Human somatic cells have essentially no telomerase activity. Telomerase is linked to tumor genesis and is a valuable marker for malignant growth. Extreme paucity of the enzyme neccessitated development of a PCR-based assay, 'telomeric repeat amplification protocol' (TRAP). Unfortunately, this method is not without difficulties. Amplification products are not related to the size of the amplified telomerase products. Furthermore, false positive results can occur, and careful control of reaction conditions is crucial. We analyzed in detail the molecular basis of artifacts. Based on these data, reverse PCR primer was changed and both problems in the TRAP assay were eliminated.
Eukaryotic chromosome termini consist of telomeres, short sequence repeats. According to the telomere hypothesis, DNA replication leads to telomere shortening, resulting in a cellular mitotic clock. Telomerase resets it by telomere synthesis. In mammals with a limited growth phase, telomerase activity in somatic tissues is restricted to stem cell derivatives with high proliferation potential. But other animals, like some fish, grow throughout their life with little senescence. All somatic cells require a high proliferation capacity and telomerase should be active in all cells, irrespective of fish age. Indeed, we detected high telomerase activities in all analyzed organs of rainbow trout (Oncorhynchus mykiss).z 1998 Federation of European Biochemical Societies.
Mammals have high growth rates in embryonic and juvenile phases and no growth in adult and senescent phases. We analyzed telomerase activity in a fundamentally different animal which grows indeterminately. Lobsters (Homarus americanus) grow throughout their life and the occurrence of senescence is slow. A modified TRAP assay was developed and the lobster telomeric repeat sequence TTAGG was determined. We detected telomerase activities which were dependent on RNA and protein components, required dGTP, dATP and dTTP, but not dCTP. Telomerase products with a five nucleotide periodicity were generated. High telomerase activities were detected in all lobster organs. We conclude that telomerase activation is a conserved mechanism for maintaining long-term cell proliferation capacity and preventing senescence, not only in cellular models or embryonic life stages but also in adult multicellular organisms.z 1998 Federation of European Biochemical Societies.
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