Complete deletion of yeast chromosomal rDNA repeats and integration of a new rDNA repeat: use of rDNA deletion strains for functional analysis of rDNA promoter elements in vivo.
Strains of Saccharomyces cerevisiae were constructed in which chromosomal rDNA repeats are completely deleted and their growth is supported by a plasmid carrying a single rDNA repeat, either a plasmid carrying the 35S rRNA gene transcribed from the native promoter by RNA polymerase I or a plasmid carrying the 35S rRNA gene fused to the GAL7 promoter for transcription by RNA polymerase II. This system has made it possible to assess the expression of rDNA by measuring the ability of synthesized rRNA to support cell growth as well as by measuring the actual rRNA synthesized rather than by the use of reporter mini-rDNA genes. Using this system, deletion analysis of the rDNA promoter confirmed the presence of two elements, the upstream element and the core promoter, and showed that basal transcription from the core promoter, if it takes place in vivo as was observed in vitro, is not sufficient to allow cell growth. We have also succeeded in integration of a rDNA repeat and its copy number expansion at the original chromosomal locus, which will allow future mutational analysis not only of rRNA but also other DNA elements involved in rRNA transcription, rDNA replication and recombination within a repeated rDNA structure.
Wai
H H
HH
Department of Biological Chemistry, University of California at Irvine, Irvine, CA 92697-1700, USA.
Vu
L
L
Oakes
M
M
Nomura
M
M
D015825:Chromosomes, Fungal; D004271:DNA, Fungal / Q000235:genetics* / Q000502:physiology; D004275:DNA, Ribosomal / Q000235:genetics* / Q000502:physiology; D018628:Gene Dosage; D005821:Genetic Techniques; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; D012319:RNA Polymerase II; D012318:RNA Polymerase I