©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Genomic Structure and Expression of the ADH7 Gene Encoding Human Class IV Alcohol Dehydrogenase, the Form Most Efficient for Retinol Metabolism in Vitro (*)

(Received for publication, September 7, 1994; and in revised form, November 2, 1994)

Mirna Zgombic-Knight Mario H. Foglio Gregg Duester (§)

From the Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human alcohol dehydrogenase (ADH) consists of a family of five evolutionarily related classes of enzymes that collectively function in the metabolism of a wide variety of alcohols including ethanol and retinol. Class IV ADH has been found to be the most active as a retinol dehydrogenase, thus it may participate in retinoic acid synthesis. The gene encoding class IV ADH (ADH7) has now been cloned and subjected to molecular examination. Southern blot analysis indicated that class IV ADH is encoded by a single unique gene and has no related pseudogenes. The class IV ADH gene is divided into nine exons, consistent with the highly conserved intron/exon structure of other mammalian ADH genes. The predicted amino acid sequence of the exon coding regions indicates that a protein of 373 amino acids, excluding the amino-terminal methionine, would be translated, sharing greater sequence identity with class I ADH (69%) than with classes II, III, or V (59-61%). Expression of class IV ADH mRNA was detected in human stomach but not liver. This correlates with previous protein studies, which have indicated that class IV ADH is the major stomach ADH but unlike other ADHs is absent from liver. Primer extension studies using human stomach RNA were performed to identify the transcription initiation site lying 100 base pairs upstream of the ATG translation start codon. Nucleotide sequence analysis of the promoter region indicated the absence of a TATA box sequence often located about 25 base pairs upstream of the start site as well as the absence of GC boxes, which are quite often seen in promoters lacking a TATA box. The class IV ADH promoter thus differs from the other ADH promoters, which contain either a TATA box (classes I and II) or GC-boxes (class III), suggesting a fundamentally different form of transcriptional regulation.


INTRODUCTION

Vertebrate alcohol dehydrogenase (EC 1.1.1.1) (ADH) (^1)exists as a family of enzymes divided into several classes. Five classes of ADH have been identified in humans including class I ADH, the typical liver ADH, which efficiently oxidizes ethanol to acetaldehyde and is responsible for most of the ethanol metabolism (Jörnvall et al., 1993). The complete amino acid sequences of all five classes of human ADH have revealed sequence identites ranging from 57 to 69% (Satre et al., 1994). The evolutionary progenitor of all vertebrate ADH classes (as well as all other ADHs found in other organisms) has been found to be class III ADH, which exists ubiquitously in all organisms analyzed (Danielsson and Jörnvall, 1992; Danielsson et al., 1994). Class III ADH does not function in ethanol metabolism but instead acts as a glutathione-dependent formaldehyde dehydrogenase needed to remove formaldehyde normally produced by some metabolic reactions (Koivusalo et al., 1989). Gene duplications from an ancestral class III ADH gene and subsequent mutational divergence have evidently given rise to several ADHs with enzymatic functions other than formaldehyde metabolism such as ethanol and retinol metabolism.

The ability of ADH to act as a retinol dehydrogenase (Boleda et al., 1993; Yang et al., 1994) implies that it may participate in the synthesis of retinoic acid, the active form of vitamin A involved in regulating epithelial cell differentiation (Connor, 1988; De Luca, 1991). Retinoic acid is derived from retinol via two oxidation steps with retinal as the intermediate (Connor and Smit, 1987; Kim et al., 1992). Of the various classes of human ADH examined for retinol dehydrogenase activity in vitro, class IV ADH has been found to have the highest efficiency, with less activity attributed to ADH classes I and II, and no activity for class III (Yang et al., 1994). The inefficiency of class IV ADH in ethanol oxidation (Moreno and Parés, 1991; Stone et al., 1993) further suggests it has evolved to perform a role in the metabolism of other alcohols such as retinol. In addition to the forms of ADH that may function as cytosolic retinol dehydrogenases, a microsomal retinol dehydrogenase distinct from ADH has also been identified that can oxidize retinol in vitro (Posch et al., 1991). The relative importance of these various retinol dehydrogenases in vivo remains to be determined.

