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Results: 6

1.
Figure 3.

Figure 3. From: Pol12, the B subunit of DNA polymerase ?, functions in both telomere capping and length regulation.

The pol12-216 and pol12-109 (unlike pol1 and other pol12 alleles) are lethal in combination with hypomorphic STN1 mutations. Single- or double-mutant strains (right) containing the YCp33-STN1-URA3 plasmid were grown either in the absence (left) or presence (center) of 5-FOA to select for the loss of the plasmid. Strains carrying the stn1-101 or stn1-154 mutations have been deleted at their STN1 chromosomal locus and contain a LEU2 plasmid with the indicated mutant STN1 allele in addition to the YCp33-STN1-URA3 plasmid (see Materials and Methods).

Simona Grossi, et al. Genes Dev. 2004 May 1;18(9):992-1006.
2.
Figure 6.

Figure 6. From: Pol12, the B subunit of DNA polymerase ?, functions in both telomere capping and length regulation.

Genetic interaction of the pol12-216 mutation with TLC1, RAD52, RIF1, RIF2, TEL1, MRE11, and RAD50 deletions. Southern blot analysis of telomeres from the indicated single- and double-mutant strains (as described in Fig. 2C). (A) The tlc1Δ and pol12-216 tlc1Δ double-mutant spores were identified by tetrad analysis, as described in Materials and Methods, and directly inoculated in liquid YPD medium for DNA preparation and Southern blot analysis. (B-E) Diploid parent strains and haploid mutant segregants were restreaked at least 10 times on YPD medium at 30°C prior to DNA preparation (and sporulation, in the case of diploids). Telomeric DNA was hybridized either to a d(TG)/d(CA) probe (A-D) or to a Y′ probe (E), as described in Materials and Methods.

Simona Grossi, et al. Genes Dev. 2004 May 1;18(9):992-1006.
3.
Figure 4.

Figure 4. From: Pol12, the B subunit of DNA polymerase ?, functions in both telomere capping and length regulation.

Physical interaction between Pol12 and Stn1. (A) Values obtained from liquid β-galactosidase assays to detect two-hybrid interactions between LexA/Pol12 and GAD/Stn1 fusion proteins. (B) In vitro binding of Stn1-HA to GST-Pol12, GST-pol12-216, GST-Pol12 Nt (N terminus), and GST-pol12-216 Nt hybrid proteins. Crude whole-cell extracts were prepared from W303-1B carrying the multicopy plasmid pMC479 (PGAL1-STN1-2HA-6HIS) and grown to exponential phase in selective medium containing either galactose (lanes 1) or glucose (lanes 2), to induce and repress the expression of Stn1, respectively. The extracts were incubated with GST-Pol12 (or any one of the fusion hybrids indicated above) bound to glutathione agarose beads. As an additional control, the W303-1B strain carrying the empty vector pE195, and grown in the presence of galactose, was included (lanes 3).

Simona Grossi, et al. Genes Dev. 2004 May 1;18(9):992-1006.
4.
Figure 5.

Figure 5. From: Pol12, the B subunit of DNA polymerase ?, functions in both telomere capping and length regulation.

Increase in telomeric single-stranded DNA in pol12-216 stn1-13 double mutants. (A) Schematic representation of the strains used in the experiment. The POL12 and STN1 alleles of each strain are indicated in B. Each strain contains an STN1-ADE2 plasmid in which the CEN/ARS sequence is flanked by two LoxP sites in the same orientation (pGS120). A second plasmid (pSH63) carries the Cre recombinase gene driven by the GAL1 promoter. Growth in galactose at 23°C leads to the “pop out” of the intervening CEN/ARS sequence and consequently to the loss of the pGS120 plasmid. (B) In-gel hybridization analysis of telomeres from POL12 STN1 wild type, pol12-216, and stn1-13 single-mutant and pol12-216 stn1-13 double-mutant strains grown in the presence of galactose for the indicated times. (Top) The native gel was hybridized to a CA-rich labeled probe. (ExoI) ExonucleaseI. (Bottom) The same gel was denatured and hybridized to a CA-rich labeled probe.

Simona Grossi, et al. Genes Dev. 2004 May 1;18(9):992-1006.
5.
Figure 2.

Figure 2. From: Pol12, the B subunit of DNA polymerase ?, functions in both telomere capping and length regulation.

Phenotypes and synthetic interactions of pol12-216 and pol1 mutants. (A) Growth and silencing phenotypes of pol12-216 and pol1 mutants. Tenfold serial dilutions of overnight cultures were spotted on YPD (30°C and 37°C), SC, SC-Ura, and SC + 5-FOA media (30°C). Relevant genotypes are indicated on the left. (B) In-gel hybridization analysis of telomeres from strains grown for several generations at 30°C. (Left) A native agarose gel was hybridized with a CA-rich labeled probe. (Right) The same gel was denatured and rehybridized with a CA-rich probe. (C) Southern blot analysis of telomeres from the indicated single- and double-mutant strains (XhoI digestion and hybridization to a d(TG)/d(CA) probe; see Materials and Methods). All the strains were restreaked at least 10 times on YPD medium at 30°C (left panel) or 23°C (right panel) before telomere length analysis was performed. (D) The same strains (five of which are shown) were spotted on YPD at 23°C and 30°C to test for viability.

Simona Grossi, et al. Genes Dev. 2004 May 1;18(9):992-1006.
6.
Figure 1.

Figure 1. From: Pol12, the B subunit of DNA polymerase ?, functions in both telomere capping and length regulation.

Instability of a modified Chromosome VII-L telomere carrying 32-misoriented Rap1-binding sites. (A) Colony color assay using the ADE2 marker to detect VII-L telomere loss from strains disomic for Chromosome VII (left). The wild-type and modified copies of Chromosome VII-L present in test (top left) and control (bottom left) strains are shown. The modified copy of Chromosome VII-L carries either 32 misoriented (black arrows pointing right; test strain, top left) or correctly oriented (black arrows pointing left; control strain, bottom left) Rap1-binding sites, the URA3 and the ADE2 genes integrated at the ADH4 locus. White arrows represent native TG1-3 sequence. The strains are ADE3 and ade2Δ at their chromosomal loci. Examples of isolated colonies generated by either strain on YPD medium are shown on the right. (B) Schematic representation of the modified VII-L telomere (as in A) in strains disomic for Chromosome VII (top). The black arrowhead (pointing right) represents a synthetic Rap1-binding site in the “incorrect” orientation. (H) HindIII. (Bottom panel) The Southern blot analysis of telomeres containing the indicated number of Rap1 sites. Following HindIII digestion, the URA3 probe detects the endogenous ura3-52 locus, a diffuse telomeric band, and a faint ladder of bands in the case of 32 misoriented Rap1-binding sites.

Simona Grossi, et al. Genes Dev. 2004 May 1;18(9):992-1006.

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