cDNA sequence of four purine nucleoside phosphorylase (Np) alleles in the mouse

The molecular basis of four electrophoretic and activity variants of purine nucleoside phosphorylase in the mouse was examined by amplification and sequence analysis of cDNA. Compared with the cDNA coding sequence for C3H/HeHa designated Npa, there were five nucleotide changes for C57BL/6J, Npb; three for MOLF/Ei, Npc; and five for SPRET-1, Npd. There was only a single codon change between Npa and Npb, the deduced substitution of threonine 176 by serine. Similarly, there was only a single codon change between Npa and Npc, resulting in substitution of methionine 258 by lysine. There were three codon changes between Npa and Npd, resulting in substitution of glutamate 22 by lysine, threonine 39 by alanine, and aspartate 152 by glutamate. These amino acid substitutions-neutral to neutral, neutral to basic, and acidic to basic--are in agreement with the electrophoretic properties of the gene products of Npa relative to Npb, Npc, and Npd previously described by isoelectric focusing. Codon differences were confirmed by PCR-RFLP or single nucleotide primer extension analysis and extended to include the assignment of other strains as Npa: C3H/HeHa, DBA/2J, CLA, Posch-2; or Npb: C57BL/6J, C57L/J, C58/J. Both RFLP analysis of amplified genomic DNA and Southern analysis are consistent with single but unique Np alleles present in the C3H/HeHa and C57BL/6J genomes. As these data do not support the previous two-loci, Np-1 and Np-2, classification, we propose and employ a new single locus multiple allele classification for Np on the basis of the sequence analysis.