In order to learn more about class IV ADH, we have initiated molecular genetic studies. A cDNA encoding human class IV ADH has been previously described (Satre et al., 1994). We have now analyzed genomic clones encoding the class IV ADH gene, which we refer to as ADH7. The intron/exon structure was determined, and the promoter region was identified. We also show that the class IV ADH gene, unlike other ADH genes, is expressed much higher in the stomach than in the liver.


EXPERIMENTAL PROCEDURES

Screening of Human Genomic DNA Library and Southern Blot Analysis

A partial cDNA for human class IV ADH described previously (Satre et al., 1994) was used to screen a human genomic DNA library prepared in EMBL3 (Clontech Laboratories, Inc.) by the plaque lift technique described previously (Sambrook et al., 1989). This human class IV ADH cDNA fragment, a 1.7-kb EcoRI fragment derived from pMS7, was gel-purified and radiolabeled with [alpha-P]dATP by random hexamer-primed labeling using the oligolabeling kit (Pharmacia Biotech Inc.). Hybridization of nitrocellulose filters was carried out for 20 h at 42 °C in 50% formamide, 5 times Denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 5 times SSC (1 times SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.6), 1% SDS, 10 µg/µl poly(A), 250 µg/µl heat-denatured herring sperm DNA, and 10^6 cpm/ml of radiolabeled probe. Final washing was at 68 °C for 1 h in 0.1 times SSC, 0.1% SDS. After three rounds of plaque purification, phage DNA was prepared using the CsCl gradient method (Sambrook et al., 1989). Purified phage DNA was subjected to restriction mapping and Southern blotting by standard single and double enzyme digests (Sambrook et al., 1989), and further restriction mapping was accomplished by partial enzyme digestion using the mapping system (Life Technologies, Inc.). Genomic insert DNA fragments were released with SalI + KpnI or BamHI and subcloned into the plasmid Bluescript II KS (Stratagene) for further restriction mapping and DNA sequence analysis.

Southern blotting hybridization conditions were as described above and was performed with a class IV ADH cDNA probe radiolabeled as described above or with oligonucleotide probes labeled on the 5` end with [-P]ATP and polynucleotide kinase as described (Sambrook et al., 1989). Human genomic DNA used for Southern blot analysis was purchased from Clontech Laboratories, Inc.

Polymerase Chain Reaction Cloning of Exon 9

Sequence information from the 3` end of the human class IV ADH genomic DNA insert in MZ9 was used to generate an upstream polymerase chain reaction (PCR) primer (GD-135) 5`-GTCGAAGGATCCCAATGTTGTGTCTTGTCTCCAC-3` located on the coding strand in intron 8 about 1.2 kb downstream of exon 8. Sequence information from the 3`-untranslated region of the partial human class IV ADH cDNA (Satre et al., 1994) was used to design a downstream PCR primer (GD-134) 5`-TGCTAGAAGCTTGGGAACTCTCACAAGAGAAAC-3` whose 3` end is located on the noncoding strand 44 bp downstream of the stop codon. The conditions for PCR were a modification of the standard methodology (Ausubel et al., 1989). PCR reactions (50 µl) contained 0.5 mM of each nucleotide (dATP, dGTP, dTTP, and dCTP), 1 times reaction buffer [10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl(2), 50 mM KCl], 2 units of Taq DNA Polymerase (Life Technologies, Inc.), 100 pmol of both upstream and downstream primers described above, and 1.0 µg of human genomic DNA (Clontech, Inc.), which had been previously heat-denatured at 95 °C for 10 min. The reaction was overlaid with 50 µl of mineral oil and then subjected to 35 cycles of PCR. Cycling conditions were 93 °C (60 s) for denaturation, 56 °C (60 s) for annealing, and 72 °C (60 s) for elongation. The sample was extracted once with chloroform to remove mineral oil and analyzed by 1% agarose gel electrophoresis. The PCR product was gel-purified, digested with BamHI and HindIII (sites engineered into the upstream and downstream primers, respectively, to generate sticky ends), and then cloned into Bluescript II KS for DNA sequence analysis.

