Search PubMed⌕ Search

Biomedical subjects

D Jahn

Publications and source records attributed to D Jahn.

At least 37 records · Page 2Linked to original sources
PubMed1997

Testosterone-regulated expression of enzymes involved in steroid and aromatic hydrocarbon catabolism in Comamonas testosteroni.

The effect of testosterone as the sole carbon source on protein expression was analyzed in Comamonas testosteroni. Testosterone simultaneously induced the expression of steroid- and aromatic hydrocarbon-catabolizing enzymes and repressed one amino acid-degrading enzyme. It is suggested that steroids play a regulative role in catabolic enzyme synthesis during adaptive growth of C. testosteroni.

Amino Acid Sequence↗
PubMed1997

Characterization of Bacillus subtilis hemN.

A recently cloned Bacillus subtilis open reading frame (hemN) upstream of the dnaK operon was identified as encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation. B. subtilis hemN functionally complemented two Salmonella typhimurium hemF hemN double mutants under aerobic and anaerobic conditions. A B. subtilis hemN mutant accumulated coproporphyrinogen III only under anaerobic conditions. Interestingly, growth experiments using the B. subtilis hemN mutant revealed normal aerobic and anaerobic growth, indicating the presence of an alternative oxygen-independent enzymatic system. Northern blot experiments identified hemN mRNA as part of an approximately 7-kb pentacistronic transcript consisting of lepA, hemN, hrcA, grpE, and dnaK. One potential start site for aerobic and anaerobic transcription was located 37 bp upstream of the translational start codon of lepA. Comparable amounts of hemN transcript were observed under aerobic and anaerobic growth conditions. No experimental evidence for the presence of hemF in B. subtilis was obtained. Moreover, B. subtilis hemY did not substitute for hemF hemN deficiency in S. typhimurium. These results indicate the absence of hemF and suggest the presence of a second hemN-like gene in B. subtilis.

Aerobiosis↗
PubMed1996

The hemA gene encoding glutamyl-tRNA reductase from the archaeon Methanobacterium thermoautotrophicum strain Marburg.

In archaea the first general tetrapyrrole precursor 5-aminolevulinic acid (ALA) is formed via the tRNA-dependent five-carbon pathway from glutamate. We have cloned the hemA gene encoding the central enzyme of the pathway glutamyl-tRNA reductase from the methanogenic archaeon Methanobacterium thermoautotrophicum by complementation of an Escherichia coli hemA mutant to ALA prototrophy. An 1194 bp open reading frame that encodes a 398 amino acid polypeptide with the calculated M, 44,509 was detected. The deduced amino acid sequence showed 20-35% amino acid identity to bacterial HemAs with the highest identity score to the Pseudomonas aeruginosa HemA. An identity of approximately 22% was found to plant HemAs. Glutamyl-tRNA reductase activity was shown for the M. thermoautotrophicum HemA after overexpression in E. coli and partial purification. The enzymatic reaction catalyzed by the partially purified enzyme revealed a temperature optimum of 65 degrees C at an optimal pH of 7.0. The reductase utilized preferentially NADPH for the reduction of the activated carboxyl group. The presence of ATP and GTP showed no obvious influence on catalysis.

Aldehyde Oxidoreductases↗
PubMed1996

[Unusual pathways and environmentally regulated genes of bacterial heme biosynthesis].

The majority of bacteria, all investigated archaea and plants form the general precursor molecule of all tetrapyrroles 5-aminolevulinic acid by a unique transformation of transfer RNA bound glutamate. Only the alpha-group of the proteobacteria, mammals and yeast synthesize 5-aminolevulinic acid via the well known condensation of succinyl-CoA and glycine. The late steps in tetrapyrrole biosynthesis also contain alternative biosynthetic pathways for the formation and oxidative decarboxylation of coproporphyrinogen III. Unusual enzymatic reactions including the utilization of two substrate molecules as cofactor by the porphobilinogen deaminase and the formation of a spiro intermediate are involved in the formation of uroporphyrinogen III. The biosynthesis of hemes in bacteria is strictly regulated at the formation of 5-aminolevulinic acid and the oxidative decarboxylation of coproporphyrinogen III. The involved heme biosynthetic genes are regulated by the environmental concentrations of oxygen, iron, nitrate, growth phase and intracellular levels of heme. The current knowledge on the various enzymatic reactions and gene regulatory mechanisms is reviewed.

Aminolevulinic Acid↗
PubMed1995

The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system.

