Báo cáo Y học: Organization of six functional mouse alcohol dehydrogenase genes on two overlapping bacterial arti®cial chromosomes pdf

Organization of six functional mouse alcohol dehydrogenase
genes on two overlapping bacterial arti®cial chromosomes
Gabor Szalai
1
, Gregg Duester
2
, Robert Friedman
1
, Honggui Jia
3
, ShaoPing Lin
3
, Bruce A. Roe
3
2
and Michael R. Felder
1
1
Department of Biological Sciences, University of South Carolina, Columbia, USA;
2
Gene Regulation Program, The Burnham
Institute, La Jolla, CA, USA;
3
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
Mammalian alcohol dehydrogenases (ADH)
3
form a com-
plex enzyme system based on amino-acid sequence, func-
tional p roperties, and gene expression pattern. At least four
mouse Adh genes are known to encode dierent enzyme
classes that share less than 60% amino-acid seq uence iden-

tity. Two ADH-containing and overlapping C57BL/6
bacterial arti®cial chromosome clones, RP23-393J8 and
-463H24, were identi®ed in a library screen, physically
mapped, and sequ enced. The gene order in the complex and
two new mouse genes, Adh5a and Adh5b, and a pseudogene,
Adh5ps, were obtained from the physical map and sequence.
The mouse genes are all in the same transcriptional orien-
tation in the order Adh4-Adh1-Adh5a-Adh5b-Adh5ps-Adh2-
Adh3. A phylogenetic tree analysis shows that adjacent genes
are most closely related suggesting a series of duplication
events resulted in the gene complex. Although mouse and
human ADH gene clusters contain at least one gene for
ADH classes I±V, the human cluster contains 3 class I genes
while the mouse cluster has two class V genes plus a class V
pseudogene.
Keywords: alcohol dehydrogenase; mouse; gene complex.
The alcohol dehydrogenases (ADHs; EC 1.1.1.1) are zinc-
containing, dimeric enzymes fo und in the cytosolic fraction
of the cell that are capable of reversible oxidation of a
spectrum of primary and secondary alcohols to the corre-
sponding aldehydes and ketones. Mammalian ADHs
currently known a re grouped into six distinct classes [1±3]
with members of different classes sharing less than 70%
amino-acid sequence identity within a species. Humans
possess classes I, II, III, IV and V [2] whereas the mouse is
known to have expressed genes encoding ADHs of class I ,
II, III and IV [4,5]. Humans have three class I genes [6]
encoding proteins with greater than 90% positional identity
whereas the mouse has a single class I g ene [7,8]. A human
class V ADH with approximately 60% positional identity a t

the amino-acid level with the other human classes has been
revealed from cDNA encoded sequence but not yet
associated with an enzyme [9]. Deermouse [10] and rat [11]
ADH c DNAs have been isolated that would e ncode
proteins most closely related to this human class V with
the deermouse ADH cDNA encoding a protein with 67%
positional identity to this class.
The ADH family of enzymes perform important meta-
bolic functions. In the mouse, class III functions as a
glutathione-dependent formaldehyde dehydrogenase
4
[12]
and is involved in S-nitrosoglutathione metabolism [13] and
retinol metabolism (G. Duester, unpublished results). Class
IV has a n important role in retinol metabolism leading to
retinoic acid p roduction. Adh4 is expressed during embry-
ogenesis [14,15] and Adh4 null mouse mutants have reduced
fetal survival during vitamin A de®ciency [16]. This obser-
vation coupled with reduced conversion of retinol to retinoic
acid in tissues of Adh4 mutant mice [12] suggests a role for
class IV enzyme i n retinoid signaling during embryogenesis.
Expressed at high levels in liver, the role of class I in ethanol
metabolism has been con®rmed by natural [17] and
engineered [12] enzyme-de®cient animals. Adh1 null mutant
mice also demonstrate a signi®cant decrease in metabolism
of retinol to retinoic acid [12]. The in vivo physiological roles
of class I, III, and IV ADHs in the mouse are being unveiled
with the development of targeted d isruptions for e ach gene.
The mouse Adh1 and Adh4 genes are lin ked [18] on
chromosome 3 at 71.2 cM (Mouse Genome Database), and

this complex is a candidate region for a quantitative trait
locus (Alcp3) for alcohol preference [19].
The genes encoding different classes of mammalian ADH
have different tissue expression patterns suggesting that
Correspondence to M. R. Felder, Department of Biological Sciences,
University of South Carolina, Columbia, SC 29208, USA.
Fax: + 803 777 4002, Tel.: + 803 777 5135,
E-mail:
Abbreviations: BAC, bacterial arti®cial chromosome; NJ,
neighbor-joining; PC, Poisson corrected; CHEF
I
, contour-clamped
homogenous electric ®eld.
De®nitions: The nomenclature for the alcohol dehydrogenase gene and
enzyme family being followed is that of Duester, G ., Farre
Â
s, J., Felder,
M.R., Holmes, R.S., Ho
È
o
È
g, J O., Pare
Â
s, X., Plapp, B.V., Yin, S J. &
Jo
È
rnvall, H. (1999) Biochem. Pharmacol. 58, 389±395. The previously
used human ADH1, ADH2, ADH3 genes encoding class I enzymes are
renamed ADH1A, ADH1B,andADH1C indicating their greater than
90% similarity. The enzyme class names and g en e names now corre-

