Concentrations Of Alpha-amanitin Research Articles

The capped U6 small nuclear RNA is transcribed by RNA polymerase III.

U6 RNA is an abundant, capped, small nuclear RNA (snRNA) species associated with heterogeneous nuclear ribonucleoproteins in eukaryotic cells. U4 RNA and U6 RNA are hydrogen bonded in a 1:1 ratio in discrete small nuclear ribonucleoprotein particles that are required in pre-mRNA processing. Previous reports have established that the mRNAs and U1 to U5 U-snRNAs are synthesized by RNA polymerase II. Evidence is presented here for synthesis of U6 RNA by RNA polymerase III. The synthesis of U6 RNA in vitro, using Novikoff hepatoma or HeLa whole cell extracts, was not inhibited at low (1 microgram/ml) concentrations of alpha-amanitin, and only 35% inhibition occurred at 10 micrograms/ml concentration. The in vitro synthesized U6 RNA, like other RNA polymerase III transcripts, was associated with La antigen. The U6 RNA synthesized in vitro by the whole cell extracts was capped, but no other internal post-transcriptional modifications were found. Uridylic acid residues were also added post-transcriptionally to the 3'-end of U6 RNA in vitro. U6 RNA, though capped on its 5'-end, is transcribed by RNA polymerase III; this is the first report of a capped RNA molecule synthesized by RNA polymerase III.

Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin.

Nuclear protein factor type 1 (NPF-1) that simulates IMR-32 primase-associated DNA polymerase alpha 1 and alpha 2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase alpha, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37 degrees C), DNase II (60 min at 37 degrees C), and heat treatment (2 min at 68 degrees C). The 125I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase alpha-primase complex (alpha 1 and alpha 2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of alpha-amanitin (400 micrograms/ml). The labeled duplex containing the poly(dC) template, [32P]-GTP, and [3H]dGTP loses 80% of the 32P label and 70% of the 3H label after treatment with 0.3 M KOH and DNase I, respectively. The products were isolated from reaction mixtures incubated with and without NPF-1 and subjected to alkaline sucrose-density-gradient sedimentation analysis. The results suggest that the rate of synthesis of DNA short chains is increased in the presence of NPF-1 without a concomitant increase in the chain length of the newly synthesized products.

Transcriptional regulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver.

This study addresses whether transcriptional control of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver plays a role in determining the level of reductase mRNA. Isolated rat liver nuclei were allowed to elongate nascent RNA transcripts in the presence of [alpha-32P]CTP, and radiolabeled nuclear reductase RNA was quantitated by filter hybridization. Rats fed a diet supplemented with the drugs cholestyramine and mevinolin and having 20-60-fold induced levels of reductase mRNA exhibited levels of reductase transcription which were 20-fold higher than in rats fed an unsupplemented diet. Over 90% of the transcription of the reductase gene was inhibited by concentrations of alpha-amanitin which selectively inhibit RNA polymerase II. Administration of mevalonolactone (the end product of the reaction catalyzed by reductase) to rats fed cholestyramine and mevinolin caused an 80% decrease in the rate of reductase transcription by approximately 1 h. We conclude that under these conditions changes in reductase transcription are primarily responsible for the regulation of reductase mRNA levels.

Ribosomal RNA Synthesis in Mammalian Cells Permealized by Diethylaminoethyl (DEAE)-Dextran Treatment1

Ehrlich ascites tumor cells were made permeable to nucleoside triphosphates by treating them with diethylaminoethyl (DEAE)-dextran. Permeable cells incorporated labeled UTP into RNA at a constant rate for at least 30 min at 37 degrees C and 100 min at 25 degrees C, whereas no incorporation was detected without DEAE-dextran treatment. This RNA synthesis is dependent on added nucleoside triphosphates and is affected differentially by different concentrations of alpha-amanitin. The results of both hybridization and S1-nuclease protection mapping experiments with pulse-labeled RNA synthesized in this system indicate that reinitiation of rRNA transcription takes place from the physiological initiation site in cells made permeable by treatment with DEAE-dextran. By using this system, we examined the kinetics of rRNA synthesis in mouse FM3A cells during inhibition of protein synthesis. The results reflected those previously obtained by analyses of nucleolar RNA synthesis in vivo during protein synthesis inhibition. This permeable cell system should provide a rapid and reproducible assay method for rRNA synthesis in mammalian cells.

