Molecular cloning and expression of telomerase reverse transcriptase (TERT) in zebrafish central nervous system

Abstract

Telomerase is a specialized reverse transcriptase that specifically elongates telomeres and thus prevents cellular senescence. The telomerase ribonucleoprotein complex contains a complementary RNA template (TR) and a reverse transcriptase catalytic subunit (TERT), which has been shown to be the key determinant of its enzymatic activity. Based on the amino acid sequence of mouse TERT (mTERT), a zebrafish genomic DNA sequence significantly similar to mTERT has been isolated from zebrafish whole genome shotgun database. Utilizing Rapid Amplification of cDNA Ends (RACE), the full-length zebrafish TERT (zfTERT) cDNA was cloned into a pDNR-CMV mammalian expression vector and its sequence was revealed. After translation, the deduced protein sequence contains 1091 amino-acid residues and a predicted molecular mass of 126 kDa. Multiple alignment reveals that the protein sequence bears the conserved motifs and residues found in TERT of other species. However, transfection of pDNRCMV-zfTERT into human fibroblast did not reconstitute telomerase activity. The lack of telomerase activity expression implies that zebrafish TERT cannot utilize human TR in mammalian cells. RT-PCR and functional assay of telomerase activity (TRAP assay) detect TERT mRNA expression and telomerase activity in all zebrafish central nervous system tissues examined, including telencephalon, optic tectum, cerebellum, brain stem, spinal cord and retina. Since continuous neurogenesis can be found in teleost nervous system, the presence of telomerase in these tissues may reflect the neurogenesis potential in zebrafish neural tissues relative to mammalian tissues. This may serve as a comparative model to mammalian central nervous system.