Telomerase gene structure and expression

The elusive telomerase gene was long sought after.  Candidates from a number of organisms came and went until investigators finally turned their attention to Euplotes, a hypotrichous ciliate.  These ciliated protozoans contain two types of nuclei:  micronuclei and macronuclei.  The macronucleus of hypotrichous ciliates contains at least 10 million small DNA molecules (about 1800 – 2500 bp long).  Moreover, each of the DNA molecules have telomeric sequences capping their ends.  (Hoffman et al. 1995)

Fig. 1. Euplotes on a droplet of water.

The ribonucleoprotein containing telomerase was purified from Euplotes macronuclei by affinity chromatography with antisense 2’-O-methyl oligonucleotides (Lingner and Cech 1996).  The active complex contained two proteins, a 123 kD and 43 kD protein, along with an RNA subunit.  The larger protein proved to be the catalytic subunit – analysis of the predicted translation of the gene sequence revealed characteristic reverse transcriptase (RTase) motifs (Lingner et al. 1997).  The requirement of the RTase motifs for telomerase activity was shown using Est2p, a yeast homolog.  Conserved amino acids in the RTase motifs of Est2p were changed by site-directed mutagenesis, and the mutants transformed into yeast lacking the est2 gene.  Expression of the mutant proteins led to senescence and shortened telomeric tracts (Lingner et al. 1997).  This provided the best evidence that the 123 kD protein from Euplotes and Est2p were the catalytic subunit of telomerase.

Despite knowledge of the DNA and protein sequences of two telomerases, direct cloning of the gene for human telomerase was not possible.  Generally, using sequence from one species to find the homolog in an evolutionarily distant second species is most successful when used to probe cDNA libraries.  Because humans have only 46 chromosomes (92 telomeres), very little telomerase protein – and very low amounts of RNA encoding telomerase – is expected, even in immortalized cells.  Fortunately, in the late 1990s, large scale EST (expressed sequence tags) projects were underway, both in industry and in academia.  A BLAST search of EST databases with the Euplotes telomerase sequence as the query sequence identified a single EST as a significant match (Kilian et al. 1997; Meyerson et al. 1997d; Nakamura et al. 1997).  The EST sequence was then used to identify cDNA clones in libraries constructed from transformed human cell lines.  Three research groups used this approach to nearly simultaneously isolate a human telomerase cDNA (Kilian et al. 1997; Meyerson et al. 1997c; Nakamura et al. 1997).  The importance of this discovery was underscored by numerous write-ups in mainstream news organizations, such as the New York Times, the Washington Post, and Associated Press.

Subsequently, telomerase gene sequences have been cloned from a plethora of other organisms.  The organisms include other mammals (e.g., macaque, mouse, rat, cattle, pig, dog), birds, amphibians (e.g., Xenopus laevis), echinoderms (e.g., sea urchin), insects (e.g., bee, silkworm, drosophila), Caenorhabditis elegansTrypanosoma brucei, plants (e.g., corn, barley), and fungi (e.g., AspergillusCryptococcus).