Human globin gene transcription in injected Xenopus oocytes: enhancement by sodium butyrate.

We have studied the expression of the human adult (beta) and foetal (gamma) globin genes by injecting a cosmid containing the complete -G gamma-A gamma-psi beta-delta-beta cluster into Xenopus oocytes. The transcripts from the gamma and beta genes were characterised with respect to their 5' termini by S1 nuclease mapping using probes which extend 5' to the cap site and 3' into the first exon. The only beta transcripts detectable in injected oocytes spanned from either -231 or -177 (cap site +1) into the first exon. The sensitivity of this transcription to inhibition by a low concentration of alpha-amanitin indicated that polymerase II was responsible. S1 analysis of gamma gene transcripts showed that the predominant transcript mapped from a site at -227, but a low level of transcription from the cap site was also detectable. Incubation of oocytes in saline solution containing sodium butyrate prior to injection resulted in a significant stimulation of transcription from the gamma gene cap site. In contrast, transcription from the beta gene upstream sites was only slightly increased. The possible role of sodium butyrate in promoting this effect is discussed in the context of its known property as a modifier of cellular chromatin.

Transcription of intracisternal A-particle genes in mouse myeloma and Ltk- cells.

In vivo- and in vitro-synthesized RNA from the murine plasmacytoma MOPC-315 was found to hybridize to all regions of a 7.2-kilobase-pair intracisternal A-particle (IAP) gene. IAP-specific transcripts were also detected in mouse Ltk- cells, but not in cells derived from normal tissues (kidney, liver, spleen) of 6-week-old BALB/c mice. Three RNA species of 7.2, 5.3, and 3.8 kilobases were identified by Northern blot analysis of MOPC-315 polyadenylated RNA. The 7.2- and 5.3-kilobase transcripts were found in greater levels in nuclear as compared with whole cell RNA, suggesting the involvement of one or more of the following mechanisms: RNA processing, preferential nuclear transport, or differential RNA stability. We show that the primary IAP transcript is initiated within the long terminal repeat by hybridization analysis with restriction digests of cloned IAP DNA and [gamma-S]pppApNp ... RNA synthesized in nuclei with [gamma-S]ATP as the RNA initiating probe. Low concentrations of alpha-amanitin (2 micrograms/ml) inhibited IAP RNA synthesis by greater than 90%, suggesting that RNA polymerase II is responsible for IAP transcription.

Transcription of alpha- and beta-tubulin genes in vitro in isolated Chlamydomonas reinhardi nuclei.

Removal of the flagella of Chlamydomonas results in increases in both flagellar protein synthesis and tubulin messenger RNA accumulation. These observations led us to examine whether flagellar protein gene sequences are transcribed differentially in nuclei isolated before and after deflagellation. A nuclear isolation protocol was developed using the cell wall-less strain of Chlamydomonas, CW 15, after cell lysis with 0.5% Nonidet P-40. Transcriptional activity of isolated nuclei was determined by incorporating [32P]UTP into TCA-precipitable and phenol-extractable RNA, and by hybridizing newly transcribed RNA to complementary DNA clones containing alpha- and beta-tubulin sequences. Nuclei from deflagellated cells are more active in transcribing sequences that hybridize with alpha- and beta-tubulin complementary DNA probes than are nuclei from nondeflagellated cells. In addition, while total [32P]UTP incorporation is inhibited 45% by alpha-amanitin concentrations of 1.0 micrograms/ml, tubulin RNA synthesis in this system is completely inhibited by this concentration of alpha-amanitin. This demonstration of differential transcription in nuclei before and after cell deflagellation provides the means to study in vitro the mechanisms that signal and regulate flagellar protein gene activity.

