©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning of the Mouse Class IV Alcohol Dehydrogenase (Retinol Dehydrogenase) cDNA and Tissue-specific Expression Patterns of the Murine ADH Gene Family (*)

Mirna gombi&;-Knight , Hwee Luan Ang , Mario H. Foglio , Gregg Duester (§)

From the (1) Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Humans possess five classes of alcohol dehydrogenase (ADH), including forms able to oxidize ethanol or formaldehyde as part of a defense mechanism, as well as forms acting as retinol dehydrogenases in the synthesis of the regulatory ligand retinoic acid. However, the mouse has previously been shown to possess only three forms of ADH. Hybridization analysis of mouse genomic DNA using cDNA probes specific for each of the five classes of human ADH has now indicated that mouse DNA cross-hybridizes to only classes I, III, and IV. With human class II or class V ADH cDNA probes, hybridization to mouse genomic DNA was very weak or undetectable, suggesting either a lack of these genes in the mouse or a high degree of mutational divergence relative to the human genes. cDNAs for murine ADH classes I and III have previously been cloned, and we now report the cloning of a full-length mouse class IV ADH cDNA. In Northern blot analyses, mouse class IV ADH mRNA was abundant in the stomach, eye, skin, and ovary, thus correlating with the expression pattern for the mouse Adh-3 gene previously determined by enzyme analysis. In situ hybridization studies on mouse stomach indicated that class IV ADH transcripts were abundant in the mucosal epithelium but absent from the muscular layer. Comparison of the expression patterns for all three mouse ADH genes indicated that class III was expressed ubiquitously, whereas classes I and IV were differentially expressed in an overlapping set of tissues that all contain a large component of epithelial cells. This expression pattern is consistent with the ability of classes I and IV to oxidize retinol for the synthesis of retinoic acid known to regulate epithelial cell differentiation. The results presented here indicate that the mouse has a simpler ADH gene family than the human but has conserved class IV ADH previously shown to be a very active retinol dehydrogenase in humans.


INTRODUCTION

The existence of multiple classes of mammalian alcohol dehydrogenase (E.C.1.1.1.1) (ADH)() has been best demonstrated in humans that have been shown to possess five classes of ADH encoded by seven genes, one gene for each class except class I ADH, which is encoded by three closely related genes (Duester et al.,1986; Von Bahr-Lindström et al.,1991; Yasunami et al.,1991; Hur and Edenberg, 1992; Satre et al.,1994). Amino acid sequence comparisons between the five classes of human ADH have revealed interclass sequence identities in the 57-69% range, indicating divergence from a common progenitor about 450 million years ago during early vertebrate evolution (Satre et al.,1994). All classes of vertebrate ADH as well as all other medium chain ADHs ( i.e. 35-40 kDa) found in bacteria, yeast, plants, and invertebrates have arisen from duplication and divergence of a primordial class III ADH gene, which encodes a glutathione-dependent formaldehyde dehydrogenase found ubiquitously in all organisms analyzed (Koivusalo et al.,1989; Danielsson and Jörnvall, 1992; Danielsson et al.,1994). Class III ADH does not function in ethanol metabolism, but other ADHs that have evolved from it have acquired this ability (Kaiser et al.,1993).

Some vertebrate ADHs have evolved to metabolize additional alcohols such as retinol (vitamin A). The ability of some classes of ADH to act as retinol dehydrogenases implies that they may participate in the synthesis of retinoic acid, the active form of vitamin A involved in regulating cellular differentiation and embryonic development (Connor, 1988; De Luca, 1991). Purified mammalian ADHs have been demonstrated to catalyze retinol oxidation in vitro including human ADH classes I, II, and IV (Yang et al.,1994), rat ADH classes I and IV (Boleda et al.,1993), and mouse ADH-C (Connor and Smit, 1987) (defined herein as class IV ADH). Of these isozymes, only class I ADH has evolved to efficiently oxidize both ethanol and retinol (Boleda et al.,1993; Yang et al.,1994). ADH classes I and IV are found in all mammals analyzed in a wide variety of epithelial tissues (Holmes, 1978; Juli et al.,1987), which are known to convert retinol to retinoic acid (Blaner and Olson, 1994). Since the only major site of ethanol metabolism is the liver (Boleda et al.,1989), the function of extrahepatic class I and IV ADH is more consistent with a role in retinoid metabolism rather than ethanol metabolism. In addition to the cytosolic alcohol/retinol dehydrogenases, a microsomal retinol dehydrogenase has also been identified that catalyzes retinol oxidation in vitro (Posch et al.,1991). The relative importance of all of these enzymes in vivo remains to be determined.

To use molecular genetics for further study of this issue, we have sought to characterize the mouse ADH gene family. The murine ADH system appears to be less complex than that seen in humans. The existence of three forms of mouse ADH encoded by three genes was early established by enzyme analyses and genetic studies, i.e. ADH-A, ADH-B, and ADH-C encoded by Adh-1, Adh-2, and Adh-3, respectively (Holmes, 1977, 1978). The sequencing of cDNAs for Adh-1 (Edenberg et al.,1985; Ceci et al.,1986) and Adh-2 (Hur et al.,1992) allowed a comparison with the known sequences of several classes of human ADH, indicating that they encode mouse class I and class III ADH, respectively. Whereas humans possess three class I ADH genes, the mouse was found to possess only one gene encoding class I ADH, i.e.Adh-1 (Ceci et al.,1987; Zhang et al.,1987). A lack of sequence data on the form of ADH encoded by the mouse Adh-3 gene has made it impossible to determine if it is homologous to one of the known classes of human ADH. The ADH enzyme encoded by Adh-3 has been shown to be expressed predominantly in the stomach (Holmes, 1978) and thus may be most closely related to the major stomach ADH of humans and rats (Yin et al.,1990; Parés et al.,1990; Moreno and Parés, 1991). Amino acid sequence data on human and rat stomach ADH defined a new class called class IV ADH (Parés et al.,1990,1992; Stone et al.,1993; Parés et al.,1994). Recently, the cloning of a cDNA for human class IV ADH was described by this laboratory (Satre et al.,1994). We have now used this human class IV ADH cDNA to verify that the mouse contains this gene and have isolated a full-length mouse class IV ADH cDNA. This has allowed a detailed comparison of the expression patterns for ADH classes I, III, and IV in mouse tissues. The results suggest a correlation of mouse class I and class IV ADH gene expression with epithelial tissues known to require retinoic acid for proper differentiation.