Reverse Transcription Polymerase Chain Reaction to Generate a Full-length Class IV ADH cDNA

First-strand cDNA synthesis was carried out with 1.0 µg of human stomach poly(A) RNA (Clontech Laboratories, Inc.) using oligo(dT) as a primer and avian myoblastosis virus reverse transcriptase as described in the cDNA cycle kit (Invitrogen Corp.). The RNA sample was heat-denatured at 80 °C for 5 min prior to reverse transcription. Oligonucleotide primers designed for PCR were based upon the sequences of the 5`-untranslated region of the human class IV ADH gene derived from the genomic clone MZ5 described herein and from the 3`-untranslated region of the partial cDNA (Satre et al., 1994). The upstream PCR primer (GD-133) was 5`-GTCGAAGGATCCCAGATCCAAGACAAAGACAGG-3`, whose 3` end is located on the coding strand 1 bp upstream of the start codon, and the downstream primer derived from the 3`-untranslated region was GD-134 described above. The conditions for PCR and cloning of the product were as described above except that the template was 1-5 µl of the above cDNA product, and the cycling conditions were 93 °C (60 s) for denaturation, 52 °C (60 s) for annealing, and 72 °C (60 s) for elongation during the first 10 cycles; then 93 °C (45 s), 55 °C (50 s), and 72 °C (45 s) during cycles 16-25; and finally 93 °C (35 s), 56 °C (45 s), and 72 °C (45 s) during cycles 26-35.

DNA Sequence Analysis of Human Class IV ADH Gene

Double-stranded plasmid DNA derived from the genomic DNA and cDNA clones was subjected to dideoxynucleotide sequence analysis using the Sequenase kit (U. S. Biochemical Corp.). DNA sequences were determined on both strands. Sequencing of exons 1 and 2 present in clone MZ5 was described previously (Satre et al., 1994). Exons 3-9 and adjacent intron sequences were similarly sequenced using exon-specific oligonucleotide primers based upon the sequence of the cDNA (Satre et al., 1994) to allow sequencing into the adjacent introns, and then intron-specific oligonucleotide primers were used to sequence across the exons. Computer analysis of nucleotide sequences was accomplished using the MacVector version 4 program (International Biotechnologies, Inc.).

Analysis of RNA by Northern Blotting

Samples (0.5 µg) of human stomach and liver poly(A) RNA (Clontech, Inc.) were separated by formaldehyde-agarose gel electrophoresis and subjected to Northern blot analysis as described (Sambrook et al., 1989). Samples (10 µg) of total mouse liver RNA containing 18 S RNA (1.87 kb) and 28 S RNA (4.72 kb) were also loaded to estimate molecular sizes. The radiolabeling of the cDNA probes and hybridization conditions were as described above for screening of the genomic library. The hybridization probes included the human class IV ADH cDNA described previously (Satre et al., 1994) and a cDNA for the human class I ADH gene ADH3 described previously (Höög et al., 1986).

Primer Extension to Map Transcription Initiation Site

A 1.0-µg sample of human stomach poly(A) RNA (Clontech, Inc.) was subjected to primer extension using avian myoblastosis virus reverse transcriptase and the following modifications of the conditions described for first strand cDNA synthesis in the cDNA cycle kit (Invitrogen Corp.). (a) The RNA sample was incubated at 80 °C for 5 min prior to use. (b) The radiolabeled nucleotide [alpha-P]dCTP replaced unlabeled dCTP, and (c) a specific primer was used (GD-108) consisting of the first 21 bp of noncoding strand sequence upstream of the ATG translation start codon in the human class IV ADH gene: 5`-CCTGTCTTTGTCTTGGATCTG-3`. For a marker, primer GD-108 was used in a DNA sequencing reaction of a plasmid containing the 5`-untranslated region of human class IV ADH. The reaction products were separated on a 6% sequencing gel as described (Sambrook et al., 1989).


