Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the rale of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate-sensitive activity was not influenced. We conclude that the DNA strand-breaks induced by low doses of X-rays. UV-light or bleomycin do not increase the total or the repair-DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand-breaking is not preceded by enhanced DNA polymerase activity.     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.     

Experimentelle Untersuchungen zur Frage der Entstehung roter Lipochrome in Vogelfedern

Zusammenfassung Das orangerote Lipochrom im Gefieder des Roten Farbenkanarienvogels und des Kapuzenzeisigs (Spinus cucullatus) ist eine Mischfarbe, die aus Canthaxanthin und Kanarienxanthophyll besteht. Auch im Eidotter ist Canthaxanthin als Begleitpigment gelber Carotinoide vorhanden. Der Entzug der Nahrungscarotinoide hat in kürzester Zeit die fast völlige Depigmentierung der Federn zur Folge.Von fünf am Rotvogel getesteten definierten Carotinoidfarbstoffen sind vier von Einfluß auf die Gefiederfarbe: während Rhodoxanthin und Carotin nur schwache Rosafärbung der Federn hervorrufen und Lutein die normale orangerote Gefiederfarbe bedingt, ist die Wirkung von Canthaxanthin am intensivsten, indem es starke Rotfärbung zur Folge hat. Die Ergebnisse am Gelbvogel sind vergleichsweise erwähnt.Es werden die Möglichkeiten der Enstehung roter Lipochrome aus gelben Nahrungscarotinoiden besprochen.     

Resonance Raman characterization of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

 Di-heme Pseudomonas stutzeri cytochrome c ₄ has been characterized by electronic absorption and resonance Raman spectroscopies in the ferric and ferrous forms at pH 7.5 and at room temperature. The data indicate that the two hemes are inequivalent. It is proposed that the N-terminal contains a more relaxed heme as a consequence of the relative orientation of the methionine and histidine ligands with respect to the N-Fe-N directions of the heme plane. This causes a weakening of the Fe-S bond with concomitant partial dissociation of the methionine and the formation of an Fe-aquo bond. Heme group relaxation is further accompanied by less distortion of the heme group than that associated with cytochrome c, expansion of the "core" and a negative shift of the redox potential. Received: 17 December 1996 / Accepted: 6 March 1997     

Microtubule arrays in the cortex and near the germinal vesicle of immature starfish oocytes

An extensive array of long, crisscrossing microtubules has been discovered in the cortex of oocytes of the starfish Pisaster ochraceus. The microtubules were visualized in cortex preparations by indirect immunofluorescence microscopy using antibodies to tubulin. The cortical array of microtubules is present in all oocytes before and for about 30 min after the application of 1-methyladenine, the hormone that induces oocyte maturation. The presence of microtubules was confirmed by electron microscopy. The microtubules in this array are depolymerized when oocytes are treated with colchicine or nocodozole and are augmented when oocytes are treated with taxol. Dihydrocytochalasin B treatment of the oocytes causes the microtubules to aggregate, presumably by altering a microfilament network also found in the cortex. The distribution of microtubules was also explored in whole oocytes stained with antitubulin. One or two aster-like structures were observed adjacent to the germinal vesicle of each oocyte.     

Example of a system which is computation universal but not effectively programmable

The incorporation of a chaotic component in a computing system is incompatible with its being effectively programmable. The example presented shows that concepts of programming suitable for biological systems may differ from those which have grown out of our experience with present day digital computers.     

Flow cytogenetics of uncloned and cloned Chinese hamster cells

Flow cytometry has greatly facilitated the routine use of DNA content as a cellular indicator of the stages of the cell cycle and ploidy. DNA content can also be used to distinguish individual chromosomes. Fluorescent staining of chromosome DNA was done with a combination of ethidium bromide and mithramycin in hypotonic solution. Subsequent detergent treatment of the cells with Triton X-100 facilitated chromosome isolation. DNA flow cytometry of chromosomes of four established uncloned Chinese master cell lines showed 10 to 12 major subpopulations of chromosomes with varying degrees of overlap in the range of low and intermediate DNA content. Cloning of B14F28 cells, the line with the largest heterogeneity in chromosome number and DNA content, considerably reduced the dispersion in chromosome number and improved the resolution of DNA content distributions. Thus, cloned cells with a relatively homogeneous karyotype permit better discrimination of chromosome subpopulations by DNA content than uncloned cells and provide a more sensitive system to study mutagenic effects.