The Gram-positive soil bacterium Bacillus subtilis, generally regarded as an aerobe, grows under strict anaerobic conditions using nitrate as an electron acceptor and should be designated as a facultative anaerobe. Growth experiments demonstrated a lag phase of 24 to 36 hours after the shift from aerobic, to the onset of anaerobic respiratory growth. Anaerobically adapted cells grew without further lag phase after their transfer to fresh anaerobic growth medium. The cells change their morphology from rods to longer filament-like structures when moved from aerobic to anaerobic respiratory growth conditions. Surprisingly, anaerobically grown B. subtilis lost the capacity for sporulation. An investigation of the molecular basis of the switch between aerobic and anaerobic growth was initiated by the cloning of the genes encoding the respiratory nitrate reductase from B. subtilis. Oligonucleotides deduced from conserved amino acid sequence regions of eubacterial respiratory nitrate reductases and related enzymes were used for the isolation of the genes. Four open reading frames with significant homology to the E. coli respiratory nitrate reductase operons (narGHIJ, narZYWV) were isolated and termed narGHJI. A chromosomal knock-out mutation of the B. subtilis nar operon totally abolished nitrate respiration.

Amino Acid Sequence↗
PubMed1995

Cloning, mapping and characterization of the Pseudomonas aeruginosa hemL gene.

The rate-limiting step in the biosynthesis of tetrapyrroles is the formation of 5-aminolevulinic acid (ALA). In Pseudomonas aeruginosa ALA is synthesized via a two-step reaction from aminoacylated tRNA(Glu) by the action of glutamyl-tRNA reductase and glutamate-1-semialdehyde-2,1-amino mutase. To initiate an investigation of the regulation of the second step in ALA formation, the hemL gene was cloned from P. aeruginosa by complementation of an Escherichia coli hemL mutant. An open reading frame of 1284 bp encoding a protein of 427 amino acids with a calculated molecular mass of 45,404 Da was identified. The hemL gene was mapped to the SpeI fragment Z and the DpnI fragment J1 of the P. aeruginosa chromosome corresponding approximately to min 0.3-0.9. One transcription start site was located 280 bp upstream of the translational start site of the hemL gene. No classical sigma 70-dependent promoter was detected. Oxygen stress induced by the addition of H2O2 to the growth medium led to an approximately 3.5-fold increase in hemL expression as determined by mRNA dot blot assays. Anaerobic denitrifying growth led to a 2-fold stimulation of hemL transcription. Two additional open reading frames were detected downstream of the hemL gene. One open reading frame (orf1) of 549 bp encodes a protein of 182 amino acids with a calculated molecular mass of 19,638 Da.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of the Escherichia coli hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase.

Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic (HemF) and one for anaerobic (HemN) conditions. Here we report the cloning of the hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase from Escherichia coli by complementation of a Salmonella typhimurium hemF hemN double mutant. An open reading frame of 1,371 bp encoding a protein of 457 amino acids with a calculated molecular mass of 52.8 kDa was identified. Sequence comparisons revealed 92% amino acid sequence identity to the recently cloned S. typhimurium hemN gene and 35% identity to the Rhodobacter sphaeroides gene. The hemN gene was mapped to 87.3 min of the E. coli chromosome and found identical to open reading frame o459 previously discovered during the genome sequencing project. Complementation of S. typhimurium hemF hemN double mutants with the E. coli hemN gene was detected under aerobic and anaerobic conditions, indicating an aerobic function for HemN. The previously cloned E. coli hemF gene encoding the oxygen-dependent enzyme complemented exclusively under aerobic conditions. Primer extension experiments revealed a strong transcription initiation site 102 bp upstream of the translational start site. DNA sequences with homology to a sigma 70-dependent promoter were detected. Expression of the hemN gene in response to changing environmental conditions was evaluated by using lacZ reporter gene fusions. Under anaerobic conditions, hemN expression was threefold greater than under aerobic growth conditions. Removal of iron from the growth medium resulted in an approximately fourfold decrease of aerobic hemN expression. Subsequent addition of iron restored normal expression.

Aerobiosis↗
PubMed1995

Regulation of the hemA gene during 5-aminolevulinic acid formation in Pseudomonas aeruginosa.

