Molecular genetic studies on alcohol and aldehyde dehydrogenase: individual variation, gene mapping and analysis of regulation.

625th MEETING, LONDON von Bahr-Lindstrom, H., Jeck, R., Woenckhaus, C., Sohn, S., Hempel. J. & Jornvall, H. (1985) Lliochernistry24.5847-585 1 von Bahr-Lindstrom, H., Hiiog, J.-O., Heden, L.-0.. Kaiser, R., Fleetwood, L., Larsson, K., Lake, M., Holmquist, B., Holrngren, A,, Hempel, J., Vallee, B. L. & Jornvall, H. (1986) Biochemistry Williamson, V. M. & Paquin, C. E. (1987) Mol. Gen. Genet. 209. Received 1 I December 1987 Molecular genetic studies on alcohol and aldehyde dehydrogenase: individual variation, gene mapping and analysis of regulation MOYRA SMlTH Department of l’ediatrics, Utiiversiy of Culijortiia, Irvitie, C‘A 9271 7, U.S.A. Introdirctioti Through analysis of alcohol and aldehyde dehydrogenase enzymes at the protein level, the number of gene loci which encode these enzymes has been determined (Smith et al., 1972; Li & Magnes, 1975; Pares & Vallee, 1981). The tissue distribution, and kinetic properties have been defined and in addition a number of allelic variants were identified (Smith et a/., 1973; Bosron et a/., 1980, 1983; Yoshida et ul., 1983). Refinement of purification procedures led to isolation of enzyme polypeptides suitable for amino-acid sequence analysis (Impraim el a/., 1982; Hempel et a/., 1984a,h,c; Buhler et ul., 1984). Availability of amino-sequence data led to the development of oligonucleotide probes to screen liver complementary (c) DNA libraries to isolate cDNA clones for alcohol and aldehyde dehydrogenases (Duester et a/., 1984; Smith, 1986; von Bahr-Lindstrom et a/., 1986). In certain instances, cDNA clones were isolated using antibodies to purified enzymes and expression libraries of liver cDNA (lkuta et a/., 1986; Hsu et a/., 1985). Subsequently, cDNA clones were used to isolate genomic DBA clones and to examine the organization of the alcohol metabolizing genes. This paper will describe studies which we and our collabora- tors have carried out over the past 3 years primarily on class 1 alcohol dehydrogenase (ADH) and, to a lesser extent, on mitochondrial aldehyde dehydrogenase. In the case of ADH, the genomic probes which we have isolated for a-, P- and y-ADH genes and the application of these probes to physical gene mapping, investigations of inherited variations and genetic linkage analysis will be described. Results of studies in human hepatomas indicate that definition of the gene mapping relationships may contribute to our under- standing of the mechanism of their origin and to our under- standing of the clinical manifestations in hepatomas. Analysis of RNA species in different tissues and detailed analysis of cDNA clones has led to insights into some of the factors which may be involved in the regulation of expression of class 1 alcohol dehydrogenases and mitochondrial alde- hyde dehydrogenases (ALDH). Physical chromosome mapping ofthe class I and class I1 A D H loci Physical mapping of the ADH loci was initially accom- plished using a P-ADH cDNA clone, pADH 12 and a series of man-rodent hybrids. Since the three class 1 ADH genes ( a , /? and y), have a high degree of sequence homology (Duester et al., 1984; Ikuta et a/., 1986, von Bahr-Lindstrom Abbreviations used: ADH, alcohol dehydrogenase; ALDH, alde- hyde dehydrogenase; RFLP, restriction fragment length poly- morphism; EGF, epidermal growth factor; IL 2. interleukin 2; lod, logarithm of the odds in favour of linkage. Vol. 16 et ul., 1986), under conditions of low and medium stringency the P-ADH cDNA probe hybridizes to restriction fragments from all three genes. On analysing Southern blots of genomic DNA of human, rodent and hybrid cell lines, we observed that all of the pADH 12 hybridizing fragments characteristic of human DNA were present in hybrids containing human chromosome 4 (Smith et ul., 1984). Analysis of DNA from hybrids containing fragments of human chromosome 4, pro- vided evidence that the class 1 ADH genes were located in the region 4q21-4q24 (Smith et a/., 1985). Confirmation of this physical assignment has been obtained in studies on the linkage relationships of ADH and other genes assigned to human chromosome 4 (see below). Derivation of A D H genomic clones The first series of ADH genomic clones were derived by screening of the Maniatis library of human genomic DNA in I phage with the P-ADH cDNA clone pADH12. A second series of clones was derived by screening of either the Mania- tis library or of a genomic DNA library prepared from a hybrid which contained chromosome 4 as the only human chromosome. In this second screening the most 5‘ region of the longest P-ADH genomic clone was used. The genomic clones were classified as being derived from the 0-, P- or y- ADH loci based on stringency of hybridization and sequence analysis in regions where amino-acid analysis had indicated that substitutions specific for a-, b-, or y-ADH occurred (Duester et ul., 1986). Based on the sizes of the genomic clones which were iso- lated and on the fact that no overlaps were detected in the restriction maps of the clones, it seems likely that the ADH genes are not immediately adjacent to each other (Duester et ul., 1986). IMuiled seqiretice utiu1y.si.s itr the 5‘ regiotr oJ‘ P - A D f l atrd its implicatiotis for stirdies 011 A D H regitlutioti Detailed sequence analysis in the 5’ ADH region by Duester et ul. (1986) led to the identification of a number of interesting features. Located 27 base pairs (bp) and 158 bp, respectively, upstream of thc transcription initiation site, the P-ADH gene contains sequences which match closely to the TATA box and CCAT box. These sequences are associated with eukaryotic promoters (Breathnach & Chambon, 198 1; Benoist et a/., 1980). At - 250 bases there is a sequence which matches in seven out of eight bases with the enhancer core sequence and which is embeddcd in a purine-rich stretch of DNA. Enhancer elements are sequences of DNA which have been shown to influence gene transcriptional activity through enhancement of RNA polymerase binding (Weiher et a/., 1983). A number of investigators have deter- mined that purine and pyrimidine asymmetry usually occurs in the region of enhancer sequences, and that this asymmetry is necessary for correct function o f the enhancer elements (Gillies et ul., 1984). In a number of other gene systcms investigators have succeeded in identifying DNA-binding