Translation of a Newly Sequenced DPSC Zinc-finger Transcription Factor

Objectives: Previous work validated the predicted gene (hmm30337) as an 8565 base pair coding region containing three, distinct Open Reading Frames (ORFs) with a 1746 base pair transcript encoding the 582 amino acid (68.1 kDa) hypothetical protein (MGC42493) that was expressed in some mesenchymal dental pulp stem cells (DPSCs). Objective: Based upon these results, the goal of this project was to express this gene in a non-DPSC cell to determine if this protein can be translated and to determine the cellular location of the protein produced.
Methods: The full-length coding sequence cDNA was produced using nested primers and confirmed by sequence analysis and gel electrophoresis. Corresponding restriction enzyme cut sites for ScaI and PvuII were identified on both pBlueScriptII plasmid and the MGC42493-cDNA, which allowed for blunt-end ligation and subsequent transfection using Calcium Phosphate.
Results: Restriction enzyme cuts and ligation were confirmed using gel electrophoresis. The pBS-MCG42493 construct was transfected into several cell lines and production was confirmed using Xgal (beta galactosidase) expression, which also revealed intranuclear protein expression.
Conclusions: Although a potential 678 amino acid protein (ZN678) was originally predicted for MGC42493, these results confirm a 582 amino acid zinc-finger transcription factor (ZNF582) with high homology to the original, predicted sequence from chromosome 1 (1q42.13). Transfection and translation of the full-length transcript demonstrates that this protein may be expressed in the nucleus, which supports the predicted function of a zinc-finger transcription factor that may be expressed in some mesenchymal and dental pulp stem cells.