RESULTS AND DISCUSSION

Isolation of Genomic DNA Clones Encoding Human Class IV ADH

In order to determine the intron/exon arrangement and exon coding sequences for the human class IV ADH gene, we screened a human genomic DNA library to isolate the ADH7 gene. A total of 300,000 plaques were screened using a partial human class IV ADH cDNA as a probe (Satre et al., 1994). Two clones were isolated (MZ5 and MZ9), and restriction mapping indicated that they overlapped by 1.1 kb and contained most of the gene (Fig. 1). The genomic DNA clones were subjected to Southern blotting using oligonucleotide probes specific for various exons to locate these regions for DNA sequence analysis. DNA fragments released by SalI + KpnI or BamHI digestion were subcloned into pBluescript II KS for DNA sequence analysis. This analysis indicated that MZ5 contained the 5` end of the gene and that MZ9 contained the 3` end, but exon 9 containing the 3`-untranslated region was not included. Since a further screen of the library did not result in the isolation of a genomic clone containing this region, we used PCR technology for its cloning. A small amount of nucleotide sequence information obtained from the 3` end of MZ9 (inside intron 8 about 1.2 kb downstream of exon 8) was used to design an upstream PCR primer, while the previously determined 3`-untranslated sequence in the human class IV ADH cDNA (Satre et al., 1994) was used to design a downstream PCR primer. Using human genomic DNA as a template for PCR with the two above primers, we were able to generate a 1.2-kb DNA fragment containing exon 9 that was cloned into pBluescript II KS to generate pPCRexon9 (Fig. 1). The region from exons 1-9 of the human class IV ADH gene was thus found to span about 23 kb.


Figure 1: Restriction map of human class IV ADH gene. Restriction mapping and sequencing of the class IV ADH gene revealed that it is divided into 9 exons covering about 23 kb. The cleavage sites for several restriction endonucleases are indicated. The 5` end of the gene containing exons 1 and 2 was cloned in MZ5, which has a 17.3-kb insert of human DNA. Exons 2-8 were cloned in MZ9 containing 15.7 kb of human DNA of which 1.1 kb overlaps with MZ5. Exon 9 was cloned (by polymerase chain reaction) in pPCRexon9 containing 1.2 kb of human DNA of which 0.1 kb overlaps with MZ9. Exon 9 and the 3`-untranslated region (which contains a PstI restriction site) are present within pMS7, a human class IV ADH cDNA (Satre et al., 1994).



Exon Nucleotide Sequences of the Human Class IV ADH Gene

Detailed sequence analysis of the genomic clones and comparison with the cDNA sequence indicated that the human class IV ADH gene was divided into 9 exons (Fig. 2). The nucleotide sequences of the exons and 3`-untranslated sequence from the human class IV ADH genomic clones matched perfectly with the previously determined cDNA sequence (Satre et al., 1994). Since this cDNA was incomplete (its 5` end started at codon 8), we used PCR to clone the full-length coding region for class IV ADH. An upstream PCR primer was synthesized based upon the sequence of the 5`-untranslated region located in exon 1 in the genomic clone MZ5, and a downstream primer was synthesized based upon the 3`-untranslated sequence lying just downstream of the stop codon in the partial cDNA. Since class IV ADH protein has been found to be most abundant in the stomach (Yin et al., 1990; Moreno and Parés, 1991), human stomach poly(A) RNA was subjected to reverse transcription and PCR with the above primers. The expected 1.2-kb PCR product was generated, cloned into pBluescript II KS, and sequenced. The sequence contained the entire coding region of human class IV ADH and matched perfectly with all nine exons in the genomic clones from position +80 in the 5`-untranslated region to position 64 of the 3`-untranslated region (Fig. 2). The sequence of the coding region of our full-length cDNA matches that described in two recent reports of full-length cDNAs for human class IV ADH (Farrés et al., 1994; Yokoyama et al., 1994).