spond. The class II human and mouse enzymes are encoded by ADH2
and Adh2, r espectively; class III enzymes by human ADH3 (old
ADH5)andmouseAdh3 (old Ad h5); class IV enzymes by human
ADH4 (old ADH7)andmouseAdh4 (old Adh3); class V enzyme by
human ADH5 (old ADH6). The deermouse c lass VI ADH is closest to
human ADH5 protein in amino -acid sequence, and based on phy-
logeny and genome organization of t he mouse presented here is now
referred to as class V with a gene symbol of Adh5. New mouse genes
reported here are named Adh5a, Adh5b,andAdh5ps.
(Received 2 0 September 2001, accepted 30 October 2001)
Eur. J. Biochem. 269, 224±232 (2002) Ó FEBS 2002
complex regulatory mechanisms control this gene family.
The mouse Adh1, Adh2, Adh3,andAdh4 genes h ave some
overlap in tissue expression pattern, but levels of expression
in different adult tissues and hormonal responsiveness of
different genes in the complex vary widely [4,20±23].
cis-Acting sequences controlling the expression pattern
of the Adh genes in mouse are not fully understood. A
minimal promoter directing expression in transfected
hepatoma cells has been de®ned for Adh1 [24,25]. How-
ever, as much as 10 kb of sequence upstream of an Adh1
minigene does not direct expression in liver of transgenic
mice [26] although kidney a nd adrenal e xpression is
promoted. An entire Adh1 gene containing 7 kb of 5¢
and21kbof3¢ ¯anking sequence in transgenic mice
expresses properly in most t issues except live r and intestine
[27]. More distal sequences must control expression of
Adh1 in these tissues, and this has stimulated an effort to
obtain mouse Adh1 bacterial arti®cial chromosome (BAC)
clones to identify these cis-acting regions. Analysis of these

BAC clones has provided a more detailed knowledge of
the genomic organization of the mouse Adh gene complex.
This is an important prerequisite to understanding the
regulatory strategy of this gene family.
In this report, we show that the previously known four
mouse Adh genes a re clustered within 250 kb of the mouse
genome. Furthermore, two additional Adh genes and one
pseudogene are found within this DNA region, and the
order of these genes on two overlapping BAC clones has
been determined. All genes are transcribed in the same
orientation, and s equence comparisons and location within
the complex suggests that the two newly identi®ed Adh
genes are most closely related to the human ADH5 gene.
MATERIALS AND METHODS
Materials
Chemicals a nd reagents were obtained f rom Sigma Chem-
ical Co., Fisher Chemicals, J. T. Baker (Phillipsburg, NJ,
USA), and New England Biolabs unless otherwise indicated.
RPCI-23 segment 2 BAC library was purchased from
BACPAC Resources of Roswell Park Memorial Institute
(Buffalo, NY, USA). Individual clones w ere also purchased
from this resource.
CDNA hybridization probes for mouse genes
The ADH cDNA probes used in this study were inserts from
pADHn1 f ordeermouse ADH5 andpCK2 for m ouse ADH1
[10]. The ADH 4 and ADH3 cDNAs have been previously
described [4]. Mouse ADH2 cDNA was prepared using
methods previously described [4] by performing RT-PCR
on mouse liver RNA with primers overlapping the start
and stop codons of the published mouse ADH2 cDNA

sequence [5]. The ADH2 cDNA was con®rmed b y nucleotide
sequence analysis. Isolated cDNA inserts were used to
prepare all probes for labeling by random priming [28].
Screening the BAC library for mouse
Adh1
-containing
clones
Five ®lters containing a total of over 90 000 clones were
screened by hybridization with
32
P-labeled mouse ADH1
full-length insert cDNA in pCK2. Probe was used in the
hybridization solution at 2.4 ´ 10
6
c.p.m.ámL
)1
. Filters were
prehybridized for ³ 2 h at 65 °Cin6´ NaCl/P
i
/EDTA
(1 ´  0.15
M
NaCl, 0.01
M
sodium phosphate, 1 m
M
EDTA, pH 7 .4)/6 ´ Denhardt's solution, 0.5% SDS, and
50 lgámL
)1
denatured salmon sperm DNA. After prehy-

bridization, the solution was discarded a nd replaced with an
identical hybridization solution except without Denhardt's
containing the radioactive probe. After overnight hybrid-
ization the ®lters were washed twice with 6 ´ NaCl/ P
i
/
EDTA/0.5% SDS at room temperature for 15 min each,
twice in 1 ´ NaCl/P
i
/EDTA/0.5% SDS at 37) 42 °Cfor
15 min each, and once in 0 .1 ´ SSPE
5
/0.5% SDS at 65 °C.
The ®lters were blotted of exc ess moisture, wrapped in
Saran wrap, and exposed to Kodak XAR ®lm at )80 °Cfor
several hours to a day depending upon intensity of signal
and level of background necessary to reveal outline of the
®elds on the ®lter.
BAC DNA isolation and restriction enzyme
digest analysis
A s ingle isolated bacterial colony containing a BAC clone of
interest was obtained from a freshly streaked plate and used
to inoculate 300 mL of Luria±Bertani media containing
20 lgámL
)1
of chloramphenicol. After overnight gr owth at
37 °C, cells were harvested by centrifugation. Isolation of
BAC DNA was by a rapid alkaline lysis method using no
organic extractions following the protocol provided by
BACPAC Resources. P ipetting of the ®nal BAC DNA

