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W Schumann

Publications and source records attributed to W Schumann.

At least 55 records · Page 3Linked to original sources
PubMed1994

Tn 5Map, a transposon for the rapid mapping of restriction sites in plasmids.

A transposon was constructed allowing the rapid restriction mapping of plasmids. This transposon, Tn 5Map, contains a cleavage site for the I-SceI endonuclease which recognizes an 18-mer. After in vivo transposition of Tn5Map into the plasmid of interest, the plasmid is isolated and linearized with I-SceI. Splinkers labelled with digoxygenin and complementary to the left and right end of the linearized molecule are added and ligated. After partial digestion of the splinkered molecules with the restriction enzyme of interest, separation of the cleavage products in an agarose gel, and Southern transfer, the labelled fragments are visualized by the addition of the chemiluminescent substrate AMPPD and alkaline phosphatase. The restriction map can be directly read from the bottom to the top of the gel.

Bacteriocin Plasmids↗
PubMed1994

CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis.

The dnaK and groESL operons of Bacillus subtilis are preceded by a potential sigma 43 promoter sequence (recognized by the vegetative sigma factor) and by an inverted repeat (IR) consisting of 9 bp separated by a 9-bp spacer. Since this IR has been found in many bacterial species, we suspected that it might be involved in heat shock regulation. In order to test this hypothesis, three different mutational alterations of three bases were introduced within the IR preceding the dnaK operon. These mutations were crossed into the chromosome of B. subtilis, and expression of the dnaK and of the unlinked groESL operons was studied. The dnaK operon exhibited increased expression at low temperature and a reduction in the stimulation after temperature upshift. Furthermore, these mutations reduced expression of the groESL operon at low temperature by 50% but did not interfere with stimulation after heat shock. These experiments show that the IR acts as a negative cis element of the dnaK operon. This conclusion was strengthened by the observation that the IR reduced expression of two different transcriptional fusions significantly after its insertion between the promoter and the reporter gene. Since this IR has been described in many bacterial species as preceding only genes of the dnaK and groESL operons, both encoding molecular chaperones (39 cases are documented so far), we designated this heat shock element CIRCE (controlling IR of chaperone expression). Furthermore, we suggest that this novel mechanism is more widespread among eubacteria than the regulation mechanism described for Escherichia coli and has a more ancient origin.

Bacillus subtilis↗
PubMed1993

Isolation of stress mutants of Bacillus subtilis by a novel genetic method.

A novel genetic procedure is described to identify stress genes in Bacillus subtilis by insertion mutagenesis. In addition, this method allows the rapid mapping of the mutation and the establishment of the DNA sequence of the gene impaired by the mutation. Small restriction fragments of chromosomal DNA of B. subtilis are inserted into a pBR322-based vector, the recombinant plasmids are transformed into B. subtilis, and integrants are selected which arise by recombination between the insert and its homologous region within the bacterial chromosome. About two dozen heat-, cold- and salt-sensitive mutants were isolated. Four mutations were mapped using PBS1 transduction, and the physiology of one salt-sensitive mutant was analysed.

Bacillus subtilis↗
PubMed1993

Molecular cloning, sequencing, and transcriptional analysis of the groESL operon from Bacillus stearothermophilus.

Using a gene probe of the Bacillus subtilis groEL gene, a 7.3-kb HindIII fragment of chromosomal DNA of Bacillus stearothermophilus was cloned. Sequencing of 2,309 bp led to the detection of two open reading frames in the order groES groEL. Primer extension studies revealed one potential transcription start site preceding the groESL operon, which was activated upon temperature upshift. Northern blot (RNA) analysis resolved two mRNA species with lengths of 2.2 and 1.5 kb; RNA slot-blot experiments revealed an at least 10-fold increase in the amount of specific mRNA from 0 to 7 min postinduction followed by a decrease. The 9-bp inverted repeat characteristic of many gram-positive bacteria was found within the 5' leader region of the mRNA. The groESL operon of B. stearothermophilus could complement E. coli groES(Ts) and groEL(Ts) mutants for growth at high temperature and for propagation of phage lambda.

