ISOLATION OF A CANDIDATE HUMAN TELOMERASE CATALYTIC SUBUNIT GENE, WHICH REVEALS COMPLEX SPLICING PATTERNS IN DIFFERENT CELL-TYPES

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a te mplate for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene h omologous to a protein from Saccharomyces cerevisiae and Euplotes aedi culatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is presen t in the human genome as a single copy sequence with a dominant transc ript of similar to 4 kb in a human colon cancer cell line, LIM1215. Th e cDNA sequence was determined using clones from a LIM1215 cDNA librar y and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We sho w that the gene is expressed in several normal tissues, telomerase-pos itive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non- immortal) cells and telomerase-negative immortal (ALT) cell lines. Mul tiple products were identified by RT-PCR using primers within the reve rse transcriptase domain. Sequencing of these products suggests that t hey arise by alternative splicing. Strikingly, various tumors, cell li nes and even normal tissues (colonic crypt and testis) showed consider able differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regu lation of telomerase activity and may give rise to proteins with diffe rent biochemical functions.