preparation was carried out with large ori®ce tips from USA
Scienti®c.
The BAC DNA was digested with various restriction
endonucleases and the fragments were resolved o n 1 %
agarose gels in 0.5 ´ Tris/borate/EDTA (1 ´ Tris/borate/
EDTA  0.089
M
Tris, 0.089
M
boric acid, 0 .002
M
EDTA)
using a contour-clamped homogenous electric ®eld (CMHF)
apparatus. Electrophoresis was conducted at 6 Vácm
)1
at
15 °C for 16 hrwith the switch time ramped from 1 to 12 s.
After completion of electrophoresis, gels were stained in
0.5 lgámL
)1
ethidium bromide in 0.5 ´ Tris/borate/EDTA
and washed for several hours in distilled water. After
photographing, the gels were treated with 0.25
M
HCl for
15 min, rinsed in distilled water, denatured with 0.5
M
NaOH/1. 0
M
NaCl, and neutralized with 0.5

M
Tris/HCl/
1.5
M
NaCl (pH 7.4). The gels w ere inverted and DNA was
transferred overnight onto Hybond Nytran membranes in
10 ´ NaCl/P
i
/EDTA by capillary movement. After trans-
fer, membranes were baked for 2 h at 80 °C.
BAC isolation, shotgun sequencing,
and custom-synthetic primer directed closure
The detailed procedures for cloned, large insert genomic
DNA isolation, random shot-gun cloning, ¯uorescent-
based DNA sequencing a nd subsequent analysis were used
as described previously [29±31]. Brie¯y, BAC DNA was
isolated free from host genomic DNA via a cleared lysate-
acetate p recipitation-based protocol [ 29]. Subsequently,
50 lg portions of puri®ed BAC DNA were randomly
sheared and made blunt ended [30,31]. After kinase
treatment and gel puri®cation, fragments in the 1- to 3-
kb range were ligated into SmaI-cut, bacterial alkaline
Ó FEBS 2002 Mouse alcohol dehydrogenase gene complex (Eur. J. Biochem. 269) 225
phosphatase-treated pUC18 (Pharmacia) and Escherichia
coli, strain XL1BlueMRF¢ (Stratagene), was transformed
by electroporation. A random library of approximately
1200 c olonies were picked fr om each transformation,
grown in t erri®c broth [32] s upplemented with 100 lgámL
7
)1

of ampicillin for 14 h at 37 °C with shaking at 250 r.p.m.,
and the sequencing templates were isolated by a cleared
lysate-based protocol [30].
Sequencing reactions were performed as previously
described [31] using Taq DNA polymerase, the Perkin
Elmer Cetus ¯uorescently labeled Big Dye Taq termina-
tors. The reactions were incubated for 60 cycles in a
PerkinElmer Cetus DNA Thermocycler 9600 and after
removal of unincorporated dye terminators by ®ltration
through Sephadex G-50, the ¯uorescently labeled nested
fragment sets were resolved by electrophoresis on an ABI
3700 Capillary DNA Sequencer. After base calling with
the ABI analysis software, the analyzed data was trans-
ferred to a Sun workstation cluster, and assembled using
the
PHRED
and
PHRAP
programs [33,34]. Overlapping
sequences and contigs were analyzed using
CONSED
[35].
Gap closure and proofreading was performed using either
custom primer walking or using PCR ampli®cation of the
region corresponding to the gap in the sequence followed
by subcloning into pUC18 and cycle sequencing with the
universal pUC-primers via Taq terminator chemistry. In
some instances, additional synthetic custom primers were
necessary to obtain at least threefold coverage for each
base.

Draft and ®nished BAC s equences were analyzed on Sun
workstations with the programs contained within the
GCG
package [36] as well as the
BLAST
[37],
BEAUTY
[38] and
BLOCKS
[39] programs. The sequences of BAC clones
RPCI23-463H24 and RPCI23-463H24 have been deposited
into GenBank and give n accession numbers AC079823 and
AC079682, respectively,
Computer analysis of DNA sequence
CLUSTAL W
(version 1.81) was used to align the amino-acid
sequences [40] for phylogenetic treatments. Any site at
which the alignment postulated a gap in any sequence was
removed from the data set for all pairwise comparisons so
that a s imilar data set was used for each comparison.
Phylogenies were constructed u sing the following methods:
(a) the neighbor-joining (NJ) method [41] based on the
Poisson corrected (PC) amino-acid distance [42] and (b) th e
NJ method based on the gamma-corrected (a  2.0) amino-
acid distance [42].
Both methods produced es sentially identical results;
therefore, only the results of the NJ tree based on PC are
presented here. The NJ method is re liable a t reconstructing
phylogenies even when evolutionary rates differ among
branches of a phylogenetic tree [42]. The reliability of

