Humans possess classes I, II, II, IV and V [2] whereas the mouse is known to have expressed genes encoding ADHs of class I, Correspondence to M.. The class I] human and mouse enzymes ar
Trang 1Organization of six functional mouse alcohol dehydrogenase
genes on two overlapping bacterial artificial chromosomes
Gabor Szalai', Gregg Duester”, Robert Friedman’, Honggui Jia*, ShaoPing Lin*, Bruce A Roe®
and Michael R Felder’
' Department of Biological Sciences, University of South Carolina, Columbia, USA; “Gene Regulation Program, The Burnham Institute, La Jolla, CA, USA; Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
Mammalian alcohol dehydrogenases (ADH) form a com-
plex enzyme system based on amino-acid sequence, func-
tional properties, and gene expression pattern At least four
mouse Adh genes are known to encode different enzyme
classes that share less than 60% amino-acid sequence iden-
tity Two ADH-containing and overlapping CS57BL/6
bacterial artificial chromosome clones, RP23-393J8 and
-463H24, were identified in a library screen, physically
mapped, and sequenced The gene order in the complex and
two new mouse genes, Adh5a and Adh5b, and a pseudogene,
AdhSps, were obtained from the physical map and sequence
The mouse genes are all in the same transcriptional orien- tation in the order Adh4-Adh1-AdhSa-Adh5b- AdhSps-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 found 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 are 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, II, IV and V [2] whereas the mouse is
known to have expressed genes encoding ADHs of class I,
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: felder@ mail.biol.sc.edu
Abbreviations: BAC, bacterial artificial chromosome; NJ,
neighbor-joining; PC, Poisson corrected; CHEF, contour-clamped
homogenous electric field
Definitions: The nomenclature for the alcohol dehydrogenase gene and
enzyme family being followed is that of Duester, G., Farreés, J., Felder,
M.R., Holmes, R.S., Hd6g, J.-O., Parés, X., Plapp, B.V., Yin, S.-J &
Jérnvall, H (1999) Biochem Pharmacol 58, 389-395 The previously
used human ADH], ADH2, ADH genes encoding class I enzymes are
renamed ADH1A, ADH/B, and ADHIC indicating their greater than
90% similarity The enzyme class names and gene names now corre-
spond The class I] human and mouse enzymes are encoded by ADH2
and Adh2, respectively; class III enzymes by human ADH3 (old
ADHS) and mouse Adh3 (old Adh5); class TV enzymes by human
ADH¢4 (old ADH7) and mouse Adh4 (old Adh3); class V enzyme by
human ADHS (old ADH6) The deermouse class VI ADH is closest to
human ADHS protein in amino-acid sequence, and based on phy-
logeny and genome organization of the mouse presented here is now
referred to as class V with a gene symbol of Adh5 New mouse genes
reported here are named AdhSa, AdhSb, and AdhSps
(Received 20 September 2001, accepted 30 October 2001)
II, UI 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 gene [7,8] A human class V ADH with approximately 60% positional identity at 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 cDNAs have been isolated that would encode 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 HI functions as a
glutathione-dependent formaldehyde dehydrogenase [12] and is involved in S-nitrosoglutathione metabolism [13] and retinol metabolism (G Duester, unpublished results) Class
IV has an important role in retinol metabolism leading to retinoic acid production Adh4 is expressed during embry- ogenesis [14,15] and Adh4 null mouse mutants have reduced fetal survival during vitamin A deficiency [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 in retinoid signaling during embryogenesis Expressed at high levels in liver, the role of class [ 1n ethanol
metabolism has been confirmed by natural [17] and engineered [12] enzyme-deficient animals Adh/ null mutant
mice also demonstrate a significant decrease in metabolism
of retinol to retinoic acid [12] The in vivo physiological roles
ofclass I, HH, and IV ADHs in the mouse are being unveiled
with the development of targeted disruptions for each gene The mouse Adhi and Adh4 genes are linked [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
Trang 2complex regulatory mechanisms control this gene family
The mouse Adh1, Adh2, Adh3, and Adh4 genes have 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 1m transfected