EXPERIMENTAL PROCEDURES

PCR Cloning of a Mouse Class IV ADH cDNA

Total RNA was isolated from mouse stomach mucosal layer tissue (6-month-old female, strain FVB/N) by the acid guanidinium thiocyanate-phenol chloroform method previously described (Chomczynski and Sacchi, 1987). cDNA cloning was performed by reverse transcription-PCR. First strand cDNA synthesis was performed on 10 µg of total mouse stomach mucosa RNA using oligo(dT) as a primer and avian myeloblastosis virus reverse transcriptase as described in the cDNA Cycle kit (Invitrogen). The single-stranded cDNA product was subjected to PCR using primers specific for mouse class IV ADH as described below.

The conditions for PCR were a modification of the standard methodology (Ausubel et al.,1989). PCR reactions (100 µl) contained 0.5 mM of each nucleotide (dATP, dGTP, dTTP, and dCTP), 1 reaction buffer (10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl, 50 mM KCl), 2 units of Taq DNA polymerase (Life Technologies, Inc.), 100 pmol of both upstream and downstream primers, and 1-5 µl of the above single-stranded cDNA product. The reaction was overlaid with 50 µl of mineral oil and then subjected to 36 cycles of PCR with increasing annealing stringency in later cycles. Cycling conditions were 94 °C (1 min) for denaturation, 51 °C (2 min) for annealing, and 72 °C (3 min) for elongation during cycles 1-10; 94 °C (1 min), 53 °C (2 min), and 72 °C (3 min) during cycles 11-20; 94 °C (1 min), 55 °C (2 min), and 72 °C (3 min) during cycles 21-35; and for the 36th cycle the elongation at 72 °C was extended to 7 min. 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 (Stratagene) for DNA sequence analysis.

A partial mouse class IV ADH PCR product of 738 bp in length extending from codons 93 to 339 was obtained using the same primers (GD-70 and GD-73) previously reported as successful for preparing a partial rat class IV ADH PCR product (Satre et al.,1994). A PCR product containing the full coding region of the mouse class IV ADH gene was obtained using additional primers determined from sequencing of genomic clones encoding mouse class IV ADH. A mouse 129 SVJ genomic DNA library (Stratagene) was screened by the plaque-lift technique previously described (Sambrook et al.,1989). The hybridization probe consisted of the 738-bp partial mouse class IV ADH PCR product described above. Radiolabeling of the probe, hybridization conditions, and phage DNA purification were performed as previously described (gombi&;-Knight et al.,1995). DNA from four independently isolated phage was subjected to nucleotide sequence analysis by linear PCR amplification as previously described (Wang et al.,1992) using primers able to hybridize to the 5`- or 3`-ends of the coding region to enable sequencing of the 5`- or 3`-untranslated regions, respectively. As a 5`-sequencing primer, we used a degenerate human class I ADH exon 1 primer (GD-88), which previously proved successful in sequencing the 5`-untranslated region of the human class IV ADH gene due to its ability to cross-hybridize to exon 1 (Satre et al.,1994). As a 3`-sequencing primer, we used GD-113, 5`-AGCATTCGAACGGTCCTGAC(A/T/G/C)TT(T/C)TGA-3`, corresponding to the coding strand sequence of codons 367-374 plus the stop codon previously determined for the human class IV ADH cDNA (Satre et al.,1994). Nucleotide sequence data of the mouse class IV ADH 5`-untranslated region obtained from primer GD-88 with phage MZm9 were used to construct an upstream PCR primer (GD-122) 5`-GTCGAAGGATCCAGAGGCAGGATGGGCACCGCTG-3` located on the coding strand overlapping the start codon. Nucleotide sequence data of the mouse class IV ADH 3`-untranslated region from primer GD-113 with phage MZm2 were used to construct a downstream PCR primer (GD-115) 5`-TGCTAGAAGCTTCAGAGAACACTGTCAGGAGCAAGG-3` located on the noncoding strand 70 bp downstream of the stop codon. PCR with GD-122 and GD-115 was carried out as described above to generate a DNA fragment of about 1.2 kb containing the full coding region of mouse class IV ADH.

PCR Cloning of a Mouse Class III ADH cDNA

A mouse class III ADH cDNA was prepared by reverse transcription-PCR as described above, except that the source of the RNA was total RNA (10 µg) from liver, and the upstream and downstream PCR primers were based upon the published nucleotide sequence of a mouse class III ADH cDNA (Hur et al.,1992). The upstream PCR primer (GD-106) 5`-GTCGAAGGATCCGCCATGGCGAACCAGGTGATC-3` was located on the coding strand at positions 37-57 bp overlapping the start codon, and the downstream PCR primer (GD-107) 5`-CCATGAAAGCTTGAAAGAGTGCAGGATGGACAG-3` was located on the noncoding strand at positions 1180-1200 bp just downstream of the stop codon.

