Figure 1.
Recombination Interval Used for Molecular Analyses
(A) The 3.5-kilobase ectopic recombination interval contains coding sequences for URA3 (gray) and ARG4 (black). DSBs form in the promoter regions of URA3 (DSB1) and ARG4 (DSB2). The lollipop in arg4 represents a palindromic sequence inserted at +9 of the open reading frame; this mutation is used to score gene conversion [16].
(B) The ectopic recombination interval is inserted at HIS4 (blue) on one copy of Chromosome III and at LEU2 (red) on the homolog. HIS4 and LEU2 are 16.7 kilobases apart. In rad50S strains, where DSBs persist, 5% of chromosomes have a DSB in his4::URA3-ARG4 and 0.7% have a DSB in leu2::URA3-ARG4 [16]. The centromere (black circle) and MAT locus (green) are also indicated. Allelic COs can be scored in the HIS4-LEU2 and LEU2-MAT intervals.
(C) Ectopic COs can occur between his4::URA3-ARG4 and leu2::URA3-ARG4.
Figure 2.
Loss of Full Sgs1 Activity Restores Spore Viability and Crossing Over to zmm Mutants
(A) Overall spore viability and patterns of spore lethality in tetrads from SK1 strains.
(B) Map distance (cM; error bars denote standard error of map distance) in three intervals on Chromosome III in SK1 (see Figure 1B and 1C for details). Values for wild-type are from [16].
(C) Map distance in two allelic intervals on Chromosome III in BR strains. Values for wild-type and sgs1Δ are from [32].
Figure 3.
Sgs1 Prevents COs in zmm Mutants
(A) Schematic representation of the ectopic URA3-ARG4 interval. Symbols are as in Figure 1. EcoRI (E) and XhoI (X) restrictions sites are indicated. To detect COs and DSBs, DNA is digested with XhoI and probed with ARG4 sequences (ArgD; [16]). To detect NCOs and a CO product (CO1′), DNA is digested with EcoRI and XhoI and probed with HIS4 sequences (HisU; [16]).
(B) Southern blot of DNA isolated from a meiotic culture of MJL3035 (TRP1:SGS1) at the indicated time after initiation of sporulation, digested, and probed to detect COs and DSBs. mw, HinDIII digest of phage λ DNA. DSBs occur in URA3-ARG4 inserted at both his4 and leu2, but are ~7-fold stronger in the his4 insert than at leu2 [16]. Palindrome cleavage, by unidentified activities, occurs at the same time as CO formation and results in a band about the size of DSB2 (T. Allers, L. Jessop, and M. Lichten, unpublished data).
(C) Southern blot of the same samples, digested and probed to detect NCO and CO1′ recombinants. HIS4, HIS4 locus lacking an insert; DSB*, DSB1 product where resection has passed the EcoRI site in HIS4; mw, BstEII digest of phage λ DNA.
(D) Molecular analysis of mutants. Color codes: TRP1:SGS1, black; sgs1ΔC795, grey; TRP1:SGS1 zmm single mutants, red; sgs1ΔC795 zmm double mutants, blue. M1+M2, percent of cells containing at least two nuclei, a sign of passage through meiosis 1. DSB, DSB1 band signal/total lane signal from Southern blots as in (B). CO, CO1′ signal/total lane signal from Southern blots as in (C). NCO, NCO band signal/total insert signal from Southern blots as in (C).
Figure 4.
Sgs1 Prevents AA in mer3Δ and zip3Δ Mutants
(A) Analysis of AAs in TRP1:SGS1 mer3Δ and mer3Δ sgs1ΔC795 SK1 strains. Nuclei from cells harvested 5 h after initiation of sporulation were surface spread and probed with anti-Zip (red) and anti-Red1 (green) antisera. Nuclei where chromosomes displayed linear Red1 were examined and classified as displaying minimal, partial, or full pseudosynapsis, as described in Materials and Methods. Nuclei with fully associated chromosomes displayed discontinuous Zip1 staining (right-hand example), Zip1 localization in polycomplexes (PC, left-hand example), or both. White bar: 2 microns.
(B) Analysis of AAs in zip3Δ and zip3Δ sgs1Δ BR strains. Nuclei from cells harvested 18 h after the initiation of sporulation were spread, stained, and analyzed as for mer3 strains. In most nuclei with full AA, at least some chromosome pairs displayed end-to-end Zip1 staining with the remainder being pseudosynapsed.