GENETIC ANALYSIS OF BACTERIAL CHROMOSOME STRUCTURE
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM027068-06 from National Institute Of General Medical Sciences IRG: MG
Abstract: We will investigate the functional significance of gene order in the chromosome. A variety of chromosomal rearrangements will be constructed and analyzed for effects on growth and viability. Studies immediately planned involve large inversions which will be selected as mutants, mapped and analyzed. Methods have also been devised for deliberate construction of inversions with predetermined endpoints, this should permit identification of lethal inversions. We ll study the effect of chromosome position on complementation responses to determine whether regions of the chromosome determine or participate in intracellular compartments. We ll continue work on regulation of Tn5 transposition and on a genus-specific transposable sequence (IS200) we discovered in Salmonella.
Keywords: GENETICS, CHROMOSOMES, CHROMOSOME REPLICATION, GENETICS, MICROBIAL, BACTERIAL, GENETICS, MUTATION, CHROMOSOME MUTATION, CHROMOSOME INVERSION, MICROBIAL GENETICS REVIEW GROUP, genetic manipulation, GENETICS, BIOCHEMICAL GENETICS, MOLECULAR CLONING, GENETICS, CHROMOSOME COMPLEMENT, GENOME, GENETICS, GENES, GENE EXPRESSION, GENETICS, GENES, GENE EXPRESSION, LETHALS, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENETICS, MUTATION, NUCLEIC ACIDS STRUCTURE, NUCLEOSIDES (TIDES) SEQUENCE, genetic mapping, BACTERIA, ENTEROBACTERIACEAE, SALMONELLA TYPHIMURIUM, CELL DIVISION, MITOSIS
Project start date: 1979-12-01
Project end date: 1985-11-30
Sponsored Links Excellgen http://Excellgen.com
GENETIC ANALYSIS OF BACTERIAL CHROMOSOME STRUCTURE
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM027068-05 from National Institute Of General Medical Sciences IRG: MG
Abstract: We will investigate the functional significance of gene order in the chromosome. A variety of chromosomal rearrangements will be constructed and analyzed for effects on growth and viability. Studies immediately planned involve large inversions which will be selected as mutants, mapped and analyzed. Methods have also been devised for deliberate construction of inversions with predetermined endpoints, this should permit identification of lethal inversions. We ll study the effect of chromosome position on complementation responses to determine whether regions of the chromosome determine or participate in intracellular compartments. We ll continue work on regulation of Tn5 transposition and on a genus-specific transposable sequence (IS200) we discovered in Salmonella.
Keywords: GENETICS, CHROMOSOMES, CHROMOSOME REPLICATION, GENETICS, MICROBIAL, BACTERIAL, GENETICS, MUTATION, CHROMOSOME MUTATION, CHROMOSOME INVERSION, MICROBIAL GENETICS REVIEW GROUP, genetic manipulation, GENETICS, BIOCHEMICAL GENETICS, MOLECULAR CLONING, GENETICS, CHROMOSOME COMPLEMENT, GENOME, GENETICS, GENES, GENE EXPRESSION, GENETICS, GENES, GENE EXPRESSION, LETHALS, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENETICS, MUTATION, NUCLEIC ACIDS STRUCTURE, NUCLEOSIDES (TIDES) SEQUENCE, genetic mapping, BACTERIA, ENTEROBACTERIACEAE, SALMONELLA TYPHIMURIUM, CELL DIVISION, MITOSIS
Project start date: 1979-12-01
Project end date: 1985-11-30
5R01GM027068-19 (1998): $280174
5R01GM027068-18 (1997): $270310
GENETIC ANALYSIS OF BACTERIAL CHROMSOME STRUCTURE
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM027068-08 from National Institute Of General Medical Sciences IRG: MBC
Abstract: The genetic maps of the enteric bacteria Salmonella and E. coli have remained remarkably similar despite the fact that these organisms have been separated for substantial evolutionary time and their sequence homology has diverged. This suggests that selective forces have prevented survival of cells that have undergone chromosome rearrangement. Despite evolutionary stability of the genome, many events are known that cause rearrangement of the chromosome. These include transposable elements and the events they induce, and spontaneous duplication and inversion events that seem to occur by recombination between repeated sequences that are separated in the chromosome. We will investigate these events by studying IS sequences and drug resistance transposons, and by devising genetic means of selecting and studying rearrangements. We ll study the mechanisms of rearrangements and the physiological consequences of these rearrangements. In particular, we will pursue the idea that gene duplication is an advantageous event for bacteria. We suspect that the structure of the bacterial chromosome includes sequences located appropriately for generation of advantageous duplications.