PCR Cloning of a Human Class V ADH cDNA

To obtain a probe for human class V ADH, we used reverse transcription-PCR to prepare a full-length class V ADH cDNA from human liver RNA, which has been shown to express this form of ADH (Yasunami et al.,1991). First-strand cDNA synthesis was carried out as above except with 1.0 µg of human liver poly(A) RNA (Clontech). The conditions for PCR amplification from the single-stranded cDNA and cloning of the PCR product are described in detail above except that primers specific for human class V ADH were used. Based upon the sequence of human class V ADH encoded by the ADH6 gene (Yasunami et al.,1991), we synthesized an upstream PCR primer (GD-123) 5`-GTCGAAGGATCCAAATCAGCATGAGTACTACAGG-3` containing sequence on the coding strand from -8 to +14 bp relative to the ATG translation start codon, as well as a downstream PCR primer (GD-103) 5`-GATGACAAGCTTTCTACTCCCAAGCCAATACACCC-3` containing sequence on the noncoding strand downstream from the stop codon at +1154 bp to +1176 bp.

DNA Sequence Analysis of ADH cDNAs

Double-stranded plasmid DNAs from ADH cDNAs cloned in pBluescript II KS were subjected to dideoxynucleotide sequence analysis using the Sequenase Kit (U. S. Biochemical Corp.). DNA sequences were determined on both strands. The standard DNA sequencing primers for pBluescript II KS were used, i.e. T3, T7, KS, and SK (Stratagene) as well as several specialized oligonucleotides, all of which were synthesized by the La Jolla Cancer Research Foundation DNA Chemistry Facility. Computer analysis of nucleotide sequences, alignment of deduced amino acid sequences, and estimation of enzyme pI values were accomplished using the MacVector version 4 program (International Biotechnologies, Inc.).

Southern Blot Analysis of Mammalian Genomic DNAs

Human, mouse, and rat genomic DNAs were purchased from Clontech. Samples (5 µg) of each were digested with EcoRI or HindIII and fractionated by electrophoresis in 1% agarose gels. DNA was denatured and transferred to filters for hybridization analysis by Southern blotting as previously described (Sambrook et al.,1989). Hybridization of filters was carried out for 20 h at 42 °C in 50% formamide, 5 Denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 5 SSC (1 SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.6), 10% dextran sulfate, 1% SDS, 10 µg/µl poly(A), 250 µg/µl herring sperm DNA, and 10 cpm/ml radiolabeled probe. Final washing was under moderate stringency conditions (to allow human/mouse cross-hybridization) at 68 °C for 1 h in 0.5 SSC, 0.1% SDS. Autoradiography was performed with intensifying screens at -70 °C for 48 h.

The hybridization probes consisted of human cDNAs representing all five known classes of human ADH. The cDNA inserts for each ADH plasmid were purified by agarose gel electrophoresis and radiolabeled with [-P]dCTP by random hexamer-primed labeling using the oligolabeling kit (Pharmacia Biotech Inc.). Plasmids containing full-length cDNAs for human class I ADH encoded by ADH3 (Höög et al.,1986), human class II ADH encoded by ADH4 (Höög et al.,1987), and human class III ADH encoded by ADH5 (Giri et al.,1989) were obtained for use as hybridization probes. A cDNA for human class IV ADH encoded by ADH7 was recently cloned in this laboratory (Satre et al.,1994). A cDNA for human class V ADH encoded by ADH6 was prepared by reverse transcription coupled to PCR as described above.

Analysis of RNA by Northern Blotting

Total RNA was isolated from mouse tissues (6-month-old males and females, strain FVB/N) by the acid guanidinium thiocyanate-phenol chloroform method previously described (Chomczynski and Sacchi, 1987). Samples (10 µg) of total mouse RNA were separated by formaldehyde-agarose gel electrophoresis and subjected to Northern blot analysis as described (Sambrook et al.,1989). The presence of mouse 18 S RNA (1.87 kb) and 28 S RNA (4.72 kb) was used to estimate the integrity of the various RNA preparations as well as controlling for lane-to-lane differences in the amount of RNA loaded and to estimate the molecular sizes of hybridizing species of RNA. The hybridization probes included a cDNA for the mouse class I ADH gene ( Adh-1) previously described (Edenberg et al.,1985; Ceci et al.,1986), and cDNAs for the mouse class III ADH gene ( Adh-2) and class IV ADH gene ( Adh-3) both prepared by reverse transcription coupled to PCR as described above. Radiolabeling of the cDNA probes and hybridization conditions were as described above for Southern blot analysis except that final washing was performed under high stringency conditions at 68 °C for 1 h in 0.1 SSC, 0.1% SDS to prevent cross-hybridization.

For some analyses, samples (10 µg) of total mouse liver RNA were run in parallel with samples (0.5 µg) of human stomach and liver poly(A) RNA (Clontech). These samples were subjected to Northern blot analysis as above except that the probes consisted of a cDNA for human class II ADH (Höög et al.,1987) and a cDNA for human class V ADH described above, with final washing being performed under moderate stringency conditions at 68 °C for 1 h in 0.5 SSC, 0.1% SDS to allow human/mouse cross-hybridization.

In Situ Hybridization of Mouse Stomach Tissue

Stomach tissue from adult mice was fixed, embedded in paraffin, sectioned, and processed for in situ hybridization according to the methods previously described (Wilkinson and Green, 1990). A S-labeled antisense RNA probe for mouse class IV ADH mRNA was synthesized using a T3 polymerase in vitro transcription reaction with [-S]UTP from the full-length cDNA encoding mouse class IV ADH cloned in pBluescript II KS described above. A control sense RNA probe was synthesized from the same plasmid using T7 polymerase. Slides were exposed to emulsion for 2-3 weeks prior to development. Staining of sections was performed with toluidine blue or neutral red, and photography was under bright field and dark field conditions.


