Ultraviolet crosslinking of DNA protein complexes via 8-azidoadenine.

The synthesis of 8-azido-2'-deoxyadenosine-5'-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography and UV spectroscopy. Its photoreactivity upon UV irradiation was studied.

Survival of microorganisms under the extreme conditions of the Atacama Desert.

Spores of Bacillus subtilis, conidia of Aspergillus niger, versicolor and ochraceus and cells of Deinococcus radiodurans have been exposed in the dark at two locations (at about 23 degrees S and 24 degrees S) in the Atacama Desert for up to 15 months. B. subtilis spores (survival approximately 15%) and A.

Response of Bacillus subtilis spores to dehydration and UV irradiation at extremely low temperatures.

Spores of Bacillus subtilis have been exposed to the conditions of extreme dehydration (argon/silica gel; simulated space vacuum) for up to 12 weeks at 298 K and 80 K in the dark. The inactivation has been correlated with the production of DNA-double strand-breaks. The temperature-dependence of the rate constants for inactivation or production of DNA-double strand-breaks is surprisingly low.

DNA stability and survival of Bacillus subtilis spores in extreme dryness.

The inactivation of Bacillus subtilis spores during long-term exposure (up to several months) to extreme dryness (especially vacuum) is strain-dependent, through only to a small degree. During a first phase (lasting about four days) monolayers of spores lose about 20% of their viability, regardless of the strain studied. During this phase loss in viability can be equally attributed both to damages of hydrophobic structures (membranes and proteins) and DNA.

Biochemical constraints for survival under Martian conditions.

A wide variety of terrestrial organisms, the so-called "anhydrobiotes," has learned to survive in a state of extreme dehydration in dry environments. Strategies for survival include the accumulation of certain polyols and nonreducing saccharides, which help to prevent damage to membranes and proteins, but at low water partial pressure DNA is also progressively damaged by various lesions, including strand breaks and cross-linking to proteins. These lesions, if they are not too numerous, can be repaired before the first replication step after rehydration, but long-term exposure to dry conditions finally diminishes the chances of survival as these lesions accumulate.

ERA-experiment "Space Biochemistry".

The general goal of the experiment was to study the response of anhydrobiotic (metabolically dormant) microorganisms (spores of Bacillus subtilis, cells of Deinococcus radiodurans, conidia of Aspergillus species) and cellular constituents (plasmid DNA, proteins, purple membranes, amino acids, urea) to the extremely dehydrating conditions of open space, in some cases in combination with irradiation by solar UV-light. Methods of investigation included viability tests, analysis of DNA damages (strand breaks, DNA-protein cross-links) and analysis of chemical effects by spectroscopic, electrophoretic and chromatographic methods. The decrease in viability of the microorganisms was as expected from simulation experiments in the laboratory.

Purification and characterization of the inhibitory subunit (delta) of the ATP-synthase from Micrococcus luteus.

Subunit delta was isolated from the ATP-synthase from Micrococcus luteus strain (ATCC 4698). delta, in the case of M. luteus F0F1-ATPase, acts as an inhibitor of ATP hydrolysis and thus resembles subunits in E.

Extreme dryness and DNA-protein cross-links.

Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125 J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography.

Survival in extreme dryness and DNA-single-strand breaks.

A wide variety of organisms (the so-called "anhydrobiotes') is able to survive long periods of time in a state of utmost dehydration and can thus survive in extremely dry environments including artificially imposed or space vacuum. Known strategies of survival include the accumulation of certain polyols, especially disaccharides, which help prevent damage to membranes and proteins. Here we report that DNA in vacuum-dried spores is damaged to a very substantial degree by processes leading to DNA strand breaks.

Structural dynamics in F1ATPase during the first reaction cycle of ATP hydrolysis.

The velocity of ATP hydrolysis, catalyzed by purified F1ATPase from Micrococcus luteus, was decelerated on decreasing the temperature. At 13 degrees C one reaction cycle is completed after 20 s. Hydrolysis was triggered upon rapid mixing of the enzyme with ATP.

DNA-strand breaks limit survival in extreme dryness.

The inactivation of the anhydrobiotic organisms Bacillus subtilis (spores) and Deinococcus radiodurans during long-term exposure (up to several weeks) to extreme dryness (especially vacuum) is correlated with an increase in the number of DNA-strand breaks and other DNA lesions. Survival finally depends on the repair of DNA damages. Exposure of anhydrobiotic organisms to extreme dryness (e.

UV-induced cross-linking of Tet repressor to DNA containing tet operator sequences and 8-azidoadenines.

The synthesis of 8-azido-2'-deoxyadenosine-5'-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography, proton resonance spectroscopy, infrared spectroscopy and UV spectroscopy. Its photolysis upon UV irradiation was studied.

3'-Arylazido-beta-alanyl-2-azido ATP, a cross-linking photoaffinity label for F1ATPases.

The synthesis of the 3'-arylazido-2-azido ATP derivative 3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino]propionyl)2-azido-adenosine 5'-triphosphate (2,3'-DiN3ATP) is described. The bifunctional photoreactive ATP analog is characterized spectroscopically. Photoaffinity labeling of F1ATPase from Micrococcus luteus by this analog results in the inactivation of the enzyme and in the formation of higher molecular weight cross-links, composed of alpha- and beta-subunits.

