Molecular cloning of bovine telomerase RNA.

Abstract

Telomerase is a ribonucleoprotein reverse transcriptase essential for the maintenance of telomere length. However, the available information concerning the biochemical and structural aspects of mammalian telomerases is scarce, primarily due to the low abundance of these enzymes and the difficulty and expense involved in its purification. To overcome these problems, we started to purify and characterize telomerase from bovine testis. Bovine telomerase was purified over columns of hydroxyapatite, DEAE-Sepharose, heparin-agarose, phenyl-agarose and spermine-agarose. In a sedimentation study performed using a 15-40% glycerol gradient, partially purified bovine telomerase exhibited an apparent molecular weight of more than 1000 kDa. One 435-bp RNA, bTR, was cloned from the most purified telomerase fraction and shown to be co-purified with telomerase activity in a glycerol gradient. bTR shares 83% similarity to the human telomerase RNA and 65% to the mouse telomerase RNA. A putative template region encompassing 10 nucleotides (5'-CUAACCCUAA-3') complementary to the mammalian telomere sequence (TTAGGG)n is located between nucleotides 38-47 of bTR. Besides, the bTR 5'-flanking region shares only three regulatory elements with that of hTR: a TATA-like sequence, a CCAAT box and an E1A-F box. Furthermore, the addition of in-vitro transcribed bTR reconstituted telomerase activity after removal of the endogenous bTR by micrococcal nuclease digestion. Northern blot analysis identified a single RNA of about 430-440 nucleotides expressed in the bovine testis and an immortalized bovine cell line. Taken together, these data strongly suggest that bTR is the RNA component of bovine telomerase.