Abstract
Lesions in hypoxanthine guanine phosphoribosyl transferase (HPRT) gene are associated with the devastating Lesch-Nyhan syndrome and forms of gouty arthritis in human patients. We are interested in developing a retroviral vector by which the human HPRT gene can be introduced into mice, and its expression can be detected by immunocytochemical methods. Due to the nearly identical amino acid sequences of mouse and human HPRT, antibodies against human HPRT cross react with mouse HPRT. To overcome this problem, we constructed a retroviral vector which codes for a mutated HPRT protein by site-directed in vitro mutagenesis. The termination codon of human HPRT gene was mutated so that six extra amino acids (glu-asp-glu-ser-ser-ser) are added to the C-terminus of normal HPRT protein during translation. In DNA transfection, the mutated protein worked as well as the normal protein in rescuing HPRT− cells during HAT selection. The isoelectrofocusing point of this mutated protein is also altered on gels due to the addition of three acidic amino acids. A polyclonal antibody, directed against the last eight amino acids of C-terminus of the mutated protein, was raised. In immunoprecipitation this antibody can bring down the mutated human HPRT protein, but not normal human HPRT protein or mouse HPRT protein.
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Yee, JK., Jolly, D. & Friedmann, T. AN ALTERED HPRT PROTEIN, CREATED BY AN IN VITRO MUTAGENESIS OF THE GENE, CAN BE RECOGNIZED SPECIF: CALLY BY AN ANTIPEPTIDE ANTIBODY: 235. Pediatr Res 19, 783 (1985). https://doi.org/10.1203/00006450-198507000-00255
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DOI: https://doi.org/10.1203/00006450-198507000-00255