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Recently published results (Nucleic Acids Res. 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5-MeC-DNA glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.  相似文献   
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Zusammenfassung Bei Kreuzungen zwischen Culex pipiens-Populationen verschiedener geographischer Herkunft werden drei Kreuzungstypen festgestellt: normale Kreuzbarkeit, reduzierte Kreuzbarkeit und Inkompatibilität (Nichtkreuzbarkeit). Die drei Kreuzungstypen sind mit Hilfe der Embryonierungsrate, der Schlüpfrate und der entstehenden Nachkommenschaft gegeneinander abgrenzbar. Bei Inkompatibilität sind 99.9% der sich entwickelnden Embryonen letal und etwa 0.1% der Tiere schlüpfen und sind fertile, diploide Weibchen. Die Aktivierung des Eies erfolgt durch das Spermium. Es liegt induzierte, meiotische Parthenogenese vor. Die diploiden, parthenogenetischen Weibchen gehen aus einer Oocyte 2. Ordnung oder aus Teilungsprodukten einer Oocyte 2. Ordnung hervor. Bei den letalen, haploiden Embryonen beteiligt sich das Spermium nicht am Aufbau des Embryos.
Genetic investigations on the incompatibility in the Culex pipiens complex
Summary In crosses between populations of the mosquito Culex pipiens of different geographical origin three crossing types have been found (1) crosses with normal offspring (2) crosses with reduced offspring and (3) crosses that show almost total incompatibility. In the case of incompatible crosses 99.9% of the developing embryos are lethal and only about 0.1% of the embryos hatch and develop into fertile diploid females. The origin of the exceptional diploid females has been investigated by means of the marker genes Kuf, r, w, var, y and ru. These females develop from a diploid oocyte or from a diploid nucleus that originated by fusion of the pronucleus and the last polar body. This has been concluded from the frequency of homozygous and heterozygous offspring from heterozygous females, the frequency of equational separation of different genes and the distribution of cross-over gametes in the exceptional females. Based on the genetical data it is argued that induced meiotic parthenogenesis takes place. The sperm does not play any part in the production of the diploid females and the lethal embryos. After the activation of the egg the sperm moves to the center of the egg but it does not succeed to fuse with the pronucleus. As a result the pronucleus starts to develop into a haploid embryo in about 99.9% and only in a few cases is the diploidy restored by a change in the meiotic process in the egg.


Teil einer Dissertation der Math.-Nat. Fakultät der Universität Mainz

Danksagung: Herrn Prof. Dr. H. Laven danke ich für die Bereitstellung des Untersuchungsmaterials sowie für die Anregungen bei der Durchführung der Arbeit.

Angenommen durch H. Stubbe  相似文献   
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In the management of neurological intensive care patients with an intracranial space-consuming process the measurement and recording of intracranial pressure together with arterial blood pressure is of special interest. These parameters can be used to monitor the treatment of brain edema and hypertension. Intracranial pressure measurement is also important in the diagnosis of the various subtypes of hydrocephalus. Not only the absolute figures, but also the recognition of specific pressure-patterns is of particular clinical and scientific interest. This new, easily installed and inexpensive system comprises a PC and a conventional monitor, which are connected by an AD-conversion card. Our software, specially developed for this system demonstrates, stores and prints the online-course and the trend of the measurements. In addition it is also possible to view the online-course of conspicuous parts of the trend curve retrospectively and to use these values for statistical analyses. Object-orientated software development techniques were used for flexible graphic output on the screen, printer or to a file. Though developed for this specific purpose, this system is also suitable for recording continuous, longer-term measurements in general.  相似文献   
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Editorial     
Circular double-stranded phage M13 DNA-containing gaps of defined size and location were obtained in preparative amounts by in vitro hybridization of plus- and minus-strands derived from different phage strains.  相似文献   
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Summary Extracellular Pseudomonas lipase is able to interact directly or indirectly with alginate as deduced from the following results: (i) During adsorption chromatography of exolipase the enzyme adsorbed quantitatively to glass beads in the absence of alginate, but not after its preincubation in the presence of the polysaccharide; pretreatment of glass beads with alginate did not prevent enzyme adsorption. (ii) In the presence of alginate exolipase was much more resistant to heat inactivation than in its absence. (iii) In the presence of alginate the increase in exolipase activity caused by the non-ionic detergent Triton X-100 was drastically reduced. (iv) Exolipase could be rapidly and almost completely harvested from cell-free culture fluid of P. aeruginosa 5940 by ethanolic coprecipitation with alginate. After dissolving the coprecipitate in detergent-containing buffer exolipase and polysaccharide could be easily separated by ion-exchange chromatography on DEAE-Sephadex A-25. The coprecipitation method was also successfully applied to exolipases produced by Pseudomonas sp., Chromobacierium viscosum and Rhizopus delamar, thus suggesting potential use of this method in biotechnology.  相似文献   
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