B.S., University of Massachusetts, 1974 Presented in partial fulfillment of the requirements for the degree of Master of Arts UNIVERSITY OF MONTANA 1980 Approved by: lUoO. Chairman, Board of Examiners De4n, Graduate Scho" A- X<\- Date UMI Number: EP33984 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent on the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted.
    [Show full text]
  • Brief Communication Population Genetic Studies on Aldehyde Dehydrogenase Isozyme DORIS MEIER-TACKMANN,I Du RUOFU,2 ULRICH BIENZL
    Am J Hum Genet 35:769-772, 1983 Brief Communication Population Genetic Studies on Aldehyde Dehydrogenase Isozyme Deficiency and Alcohol Sensitivity H. WERNER GOEDDE,1 DHARAM P. AGARWAL,' SHOJI HARADA, DORIS MEIER-TACKMANN,I Du RUOFU,2 ULRICH BIENZLE , AXEL KROEGER,4 AND LAILA HUSSEIN5 SUMMARY Population genetic studies on aldehyde dehydrogenase polymorphism using hair-root samples were pertormed on Europeans, Liberians, Su- danese, Egyptians, Kenyans, Vietnamese, Japanese, Indonesians, Chinese, Thais, and South American Indians. A possible correlation between ALDH I deficiency and sensitivity to alcohol in Oriental pop- ulations is discussed. INTRODUCTION The NAD-dependent aldehyde dehydrogenase (ALDH, E.C. 1.2.1.3) catalyzes the oxidation of various aldehydes in humans. Greenfield and Pietruszko [1] found two isozymes of ALDH, a faster-migrating isozyme with a low Km for acetaldehyde and a slower-migrating one with a high Km for acetaldehyde in human liver. The existence of two additional isozymes of ALDH with a high Km for acetal- dehyde in various organs and tissues was reported by us [2, 3]. The four isozymes are termed ALDH I, II, III, and IV according to their decreasing electrophoretic migration and increasing isoelectric point. Received September 8, 1982; revised November 1, 1982. This work was supported by the Stiftung Volkswagenwerk, Hannover, Deutsche Forschungsge- meinschaft, Bonn, West Germany, and the Fritz Thyssen-Stiftung, Koin, West Germany. 1 Institut fur Humangenetik der Universitat Hamburg, West Germany. 2 Institute of Genetics, Academia Sinica, The People's Republic of China. 3Landesimpfanstalt, West Berlin, West Germany. 4 Institut fur Tropenhygiene und offentliches Gesundheitswesen am Sudasien-Institut, Universitat Heidelberg, West Germany.
    [Show full text]
  • Glucokinase Regulatory Protein As the Allosteric Switch for Glucokinase
    Molecular basis for the role of glucokinase regulatory protein as the allosteric switch for glucokinase Jung Min Choia,1, Moon-Hyeong Seoa,1, Hyun-Ho Kyeonga, Eunkyung Kima,2, and Hak-Sung Kima,b,3 aDepartment of Biological Sciences and bGraduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea Edited* by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, and approved May 6, 2013 (received for review January 9, 2013) Glucokinase (GK) is a monomeric allosteric enzyme and plays between GK and GKRP and a modulation through the effectors a pivotal role in blood glucose homeostasis. GK is regulated by GK (17, 18). Despite the intensive studies on GK and GKRP, the regulatory protein (GKRP), and indirectly by allosteric effectors of molecular basis for the allosteric regulation mechanism of GK by GKRP. Despite the critical roles of GK and GKRP, the molecular basis GKRP remains poorly understood because of the lack of struc- for the allosteric regulation mechanism of GK by GKRP remains tural information of the GK/GKRP complex. unclear. We determined the crystal structure of Xenopus GK and Here, to demonstrate the molecular mechanism for the allo- GKRP complex in the presence of fructose-6-phosphate at 2.9 Å. steric regulation of GK by GKRP and effectors, we determined GKRP binds to a super-open conformation of GK mainly through the crystal structure of a Xenopus laevis GK and GKRP complex hydrophobic interaction, inhibiting the GK activity by locking a small in the presence of F6P. Structural analysis of the complex and domain of GK.
