Isolation of a Candidate Human Telomerase Catalytic Subunit Gene, Which Reveals Complex Splicing Patterns in Different Cell Types

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.

Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation

“…This notion is supported by the observation that TERT transcripts are low or undetectable in most human primary somatic cells and tissues, but readily observable in the same cell or tissue type following neoplastic transformation (Nakamura et al, 1997;Meyerson et al, 1997). Moreover, in approximately 90% of all tumors, telomerase activity (Kim et al, 1994) as well as TERT expression (Nakamura et al, 1997;Meyerson et al, 1997;Harrington et al, 1997;Kilian et al, 1997;Nakayama et al, 1998) are detected. Together, these ®ndings are consistent with the hypothesis that either activation of oncogene or loss of tumor suppressor function serves to override the strict repression of TERT in primary somatic cells.…”

“…PCR was performed on 2 mL of the reverse transcriptase reaction with primers HT1553F and HT1893R (Kilian et al, 1997) for 35 cycles of 948C for 45 s, 608C for 45 s, and 728C for 90 s. PCR products were run on a 1.5% agarose TBE gel and transferred to nitrocellulose. Blots were probed with a random-primed 32 P-labeled hTERT fragment (nucleotides 1680 ± 2700) in hybridization solution containing 50% formamide at 428C for 6 h. Blots were exposed to Biomax MS ®lm (Kodak) for 3 h. GAPDH controls were coampli®ed with primers K136 and K137 as previously described (Nakamura et al, 1997), run on 1.4% agarose TBE gels and visualized by ethidium bromide staining.…”

“…Growth beyond the replicative senescence checkpoint (Hay¯ick limit) correlates well with genetic lesions that interfere with one or more key cellular mortality pathways, most prominently Myc, Rb and/or p53 Wright and Shay, 1995). Studies addressing the relationship between telomere length, telomerase regulation and replicative capacity have established a critical role for the telomerase catalytic protein component or TERT (for Telomerase Reverse Transcriptase) (Bodnar et al, 1998;Vaziri and Benchimol, 1998;Nakamura et al, 1997;Meyerson et al, 1997;Harrington et al, 1997;Kilian et al, 1997;Nakayama et al, 1998) in immortalization. Speci®cally, normal human somatic cells can acquire the ability to maintain telomeres and replicate well beyond the Hay¯ick limit by stable enforced expression of TERT (Bodnar et al, 1998;Vaziri and Benchimol, 1998).…”

Expression of mouse telomerase reverse transcriptase during development, differentiation and proliferation

“…Telomeres consist of lengthy G-rich simple repeat sequences that are synthesized de novo by a specialized reverse transcriptase known as telomerase (Greider and Blackburn, 1985;Yu et al, 1990;Singer and Gottschling, 1994). The telomerase core enzyme consists of an RNA component and a polypeptide that bears classical structural motifs and functional activities of a reverse transcriptase Nakamura et al, 1997Nakamura et al, , 1998Meyerson et al, 1997;Harrington et al, 1997;Kilian et al, 1997;. As conventional DNA-dependent DNA polymerases fail to fully replicate the ends of chromosomes, telomerase has evolved to serve as the primary means by which eukaryotic cells maintain telomere length with each cell division cycle.…”

“…The human telomerase reverse transcriptase protein (previously hTRT, now known as hTERT) has recently been cloned by Nakamura et al (1997) and by others under di erent names (hEST2, hTLP2, hTCS1, hTRT) (Meyerson et al, 1997;Harrington et al, 1997;Kilian et al, 1997;Nakayama et al, 1998). These studies, using primary and immortalized human cell lines, have established that an additional level of telomerase activity regulation is achieved through mechanisms governing expression of hTERT Nakamura et al, 1997Nakamura et al, , 1998Meyerson et al, 1997;Harrington et al, 1997;Kilian et al, 1997). In this study, we have isolated and characterized the mouse ortholog of hTERT with the goals of understanding the regulation of telomerase activity in normal and neoplastic processes in the mouse, providing insights into the immortalization behavior of mouse and human cells, and characterizing the catalytic activities of the mouse protein.…”