To isolate murine purine nucleoside phosphorylase-encoding cDNA sequences (PNP), a murine BALB/c liver cDNA library in λgt10 was screened for recombinants hybridizing to a human PNP cDNA probe. Two of three clones recovered included inserts large enough to contain the full-length coding sequence. Sequence analysis of the largest clone revealed an 867-nucleotide open reading frame encoding 289 amino acids with 84% residue identity to that encoded by human PNP and 351 bp of 3′-untranslated region. The 5′ end of the murine PNP message was specifically amplified by PCR using the RACE (rapid amplification of cDNA ends) protocol, revealing a 5′-untranslated region of 78 bp. Northern hybridization using the murine PNP cDNA sequence as a probe identified a message of approx. 1.6 kb in mouse NIH3T3 cells which was slightly smaller than the human message observed in HeLa cells. The cloned murine PNP cDNA coding sequence was inserted into a mammalian expression vector under transcriptional regulation of the Moloney murine leukemia virus long terminal repeat. Transfection of this plasmid into human 293 cells resulted in the expression of PNP activity which co-focused with murine PNP activity extracted from NIH3T3 cells, verifying that the isolated murine PNP cDNA clone encoded catalytically active PNP protein.
- Mammalian expression
- cDNA sequence
- rapid amplification of cDNA ends