Figure 2: Nucleotide sequences of exons for the human class IV ADH gene ADH7. The nucleotide sequences for all nine exons are shown as well as some sequence upstream and downstream of each. The locations of the eight introns are indicated with their approximate sizes in parentheses. The 5` and 3` ends of each intron contained the conserved GT/AG splice site sequences indicated with asterisks. The transcription initiation site, inferred from primer extension analysis in Fig. 5, is shown at position +1 at an adenine labeled with a closed circle. Upstream of the transcription start site, two potential transcription factor binding sites in the promoter (AP-1 and C/EBP) are indicated based upon consensus sequence matches. Downstream of the transcription start site, a TATA box in the reverse orientation (rev TATA box) is shown. The predicted amino acid sequence of the class IV ADH coding region (downstream of the initiator methionine at position +101) is numbered according to the homology with class I ADH, which is used for numbering all vertebrate ADH sequences (Jörnvall et al., 1987). The region downstream of the initiator methionine is actually 373 amino acids in length, one shorter than class I ADH due to the apparent deletion of codon 118, which is noticed when the sequences of all classes of human ADH are aligned (Satre et al., 1994). The stop codon is indicated by a triangle, and 64 bp of the 3`-untranslated region are shown.




Figure 5: Primer extension analysis on 5` end of human class IV ADH mRNA. A 1.0-µg sample of human stomach poly(A) RNA (St) was subjected to primer extension using avian myoblastosis virus reverse transcriptase and a specific primer consisting of the first 21 bp of noncoding strand sequence upstream of the ATG translation start codon in the human class IV ADH gene. This primer was also used to sequence the noncoding strand of a genomic clone containing the 5`-untranslated region and exon 1 (G, guanine; A, adenine; T, thymine; C, cytosine). The major primer extension product from stomach RNA, indicated by an arrow, is shown by the DNA sequencing ladder to fall at a T residue on the noncoding strand, corresponding to an A residue on the coding strand. This A residue (labeled +1 in Fig. 2) is located exactly 100 bp upstream of the ATG translation start codon.



The sequences of all of the introns at the intron/exon junctions conformed to the GT/AG rule (Breathnach and Chambon, 1981). The positions at which the introns interrupted the coding region were identical to those observed in the other human ADH genes previously analyzed (Duester et al., 1986; Matsuo and Yokoyama, 1989; Von Bahr-Lindström et al., 1991; Yasunami et al., 1991; Hur and Edenberg, 1992). The sizes of the introns for the human class IV ADH gene as well as the other known mammalian ADH genes are summarized (Table 1). Except for human class V, which lacks intron 8 (and exon 9), all the genes contain 8 introns that interrupt the coding sequence at the same locations. This indicates that all classes of mammalian ADH are derived from duplication of an ancient gene that already possessed the intron/exon structure shown in the class IV ADH gene. Presumably, the class V ADH gene lost exon 9 subsequent to its divergence from other ADH genes.



The amino acid sequence of human class IV ADH predicted from the coding region indicated that the translated protein would contain 373 amino acids downstream of the initiator methionine (Fig. 2). The predicted amino acid sequence of human class IV ADH was aligned with the recently determined full-length sequence of the rat class IV ADH enzyme (Parés et al., 1994), indicating a rat/human interspecies sequence identity of 87.1%. The human class IV ADH sequence was also aligned with the sequences of the other human classes. The human interclass sequence identity was greater with class I ADH (69%) than with classes II, III, or V (59-61%). This greater interclass sequence identity between classes I and IV was also noticed in a comparison of rat class IV ADH with the other classes of rat ADH (Parés et al., 1994). Thus, it is reasonable to propose that ADH classes I and IV may have diverged from a common ancestral gene subsequent to its divergence from the other classes. Since classes I and IV do share a common catalytic function as retinol dehydrogenases in several mammalian species (Connor and Smit, 1987; Boleda et al., 1993; Yang et al., 1994), this further suggests a common origin.