The general tetrapyrrole precursor 5-aminolevulinic acid is formed in bacteria via two different biosynthetic pathways. Members of the alpha group of the proteobacteria use 5-aminolevulinic acid synthase for the condensation of succinyl-coenzyme A and glycine, while other bacteria utilize a two-step pathway from aminoacylated tRNA(Glu). The tRNA-dependent pathway, involving the enzymes glutamyl-tRNA reductase (encoded by hemA) and glutamate-1-semialdehyde-2,1-aminomutase (encoded by hemL), was demonstrated to be used by Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Comamonas testosteroni, Azotobacter vinelandii, and Acinetobacter calcoaceticus. To study the regulation of the pathway, the glutamyl-tRNA reductase gene (hemA) from P. aeruginosa was cloned by complementation of an Escherichia coli hemA mutant. The hemA gene was mapped to the SpeI A fragment and the DpnIL fragment of the P. aeruginosa chromosome corresponding to min 24.1 to 26.8. The cloned hemA gene, coding for a protein of 423 amino acids with a calculated molecular mass of 46,234 Da, forms an operon with the gene for protein release factor 1 (prf1). This translational factor mediates the termination of the protein chain at the ribosome at amber and ochre codons. Since the cloned hemA gene did not possess one of the appropriate stop codons, an autoregulatory mechanism such as that postulated for the enterobacterial system was ruled out. Three open reading frames of unknown function transcribed in the opposite direction to the hemA gene were found. hemM/orf1 and orf2 were found to be homologous to open reading frames located in the 5' region of enterobacterial hemA genes. Utilization of both transcription start sites was changed in a P. aeruginosa mutant missing the oxygen regulator Anr (Fnr analog), indicating the involvement of the transcription factor in hemA expression. DNA sequences homologous to one half of an Anr binding site were detected at one of the determined transcription start sites.

Acinetobacter calcoaceticus↗
PubMed1994

Transposon Tn5 mutagenesis of pseudomonas fluorescens to isolate mutants deficient in antibacterial activity.

Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn5-GM into the chromosome of P. fluorescens was achieved by heat treatment of the transformed cells at 42 degrees C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens. One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis. These results introduce Tn5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens.

Antibiosis↗
PubMed1994

Isolation of the hemF operon containing the gene for the Escherichia coli aerobic coproporphyrinogen III oxidase by in vivo complementation of a yeast HEM13 mutant.

Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic and one for anaerobic conditions. Here we report the cloning of the hemF gene, encoding the aerobic coproporphyrinogen III oxidase from Escherichia coli, by functional complementation of a Saccharomyces cerevisiae HEM13 mutant. An open reading frame of 897 bp encoding a protein of 299 amino acids with a calculated molecular mass of 34.3 kDa was identified. Sequence comparisons revealed 43% amino acid sequence identity with the product of the S. cerevisiae HEM13 gene and 90% identity with the product of the recently cloned Salmonella typhimurium hemF gene, while a structural relationship to the proposed anaerobic enzyme from Rhodobacter sphaeroides was not obvious. The hemF gene is in an operon with an upstream open reading frame (orf1) encoding a 31.7-kDa protein with homology to an amidase involved in cell wall metabolism. The hemF gene was mapped to 52.6 min of the E. coli chromosome. Primer extension experiments revealed a strong transcription initiation site upstream of orf1. A weak signal, possibly indicative of a second promoter, was also identified just upstream of the hemF gene. A region containing bent DNA (Bent 111), previously mapped to 52.6 min of the E. coli chromosome, was discovered in the 5' region of orf1. Two potential integration host factor binding sites were found, one close to each transcription start site. An open reading frame (orf3) transcribed in a direction opposite that of the hemF gene was found downstream of the hemF gene. It encodes a protein of 40.2 kDa that showed significant homology to proteins of the XylS/AraC family of transcriptional regulators.

Amino Acid Sequence↗
PubMed1994

Financing health services for children in Texas.

In Texas, children's health services are financed by a bewildering combination of private and public sources, each of which employs different eligibility rules, benefit packages, and reimbursement methods. This article describes these financing mechanisms, underscoring their complexity and the need for consolidation and reform. As private coverage for children has declined, it has become less comprehensive with increased coinsurance and deductibles. Concurrently, due to a number of federal initiatives, Medicaid coverage has increased rapidly with new benefits, also now mandated. Levels and structure of payment have influenced variable participation in the program by physicians, rural health clinics, federally qualified health centers, and children's hospitals. The Texas Department of Health and the Texas Department of Mental Health and Mental Retardation have undertaken a number of initiatives to fill in the gaps and to serve mandated categorically eligible populations. Continuity of care is difficult to achieve. Comprehensive community-based care should be promoted with the goal of expanding the role of all types of providers.