hepatoma cells has been defined for Adh/ [24,25] How-
ever, as much as 10 kb of sequence upstream of an Adh/
minigene does not direct expression in liver of transgenic
mice [26] although kidney and adrenal expression is
promoted An entire Adh/ gene containing 7 kb of 5’
and 21 kb of 3’ flanking sequence in transgenic mice
expresses properly in most tissues except liver and intestine
[27] More distal sequences must control expression of
Adh! in these tissues, and this has stimulated an effort to
obtain mouse Adh/ bacterial artificial 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 are 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 sequence comparisons and location within
the complex suggests that the two newly identified Adh
genes are most closely related to the human ADHS5 gene
MATERIALS AND METHODS
Materials
Chemicals and reagents were obtained from 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 were also purchased
from this resource
CDNA hybridization probes for mouse genes
The ADH cDNA probes used in this study were inserts from
pADHn | for deermouse ADH5 and pCK2 for mouse ADH1
[10] The ADH4 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 confirmed by nucleotide
sequence analysis Isolated cDNA inserts were used to
prepare all probes for labeling by random priming [28]
Screening the BAC library for mouse Adh/7-containing
clones
Five filters containing a total of over 90 000 clones were
screened by hybridization with *’P-labeled mouse ADHI1
full-length insert cDNA in pCK2 Probe was used in the hybridization solution at 2.4 x 10°c.p.m.mL™"' Filters were prehybridized for >2h at 65 °C in 6 x NaCl/P;/EDTA (1 x = 0.15 m NaCl, 0.01 mM sodium phosphate, 1 mm EDTA, pH 7.4)/6 x Denhardt’s solution, 0.5% SDS, and
50 p:gmL~! denatured salmon sperm DNA After prehy- bridization, the solution was discarded and replaced with an identical hybridization solution except without Denhardt’s containing the radioactive probe After overnight hybrid- ization the filters were washed twice with 6 x NaCl/P;/ EDTA/0.5% SDS at room temperature for 15 min each, twice in | x NaCl/P;/EDTA/0.5% SDS at 37- 42 °C for
15 min each, and once in 0.1 x SSPE/0.5% SDS at 65 °C The filters were blotted of excess moisture, wrapped in Saran wrap, and exposed to Kodak XAR film at —80 °C for several hours to a day depending upon intensity of signal and level of background necessary to reveal outline of the fields on the filter
BAC DNA isolation and restriction enzyme digest analysis
A single 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 ug-mL of chloramphenicol After overnight growth 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 Pipetting of the final BAC DNA preparation was carried out with large orifice tips from USA Scientific
The BAC DNA was digested with various restriction endonucleases and the fragments were resolved on 1% agarose gels in 0.5 x Tris/borate/EDTA (1 x Tris/borate/
EDTA = 0.089 o Tris, 0.089 m boric acid, 0.002 Mm EDTA)
using a contour-clamped homogenous electric field (CMHF) apparatus Electrophoresis was conducted at 6 V-cm™! at
15 °C for 16 hrwith the switch time ramped from | to 12 s After completion of electrophoresis, gels were stained in 0.5 wg-mL! ethidium bromide in 0.5 x 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 0.5m NaOH/1.0 m NaCl, and neutralized with 0.5 m Tris/HCl/ 1.5m NaCl (pH 7.4) The gels were inverted and DNA was transferred overnight onto Hybond Nytran membranes in
10 x NaCl/P;/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, fluorescent- based DNA sequencing and subsequent analysis were used
as described previously [29-31] Briefly, BAC DNA was isolated free from host genomic DNA via a cleared lysate- acetate precipitation-based protocol [29] Subsequently,
50 ug portions of purified BAC DNA were randomly sheared and made blunt ended [30,31] After kinase treatment and gel purification, fragments in the I- to 3-
kb range were ligated into Smal-cut, bacterial alkaline
Trang 3phosphatase-treated pUC18 (Pharmacia) and Escherichia
coli, strain XLIBlueMRF’ (Stratagene), was transformed
by electroporation A random library of approximately
1200 colonies were picked from each transformation,
grown in terrific broth [32] supplemented with 100 pgmL™!