Keywords: GENETICS, BIOCHEMICAL GENETICS, GENETICS, CHROMOSOME COMPLEMENT, GENOME, GENETICS, MICROBIAL, BACTERIAL, GENETICS, MUTATION, CHROMOSOME MUTATION, CHROMOSOME INVERSION, GENETICS, MUTATION, CHROMOSOME MUTATION, GENE DUPLICATION, GENETICS, RECOMBINATION, MICROBIAL GENETICS REVIEW GROUP, NUCLEIC ACIDS STRUCTURE, NUCLEOSIDES (TIDES) SEQUENCE, NUCLEIC ACIDS, DNA INSERTION ELEMENTS, genetic manipulation, genetic mapping, GENETICS, CHROMOSOMES, CHROMOSOME REPLICATION, GENETICS, GENES, GENE AMPLIFICATION, GENETICS, GENES, GENE EXPRESSION, GENETICS, GENES, GENE EXPRESSION, LETHALS, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENETICS, MUTATION, BACTERIA, ENTEROBACTERIACEAE, SALMONELLA TYPHIMURIUM, CELL DIVISION, MITOSIS, GENETICS, BIOCHEMICAL GENETICS, MOLECULAR CLONING
Project start date: 1979-12-01
Project end date: 1990-11-30
GENETIC ANALYSIS OF BACTERIAL CHROMOSOME STRUCTURE
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM027068-15 from National Institute Of General Medical Sciences IRG: MBC
Abstract: The general goal of this project is to understand the molecular mechanisms and selective forces that determine the arrangement of genes in bacterial chromosomes. work includes characterization Of chromosomal rearrangements, their genetic behavior and consequences for bacterial growth. We are also studying the mechanisms that contribute to chromosome rearrangements including both legitimate recombination events and transposon mediated events. We hope to understand what selective or mechanistic factors prevent the recovery of inversion mutations for some (but not all) segments of the bacterial chromosome. We will continue to pursue evidence that chromosomal duplications are a valuable means by which bacteria adapt to environmental stress. This will include work to elucidate why particular duplications convey a greatly enhanced ability of cells to grow on limiting carbon sources. We will continue to characterize two repeated sequences found in Salmonella. One is the Salmonella-specific insertion sequence, IS200; we hope to learn why this element is limited to Salmonella and is not found in the related enteric bacteria which exchange plasmids and phages with Salmonella. The second element is the tiny REP sequence which is shared by both Salmonella and E. coli. Distribution of REP suggests that it is a degenerate tranposable element that may have acquired selective value to bacteria. We are most interested in the possibility that this element serves to mediate selectively valuable chromosomal rearrangements. Finally, we will initiate work on the mechanism of transductional recombination and the contribution of phage and host to this process. This work will be done in conjunction with other work on the recombination mechanisms and pathways involved in chromosomal rearrangement.
Keywords: Salmonella typhimurium, bacterial genetics, chromosome, gene rearrangement, molecular genetics, nucleic acid sequence, protein structure function, DNA replication, chromosome inversion, gene duplication, gene expression, genetic manipulation, genetic mapping, genetic model, genetic recombination, genetic translation, genome, microorganism genetics, model design /development, mutant, transposon /insertion element
Project start date: 1979-12-01
Project end date: 1994-11-30
5R01GM027068-15 (1994): $193543
5R01GM027068-14 (1993): $182929
5R01GM027068-13 (1992): $175316
Sponsored Links Excellgen http://Excellgen.com
5R01GM027068-23 (2002): $378990
5R01GM027068-22 (2001): $367929
5R01GM027068-21 (2000): $357408
John R Roth
University Of California Davis
Project start date: 1979-12-01
Project end date: 2015-03-31
GENETIC ANALYSIS OF BACTERIAL CHROMOSOME STRUCTURE
John R Roth, Distinguished Professor
University Of California Davis, Office Of Research - Sponsored Programs, Davis, Ca 95618
Grant 5R01GM027068-31 from National Institute Of General Medical Sciences
Keywords: Aging; Assay; Bacterial Chromosomes; Bioassay; Biologic Assays; Biological Assay; Cancers; Cells; Cellular Expansion; Cellular Growth; Chemotherapy Protocol; Chemotherapy Regimen; Chemotherapy, Cancer, General; Chemotherapy-Oncologic Procedure; Chromosomal Organization; Chromosomal Structure; Chromosome Organization; Chromosome Structures; Chromosome inversion; Chromosomes; Chromosomes, Bacterial; Clonal Expansion; Combination Chemotherapy Regimen; DNA; DNA Recombination; DNA Sequence Rearrangement; DNA recombination (naturally occurring); Defect; Deletion Mutation; Deoxyribonucleic Acid; Detection; Development; Disease; Disorder; Duplicate Genes; E coli; Enzymes; Escherichia coli; Event; Frequencies (time pattern); Frequency; Gene Amplification; Gene Duplication; Generalized Growth; Genes; Genes, Duplicate; Genetic; Genetic Alteration; Genetic Change; Genetic Processes; Genetic Recombination; Genetic analyses; Genetic defect; Genetics-Mutagenesis; Genomics; Growth; Home; Home environment; Inversion, Chromosomal; Inversion, Sequence; Investigation; Learning; Malignant