clustering patterns in NJ trees was tested by bootstrapping
[43], which involved clustering of trees based on pseudos-
amples of sites sampled in the data set (with rep lacement).
Five-hundred bootstrap pseudosamples were used.
GenBank and EST database searches were performed
using
BLAST
programs [44] (available at http://
www.ncbi.nlm.nih.gov/BLAST website). Sequence analysis
and manipulation
8
were carried out using the
GCG
software
package.
RESULTS
Identi®cation of
Adh1
-containing BAC clones
DNA was i solated from all BAC clones hybridizing to the
ADH1 cDNA probe. Puri®ed DNA was digested with
EcoRI and analyzed by Southern blotting and hybridization
to ADH1 cDNA. Finally, 13 positive clones were identi® ed
and among these were found all EcoRI restriction fragments
detectable in genomic DNA with an ADH1 cDNA probe.
The Adh1-containing EcoRI fragments in C57BL/6 DNA
are 6.8-, 3.8-, 2.5- and 1.1 kb. In addition, more faintly
hybridizing 2.0- and 2.3 kb EcoRI fragments present in the
genome were identi®ed among the BAC clones. Of these 13
clones, tw o were chosen for e xtensive analysis because they

overlap within the Adh1 gene. BAC 463H24 contains the
entire Adh1 gene while 393J8 contains only t he most 3¢ end,
the 3.8- a nd 1.1-kb Eco RI [7] h ybridizing fragmen ts
(Fig. 2 A). T herefore, 393J8 extends farther downstream
than 463H24 relative to Adh1 orientation. 393J8, in contrast
to 463H24, includes the 2.0 k b non-Adh1 [7] hybridizing
fragment as does another BAC clone 461A12. As 461A12
contains no Adh1 sequence, this con®rms that the cross-
hybridizing 2.0-kb EcoRI fragment is downstream of the
Adh1 g ene. T wo additional B AC clones, 388D7 a nd
434F16, were identi®ed that contained the 2.3-kb EcoRI
Adh1-crosshybridizing fragment [7], but n o Adh1 sequence,
as the only species to hybridize to ADH1 cDNA.
Adh
genes on 463H24
BAC 463H24 restriction endonuclease digests were pro-
duced, and the resulting fragments were resolved by
9
CHEF
analysis. A s estimated by the sizes of the r estriction
fragments, 463H24 contains an insert of over 165 kb
(Fig. 1 A). The blots prepared from CHEF separation of
restriction fragments were hybridized to several mouse ADH
cDNA clones. Only ADH1 and ADH4 cDNAs hybridize to
restriction fragments in 463H24 (Fig. 1A). The results of
several restriction digests revealed that the positions of Adh1
and Adh4 are resolved on this clone. For example, Adh1 is
locatedonthe60-kbRsrII fragment while Adh4 is found on
the 100-kb fragment (Fig. 1 A, N/R). Either because of
context or the sequence recognized by RsrII [CGG

(A/T)CCG] t here was never complete digestion a t t his s ite
as revealed by the remaining 160-kb undigested insert
present in a nonstoichiometric amount. As expected, this
undigested fragment hybridizes to both ADH1 and ADH4
cDNAs. Adh4 is found on the 135-kb Eag I fragment while
Adh1 is found on the 135- and 25-kb fragments (Fig. 1A,
N/E). Within t he Adh1 gene a single EagI site is f ound near
the 3¢ end of exon 6 accounting for the two hybridizing
fragments. This also localizes the 3 ¢ end of Adh1 approxi-
mately 21 kb from one end of the clone. Adh1 and Adh4 are
on the large SalIfragmentsuggestingthissiteisnearerthe
opposite end of the clone th an EagI. As EagI, Rsr II, and SalI
all cut the clone once and EagIandSalI are nearer the ends
of the BAC, other double digests were performed to
construct a more detailed map o f 463H24 (Fig. 2A).
Two PmeI sites are present (Fig. 1A, N/P) and both Adh1
and Adh4 are found on the large 90-kb fragment. The
location of the additional PmeI site was not determined
from the digests performed. The RsrII/PmeI double digest
226 G. Szalai et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 1 A, N/R/P) delineates t he position of Adh1 and Adh4
(Fig. 2 A) on th e BAC.
Adh
genes on BAC 393J8
The p resence of the 3¢ end of Adh1 near one end of 393J8
provides a useful approach to determine the location of each
restriction enzyme site located nearest this end by probing
with ADH1 cDNA. Sizes of fragments produced by
digestion of 393J8 with various restriction enzymes were
determined by CHEF (Fig. 1B). The results of probing a

