Interaction of several class II and III RNA polymerases with two families of 7'-substituted .alpha.-amanitin

Biochemistry ◽  
1982 ◽  
Vol 21 (21) ◽  
pp. 5164-5170 ◽  
Author(s):  
Erik Falck-Pedersen ◽  
Wilma Neuman ◽  
Paul W. Morris
Keyword(s):  
Class Ii ◽  
Rna Polymerases ◽  
Alpha Amanitin

scholarly journals Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of α-amanitin-resistant and -sensitive enzymes

1987 ◽  
Vol 241 (3) ◽  
pp. 649-655 ◽  
Author(s):  
D L Earnshaw ◽  
T J C Beebee ◽  
W E Gutteridge
Keyword(s):  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Gel Electrophoresis ◽  
Protozoan Parasite ◽  
Rna Polymerases ◽  
Subunit Structure ◽  
Major Band ◽  
Native Gel ◽  
Alpha Amanitin

We have isolated, characterized and substantially purified two distinct RNA polymerase activities from the flagellate protozoan parasite Trypanosoma brucei. RNA polymerases from this organism were resolved poorly on DEAE-Sephadex, but could be separated with CM-Sephadex. One form was totally resistant to alpha-amanitin, whereas the second was 50% inhibited by 10-20 micrograms of the drug/ml. The enzymes had different salt optima, but both were of high Mr (greater than 480,000) and demonstrated the template preference: poly[d(A-T)] greater than denatured DNA greater than native DNA, and both were more active with Mn2+ than with Mg2+. The amanitin-resistant enzyme, polymerase R, was partially purified by chromatography on CM-Sephadex, DEAE-Sephadex and heparin-Sepharose. This enzyme was very labile, and activity yields were around 9%; after purification, one or two protein bands could be discerned after electrophoresis under non-denaturing conditions, but about 20 polypeptides were resolved on denaturing gels, including a major component (not thought to be part of the enzyme) of Mr 65,000. Polymerase S, sensitive to low alpha-amanitin concentrations, was more extensively purified, with an 18% recovery, and yielded a single major band with two minor ones after native gel electrophoresis. Analysis under denaturing conditions permitted a possible subunit structure for this enzyme to be ascribed.

mRNA transcription in nuclei isolated from Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1633-1639
Author(s):  
J F Jerome ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Blot Hybridization ◽  
Single Copy ◽  
Rna Polymerases ◽  
Slot Blot Hybridization ◽  
Genes Encoding ◽  
Diploid Cells ◽  
Alpha Amanitin

We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

scholarly journals mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (5) ◽  
pp. 1633-1639 ◽  
Author(s):  
J F Jerome ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Blot Hybridization ◽  
Single Copy ◽  
Rna Polymerases ◽  
Slot Blot Hybridization ◽  
Genes Encoding ◽  
Diploid Cells ◽  
Alpha Amanitin

We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

Electron microscopy analysis of degenerating pancreatic ß cells in transgenic mice expressing the MHC Class I antigen Dd

1990 ◽  
Vol 48 (3) ◽  
pp. 548-549
Author(s):  
T. A. Stewart ◽  
D. Liggitt ◽  
S. Pitts ◽  
L. Martin ◽  
M. Siegel ◽  
...  
Keyword(s):  
Type I Diabetes ◽  
Disease Process ◽  
Class Ii ◽  
Class I ◽  
Type I ◽  
Aberrant Expression ◽  
Mhc Molecules ◽  
Endogenous Insulin ◽  
Class Ii Mhc ◽  
Ifn Γ

Insulin-dependant (Type I) diabetes mellitus (IDDM) is a metabolic disorder resulting from the lack of endogenous insulin secretion. The disease is thought to result from the autoimmune mediated destruction of the insulin producing ß cells within the islets of Langerhans. The disease process is probably triggered by environmental agents, e.g. virus or chemical toxins on a background of genetic susceptibility associated with particular alleles within the major histocompatiblity complex (MHC). The relation between IDDM and the MHC locus has been reinforced by the demonstration of both class I and class II MHC proteins on the surface of ß cells from newly diagnosed patients as well as mounting evidence that IDDM has an autoimmune pathogenesis. In 1984, a series of observations were used to advance a hypothesis, in which it was suggested that aberrant expression of class II MHC molecules, perhaps induced by gamma-interferon (IFN γ) could present self antigens and initiate an autoimmune disease. We have tested some aspects of this model and demonstrated that expression of IFN γ by pancreatic ß cells can initiate an inflammatory destruction of both the islets and pancreas and does lead to IDDM.

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