Figure 2.

Southern analysis of stabilized clones. (A) Structure of chromosomes stabilized by telomere addition within URA3. The structure of the left telomere of the test chromosome is shown. The open arrow head represents the ∼350-base pair tract of C_1–3A/TG_1–3 DNA. The solid black rectangle is the 1.1-kb HindIII-SmaI fragment that contains the URA3 open reading frame; the bent arrow denotes the translational start site for URA3. The region between the telomeric tract and the end of the HindIII-SmaI URA3 fragment is a 308-base pair EcoRI-HincII fragment containing the MAT locus with its HO recognition site. There are 50 bps between the HO cut site and the start of the telomeric tract. Sites for relevant restriction enzymes and for the HO endonuclease are indicated. DNA from two stabilized clones (lanes 1 and 2) or the starting disome strain (lane 3) was digested with StuI or NcoI, separated on a 1% agarose gel, and analyzed by Southern blotting with the use of a probe for the entire URA3 gene. Asterisks indicate telomeric restriction fragments. The other bands that hybridize to the probe were from either the ura3-52 locus or the portion of the telomeric URA3 that was proximal to the StuI (or NcoI) site. The sites of telomere addition as deduced from this analysis are indicated on the diagram (labeled 1 and 2, for clones in lanes 1 and 2, respectively). M, molecular weight markers (1-kb ladder). (B) PFGE mapping of stabilized clones. Positions of hybridization probes and the relevant NotI site are shown in Figure 1. Probe 1 (detects both copies of chromosome VII) was used in C and D; probe 2 (detects only the test chromosome) was used in B. Lanes 1–7 in B contain undigested DNA from independent stabilized clones isolated in the rad52 strain. NotI-digested DNA from the same clones is shown in C. Lanes 1 and 7 contain DNA from clones stabilized by de novo telomere addition within URA3; lanes 2, 3, and 4 contain clones stabilized by recombination with the endogenous copy of chromosome VII; lanes 5 and 6 contain DNA from clones stabilized near the (CA)₁₇ tract. Lane 8 contains DNA from the starting disome strain; lane 9 has DNA from a haploid strain containing only the endogenous copy of chromosome VII. Lanes 1–8 in D contain NotI-digested DNA from independent stabilized clones isolated in the rad52 pif1-m2 strain. Each clone was stabilized by telomere addition internal to URA3. Lane 9 contains DNA from the starting disome strain, and lane 10 contains DNA from a haploid strain containing the endogenous chromosome VII.