RESULTS

Analysis of the Murine ADH Gene Family

To determine whether the mouse possesses ADH genes in addition to the previously characterized class I and class III ADH genes, cDNAs for each of the five classes of human ADH were used as cross-hybridization probes to examine mouse genomic DNA for the presence of the homologous genes. The human/mouse intraclass amino acid sequence identity for class I ADH is 85% (Zhang et al.,1987) and for class III ADH is 93% (Hur et al.,1992), whereas the interclass amino acid sequence identity among the five known human ADH classes ranges between 57 and 69% (Satre et al.,1994). Therefore, hybridizations were able to be carried out under conditions that would easily allow detection of human/mouse intraclass cross-hybridization but not human/mouse interclass cross-hybridization. When cDNAs for human ADH classes I, II, III, IV, and V were used as hybridization probes for human DNA, we detected for each probe a unique set of human DNA fragments (Fig. 1, A-E), thus indicating that the hybridization conditions were sufficiently stringent to prevent interclass cross-hybridization among any of the classes.


Figure 1: Analysis of the ADH gene family in human, mouse, and rat genomic DNA. Southern blot analysis was performed on 5-µg samples of human, mouse, or rat genomic DNA digested with EcoRI or HindIII. Hybridizations were carried out with human cDNA probes for class I ADH ( A), class II ADH ( B), class III ADH ( C), class IV ADH ( D), and class V ADH ( E). Each autoradiographic exposure shown was 48 h, this being most useful for observation of the mouse and rat DNA fragments, which hybridize less efficiently than the human fragments. Upon shorter autoradiographic exposure, closely migrating human fragments could be more easily differentiated (data not shown). Two regions of nonspecific binding are observed near the rat class I ADH fragments of 3.0 and 1.0 kb in panelA, and they were not totally removed in the stripping of the filter for rehybridization to probes for ADH classes II and III in panelsB and C. Also shown are the sizes (kb) of DNA fragments from the 1-kb ladder DNA gel markers (Life Technologies, Inc.).



The numerous human DNA fragments detected with the class I ADH probe represent the previously reported EcoRI and HindIII fragments observed in genomic clones of the three human class I ADH genes ( i.e.ADH1, ADH2, and ADH3 encoding enzymes sharing about 94% sequence identity), which cross-hybridize under these conditions (Duester et al.,1986). The rodent DNA fragments detected with the human class I ADH cDNA probe represent those present in the previously analyzed genes for mouse class I ADH (Ceci et al.,1987; Zhang et al.,1987) and rat class I ADH (Crabb et al.,1989). As expected, the degree of hybridization of the human class I ADH cDNA with mouse and rat DNA fragments was much less than that observed with human DNA fragments due to species-specific sequence differences.

With the human class II ADH probe, we detected five EcoRI fragments (14.4, 6.8, 4.4, 3.7, and 1.0 kb) previously shown to be present in the cloned gene (Von Bahr-Lindström et al.,1991). When rat genomic DNA was analyzed with the human class II ADH cDNA probe, a clearly discernible 6.0-kb HindIII DNA fragment was detected (Fig. 1 B). The observation of a strongly cross-hybridizing rat DNA fragment is consistent with previous studies describing a class II ADH gene cloned from a rat liver cDNA library (Höög, 1991; Estonius et al.,1993). In mouse DNA, we detected a very weakly cross-hybridizing 11.0-kb HindIII fragment, but no EcoRI fragments were detected (Fig. 1 B). To provide further insight into whether the mouse has a class II ADH gene, we screened a mouse liver cDNA library as well as a mouse genomic DNA library with the human class II ADH cDNA probe. In both libraries, we failed to detect any positively hybridizing clones (data not shown). Thus, the nature of the 11.0-kb mouse HindIII fragment that hybridized weakly is unknown, but it probably does not represent a class II ADH gene. The inability to detect a hybridizing EcoRI fragment also suggests that the class II ADH gene is missing from mouse or has undergone extensive divergence from the human gene.

When a human class III ADH cDNA probe was used (Fig. 1 C), we detected in human DNA three EcoRI fragments (8.7, 6.5, and 4.1 kb) and three HindIII fragments (11.0, 8.5, and 4.1 kb) present in the previously cloned gene (Hur and Edenberg, 1992). We also detected a 12.3-kb EcoRI fragment and a 2.0-kb HindIII fragment, which correspond to a previously characterized human class III ADH processed pseudogene (Matsuo and Yokoyama, 1990). In mouse genomic DNA, there was cross-hybridization to EcoRI fragments of 11.0, 8.5, and 3.0 kb as well as HindIII fragments of 14.0, 5.3, 2.8, and 1.7 kb (Fig. 1 C). Rat EcoRI fragments of 6.0 and 3.1 kb hybridized to the human class III ADH cDNA. Both mouse and rat have previously been shown to possess the class III ADH enzyme, and a cDNA for the mouse form has been cloned (Hur et al.,1992).

A human class IV ADH cDNA probe detected four EcoRI fragments of 15.0, 13.5, 8.0, and 7.0 kb as well as three HindIII fragments of 13.5, 4.0, and 2.7 kb (Fig. 1 D), all accounted for in the restriction maps of the cloned gene (Yokoyama et al.,1994; gombi&;-Knight et al.,1995); the additional HindIII fragments observed (12.2 and 1.3 kb not in the cloned gene) may be due to a restriction fragment length polymorphism in the 13.5-kb HindIII fragment. In mouse genomic DNA, there was cross-hybridization to EcoRI fragments of 12.5 and 10.0 kb as well as HindIII fragments of 11.0, 4.5, and 1.8 kb (Fig. 1 D). Rat EcoRI fragments of 12.0, 4.5, and 3.2 kb hybridized to the human class IV ADH cDNA. This clearly indicates the presence of a class IV ADH gene in both mouse and rat.

