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B Hutter

Publications and source records attributed to B Hutter.

12 recordsLinked to original sources
PubMed2006

Repetitive elements in imprinted genes.

Genomic imprinting in mammals results in mono-allelic expression of about 80 genes depending on the parental origin of the alleles. Though the epigenetic mechanisms underlying imprinting are rather clear, little is known about the genetic basis for these epigenetic mechanisms. It is still rather enigmatic which sequence features discriminate imprinted from non-imprinted genes/regions and why and how certain sequence elements are recognized and differentially marked in the germlines. It seems likely that specific DNA elements serve as signatures that guide the necessary epigenetic modification machineries to the imprinted regions. Inter- and intraspecific comparative genomic studies suggest that the unusual occurrence and distribution of various types of repetitive elements within imprinted regions may represent such genomic imprinting signatures. In this review we summarize the various observations made and discuss them in light of experimental data.

Animals↗
PubMed2000

Analysis of the dormancy-inducible narK2 promoter in Mycobacterium bovis BCG.

Upon depletion of oxygen, the obligate aerobe mycobacteria switch from growth to a state of non-replicating persistence or dormancy. Here, we report the first functional analysis of a dormancy-dependent mycobacterial promoter in Mycobacterium bovis BCG. Promoter probing using a 'lacZ reporter detected a dormancy-inducible promoter activity upstream of the coding sequence for the putative nitrite extrusion protein NarK2. Primer extension analysis mapped a transcriptional start point 47 bp upstream of the narK2 start codon. Deletion analysis revealed that the sequence -222 to -133 bp upstream from the transcriptional start point was required for basal and dormancy-inducible reporter expression. The sequence +1 to +47 downstream of the transcriptional start point had a strong inhibitory effect on the level of dormancy-induced beta-galactosidase activity. The identification of apparent activating and inhibiting regions suggests that the narK2 promoter is at least under dual control.

Anaerobiosis↗
PubMed1999

Properties of the 40 kDa antigen of Mycobacterium tuberculosis, a functional L-alanine dehydrogenase.

The 40 kDa antigen of Mycobacterium tuberculosis is the first antigen reported to be present in the pathogenic M. tuberculosis, but not in the vaccine strain Mycobacterium bovis BCG. It is a functional L-alanine dehydrogenase (EC 1.4.1.1) and hence one of the few antigens possessing an enzymic activity. This makes the 40 kDa antigen attractive for potential diagnostic and therapeutic interventions. Recently, we developed a strategy to purify quantities of the recombinant protein in active form, and here we describe the biochemical properties of this enzyme. In the oxidative-deamination reaction, the enzyme showed K(m) values of 13. 8 mM and 0.31 mM for L-alanine and NAD(+), respectively, in a random-ordered mechanism. K(m, app) values in the reductive-amination reaction are 35.4 mM, 1.45 mM and 98.2 microM for ammonium, pyruvate and NADH, respectively. The enzyme is highly specific for all of its substrates in both directions. The pH profile indicates that oxidative deamination virtually may not occur at physiological pH. Hence L-alanine most likely is the product of the reaction catalysed in vivo. The enzyme is heat-stable, losing practically no activity at 60 degrees C for several hours.

Alanine Dehydrogenase↗
PubMed1999

Up-regulation of narX, encoding a putative 'fused nitrate reductase' in anaerobic dormant Mycobacterium bovis BCG.

Mycobacterium tuberculosis and its closely related but non-pathogenic relative M. bovis Bacille Calmette-Guèrin (BCG) have the capability to adapt to anaerobiosis by shifting down from aerobic growth to a state of non-replicating persistence or dormancy. Here, we report the results of a comparative Northern analysis of 23 genes identified in the tubercle bacillus genome project that might play a role in the energy metabolism under anaerobic conditions. The expression of a majority of the genes was found to be down-regulated in the dormant BCG culture. However, the mRNA level for narX, a putative 'fused nitrate reductase' not found in other bacteria, was strongly up-regulated in anaerobic dormant bacilli. narX is the first transcriptionally induced gene in anaerobic dormant mycobacteria and might be a useful marker for monitoring the dormancy response in infected animals.

Anaerobiosis↗
PubMed1999

Molecular genetic characterisation of whiB3, a mycobacterial homologue of a Streptomyces sporulation factor.

WhiB is an essential sporulation factor in Streptomyces coelicolor. We report here the molecular genetic characterisation of whiB3, a whiB-like gene in the nonspore-forming Mycobacterium smegmatis mc(2)155. M. smegmatis whiB3 encodes a 96-amino-acid protein with 81% similarity to its M. tuberculosis counterpart identified in the genome project, and 35% similarity to S. coelicolor WhiB. In both mycobacteria, whiB3 is flanked by the same upstream gene, Rv3415c, and appears to be monocistronic. Promoter probe analyses suggest that the whiB3 gene is expressed constitutively. Disruption of whiB3 did not affect growth or the dormancy response of M. smegmatis.

Amino Acid Sequence↗
PubMed1999

Oxygen depletion-induced dormancy in Mycobacterium bovis BCG.

