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. 2004 Mar 26;32(6):1917–1927. doi: 10.1093/nar/gkh511

Figure 1.

Figure 1

Single-stranded overhangs and sequence composition of telomeres in yeast strains expressing Tlc1h. Genomic DNA from yeast strains expressing wild-type TLC1 and strains expressing the humanized RNA (Tlc1h) was extracted after the strains had been cultured for the indicated numbers of generations (40 to ∼2500). The DNA was digested with XhoI and analyzed by non-denaturing in-gel hybridization. Lanes 1, 3, 5, 7, 9, 11 and 13: strains expressing TLC1wt. Lanes 2, 4, 6, 8, 10, 12 and 14: strains expressing Tlc1h. (A) The native gel was first hybridized to an end-labelled human telomeric probe [32P](C3TA2)3. (B) DNA in the gel shown in (A) was denatured and rehybridized to the same probe. (C) The probe in (B) was washed off and the gel rehybridized to an end-labelled yeast telomeric probe [32P](C1-3A). (D) The same gel as shown in (A)–(C) was rehybridized to a probe specific for the CEN4 region to show about equal loading of the lanes. (E) Non-denaturing in-gel analysis of the same DNA as shown in (A)–(D), but using the yeast-specific C-strand probe first. Lanes 1–7: strains expressing Tlc1h. Lanes 8–14: strains expressing TLC1wt. Lane 15: genomic DNA derived from a wild-type strain (BY4705) without any plasmids. (F) The DNA in the gel shown in (E) was denatured and rehybridized to a probe specific for CEN4 to control for DNA loading. Lanes 15–17 of the gel shown in (A)–(C) and lanes 16–18 of the gel shown in (E) contain control DNAs harbouring human telomeric repeats in single-stranded form (H, ss), human telomeric repeats in double-stranded from (H, ds) or a mixture of yeast telomeric repeats in single- and double-stranded form (Y, ss+ds). The DNA size standard was end-labelled 1 kb ladder DNA and sizes are indicated on the left of the gels.