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First published online 6 May 2003
doi: 10.1242/jcs.00453

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Increased ploidy and KAR3 and SIR3 disruption alter the dynamics of meiotic chromosomes and telomeres

¹ Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany
² Institute of Botany, University of Vienna, Vienna, Austria

View larger version (28K): [in a new window]   Fig. 2. (A) Representative centromere (green) and (B) XY' telomere FISH signal patterns (red) in mildly spread nuclei (DAPI, blue) obtained from synchronized meiotic cultures of meiosis-proficient haploid sir3 spo13 control strain (haploid), a corresponding haploid kar3 sir3 spo13 strain (haploid kar3), a diploid sir3 strain (diploid sir3) and a tetraploid strain (tetraploid). All strains were of identical SK1 background. The FISH patterns observed are ordered according to their deduced sequential occurrence and match those observed in diploid wild-type SK1 meiosis (Trelles-Sticken et al., 2000). (A, left) Premeiotic nuclei (0 minutes, fixed at transfer to SPM) with a single centromere signal cluster (green). (A, right) Meiocyte nuclei (taken at 300 minutes) exhibit dispersed centromere signals. Notice the increase in cen-cluster size from haploid to tetraploid. (Bi) Premeiotic nuclei (0 minutes) of all strains display a few telomere FISH signal clusters (red). (Bii) Early meiocytes exhibiting a meiosis-specific rim-like telomere distribution (Hayashi et al., 1999; Trelles-Sticken et al., 2000). (Biii) Bouquet nuclei of all strains, which exhibit telomere FISH signals clustered at a limited region of the nuclear periphery. (Biv) Meiocyte nuclei showing dispersed telomere FISH signals, which is typical for pachytene (Trelles-Sticken et al., 1999). (C) Colocalization of clustered telomeres (red) and the SPB (green). In the diploid sir3 and tetraploid SK1 strain, telomeres and SPB were visualized by XY' telomere FISH in combination with Tub4 IF, whereas telomeres in the haploid control and haploid kar3 strain were stained by Ndj1-HA IF. Notice the rise in nuclear diameter and signal numbers with increase in ploidy. Bar, 5 µm.

View larger version (14K): [in a new window]   Fig. 1. Occurrence of meiotic divisions after induction of sporulation in diploid wild-type (2n WT), tetraploid (4n) and sir3 diploid (2n (sir3)), and in sir3 spo13 haploid (1n (sir3)) SK1 strains. Bi- and tetranucleated cells (100-200 per time point) were scored as divisions at the indicated time points in sporulation medium (SPM). The diploid and tetraploid time courses are given as the mean (±S.D.) of three experiments. Meiosis in diploid wild-type SK1 progresses significantly faster than in the tetraploid strain. Divisions in the sir3 spo13 haploid and sir3 diploid occur at similar times after induction of meiosis and are delayed with respect to diploid wild-type meiosis.

View larger version (16K): [in a new window]   Fig. 3. Frequencies of mildly spread nuclei that display a single centromere FISH signal (cen cluster) at the indicated time points after induction of meiosis in a diploid wild-type strain (2n WT), a tetraploid (4n), a sir3 diploid (2n sir3D) and a sir3 spo13 haploid (1n sir3D) SK1 strain. In the diploid wild-type and the 1n sir3D strain, centromere clustering is resolved at a similar pace. In tetraploid meiosis, cen clustering resolves only gradually, as is the case for the 2n sir3D strain after 180 minutes in sporulation medium (SPM; compare Fig. 5).

View larger version (16K): [in a new window]   Fig. 4. Relative frequencies of nuclei with a single telomere FISH cluster (bouquet) obtained in limited nuclear spreads of the following SK1 strains. Diploid wild-type (2n WT; data taken from Trelles-Sticken et al., 1999), tetraploid (4n) and sir3 diploid (2n sir3D) and haploid sir3 spo13 (1n sir3D). The data shown are corrected for the initial frequencies in that the t=0 values (6% in diploid WT, 2% in 2n sir3D, 0% in tetraploid and 2% in haploid) were subtracted from all values of the subsequent time points. Initial increase in bouquet frequencies is similar in diploid WT, haploid and diploid sir3 meioses, whereas tetraploid meiosis displays a substantial delay in bouquet occurrence. In sir3, haploid and diploid meiosis a further increase in bouquet frequency pauses after 180 minutes Two peaks in the mutant and tetraploid time course suggest that physiological sub-populations progress through meiosis consecutively.