blot of these CHEF separated restriction endonuclease
fragments o f 393J8 with four ADH cDNA probes are
shown in Fig. 1B. The single SalI site is located approxi-
mately 30 kb from the Adh1-tagged end of the clone
con®rmed by the strong hybridization signal of the 30-kb
fragment when probed with ADH1 cDNA (Fig. 1B, N/S).
The faint signal observed from t he large fragment must be
due to cross-hybridization with other ADH genes. The only
RsrII site is located a bout 100 kb from the Adh1 end of the
clone (Fig. 1B, N/R). The single SgrAI site is locate d about
135 k b from the Adh1 end (Fig. 1B, N/Sg), and the EagIsite
Fig. 1. CHEF analysis of restriction enzyme
digests of BAC clone. The r estriction enzymes
used to prepare BAC digests loaded into each
lane ar e indicated at the top. The enzymes
used were N, NotI; B, BssHII; E, EagI; P,
PmeIR,RsrII; S, SalI; and Sg, SgrAI. The top
panel in both (A) an d (B) shows the fragments
detected by ethidium bromide staining.
(A) Identi®cation of ADH-containing restric-
tion fragments in BAC 463H24. Blots probed
with ADH1 or ADH4 cDNAs are indicated.
(B) Identi®cation of ADH-containing restric-
tion fragments in BAC 393J8. Autoradiogram
resulting from p robing identically prepared
blots with ADH4, ADH2, ADH3 or deer-
mouse ADH5 (mammalian class V) cDNAs
are shown.
Ó FEBS 2002 Mouse alcohol dehydrogenase gene complex (Eur. J. Biochem. 269) 227
located nearest Adh1 is over 60 kb (Fig. 1B, N/E) from the

end (the largest EagI f ra gmen t). A DH1 c DNA a ls o
hybridizes to a 75-kb PmeI fragment (Fig. 1B, N/P), but
this band is actually composed of two nearly identical
fragments. The smaller 12-kb PmeIfragmentisfromthe
middle of this c lone. The observation t hat SalI, RsrII, and
SgrAI digests not only have a strong signal from the
fragment containing the 3¢ end of Adh1 but also a weak
hybridization signal from the other fragment in t he digest
suggests other Adh genes that weakly cross-hybridize to
Adh1 are found on this clone. In EagI digests, ADH1 cDNA
hybridizes to the 5-kb f ragment w hich contains the portion
of the Adh1 gene located 5¢ of this site to the end of the
clone, t o t he 60-kb fragment a lready mentioned, and t o the
slightly smaller, weakly hybridizing fragment, which must
contain other Adh genes.
The ability to precisely locate the position of several
restriction enzyme sites relative to the Adh1 end of the clone
enabled a more detailed map to be constructed by analyzing
additional double digests. The resulting blots were also
hybridized with ADH2, ADH3 and deermouse ADH5
(Fig. 1B) cDNAs. ADH1 and deermouse ADH5 cDNAs
both gave strong and weak signals suggesting that these
probes cross-hybridize to other ADHs. For instance, ADH1
probe gives strong and w eak signals with the 30-kb and 140-
kb NotI/ SalI fragments, respectively. However, deermouse
ADH5 probe gives approximately equal signals in both
fragments. The d eermouse ADH5 probe does c ross-hybrid-
ize to Adh1 sequences, but the strong signal suggests there
may be other cross-hybridizing sequences in the small
fragment. Both mouse ADH2 and ADH3 cDNA probes

hybridize only to the large fragment. Resolution of the
positions of the genes on the BAC was obtained from single
and double digests. The Adh2 gene is found on the large
NotI/S grAI fragment whereas Adh3 is on the small
fragment (Fig. 1 B, N/Sg).
A map (Fig. 2A) of the two overlapping BACs was
generated based upon estimated fragment sizes generated in
single and double digests, hybridization of the different
probes, and the ability to determine precisely the location of
restriction sites relative to the Adh1 end of the BAC. The
positions of Adh4, Adh2,andAdh3 are arbitrarily placed in
the middle of the ¯anking restriction sites. Adh1 is anchored
by the presence of the EagI site in the gene. T he deermouse
ADH5 cross-hybridizing sequence is positioned encompass-
ing restriction sites thought to reside in the hybridizing
sequence. However, additional Adh sequences may reside
between the position of Adh5 and Adh1 base d upon weak
cross-hybridization signals observed with both cDNAs as
probe and the fact that the EagI i s p laced w ithin Adh5, but
could b e betw een relate d ge nes. However, the other nearby
EagI site may be able to resolve t wo Adh5 cross-hybridizing
species. The precise location of the mouse ortholog of
deermouse Adh5 cannot be determined from mapping alone
but was determined from sequence data (see below).
Transcriptional orientation of
Adh4
,
Adh1
,
Adh2

and
Adh3
genes
The draft sequence of 463H24 (AC 079682.14) consists of
four unordered pieces and is s uggested to be approximately
182084 bp in length.
BLAST
analysis reveals that the Adh4
gene is on the 32 589±106 087 bp contig in that transcrip-
tional orientation. The contig also contains three SalIsites,
all located within 7-kb of each other, upstream from t he 5¢
end of the gene. These sites are too close to resolve on
CHEF gels and must represent the single SalI site shown on
the map (Fig. 2A). Based on the sequence, the PmeIsite
on the m ap is loca ted upstream o f Adh4. T he sequence also
identi®es the location of the add itional PmeI site which is
near the 5¢ end of Adh4.TheRsrII site on the map is on a
different contig (1 82 084±106 188) and is upstream of the 5¢
end of the Adh1 gene located on this contig. The single Eag I
site located near the 3¢ end of exon 6 in the Adh1 gene
localizes this gene on 463H24 and the draft sequence
con®rms t he 5¢ to 3¢ orientation o f t his g ene. As one end of
393J8 begins in the 3¢ end of Adh1, this con®rms that Adh4
and Adh1 are transcribed in the same orientation .
BLAST
analysis of 393J8 complete sequence (AC
079832.16) with ADH1, ADH2, and ADH3 cDNAs also
revealed that these genes have the same transcriptional
orientation. The o verall length of the sequence is 194 850 bp.
Identi®cation of

