Replication Pausing and Imprinting Figure 3. Identification of a cis-acting ‘‘spacer’’ element in mat1M. (A) Left, line drawing representation of the strains used is given. The grey line segment indicates the fragment that is substituted by a random stretch of DNA. Right, the names of the strains and quantification of the DSB products (including standard error) and of the pause signals are given. (B) Southern analysis of the strains shown in panel A. See Figure 2 legend for method. (C) 2D-gel analysis of the strains shown in panel A. See Figure 2 legend for method. (D) Schematic representation of the mating-type region of the strain used for analyzing the spacer deletion in vivo. The largest spacer deletion (same as in SS13, see A) was introduced into the donor locus mat3M to yield a mutant form of the donor M information (denoted as M*). The ura4+ gene linked to the mat3M locus, which was used during strain construction, is marked by a triangle. (E) Sporulation phenotype caused by the spacer deletion. The h90 wild-type strain and the mutant described in panel D (SS56) were allowed to form single colonies on sporulation media (PMA+), which were subsequently stained by iodine vapours. Iodine reacts with the starch found in spores to yield dark-coloured colonies. (F) Southern analysis of the strain SS56. Genomic DNA from h90 wild type and SS56 strains was digested with HindIII and probed for the mat1 locus. Because of the presence of homology, the donor loci are also visualized. The positions of the bands corresponding to mat1, mat2P, mat3M, DSB products, and mat3M linked to the ura4+ gene (mat3::ura4+) are indicated. doi:10.1371/journal.pgen.1001328.g003 Figure 4. Transcription of the mating-type-specific gene Mc does not affect imprinting. (A) The names of the strains and quantification of the DSB products generated during growth in either low-nitrogen sporulation (PMA+) or rich (YEA) media are given. The levels of imprinting, as assessed by DSB products, are similar for both growth conditions. (B) The effect of abolishing Mc transcription on the spacer deletion phenotype. Left, schematic representation of the mat1M cassette and the strains used is given. The positions of the Mi and Mc transcripts are shown by horizontal arrows. Deletion of the promoter region between the two transcripts (in strains SS66 and SS63) was performed, thus abolishing transcription. Right, the names of the strains and quantification of the DSB products (including standard error) are given. (C) Southern analysis of the strains shown in panel B. See Figure 2 legend for method. (D) Northern analysis of total RNA from the strains shown in panel B. The blot was probed for Mc using a double-stranded fragment generated by PCR. (E) Characterization of the strain (SV20) carrying a deletion of most of the spacer region and the region containing the previously mapped priming site. See Figure 2 legend for method. The position of the deletion is shown as a line drawing. Please note that fork-regression at MPS1 is observed in the 2D-gel displayed, in the absence of the imprint. This observation is fully analyzed in a related study (Vengrova and Dalgaard, in preparation). doi:10.1371/journal.pgen.1001328.g004 PLoS Genetics | www.plosgenetics.org 7 March 2011 | Volume 7 | Issue 3 | e1001328