When the human class V ADH cDNA probe was used with human genomic DNA (Fig. 1 E), we detected four EcoRI fragments (17.5, 10.6, 3.2, and 0.8 kb) and four HindIII fragments (5.7, 3.3, 3.0, and 0.8 kb), which all correspond to the previously cloned gene (Yasunami et al.,1991). Two other weakly hybridizing human DNA fragments were also detected (a 4.5-kb EcoRI fragment and a 13.0-kb HindIII fragment), which do not correspond to the cloned gene (Fig. 1 E). For the mouse, there was no detectable hybridization in genomic DNA digested with either EcoRI or HindIII, suggesting the absence of a class V ADH gene. In rat genomic DNA, only one very weakly hybridizing 3.1-kb EcoRI fragment was detected (Fig. 1 E). The weak hybridization signal detected between rat DNA and the class V ADH probe, compared with the much stronger signals observed with rat DNA and the class I, II, III, and IV ADH probes (Fig. 1, A-D), suggests that the rat DNA fragment detected may not represent a class V ADH gene. This contention is further strengthened by a report of the cloning of a novel rat liver ADH cDNA using the human class V ADH cDNA as a probe, i.e. class VI ADH sharing only 65% sequence identity with human class V ADH (Höög and Brandt, 1995). This rat class VI ADH shares 79% sequence identity with a previously identified novel deer mouse ADH (Zheng et al.,1993), suggesting that the deer mouse possesses class VI ADH. Thus, the rat 3.1-kb EcoRI fragment (Fig. 1 E) may represent the class VI ADH gene recently cloned. In the mouse, the lack of a Southern blot hybridization signal provides no support for the presence of either class V or class VI ADH genes. However, we cannot discount the possibility that the mouse possesses a form that has significantly diverged and does not cross-hybridize to the human class V ADH cDNA probe under the conditions used.

Expression of ADH Classes II and V

Since Southern blot studies failed to provide evidence for the presence of genes encoding ADH classes II and V (or VI) in the mouse, we analyzed mouse and human RNA for expression of these genes. Northern blot analysis was carried out under the same conditions used for Southern blotting to allow detection of mouse/human intraclass cross-hybridization. Human liver RNA (but not stomach RNA) possessed two very abundant mRNAs for class II ADH of 2.1 and 1.6 kb, but mouse liver RNA failed to hybridize to the class II ADH probe (Fig. 2). The 2.1-kb mRNA matches the size of the reported full-length cDNA for human class II ADH (Höög et al.,1987), and the expression of two mRNAs for class II ADH (2.4 and 1.6 kb) in human liver RNA has previously been reported (Giri et al.,1989). Failure to detect class II ADH mRNA in mouse liver lends further support to the possibility that this form of ADH is absent in the mouse.


Figure 2: Analysis of class II and class V ADH mRNA. Northern blot analysis was performed on samples (10 µg) of total mouse liver RNA run in parallel with samples (0.5 µg) of human stomach ( St) and liver ( Li) poly(A) RNA. Hybridizations were carried out with human cDNA probes for class V ADH ( upperleftpanel) or class II ADH ( upperrightpanel) under the same conditions used for the Southern blots in Fig. 1. The bottompanel shows an ethidium bromide-stained image of the agarose gel before Northern blot analysis, indicating the 28 and 18 S rRNAs present in the total RNA preparations from mouse liver. The numbers in the upperpanels refer to sizes in kb of the detected mRNAs using the rRNAs in the bottompanel as molecular size standards.



In Northern blot studies of human liver RNA with the class V ADH cDNA probe, we detected an abundant mRNA species of 3.1 kb accompanied by a minor species of 1.5 kb (Fig. 2). The 1.5-kb class V ADH mRNA matches the size of the previously cloned cDNA, which contains a poly(A) signal (Yasunami et al.,1991). Class V ADH was previously reported to be expressed in both human liver and stomach by PCR analysis (Yasunami et al.,1991), but in our studies no mRNA was detected in human stomach RNA (Fig. 2). However, hybridization of the same sample with a human class IV ADH cDNA probe detected a 2.3-kb mRNA, indicating the integrity of the stomach RNA (data not shown; gombi&;-Knight et al., 1995). The hybridization conditions used here prevented interclass hybridization among classes II and V, as evidenced by the lack of detection of class II ADH mRNA in human liver RNA with a class V ADH probe and the lack of detection of class V ADH mRNA in human liver RNA with a class II ADH probe (Fig. 2).

In mouse liver RNA, we did detect a minor mRNA species of 1.5 kb hybridizing to the class V ADH probe, matching the size of the smaller human class V ADH mRNA (Fig. 2). However, since the mouse genomic DNA analysis indicated the lack of a class V ADH gene (Fig. 1 E), the mouse liver mRNA detected with the human class V ADH probe cannot confidently be placed in the class V (or VI) family.

Cloning and Nucleotide Sequencing of a Full-length Mouse Class IV ADH cDNA

The above hybridization studies clearly indicated that the mouse possesses a gene for class IV ADH in addition to the previously known genes encoding classes I and III. No clear support for the presence of class II or class V ADH genes was obtained. Thus, to compare the expression patterns of the three known murine ADH genes, we cloned a cDNA for the mouse class IV ADH gene.

The partial amino acid sequence of purified rat stomach class IV ADH (Parés et al.,1990,1992) was used to predict portions of the mRNA nucleotide sequence for mouse class IV ADH and thus allowed the synthesis of degenerate oligonucleotides able to clone a cDNA via reverse transcription-PCR. A PCR product of 738 bp stretching from codons 93 to 339 was generated and cloned as described under ``Experimental Procedures.'' To isolate a full-length cDNA via PCR, sequence data from genomic clones were sought to be used to design additional PCR primers. The partial mouse class IV ADH cDNA described above was used as a hybridization probe to screen a mouse genomic DNA library. Out of 300,000 plaques screened, we isolated four genomic clones, which were then subjected to nucleotide sequence analysis as described under ``Experimental Procedures.'' These data were used to construct an upstream PCR primer located on the coding strand overlapping the start codon and a downstream PCR primer located on the noncoding strand just downstream of the stop codon. Reverse transcription-PCR of mouse stomach RNA with these two primers generated the expected DNA fragment of about 1.2 kb (containing the full coding region of mouse class IV ADH), which was then cloned for sequence analysis.