Neoplasms; Malignant Tumor; Methods; Modeling; Molecular Biology, Mutagenesis; Mutagenesis; Mutation; Population; Population Biology; Position; Positioning Attribute; Process; Production; Quimioterapia; Qui Compound; Quis; Racial Segregation; Rearrangement; Recombination; Recombination, Genetic; Resistance; Salmonella; Senescence; Stress; Structure; System; System, LOINC Axis 4; Testing; Thinking; Thinking, function; Tissue Growth; Yang; base; cancer chemotherapy; cell growth; disease/disorder; experiment; experimental research; experimental study; genetic analysis; genetic evolution; genome mutation; improved; insight; malignancy; mutant; natural gene amplification; neoplasm/cancer; null mutation; ontogeny; pathogen; research study; resistant; segregation; senescent; theories
Project start date: 1979-12-01
Project end date: 2010-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
5R01GM027068-31 (2010): $484858
5R01GM027068-27 (2006): $455592
5R01GM027068-26 (2005): $456010
Sponsored Links Excellgen http://Excellgen.com
5R01GM027068-25 (2004): $445771
5R01GM027068-17 (1996): $260826
Grants awarded to John R Roth
TRANSFER RNA PRODUCTION AND ROLE IN GENE REGULATION
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM023408-10 from National Institute Of General Medical Sciences IRG: MG
Abstract: We propose a genetic analysis of tRNA structure and function including work on synthesis and function of modified bases, and the mechanims of tRNA s role in protein synthesis and in gene regulation. Three main lines of work will be undertaken 1. Analysis of suppressor mutations which affect tRNA sequence and base modification. Projects will include analysis of codon context effects on tRNA efficiency, new UGA suppressors and genetic analysis of the biosynthetic pathway for a modified base, the methyl ester of uridine-5-oxyacetic acid. 2. Analysis of the regulatory mechanism for the histidine operon. We will determine the DNA sequence changes for more mutants in the control (his0) region in order to refine the current model for tRNA s involvement in this mechanism. This will involve study of the derepressive effect of rif(r) and str(r) metations on operon expression. We also hope to learn how the high basal levels of operon expression are set. We will try to determine the mechansim where by the operon is subject to metabolic (ppGpp) control. The possibility of regulatory interactions between the control mechanisms for the histidine and purine pathways will be investigated. 3. Analysis of the regulatory mechanism for proline degradation. We will determine whether or not tRNA(PRO) is involved in regulation of this system and how the degradative enzyme (a bifunctional oxidase-dehydrogenase) manages to act in a regulatory capacity and also be a bifunctional, membrane-associated enzyme.
Keywords: GENETICS, GENES, GENETICS, MICROBIAL, BACTERIAL, MICROBIAL GENETICS REVIEW GROUP, NUCLEIC ACIDS, TRNA, AMINO ACIDS BIOSYNTHESIS, CYCLIC AMINO ACIDS, HISTIDINE, CYCLIC AMINO ACIDS, PROLINE, DRUGS RESISTANCE, MICROBIAL, GENETICS, CHROMOSOMES, GENETICS, GENES, OPERON, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENETICS, MUTATION, MUTANTS, MICROORGANISMS METABOLISM, PROTEINS, PROTEINS BIOSYNTHESIS, genetic manipulation, BACTERIA, ENTEROBACTERIACEAE, ESCHERICHIA COLI, BACTERIA, ENTEROBACTERIACEAE, SALMONELLA, BIOMEDICAL SYSTEMS AUTOMATED, COMPUTER PROCESSING OF LABORATORY DATA (GENERAL)
Project start date: 1977-01-01
Project end date: 1986-12-31
BIOSYNTHESIS OF VITAMIN B12 AND ANAEROBIC METABOLISM
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM034804-05 from National Institute Of General Medical Sciences IRG: MBC
Abstract: Vitamin B12 is an essential nutrient for humans, that is only synthesized by microbes. Little is known about its biosynthesis or its physiological importance to the microbes that produce it. The bacterium Salmonella typhimurium is capable of a complete synthesis of B12, but does so only under anaerobic conditions. Since sophisticated genetics can be done in this bacterium, we propose a genetic approach to understanding the biosynthesis of vitamin B12 in this organism. We will identify the genes involved (probably an excess of thirty) and will pursue how this pathway is regulated with special emphasis on how 02 is involved. The genetic and biochemical study of enteric bacteria has been done largely under aerobic conditions. Relatively little is known about the details of the anaerobic life-style of these organisms even though virtually all their interactions with human hosts (colonization and pathogenesis) proceed in the absence of oxygen. We suspect that production of B12 anaerobically indicates the existence of a substantial body of metabolic activity that occurs only anaerobically and is totally unknown. We propose to approach these aspects of metabolism by studying the role of B12 in the anaerobic life of Salmonella.
