Pteridines (Feb 1996)
The Sequence of Mouse Dihydropteridine Reductase cDNA, and Comparison with Human and Rat Sequences
Abstract
A mouse liver eDNA library in λZAP was screened using a 627 bp portion of 32p randomly labeled human dihydropteridine reductase (731 bp) cDNA and seven positive clones were obtained. Six of these clones were sequenced using T3 and T7 primers as well as five selected 18-mer oligonucleotides in a "primer walking" strategy. From the data a consensus sequence was obtained for the coding region, the 5 ' flanking region and the 3' flanking region up to the poly-A tail. Of the 720 nucleotides in the coding region of mouse dihydropteridine reductase only 89 were different from those in the human reductase and only 31 nucleotides were different from those in the rat reductase. The amino acid sequence of the mouse reductase on the other hand differed from that of the human reductase by 15 residues in addition to the three extra alanine residues at positions 4, 5 and 6 from the N terminus; and from the rat reductase by 6 residues, many of which were conservative changes. The rat enzyme differed from the human enzyme in 9 residues in addition to the three alanines. The similarity of the eDNA coding regions and the reductases produced were characteristic of, and essential for, a “house-keeping” enzyme. The DNA in the 5′ and 3′ flanking regions of the three cDNAs, on the other hand, were very dissimilar although the human DNA had diverged a lot more from the mouse DNA than the rat DNA. Initiation, termination and poly-A tail consensus sequence signals were identified in all three cDNAs. A vector was engineered that expressed mouse dihydropteridine reductase which had kinetic parameters (Km for q-6MeDHP 17.4µM, NADH 7.3µM and k 170 sec1 and Km for natural q-6R-BH2 8 .7µM, NADH 7.7µM and k 44.3 sec1 ) similar to those for the natural mouse reductase (Km for q-6MeDHP 16.5 µM, NADH 16.0 µM and k 214 sec1; and Km for natural q-6R-BH2 4.7 µM, NADH 9 .1µM and k 81.9 sec1 ) which was purified from mouse liver.
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