Sensitivity to methylmethanesulfonate and nitrous acid of ultraviolet light-sensitive mutants in Saccharomyces cerevisiae

Summary Twenty two ultraviolet light-sensitive mutants isolated byParry andCox (1968) were tested for a possible cross-sensitivity to nitrous acid and methylmethanesulfonate. Eighteen of these mutants showed, in comparison to wild type, an increased sensitivity to methylmethanesulfonate and 16 mutants were cross-sensitive to nitrous acid. Cross-sensitivity between these two chemicals was good even when the degrees of sensitivity were compared; only two major exceptions were found. The degree of sensitivity to the two chemicals on one side and to ultraviolet light was not always the same. Two mutants of extreme sensitivity to chemicals were only weakly sensitive to ultraviolet light whereas in another mutant an extreme sensitivity to ultraviolet light was combined with a low sensitivity to chemicals. These observations are discussed in the light of current views on excision repair models.     

Negative complementation among ad2 mutants of yeast

Summary Ad ₂ remedial mutants of Saccharomyces cerevisiae which showed conditional growth at 35°C or with 1 M KCl at 25° C or 35° C, were crossed with other remedial and non-remedial mutants in all pairwise combinations. 19 remedials (11 K ₂₅, 5 temp. and 3 K ₃₅) and 189 non-remedial mutants were used. The standard conditions used were incubation at 25° C in the absence of adenine.Among 1625 combinations there were 367 (22.6%) cases of negative complementation. In these cases the diploids were unable to grow under the same conditions which permitted a strong growth of the remedial haploid.No negative complementation was observed among K ₂₅×K ₂₅ combinations. When only one haploid partner was remedial the temp. ¢ non-remedial combinations showed a rate 2.5 times higher than that observed among K ₂₅ × non-remedial combinations. When the remedial haploid was a K ₃₅ the incidence was the same as that noted with temp. × non-remedial combinations. The high incidence observed in the latter temp. combinations was not subject to influence attributable to strain; the incidence being the same whether the remedial partner belonged to the a or to the mutant strains. The K ₂₅ × non-remedial combinations, on the other hand, showed such an influence.For an explanation of the mechanism involved in negative complementation it was postulated that, assuming non-random monomer aggregation, some mutant pairs would form only active heterologous multimers while others, due to the nature of their mutation, would form only inactive aggregates which entrap all the remedial monomers. It is thought that this postulate may have some application in explaning the complementation mechanism in general.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Feldversuchswesen: die Gitterquadratmethode in praktischer Anwendung

Zusammenfassung Die Grundlagen der Gitterquadratmethode werden soweit erörtert, wie es für die praktische Anwendung erforderlich ist. In Tabellen werden die Teilstückzahlen, welche die einzelnen Versuchsgrößen notwendig machen, gegeben.Es wird darauf hingewiesen, daß durch Verwendung von Standortnummern die technische Durchführung der Versuche wesentlich erleichtert wird.Zur Verrechnung wird auf Schemata hingewiesen, von denen im Rahmen dieser Erörterung nur ein Beispiel gebracht werden kann.Die in der Futterpflanzenabteilung des Instituts für Acker- und Pflanzenbau, Müncheberg, seit 1954 nach der Gitterquadratmethode angelegten und ausgewerteten Versuche sind in einer Tabelle zusammengefaßt. Eine Besprechung der einzelnen Versuche ergab, daß die erzielten Grenzdifferenzen dem Normalen entsprechen.Es kann gesagt werden, daß die technischen und verrechnungsmäßigen Schwierigkeiten der GM bei weitem nicht so groß sind, wie bisher angenommen wurde, und es wird besonders dem Pflanzenzüchter empfohlen, die Methode in stärkerem Maße für die Prüfung von zahlreichen Zuchtstämmen anzuwenden.     

Vier Jahre Arbeiten an Luzerne in Müncheberg 1948–1952

Ohne Zusammenfassung Hans Lembke zum 75. Geburtstag.     

