Saccharomyces cerevisiae killer virus transcripts contain template-coded polyadenylate tracts.

Abstract

The M double-stranded RNA component of type 1 killer strains of the yeast Saccharomvces cerevisiae contains an internal 200-base pair adenine-and uracil-rich region. The Virus particles from both killer and nonkiller strains have been shown to copurify with a DNA-independent RNA polymerase activity which catalyzes the synthesis of fulllength (as judged by denaturing gel electrophoresis), asymmetric positive polarity transcripts of L and M dsRNAs (13, 51, 53), designated I and m, respectively. Denatured L dsRNA and I transcript can be translated in vitro to produce the major capsid protein of L-and M-containing virions (10,13,29). Denatured M dsRNA and m transcript encode Mp32, a 32,000-dalton putative toxin precursor (9, 52). L dsRNA has recently been found to consist of at least three distinct forms, LA, LB, and Lc, which are present in various combinations in different yeast strains (21,23,45).Electron microscopy of M dsRNA has shown an internal, readily denaturable region of approximately 200 base pairs, which appears to be almost 100% adenine plus uracil (A+U) base pairs (26). M dsRNA can be selectively cleaved at this internal region by Si nuclease treatment or high temperature (48,52) to yield two double-stranded fragments, designated M-1 (1,000 base pairs) and M-2 (630 base pairs). The A,Urich region lies between M-1 and M-2. Denatured M-1 encodes M-p32, and denatured M-2 encodes variable amounts of a 19,000-dalton protein (M-p19) in a rabbit reticulocyte lysate protein synthesis system (52). S3 dsRNA, an internal deletion mutant of M dsRNA, lacks the 200-base pair, A,U-rich region along with flanking sequences (26;

tRNA genes as transcriptional repressor elements.

“…Expression of ,B-galactosidase from the CUPJ UAS/promoter constructs (Fig. 5) was assayed in triplicate in at least two separate experiments as described previously (30,35). Although absolute numbers of units varied between experiments, triplicate assays within an experiment were reproducible (±5%), and the degree of repression by tRNA genes was consistent between experiments.…”

Upstream activation and repression elements control transcription of the yeast COX5b gene.

“…RNA was prepared by a modification of the method described by Hannig et al (19). Approximately 40 ml of cells from a logarithmic culture was collected by low-speed centrifugation, washed with extraction buffer (50 mM Tris hydrochloride [pH 7.5], 10 mM EDTA, 0.1 M NaCl), and suspended in 250 ,ll of ice-cold extraction buffer plus 1% sarcosyl.…”

Tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene.

“…Cultures (400 ml) for RNA analyses were grown and induced as described for galactokinase assays. Cells were harvested at 4°C and washed with 25 ml of ice-cold sterile H20, and total RNA was isolated as described previously (11). Si nuclease mapping was performed as described previously (23) with 25 ,ug of total RNA per sample.…”