Amino Acid Sequence↗
PubMed1992

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis.

By using an internal part of the dnaK gene from Bacillus megaterium as a probe, a 5.2-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf39-grpE-dnaK-dnaJ. orf39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orf39 (S1 and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in alpha-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orf39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 223 degrees on the B. subtilis genetic map.

Amino Acid Sequence↗
PubMed1992

Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis.

Using a gene probe of the Escherichia coli groEL gene, a 1.8-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Upstream sequences were isolated as a 3-kb PstI fragment. Sequencing of 2,525 bp revealed two open reading frames in the order groES groEL. Alignment of the GroES and GroEL proteins with those of eight other eubacteria revealed 50 to 65% and 72 to 84% sequence similarity, respectively. Primer extension studies revealed one potential transcription start site preceding the groESL operon (S) which was activated upon temperature upshift. Northern (RNA) analysis led to the detection of two mRNA species of 2.2 and 1.5 kb. RNA dot blot experiments revealed an at least 10-fold increase in the amount of specific mRNA from 0 to 5 min postinduction, remaining at this high level for 10 min and then decreasing. A 9-bp inverted repeat within the 5' leader region of the mRNA might be involved in regulation of the heat shock response. By using PBS1 transduction, the groESL operon was mapped at about 342 degrees.

Amino Acid Sequence↗
PubMed1991

Tn5cos: a transposon for restriction mapping of large plasmids using phage lambda terminase.

A method for the rapid restriction mapping of large plasmids has been developed. A 400-bp fragment of phage lambda DNA containing the cos region has been inserted into Tn5. After in vivo transposition of this Tn5cos element into the plasmid of choice, the plasmid is isolated and linearized at its cos site with phage lambda terminase (Ter). Such Ter linearization was about 70% efficient. After partial digestion of the linear molecules with the appropriate restriction enzyme, the products are selectively labelled at the right or left cohesive phage lambda DNA termini by hybridization with digoxygenin (DIG)-11-dUTP-labelled (using terminal transferase) oligodeoxyribonucleotides complementary to the single-stranded cos ends. After pulsed field gel electrophoresis, the labelled fragments are visualized in the dried gel using a DIG-detection kit. The restriction map can be directly determined from the 'ladder' of partial digestion products.

DNA Transposable Elements↗
PubMed1991

Construction and evaluation of a cea-lacZ gene fusion for the detection of environmental mutagens and carcinogens.

The cea-kil operon of the ColE1 plasmid is negatively regulated by the LexA-repressor and therefore, it is under the control of SOS regulation. We constructed a gene fusion between the cea and lacZ genes. Expression of the translational fusion can be easily detected by monitoring the levels of beta-galactosidase. Since the whole detection system is plasmid-based, it can be used in both Escherichia coli and Salmonella typhimurium strains. The SOS-function-inducing activities of 14 chemical mutagens were investigated in E. coli K12 and in two S. typhimurium Ames-strains and compared with results obtained by the SOS-chromotest and by the Umu-test. To correct for the inhibitory effects of test chemicals on mRNA and/or protein synthesis, the level of the constitutive chloramphenicol acetyl transferase was assayed in parallel.

Bacillus subtilis↗
PubMed1991

[The pharmacokinetics of Oral-Turinabol in humans].