View larger version (17K): [in a new window]   Fig. 5. Frequency of nuclei with one centromere cluster (cen cluster) in repeated time course experiments in the WT diploid (2n WT) and sir3 diploid (2n sir3D) SK1 strains. The values are the mean of four experiments in the wild-type and three experiments in the sir3 diploid. The standard deviation reflects considerable variability between the time courses in the mutant, whereas cen cluster resolution in the diploid WT is less variable, particularly at early time points.

View larger version (12K): [in a new window]   Fig. 6. FISH analysis of homologue pairing during meiotic time courses of wild-type (2n WT) and sir3 (2n sir3D) SK1 strains. Cosmid probe h, which maps near centromere XI (Trelles-Sticken et al., 2000), was hybridized to spread preparations obtained at various times after transfer to sporulation medium (Minutes in SPM). Pairing values (%) reflect the proportion of nuclei containing cosmid signals that touched each other or showed an enlarged coalesced signal. More than 100 FISH signal pairs were scored per time point and strain. The frequencies of nuclei with paired signals increased more gradually in the absence of Sir3p, reaching nearly wild-type levels with a 3 hour delay. In the wild type, maximal signal pairing values were reached at 300 minutes and remained nearly constant up to 420 minutes The occurrence of meiotic divisions after 240 minutes and 300 minutes in 2n WT and 2n sir3D meiosis, respectively, might explain why pairing values never exceeded 60%.

View larger version (18K): [in a new window]   Fig. 7. Centromere and telomere analysis of Ndj1-HA-expressing haploid control and kar3 SK1 strains after transfer to sporulation medium (SPM). The frequencies (based on >100 nuclei per time point) of nuclei with one centromere FISH cluster in meiotic time courses of haploid sir3 spo13 control [cen cluster (1n control)] and haploid kar3 sir3 spo13 [cen cluster (1n kar3)] drop at similar rates after the induction of meiosis. In the 1n kar3 time course, cen-cluster dissolution stalled between 180 and 240 minutes. All values were normalized with the values at 0 minutes (58% in WT; 56% in kar3) set to 100%. The frequency of bouquet nuclei with one peripheral Ndj1-HA IF telomere cluster peaks at 300 minutes in the haploid control [Ndj1-HA bouquets (1n control)] and kar3 [Ndj1-HA bouquets (1n kar3)] SK1 strain. Ndj1-HA bouquet nuclei are more abundant at all time points in kar3 meiosis. The bouquet frequencies shown are given as the percentage of haploid Ndj1-HA-expressing (meiotic) cells (74% in the WT; 48% in kar3 mutant; Fig. 8). It appears that bouquet nuclei accumulate in absence of Kar3p.

View larger version (15K): [in a new window]   Fig. 8. The frequency of nuclei exhibiting Ndj1-HA fluorescence in meiotic time courses of haploid sir3 spo11 control (Ndj1-HA+ hapl. control) and kar3 sir3 spo11 (Ndj1-HA+ hapl. kar3) SK1 strains. In the haploid control meiosis, fewer Ndj1p-HA-expressing cells (Ndj1p-HA+) are present at early time points, whereas most cells rapidly enter meiosis after 180 minutes in sporulation medium (SPM). A high frequency of kar3 cells expressing Ndj1-HA are seen before 180 minutes in SPM, whereas the increase of Ndj1-HA+ cells occurs only gradually over the entire time course. Similar observations are seen when the expression of Zip1p is monitored by IF and all cells displaying Zip1p signals of any nature were scored as Zip1p+. As in the Ndj1-HA study, induction of meiosis in the kar3 strain failed to induce a significant increase in Zip1p-expressing cells. The Ndj1-HA+ cells at 0 minutes probably result from ectopic sporulation of kar3 meiocytes.