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In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining
Published on: October 15, 2016
[RNA-Synthesis in telotrophic meroistic ovarioles of dysdercus intermedius DIST. (heteroptera, pyrrhoc.)]
Franz Duspiva1, Klaus Scheller1, Doris Wei1
1Physiologischer Lehrstuhl, Zoologisches Institut der Universität Heidelberg, Deutschland.
Abstract:
RNA in insect ovaries was investigated by polyacrylamide gel electrophoresis to study site of synthesis, transportation and incorporation in mature eggs. Telotrophic-meroistic ovarioles ofDysdercus intermedius were selected for this work since they could be dissected in distinct portions: the apical one with polyploid nurse cells and the vitellarium with oocytes covered by a follicular epithelium. RNA was labeled by injecting radioactive precursors or incubating isolated ovarioles in vitro.In nurse cells labeled rRNA, tRNA and non-ribosomal RNA were found and evidence was presented for the processing of a 39s rRNA precursor molecule in 2 steps (36s and 32s) into 28s rRNA while the 18s rRNA was directly derived from the 36s molecule. A low concentration (0.5 Μg/ml) of α-Amanitin inhibited synthesis of non-ribosomal RNA thereby revealing a more distinct processing pattern for rRNA, while a high concentration (5 Μg/ml) blocked processing at the 36s molecule and led to its accumulation. Appropriate concentrations of Actinomycin D inhibited rRNA synthesis while distinct peaks of non-ribosomal RNA became apparent.RNA synthesized in the follicular cells was similar to the RNA made in the nurse cells only in the high molecular weight RNA. Some material, tentatively named "nucleotide fraction", with a mobility higher than tRNA was detected and could be localized in yolk granules. It was separable from RNA by chromatography on Sephadex G 100 and had an absorption maximum at 255 run. The "nucleotide fraction" could be rapidly labeled. It seemed to be utilized in RNA synthesis during embryogenesis. Mature eggs contained a relatively high amount of the "nucleotide fraction". If a single injection with precursor was done 4 days before analysing the RNA of mature eggs most of the label was found in the "nucleotide fraction". Eggs collected 5-8 days after injection contained only high molecular labeled RNA. This observation correlated well with the timing of egg maturation. It takes 4 days for a group of oocytes to reach the distal part of the vitellarium where yolk production and maturation go on for another 4 days. It is apparent, therefore, that the follicular epithelium contributes the "nucleotide fraction" to the oocyte, while most of the other RNA, including a stable polydisperse class of RNA, is made in the nurse cells and is transported into the oocyte.
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