SORBITOL DEHYDROGENASE - FULL-LENGTH CDNA SEQUENCING REVEALS A MESSENGER-RNA CODING FOR A PROTEIN CONTAINING AN ADDITIONAL 42 AMINO-ACIDS AT THE N-TERMINAL END

A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated fr om a rat testis lambdaZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be ini tiated. A second ATG codon, however, was found 126 bp upstream, aligne d in the same reading frame as that of the active enzyme. Therefore, t he coding sequence for SDH can be translated into an additional 42-ami no-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate t hat the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH th an the ATG codon preceding the nucleotide sequence for SDH. Since no k nown SDH starts with the additional 42 amino acids, it may be that pos t-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA conta ined a non-coding 1021 bp downstream from the TAA stop codon. The latt er sequence included three putative poly(A) signals: one at nucleotide s 1362-1367, the second at nucleotides 1465-1470, and the third at nuc leotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, w here threonine is coded for instead of aspartic acid; in the rat testi s SDH cDNA, we find the sequence is ACG instead of GAC, as was reporte d for the rat liver SDH cDNA. Northern-blot hybridization analysis sho wed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3 - 2.4 kb, in both the rat liver and the rat lens, further confirming t hat the isolated SDH cDNA constituted a full-length cDNA.