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 Tag DNA polymerase, the Perkin
Elmer Cetus fluorescently 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 filtration
through Sephadex G-50, the fluorescently 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 amplification of the
region corresponding to the gap in the sequence followed
by subcloning into pUC18 and cycle sequencing with the
universal pUC-primers via Tag terminator chemistry In
some instances, additional synthetic custom primers were
necessary to obtain at least threefold coverage for each
base
Draft and finished BAC sequences 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 given 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 similar data set was used for each comparison
Phylogenies were constructed using the following methods:
(a) the neighbor-joining (NJ) method [41] based on the
Poisson corrected (PC) amino-acid distance [42] and (b) the
NJ method based on the gamma-corrected (a = 2.0) amino-
acid distance [42]
Both methods produced essentially identical results;
therefore, only the results of the NJ tree based on PC are
presented here The NJ method is reliable at 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 replacement)
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 were carried out using the GcG software
package
RESULTS
Identification of Adh7-containing BAC clones DNA was isolated from all BAC clones hybridizing to the ADHI cDNA probe Purified DNA was digested with EcoRI and analyzed by Southern blotting and hybridization
to ADH1 cDNA Finally, 13 positive clones were identified and among these were found all EcoRI restriction fragments detectable in genomic DNA with an ADH1 cDNA probe The Adh/-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 identified among the BAC clones Of these 13 clones, two were chosen for extensive analysis because they overlap within the Adh/ gene BAC 463H24 contains the entire Adh/ gene while 393J8 contains only the most 3’ end, the 3.8- and 1.1-kb EcoRI [7] hybridizing fragments (Fig 2A) Therefore, 393J8 extends farther downstream
than 463H24 relative to Adh/ orientation 393J8, in contrast
to 463H24, includes the 2.0 kb non-Adhi [7] hybridizing fragment as does another BAC clone 461A12 As 461A 12 contains no AdhI1 sequence, this confirms that the cross- hybridizing 2.0-kb EcoRI fragment is downstream of the AdhI gene Two additional BAC clones, 388D7 and
434F 16, were identified that contained the 2.3-kb EcoRI
Adh1-crosshybridizing fragment [7], but no 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 CHEF analysis As estimated by the sizes of the restriction fragments, 463H24 contains an insert of over 165 kb (Fig 1A) 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 Adhd are resolved on this clone For example, Adh/ is located on the 60-kb RsrII fragment while Adh4 is found on the 100-kb fragment (Fig [A, N/R) Either because of context or the sequence recognized by RsrII [CGG (A/T)CCG] there was never complete digestion at this site
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 EagI fragment while AdhT 1s found on the 135- and 25-kb fragments (Fig 1A, N/E) Within the Adh/ gene a single Eagl site is found near the 3’ end of exon 6 accounting for the two hybridizing fragments This also localizes the 3’ end of Adh/ approxi- mately 21 kb from one end of the clone Adh] and Adh4 are
on the large Sa/I fragment suggesting this site is nearer the opposite end of the clone than Eagl As Eagl, RsrII, and Sail all cut the clone once and EagI and SalI are nearer the ends
of the BAC, other double digests were performed to construct a more detailed map of 463H24 (Fig 2A) Two Pmel 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 Pmel site was not determined from the digests performed The RsrII/Pmel double digest
Trang 4
-
45 _~ 910- - 231- " = 48 5 - — ¬ — thang a
6.55 -
SS - ix
Fig 1 CHEF analysis of restriction enzyme 4a5-
digests of BAC clone The restriction enzymes 23.1
used to prepare BAC digests loaded into each 9.42 s
used were N, Wøil; B, BssHII; E, Eagl; P,
Pmel R, RsrII; S, Sail; and Sg, SgrAI The top
panel in both (A) and (B) shows the fragments
detected by ethidium bromide staining AdhS
(A) Identification of ADH-containing restric- ‘Ss-— w
tion fragments in BAC 463H24 Blots probed - — Af, @
(B) Identification of ADH-containing restric-
tion fragments in BAC 393J8 Autoradiogram
resulting from probing identically prepared
blots with ADH4, ADH2, ADH3 or deer-
mouse ADH5 (mammalian class V) cDNAs
are shown
(Fig 1A, N/R/P) delineates the position of AdhI and Adh4
(Fig 2A) on the BAC
Adh genes on BAC 393J8
The presence of the 3’ end of Adh/ 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 ADHI1 cDNA Sizes of fragments produced by
digestion of 393J8 with various restriction enzymes were
determined by CHEF (Fig 1B) The results of probing a
2.1 - ~ ~
942- 65S