Effects of ethidium bromide,tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ

Summary Several investigators have described the ultrastructural changes that occur in the mitochondria of cells in tissue cultures after treatment with the drug ethidium bromide (E). The mitochondria swell and the cristae become greatly altered and finally disappear; in the cristae-free region of the matrix electron-dense granules can be observed. It has been assumed that intercalation of E between the base pairs of the mitochondrial DNA induces the formation of the granular inclusions. To investigate whether intercalation is really the initial step in the generation of dense granules inside the matrix, we performed a comparative incubation study of HeLa-cell mitochondria in situ using three closely related dyes (D), i.e. E, tetramethylethidium bromide (TME) and betaine B (B). They strongly differ with regard to their affinity for DNA and their ability to cross membranes. E was used as a reference dye. TME does not intercalate, but is externally bound to DNA only weakly. The neutral B is not bound at all, but can cross membranes more easily than the cation E. Moreover, in aqueous solutions at pH7.0, B is in equilibrium with its protonated cation BH. BH and E have almost equal affinities for DNA. Therefore B may quickly pass the inner mitochondrial membranes and the cristae, and should then be bound inside the matrix, thus forming a BH-DNA complex. On the assumption that intercalation is necessary for the generation of intramitochondrial electron-dense bodies, we predicted that BH/B should be more efficient than E, while TME should be relatively ineffective. In experiments using HeLa cells, these predictions were found to be inaccurate. E, TME and BH/B produced almost the same mitochondrial alterations, but at different concentrations and after different incubation periods. In contrast to our expectations TME was much more effective than E and BH/B, with the last two behaving rather similarly.Therefore, it seems unlikely that the drugs penetrate the inner mitochondrial membrane system by simple physical diffusion or that intercalation is the preliminary step for the generation of dense granules inside the matrix. Instead, we assume that hydrophobic interaction between the dye cations E, BH and TME and the cristae is the main cause of the mitochondrial changes. The favoured binding partner of the dye cations may be the divalent anion, cardiolipin: this phospholipid is an essential part of the inner membrane system but is absent in other membranes of cells. By distributing the dyes between a lipophilic phase and water, it was shown that TME is more lipophilic than E and BH; this may explain the greater effectiveness of TME. The bound dye cations disturb the organization of the cristae, which become altered and finally disappear. We assume that the electron-dense granules in the matrix are mainly composed of the dyes and former membrane materials such as phospholipids and proteins, as well as perhaps some other hydrophobic matrix materials. This would also explain why it was impossible to digest the dense granules by DNase treatment. The drugs enter the mitochondrial matrix by disordering and finally destroying the cristae.     

Characterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine

Four serologically unrelated closterovirus-like particles (GLRV-1, GLRV-2, GLRV-3 and GLRV-4) were isolated in our laboratory from leafroll diseased grapevines. Polyclonal antibodies raised against these particles were useful for their characterization and their detection in infected plants. The coat proteins of these four serotypes were characterized by a SDS-PAGE after denaturation, followed by a transfer on nitrocellulose sheet and immunoprinting using the specific polyclonal antibodies. The capsid of GLRV-1, GLRV-2, GLRV-3 and GLRV-4 contains a single protein species with molecular weight of about 39 Kd, 26 Kd, 43 Kd and 36 Kd respectively. No serological relation was found between these four filamentous particles either by ELISA, immuno electron microscopy or immunoblotting experiments. Serological analysis of many grapevines originating from the Middle East and Europe showed a very close association between the presence of GLRV-1, GLRV-2, GLRV-3 and GLRV-4 antigens, and leafroll symptoms on Vitis vinifera Pinot Noir. This association was confirmed by testing symptomless and diseased grapevines collected in the field, and by serological analysis of heat treated plants originally infected by GLRV-1 and GLRV-3, which are the most widespread antigens detected in leafroll infected grapevines.     

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.     

Über die Vitalfluorochromierung des Kernchromatins und der Chromosomen von HeLa-Zellen mit AMHA und die Bindung des Acridinfarbstoffs an DNA

Summary The fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations C _F10^–4 M and short incubation periods t _I2 h cell growth is not affected by the drug. But at higher C _F and longer t _I the population doubling time of the cell cultures rapidly increases, and finally the cells die.In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm^–1 between the 0-0-transitions of the long wave length ¹ L _a absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm^–1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission.Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nuceleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant K _F1=1,1·10⁵ M ^–1, binding parameter n ₁=0,15; the pre-intercalative or external binding 2, K _F2=6,9·10⁵ M ^–1, n ₂=0,21; the external binding 3, K _F3=2,8·10⁵ M ^–1, n ₃=0,55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant K _F1=1,1·10⁵ M ^–1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, K _F1=(1–4)·10⁴ M ^–1. Therefore, the amount of intercalated AMHA is also relatively high, and it is possible to visualize the DNA-bound fluorochrome in the nuclei and chromosomes of the living cells under the fluorescence microscope.