Assignment 2

This assignment is worth 5% of the course grade.

Due: Thur. Oct 26.

1.  (4 points)

Figure 1A shows a map of the subtelomeric region (ie. region right next to the telomere) for one of the chromosomes of yeast. In most yeast strains, all chromosomes have essentially the same four sequences, labeled Telomere, TAS1, TAS2 and TAS3 in the same order. Yeast mutants homozygous for mutations in the telomere terminal transferase gene trt begin to lose viability after several generations in culture.  Figure 1B shows a southern blot of HindIII-digested yeast DNA from either wild type (trt+) or mutants (trt-).


a) Why is more than one band seen with HindIII digested DNA from trt+ cells? That is, not all chromosomes yield the same band in HindIII digestion.
b) When southerns of DNA from trt- colonies are probed with either Telomere, TAS1, or TAS2 sequences, no bands are seen (results only shown for TAS2). However, bands are seen with the TAS3 probe.  What might be the cause of this result?

2. (1 point)
Although trt-  yeast generally become senescent and die, researchers have observed a small percentage of clones that, after several generations of poor growth, begin to grow prolifically. These clones are referred to as "trt- survivors".  The map in Figure 2A shows restriction sites for NotI (5'GCGGCCGC3') on yeast chromosomes I & II (chromosome III has no NotI sites). Terminal fragments I&L on chromosome I, and C&M on chromosome II, are shown in black.

In Figure 2B, undigested yeast chromosomal DNA was run on a pulsed field gel stained with Ethidium Bromide.  In trt- survivors, the chromosomal DNA stays at the origin, rather than running into the gel. 

In Figure 2C, NotI-digested chromosomal DNA from trt+  and trt- survivors is visualized on a pulsed-field gel (left). The gel was blotted and hybridized with TAS1 (middle), and probe washed off and rehybridized with a mixed probe containing both C and I sequences (right).

Wild type (trt+)  show bands for fragment I (530 kb) and fragment C (1525 kb). Although the complementary experiment is not shown, terminal fragments L&M,  should produce 380 kb and 240 kb bands if a mixed L&M probe was used.  trt- survivors have lost these bands, but show new bands at 920 kb and 1765 kb.

What changes have occurred in the chromosomes of the trt- survivors?

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