Adolescent↗
PubMed1993

Yeast seryl-tRNA synthetase expressed in Escherichia coli recognizes bacterial serine-specific tRNAs in vivo.

The Saccharomyces cerevisiae serS gene which encodes seryl-tRNA synthetase (SerRS) was expressed in Escherichia coli from the promoter and the ribosome binding sequences contained in its own 5'-flanking region. The low level of yeast SerRS in the prokaryotic host was sufficient to permit in vivo complementation of two temperature-sensitive E. coli serS mutants at the nonpermissive temperature. Thus, yeast SerRS can aminoacylate E. coli tRNA(Ser) species in vivo. Yeast SerRS, isolated from an overexpressing E. coli strain by a rapid two-step purification on FPLC, aminoacylated E. coli tRNA with serine much more poorly (relative kcat/Km = 2 x 10(-4)) than its homologous tRNAs. DL-Serine hydroxamate, an inhibitor of E. coli SerRS, inhibits yeast SerRS in vivo and in vitro with an inhibition constant (Ki) of 2.7 mM, a value 90-fold higher than that for E. coli SerRS.

Amino Acid Sequence↗
PubMed1992

Complex formation between glutamyl-tRNA synthetase and glutamyl-tRNA reductase during the tRNA-dependent synthesis of 5-aminolevulinic acid in Chlamydomonas reinhardtii.

The formation of a stable complex between glutamyl-tRNA synthetase and the first enzyme of chlorophyll biosynthesis glutamyl-tRNA reductase was investigated in the green alga Chlamydomonas reinhardtii. Apparently homogenous enzymes, purified after previously established purification protocols were incubated in various combinations with ATP, glutamate, tRNA(Glu) and NADPH and formed complexes were isolated via glycerol gradient centrifugation. Stable complexes were detected only after the preincubation of glutamyl-tRNA synthetase, glutamyl-tRNA reductase with either glutamyl-tRNA or free tRNA(Glu), ATP and glutamate, indicating the obligatory requirement of aminoacylated tRNA(Glu) for complex formation. The further addition of NADPH resulting in the reduction of the tRNA-bound glutamate to glutamate 1-semialdehyde led to the dissociation of the complex. Once complexed to the two enzymes tRNA(Glu) was found to be partially protected from ribonuclease digestion. Escherichia coli, Bacillus subtilis and Synechocystis 6803 tRNA(Glu) were efficiently incorporated into the protein-RNA complex. The detected complexes provide the chloroplast with a potential channeling mechanism for Glu-tRNA(Glu) into chlorophyll synthesis in order to compete with the chloroplastic protein synthesis machinery.

Adenosine Triphosphate↗
PubMed1992

Expression of the Chlamydomonas reinhardtii chloroplast tRNA(Glu) gene in a homologous in vitro transcription system is independent of upstream promoter elements.

Chloroplast tRNA(Glu) is a bifunctional molecule involved in both the early steps of chlorophyll synthesis and chloroplast protein biosynthesis. Recently the enzymes involved in these processes have been characterized from the green alga Chlamydomonas reinhardtii. In order to investigate whether transcription of the gene for the tRNA(Glu) cofactor would be a possible point of regulation for the biosynthesis of chlorophyll, a homologous in vitro transcription system for C. reinhardtii chloroplast RNA polymerase was developed. The enzymatic activity was partially purified by ion-exchange chromatography to separate it from nuclear RNA polymerases. The highest rate of synthesis was found at pH 7.9, 40 mM KCl, 9 mM MgCl2 and with 25 micrograms plasmid DNA containing the chloroplast tRNA gene per milliliter. The activity was not sensitive to high amounts of alpha-amanitin (500 micrograms/ml) and rifampicin, but was clearly inhibited by heparin. This system was used to undertake a promoter analysis of one of the two identical tRNA(Glu) gene copies found in the C. reinhardtii chloroplast genome (trnE1). The analyzed tRNA gene behaved like a single transcription unit driven by its own promoter. The transcript terminated in a run of four consecutive T residues downstream of the gene. The nucleotide sequence in the 5' region of the gene revealed several potential promoter elements with homology to known chloroplast promoters of the "-10 and -35 region" and the "Euglena promoter" types. Surprisingly, deletion of the complete 5' region did not affect in vitro transcription, while partial deletions of the 5' and 3' coding region totally abolished transcription. This indicates the presence of an internal control region previously found for genes transcribed by nuclear RNA polymerase III. Protein binding studies with the coding region of trnE1 using gel retardation assays demonstrated the formation of two differently sized complexes. In vitro transcription of the tRNA(Glu) gene in extracts prepared from light and dark grown algae failed to demonstrate any significant influence of light on the transcription reaction.