Adh5a
,
Adh5b
,
and
Adh5ps
between
Adh1
and
Adh2
Sequences in 393J8 hybridizing to the deermouse ADH5
cDNA maps between Adh1 and Adh2 but closer to Adh2
(Fig. 2 A). It is possible that other sequences in this region
may cross hybridize to ADH1 or deermouse ADH5 cDNA
probes.
BLASTN
analysis of the region revealed that the
exon 2±exon 6 deermouse ADH5-like sequence is found at
position 97 694±110 93 4 in the BAC. Exon 6 sequence
containsa9-bpdeletionfollowedbya7-bpstretchandthen
a single b p deletion
10
when compared to the deermouse
ADH5 or human ADH5 cDNAs. After 22 codons in the
altered reading frame a stop codon is encountered strongly
suggesting this is a nonfunctioning pseudogene, Adh5ps.
Before the deletion, the encoded sequence has a p ositional
identity of 24/26 amino-acid residues with human ADH5
and there is an identity of 29/57 after the deletion by
returning to the proper reading frame. No coding regions

beyond exon 6 were found by
BLASTN
searches with other
ADH cDNAs. However, a perfect match w as found
between nucleotide positions 7±95 of an adult male liver
cDNA clone (GI: 12836366) and nucleotide position
89 989±90 077 in the BAC. This cDNA has a start codon
at position 7 6 and encodes s ix amino acids with 3/6 identity
with human ADH5 exon 1 encoded sequence. Also, this
cDNA at position 511±2407 has a 99% identity with
position 101 875±103 771 in 393J8 that includes potential
exons 4 and 5. Because so much of the cDNA extends
beyond these potential exons, it is probable t hat this cDNA
represents partially spliced mRNA. Nucleotides 2513±2795
of this same cDNA have a 99% sequence identity with
position 103 877±104 159 in the BAC.
TBLASTN
searches in
the 75 kb of sequence b etween Adh1 and Adh5ps for
sequences homologous to deermouse ADH5 or human
ADH5 protein sequence revealed two probable complete
genes each with nine exons. These genes are de®ned as
Adh5a and Adh5b based on location and phylogeny (see
below).
TBLASTN
and
BLASTN
analyses de®ne the location of
Adh5a as encompassing 29 317±47 164 in the 5¢ to 3¢
orientation. Adh5b is located from 57 150 to 74 874. A

TBLASTN
search of the GenBank database failed to locate the
®rst and last exons of the Adh5 gene.
228 G. Szalai et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Adh5a and Adh5b have available complete cDNA or EST
sequence represented in the database, respectively. T his h as
allowed the last exon of Adh5b to be de®ned by an EST
expressed in mouse skin (GI: 4404412). A full-length cDNA
(GI: 12840922) obtained from 10-day-old male pancreas
represents the expressed product of Adh5a and has been
used to determine g ene structure. The complete gene
structures of Adh5a and Adh5b are fully annotated in
GenBank (GI: 15383846). Both g enes consist of nine exons
and e ncode 374 amino acids excluding the initiation
methionine which is the same as the mouse Adh4 and
Adh1 protein products. Exon 1 of Adh5b is tentatively
identi®ed as a sequence encoding the i ntiation met and ®ve
additional amino acids before encountering a gt splice site.
The nucleotide sequence of the Adh2 gene is fully
annotated in GenBank (GI: 1538346). This gene has nine
exons a nd encodes a p olypeptide of 376 am ino a cids, again
exclusive of the inititation methionine. All three genes,
Adh2, Adh5a and Adh5b have consensus gt and ag
nucleotides ¯anking the intron sequences. The translation
initiation codons for Adh5a and Adh2 have 8/10 and 6 /10
nucleotide identity with the consensus initiator sequence,
respectively. The Adh5b initiator shares 5/10 identity with
the consensus sequence and does not have, in contrast to
Adh2 and Adh5a, the important G residue after the ATG or
the A/G three nucleotides upstream from t he ATG.