blot of these CHEF separated restriction endonuclease fragments of 393J8 with four ADH cDNA probes are shown in Fig 1B The single Sa/I site is located approxi- mately 30 kb from the AdhI-tagged end of the clone confirmed by the strong hybridization signal of the 30-kb fragment when probed with ADH1 cDNA (Fig 1B, N/S) The faint signal observed from the large fragment must be due to cross-hybridization with other ADH genes The only RsrIlI site is located about 100 kb from the Adh/ end of the clone (Fig 1B, N/R) The single SgrAI site is located about
135 kb from the Adh/ end (Fig 1B, N/Sg), and the Eagl site
Trang 5located nearest Adh/ is over 60 kb (Fig 1B, N/E) from the
end (the largest Eagl fragment) ADHI cDNA also
hybridizes to a 75-kb Pmel fragment (Fig 1B, N/P), but
this band is actually composed of two nearly identical
fragments The smaller 12-kb PmelI fragment is from the
middle of this clone The observation that Sa/I, RsrII, and
SgrAlI digests not only have a strong signal from the
fragment containing the 3’ end of Adh/ but also a weak
hybridization signal from the other fragment in the 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 fragment which contains the portion
of the Adh/ gene located 5’ of this site to the end of the
clone, to the 60-kb fragment already mentioned, and to 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 Adh/ 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 weak signals with the 30-kb and 140-
kb Notl/Sall fragments, respectively However, deermouse
ADHS probe gives approximately equal signals in both
fragments The deermouse ADHS probe does cross-hybrid-
ize to AdhI 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
Notl/SgrAI fragment whereas Adh3 is on the small
fragment (Fig 1B, 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 AdhI end of the BAC The
positions of Adh4, Adh2, and Adh3 are arbitrarily placed in
the middle of the flanking restriction sites Adh/ is anchored
by the presence of the EagI site in the gene The deermouse
ADHS 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 Adh! based upon weak
cross-hybridization signals observed with both cDNAs as
probe and the fact that the Eagl is placed within Adh5, but
could be between related genes However, the other nearby
EaglI site may be able to resolve two AdhS 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, Adh†,
Adh2 and Adh3 genes
The draft sequence of 463H24 (AC 079682.14) consists of
four unordered pieces and is suggested 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 SalI sites, all located within 7-kb of each other, upstream from the 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 Pmel site
on the map is located upstream of Adh4 The sequence also identifies the location of the additional Pmel site which is near the 5’ end of Adh4 The RsrII site on the map is on a different contig (182 084-106 188) and is upstream of the 5’ end of the Adh/ gene located on this contig The single EagI site located near the 3’ end of exon 6 in the Adh/ gene localizes this gene on 463H24 and the draft sequence confirms the 5’ to 3’ orientation of this gene As one end of 393J8 begins in the 3’ end of Adh/, this confirms that Adh4 and Adh/ 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 overall length of the sequence is 194 850 bp
Identification of Adh5a, Adh5b, and Adh5ps between Adh7 and Adh2 Sequences in 393J8 hybridizing to the deermouse ADHS5 cDNA maps between Adhi and Adh2 but closer to Adh2 (Fig 2A) 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 97 694-110 934 in the BAC Exon 6 sequence contains a 9-bp deletion followed by a 7-bp stretch and then
a single bp deletion when compared to the deermouse ADHS or human ADH5 cDNAs After 22 codons in the altered reading frame a stop codon is encountered strongly suggesting this is a nonfunctioning pseudogene, AdhSps Before the deletion, the encoded sequence has a positional 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 was 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 76 and encodes six amino acids with 3/6 identity with human ADH5 exon | 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 that 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 between AdhI and Adh5Sps for sequences homologous to deermouse ADH5 or human ADHS protein sequence revealed two probable complete genes each with nine exons These genes are defined as AdhSa and AdhSb based on location and phylogeny (see below) TBLASTN and BLASTN analyses define the location of AdhSa as encompassing 29 317-47 164 in the Y to 3’ orientation AdhSb is located from 57 150 to 74 874 A TBLASTN search of the GenBank database failed to locate the first and last exons of the Adh5 gene
Trang 6
463H24
393J8
re 10 kb
Adhd Adh! Adh3a Adh$h Adh3ps Adh2 Adh3
Adhd a s +—+# 2 24 +
