The promoter of the Streptomyces glaucescens mel operon.
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Amino Acid Sequence↗
Biomedical subjects
R Hütter
Publications and source records attributed to R Hütter.
Tryptophan accumulation in Saccharomyces cerevisiae under the influence of an artificial yeast TRP gene cluster.
Plasmid pME559, carrying all five yeast TRP genes, was constructed. This plasmid is a yeast/Escherichia coli shuttle vector based on pBR322 and 2 micron-DNA sequences derived from plasmid pJDB207. We studied in yeast (i) the stability of the plasmid under selective and non-selective conditions, (ii) expression of all five TRP genes and (iii) tryptophan accumulation in yeast transformants. These studies were conducted in comparison with an earlier construction, pME554, which differs from plasmid pME559 in the expression of the TRP1 gene and which carries the TRP2 wild type instead of the TRP2fbr mutant allele. For stable maintenance of the plasmids in yeast a selection was necessary. Plasmid pME559 displayed normal expression of all TRP genes, and enzyme levels on average 23-fold higher than in the wild type strain were found. In comparison, the maximal tryptophan flux observed in such a plasmid-carrying strain was about ten-fold higher than the maximal flux capacity in the wild type strain.
Alleles↗
Site-specific integration and excision of pMEA100 in Nocardia mediterranei.
Nocardia mediterranei strain LBG A3136 contains the 23.7 kb element pMEA100 in a chromosomally integrated form as well as in the free state (Moretti et al. 1985). The integrated form of this element can be excised precisely from the Nocardia chromosome without any accompanying rearrangements in flanking chromosomal DNA. After transfer into plasmid-free mutant strains, pMEA100 reintegrates site specifically into its original chromosomal locus. The exact mapping of the pMEA100 integration site was accomplished by restriction analysis and DNA sequencing. The attachment site of pMEA100, the junctions of its integrated form and plasmid-free chromosomal DNA of N. mediterranei contain an identical 47 bp long sequence which is probably required for site-specific recombination connected with integration and excision of pMEA100. Only one such sequence was found in the chromosome of pMEA100-free N. mediterranei derivatives as suggested by the single integration locus.
Base Sequence↗
Characterisation of the hydroxystreptomycin phosphotransferase gene (sph) of Streptomyces glaucescens: nucleotide sequence and promoter analysis.
The nucleotide sequence of a 1384 bp fragment containing the coding and promoter sequences of the streptomycin phosphotransferase gene (sph) of the hydroxystreptomycin-producing Streptomyces glaucescens was determined. Evidence for an ATG as translation start codon for sph was derived from a comparison with the amino-terminal amino acid sequence of an aminoglycoside phosphotransferase (aphD gene product) of S. griseus, exhibiting a high degree of amino acid homology to the deduced amino acid sequence of the S. glaucescens sph gene product. Transcriptional start and termination sites for the sph gene were identified by primer extension and/or nuclease S1 mapping experiments. The promoter region of the sph gene appears to be complex since tandemly arranged promoters (orfIp1, orfIp2) initiating transcription of a likely coding region (ORFI) in the opposite direction overlap sph promoter sequences. The presumptive sphp and orfIp1 promoters show considerable sequence similarities in the -10 region to Escherichia coli consensus promoter sequences but no homology to E. coli or Streptomyces -35 regions.
Amino Acid Sequence↗
Specificity of Bacillus thuringiensis Delta-Endotoxin.
The insecticidal activity of the delta-endotoxins of 14 Bacillus thuringiensis strains belonging to 12 subspecies was determined against Pieris brassicae, Heliothis virescens, and Spodoptera littoralis. Larvae of P. brassicae were highly susceptible to purified crystals of strains of B. thuringiensis subsp. thuringiensis and B. thuringiensis subsp. morrisoni, whereas H. virescens responded best to B. thuringiensis subsp. kenyae and B. thuringiensis subsp. kurstaki. The crystals of the B. thuringiensis subsp. entomocidus strain were the most potent against S. littoralis. It was shown that the solubility of the crystals within the gut of the three insect species is a first important step in the mode of action. Predissolution of the crystals especially enhanced the insecticidal activity against H. virescens. When in vitro-activated toxins were applied, the relative potency range varied greatly from one insect species to another. It can be concluded that at least three factors influence the potency of B. thuringiensis delta-endotoxins: the strain-related origin of the toxin, the degree of solubility of the crystals in the gut juice, and the intrinsic susceptibility of the insect to the toxin.