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells.

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to alpha-amanitin concentrations up to 100 micrograms/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be alpha-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.

RNA polymerase II from wild type and alpha-amanitin-resistant strains of Caenorhabditis elegans.

DNA-dependent RNA polymerases I, II, and III have been isolated from the soil nematode, Caenorhabditis elegans, and RNA polymerase II has been partially purified. The sensitivities of these enzymes to alpha-amanitin resemble those of the cognate enzymes from vertebrates. RNA polymerase II from C. elegans is 50% inhibited by 7 ng/ml of the amatoxin and RNA polymerase III by 80 micrograms/ml, whereas RNA polymerase I is insensitive to 500 micrograms/ml. We have obtained mutants of C. elegans which can grow and reproduce in concentrations of alpha-amanitin which arrest development of wild type animals. One of these mutants (DR432) has an altered RNA polymerase II which in partially purified extracts is 150 times less sensitive to the drug than the wild type enzyme. The mutation, ama-1(m130), in DR432 is dominant and maps near dpy-13 on linkage group IV. RNA polymerase II isolated from ama-1/+ heterozygotes contains equal proportions of two components, corresponding in alpha-amanitin sensitivity to the enzymes from DR432 and wild type. Thus, ama-1 appears to affect a subunit of RNA polymerase II.

Use of adenosine 5'-0-(2-thiotriphosphate) for revealing of newly initiated transcripts in isolated rat liver nuclei.

Initiation of DNA-dependent RNA synthesis in isolated rat liver nuclei was studied with adenosine 5'-0-(2-thiotriphosphate), (beta-S-ATP), as a precursor. The newly made RNA labelled with sulfur at 5'-triphosphate termini (thio-RNA) was isolated by affinity chromatography on a mercury-agarose column. Sulfur label can be removed from thio-RNA by digestion with phosphodiesterase I and nucleotide pyrophosphatase. Gel electrophoresis revealed that thio-RNA synthesized during 30 min was composed of 4S-35S molecules with three prevailing classes grouped around 4S-5S, 16S and approximately 35S. Differential sensitivity of the thio-RNA classes to low (1 microgram/ml) and high (200 micrograms/ml) concentrations of alpha-amanitin disclosed that beta-S-ATP was used for initiation of transcription by all three classes of RNA polymerases, and that thio-RNA included molecules as large as 18S initiated by RNA polymerase II. Thio-RNA resistant even to high doses of alpha-amanitin represents probably a product of RNA polymerase I which was initiated and elongated up to 35S.

Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by alpha-amanitin.

The template-directed synthesis of a single phosphodiester bond by highly purified calf thymus RNA polymerase B is not inhibited by high concentrations of alpha-amanitin (10(-6) M). However, a subsequent internucleotide bond is not synthesized in the presence of alpha-amanitin. These results suggest that translocation of the nascent RNA and RNA polymerase B along the DNA template is the enzymatic process inhibited by alpha-amanitin. It is also shown that the formation of a single phosphodiester bond by RNA polymerase B results in a stable ternary transcription complex, i.e., between the enzyme, the DNA, and the nascent RNA. Under reaction conditions which normally favor the elongation of RNA, the transcriptional process is arrested at initiation by alpha-amanitin. Such ternary initiation complexes have been isolated by agarose gel electrophoresis.

High salt effect on RNA synthesis in Krebs ascites tumor cells.

