Somatic pairing of homologs in budding yeast: existence and modulation

Abstract

FISH analysis of well-spread chromosomes reveals that homologs are paired in vegetatively growing budding yeast diploid cells, via multiple interstitial interactions, and independent of recA homologs and mating type heterozygosity. Pairing is present during G1 and G2, and in cells arrested at G1 by mating pheromone, but is disrupted during S phase. Thus, somatic pairing is qualitatively analogous to premeiotic and early meiotic pairing. S-phase pairing disruption occurs by a complex intranuclear program involving regional, nucleus-wide, and temporal determinants. Pairing is also disrupted in two G2-arrest conditions (cdc13ts and nocodazole). Together these findings suggest that cell cycle signals may provoke pairing disruption by modulating underlying chromosome and/or chromatin structure. Whether the cell chooses to disrupt pairing contacts or not (e.g., S phase and G2 arrest, but not G1 arrest or normal G1 or G2), could be dictated by functional considerations involving homolog/sister discrimination.

Figure 1

FISH analysis. (A) Images obtained by hybridization of two probes labeled with digoxigenin (green probe, shown in white) or dinitrophenyl (red probe, shown in black) to DNA in spread nuclei from hypotonically lysed cells. DNA was visualized by DAPI staining (demarcated by broken line). Images were digitally collected and processed to determine the distances between all pairs of hybridizing signals (RR, GG, and GR1–GR4). Examples of the four observed classes of nuclei and corresponding measured distances (μm) between signals are given. Homologs separated by ≤0.7 μm are considered to be paired (Weiner and Kleckner 1994). Bar, 2 μm. (B) Probes used.

Figure 2

Ranked homologous and nonhomologous foci distances from premeiotic, pheromone-arrested, and cycling cells. In each panel, the six distances among pairs of hybridization signals (Fig. 1A) are individually ranked from the smallest to largest. (□) RR; (●) GG; (♦) GR1–GR4. The cutoff limit for pairing (d ≤ 0.7 μm) is indicated by a horizontal line for each plot. R_obs and G_obs are indicated for A by open and solid arrowheads, respectively. All cultures are SK1 MATa/MATα unless otherwise noted; all data are from Table 1. Loci probed are listed within each panel.

Figure 3

Homolog pairing in synchronously dividing cells. Diploid MATa/MATa cells were released from pheromone arrest at t= 0 min. (A) Percentage of cells in each morphological class. (B) Cumulative curves showing the entry into and exit from each morphological category were calculated from the plots shown in A. The cumulative curve describing the exit from G₁ was calculated from the noncumulative plot of [1 − (%G₁)] (not shown). (C) FACS and FISH analysis with probes to two chromosomes at representative time points (□ red probe q; ● green probe g). Solid and open arrows indicate the 40- and 45-min time points, when most cells are in S phase.

Figure 4

Pairing in synchronously dividing cells. (Arrows) S phase as in Fig. 3; (broken lines) onset of late anaphase according to nuclear morphology. (A) Observed pairing levels (Table 1). (B) Nucleus types (Fig. 1A). (C) Percentages of nuclei not engaged in pairing and the calculated duration of S-phase unpairing (see text). (D) Cumulative curves for entry into and exit from unpairing (see text). Shaded region shows entry into and exit from the small bud morphology stage (relevant curves in Fig. 3). At t=0 min, probes R- (r) and G- (o) were used.

Figure 5

Pairing in G₂/M-arrested cells. (A, a–c) FISH analysis of exponentially growing cells from culture 11 (MATa/MATα, SK1; Table 1) before and after treatment with nocodazole. (B) Parallel analysis of wild-type and cdc13-1ts cultures grown to mid-log phase at 23°C (d,f) and shifted to 37°C for 2 hr (e,g). (C) Distribution of cells among morphological classes for all samples analyzed in A and B. (##) In cdc13-1ts arrested cells, no true late anaphase cells were seen; instead, a thin thread of DAPI-stained material from the bulk nuclear mass extended through the bud neck and into the bud cell.