Chapter 19 Tetrahymena Telomerase Activity, Purification, and Reconstitution

Abstract

Telomerase is a DNA replication enzyme responsible for the synthesis of telomeres, specific G-rich sequences at chromosome ends. A specific sequence within the RNA component of telomerase serves as a template for the synthesis of telomeric repeats, characterizing telomerase as a reverse transcriptase. Tetrahymena , like other ciliates such as Oxytricha and Euplotes , provides an abundant source of telomerase. The extensive chromosome fragmentation and the amplification that occur during macronuclear development in Tetrahymena result in the addition of telomeric repeats onto approximately 20,000 chromosome ends. Unlike other ciliates such as Oxytricha and Euplotes , Tetrahymena is cultured easily in defined media (10 5 cells/ml). In culturing Tetrahymena , soap residue left on glassware may inhibit cell growth. Thus, all glassware is kept soap free. All manipulations are performed using sterile techniques and sterile glassware, buffers, and media. Strains SB210 or CU428 are examples of strains routinely grown for preparation of Tetrahymena protein extracts. If the extract is prepared from mated cells, then two strains of different mating type are required.