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Journal of Bacteriology Mar 1982Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more...
Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.
Topics: Adenine; Amino Acids; Caseins; Culture Media; Glucose; Phosphates; Potassium; Potassium Compounds; Protein Hydrolysates; Purines; Pyrimidines; Quaternary Ammonium Compounds; Spores, Bacterial; Streptomyces
PubMed: 7061380
DOI: 10.1128/jb.149.3.1102-1111.1982 -
Microbiology Resource Announcements Jul 2020Here, we describe the genome of DSM 40503, an 8-azaguanine-producing strain. The genome is the basis for future study and presents an underexplored taxonomy and...
Here, we describe the genome of DSM 40503, an 8-azaguanine-producing strain. The genome is the basis for future study and presents an underexplored taxonomy and biosynthetic potential, which expands our understanding of the diversity of microorganisms that produce nitrogen heterocyclic compounds.
PubMed: 32732231
DOI: 10.1128/MRA.00518-20 -
Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis.BMC Microbiology Jun 2003Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One...
BACKGROUND
Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase.
RESULTS
The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM.
CONCLUSION
The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.
Topics: Azaguanine; Azathioprine; Cloning, Molecular; Enzyme Inhibitors; Gene Expression; Methionine Adenosyltransferase; Mycobacterium smegmatis; Mycobacterium tuberculosis; Species Specificity
PubMed: 12809568
DOI: 10.1186/1471-2180-3-12 -
Proceedings of the National Academy of... Jun 1963
Topics: Azaguanine; Cyanides; Learning; RNA; Ristocetin
PubMed: 14019945
DOI: 10.1073/pnas.49.6.918 -
Molecular Microbiology Jan 2017NCS1 proteins are H or Na symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are...
NCS1 proteins are H or Na symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur- and Fcy-like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5-fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate-affinity, low-capacity, highly specific adenine transporter, whereas FcyE contributes to 8-azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.
Topics: Amino Acid Sequence; Aspergillus nidulans; Biological Evolution; Biological Transport; Conserved Sequence; Fungal Proteins; Gene Duplication; Nucleobase Transport Proteins; Phylogeny; Protein Structure, Tertiary; Purines; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 27741561
DOI: 10.1111/mmi.13559 -
Journal of Bacteriology Jan 1962Mangalo, R. (University of Illinois, Urbana) and J. T. Wachsman. Effect of 8-azaguanine on growth and viability of Bacillus megaterium. J. Bacteriol. 83:27-34. 1962.-The...
Mangalo, R. (University of Illinois, Urbana) and J. T. Wachsman. Effect of 8-azaguanine on growth and viability of Bacillus megaterium. J. Bacteriol. 83:27-34. 1962.-The addition of 8-azaguanine to exponentially growing cells of Bacillus megaterium results in an inhibition of growth after a lag of approximately 30 min. However, 8-azaguanine-2-C(14) is incorporated into the nucleic acids in a linear fashion without a detectable lag. The inhibitory action is reversed by purines and their derivatives, but not by uridine, thymidine, or cytidine. 8-Azaguanine is bactericidal, especially under conditions where growth (ribonucleic acid synthesis) is possible. Growth in the presence of a complete amino acid mixture, either before or during exposure to 8-azaguanine, increases the rate of killing. Chloramphenicol has little or no effect on the bactericidal action of the analogue.
Topics: Amino Acids; Antimetabolites; Azaguanine; Bacillus; Bacillus megaterium; Nucleic Acids; Purines; RNA; Thymidine; Uridine
PubMed: 14469259
DOI: 10.1128/jb.83.1.27-34.1962 -
Atherosclerosis Sep 1986Murine hybridomas were prepared by fusion of azaguanine-resistant plasmacytoma cells and spleen cells from BALB/c mice immunized with intact rat lipoproteins (Lp)....