Keywords: BACTERIA, ENTEROBACTERIACEAE, SALMONELLA TYPHIMURIUM, GENETICS, BIOCHEMICAL GENETICS, GENETIC CODING, GENETICS, MICROBIAL, BACTERIAL, MICROORGANISMS METABOLISM, RESPIRATION INTERNAL, ANAEROBIOSIS, VITAMINS BIOSYNTHESIS, VITAMINS, VITAMIN B12, metabolism, AMINO ALCOHOLS, ETHANOLAMINES, AMMONIA-LYASES, BACTERIA, ENTERIC BACTERIA, GENETICS, BIOCHEMICAL GENETICS, MOLECULAR CLONING, GENETICS, GENES, GENE INTERACTION, COMPLEMENTATION, GENETICS, GENETIC REGULATION, TRANSCRIPTION, GENETICS, MUTATION, MUTANTS, MICROORGANISMS, ANAEROBES, NUCLEOTIDES, RIBONUCLEOTIDES, OXIDOREDUCTASES, PORPHYRINS BIOSYNTHESIS, RESPIRATION INTERNAL, AEROBIOSIS, RESPIRATORY GASES, OXYGEN, VITAMIN B12 COENZYMES, genetic manipulation, genetic mapping, heme
Project start date: 1985-07-01
Project end date: 1990-06-30
5R01GM034804-14 (1998): $311719
5R01GM034804-13 (1997): $300329
Sponsored Links Excellgen http://Excellgen.com
Genetic Analysis Of Bacterial Chromosome Structure
John R Roth, Professor
University Of California Davis Office Of Research - Sponsored Programs Davis, Ca 95618
Grant 2R01GM027068-24 from National Institute Of General Medical Sciences IRG: MBC
Abstract: This project proposes multiple approaches to the general question of the role of recombination in repair, mutagenesis and genetic adaptation of bacteria. Recent results for provided evidence that the phenomenon know as "adaptive mutation" (Cairns) can be explained purely by standard genetic events that occur during selective growth of cells carrying an amplification of the mutational target. The Amplification model proposes a sequence of genetic events occurring within the developing revertant clone - amplification - mutation - segregation - haploid overgrowth. It argues against the widely circulated ideas of stress-regulated mutation (directed or general), stationary phase mutagenesis or mutagenic recombination. We will continue to characterize this system and test detailed hypotheses to explain the remaining questions, which include the following. What are the exact mechanisms by which amplification induces SOS and activates DinB-dependent rnutagenesis? Why does selection-stimulated reversion require that the gene under selection (lac) be located on an F plasmid? Why does non-essential, reversion-associated general mutagenesis require that lac be located on the specific plasmid (F 128)? Why is growth of the amplification clone inhibited after appearance of the lac reversion? Why do some amplification clones reach maturity (full-sized) revertants without ever achieving lac reversion? We will experimentally measure rates for each step proposed by the model try to mathematically describe the process by which the Cairns system completes a long series of genetic steps within on week of growth under selection. The model promises to shed light on the process of gene evolution, adaptation of pathogens to their hosts and multi-mutational origins of cancer. While mechanisms of replication, recombination, and repair are understood in great detail, it is less clear how these systems interact and how they achieve the amazingly low rate of mutation seen in bacteria. We will continue to pursue a set of assays that measure aspects of recombination as it occurs within the chromosome of growing bacteria. These assays do not involve crosses and require endogenous sources of strand ends to initiate recombinational repair. Thus they can be used to identify endogenous metabolic sources of DNA damage and the role of long-range replication in completion of a recombination event.
Keywords: DNA repair, bacterial genetics, chromosome, gene mutation, genetic recombination, DNA damage, bacterial protein, directed evolution, molecular genetics, Escherichia coli, Salmonella
Project start date: 1979-12-01
Project end date: 2006-11-30
2R01GM027068-24 (2003): $449206
AN ANALYSIS OF GENES INVOLVED IN TRNA PRODUCTION
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM023408-03 from National Institute Of General Medical Sciences IRG: MBC
Abstract: This project involves several lines of work (1) The role of anticodon loop pseudouridine in tRNA function. Mutants unable to form this modified base (hisT) are defective in regulation of several operons and show greatly diminished activity for several informational suppressors. The latter fact is being exploited to ascertain the biological importance of this modification. (2) Regulation of the histidine operon. This mechanism responds to the level of his-tRNA in the cell. This level is apparently perceived by the translation of a small peptide (16 amino acids) including seven adjacent his codons. We are pursuing a genetic analysis of the control region which includes the gene for this small peptide. (3) Work is continuing on regulation of proline degradative genes.