Animals↗
PubMed1992

Activity and spectroscopic properties of the Escherichia coli glutamate 1-semialdehyde aminotransferase and the putative active site mutant K265R.

Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,1-aminomutase; EC 5.4.3.8; GSA-AT) catalyzes the transfer of the amino group on carbon 2 of glutamate 1-semialdehyde (GSA) to the neighboring carbon 1 to form delta-aminolevulinic acid (ALA). To gain insight into the mechanism of this enzyme, possible intermediates were tested with purified enzyme and the reaction sequence was followed spectroscopically. While 4,5-dioxovaleric acid (DOVA) was efficiently converted to ALA by the pyridoxamine 5'-phosphate (PMP) form of the enzyme, 4,5-diaminovaleric acid (DAVA) was a substrate for the pyridoxal 5'-phosphate (PLP) form of GSA-AT. Thus, both substances are reaction intermediates. The purified enzyme showed an absorption spectrum with a peak around 338 nm. Addition of PLP led to increased absorption at 338 nm and a new peak around 438 nm. Incubation of the purified enzyme with PMP resulted in an additional absorption peak at 350 nm. The reaction of the PLP and PMP form of the enzyme with GSA allowed the detection of a series of peaks which varied in their intensities in a time-dependent manner. The most drastic changes to the spectrum that were observed during the reaction sequence were at 495 and 540 nm. Some of the detected absorption bands during GSA-AT catalysis were previously described for several other aminotransferases, indicating the relationship of the mechanisms. The reaction of the PMP form of the enzyme with DOVA resulted in a similar spectrum as described above, while the spectrum for the conversion of DAVA by the PLP form of the enzyme indicated a different mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Glutamyl-tRNA reductase from Escherichia coli and Synechocystis 6803. Gene structure and expression.

In the cyanobacterium Synechocystis sp. PCC 6803 and in the enterobacterium Escherichia coli delta-amino-levulinic acid (ALA) is formed from glutamyl-tRNA by the sequential action of two enzymes, glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase. E. coli has two GluTR proteins with sizes of 45 kDa (GluTR45) and 85 kDa (GluTR85) (Jahn, D., Michelsen, U., and Söll, D. (1991) J. Biol. Chem. 266, 2542-2548). The hemA gene, isolated from E. coli and several other eubacteria, has been proposed to encode a structural component of GluTR. Because of the inability to overexpress this gene in E. coli, we demonstrate directly GluTR function for the E. coli hemA gene product by its expression and functional analysis in yeast, which does not form ALA from Glu-tRNA. Gel filtration experiments demonstrated definitively that the yeast-expressed HemA protein corresponded to GluTR45. Furthermore, analysis of GluTR activity in an E. coli strain with a disrupted hemA gene displayed GluTR85, but not GluTR45 activity. The hemA gene from Synechocystis 6803 was cloned by functional complementation in E. coli. DNA sequence analysis revealed an open reading frame capable of encoding a 427-amino acid polypeptide (molecular mass of 47,525 Da). The Synechocystis 6803 amino acid sequence shows significant similarity upon alignment with HemA sequences from E. coli, Bacillus subtilis, Salmonella typhimurium, and Chlorobium vibrioforme but does not contain the amino acid sequence derived from the N terminus of the previously purified GluTR protein (Rieble, S., and Beale, S. I. (1991) J. Biol. Chem. 266, 9740-9745). These experiments are the first direct demonstration of GluTR activity of the HemA protein and provide further evidence for two pathways of ALA formation in prokaryotes.

Aldehyde Oxidoreductases↗
PubMed1992

Glutamyl-transfer RNA: a precursor of heme and chlorophyll biosynthesis.

In green plants, archaebacteria and many eubacteria, the porphyrin ring that is common to both chlorophyll and heme is synthesized from 5-aminolevulinic acid (ALA) via an interesting pathway. This two-step process involves the unusual enzymes glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase. Interest in this pathway has increased since it was discovered that a tRNA cofactor was required for the formation of ALA. This tRNA(Glu) is common to the biosyntheses of both porphyrins and proteins.

Aldehyde Oxidoreductases↗