Southern Blot Analysis of Class IV ADH in Human Genomic DNA

Human genomic DNA was digested with KpnI or Pst I and subjected to Southern blot analysis using as a probe the human class IV ADH cDNA described previously (Satre et al., 1994). This cDNA probe should be able to hybridize to exons 2-9 as well as the 3`-untranslated region. Under the high stringency conditions used for hybridization and washing, the probe should not be able to cross-hybridize to the genes encoding the other classes of ADH, which share less than 70% sequence identity to class IV ADH as described above. The DNA fragments that hybridized are shown (Fig. 3). The sizes of detected fragments match closely with the sizes predicted from the KpnI and Pst I restriction maps of the genomic clones shown above (Fig. 1). This indicates that no major rearrangements occurred during the cloning process. With KpnI digestion (Fig. 3A), the 5.3-kb fragment observed correlates with the size of a region in the cloned DNA containing exons 2-5; the 3.4-kb fragment observed correlates with a fragment in the cloned DNA containing exons 6 and 7; and the 16.0-kb fragment observed presumably contains exons 8 and 9 plus the 3`-untranslated region that would be on a fragment of greater than 4.5 kb based upon the cloned DNA. With PstI digestion (Fig. 3B), the sizes of the hybridizing fragments also correlated with the restriction map; a fragment of 19.6 kb correlates in size with cloned DNA fragments containing exons 2-8, a fragment of 2.2 kb correlates with cloned DNA containing exon 9 plus the proximal end of the 3`-untranslated region, and a fragment of 10.0 most likely contains the distal end of the 3`-untranslated region (downstream of the PstI site), which should be detected with the cDNA probe used.


Figure 3: Analysis of class IV ADH gene structure in human genomic DNA. Human genomic DNA (5 µg) was digested with KpnI (A) or PstI (B) and analyzed by Southern blot analysis using as a probe the human class IV ADH cDNA containing the region from codon 8 to the 3`-untranslated region (Satre et al., 1994). The sizes in kilobase pairs of the DNA fragments able to hybridize to the class IV ADH cDNA are indicated. A shorter autoradiographic exposure allowed a clearer definition of the three KpnI fragments in laneA (data not shown).



Since all hybridizing DNA fragments in human genomic DNA digested with either KpnI or PstI correlated with the class IV ADH gene cloned, we assume that class IV ADH is encoded by a single unique gene and has no closely related pseudogenes. No pseudogenes were detected in the genomic clones analyzed as well. Of the five classes of mammalian ADH that have been analyzed, only class I ADH has been shown to be encoded by more than one gene in some species; i.e. human class I ADH is encoded by three closely related genes ADH1, ADH2, and ADH3, which cross-hybridize under high stringency (Duester et al., 1986). Only class III ADH (the most ancient form of ADH) has been demonstrated to possess pseudogenes; these were found to be processed pseudogenes (Matsuo and Yokoyama, 1990; Hur and Edenberg, 1992).

Expression of Class IV ADH mRNA

Human class IV ADH protein has been previously shown to be most abundant in the stomach and esophagus and undetectable in the liver (Yin et al., 1990; Moreno and Parés, 1991; Yin et al., 1993; Stone et al., 1993). To examine the mRNA for class IV ADH, we performed Northern blot analysis on human adult stomach and liver poly(A) RNA (Fig. 4). Using the class IV ADH cDNA as a probe, a single mRNA species of 2.3 kb was detected in human stomach RNA (Fig. 4D), whereas no mRNA was detected in liver RNA (Fig. 4C). As a control for liver RNA, we used a class I ADH cDNA as a probe (the human ADH3 gene), and detected three mRNA species of 2.6, 1.9, and 1.6 kb present at high levels (Fig. 4A). The expression of class I ADH mRNA we observed in liver correlates with previous studies on human liver RNA, which indicated high levels of mRNA for three forms of class I ADH, i.e. alpha, beta, and ADH encoded by ADH1, ADH2, and ADH3, respectively (Bilanchone et al., 1986; Ikuta and Yoshida, 1986). ADH1 and ADH2 mRNAs are detected by cross-hybridization with the ADH3 probe, with all three genes expressing mRNAs of approximately 1.6 kb as well as ADH2 also expressing mRNAs of 1.9 and 2.6 kb by differential polyadenylation (Von Bahr-Lindström et al., 1986; Heden et al., 1986; Höög et al., 1986). We also detected the same three class I ADH mRNAs at lower levels in stomach RNA (Fig. 4B). This correlates with previous studies showing that the stomach mucosal layer contains -ADH, and the muscular layer contains beta-ADH (Yin et al., 1988; Moreno and Parés, 1991). The presence of class IV ADH mRNA in the stomach but not the liver suggests that its gene is regulated much differently than those encoding the other classes of ADH (classes I, II, III, and V), which are all expressed in the liver (Duester et al., 1986; Höög et al., 1987; Giri et al., 1989; Yasunami et al., 1991).


