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.     

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.     

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.     

Variabilin,a 6a-hydroxypterocarpan from Dalbergia variabilis

Bark and wood of the creeper Dalbergia variabilis contain the previously described friedelin, O-acetyl-oleanolic acid, formononetin, 8-O-methylretusin, (+)-vestitol, (±)-mucronulatol, (+)- and (±)-medicarpin, besides (+)-variabilin [(6aR,11aR)-6a-hydroxy-3,9-dimethoxypterocarpan]. This structure was confirmed by the conversion of (+)-variabilin into di-O-methylcoumestrol.     

Repeated characterization, analysis of variance limitation and discrimination analysis classification of EEG-activity patterns in human sleep]

1. To describe quantitatively and to deliminate nine EEG sleep patterns, mean values and standard deviations of abundances of the frequencies 0.8 ... 1.8 c/sec, 2...3.5 c/sec, 4...13c/sec, 14 to 17 c/sec, 18 to 22 c/sec, and 23 to 40 c/sec as well as of the average amplitudes in selected frequency ranges were calaculated and the distributions represented. 2. All nine EEG activity patterns could be separated by means of univariate and multivariate analyses of variance on the basis of all 28 as well as the 17 indispensable variables. 3. In the course of a stepwise reduction of variables within the framework of a linear discriminant analysis an optimal set of 17 variables was determined for the separation of the patterns, comprising: the percent quantity of the frequencies 0.8 ... 3.5 c/sec, 7 ... 9 c/sec and 18 to 40 c/sec as well as the average amplitudes in the frequency ranges 0.8 to 3.5 c/sec and 7.5 to 40 c/sec. 4. By linear regression analyses it could be shown that the sleep scording system used, can be reflected on an interval scale with the aid of discriminant functions; this can be achieved on the basis of the optimal set of variables as well as of the five most indispensable variables. 5. Finally the degree of the objectivity of the scoring procedures was demonstrated. Advantages and disadvantages of sleep scoring systems were discussed and possibilities of the utilization of results suggested, also in respect to the further development of the automatic recognition of EEG activity patterns.     

Genetic and biochemical characterization of a new chymotrypsin isozyme of the house mouse,CTRA-1

Genetic variation of a codominantly inherited pancreas protease, designated CTRA-1, was discovered in the house mouse by isoelectric focusing in polyacrylamide gels. Phenotype CTRA-1A was found in MOLH/Fre and in the majority of common laboratory mouse strains. Phenotype CTRA-1B was found in PWD/Ph. It was characterized by the absence of a corresponding protease band. A third phenotype, CTRA-1C, was observed in IS/Cam and a fourth phenotype, CTRA-1D, was detected in SEG/1. CTRA-1 was found only in the pancreas and may represent the A form of chymotrypsin. The enzyme was shown to be controlled by the presumed structural locus Ctra-1 located on chromosome 8. From two backcross series, including a total of 274 animals, the gene order (Es-1, Es-9)-3.9 +/- 1.7%-Got-2-3.9 +/- 1.7%-(Es-2, Es-7, Es-23)-0.7 +/- 0.5%- Ctra-1-6.3 +/- 2.2%-Prt-2 was established.     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.