Keywords: GENETICS, GENES, MICROBIAL CHEMISTRY STUDY SECTION, NUCLEIC ACIDS, TRNA, AMINO ACIDS BIOSYNTHESIS, CYCLIC AMINO ACIDS, HISTIDINE, CYCLIC AMINO ACIDS, PROLINE, DRUGS RESISTANCE, MICROBIAL, GENETICS, CHROMOSOMES, GENETICS, GENES, OPERON, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENETICS, MICROBIAL, GENETICS, MUTATION, MUTANTS, PROTEINS, genetic manipulation
Project start date: 1977-01-01
Project end date: 1981-12-31
POPULATION BIOLOGY AND EVOLUTION OF MICROORGANISMS
John R Roth, Professor
Gordon Research Conferences
west Kingston, Ri 02892
Grant 1R13AI034518-01 from National Institute Of Allergy And Infectious Diseases IRG: MID
Abstract: This application seeks partial support for a Gordon Research Conference on the Population Biology and Evolution of Microorganisms. This will be the fifth such conference, which has been held biennially since its first meeting in 1985. The conference brings together scientists working in molecular genetics, population biology, microbiology and infectious disease, all of whose disciplines interact to define the main topic of this conference. There are virtually no conferences that assemble this blend of expertise. A detailed of the aims of the conference, its significance and the plans for the next meeting are attached
Project start date: 1993-06-25
Project end date: 1994-06-24
1R13AI034518-01 (1993): $1000
Genetic Analysis Of Bacterial Chromosome Structure
John R Roth, Professor
University Of California Davis Office Of Research - Sponsored Programs Davis, Ca 95618
Grant 2R01GM027068-28 from National Institute Of General Medical Sciences IRG: PCMB
Abstract: This application proposes an investigation of the processes by which genes duplicate and amplify further during growth under selection. We ve applied this process to explain the phenomenon of "adaptive mutation" and have shown that selective stress is not actually mutagenic (as claimed) but appears so because selection favors growth of cells with additional copies of the rate-limiting gene formed by duplication and amplification. Selection enhances the frequency of mutants by increasing the number of mutation targets and can do so with no change in the mutation rate. Our model is general for all genetic systems and leads to an extremely powerful process for genetic adaptation-nested serial clonal expansions. This process is highly relevant to the origins of cancer and adaptation of pathogens to hosts-2 situations in which populations of single cells adapt genetically during the course of a disease. Gene amplifications are proving important to development of cancer and resistance to cancer chemotherapies. We suggest that the approach taken here is important because it investigates the interface between recombination mechanism and population biology. We will characterize 2 bacterial systems (in addition to Cairns ) that have been used (incorrectly, we believe) to support the idea of stress-induced mutagenesis. Our hope is to reveal new mechanisms of genetic adaptation and to resolve the controversy surrounding "adaptive mutation". Thus far, we ve learned that amplifications are remodeled during growth under selection to shorten their repeated unit and increase their copy number. We ve discovered a new form of amplification (inversion-duplication) and will test a model for how these rearrangments arise and remodel under selection. A new recombination-independent assay shows that RecA is not essential for duplication formation; this assay should help characterize the functional requirements of duplication. We ve found that the reversibiltiy of duplications causes their frequency in a population to approach a steady state level. The segregation of duplications provides an assay for internal recombination that does supply DNA ends (as does sexual exchange) but relies on spontaneous production of initiating structures. Therefore, this assay allows study of spontaneous events that initiate recombination. We ve developed an assay for duplication segregation rate that avoids growth rate problems and shows that palindromic sequence stimulates duplication formation.
Keywords: chromosome, gene mutation, genetics, model, DNA, Salmonella, aging, biology, cell, copying, enzyme, evolution, gene, gene duplication, insight, lead, learning, mutant, neoplasm /cancer, neoplasm /cancer chemotherapy, quinone, social integration, stress, thinking
Project start date: 1979-12-01
Project end date: 2010-12-31
2R01GM027068-28 (2007): $471614
3R01GM027068-30S1 (2009): $284941
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM023408-22 from National Institute Of General Medical Sciences IRG: MBC
Abstract: The project proposes an investigation of the control of synthesis, assimilation and recycling of the central metabolic cofactors, NAD and NADP. Emphasis is on pursuing preliminary evidence that cells protect themselves from oxidative stress by reducing their NADH pool. This minimizes production of the damaging -OH radical from H2O2 via the nonenzymatic Fenton reaction, which requires Fe+2 and a reducing agent. NADH, but not NADPH, can serve as the required reducing agent. By reducing NAD levels temporarily, cells can freeze metabolism, allowing time for DNA repair and destruction of H2O2. The model proposes that NAD levels are reduced by conversion of NAD to NADP, followed by destruction of excess NADP. The NMN produced may be excreted to the periplasm for later uptake when growth resumes. The second product of NAD(P) breakdown, 2 5 ADP, may play a regulatory role in control of the response to oxidative stress. Control of internal iron levels may also be involved in the stress responses; one of the NAD kinases (NAD-more than NADP) has already been shown to be activated by Fe. Other enzymes that initiate the NAD cycle may also be controlled by either O2 or Fe. It is not clear how cells control the relative size of their NAD and NADP pools. We will sequence the two NAD kinase genes, make lac fusions and expression plasmids. Using the fusions, the transcriptional regulation of these genes will be pursued. The purified enzymes will be analysed for modulation of their activity in response to oxidative stress, iron and pyridine precursor limitation. The essential NMN deamidase maintains a low internal level of the toxic intermediate, NMN; we will study the control of this step. Excess NMN may be excreted by the pnuC transporter which appears to contribute to both import and excretion of NMN. The energetics of this transporter will be investigated to determine whether NAD serves to dictate the direction of flow. We will continue the search for transporters of Nm or Na have been described. It seems likely that there must be some control on assimilation of these compound. Several candidates for Nm and Na transporters are in hand which will be pursued to determine how many transporters exist and whether any are controlled. The nadD gene will be tested as a likely candidate for a step that might be regulated to control assimilation and recycling. This compound is a synthetic intermediate, the entry point of assimilated Nm and Na, the entry point of recycled NMN and the source of ribose for synthesis of cobalamin. A missense suppressor will be investigated that may act by unbalancing rNTP pools and increasing the transcriptional error frequency. We pursue this because it is part of a novel idea regarding a possible chemotherapy for AIDS. The idea involves increasing the error rate in transcriptional replication of HIV by attacking host targets, which control purine and pyrimidine pool sizes.