Figure 4: Expression of class IV ADH mRNA in human tissues. Samples (0.5 µg) of poly(A) RNA from human liver (A and C) and human stomach (B and D) were separated by formaldehyde-agarose gel electrophoresis and subjected to Northern blot analysis. LanesA and B were hybridized to the human ADH3 cDNA (a class I ADH gene), whereas lanesC and D were hybridized to the human class IV ADH cDNA. The sizes of the RNAs detected are in kilobases.



The size of the mRNA for human stomach class IV ADH, estimated here at 2.3 kb by Northern blotting, correlates approximately with the size of the cDNA reported by Farrés et al.(1994), which is 2055 bp in length with a putative poly(A) signal near the 3` end but no poly(A) stretch. A 5`-untranslated region of 60 bp was reported in that cDNA, but our primer extension studies (discussed below) indicate that the mature mRNA contains 100 bp in the 5`-untranslated region. This would increase the predicted size of the mRNA to about 2095 bp plus the poly(A) stretch, which is generally 100-200 residues.

Identification of Transcription Initiation Site for Class IV ADH Gene

In order to determine where the promoter for the class IV ADH gene lies, we performed primer extension analysis on human stomach poly(A) RNA, which was shown above to hybridize to a class IV ADH cDNA probe. The primer consisted of the first 21 bases upstream of the methionine start codon on the noncoding strand. The main primer extension product was extended by reverse transcriptase to an adenine residue lying 100 bp upstream of the methionine start codon (Fig. 5). The sequence surrounding this adenine is pyrimidine-rich (5`-TCTATGT-3`), thus conforming to the consensus of known eukaryotic transcription initiation sites (Breathnach and Chambon, 1981).

The sequence downstream of this transcription initiation site (designated as position + 1 bp) contains two ATG triplets (one at +1 and one at +65 bp) prior to the ATG designated as the translation initiation codon at +101 (Fig. 2). Both of the upstream ATGs are flanked by thymines at critical positions (TNNATGT) and are thus classified as nonfunctional for translation initiation (Kozak, 1987). The ATG at position +101 is flanked by an adenine and guanine at these critical positions (ANNATGG) and is one of the most common functional translation initiation codons. The ATG at position +1 is also part of the transcription initiation site and is probably subjected to 5`-capping with 7-methylguanosine, making it even further less likely to be involved in translation initiation. Interestingly, the transcription initiation site for class IV ADH (5`-TCTATGT-3`) has a very similar sequence to the initiation sites previously observed in the three human class I ADH genes (ADH1, ADH2, and ADH3), which were all shown to initiate at an adenine within an ATG surrounded by pyrimidines: (5`-TTTATGC-3`) (Stewart et al., 1990).

