CHARACTERIZATION OF THE FUNCTIONAL GENE ENCODING MOUSE CLASS-III ALCOHOL-DEHYDROGENASE (GLUTATHIONE-DEPENDENT FORMALDEHYDE DEHYDROGENASE) AND AN UNEXPRESSED PROCESSED PSEUDOGENE WITH AN INTACT OPEN READING FRAME

Multiple forms of vertebrate alcohol dehydrogenase (ADH) have been ide ntified, but only one form, class III ADH, has been conserved in all o rganisms studied, Class III ADH functions in vitro as a glutathione-de pendent formaldehyde dehydrogenase, which suggests that this was the o riginal function that drove the evolution of ADH. Genetic analysis of class III ADH in yeast supports this view, but such studies are lackin g in higher eukaryotes. The mouse ADH family has been previously analy zed and it contains three forms of ADH including the class III enzyme. We have initiated a molecular genetic analysis of the mouse class III ADH gene (Adh-2) by screening a genomic library with a full-length cD NA. Two overlapping clones contained the complete Adh-2 gene composed of nine exons in a 12-kb region, with the placement of introns matchin g that observed in other mammalian ADH genes, In this screening, we al so isolated a clone (psi Adh-2) that lacks introns and which resembles a processed pseudogene. psi Adh-2 contained 25 point mutations relati ve to the previously analyzed Adh-2 cDNA, but still retained an intact open reading frame, Northern blot analysis using gene-specific probes provided evidence that psi Adh-2 does not produce a mRNA in either li ver or kidney, whereas Adh-2 does. The functionality of the two gents was also compared by fusion of their 5'-flanking regions to a lacZ rep orter gene, Reporter gene expression following transfection into mouse F9 embryonal carcinoma cells indicated that only Adh-2 possesses prom oter activity.