Disposition and excretion of the anabolic steroid Oral-Turinabol (1;4-chloro-17 alpha-methyl-androsta-1,4-diene-17 beta-hydroxy-3-one) were investigated in male volunteers. Following single p.o. and i.v. administration of the tritium-labelled compound the plasma concentration courses of total radioactivity (1 and 1-metabolites) and of the unchanged parent drug as well as the urinary excretion were estimated. From these data model independent pharmacokinetic parameters based on statistical moments were calculated. 1 is almost completely absorbed after p.o. administration of 10 mg per volunteer. Peak concentrations of total radioactivity and of 1 in plasma were reached about 3 h p.a. Irregularities observed in the plasma level profile following both p.o. and i.v. administration of 1 are due to a marked enterohepatic circulation. Orally given 1 is subject to a first-pass effect, resulting in a diminished systematic availability. The AUC-ratio of the unchanged drug and the total radioactivity of 1 : 13 shows the predominance of metabolites in plasma. After i.v. administration the disposition of unchanged 1 was found biphasically with a terminal half-life of 16 h. 1 and its metabolites are preferentially excreted via the kidneys. The urinary total radioactivity represented about 60% of the dose following both administrations. Due to its affinity to SHBG 1 is able to compete for the protein binding of testosterone, resulting in an increased plasma level of non protein-bound testosterone.

Administration, Oral↗
PubMed1990

Promoters of major Escherichia coli heat shock genes seem non-functional in Bacillus subtilis.

To find out whether Escherichia coli heat shock promoters are recognized in Bacillus subtilis, the regulatory regions including the heat shock promoters of the main heat shock genes dnaKJ, lon, groES, and htpG were fused to the indicator genes lacZ and cat. Whereas all transcriptional fusions were expressed in E. coli at low temperature and transient increases of beta-galactosidase activity could be measured at the inducing temperature, no enzymatic activity was found in B. subtilis. This indicates that E. coli heat shock promoters are nonfunctional in B. subtilis.

Bacillus subtilis↗
PubMed1989

Holographic interferometry applied to the case of large deformations.

This investigation in holographic interferometry concerns an approach to a systematic quasi-compensation by appropriate optical modifications at the reconstruction in order that the fringes of interference become visible in the case of large unknown object deformations. The relevant relations are established by using the aberration theory for the image formation in combination with elementary intrinsic differential geometry.

Algorithms↗
PubMed1988

Isolation of mutations of the phage Mu ner gene.

A method was devised which allows the easy detection of mutations within the ner gene of Mu DNA. This method is based upon the observation that a transcriptional gene A-galK fusion containing the complete ner gene and the cts62 allele does not express the galK gene in an Escherichia coli strain lacking functional integration host factor under inducing conditions (white colonies on MacConkey galactose plates at 42 degrees) In contrast, a gene ner-galK fusion which lacks part of the ner gene exhibits GalK activity (red colonies) on MacConkey galactose plates at 42 degrees. After mutagenesis of a plasmid carrying a transcriptional gene A-galK fusion, putative ner mutants could be identified on indicator plates. Cloning experiments locate the mutation(s) to the right of the HindIII site which is situated within the early promoter of Mu DNA. One of the mutants was sequenced and revealed two substitutions: one within the-10 region of the early promoter, and another near the end of the ner gene. The former lesion was shown to be pleiotropic.

Bacteriophage mu↗
PubMed1988

[The biotransformation of STS 267 [16 alpha-azido-3-methoxyestra-1,3,5(10)-triene-17-one]. Epimerization of a 16 alpha-azido group--a new metabolic reaction in the rat liver].

STS 267 [1; 16 alpha-azido-3-methoxy-estra-1,3,5(10)-trien-17-one] was found to have lipid shifting and fertility inhibitory effects in rats. In preclinical studies the metabolic fate of 1 was investigated in the rat. Demethylation in 3-position, reduction of the 17-carbonyl to the 17 beta-hydroxyl group, and for the first time the epimerization of the 16 alpha-azido to the 16 beta-azido group were established following perfusion of STS 267 in the isolated rat liver. From the perfusion medium 16 alpha-azido-estra-1,3,5(10)-trien-3-ol-17-one (2), 16 alpha-azido-estra-1,3,5(10)-trien-3,17 beta-diol (3), 16 beta-azido-3-methoxy-estra-1,3,5(10)-trien-3,17 beta-diol (4) and 16 beta-azido-estra-1,3,5(10)-trien-3,17 beta-diol (5) as the main metabolite were isolated by TLC and identified by comparison with authentic samples. The extent of the hepatic extraction of 1 in the rat liver was significant lower compared with mestranol.

Animals↗