Abstract
Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template 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 homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive 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. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.
Highlights
Telomerase is a multi-subunit reverse transcriptase enzyme that adds DNA to telomeres, using its RNA component as a template for synthesis of the DNA repeat sequences [1]
The gene, which we designate hTCS1, is present in single copy in the human genome but is expressed in a complex splicing pattern that gives rise to a number of potential proteins. While this manuscript was in preparation, Nakamura et al reported the cloning of the same gene, which they referred to as hTRT [32].We find that expression of hTRT/hTCS1 positively correlates with the known telomerase status of tissues and cell lines and occurs preferentially in in vitro cell lines after they had undergone crisis, in normal tissues with a significant stem cell component and in a range of tumors
We hybridized DNA obtained from LIM1215 cells to determine whether there were any changes in the hTCS1 gene in this cell line that could account for activation of telomerase expression
Summary
Telomerase is a multi-subunit reverse transcriptase enzyme that adds DNA to telomeres, using its RNA component as a template for synthesis of the DNA repeat sequences [1]. This activity of telomerase is capable of compensating for the loss of terminal sequence that occurs during the normal replication of linear DNA molecules. In mammalian cells loss of telomeric DNA occurs due to a putative 5′-3′ exonuclease activity that produces a long G-rich overhang at both telomeres [2]. In the absence of telomerase, the combined effect of these processes is progressive loss of telomeric sequence with each round of cellular replication. It has been proposed that in normal somatic cells this telomeric attrition acts as a cell division counting mechanism that eventually dictates that the cell must enter the state of senescence, in which cellular replication permanently ceases [3,4,5]
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