Computer analysis of the DNA sequence from -496 to +100 bp was performed to scan for potential binding sites of several common transcription factors that may be part of the promoter (Fig. 2). A TATA box was not observed just upstream of the transcription initiation site near position -25 bp, which is its usual location if present (Breathnach and Chambon, 1981). However, we did observe the sequence TATATAA located from +28 to +22 bp downstream of the start site in the reverse orientation on the noncoding strand. Since the adenovirus IVa2 promoter has been shown to have a functional TATA box located about 20 bp downstream of the start site in the reverse orientation (Carcamo et al., 1990), the downstream TATA we observe in class IV ADH may also function in an unusual type of initiation event. We did not observe any GC-box binding sites (GGGCGG) for the transcription factor Sp1, which are often present in genes lacking a TATA box, nor other common sites such as those binding transcription factor CTF/NF1 (GCCAAT) and octamer-binding proteins (ATTTGCAT) (Mitchell and Tjian, 1989). This promoter also does not possess the initiator element (YAYTCYYY) often found overlapping with the site of transcription initiation, which can be found in genes that either contain or lack a TATA box (Roeder, 1991).

The class IV ADH promoter was found to contain a site at -43 bp matching the TGA(C/G)TCA consensus sequence for the transcription factor AP-1 (Mitchell and Tjian, 1989), as well as a site at -35 bp matching the T(T/G)NNG(T/C)AA(T/G) consensus sequence for C/EBP (De Simone and Cortese, 1992) (Fig. 2). The lack of an upstream TATA box at -25 bp or GC boxes to help direct the site of transcription initiation suggests that the AP-1 and C/EBP sites lying adjacent to each other between -43 and -27 bp may function together or separately to help direct transcription initiation for class IV ADH. Alternatively, this promoter may use the downstream TATA box or a novel mechanism. In this respect, the class IV ADH promoter is unlike the other ADH promoters characterized, which have either an upstream TATA box, i.e. the promoters for class I ADH (Duester et al., 1986; Stewart et al., 1990) and class II ADH (Von Bahr-Lindström et al., 1991), or GC boxes clustered near the start site, i.e. the promoter for class III ADH (Hur and Edenberg, 1992).

The presence of a C/EBP site in the class IV ADH promoter between -35 and -27 bp is similar to what was noticed for the three human class I ADH promoters that have previously been demonstrated to possess functional C/EBP sites between -45 and -37 bp (Van Ooij et al., 1992). However, the class I ADH promoters also contain a TATA box just downstream of this C/EBP site, and they contain additional C/EBP sites further upstream. Thus, if C/EBP plays a role in class IV ADH transcription, it may function differently than it does in class I ADH transcription where several related forms of C/EBP have been demonstrated to play a role in liver transcription (Van Ooij et al., 1992). The lack of class IV ADH expression in the liver further suggests a fundamentally different form of regulation than that seen for class I ADH genes. Finally, the class IV ADH promoter did not possess a retinoic acid response element in the region analyzed, thus differing from the human class I ADH gene ADH3, which was found to possess this element at -300 bp (Duester et al., 1991).

In summary, human class IV ADH was shown to be encoded by a single gene whose coding region is most closely related to that of class I ADH. However, the expression pattern of class IV ADH differs markedly from class I ADH. The mRNA for class IV ADH was shown to be much more abundant in stomach than liver, thus differing from the mRNA for class I ADH and other known ADHs, which are all actively expressed in liver. The promoter for class IV ADH is much different than those seen in other ADH genes, lacking many of the common sequence elements that direct transcription initiation and thus representing an unusual type of promoter. The cloning of the class IV ADH gene now enables us to use molecular genetic techniques to further study its expression patterns, gene regulation, and function, particularly its role in retinoid metabolism as a retinol dehydrogenase.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant AA07261 and by a grant from the Alcoholic Beverage Medical Research Foundation (to G. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16286[GenBank]-U16293[GenBank].

§
To whom correspondence should be addressed: Cancer Research Center, La Jolla Cancer Research Foundation, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-6480; Fax: 619-455-1048.

(^1)
The abbreviations used are: ADH, alcohol dehydrogenase; ADH, human gene encoding ADH; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; C/EBP, CCAAT/enhancer-binding protein.


ACKNOWLEDGEMENTS

We thank J. Knight of the La Jolla Cancer Research Foundation DNA Chemistry Facility for oligonucleotide synthesis and H. Jörnvall for the human ADH3 cDNA.


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