Gradual depletion of oxygen causes the shift-down of aerobic growing Mycobacterium bovis BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and alpha-crystallin-like protein and is sensitive to metronidazole.

Amino Acid Oxidoreductases↗
PubMed1998

Increased alanine dehydrogenase activity during dormancy in Mycobacterium smegmatis.

The aerobic fast-growing Mycobacterium smegmatis has, like its slow-growing pathogenic counterpart M. tuberculosis, the capability to adapt to anaerobiosis by shifting down to a drug resistant dormant state. Here, we report the identification of the first enzyme, L-alanine dehydrogenase, whose specific activity is increased during dormancy development in M. smegmatis. This mycobacterial enzyme activity was previously identified as the 40-kDa antigen in M. tuberculosis and shows a preference for the reductive amination of pyruvate to alanine at physiological pH. The determination of the temporal profile of alanine dehydrogenase activity during dormancy development showed that the activity stayed at a low baseline level during the initial aerobic exponential growth phase (0.7 mU mg-1 min-1). After termination of aerobic growth, alanine dehydrogenase activity increased rapidly 5-fold. As oxygen becomes more and more limiting, the enzyme activity declined until it reached a level about two-third that of the peak value. The strong induction immediately after deflection from aerobic growth suggests that alanine might be required for the adaptation from aerobic growth to anaerobic dormancy. As alanine synthesis is coupled to NADH oxidation, we propose that the induction of alanine dehydrogenase activity might also support the maintenance of the NAD pool when oxygen as a terminal electron acceptor becomes limiting.

Adaptation, Physiological↗
PubMed1998

Host vector system for high-level expression and purification of recombinant, enzymatically active alanine dehydrogenase of Mycobacterium tuberculosis.

The 40-kDa antigen of M. tuberculosis, which is an alanine dehydrogenase, is a species-specific antigen that is potentially useful for strain identification. Large quantities of the purified protein are required for immunological, as well as for detailed biochemical and structural, characterization. The AlaDH gene was cloned by PCR from H37Rv (virulent) and H37Ra (partially attenuated) strains of M. tuberculosis, and their DNA sequence was determined. A host-vector system suitable for the production of sufficient quantities of the recombinant AlaDH antigen was developed. The AlaDH gene was expressed under the control of strong, transcriptional (bacteriophage pLpR) and translational (atpE) signals. High-level expression of soluble AlaDH was obtained using the recombinant E. coli K-12 strain CAG629 [pMSK12], which is deficient in Lon protease and the heat-shock response. A simple two-step procedure for the rapid purification of the recombinant protein was developed. The protein was purified to near homogeneity, and the purified AlaDH showed a specific enzyme activity comparable to the native protein isolated from M. tuberculosis. In addition, the product showed an expected amino acid sequence and reacted strongly to the 40-kDa (AlaDH)-specific mAb HBT-10. Furthermore, the epitope of the mAb HBT-10 was mapped to a 12-amino-acid region. Contrary to the published results, we show that the AlaDH and the PNT (pyridine nucleotide transhydrogenase) of M. tuberculosis do not share common epitopes reacting to the species-specific mAb HBT-10. The availability of highly purified AlaDH should now enable a detailed biochemical and structural characterization of this important enzyme of M. tuberculosis.

Alanine Dehydrogenase↗
PubMed1997

A study of combined filtration and adsorption on nylon-based dye-affinity membranes: separation of recombinant L-alanine dehydrogenase from crude fermentation broth.

Dextran, hydroxyethylcellulose (HEC), and poly(vinyl alcohol) PVA were covalently linked to bisoxirane-activated nylon membranes. Cibacron Blue F3G-A was immobilized on to these membranes to yield a dye-affinity membrane. The hydrodynamic permeability of affinity membranes was reduced to approximately 50% of that of the original Nylon membrane due to extension of polymer coils into flow-through pores. Adsorption of pre-purified human serum albumin (HSA) and malate dehydrogenase (MDH) displayed highest maximum binding capacities on HEC-coated dye-ligand-affinity membranes, ranging from (163 micrograms/cm2 for HSA to 316 micrograms/cm2 for MDH. The protein recovery of HSA was 100% on dextran-coated membranes compared with 70% on PVA-coated membranes, whereas almost 100% recovery was found for MDH, independent of the polymer. Application of crude supernatant from recombinant Escherichia coli yielded purification factors of 7.4, 8.9 and 11.2 for recombinant alanine dehydrogenase from Mycobacterium tuberculosis for HEC-, dextran- and PVA-coated membranes respectively. Dynamic capacities decreased remarkably to approximately 3 micrograms/cm2 due to co-adsorption of host proteins. The presence of cell debris caused only a slight decrease of purification factors, but a dramatic decrease of the permeability of affinity membranes due to development of a particle layer in front of the membranes. Although enzyme recoveries were up to 90% using cell-free supernatant, more than 50% of the product was lost due to polarization, concentration and rejection at particle layers when using crude homogenates. In order to further improve this integrated downstream process, sophisticated membrane techniques are required by which the formation of a filter cake is circumvented. Further refinement of polymer-coated membranes would not help one to avoid this problem.

Alanine Dehydrogenase↗