Carol Greider and Liz Blackburn (Cell 43:405, 1985) A telomere-extending enzyme would solve the end-replication problem. But does one really exist? Seeking.

Carol Greider and Liz Blackburn (Cell 43:405, 1985)

A telomere-extending enzyme would solve the end-replication problem.But does one really exist?

Seeking biochemical evidence of telomerase from Tetrahymena extracts

Formation of the macronucleus includes extensive breaking of chromosomes to make new, small chromosomes.

These will need new telomeres, so high levels of telomerase would be expected!

Tetrahymena telomeres are (TTGGGG)n

Simple assay:Tetrahymena extract

(TTGGGG)4 as one substrate

some combination of dNTPs

some are 32P-a-dNTP

run products on a gel.

With an assay, we can isolate the enzyme and characterize the protein & RNA

Andrea Bodnar et al. (Science 1998 279:349)

Will activation of telomerase prevent senescence?

Normal retinal pigment epithelial (RPE) cells lack telomerase activitydue to lack of TERT (protein) expressionhas low expression of TR (RNA)

Stably transfect RPE cells with TERT expressing plasmid

Telomerase assay:

Effect on time to senescence?

Population Doublings

Circles are control transfected RPE cells.Triangles are TERT-transfected RPE cells.

Black shapes represent cell lines that have senesced.Open shapes represent cell lines that have not senesced.