Journal Article↗
The TRP4 gene of Saccharomyces cerevisiae: isolation and structural analysis.
The TRP4 gene of Saccharomyces cerevisiae, encoding the anthranilate phosphoribosyl transferase, was isolated and subcloned by functional complementation in yeast. A 2 kb fragment containing information for a polypeptide of 380 amino acids and the 5'- and 3'-flanking regions was sequenced. The TRP4 transcript was identified and mapped with S1 nuclease. Homologies to two prokaryotic genes encoding the same function, and sequences potentially involved in transcription start and termination and in regulation of TRP4 gene expression are discussed.
Amino Acid Sequence↗
Identification and characterization of four new GCD genes in Saccharomyces cerevisiae.
Mutant strains, resistant against the amino acid analogues 5-methyltryptophan, 5-fluorotryptophan and canavanine were isolated, starting with a trp2 leaky auxotrophic strain. Of 10 such strains, only four turned out to be of the "general control derepressed" (gcd) mutant type. Three other isolates were shown to be defective in the general amino acid permease system, while the remaining three strains displayed low spore viability and were not further investigated. Complementation tests amongst the four new gcd-mutant strains, including strain RH558 gcd2-1 isolated earlier, yielded five complementation groups: GCD2, GCD3, GCD4, GCD5, and GCD6. All mutant strains showed a dual phenotype, which was not separable by wild type backcrosses: "constitutive derepression" and "slow growth". Epistasis of all gcd mutations over gcn1-1, gcn2-1 and gcn3-1 was found with respect to both phenotypes, except for gcd5-1, which was lethal in these combinations. On the other hand gcn4-101 was found to be epistatic over all gcd mutations, but only with respect to the "constitutive derepression" phenotype, and not to "slow growth"; again the combination with gcd5-1 was lethal. Mutation gcd2-1 was mapped on chromosome VII, 50 cM from leu1 and 22 cM from ade6. A new model is discussed, in which GCD-genes are involved in the amino acid uptake into the vacuoles.
Amino Acids↗
Cloning of the ARO3 gene of Saccharomyces cerevisiae and its regulation.
Regulation of the two isozymes of 3-deoxy-D-arabino-heptulosonate-7phosphate synthase (DAHP synthase; EC 4.1.2.15) encoded by the genes ARO3 and ARO4 of Saccharomyces cerevisiae was studied. Both genes were shown to respond equally well to the general control of amino acid biosynthesis. Strains with mutations in these two genes were obtained by selecting first for a single aro3 mutation and afterwards for a double aro3 aro4 mutation. Gene ARO3, coding for the phenylalanine-dependent isozyme of DAHP synthase was cloned on the 2 micron multicopy vector pJDB207 by complementation of mutation aro3-1 in yeast. The ARO3 gene, carried originally on a 9.6 kb BamHI fragment (plasmid pME541A), was subcloned on a 1.9 kb HindIII-XbaI fragment (plasmid pME543). A transcript of about 1.5 kb was shown to proceed from the HindIII towards the XbaI site. Expression from the 9.6 kb as well as from the 1.9 kb fragment was normal on a multicopy vector, since in both cases DAHP synthase levels of about 50-fold the wild-type level were observed.
3-Deoxy-7-Phosphoheptulonate Synthase↗
Plasmid curing and generation of mutations induced with ethidium bromide in streptomycetes.
The DNA-intercalating agent ethidium bromide was used to eliminate plasmid DNA from streptomycetes. Other mutational events associated with chromosome changes occurred at high frequency; they resulted in phenotypic changes such as loss of enzyme activity, antibiotic resistance or ability to sporulate.
Colony-Forming Units Assay↗
Tryptophan biosynthetic genes in eukaryotic microorganisms.
