mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

Abstract

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 ...

A region internal to the coding sequences is essential for transcription of the yeast Ty-D15 element.

“…Second, the inserted SP016 fragment in pK-54 and pK-55 blocks the activating effect of the IAR so that initiation in the 8 and the inserted DNA is reduced. (20), Ty transcription in isolated nuclei is inhibited by a-amanitin concentrations characteristic of a gene transcribed by RNA polymerase TI (22), and a strain with a temperature-sensitive mutation in a gene for a subunit of RNA polymerase IT stops transcribing Ty at the restrictive temperature (31). Therefore, Ty elements are transcribed by RNA polymerase II as one would expect for the large, polyadenylated Ty RNAs.…”

Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa.

“…5). Nuclear transcription assays have been used to provide indications of RNA polymerase loading levels for a gene (13,16,18,43). To further examine the level of regulation, a nuclear transcription assay was performed.…”

“…Nuclei were isolated and assayed by modifications of previously described procedures (2,15,18). All samples were processed after no more than 10 h of growth after an inoculation of conidia.…”

“…Incubations were for 20 min at 30°C. The synthesized RNA was extracted and ethanol precipitated (18,46), and 5 x 107 cpm was used for hybridization to DNA bound on nitrocellulose filters. To prepare the filters, 5 ,ug of DNA was alkali denatured and bound to nitrocellulose (13 …”

Some of the signals for 3'-end formation in transcription of the Saccharomyces cerevisiae Ty-D15 element are immediately downstream of the initiation site.

“…The 8's, direct repeats of about 340 bp at each end of the element, flank the 5.2-kb E internal region (26,45). Ty elements are heavily transcribed by RNA polymerase 11 (13) to produce 5 to 10% of the poly(A)' RNA in haploid yeast cells (8). The most abundant Ty transcript is a 5.7-kb RNA with its 5' end in one 6 and its 3' end in the opposite 6.…”