FIG. 4.

CHEF analysis of chromosomes from strains carrying single and expanded rDNA* integrated at the native (RDN1) and ectopic sites and expression of rRNA from these ectopic loci. Chromosomal DNA was isolated from NOY388 (WT, lane 1); NOY986 (rdnΔΔ, lane 2); single rDNA* integrants NOY2048, NOY987, NOY988, and NOY2047 (lanes 3, 5, 7, and 9, respectively); and expanded rDNA* integrants NOY2029, NOY2030, NOY2031, and NOY2032 (lanes 4, 6, 8, and 10, respectively). Lane M, marker yeast chromosomes (obtained from Bio-Rad, Richmond, CA). (A) Chromosomal pattern revealed by staining with EtBr. The positions of native chromosomes V, VI, and XII are indicated. (B) Autoradiogram obtained after hybridization with a radioactive rRNA gene probe. Copy numbers of integrated rDNA* after expansion estimated from the chromosomal sizes are shown below chromosome bands carrying rDNA*. Expansion of the rDNA* integrated near TEL VI-R can be clearly recognized by comparing the bands indicated with diamonds in lanes 9 and 10 (in both panels A and B). The chromosome XII bands in rdnΔΔ with or without a single rDNA* copy at the native site (in lanes 2 and 3) overlap with the chromosome XV and VII bands, and the chromosome V bands with a single rDNA* (in lanes 5 and 7) overlap with the chromosome VIII band. Thus, the positions of chromosome XII or V before expansion of rDNA* at the native (rdn1) site, CEN5, or mid V-R cannot be clearly recognized in lanes 3, 5, or 7. C. RNA was prepared from the four expanded rDNA* integrants and NOY388 (WT), and equal amounts were analyzed by primer extension as in Fig. 2B. An autoradiogram is shown. The values obtained were normalized to the value obtained for the control WT strain (NOY388) and are indicated above the bands.