Keywords: NAD(H) analog, NAD(H) phosphate, bacterial genetics, gene, genetic regulation, transfer RNA, aminoacid biosynthesis, chromosome, drug resistance, genetic transcription, histidine, membrane transport protein, microorganism metabolism, mutant, operon, oxidative stress, proline, protein, protein biosynthesis, Escherichia coli, Salmonella, computer processing of laboratory data, genetic manipulation
Project start date: 1977-01-01
Project end date: 1999-12-31
5R01GM023408-22 (1998): $271006
5R01GM023408-21 (1997): $262355
AN ANALYSIS OF GENES INVOLVED IN TRNA PRODUCTION
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM023408-05 from National Institute Of General Medical Sciences IRG: MBC
Abstract: The objective of this work is to elucidate the role of tRNA in cellular regulatory mechanisms and to determine the function of the many minor, non-vital tRNA species which have been observed. We intend to identify and characterize as many genes as possible which affect tRNA structure. These genes will include both tRNA structural genes and genes involved in the maturation or modification of tRNA. We will determine the effects on cellular physiology that result from destruction or alteration of these genes. In addition, several gene regulatory mechanisms including that for histidine biosynthesis and for proline degradation will be investigated in hopes of demonstrating the role of tRNA.
Keywords: GENETICS, GENES, MICROBIAL PHYSIOLOGY STUDY SECTION, NUCLEIC ACIDS, TRNA, AMINO ACIDS BIOSYNTHESIS, CYCLIC AMINO ACIDS, HISTIDINE, CYCLIC AMINO ACIDS, PROLINE, DRUGS RESISTANCE, MICROBIAL, GENETICS, CHROMOSOMES, GENETICS, GENES, OPERON, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENETICS, MICROBIAL, GENETICS, MUTATION, MUTANTS, PROTEINS, genetic manipulation
Project start date: 1977-01-01
Project end date: 1981-12-31
Sponsored Links Excellgen http://Excellgen.com
GENETIC ANALYSIS OF BACTERIAL CHROMOSOME STRUCTURE
John R Roth, Professor
Biologyuniversity Of Utah
75 South 2000 East
salt Lake City, Ut 84112
Grant 2R01GM027068-20 from National Institute Of General Medical Sciences IRG: MBC
Abstract: The investigator will study genetic recombination using assay systems that monitor recombination between repeated sequences in the bacterial chromosome. Unlike crosses, these assays provide no DNA ends; spontaneous DNA damage initiates the exchange. In a duplication segregation assay, the main source of initiating structures appears to be damage to DNA caused by reactive oxygen species. The metabolic steps involved in generating reactive oxygen species will be identified. Oxidative DNA damage is thought to play a major role in carcinogenesis and aging. Chromosome inversions can form by recombination between separated inverse order repeats. For particular chromosome regions, inversion is impossible in a wild type strain but is allowed by a mutation in the tus gene, whose product terminates replication at Ter sites. It is proposed that non-permissive replication must cross an active Ter site; such replication is possible only in the absence of Tus protein. An assay for reciprocality of recombination suggests that 25% of the exchanges which form a duplication are reciprocal; they also form the corresponding deletion. This apparent reciprocality may be due to serial half reciprocal exchanges which occur by initiation of replication forks in the course of recombination. These studies provide an experimental approach for the role of replication in recombination. A model will be tested which explains the Cairns phenomenon of apparent adaptive mutability without requiring any increase in intrinsic mutation rate. The model involves selective amplification of the mutant allele and includes (but does not rely on) a hypothesized new mutation type (the do-loop) by which short DNA sequences can by highly amplified by rolling circle replication initiated by the repair process. This model offers a way of explaining the genetic basis of triplet expansion diseases and the evolution of new genes. This proposed work investigates illegitimate double strain break repair, a process by which sequences can transpose into spontaneous breaks without involvement of any transposable element or transposase. This process may be a major cause of spontaneous chromosome rearrangements. In addition, chromosome position effects on recombination, transposition and mutation will be addressed; preliminary observations suggest that chromosome context can have a big effect on all of these processes. Finally, the proposal includes studying the behavior of the unusual transposable element IS200, which is found in virtually all Salmonella strains, but transposes very rarely
Keywords: bacterial genetics, chromosome, molecular genetics DNA damage, DNA replication, free radical oxygen, gene mutation, oxidative stress, transposon /insertion element Salmonella, genetic mapping, genetic recombination
