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Biomedical subjects

B Wasylyk

Publications and source records attributed to B Wasylyk.

96 records · Page 6Linked to original sources
PubMed1979

Transcription by eukaryotic RNA polymerases A and B of chromatin assembled in vitro.

Chromatin was assembled in vitro from simian virus 40 DNA form I and the calf-thymus four histones H2A, H2B, H3 and H4. Transcription with calf thymus RNA polymerases A and B (I and II) was greatly inhibited. Nucleosomes were found to inhibit both RNA chain initiation and elongation. The inhibition of elongation could be relieved by increasing ionic strength, suggesting that electrostatic interactions between histone octamer and DNA have to be broken for RNA polymerase to transcribe DNA organised into nucleosomes.

Animals↗
PubMed1978

Effect of histone acetylation on structure and in vitro transcription of chromatin.

n-Butyrate treatment of growing Hela cells produces a dramatic increase in the levels of histone acetylation. We have exploited this system to study the effect of histone acetylation on chromatin structure. Chromatin containing highly acetylated histones is more rapidly digested to acid-soluble material by DNase I, but not by micrococcal nuclease. The same pattern of nuclease sensitivity was exhibited by in vitro-assembled chromatin consisting of SV40 DNA Form I and the 2 M salt-extracted core histones from butyrate-treated cells. Using this very defined system, it was possible to demonstrate that acetylated nucleosomes do not have a greatly diminished stability. Stability was measured in terms of exhange of histone cores onto competing naked DNA or sliding of histone cores along ligated naked DNA. Finally, it was shown that acetylated nucleosomes are efficient inhibitors of in vitro RNA synthesis by the E. coli holoenzyme as well as by the mammalian polymerases A and B.

Acetylation↗
PubMed1975

The activation of RNA polymerase by alkylammonium ions.

Tetramethylammonium and tetraethylammonium ions both lower the melting temperature of DNA. We have shown that these ions increase the activity of E. Coli RNA polymerase. When the base composition of the DNA template was varied, there was a correlation between the decrease in melting temperature and increase in RNA polymerase activity. No stimulation was observed when heat denatured DNA was used as template. It was shown that initiation of RNA synthesis was stimulated but to a smaller degree than was total RNA synthesis. The lag observed at low temperatures before RNA synthesis commences was found to be shorter in the presence of these alkyl ammonium ions. All these results are consistent with an unwinding of the double helix being an important step in transcription.

Animals↗
PubMedDate not supplied

Targetting of the N-terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric ScFvs blocks the E6-mediated degradation of cellular p53.

The E6 protein of cancer-associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway. In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6-mediated p53 in vitro degradation. The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N-terminal end of 16E6. The variable regions of these antibodies were cloned and expressed in E. coli as single chain Fvs (scFvs). Purified scFvs were present in monomeric form and totally abolished 16E6-mediated p53 degradation by preventing the formation of E6/p53 protein complexes. Our results demonstrate that monovalent binding of scFvs to the N-terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein.

Amino Acid Sequence↗