From the
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.
The existence of multiple classes of mammalian alcohol
dehydrogenase (E.C.1.1.1.1) (ADH)
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
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
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
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
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 [
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)
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.
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.
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.
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
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
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
The mouse ADH gene family was early defined as consisting of
three genes Adh-1, Adh-2, and Adh-3 encoding
the ADH-A
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
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
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
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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).
(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.
, 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.
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.
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.
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.
-
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.
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.
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.
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).
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.
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 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.
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).
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.
, 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.
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.
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 K
for ethanol in the high mM range and a
K
for 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.
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.
/EMBL Data Bank with accession number(s) U20257.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.