In recent years more information about tryptophan biosynthesis in eukaryotic microorganisms has become available. The emphasis has been on genetics and biochemistry of the pathway. Eukaryotes manifest a trend toward fewer genes and toward multifunctional proteins, while prokaryotes have a greater tendency toward separate activity domains but the genes tend to be clustered genetically. Cloning of various structural tryptophan biosynthetic genes and studies on their expression in homologous and heterologous hosts have made it possible to analyze promoter structures in detail and to define structural elements involved in regulated gene expression. Comparisons of homologous genes from different organisms have highlighted the conservation of the activity domains or parts therefrom involved in the catalysis of single steps. These studies also point to a stringent maintenance of domains responsible for protein-protein aggregation. Physiological studies will be facilitated by the availability of single cloned genes and especially the artificial gene cluster containing all five TRP genes from yeast. The range of physiological manipulation has thus been enormously broadened. With chromosomal mutations it has been possible to study primarily downward modulation of a pathway. We can now initiate studies on upward modulation, since enzyme levels appear to increase in proportion to gene dose. The new range of downward and upward modulation in the levels of single enzymes and combinations of enzymes may contribute to a better understanding of flux regulation and its influence on the overall physiology of an organism.
Escherichia coli↗
Ring cleavage and degradative pathway of cyanuric acid in bacteria.
The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.
Ammonia↗
Certain chromosomal regions in Streptomyces glaucescens tend to carry amplifications and deletions.
Streptomycetes are subject to a high degree of genetic instability. One manifestation of this phenomenon is the occurrence of tandemly reiterated DNA stretches within the chromosome. We describe the analysis of ten reiterated sequences observed in various ethidium bromide-treated streptomycin-sensitive and melanin-negative mutant strains of Streptomyces glaucescens. The repeated DNA units were 2.9 to 35 kb in length. No two sequences were identical. The amplified sequences occupied up to 45% of the total genomic DNA. Structural analysis of the cloned repeated DNA stretches by means of restriction enzymes and by cross hybridization revealed the presence of two chromosomal areas rich in DNA reiterations. In some cases reiterated regions were accompanied by nearby rearrangements.
Chimera↗
Cloning and expression of the genetically unstable tyrosinase structural gene from Streptomyces glaucescens.
The gene from Streptomyces glaucescens coding for an inducible tyrosinase was cloned using the low copy vector pIJ41 and the melanin-negative strain Streptomyces lividans TK23 as host. Hybridisation experiments as well as complementation studies showed that melC mutant strains carry large deletions of more than 10.5 kb, comprising the structural gene for tyrosinase, while melA and melB strains carry mutations in genes involved in the expression of tyrosinase activity. Strong DNA homology was found between the Streptomyces antibioticus and the S. glaucescens tyrosinase structural genes and both genes showed a similar regulation when introduced into melanin-negative hosts. While both tyrosinases exhibited clear induction in S. glaucescens, constitutive expression was observed in S. lividans. Northern blot experiments showed that tyrosinase expression is regulated at the transcriptional level and that the gene (822 bp) is part of a 2.3 kb transcript. The main start of the mRNA at about 475 bp upstream from the tyrosinase N-terminus was located by S1-mapping experiments.
Catechol Oxidase↗
Arrangement of genes TRP1 and TRP3 of Saccharomyces cerevisiae strains.
The tryptophan biosynthetic genes TRP1 and TRP3 and partly also TRP2 and TRP4 have been compared by the technique of Southern hybridization and enzyme measurements in twelve wild isolates of Saccharomyces cerevisiae from natural sources of different continents, in the commonly used laboratory strain S. cerevisiae X2180-1A and in a Kluyveromyces marxianus strain. We could classify these strains into four groups, which did not correlate with their geographical distribution. In no case are the TRP3 and TRP1 genes fused as has been found in other ascomycetes. Two strains were found which, in contrast to strain X2180-1A, show derepression of gene TRP1. Two examples are discussed to demonstrate the usefulness of Southern hybridizations for the identification of closely related strains.
DNA Restriction Enzymes↗
Isolation and characterization of an extrachromosomal element from Nocardia mediterranei.
Strain LBG A3136 of Nocardia mediterranei (ETH Collection) was found to contain a low-copy-number covalently closed circular extrachromosomal element, pMEA 100, which could only be isolated from mycelium grown on agar plates. pMEA 100 could not be isolated from the closely related strain ATCC 13685. Hybridization experiments showed that pMEA 100 is present in strain LBG A3136 in the free as well as in the integrated form whereas in strain ATCC 13685 only an integrated form was detected. Excision and reintegration in strain LBG A3136 seemed to be site specific. pMEA100 was found to be self-transmissible, eliciting the lethal zygosis phenotype, and is possibly involved in fertility in N. mediterranei.
Chromosome Mapping↗
Bacterial degradation of N-cyclopropylmelamine. The steps to ring cleavage.