Although t he ®rst intron in the Adh5b gene begins with gt
the match with the consensus is poor only having the AG
at the end of the exon before the gt in the intron. The 5¢
end o f the ®rst intron matches the consensus much more in
Adh5a and Adh2.
The locations of the intron interruptions in the coding
sequence of the mouse genes in the cluster are shown in
Table 1 . The mo lecular map of the mouse Adh complex is
shown in Fig. 2B along with a comparison with the human
ADH complex. The s izes of introns in all genes including the
pseudogene are shown in Fig. 2C.
Phylogeny of mouse and human
Adh
genes
In the phylogen y of the mouse and human genes, there were
®ve major clusters (Fig. 3), each separated by a bootstrap
Fig. 2. Structural org anization of the genes in the mouse Adh complex. (A) Restriction map o f the area encompassed by the two BACs indicating
positions of the mapped Adh genes. Restriction e nzyme symbols are the same as in Fig. 1. Genes known to lie between two restriction sites a re
arbitrarily placed i n the middle. The location of the two BAC clones a re shown b elow t he physical map. (B) Molecular map of the mouse Adh
complex (top) comp ared to th e human ADH complex (bottom, based on the sequence in NT 022863). Arrows indicate transcriptional orientation.
The draft sequence of 463H24 (GI 15042854) consists of four unordered contigs, b ut molecular distances and orientation wer e derived based on
BAC end sequence from the d atab ase, restriction sit es located w ithin contigs compared to the physical map , and paire d
BLASTN
analysis of the BAC
against ADH4 and ADH1 cDNA. The order of contigs in 463H24 relative to the 5¢ to 3¢ tra nscriptional orientation of the Adh4 and Adh1 genes on
the BAC is (32489/5054)-(32589/106087; Adh4)-(182084/106188; Adh1). One contig (1/4953) cannot be placed on the map, but is not at either e nd of
the BAC. ( C) Organization of intron s an d exon s in the mouse g enes. Introns are lines and exons are rectangles. The genes are given in the ir order
within t he complex.
Ó FEBS 2002 Mouse alcohol dehydrogenase gene complex (Eur. J. Biochem. 269) 229
value of 87 or greater: ( a) h ADH1A, hADH1B, hADH1C,

and mADH1 cluster signi®cantly with a bootstrap value of
100; (b) hADH4 and mADH4 cluster (100); (c) hADH5,
mADH5A and mADH5B cluster (87); (d) hADH2 and
mADH2 cluster (100); and (e) hADH3 and mADH3 cluster
(100). The proteins encoded by the newly identi®ed mouse
Adh5a and Adh5b genes are most closely related to the
human ADH5 andtoeachother,whichisthebasisfortheir
nomenclature.
DISCUSSION
In this report, we have isolated a series of mouse BAC
clones using a mouse ADH1 cDNA as a probe. As two of
these BACs overlap at the Adh1 gene, they could be
orientated relative to the 5¢ to 3¢ orientation of this gene.
Using cDNAs for three other mouse ADH genes and a
deermouse gene to probe restriction digests of these clones,
we were able to determine the order of the four mouse genes
and the sequences cross-hybridizing to the deermouse
ADH5 cDNA probe. Sequence of the two overlapping
BAC clones combined with the physical map allowed u s t o
determine t hat the previously known mouse genes are found
in the order Adh4-Adh1-Adh2-Adh3 in less than 250 kb of
DNA, a nd all genes are in t he same transcriptional
orientation being 5¢ to 3¢ from the Adh4 to Adh1 orientation.
This is the same order and transcriptional orientation as
found for the human genes except the human has three
ADH1 genes (ADH1C, ADH1B and ADH1A) instead of the
single mouse Adh1 ortholog. The mouse sequence cross-
hybridizing to deermouse ADH5 cDNA was mapped
physically to a region b etween Adh1 and Adh2 correspond-
ing to the location of the human ADH5 gene. The amino-

acid similarity of 67% between the deermouse encoded
protein and the human ADH5 protein [10] previously
provided the rationale for suggesting that the deermouse
sequence represents a separate class VI. A similar s equence
has been identi®ed in the rat [11]. However, the identity of
67% is an intermediate value between members of d ifferent
classes and members of the same class between species. The
orthologous physical location and this intermediate value
suggests this represents sequence most related to human
ADH5. Pairwise
BLASTN
comparisons between the draft
sequence of the BAC in this area and the deermouse and
human ADH5 cDNAs allowed the identi®cation of poten-
tial exons 2±6 in this physically mapped r egion. However,
exon 6 was found to contain deletions that altered the
reading frame leading to a termination codon. Although
available cDNA indicates this region is expressed with
spliced exons 1±3, the d eletion in exon 6 strongly suggests
that no functio nal protein is made. A partial sequence from
exon 6 of an unidenti®ed mouse Adh gene from a C 57BL/6
library contains an identical deleted sequence in the exon 6
region [46] (D. Dolney
11
, University of South Carolina,
Columbia, SC, USA, unpublished results). Because the
location of this sequence is similar r elative to other genes i n
human, it is de®ned as Adh5ps.
TBLASTN
pairwise searches between deermouse and