Project start date: 1979-12-01
Project end date: 2002-11-30
2R01GM027068-20 (1999): $359925
BIOSYNTHESIS OF VITAMIN B12 AND ANAEROBIC METABOLISM
John R Roth, Professor
University Of California Davis Office Of Research - Sponsored Programs Davis, Ca 95618
Grant 3R01GM034804-19S1 from National Institute Of General Medical Sciences IRG: MBC
Abstract: Salmonella dedicates 1 percent of its genome to synthesis of vitamin B12 (cobalamin) a large cofactor (MW 1570) synthesized de novo by Salmonella, but not E. coli. Another 1 percent of the Salmonella genome encodes metabolic pathways (including degradation of ethanolamine and propanediol) that require the B12. We propose work on several aspects of B12 synthesis import and use, including the source of the lower ligand of B12 (dimethylbenzimidazole). We have genetic evidence for a periplasmic cobalamin binding protein involved in transport and have mutants that suggest the transport proteins may contribute to repression of the cob (biosynthetic) operon beyond mere important of the effector. Analysis of the B12-dependent degradative pathways has revealed several unexpected features. (1) Physiological importance of B12 is likely to be anaerobic (the only conditions under which B12 is made). Cobalamin- dependent anaerobic growth on propanediol and ethanolamine occurs with the electron acceptor tetrathionate, but not with fumarate or nitrate. We will pursue the genetics, biochemistry and regulation of tetrathionate reduction and will try to learn why this acceptor is unique and where it appears in nature. (2) Very large operons encode enzymes for use of ethanolamine (eut; 17 genes) and propanediol (pdu; greater than 20 genes). (3) Most of the genes of these operons have no mutant phenotype under the conditions used, suggesting that they function under novel conditions or are redundant to other metabolic functions. (4) Both operons encode multiple proteins homologous to the shell proteins of carboxysomes, organelles, which we have recently visualized. This suggests that the eut and pdu pathways could involve CO2 fixation. Cells grown with high CO2 have a second pathway for ethanolamine use which requires only one (EutE) enzyme produced by the 17-gene (eut) operon; the CO2-pathway depends on B12 but does not require the B12-dependent enzyme ethanolamine ammonia lyase(EutBC). We are characterizing these pathways. We feel that B12 metabolism underlies major differences between Salmonella (a pathogen) and its sister- species E. coli. While these organisms appear very similar in the lab, they are easily distinguished taxonomically. Synthesis of B12 (cob, cbi), use of propanediol (pdu) and reduction of polysulfide (ttr, phs, asr) are all properties used to distinguish Salmonella from E. coli. Understanding this metabolism may contribute to understanding the natural lifestyle of Salmonellae.
Keywords: anaerobiosis, microorganism metabolism, vitamin B12, vitamin biosynthesis, bacterial genetics, carbon dioxide, cobalt, ethanolamine, gene expression, iron, mutant, operon, propylene glycol, transposon /insertion element, Salmonella typhimurium
Project start date: 1985-07-01
Project end date: 2005-08-31
3R01GM034804-19S1 (2004): $105283
5R01GM034804-18 (2002): $399224
5R01GM034804-17 (2001): $398121
5R01GM034804-16 (2000): $385084
2R01GM034804-15 (1999): $379653
John R Roth, Professor
University Of Utah
75 South 2000 East
salt Lake City, Ut 84112
Grant 5R37GM023408-17 from National Institute Of General Medical Sciences IRG: NSS
Abstract: Detailed information is available regarding the mechanisms of regulating specific genes in bacteria. Much less is known about how these individual control systems interact so as to coordinate the functions of multigene systems. We propose to genetically approach control of several multigene systems and how they might interact. We will work on 1) the histidine operon and how it interacts with the related purine pathway and the cells control of cell division, 2) the purine pathway per se will be investigated to determine how the many genes in its separate branches are controlled, 3) a new system of gene regulation will be investigated which we believe is related to purine metabolism and involves structural alteration of DNA. The system seems to cause reversible inactivation of cryptic prophages, 4) the NAD pathway involves not only biosynthesis but also recycling pathway for salvage of pyridine nucleotides. This system is involved in DNA repair as well as oxidation/reduction reactions. The control of this complex pathway and its integration with cellular regulatory mechanisms will be pursued. All of these endeavors will involve a heavily genetic approach employing transposable elements, some of which form operon fusions of the lac operon to the promoter of the target gene. Many of the methods used are one developed in this lab
