QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fuchs, J. Articles by Loidl, J. Search for Related Content PubMed PubMed Citation Articles by Fuchs, J. Articles by Loidl, J.

Chromosome associations in budding yeast caused by integrated tandemly repeated transgenes

Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria

View larger version (29K): [in a new window]   Fig. 1. Maps of tetO integration sites and FISH probes on chromosome V. (A) One tetO tract is integrated 35 kb away from the centromere on the short arm at the URA3 locus. The other integration site is 30 kb away from the right telomere at the BMH1 locus. (B) Yeast strains with different tetO inserts used in the experiments. Diploid strains are homozygous for CEN tetO or TEL tetO or heterozygous for the two inserts on chromosomes V. A haploid strain has both inserts in the same chromosome (cis). These strain constructs exist both as versions expressing and not expressing TetR-GFP (grey ovals, centromeres; green rectangles, integrated tetO sites in the presence of TetR; grey rectangles, integrated tetO sites in the absence of TetR). (C) Map of FISH probes for regions adjacent to centromeric (1), telomeric (2) tetO insertion sites, and for the tetO sequence (3).

View larger version (120K): [in a new window]   Fig. 2. Association and intranuclear organization of chromosomal tetO inserts in yeast nuclei. (A) In a diploid strain, two tetO repeats associate in the presence of TetR molecules and form a single signal in most cells, irrespective of their chromosomal positions. (B) In hydroxyurea-arrested nuclei, tetO associations are reduced and two separate GFP signals are seen in most nuclei. (C-E) Association behaviour of tetO repeats during anaphase of diploid cells. (C) Centromerenear tetO repeats (CEN/CEN) remain mostly associated and form a single GFP spot near the two spindle poles. (D) Telomeric regions (TEL/TEL) also remain mostly associated and appear as a single signal in the two daughter nuclei. (E) Ectopic tetO inserts (one near the centromere, the other near the telomere: trans-CEN/TEL), which are frequently associated in interphase, are mostly separate in anaphase cells and appear as two GFP signals in both daughter nuclei. (F,G) Recruitment of the telomere-near tetO repeat to the centromeric pole in trans-CEN/TEL nuclei. FISH with probes for the centromere-near (F, probe 1) and telomere-near (G, probe 2) tetO insertion site in wildtype (left) and the trans-CEN/TEL strain (right). Centromeres are always close to the SPB. In the wildtype, both telomeres are far from the SPB, whereas in the trans-CEN/TEL strain, one telomere is usually close to the SPB (see also Fig. 5). green, GFP; red, immunostained spindle and SPBs; orange, FISH signals. Bar, 2 µm.

View larger version (23K): [in a new window]   Fig. 3. Frequencies of tetO association in different GFP-TetR-expressing yeast strains (see Fig. 1B) in exponential (light grey bars) and stationary (dark grey bars) cultures. For each strain the experiment was repeated 14 times with 100 nuclei evaluted in each.

View larger version (17K): [in a new window]   Fig. 4. Association frequencies of the centromere-near with the telomere-near region in haploid strains (see Fig. 1B), monitored by adjacent FISH probes (probes 1 and 2, see Fig. 1C). Only in the presence of both the non-allelic tetO repeats and the TetR protein is the association high. With tetO only, or after inhibition of TetR binding to tetO by addition of tetracycline, association is as low as in the wildtype. The experiments were repeated five times and 50 nuclei were evaluated in each.

View larger version (23K): [in a new window]   Fig. 5. Recruitment of telomeric chromosome regions to the centromeric pole of the nucleus due to tetO-mediated centromeretelomere association. The positions of centromeres and telomeres of chromosomes V were highlighted by site-specific FISH probes (probes 1 and 2, see Fig. 1C) in different colours, and the SPB (marking the centromeric pole) by simultaneous immunostaining (see Fig. 2F,G). Distances of signals to the SPB were measured and given as a percent of the nuclear diameter.