Both the partial and full-length mouse class IV ADH cDNAs obtained by PCR were subjected to nucleotide sequence analysis. The sequence of the partial cDNA matched perfectly to an internal stretch of the full-length cDNA, the latter consisting of a 1204-bp region containing an open reading frame that translated into a polypeptide of 373 amino acids following the methionine initiator codon (Fig. 3). 9 bp of the 5`-untranslated region and 70 bp of the 3`-untranslated region were also included. No polyadenylation signal was noticed in the 3`-Region.


Figure 3: Nucleotide sequence of mouse class IV ADH cDNA. The 1204-bp nucleotide sequence of the full-length cDNA is shown including the full coding region as well as some untranslated sequences from both 5`- and 3`-ends. The methionine ATG codon at position 10 is well suited to be a translation start codon with the expected purines at positions -3 and +4 relative to the ATG (Kozak, 1987). This ATG is followed by the complete mouse class IV ADH coding region and a TGA stop codon (indicated by an asterisk). The numberingscheme for the amino acid residues (1-374) follows that previously established for class I ADH (Jörnvall et al.,1987); indicated is a deletion of the codon for amino acid 118 in class IV relative to class I.



The predicted amino acid sequence of mouse class IV ADH was aligned with that of human class IV ADH previously reported (Satre et al.,1994). Both sequences contain 373 residues, and there were 41 differences between mouse and human class IV ADH resulting in 89% amino acid sequence identity. This high level of sequence identity is what is expected between human and mouse ADHs of the same class, thus indicating that the cDNA cloned encodes the mouse version of class IV ADH. Relative to the class I ADH sequence originally determined and now used as a reference to number the amino acids in all other vertebrate ADHs (Jörnvall et al.,1987), the mouse class IV ADH sequence like the human lacks the codon for amino acid 118 and is thus 373 residues in length rather than 374 present in class I ADH.

The predicted mouse class IV ADH polypeptide has a molecular weight of 39,703 and an estimated isoelectric point (pI) of 8.44. This pI matches closely to that of mouse ADH-C (the Adh-3 gene product) observed in previous zone electrophoresis studies of mouse tissue homogenates performed at pH 8.5 (Holmes, 1978). At this pH, ADH-C migrates very little from the origin, indicating that its pI is close to 8.5, but ADH-A (the Adh-1 gene product, class I ADH, predicted pI = 8.99) migrates to the cathode and ADH-B (the Adh-2 gene product, class III ADH, predicted pI = 8.03) migrates to the anode. This provides further evidence that mouse class IV ADH is ADH-C encoded by Adh-3.

Localization of Class IV ADH mRNA in Mouse Stomach Tissues

The class IV ADH cDNA was used as a probe for in situ hybridization of mouse stomach sections. A strong hybridization signal was observed in the stomach mucosal layer, but little or no mRNA was detected in the muscular layer (Fig. 4, A and B). Under higher magnification it was observed that class IV ADH mRNA was localized to the cells of the mucus-secreting epithelium, including the mucosal cells that line the gastric pits (Fig. 4, C and D). This finding correlates with previous studies in humans indicating that class IV ADH enzyme activity is present in the mucosal layer and absent in the muscular layer (Yin et al.,1993). Since the ADH-C enzyme encoded by Adh-3 has previously been shown to be the predominant form of ADH in the mouse stomach (Holmes, 1978), the detection of large amounts of class IV ADH mRNA in the mouse stomach provides further support that Adh-3 encodes class IV ADH. These studies also demonstrate the restriction of class IV ADH expression to epithelial cells.


Figure 4: Localization of class IV ADH mRNA in mouse stomach tissue. Whole mouse stomach tissue was sectioned and subjected to in situ hybridization using the radiolabeled mouse class IV ADH cDNA as a probe. A and C, sections stained with toluidine blue and photographed under bright field to indicate the stomach morphology. The stomach is shown turned inside out (mucosal layer out) after incision and removal of the contents prior to fixation. B and D, sections adjacent to A and C, respectively, subjected to hybridization and photographed at the same magnification under dark field to observe the silver grains. The gastric pits appear as loops in panel D. Scalebars represent 500 µm.



Tissue-specific Expression of mRNAs for Mouse ADH Classes I, III, and IV

The distribution of ADH mRNAs in total RNA preparations from 15 different adult mouse tissues was examined by Northern blot analysis using cDNA probes for mouse ADH classes I, III, and IV (Fig. 5). Hybridizations were carried out at very high stringency to prevent interclass cross-hybridization.


Figure 5: Expression pattern of ADH classes I, III, and IV in mouse tissues. Northern blot analysis was performed on 10-µg samples of total RNA from the mouse tissues listed. Hybridizations were carried out at high stringency with mouse cDNA probes for class I ADH ( toppanel), class III ADH ( secondpaneldown), and class IV ADH ( thirdpaneldown). To more easily compare the results between the classes, the same Northern blot filters were sequentially hybridized to all three probes after stripping of the previous probe. The bottompanels show ethidium bromide-stained images of the agarose gel before Northern blot analysis, indicating the integrity of the 28 and 18 S rRNAs present in the total RNA preparations, as well as an indication of the amount of RNA actually present in each lane. A few loading differences were noted ( i.e. low amounts of skin and uterus RNA), but the integrity of the RNA in each lane is evidenced by the expression of class III ADH in all samples. A sample of stomach mucosa RNA is present in both the left and rightpanels to allow a comparison between them. The numbers in the upperpanels refer to sizes in kb of the detected mRNAs using the rRNAs in the bottompanel as molecular size standards.