The s-triazine cyclopropylmelamine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine) was degraded to about 6 mol of NH4+/mol of substrate by a mixture of two bacteria (strains A and D, both Pseudomonas spp.) Only strain A grew with cyclopropylmelamine as sole and limiting source of nitrogen. The organism obtained 2 mol of nitrogen/mol of substrate and excreted a product that was identified as cyclopropylammelide [6-cyclopropylamino-1,3,5-triazine-2,4(1 H,3 H)-dione]. Proteins in extracts from strain A were separated on a Sephadex G-200 column. Cyclopropylmelamine was found to be deaminated in two separable steps to cyclopropylammelide via cyclopropylammeline [4-amino-6-cyclopropylamino-1,3,5-triazine-2(1 H)-one], which was identified. Strain D could not utilize cyclopropylmelamine or cyclopropylammeline, but could utilize cyclopropylammelide (or homologue) as sole and limiting source of nitrogen and obtain about 4 mol of nitrogen/mol of substrate. Proteins in cell extracts from strain D were separated on a DEAE-cellulose column. Alkylammelides were degraded quantitatively by one enzyme fraction to 1 mol of cyanuric acid plus 1 mol of alkylamine/mol of substrate. The specific activities of enzymes in extracts of the two strains were as high as the activities observed during growth. The three activities studied in the two strains were all active under aerobic and oxygen-free conditions. The reactions appear to be hydrolytic, yielding 2 mol of NH4+ plus 1 mol of cyclopropylamine and 1 mol of cyanuric acid/mol of substrate.
Chemical Phenomena↗
HLA-DR-antigen-associated restriction of EBV-specific cytotoxic T-cell colonies.
EBV-specific cytotoxic T cells can be generated in vitro in a secondary response. Several previous studies with bulk cultures provided evidence that cytotoxicity was restricted by HLA-A,B-related antigens. In the present family study, the EBV-specific cytotoxic T-cell response of a normal EBV-seropositive donor was analysed in detail by T-cell colony formation. Peripheral blood mononuclear cells were stimulated by the gamma-irradiated autologous lymphoblastoid cell line (LCL) and 3 days later seeded into agarose. Colonies were harvested, amplified by addition of interleukin-2 (IL-2), and analysed for T-cell markers and specificity in 51Cr-release assays. Twenty-two colonies were studied: all colonies were OKT3+, five were predominantly OKT4+, 9 were OKT8+ and 8 were mixtures. As expected from previous work, the OKT8+ colonies were cytotoxic for the autologous LCL target and cytotoxicity was blocked by monoclonal antibody (W6/32) to the nonpolymorphic determinants of HLA-A,B,C antigens. Significantly, the OKT4+ colonies tested also showed specific cytotoxicity, but lysis of the autologous LCL was blocked by the monoclonal antibody (OKlal) to the non-polymorphic determinants of HLA-DR antigens. Two interesting patterns of specificity were seen in cytotoxicity tests on sibling LCL targets. In one pattern, targets bearing the A11, B5, DR7 haplotype were lysed, while those bearing the A1, B8, DR3 were not, indicating haplotype preference. In the other pattern, there was lysis of the autologous cell line but not of the sibling targets. These results including HLA-DR-associated restriction, haplotype preference and strict self-preference, further illustrate the complexity of the EBV-cytotoxic T-cell response.
Adult↗
Expression of an artificial yeast TRP-gene cluster in yeast and Escherichia coli.
All five tryptophan biosynthetic genes of Saccharomyces cerevisiae were unified on plasmid pME554, which is based on 2 micrometer DNA and pBR322 sequences allowing for autonomous replication in yeast and E. coli. Homologous and heterologous expression of this artificial yeast TRP-gene cluster was studied. Plasmid pME554 allowed for nearly normal growth of a yeast strain bearing auxotrophic mutations in all five TRP-genes. The plasmid-borne genes TRP2 to TRP5 were expressed and regulated normally in the frame of the general control. Gene TRP1, carried on an EcoRI/Bg/II fragment lacking the ARS1 function, was expressed poorly and did not respond to the general control like the chromosomally-borne TRP1 gene. Plasmid pME554 allowed for poor growth of E. coli strain W3110 tna- delta trpEA2 on minimal medium. Marked stimulation was observed, however, when anthranilic acid or indole were added. Accordingly, poor expression of the first Trp-enzyme anthranilate synthase and the last enzyme tryptophan synthase was found, whereas the other three genes were moderately well expressed in E. coli.
Base Sequence↗