Tetrahymenamicronuclear sequences that function as telomeres in yeast

Abstract

We explored the ability of S. cerevisiae to utilize heterologous DNA sequences as telomeres by cloning germline (micronuclear) DNA from Tetrahymena thermophila on a linear yeast plasmid that selects for telomere function. The only Tetrahymena sequences that functioned in this assay were (C4A2)n repeats. Moreover, these repeats did not have to be derived from Tetrahymena telomeres, although we show hat micronuclear telomeres (ike macronuclear telomeres) of Tetrahymena trminate in (C4A2)n repeats. Chromosome-internal restriction fragments carrying (C4A2)n repeats also stabilized linear plasmids and were elongated by yeast telomeric repeats. In one case, the C4A2 repeat tract was approximately 1.5 kb from the end of the genomic Tetrahynena DNA fragment that was cloned, but this 1.5 kb of DNA was missing from the linear plasmid. Thus, yeast can udlize internally located tracts of telomere-like sequences, after the distal DNA is removed. The data provide an example of broken chromo-some healing, and underscore the importance of the telomeric repeat structure for recognition of functional telomeric DNA in vivo.

New telomeres in yeast are initiated with a highly selected subset of TG1-3 repeats.

“…On the basis of our data and that of others (Murray et al 1988;Shampay and Blackburn 1989), we propose that telomerase in yeast is incapable, itself, of establishing a new telomere. It requires the presence of an organizer sequence such as (T2G4) n nearby.…”

“…It is well documented that Tetrahymena T~G4 is a good substrate for the de novo addition of yeast TGI_3 sequences (Szostak and Blackburn 1982;Shampay et al 1984;Murray et al 1988;Shampay and Blackburn 1989). We therefore chose to look at the cases where nontelomeric DNA was used as the addition site.…”

An unusual sequence arrangement in the telomeres of the germ-line micronucleus in Tetrahymena thermophila.

“…Previous results from T. thermophila (Shampay and Blackburn 1989) and the hypotrichous ciliate Oxytricha fallax (Dawson and Herrick 1984)have indicated the probability that ciliate micronuclear telomeres contain the same repeat sequence as their macronuclear counterparts. We report that the distal telomeric repeat sequence in the T. thermophila micronucleus is the same as that found in the macronucleus, but the micronuclear telomere sequence is nonetheless unique in several respects.…”

“…Initial hybridization studies indicated that micronuclear telomeres contain the same repeat sequence (Shampay and Blackburn 1989). However, these telomeres have been very difficult to identify largely because of two reasons.…”

“…Second, the micronucleus and macronucleus contain 20 and -4 x 10 4 chromosome ends, respectively. Hence, even greatly purified micronuclei still contain high levels of contaminating macronuclear telomeres; a previous attempt to clone micronuclear telomeres by functional analysis in yeast yielded only internal micronuclear G 4 T 2 t r a c t s and macronucelar telomeres (Shampay and Blackburn 1989).…”