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H G Stunnenberg

Publications and source records attributed to H G Stunnenberg.

61 records · Page 4Linked to original sources
PubMed1983

Biochemical complementation with RNA in the Xenopus oocyte: a small RNA is required for the generation of 3' histone mRNA termini.

When the cloned h22 histone gene repeat unit of the sea urchin Psammechinus miliaris is injected into the frog oocyte nucleus, transcription initiates faithfully in the promoter region of the H3 gene, but transcription termination or RNA processing to yield genuine H3 mRNA 3' ends is very inefficient. The generation of 3' ends can be enhanced by the injection of a small sea urchin poly (A)- RNA into the oocyte cytoplasm one day prior to injection of the histone gene repeat unit. This RNA is approximately 60 nucleotides in length and appears to be present in relatively low abundance in embryos of both Psammechinus and Paracentrotus. RNA extracted from a nuclear DEAE fraction, previously shown to complement the lesion in H3 gene expression, also causes the appearance of true 3' ends of H3 mRNA. Furthermore, RNA extracted from our 12S "termination" factor includes an RNA species comigrating with the 60 nucleotide RNA from poly (A)- RNA. We suggest that the 12S nuclear "termination" component is in fact a small nuclear RNP.

Animals↗
PubMed1982

Bioassay for components regulating eukaryotic gene expression: a chromosomal factor involved in the generation of histone mRNA 3' termini.

We have adapted the oocyte injection procedure for the detection of regulatory components involved in the transcription of a eukaryotic mRNA gene. Injection of the histone gene repeat h22 DNA of Psammechinus miliaris into the Xenopus oocyte nucleus results in correct initiation of the histone mRNAs, but readthrough by RNA polymerase occurs at the 3' end of the H3 histone gene (Hentschel, C. C., Probst, E. & Birnstiel, M. L. (1980) Nature (London) 288, 100-102). Coinjection into the oocyte of a chromosomal salt wash fraction derived from sea urchin embryos results in the generation of authentic 3' termini of the histone H3 mRNA. We have partially purified the protein component by column chromatography and density gradient centrifugation. The regulatory factor binds to heparin columns and, hence, has the properties anticipated of an RNA- or DNA-binding protein. The sedimentation coefficient of the active component was determined to be about 12 S, suggesting a molecular weight of 200,000-250,000.

Animals↗
PubMed1982

Effect of alpha-amanitin and actinomycin D on RNA synthesis in the protoplasts of Aspergillus nidulans.

RNA synthesis was studied in protoplasts from Aspergillus nidulans, which remained metabolically active for several days under the appropriate conditions. It is concluded, that at certain actinomycin D concentrations the ribosomal RNA synthesis can be selectively inhibited. However, alpha-amanitin at concentrations up to 400 micrograms/ml neither influenced the total nor the RNA polymerase II directed RNA synthesis. It was possible to discriminate between the RNA polymerase I or II directed RNA synthesis due to the presence of poly(A) tails attached to at least part of the RNA, presumably messenger RNAs, or their precursors.

Amanitins↗
PubMed1981

An alpha-amanitin-resistant DNA-dependent RNA polymerase II from the fungus Aspergillus nidulans.

An alpha-amanitin-resistant DNA-dependent RNA polymerase II has been purified from the lower eukaryote Aspergillus nidulans to apparent homogeneity by extraction of the enzyme at low salt concentration, polymin P (polyethylene imine) fractionation, binding to ion-exchangers and density gradient centrifugation. By this procedure 0.4 mg of RNA polymerase II can be purified over 6,000-fold from 500 g (wet weight) of starting material with a yield of 25% and a specific activity of 550 units/mg. The subunit composition has been resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by two-dimensional gel electrophoresis using a non-denaturing gel in the first dimension and a dodecylsulphate slab gel in the second dimension. The putative subunits have molecular weights of 170,000, 150,000, 33,000, 27,000, 24,000, 19,000, 18,000 and 16,000. Only one form of RNA polymerase II could be resolved by electrophoresis. The chromatographic and catalytic properties and the subunit composition of the purified RNA polymerase II are clearly different from RNA polymerase I from A. nidulans but throughout comparable with other class II enzymes. It differs from all other class II enzymes by its insensitivity towards the toxin alpha-amanitin, even at concentrations up to 400 micrograms/ml, and appears to be unable to bind O-[14C]methyl-gamma-amanitin at a concentration of 10 micrograms/ml of the toxin. We conclude that the purified RNA polymerase from A. nidulans is a real, but exceptional, type of the class II RNA polymerases.

Amanitins↗
PubMed1979

RNA polymerase from the fungus, Aspergillus nidulans. Large-scale purification of DNA-dependent RNA polymerase I (or A).

The DNA-dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio-Gel A-1.5m, binding to ion-exchangers and DNA-cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000-fold from 500 g wet weight of starting material with a yield of 30--35%. The isolated RNA polymerase I is stable for several months at -20 degrees C. The subunit compostion has been resolved by polyacrylamide gel electrophoresis on two-dimensional gels, using either non-denaturing of 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190,000, 135,000, 63,000, 62,000, 43,000, 29,000, (28,000), 16,000 and probably 13,000 and 12,000. Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE-Sephadex A-25 chromatography showing ample differences in enzymatic properties and subunit pattern. Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to alpha-amanitin at concentrations up to 400 micrograms/ml.

Aspergillus nidulans↗
PubMed1979

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.

Aspergillus nidulans↗