In this paper, the cloning and nucleotide sequence of the
cDNA of the rat gene coding for
hypoxanthine-guanine phosphoribosyltransferase (
hprt) is reported. Knowledge of the
cDNA sequence is needed, among other reasons, for the molecular analysis of
hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the
granuloma pouch assay. The rat
hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose,
oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster
hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat
hprt cDNA showed a single open reading frame of 654 bp, encoding a
protein of 218
amino acids. In the predicted rat
hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and
nucleotide binding in phosphoribosylating
enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian
hprt proteins. Analysis of
hprt amino acid sequences of 727 independent
hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of
amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the
hprt gene as target for mutational analysis is demonstrated by the fact that
amino acid changes in at least 151 of the 218
amino acid residues of the
hprt protein result in a 6-thioguanine-resistant phenotype.