The incubation of Krebs ascites tumor cells in medium with a high salt concentration resulted in a partial inhibition of nuclear RNA synthesis. The residual RNA polymerase activity in such nuclei was only slightly inhibited by low concentrations (50 nM) of alpha-amanitin. This finding suggested an inhibition of RNA polymerase II activity under conditions of medium hypertonicity. Indeed, RNA polymerase II, isolated from the nuclei of cells exposed to hypertonicity, revealed only half of the control activity. On the other hand, RNA polymerase I was not affected by hypertonicity. Moreover, chromatin fractions isolated from cells incubated in hypertonic or isotonic medium were equally template-active in RNA synthesis.

RNA polymerase II transcribes all of the heat shock induced genes of Drosophila melanogaster.

Heat shock of Drosophila melanogaster induces the transcription of a small number of RNAs. Some of these encode protein products, but not all. We have investigated whether the several induced RNAs are transcribed by RNA polymerase II or by some other RNA polymerase. Immunochemical staining of polytene chromosomes indicates that, on heat shock, RNA polymerase II is relocalized; it "migrates" from previously-active transcription sites to the heat shock induced loci. All heat shock induced puffs show immunochemical staining. Such staining correlates with RNA polymerase II activity as judged by the sensitivity of RNA synthesis at these sites to low concentrations of alpha-amanitin. Thus the protein-coding and non-protein-coding heat shock-induced RNAs are transcribed by this polymerase specifically. We have also identified several non-puffed chromosomal sites at which RNA synthesis is induced by heat shock.

Removal of alpha-amanitin from blood by hemoperfusion over uncoated charcoal. Experimental results.

The sorbent capacity of uncoated charcoal column (Detoxyl 2, Sorin Biomedica) on alpha-amanitin was studied by adding known amounts of alpha-amanitin to fresh human blood, which was recirculated through a clinical hemoperfusion circuit over a period of 30 min. The column was washed with saline to remove loosely bound alpha-amanitin, and eluted with 0.1 N HCl to detach absorbed alpha-amanitin. Alpha-amanitin concentration was measured in the saline washings and in the acid eluates by means of a radioimmunoassay. Only negligible amounts of amanitin were recovered in the saline washings. Suitable amounts of alpha-amanitin were present in the acid eluates, showing an adsorption optimum for blood concentrations ranging from 50 to 200 ng . ml-1. The results were discussed as predictable of the clinical usefulness of charcoal hemoperfusion in A. phalloides poisoning.

An oligoribonucleotide polymerase from SV40-infected cells with properties of a primase.

A transient decaribonucleotide (iRNA) is covalently linked to nascent eukaryotic DNA chains at their 5' end. Searching for the putative iRNA polymerase (primase), we detected in extracts from SV40-infected cells a DNA-dependent incorporation of UMP residues from UTP into free and DNA linked deca- or similarly sized ribonucleotides. Denatured salmon sperm DNA served as the standard template in this reaction. SV40 FIII DNA was also an effective template, SV40 FII DNA was ineffective while FI yielded mainly free decaribonucleotides. The incorporation depended on the other rNTPs and was resistant to high concentrations of alpha-amanitin and rifamycin AF/013, drugs inhibitory to RNA polymerases I, II and III. The results implicate the decaribonucleotide polymerase in the priming of nascent DNA chains and suggest that the unique size of iRNA is encoded within its primase.

In vitro RNA synthesis in oocyte nuclei of the newt Notophthalmus.

An incubation medium is described which supports RNA synthesis in isolated oocyte nuclei of the newt Notophthalmus, and which permits subsequent autoradiographic examination of the lampbrush chromosomes and nucleoli. By using different concentrations of alpha-amanitin we distinguish RNA synthesis due to RNA polymerases I, II and III. All RNA synthesis on loops is inhibited by 0.5 microgram/ml of alpha-amanitin and is therefore due to polymerase II. Polymerase III is responsible for RNA synthesis at a small number of discrete sites in condensed chromatin. These include the centromere bars of three of four chromosomes, which probably represent 5S RNA synthesis, as well as 15-20 lesser sites scattered elsewhere. Polymerase I activity is confined to the nucleoli.

