Electron Microscope Visualization of RNA Transcription and Processing in Saccharomyces cerevisiae by Miller Chromatin Spreading

Abstract

The Miller chromatin spreading technique for electron microscopic visualization of gently dispersed interphase chromatin has proven extremely valuable for analysis of genetic activities in vivo. It provides a unique view of transcription and RNA processing at the level of individual active genes. The budding yeast Saccharomyces cerevisiae has also been an invaluable model system for geneticists and molecular biologists. In this chapter, we describe methods for applying the Miller chromatin-spreading method to Saccharomyces cerevisiae. This allows one to use electron microscopic visualization of a gene of interest to study effects of specific mutations on gene activity. We are applying the method to study transcription and processing of ribosomal RNA.

Distinguishing the Roles of Topoisomerases I and II in Relief of Transcription-Induced Torsional Stress in Yeast rRNA Genes

“…4A, left panel), but typical Miller-spread conditions are known to remove some nucleic-acid-bound proteins. When we used more physiological spreading conditions (56,57), both strands of DNA within the bubble still appeared similar, but were now thicker than the flanking DNA (Fig. 4A right).…”

“…EM visualization of nucleolar chromatin dispersed by Miller spreading allows quantitative analysis of many rRNA genes from multiple nucleoli (25,57), thus combining the advantages of studying both individual genes and gene populations. The method was used to analyze rDNA from control yeast strains and strains lacking Top1 (top1⌬).…”

“…Control and experimental strains for RNase H under-and overexpression were YAEH271 Miller chromatin spreading and EM analysis. Cells were prepared for electron microscopy (EM) analysis, mainly using method 1 as described previously (57). Briefly, yeast strains were grown at 30°C in yeast extract-peptone-dextrose (YPD) for 5 to 6 h before being diluted ϳ1:100 into YPD plus 1 M sorbitol and grown to mid-log phase (A 600 ϭ 0.3 to 0.5).…”

Rpd3- and Spt16-Mediated Nucleosome Assembly and Transcriptional Regulation on Yeast Ribosomal DNA Genes

“…Miller spreads were made as described previously (27), using either method 1 for typical chromatin spreads (see Fig. 5) or method 2 for improved retention of nucleosomes (see Fig.…”

RNA polymerase I activation and hibernation: unique mechanisms for unique genes

“…Second, promoters contain two main regions termed upstream element (UE) and core element (CE) in yeast, with the latter overlapping with the transcription start site and the former extending to about 150 bp upstream of this site [2]. Third, in actively growing cells rDNA transcription represents 60% of the total transcriptional activity [3], which correlates with a high content of RNA polymerase occupancy on rDNA genes [4]. As a consequence, rDNA transcription is a major point for the regulation of cell growth and, thus, misregulation of the system is related with tumour development [5].…”