Class I ADH mRNA was observed to be differentially expressed, being detected at very high levels in liver, small intestine, and eye, which all contained a major species of 1.5 kb accompanied by two minor species of 3.1 and 6.2 kb (Fig. 5). Moderate amounts of the 1.5-kb class I ADH mRNA were detected in kidney, ovary, and uterus, and lower amounts were detected in the spinal cord, thymus, heart, stomach mucosa, skin, and testis. No class I ADH mRNA was detected in the brain or spleen. These results correlate well with previous studies on the distribution of class I ADH enzyme activity (ADH-A encoded by Adh-1) in mouse tissues where it was found that highest levels were in the liver, intestine, kidney, and uterus (Holmes, 1978). The presence of a 1.5-kb class I ADH mRNA in mouse liver and kidney has been previously reported (Ceci et al.,1986). The cDNA clones reported for mouse class I ADH include the poly(A) region and thus indicate a mRNA size near 1.5 kb (Edenberg et al.,1985; Ceci et al.,1986). The larger 3.1- and 6.2-kb species detected here may be due to differential polyadenylation or the presence of incompletely spliced transcripts.

Class III ADH mRNA was detected in all 15 tissues analyzed with a small variation in levels between tissues (Fig. 5). This variation was clearly not as great as that seen for class I ADH mRNA above, indicating that the class III ADH gene is not subject to major differential tissue-specific regulation. All tissues expressed only a 1.6-kb class III ADH mRNA. The ubiquitous expression of class III ADH noted here correlates with Northern blot analysis of a more limited set of tissues reported earlier, which also indicated the presence of a 1.6-kb class III ADH mRNA in all tissues examined (Hur et al.,1992). The ubiquitous mRNA expression pattern reported here closely parallels the previously reported analysis of mouse class III ADH enzyme activity (ADH-B encoded by Adh-2) in which the enzyme was found in all 16 mouse tissues analyzed with only small variations in the level of activity (Holmes, 1978).

Mouse class IV ADH mRNA was observed to have a high degree of differential expression in a pattern that was quite different from that observed for either class I or class III ADH mRNA. Very high expression of class IV ADH mRNA was noticed only in the stomach mucosa, with a moderate level of expression in the eye, thymus, skin, and ovary (Fig. 5). Very low levels of class IV ADH mRNA were also detected in the small intestine, liver, and uterus, but no expression was noticed in the brain, spinal cord, spleen, heart, kidney, or testis. In stomach mucosa, five species of class IV ADH mRNA were observed (3.7, 3.0, 2.1, 1.6, and 1.5 kb) with the 2.1-kb mRNA being most abundant (Fig. 5). This indicates that mouse class IV ADH mRNA probably has a large number of differentially polyadenylated or spliced variants. However, in human stomach RNA, only one class IV ADH mRNA of 2.3 kb has been detected (gombi&;-Knight et al.,1995). The mouse class IV ADH cDNA isolated herein is about 1.2 kb in length but must be derived from a larger mRNA since it does not contain the poly(A) region.

The expression pattern observed here for class IV ADH mRNA matches very well to the distribution reported for mouse ADH-C enzyme activity, which is highest in the stomach, ovary, and uterus (Holmes, 1978) as well as the eye (Rout and Holmes, 1991) and the skin (epidermis) (Connor and Smit, 1987). The previous studies also indicated that ADH-C is absent from the liver, brain, spleen, heart, and testis (Holmes, 1978), thus correlating with the lack of class IV ADH mRNA in these tissues observed by our Northern blot studies. This provides further evidence that mouse ADH-C (encoded by Adh-3) is a class IV ADH.


DISCUSSION

The mouse ADH gene family was early defined as consisting of three genes Adh-1, Adh-2, and Adh-3 encoding the ADH-A, ADH-B, and ADH-C isozymes, respectively (Holmes, 1977, 1978). After those initial studies in the mouse, five classes of ADH were identified in the human encoded by seven genes, which have all been cloned (Satre et al.,1994). Also, cDNAs for a sixth class of ADH have recently been identified in the deer mouse and rat (Zheng et al.,1993; Höög and Brandt, 1995). Of the three known mouse ADH genes, only two were previously cloned, i.e.Adh-1 encoding class I ADH (Edenberg et al.,1985; Ceci et al.,1986) and Adh-2 encoding class III ADH (Hur et al.,1992). Thus, we have attempted to more fully define the extent of the mouse ADH gene family as well as clone the form corresponding to Adh-3.

Southern blot analysis of mouse genomic DNA using cDNA probes for each of the five classes of human ADH revealed that the mouse possesses a gene for class IV ADH in addition to the class I and class III ADH genes previously known. These studies also indicated that the mouse either lacks genes for class II and class V ADH or it possesses forms that have diverged so far from the human that they are unable to efficiently cross-hybridize under our conditions. This possibility is reinforced by Northern blot studies, which indicated that mouse liver does not contain class II ADH mRNA, despite this being a major site of class II ADH expression in humans. Since the rat is known to have a class II ADH gene (Höög, 1991), which we also detected in our Southern blot studies of rat genomic DNA, this means that mice have not conserved class II ADH in the same manner as have rats and humans. The Southern blot studies also suggest that both mouse and rat lack a gene well conserved with human class V ADH. The mouse totally lacked a hybridization signal with class V ADH, and the weakly hybridizing 3.1-kb EcoRI fragment detected in rat DNA probably represents a portion of the gene encoding the recently cloned rat class VI ADH cDNA that was detected by cross-hybridization to the human class V ADH cDNA (Höög and Brandt, 1995). Thus, our studies indicate that the mouse ADH gene family, initially defined as containing only three members, cannot yet be expanded to include any additional members beyond classes I, III, and IV.