In vitro transcription of a cloned mouse ribosomal RNA gene.

An in vitro transcription system which utilizes cloned mouse ribosomal RNA gene (rDNA) fragments and a mouse cell extract has been developed. RNA polymerases I is apparently responsible for this transcription as evidenced by the complete resistance to a high concentration (200 micrograms/ml) of alpha-amanitin. Run-off products obtained with three different truncated rDNA fragments indicated that RNA was transcribed from a unique site of rDNA. The S1 nuclease protection mapping of the in vitro product and of in vivo 45S RNA confirmed this site, indicating that, in this in vitro system, transcription of rDNA started from the same site as in vivo. This site is located at several hundred nucleotides upstream from the putative initiation site reported by us (1) and by others (2). Some sequence homology surrounding this region was noted among mouse, Xenopus laevis and Drosophila melanogaster. The data also suggest that some processing of the primary transcript occurs in this in vitro system.

Transcription of T-DNA in octopine and nopaline crown gall tumours is inhibited by low concentrations of alpha-amanitin.

Highly purified and physiologically active nuclei were isolated from four different octopine and nopaline crown gall lines. These nuclei exhibited a high endogenous RNA synthesizing activity involving all three RNA-polymerases I, II and III. Isolated nuclei were shown by Southern blotting to synthesize T-DNA specific RNA. This synthesis was shown to be sensitive to actinomycin D and therefore to be DNA-dependent. The transcription of the T-DNA was also inhibited for more than 90% by low concentrations of alpha-amanitin (0.7 micrograms/ml) indicating that the T-DNA, although from bacterial origin, is transcribed by the host RNA polymerase II.

Nucleolar ultrastructure and transcription of preribosomal RNA in Rous Sarcoma Virus-Transformed Chick Embryo Fibroblasts Treated With α-Amanitin<xref ref-type="fn" rid="fn2">2</xref>

Uninfected and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts (CEF) were exposed to various concentrations of alpha-amanitin for different lengths of time. At a concentration of 4 micrograms alpha-amanitin/ml, RSV-transformed cells were shown to maintain a normal rate of transcription of all classes of RNA, whereas in uninfected cells transcription was reduced to a very low level. These observations cannot be accounted for by a difference in the penetration rate of alpha-amanitin through the plasma membrane. Investigation of the ultrastructure showed that the degree of nucleolar fragmentation induced by alpha-amanitin was comparable in both types of cells. Persistence of transcription with concomitant nucleolar fragmentation in alpha-amanitin-treated RSV-transformed CEF is not in accord with the hypothesis that nucleolar integrity is required for preribosomal RNA transcription.

RNA synthesis in rabbit endometrial nuclei. Hormonal regulation of transcription of the uteroglobin gene.

Endometrical nuclei, prepared from rabbits subjected to different hormonal treatments, were used for the cell-free synthesis of RNA. Optimal conditions for the incorporation of [3H]UMP into RNA are described, leading to the synthesis of relatively undegraded RNA molecules. Under these conditions there is virtually no initiation of new RNA chains in vitro, and RNA chain elongation is inhibited up to 60% by low concentrations of alpha-amanitin and up to 90% by actinomycin D. The synthesis of RNA is slightly inhibited in the presence of Hg-CTP and monothioglycerol, but newly synthesized mercurated RNA can be efficiently separated from endogenous RNA upon chromatography on sulfhydryl-Sepharose under stringent conditions. The RNA synthesized in vitro by endometrial nuclei from pseudopregnant rabbits contains RNA sequences transcribed from the uteroglobin gene, as demonstrated by hybridization to an excess of purified preuteroglobin cDNA. In endometrial cells from pseudopregnant animals the number of RNA polymerase II molecules transcribing the uteroglobin gene is 12-fold higher than in control animals, demonstrating that at least part of the hormonally induced accumulation of preuteroglobin mRNA is due to an increased rate of transcription of the uteroglobin gene.