Murine hybridomas were prepared by fusion of azaguanine-resistant plasmacytoma cells and spleen cells from BALB/c mice immunized with intact rat lipoproteins (Lp). Seventeen stable hybridomas from 5 fusions producing antibody against rat apolipoprotein B (apo B)-containing Lp were prepared. The distribution of antigenic determinants of rat apo B variants defined by these antibodies was similar to that found for human apo B. Thirteen mAbs were reactive to epitopes found on both the higher (apo BH) and lower (apo BL) molecular weight forms of rat apo B; two were reactive to apo BH-specific epitopes and two were reactive to apolipoprotein E. No antibodies reactive to unique apo BL epitopes were found. One anti-rat apo B mAb (1.34.3) of high affinity and avidity was reactive to denatured and intact Lp-apo B. Reactivity towards Lp-apo B was independent of variant composition and density class of Lp. The mAb was subsequently used to develop a radioimmunoassay (RIA) which was sensitive down to 5 ng apo B. The assay was used successfully to measure total nascent Lp-apo B secreted by hepatocytes in culture.
Topics: Animals; Antibodies, Monoclonal; Apolipoproteins B; Epitopes; Hybridomas; Lipoproteins, HDL; Lipoproteins, LDL; Lipoproteins, VLDL; Radioimmunoassay; Rats
PubMed: 2429674
DOI: 10.1016/0021-9150(86)90139-5 -
The Biochemical Journal Mar 19671. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose...
1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.
PubMed: 16742482
DOI: 10.1042/bj1020691 -
World Journal of Gastroenterology May 2003To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis.
AIM
To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis.
METHODS
The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3(-) by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3(-) and FL-REV3(-). The blockage effect of REV3 was measured by combination of reverse transcription-polymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3 protein level.
RESULTS
The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27 fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P<0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P<0.05). In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein. MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P<0.05) and 5.8 fold (P<0.01) respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3 was blocked by antisense RNA, and almost recovered to their spontaneous mutation levels. The spontaneous HPRT mutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66 x 10(-6) in FL cells to 0.14 x 10(-6) in transgenic cells as well (P<0.01).
CONCLUSION
The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.
Topics: Base Sequence; Carcinogens; Cell Line; DNA, Antisense; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Humans; Hypoxanthine Phosphoribosyltransferase; Methylnitronitrosoguanidine; Mutagenesis; Plasmids; Stomach Neoplasms; Transfection
PubMed: 12717825
DOI: 10.3748/wjg.v9.i5.888 -
Proceedings of the National Academy of... Feb 1976The mutagenicity of benzo[a]pyrene and 15 of its derivatives, which included phenols, the benzo[a]yrene-4,5-epoxide (the K-region epoxide), dihydrodiols, two isomeric...
The mutagenicity of benzo[a]pyrene and 15 of its derivatives, which included phenols, the benzo[a]yrene-4,5-epoxide (the K-region epoxide), dihydrodiols, two isomeric 7,8-diol-9,10-epoxides, a 6-methyl derivative, and a 6-hydroxymethyl derivative, were tested with Chinese hamster V79 cells in order to identify the mutagenic metabolites of benzo[a]pyrene. Mutations were characterized by resistance to ouabain or 8-azaguanine. Since V79 cells do not metabolize polycyclic hydrocarbons, mutagenesis was tested both in the presence and absence of benzo[a]pyrene-metabolizing normal golden hamster cells. All the tested phenols, 4,5-diols, trans-9,10-diol, 6-methyl, and 6-hydroxymethyl derivatives of benzo[a]pyrene showed little or no mutagenicity for both genetic markers. The (+/-)7alpha,8beta-dihydroxy-9alpha,10alpha-epoxy-7,8;9,10-tetrahydrobenzo[a]pyrene and K-region 4,5-epoxide exhibited similar and moderate mutagenicity in the absence of benzo[a]pyrene-metabolizing cells, but the (+/-)7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene showed a 2000- and 270-fold higher mutation frequency for ouabain and 8-azaguanine resistance, respectively, than did the K-region 4,5-epoxide. The trans-7,8-diol which was not mutagenic in the absence of benzo[a]pyrene-metabolizing cells was more mutagenic than benzo[a]pyrene after metabolism and mutagenesis by trans-7,8-diol in these cells was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Metabolically formed trans-7,8-diol was isolated and incubated with rat liver microsomes in the presence of co-factors. High-pressure liquid chromatography analysis indicated that the major metabolite of trans-7,8-diol is 7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. The results indicate that the latter compound is metabolically formed and the major mutagenic intermediate of benzo[a]yrene metabolism.
Topics: Benzopyrenes; Cell Line; Microsomes; Mutation; Structure-Activity Relationship
PubMed: 1061161
DOI: 10.1073/pnas.73.2.607