To clone the mouse class IV ADH gene, we performed reverse transcription-PCR on mouse stomach RNA since this tissue had previously been shown to have high activity for the ADH-C enzyme, which shares many properties in common with rat class IV ADH (Holmes, 1978). A cDNA containing the entire coding region of mouse class IV ADH was obtained, and the predicted amino acid sequence was shown to share 89% sequence identity with the full-length human class IV ADH sequence previously reported (Satre et al.,1994). Mouse class IV ADH also shares about 93% sequence identity with the complete rat class IV sequence, which was previously shown to share 88% sequence identity to human class IV (Parés et al.,1994). The human versus rodent (mouse and rat) interspecies sequence conservation of class IV ADH (88-89% identity) thus falls halfway between that of the highly conserved class III ADH (93-94% identity) and the lesser conserved class I ADH (82-85% identity). If a higher degree of interspecies sequence identity is indicative of a more conserved enzymatic function, class IV ADH is more likely than class I ADH to play a role common to both humans and rodents.

The mouse class IV ADH cDNA was used as a hybridization probe to determine if its expression pattern correlated with that of the mouse Adh-3 gene encoding the ADH-C isozyme. In situ hybridization studies indicated that class IV ADH mRNA was quite abundant in the mucosal layer of mouse stomach tissue, the major site of ADH-C enzyme activity (Holmes, 1978). Upon comparison of a wide variety of mouse tissues, expression of class IV ADH mRNA was observed to be highest in stomach but also present in ovary, eye, thymus, and skin. This is consistent with previous reports of the distribution of ADH-C enzyme activity, which indicated that the major site of activity is the stomach, with secondary sites in the ovary and uterus (Holmes, 1978), eye (Rout and Holmes, 1991), and epidermal layer of skin (Connor and Smit, 1987). ADH-C has also been purified from mouse stomach (Algar et al.,1983) and epidermis (Connor and Smit, 1987) and shown to have enzymatic properties similar to purified class IV ADH from rat (Boleda et al.,1993), such as a Kfor ethanol in the high mM range and a Kfor retinol in the low µM range. Thus, ADH-C encoded by the mouse Adh-3 gene can now be regarded as mouse class IV ADH.

We compared the expression of class IV ADH mRNA with that of class I and class III ADH mRNA in mouse tissues. Class III ADH mRNA was found ubiquitously with only minor variations in the level of expression. The ubiquitous expression of class III ADH correlates with its role as a glutathione-dependent formaldehyde dehydrogenase, which performs a housekeeping function needed by all cells, i.e. the removal of metabolically generated formaldehyde (Koivusalo et al.,1989). On the other hand, both class I and class IV ADH mRNAs displayed a large degree of differential expression with overlap in some tissues. The pattern for class I ADH mRNA was, however, different from that of class IV ADH mRNA, suggesting that their genes are regulated uniquely. Class I ADH was expressed primarily in the liver, small intestine, eye, kidney, ovary, and uterus, whereas class IV ADH was expressed primarily in the stomach, ovary, eye, thymus, and skin. Thus, class I and class IV ADH expression significantly overlapped in the ovary and eye but not other tissues. The large amount of class I ADH expression in the liver correlates with its function as an ethanol dehydrogenase needed to remove ethanol ingested or ethanol produced endogenously by intestinal microbes (Krebs and Perkins, 1970). The high level of class IV ADH expression in the stomach may correlate with first-pass metabolism of ingested ethanol as reported in rats (Lim et al.,1993), but this conclusion has been challenged by studies indicating that the liver rather than the stomach accounts most first-pass ethanol metabolism (Derr and Goon, 1994; Levitt et al.,1994). The other sites of class I ADH and class IV ADH expression are clearly not major sites of ethanol metabolism (Boleda et al.,1989), suggesting that these enzymes are providing another function for those tissues.

The tissues we observed to express class I or class IV ADH mRNA (liver, gut, skin, kidney, reproductive organs) possess large numbers of epithelial cells, which are known to be capable of oxidizing retinol to form retinoic acid needed to regulate epithelial cell differentiation (Blaner and Olson, 1994). In the mouse, this has been previously studied in the epithelial layer of the skin (epidermis), which has been shown to oxidize retinol to retinoic acid in a two-step reaction requiring ADH-C enzyme activity (now defined as class IV ADH) for oxidation of retinol to retinal, as well as an unspecified aldehyde dehydrogenase for oxidation of retinal to retinoic acid (Connor and Smit, 1987). In our studies, in situ hybridization analysis of mouse stomach tissue has now clearly shown that class IV ADH mRNA is restricted to the mucosal epithelium, consistent with a role for the enzyme as a retinol dehydrogenase. Tissues found not to express class I and class IV ADH mRNA include brain, heart, and spleen, which do not have major populations of epithelial cells. Thus, the tissue distributions of class I and class IV ADH are consistent with these enzymes functioning in retinoic acid synthesis for epithelial cell differentiation. The relative importance of class I and class IV ADH in the oxidation of retinol to form retinoic acid in vivo remains to be determined. The cloning of the mouse class IV ADH gene described here opens up the use of mouse molecular genetics to analyze the role of this class of ADH in retinoid metabolism.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AA07261 (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/EMBL Data Bank with accession number(s) U20257.

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

The abbreviations used are: ADH, alcohol dehydrogenase; Adh, mouse gene encoding ADH; ADH, human gene encoding ADH; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank M. Felder for the mouse class I ADH cDNA, H. Jörnvall for the cDNAs for human class I ADH ( ADH3) and human class II ADH, D. Goldman for the human class III ADH cDNA, and J. Knight for oligonucleotide synthesis.


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