(A) The 3.5-kb silenced HMR domain
can be expanded to 7 kb. sirΔ strains carrying
insertions (at SGD coordinate 293032) of either (1) 1-kb of the
TRP1 gene (ROY49 and ROY1075) or (2) 1 kb of the TRP1
gene plus 2.5 kb of the HMG2 coding sequence (ROY55 and
ROY1080) were generated. Strains ROY803, ROY84, ROY1076, and ROY 1079
are Sir+ derivatives of strains ROY49, ROY55, ROY1075, and ROY1080,
respectively. All cells were grown in liquid media and 3 μl of
10-fold serial dilutions were spotted on either YPD plates (+Trp) or on
YMD plates lacking tryptophan (−Trp) to assay for TRP1
expression. Strain numbers are shown in parentheses. (B)
Differential restriction endonuclease digestion analysis of the
expanded silenced HMR domain. Nuclei isolated from wild-type
and sirΔ strains were digested with various
restriction endonucleases. The DNA following purification was digested
with a second restriction endonuclease and analyzed by DNA blot
hybridization. For each site tested the band corresponding to wild-type
cells is present on the left and the
sirΔ strain is on the right.