Fig 2 Structural organization of the genes in the mouse ⁄4##' complex (A) Restricion map of the area encompassed by the two BACs indicating positions of the mapped Adh genes Restriction enzyme symbols are the same as in Fig 1 Genes known to lie between two restriction sites are arbitrarily placed in the middle The location of the two BAC clones are shown below the physical map (B) Molecular map of the mouse Adh complex (top) compared to the 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, but molecular distances and orientation were derived based on BAC end sequence from the database, restriction sites located within contigs compared to the physical map, and paired BLASTN analysis of the BAC against ADH4 and ADH1 cDNA The order of contigs in 463H724 relative to the 5’ to 3’ transcriptional orientation of the Adh4 and 447 genes on the BAC is (32489/5054)-(32589/106087; Adh4)-(182084/106188; AdhJ) One contig (1/4953) cannot be placed on the map, but is not at either end of the BAC (C) Organization of introns and exons in the mouse genes Introns are lines and exons are rectangles The genes are given in their order within the complex
AdhSa and AdhSb have available complete cDNA or EST
sequence represented in the database, respectively This has
allowed the last exon of AdhSb to be defined 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 AdhSa and has been
used to determine gene structure The complete gene
structures of AdhSa and Adh5Sb are fully annotated in
GenBank (GI: 15383846) Both genes consist of nine exons
and encode 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
identified as a sequence encoding the intiation met and five
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 and encodes a polypeptide of 376 amino acids, again
exclusive of the inititation methionine All three genes,
Adh2, Adhs5a and AdhSb have consensus gt and ag
nucleotides flanking the intron sequences The translation
initiation codons for AdhSa and Adh2 have 8/10 and 6/10
nucleotide identity with the consensus initiator sequence, respectively The Adh5Sb initiator shares 5/10 identity with the consensus sequence and does not have, in contrast to Adh2 and AdhSa, the important G residue after the ATG or the A/G three nucleotides upstream from the ATG Although the first intron in the AdhSb 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 of the first 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 molecular map of the mouse Adh complex is shown in Fig 2B along with a comparison with the human ADH complex The sizes of introns in all genes including the pseudogene are shown in Fig 2C
Phylogeny of mouse and human Ad genes
In the phylogeny of the mouse and human genes, there were five major clusters (Fig 3), each separated by a bootstrap
Trang 7Table 1 Amino-acid positions encoded by each exon in six mouse Adh 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 The human ADH2 exon coding regions are shown as this gene is most closely related to the mouse Adh2 Exons for human AD#H2 are from [48], and human ADH5 organization is from [9,47] Sources for organization of other mouse genes are: Adh4 [45], Adh/ [7] and Adh3 [49]
Exon
Adh4 Metl—5 6-39 40-86 86-115 115-188 189-275 276-321 321-368 368-374 Adhl
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
70/7 ADH IC in the order Adh4-AdhI-Adh2-Adh3 in less than 250 kb of
100 RADH1A orientation being 5’ to 3’ from the Adh4 to Adh/ orientation
This is the same order and transcriptional orientation as
found for the human genes except the human has three
oo mADH6B ing to the location of the human ADH5 gene The amino-
acid similarity of 67% between the deermouse encoded
protein and the human ADHS protein [10] previously
1007 NADHS sequence represents a separate class VI A similar sequence
0.1
PC
Fig 3 Phylogeny of mouse and human Adh genes constructed by the
neighbor-joining method Numbers on the branches are percentages of
500 bootstrap samples that support the branch
value of 87 or greater: (a) hADH1IA, hADHI1B, hADHIC,
and mADHI cluster significantly with a bootstrap value of
100; (b) hADH4 and mADH64 cluster (100); (c) hADHS,
mADHSA and mADHSB cluster (87); (d) hADH2 and
mADH2 cluster (100); and (e) hADH3 and mADHS3 cluster
(100) The proteins encoded by the newly identified mouse
AdhSa and AdhSb genes are most closely related to the
human ADHS5 and to each other, which is the basis for their
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 Adhi 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
ADHS5 cDNA probe Sequence of the two overlapping
BAC clones combined with the physical map allowed us to
determine that the previously known mouse genes are found
67% is an intermediate value between members of different 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 ADHS Pairwise BLASTN comparisons between the draft sequence of the BAC in this area and the deermouse and human ADHS5 cDNAs allowed the identification of poten- tial exons 2-6 in this physically mapped region 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 I-3, the deletion in exon 6 strongly suggests that no functional protein is made A partial sequence from exon 6 of an unidentified mouse Adh gene from a C57BL/6 library contains an identical deleted sequence in the exon 6 region [46] (D Dolney, University of South Carolina,