human ADH5 cDNA translation products and the BAC
sequence located between this mouse Adh5ps and Adh1
identi®ed two additional genes. The newly de®ned Adh5a
and Adh5b genes reside between Adh1 and Adh2 ind icating
an overall order of Adh4-Adh1 -Adh5a-Adh5b-Adh5ps-Adh2-
Adh3. The mouse Adh5a and Adh5b reside in the same
relative position as the human ADH5 gen e. The structures
of bo th genes a re very similar to the human ADH5 gene [9]
except an additional amino-acid residue is encoded by
exon 4 in the human sequence compared to the mouse. The
Table1. Amino-acidpositionsencodedbyeachexoninsixmouseAdh genes. Codons split between exons are shown at the end and beginning of
adjacent exons. Blanks indicate exon coding is the same as in Adh4.ThehumanADH2 exon co ding regions are shown as this gene is most closely
relatedtothemouseAdh2. Exons for hum an ADH2 are from [48], and human ADH5 organization is from [9,47]. Sources for organization of other
mouse genes are: Adh4 [45], Adh1 [7] and Adh3 [49].
Gene
Exon
123456789
Adh4 Met1±5 6±39 40±86 86±115 115±188 189±275 276±321 321±368 368±374
Adh1
Adh5a
Adh5b
Adh3 Met3±5
Adh2 115±192 193±279 280±323 323±369 369±376
ADH2 40±87 87±116 116±193 194±280 281±326 326±372 372±379
ADH5 40±87 87±116 116±188 189±275 276±321 321±368 368±375
hADH1C
hADH1B
hADH1
A
mADH1

hADH7
mADH7
hADH6
mADH6A
mADH6B
hADH4
mADH4
hADH5
mADH5
100
100
100
100
70
100
100
96
87
72
0.1
PC
Fig. 3. Phylogeny of mouse and human Adh genes constructed by the
neighbor-joining meth od. Numbers on the branch es are perce ntages of
500 bootstrap samples t hat support the branch.
230 G. Szalai et al. (Eur. J. Biochem. 269) Ó FEBS 2002
positions where intron sequence disrupts coding sequence in
Adh5a and Adh5b genes is identical to Adh4 [45] and Adh1
[7,46] However, Adh5a, Adh5b,andAdh2 are remarkably
similar to human ADH5 at the exon 8/exon 9 boundary. I n
all cases there is a potential stop codon just downstream o f

exon 8 that would produce a truncated protein if splicing
between exons 8 and 9 fails to oc cur. Recently, alternative
splicing of exons 8 and 9 has been reported for human
ADH5 [47]. All t he mouse genes except Adh4 [45] contain a
potential translational stop codon in frame just downstream
of exon 8, but it is unknown with what frequency a
transcription product lacking exon 9 is produced for any of
these genes. All genes in the complex are very similar in
location of intron positions within the coding r egion of t he
gene with the exception of the Adh2 gene that encodes four
additional amino acids in exon 5, but two less in exon 7.
This ADH2 protein contains 376 amino acids [5], but is not
as large as the human ortholog that contains 379 amino
acids [48]. An overview of the gene structure within the
complex suggests that genes in the middle of the complex are
larger than the ones at the ends. The Adh5ps even with only
six exons is the largest in the complex. The genes (Adh4,
Adh1, Adh5 a, Adh5b, Adh5ps)atthe5¢ end of the complex
relative to transcriptional orientation characteristically have
small introns between exons 4 a nd 5, but genes at t he 3¢ end
(Adh2 and Adh3) have a larger intron 4.
This report presents a detailed map of the mouse Adh
gene complex a nd ®nds that six genes in the same
transcriptional o rientation are found within 250 kb o f
DNA s equence. A pseudogene located in a transcribed
region is also detected in this locus. The ®rst gene in the
complex at the 5 ¢ end relative to transcriptional orientation,
Adh4, is expressed at high level in adult stomach, esophagus
and skin with lower levels produced in ovary, uterus,
seminal vesicle [4,23]. The next gene, Adh1, is expressed at

highest levels in liver, adrenal, and small intestine [4,21] with
somewhat smaller amounts being found in kidney and still
smaller amounts detectable in several tissues including
ovary, uterus, seminal vesicle. Expression of Adh2 occurs in
liver with lesser expression in kidney [5] although an
extensive expression pattern at t he RNA level has not been
established, while the Adh3 gene is widely expressed in
mouse tissues [4]. The expression pattern of Adh5a and
Adh5b is still to be de®ned. There is some order in the liver
expression pattern of the different genes as related to their
position on t he chromosome progressing from the 5¢ end of
the complex in which Adh4 expression is totally absent from
liver, to the middle and 3¢ end of the complex where Adh1,
Adh2,andAdh3 are h ighly expressed in liver. At this time, it
is unknown if individual regulatory elements between the
genes c ontrol e xpression o f each gene during development
and differentiation, or if a locus control region in combi-
nation with local elements control expression of the
complex. The knowledge of the organization of this locus
will be useful in addressing these questions in transgenic
mouse expression studies.
ACKNOWLEDGEMENTS
This work was supported by NIH grants AA 11823 ( M. R. F), AA
09731 (G. D), and HG 00313 (B. A. R.). The contribution of
R. Friedman in constructing the phylogenetic tree was supported
through NIH grant GM 43940 to A. L. Hughes. We are grateful to the
NIH Mouse BAC Sequencing Program for generating the seque nce of
RP23-393J8 and RP23-463H24 BAC clones.
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