Ribonucleic acid synthesis in isolated Drosophila nuclei

Gross, Robert; Ringler, J.

doi:10.1021/bi00589a021

Cited by 25 publications

(16 citation statements)

References 19 publications

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“…In both of these systems bovine serum albumin had no significant effect on the early stages of RNA synthesis, but rather enhanced the long-term stability of the RNA (9) or resulted in increased total synthesis (7). We found that the addition of bovine serum albumin to levels comparable to those used in the studies described above (8,10) had no affect on apparent degradation (data not shown).…”

Section: Resultsmentioning

confidence: 77%

“…4; in this experiment the KCl concentration was 100 mM, and the MgCl2 concentration was varied from 0.5 to 40 mM. Total incorporation was maximal between 10 and 20 mM MgCl2; higher levels of MgCl2 result in decreased total incorporation, but the percentage of a-amanitin- sensitive activity increased (data not shown). When individual transcripts were analyzed by hybridization (Fig.…”

Section: Resultsmentioning

confidence: 88%

“…Under optimal cohditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the a-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 ,g of a-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.Nuclei isolated from a number of cell types have been shown to accurately transcribe RNA in vitro (8,10,13,14,17,19). In a subset of these systems there is reinitiation of RNA synthesis (10,13,17), and in some cases polyadenylation occurs in the isolated nuclei (8,10,14).…”

mentioning

confidence: 99%

“…In a subset of these systems there is reinitiation of RNA synthesis (10,13,17), and in some cases polyadenylation occurs in the isolated nuclei (8,10,14). In one example nuclei have been shown to accurately initiate transcription of exogenous DNA added to the incubation mixture (13).…”

mentioning

confidence: 99%

“…Nuclei isolated from a number of cell types have been shown to accurately transcribe RNA in vitro (8,10,13,14,17,19). In a subset of these systems there is reinitiation of RNA synthesis (10,13,17), and in some cases polyadenylation occurs in the isolated nuclei (8,10,14).…”

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confidence: 99%

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mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

Jerome¹,

Jaehning²

1986

Mol. Cell. Biol.

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We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with a-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, tRNAT"r, and GAL7, URA3, TYI 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 cohditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the a-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 ,g of a-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.Nuclei isolated from a number of cell types have been shown to accurately transcribe RNA in vitro (8,10,13,14,17,19). In a subset of these systems there is reinitiation of RNA synthesis (10,13,17), and in some cases polyadenylation occurs in the isolated nuclei (8,10,14). In one example nuclei have been shown to accurately initiate transcription of exogenous DNA added to the incubation mixture (13). Recently, runoff transcription in isolated nuclei has been used to study the role of enhancer elements (24). The environtnent of the isolated nuclei can be precisely controlled so it is possible that these nuclei can be used to study the effects of soluble factors on transcription.The abundance of genetic and molecular biological data available for the yeast Saccharomyces cerevisiae make it especially desirable to develop a yeast nuclear isolation procedure capable of accurately reconstructing in vivo events. The first step in achieving this goal is to demonstrate that yeast nuclei contain active RNA polymerases I, II, and III. Yeast RNA polymerases I and II are 50% inhibited at a-amanitin concentrations of 300 to 600 and 1 ,ug/ml, respectively (11,18), and RNA polymerase III is insensitive to a-amanitin concentrations of up to 2.4 mg/ml (18,19). Previous studies have shown that oa-amanitin inhibits the synthesis of some size classes of RNA in isolated nuclei (14,19), but the RNAs were not positively identified. We used these unique drug sensitivities to detect the presence and activity of the three RNA polymerases by measuring the sensitivity of rRNA synthesis, tRNA synthesis, and rnRNA synthesis to a-amanitin. The low levels of most mRNAencoding genes make detection of a unique message difficult. To overcome this problem, a strain containing a high-copynumber plasmid encoding the GAL7 gene was used for the initial ...

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Section: Resultsmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 88%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

Jerome¹,

Jaehning²

1986

Mol. Cell. Biol.

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Functional analysis of the transcriptional control regions of the copia transposable element

et al. 1986

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The introduction of copia‐based vectors in Drosophila hydei cells results in their high‐level transient expression and the subsequent establishment of stably transformed cell lines containing multiple copies of vector integrated into host genomic DNA. Using transformation frequency and transient expression analysis as assays of promoter strength, we have defined the regions of copia essential for expression. We find that the essential sequences reside within the long terminal repeat, but 3′ to the site of initiation of copia RNA. Deletion of the consensus enhancer‐like sequences from copia appears to have no effect on vector expression.

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Reduced RNA synthesis levels in isolated mouse liver nuclei following reaction with [(H2O)(NH3)5Ru(II)]2+

1989

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The goal of this study is to establish the effect of [(H2O)(NH3)5Ru(II)]2+ reaction of nuclei on their RNA transcription levels. This question is important because ammineruthenium compounds share chemical and biological properties with the chemotherapeutic agent cis-dichlorodiammineplatinum(II) or cisplatin. First we demonstrate that mouse liver nuclei are active in RNA transcription in vitro and characterize the optimum conditions for in vitro transcription. Synthetic rates in the presence of inhibitors actinomycin D and alpha-Amanitin and measurements of oligo(dT)-cellulose RNA binding levels suggest that all three RNA Polymerases are active in synthesis at about the following percentages-RNA Polymerase I(30%), II(50%) and III(20%). Mouse liver nuclei reacted with [(H2O)(NH3)5Ru(II)]2+ and then oxidized had (NH3)5 Ru(III)3+n-DNA adduct levels inversely related to total RNA synthetic rates. Oligo(dT) cellulose RNA binding levels did not vary with DNA adduct density. These data suggest that direct DNA lesions rather than [(NH3)5Ru(III)]3+ effects on other aspects of the transcription system are responsible for the diminished RNA synthesis levels. Ammineruthenium complexes remain desirable candidates for chemotherapeutic agents that may be safely administered in the unreactive ruthenium(III) state and be activated toward DNA binding by reduction in the hypoxic environment of many tumour cells.

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Ribonucleic acid synthesis in isolated Drosophila nuclei

Cited by 25 publications

References 19 publications

mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

Functional analysis of the transcriptional control regions of the copia transposable element

Reduced RNA synthesis levels in isolated mouse liver nuclei following reaction with [(H2O)(NH3)5Ru(II)]2+

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