Keywords: bacterial genetics, gene, transfer RNA aminoacid biosynthesis, chromosome, drug resistance, genetic manipulation, genetic transcription, histidine, microorganism metabolism, mutant, operon, proline, protein, protein biosynthesis Escherichia coli, Salmonella, computer processing of laboratory data
Project start date: 1977-01-01
Project end date: 1994-12-31
5R37GM023408-17 (1993): $231597
4R37GM023408-16 (1992): $230587
BIOSYNTHESIS OF VITAMIN B12 AND ANAEROBIC METABOLISM
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5R01GM034804-10 from National Institute Of General Medical Sciences IRG: MBC
Abstract: Cobalamin (B-12) is an essential nutrient for humans that is only synthesized by microbes and some protists. The biosynthesis of B-12 is rather poorly understood. The complexity and instability of intermediates has made biochemical approaches difficult and most study has been focused on bacteria with very poor genetic systems. We have found that the genetic organism, Salmonella typhimurium, is able to synthesize B-12 de novo when grown under anaerobic conditions. We have initiated a primarily genetic approach to the synthesis of B-12 and how that synthesis is regulated. We hope to identify the structural genes for all of the synthetic enzymes and elucidate the mechanisms whereby these genes are regulated in response to the end product (probably adenosylcobalamin), cAMP, and redox state of the cell. Synthesis, transport, and recycling of B-12 appears to require about 1% of the Salmonella genome. Despite the size of this genetic investment in B- 12, only four enzymes are known in Salmonella that require B-12 as a cofactor; none of these functions appears to be fundamentally important. The B-12 dependent functions include on (of two) routes to methionine, synthesis of a nonessential tRNA modification, and ability to use ethanolamine and propanediol as carbon sources. Which of these, or what undiscovered function, accounts for the importance of B-12 to Salmonella? Why does Salmonella synthesize B-12 only under anaerobic conditions? (Many obligate aerobes are known to be able to synthesize B-12.) What physiological factors account for the distribution of B-12 in nature? (It is absent from plants, required by animals and some protists, and synthesized primarily by microbes.) To approach questions of biological significance of B-12, we will investigate the anaerobic metabolism of Salmonella. Despite the great interest that has been focused on genetics and metabolism of Salmonella and its relative E. coli, anaerobic behavior has received rather little attention. We have to contribute to a better understanding of anaerobic metabolism and its regulation, with special attention to the role of B-12.
Keywords: Salmonella typhimurium, anaerobiosis, bacterial genetics, microorganism metabolism, nucleic acid sequence, vitamin B12, vitamin biosynthesis, anaerobic bacteria, cobalt, ethanolamine, gene expression, gene induction /repression, genetic promoter element, genetic transcription, mutant, nitrogen, open reading frame, operon, protein engineering, regulatory gene, vitamin B12 coenzyme, gene complementation, genetic mapping, molecular cloning, mutagen, nutrition related tag, point mutation
Project start date: 1985-07-01
Project end date: 1995-06-30
5R01GM034804-10 (1994): $142021
5R01GM034804-09 (1993): $136659
Sponsored Links Excellgen http://Excellgen.com
5R01GM034804-08 (1992): $130627
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5T32GM007464-22 from National Institute Of General Medical Sciences IRG: BRT
Project start date: 1977-07-01
Project end date: 2002-06-30
5T32GM007464-22 (1998): $211582
2T32GM007464-21 (1997): $208316
John R Roth, Professor
Biologyuniversity Of Utah
75 South 2000 East
salt Lake City, Ut 84112
Grant 5R01GM023408-20 from National Institute Of General Medical Sciences IRG: MBC
Project start date: 1977-01-01
Project end date: 1998-12-31
5R01GM023408-20 (1996): $254037
John R Roth, Professor
Cellular, Viral/molecular Bioluniversity Of Utah
75 South 2000 East
salt Lake City, Ut 84112
Grant 5T32GM007464-20 from National Institute Of General Medical Sciences IRG: GBD
Project start date: 1977-07-01
Project end date: 1997-06-30
5T32GM007464-20 (1996): $159069
BIOSYNTHESIS OF VITAMIN B12 AND ANAEROBIC METABOLISM
John R Roth, Professor
Biologyuniversity Of Utah
75 South 2000 East
salt Lake City, Ut 84112
Grant 5R01GM034804-12 from National Institute Of General Medical Sciences IRG: MBC
Project start date: 1985-07-01
Project end date: 1999-06-30
5R01GM034804-12 (1996): $289377
John R Roth, Professor
University Of Utah 75 South 2000 East Salt Lake City, Ut 84112
Grant 5T32GM007464-19 from National Institute Of General Medical Sciences IRG: GBD
Project start date: 1977-07-01
Project end date: 1997-06-30
5T32GM007464-19 (1995): $132750