Genetic analysis of candidate genes modifying the age-at-onset in Huntington’s disease

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington’s disease (HD) and determines 42–73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

RanBP10 is a cytoplasmic guanine nucleotide exchange factor that modulates noncentrosomal microtubules

Microtubule spindle assembly in mitosis is stimulated by Ran.GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including megakaryocytes. In these cells, platelet biogenesis requires massive mobilization of microtubules in the cell periphery, where they form proplatelets, the immediate precursors of platelets, in the apparent absence of centrioles. Here we identify a cytoplasmic Ran-binding protein, RanBP10, as a factor that binds beta-tubulin and associates with megakaryocyte microtubules. Unexpectedly, RanBP10 harbors GEF activity toward Ran. A point mutation in the candidate GEF domain abolishes exchange activity, and our results implicate RanBP10 as a localized cytoplasmic Ran-GEF. RNA interference-mediated loss of RanBP10 in cultured megakaryocytes disrupts microtubule organization. These results lead us to propose that spatiotemporally restricted generation of cytoplasmic Ran.GTP may influence organization of the specialized microtubules required in thrombopoiesis and that RanBP10 might serve as a molecular link between Ran and noncentrosomal microtubules.     

Single nucleotide specific detection of DNA by native chemical ligation of fluorescence labeled PNA-probes

DNA-directed chemical ligations provide the opportunity to diagnose DNA sequences with very high sequence specificity. Fluorescent labels have been attached to reactive probes to enable the homogeneous detection of DNA and RNA. However, it has frequently been found that the attachment of fluorescent labels results in decreases of ligation fidelity. Herein we describe the development of a fluorogenic ligation reaction that provides for 10(2)-fold to perfect sequence selectivity. The reaction is based on the isocysteine-mediated native chemical PNA ligation. It is shown that DNA-induced rate accelerations of approximately 43.000-fold can be obtained through subtle variations of the ligation conditions. PNA-thioesters and isocysteine-PNA conjugates were labeled with FAM and TMR fluorophores, respectively. For gaining rapid synthetic access, a convenient on-resin labeling approach was developed. A new PNA monomer featuring an Alloc-protected lysine side chain was synthesized and coupled in solid-phase PNA synthesis. In the event of a ligation reaction the two fluorophores are brought into proximity. It is shown that fluorescence resonance energy transfer provides a positive fluorescence signal which is specific for product formation rather than for loss of starting materials. Single base mutations can be detected within minutes and with very high sequence selectivity at optimized conditions.     

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. It was possible to isolate them in an absolutely pure state. The described method is operationally simple and very efficient.     

Pump and displacement currents of reconstituted ATP synthase on black lipid membranes

Summary Purified ATP synthase (F ₀ F ₁) fromRhodospirillum rubrum was reconstituted into asolectin liposomes which were than adsorbed to a planar lipid bilayer. After the addition of an inactive photolabile ATP derivative (caged ATP), ATP was released after illumination with UV light, which led to a transient current in the system. The transient photocurrent indicates that the vesicles and the planar membrane are capacitatively coupled. Stationary pump currents were obtained after addition of protonophores. These currents are specifically inhibited by oligomycin and stimulated threefold by inorganic phosphate (P _i ). In analogy oligomycin-sensitive pump currents in the reverse direction coupled to net ATP synthesis were induced by a light-induced concentration jump of ADP out of caged ADP, demonstrating the reversibility of the pump. For this, a preformed proton motive force and P _i were necessary.In a second series of experiments, proteoliposomes containing both ATP synthase and bacteriorhodopsin were adsorbed to a planar bilayer. The system was excited by a laser flash. The resulting photocurrents were measured with a time resolution of 2 sec. In the presence of ADP, the signal was modulated by the electrical activity of ATP synthase. ADP-induced charge displacements in ATP synthase, with time constants of 11 and 160 sec were obtained. The kinetics of the charge movements were slowed down byF ₀ specific inhibitors (DCCD or oligomycin) and were totally absent if ADP binding toF ₁ is prevented by the catalytic site-blocking agent NBD-Cl. The charge displacement of ATP synthase is coupled only to the membrane potential induced by the electrical activity of bacteriorhodopsin. The charge movements are interpreted as conformational transitions during early steps of the reaction cycle of ATP synthase.     

Experimental retracement of the origins of a protocell

Although Oparin used coacervate droplets from two or more types of polymer to model the first cell, he hypothesized homacervation from protein, consistent with Pasteur and Darwin. Herrera made two amino acids and numerous cell-like structures (sulfobes) in the laboratory, which probably arose from intermediate polymers. Our experiments have conformed with a homoacervation of thermal proteinoid, in which amino acid sequences are determined by the reacting amino acids themselves. All proteinoids that have been tested assemble themselves alone in water to protocells. The protocells have characteristics of life defined by Webster's Dictionary: metabolism, growth, reproduction and response to stimuli in the environment. The protocells are able also to evolve to more modern cells including the initiation of a nucleic acid coding system.Principal spinoffs from the results are revised evolutionary theory, models for protoneurons and networks thereof, and numerous industrial applications of thermal polyamino acids. Life itself has thus been reaffirmed to be rooted in protein, not in DNA nor RNA, which are however crucial to inheritance in modern life as instruction manual (Kornberg).Recognition of the advances have been considerably delayed by the deeply held assumption that life began by chance from random polymerization of amino acids, in contrast to the experimental findings. The concepts of DNA/RNA-first and protein-first are reconciled by a rise-and-fall progression as often seen in biochemical and biological evolution.The fact that amino acids order themselves explains in turn that thermal copolyamino acids are finding numerous applications. The entire sequence of processes in the proteinoid origins theory is now seen to be highly deterministic, in close accord with Einstein.     