    [Show full text]
  • X-ADH Is the Sole Alcohol Dehydrogenase Isozyme of Mammalian Brains: Implications and Inferences (Class III Alcohol Dehydrogenase/Tissue-Specific Enzyme) THOMAS B
    Proc. Natl. Acad. Sci. USA Vol. 82, pp. 8369-8373, December 1985 Biochemistry X-ADH is the sole alcohol dehydrogenase isozyme of mammalian brains: Implications and inferences (class III alcohol dehydrogenase/tissue-specific enzyme) THOMAS B. BEISSWENGER, BARTON HOLMQUIST, AND BERT L. VALLEE* Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115 Contributed by Bert L. Vallee, August 14, 1985 ABSTRACT Class m (X) is the only alcohol dehydrog- occurrence and function of ADH in that tissue has always enase (ADH) in human, equine, bovine, simian, canine, and been of particular interest, but efforts to uncover facts rodent brain and is the first to be identified, purified, and relevant to ethanol have been singularly disappointing. Most characterized from the brain of humans or other vertebrates. of those studies antedate the awareness of the remarkable Like the corresponding isozymes from human placenta and number and diversity of structure and specificity of ADH liver, the X-ADH isozymes purified from mammalian brain are isozymes (4), first noted much earlier (5). neither inhibited by nor do they bind to immobilized pyrazole, Experimental work failed to reveal any significant ADH and they oxidize ethanol only very poorly (Km > 2.5 M). activity in brain (6). The level ofethanol oxidation in rat brain Indeed, it would be incorrect to classify them as "ethanol is so vanishingly low that only assays of exceptionally high dehydrogenases." They contain 4 g.atom of zinc/mol, bind 2 sensitivity could detect it (7). A number of contemporaneous moles of NAD, and readily oxidize long-chain aliphatic and studies employing [14C]ethanol concluded that the perfused aromatic primary alcohols.
    [Show full text]
  • Low-Temperature Carbon Utilization Is Regulated by Novel Gene Activity in the Heart of a Hibernating Mammal
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 8392–8397, July 1998 Physiology Low-temperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal MATTHEW T. ANDREWS*, TERESA L. SQUIRE,CHRISTOPHER M. BOWEN, AND MARTHA B. ROLLINS Department of Genetics, North Carolina State University, Raleigh, NC 27695-7614 Communicated by Donald D. Brown, Carnegie Institution of Washington, Baltimore, MD, May 6, 1998 (received for review March 2, 1998) ABSTRACT Hibernation is a physiological adaptation 2). We have begun to address the genetic control of this process characterized by dramatic decreases in heart rate, body by using a differential gene expression screen (4) to identify temperature, and metabolism, resulting in long-term dor- genes that are responsible for the physiological characteristics mancy. Hibernating mammals survive for periods up to 6 mo of hibernation in the heart of the thirteen-lined ground squirrel in the absence of food by minimizing carbohydrate catabolism (Spermophilus tridecemlineatus). Our study focused on the and using triglyceride stores as their primary source of fuel. heart because it is a contractile organ that must continue to The cellular and molecular mechanisms underlying the work despite suboptimal conditions of temperature and oxy- changes from a state of activity to the hibernating state are gen supply. In this paper, we report on the identification and poorly understood; however, the selective expression of genes differential expression of two genes associated with metabolic offers one level of control. To address this problem, we used rate depression and low-temperature carbon utilization during a differential gene expression screen to identify genes that are hibernation.
    [Show full text]
  • Model-Driven Discovery of Underground Metabolic Functions in Escherichia Coli
    Model-driven discovery of underground metabolic functions in Escherichia coli Gabriela I. Guzmána, José Utrillaa, Sergey Nurkb, Elizabeth Brunkc, Jonathan M. Monkd, Ali Ebrahima, Bernhard O. Palssona,e,f, and Adam M. Feista,e,1 Departments of aBioengineering, dNanoEngineering, and fPediatrics, University of California, San Diego, La Jolla, CA 92093; bAlgorithmic Biology Laboratory, St. Petersburg Academic University, Russian Academy of Sciences, St. Petersburg, Russia; cJoint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA 94608; and eNovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark Edited by Marc W. Kirschner, Harvard Medical School, Boston, MA, and approved December 5, 2014 (received for review August 5, 2014) Enzyme promiscuity toward substrates has been discussed in of other proteins based on sequence homology or enzyme familial evolutionary terms as providing the flexibility to adapt to novel relationships. In the present study, this task is approached from environments. In the present work, we describe an approach to- a different perspective by taking advantage of in vivo experimental ward exploring such enzyme promiscuity in the space of a meta- techniques to gain insight into activities that are more physiologi- bolic network. This approach leverages genome-scale models, cally relevant. In this way, as has been demonstrated in other in which have been widely used for predicting growth phenotypes vivo studies, many of the challenges associated with removing in various environments or following a genetic perturbation; how- enzymes from their native environment are circumvented (8). ever, these predictions occasionally fail. Failed predictions of gene Specifically, the present study focuses on examination of the reg- essentiality offer an opportunity for targeting biological discovery, ulatory and evolutionary capacity of a cell in vivo.
    [Show full text]