IN-VITRO AND IN-VIVO RECONSTITUTION AND STABILITY OF VERTEBRATE CHROMOSOME ENDS

Telomeres are essential repetitive sequences at the ends of chromosome s that prevent chromosome fusiorn and degradation. We have successfull y recapitulated these two protective functions in vivo and in vitro by incubating blunt-end DNA constructs having vertebrate telomeric ends in Xenopus eggs and egg extracts. Constructs with telomeric ends are s table as linear molecules; constructs with non-telomeric ends undergo intramolecular fusion. In extracts, 99.8% of the telomeric constructs from 78 to 700 bp in length are assembled into 'model telomeres' in <5 min and have an extpapolated half-life of >3.5 years. Non-telomeric c onstructs circularize with first order kinetics and a half-life of 4 h . In living eggs the telomeric constructs are protected from fusion an d degradation. The stability of the telomeric constructs is not due to covalent processing, Extract can protect similar to 100 pM telomeric ends (equivalent to 1.7 x 10(7) ends/egg) even in the presence of a 20 -fold excess of double-stranded telomeric DNA, suggesting that protect ion requires end-specific factors. Constructs with (TTGGGG)(n) repeats are unstable, suggesting that short tracts of this and other telomere -like sequences found within human telomeres could lead to genome inst ability if exposed by partial telomere erosion during aging.