Isolation and partial characterization of a cytochrome-o complex from chromatophores of the photosynthetic bacterium Rhodospirillum rubrum FR1.

A cytochrome-o complex was isolated from chromatophores of photoheterotrophically grown Rhodospirillum rubrum FR1. The enzyme was extracted with the non-denaturating detergent taurodeoxycholate and subsequently purified by sucrose-density-gradient centrifugation and gel-permeation HPLC. The complex contains two types of cytochromes, one of them cytochrome o, and two copper atoms.

UV-induced cross-linking of proteins to plasmid pBR322 containing 8-azidoadenine 2'-deoxyribonucleotides.

An efficient method of cross-linking DNA to protein is described. The method is based on the incorporation of photoactive 8-azidoadenine 2'-deoxyribonucleotides into DNA. We have found that 8-N3dATP is a substrate for E.

Production and characterization of a monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol.

A monoclonal antibody was obtained by the fusion of mouse myeloma cells with splenocytes isolated from Balb/c mice, which had been immunized with diacetoxyscirpenol-hemiglutarate (DAS-hemiglutarate) and verrucarol-hemiglutarates covalently bound to ethylenediamine-modified bovine serum albumin. The anti-DAS-antibody that could be induced was of the IgM type with kappa-chains. The titer of the monoclonal anti-DAS-antibody in ascites fluid obtained from mice injected the selected cell line was much higher than those of conventional antisera.

Coreconstitution of bacterial ATP synthase with monomeric bacteriorhodopsin into liposomes. A comparison between the efficiency of monomeric bacteriorhodopsin and purple membrane patches in coreconstitution experiments.

The conditions for coreconstitution of a bacterial ATP synthase and bacteriorhodopsin into lecithin liposomes and for light driven ATP synthesis have been optimized. A rate of maximally 280 nmol ATP min-1 mg ATP synthase-1 was achieved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min-1 mg-1 found for proteoliposomes containing bacteriorhodopsin in the form of purple membrane patches. The different rates are explained by the finding that monomeric bacteriorhodopsin is more homogeneously distributed among the liposomes than the purple membrane patches.

Survival under space vacuum--biochemical aspects.

Exposure to vacuum predominantly causes the removal of water. As a consequence hydrophobic bonds (e.g.

Hypotheses on the appearance of life on Earth (review).

It is generally accepted within the natural sciences that life emerged on Earth by a kind of proto-Darwinian evolution from molecular assemblies that were predominantly formed from the various constituents of the primitive atmosphere and hydrosphere. Evolutionary stages under discussion are: the self-organization of spontaneously formed biomolecules into early precursors of life (protobionts), their stepwise evolution via (postulated) protocells to (postulated) progenotes and the Darwinian evolution from progenotes to the three kingdoms of contemporary organisms (archaebacteria, eubacteria and eukaryotes). Considerable discrepancies between scientists have arisen because all evolutionary stages from prebiotic molecules to progenotes are entirely hypothetical and so are the postulated environmental conditions.

Vitamin K epoxide reductase activity in the metabolism of epoxides.

The importance of vitamin K epoxide reductase for the metabolism of a range of structurally diverse epoxides has been investigated. Vitamin K1 epoxide is reduced by rat liver microsomes at a rate of 0.47 nmoles/g liver/min.

Simultaneous isolation of xanthomegnin, viomellein, rubrosulphin, viopurpurin, and brevianamide A by preparative HPLC.

A method for the isolation of xanthomegnin, viomellein, rubrosulphin, viopurpurin, and brevianamide A from Penicillium viridicatum (DSM 2447) is described. After extraction, HPLC was performed with a preparative silicagel column, eluted with toluene / ethyl acetate / formic acid (27/9/1, v/v/v) and dichloromethane / acetic acid (9/1, v/v). The toxins were detected with a UV-monitor.

Photoaffinity cross-linking of the coupling factor 1 from Micrococcus luteus by 3'-arylazido-8-azido-ATP.

The vicinity of nucleotide binding sites and the mechanism of ATP synthesis/hydrolysis have been studied with the bifunctional photosensitive ATP analog 3'-arylazido-8-azido-ATP. 3'-Arylazido-8-azido-ATP is hydrolyzed by the F1-ATPase from Micrococcus luteus in the absence of ultraviolet light. Irradiation, by ultraviolet light, of F1-ATPase in the presence of 3'-arylazido-8-azido-ATP results in the specific formation of cross-links between alpha and beta subunits.

Life sciences: microorganisms in the space environment.

Preliminary results of the Spacelab 1 experiment on the response of Bacillus subtilis spores to conditions of free space are presented. Exposure to the vacuum of space on the Spacelab pallet reduced viability counts about 50 percent and increased mutation frequencies by a factor of about 10. Interpretation of apparent differences in the photobiological and photochemical data between flight and ground simulation experiments will require more statistical analyses and data from actual fluence measurements.