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. After incorporation of this dATP analog into DNA containing the tet operator sequence the investigation of the interactions between tet operator DNA and Tet repressor protein by UV photocross-linking becomes possible. Photocross-linking of protein to DNA was demonstrated by the reduced migration of the DNA in SDS polyacrylamide gel electrophoresis. Addition of the inducer tetracycline prior to UV irradiation significantly reduces the DNA-protein cross-linking rate. The long wave UV light applied here does not significantly alter the DNA or the protein under the photocross-linking conditions.     

Ein Vergleich der Wirkung von ionisierender Strahlung auf peptidgebundene und freie Aminosäuren in festem Zustand

Zusammenfassung Untersuchungen an thermischen Polymeren von-Aminosäuren in festem Zustand zeigen, daß in diesen insbesondere Tryptophan, Histidin, Cystin, Lysin und Methionin eine höhere Strahlenempfindlichkeit als in den bisher untersuchten Proteinen aufweisen. Diese Ergebnisse werden verglichen mit ähnlichen Untersuchungen an Filmen von Aminosäuremischungen, die in noch stärkerem Umfang auf einen beträchtlichen Energietransfer oder Chargetransfer in Richtung auf die vier genannten Aminosäuren schließen lassen. Die Ergebnisse werden auch in Hinsicht auf die Strahlenempfindlichkeit von Aminosäuren in Proteinen und auf die Inaktivierung von Enzymen diskutiert.

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A comparison of the effect of ionizing radiation on peptide- bound and free amino acids in the solid state

Summary Irradiation of thermal polymers of-amino acids with X-rays in the solid state produces a significantly increased destruction of tryptophan, histidine, cystine, lysine and methionine as compared with the response of constituent amino acids in proteins. These results are discussed with respect to related results obtained by irradiation of dry films of amino acid mixtures which indicate an even stronger energy or charge transfer towards the four amino acids mentioned. The results are also discussed with respect to the radiation sensitivity of constituent amino acids in proteins and the inactivation of enzymes.

     

Veränderung von Aminosäuren und Aktivität bei Röntgenbestrahlung von krystallisiertem Lysozym

Zusammenfassung Kristallisiertes Lysozym wurde mit Dosen zwischen 1 und 100 Mrad Röntgenstrahlen bestrahlt. Die kleineren Dosen haben keine meßbare Wirkung auf Aktivität oder quantitative Aminosäurezusammensetzung. Da das Molekül durch größere Strahlendosen in andere Modifikationen umgewandelt wird und die entsprechenden Vorgänge komplex sind, läßt sich die naktivierungdes Enzyms nicht ohne weiteres durch die lassische Treffertheorie interpretieren. Hiermit zusammenhängende Probleme werden diskutiert. Erst Strahlendosen von der Größenordnung 100 Mrad erzeugen in einem automatischen Aminosäureanalysator sicher analysierbare Aminosäureveränderungen. Die G-Werte liegen zwischen 3 und 13. Die größere Strahlenempfindlichkeit haben vor allem die aromatischen und die schwefelhaltigen Aminosäuren. Vergleiche mit anderen Ergebnissen führen zu dem Schluß, daß diese Strahlenempfindlichkeit der Aminosäuren durch Milieufaktoren nur wenig beeinflußt wird.

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Summary Crystallized lysozyme was X-irradiated with doses between 1 and 100 Mrads. Doses of a few Mrads have no measurable effect on activity and quantitative amino acid composition of lysozyme. However, increasing doses successively convert the original protein into a large number of modified proteins showing more or less lysozyme activity. These processes are of a complex nature. Consequently can the inactivation not be described in terms of the classical target theory. Only X-ray doses of the order of magnitude of 100 Mrads produce amino acid changes which can clearly be determined with an automatic amino acid analyzer. The G-values for the destruction of the various constituent amino acids are between 3 and 13. The larger sensitivities have the S-containing and aromatic amino acids as well as the long chain aliphatic amino acids. A comparison with other results shows that the radiation sensitivity of the constituent amino acids of solid proteins is little influenced by other factors (O₂-pressure, water content, protein structure etc.).

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Auszugsweise vorgetragen während des ESR-Symposiums Gatlinburg, Tenn., Mai 1965, und während eines Chemischen Kolloquiums an der University of Colorado in Boulder am 21. Oktober 1965.