Zebrafish telomerase catalytic subunit: Molecular cloning and characterization of a ribonucleoprotein reverse transcriptase gene highly expressed in the retina

Abstract

Abstract: : Purpose: To clone and sequence the zebrafish TERT gene and to study the expression pattern of TERT and telomerase activity in zebrafish retina. Methods: Primers identical to those used originally to amplify and clone the mouse TERT sequence were retrieved from tBLASTn, and were used to amplify zebrafish genomic DNA by PCR. The amplified product was then cloned into the pDNR–CMV vector using the In–Fusion PCR cloning kit (BD Bioscience). All sequencing analyses were carried out at the Genome Research Centre, at the University of Hong Kong. Western blotting, immunohistochemistry, RT–PCR and telomeric repeats amplification protocol (TRAP) assay are performed to study TERT mRNA and protein expression and telomerase activity in zebrafish retina. Results: Based on the amino acid sequence of mouse TERT, a full–length telomerase reverse transcriptase cDNA of zebrafish has been isolated and cloned. The deduced protein sequence contains 1091 amino–acid residues and a predicted molecular mass of 126 kDa. Multiple alignments reveal that the protein sequence bears the conserved motifs and residues found in TERT of other species. RT–PCR and TRAP assay detect TERT mRNA expression and telomerase activity in all tissues examined, including retina and brain. Presence of telomerase activity indicates that a fully functional form of telomerase can be found in retina. Through immunohistochemistry, the protein was localized to cells in the ganglion cell layer, and to inner and outer nuclear layers of adult zebrafish retina. Conclusions: The presence of telomerase in most somatic tissues in zebrafish is consistent with the high proliferation capacity of cells in organs of teleost fish which grow throughout their life with little senescence. However, retinal neurons in adult teleost are post–mitotic cells and do not divide under normal situation. The expression of TERT and telomerase activity in the retina suggests that telomerase has functions other than elongation of telomere. Functional studies can now be carried out to shed further light on the role of TERT in vision.