Columbia, SC, USA, unpublished results) Because the
location of this sequence is similar relative to other genes in human, it is defined as AdhSps
TBLASTN pairwise searches between deermouse and human ADH5 cDNA translation products and the BAC sequence located between this mouse AdhSps and Adhl identified two additional genes The newly defined AdhSa and Adh5Sb genes reside between Adh/ and Adh2 indicating
an overall order of Adh4-Adh1-AdhSa-Adh5b-AdhSps-Adh2- Adh3 The mouse AdhSa and AdhSb reside in the same relative position as the human ADH)5 gene The structures
of both genes are 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
Trang 8positions where intron sequence disrupts coding sequence in
AdhSsa and Adh5b genes is identical to Adh4 [45] and Adh/
[7,46] However, AdhSa, Adh5b, and Adh2 are remarkably
similar to human ADHS5 at the exon 8/exon 9 boundary In
all cases there is a potential stop codon just downstream of
exon 8 that would produce a truncated protein if splicing
between exons 8 and 9 fails to occur Recently, alternative
splicing of exons 8 and 9 has been reported for human
ADHS [47] All the 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 region of the
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 AdhSps even with only
six exons is the largest in the complex The genes (Adh4,
Adh1, AdhSa, AdhSb, AdhSps) at the 5S’ end of the complex
relative to transcriptional orientation characteristically have
small introns between exons 4 and 5, but genes at the 3’ end
(Adh2 and Adh3) have a larger intron 4
This report presents a detailed map of the mouse Adh
gene complex and finds that six genes in the same
transcriptional orientation are found within 250 kb of
DNA sequence A pseudogene located in a transcribed
region is also detected in this locus The first 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, Adh/, 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 the RNA level has not been
established, while the Adh3 gene is widely expressed in
mouse tissues [4] The expression pattern of Adh5a and
Adh5Sb is still to be defined There is some order in the liver
expression pattern of the different genes as related to their
position on the 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 Adh/,
Adh2, and Adh3 are highly expressed in liver At this time, it
is unknown if individual regulatory elements between the
genes control expression of 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 sequence of RP23-393J8 and RP23-463H24 BAC clones
REFERENCES
1 Duester, G., Farrés, J., Felder, M.R., Holmes, R.S., Hưưg, J.-O., Parés, X., Plapp, B.V., Yin, S.-J & Jưrnvall, H (1999) Recom- mended nomenclature for the vertebrate alcohol dehydrogenase gene family Biochem Pharmacol 58, 389-395
2 Jérnvall, H & Hữưg, J.-O (1995) Nomenclature of alcohol dehydrogenases Alcohol Alcohol 30, 153-161
3 Jérnvall, H., Shafqat, J., el-Ahmad, M., Hjelmquist, L., Persson,
B & Danielsson, O (1997) Alcohol dehydrogenase variability Evolutionary and functional conclusions from characterization of further variants Adv Exp Med Biol 414, 281-289
4 Zgombic-Knight, M., Ang, H.L., Foglio, M.H & Duester, G (1995) Cloning of the mouse class IV alcohol dehydrogenase (retinol dehydrogenase) cDNA and _ tissue-specific expression patterns of the murine ADH gene family J Biol Chem 270,
10868-10877
5 Svensson, S., Strémberg, P & H6ég, J.-O (1999) A novel subtype
of class II alcohol dehydrogenase in rodents J Biol Chem 274,
29712-29719
6 Duester, G., Smith, M., Bilanchone, V & Hatfield, G.W (1986) Molecular analysis of the human class I alcohol dehydrogenase gene family and nucleotide sequence of the gene encoding the B subunit J Biol Chem 261, 2027-2033
7 Ceci, J.D., Zheng, Y.-W & Felder, M.R (1987) Molecular anal- ysis of mouse alcohol dehydrogenase: nucleotide sequence of the Adh-1 gene and genetic mapping of a related nucleotide sequence
to chromosome 3 Gene 59, 171-182
8 Edenberg, H.J., Zhang, K., Fong, K., Bosron, W.F & Li, T.-K (1985) Cloning and sequencing of cDNA encoding the complete mouse liver alcohol dehydrogenase Proc Natl Acad Sci USA 82,
2262-2266
9 Yasunami, M., Chen, C.-S & Yoshida, A (1991) A human alcohol dehydrogenase gene (ADH6) encoding an additional class
of isozyme Proc Natl Acad Sci USA 88, 7610-7614
10 Zheng, Y.-W., Bey, M., Liu, H & Felder, M.R (1993) Molecular basis of the alcohol dehydrogenase-negative deer mouse Evidence for deletion of the gene for class I enzyme and identification of a possible new enzyme class J Biol Chem 268, 24933-24939
II Hưưðg, J.-O & Brandt, M (1995) Mammalian class VI alcohol dehydrogenase: novel types of the rodent enzymes Adv Exp Med
Biol 372, 355-365
12 Deltour, L., Foglio, M.H & Duester, G (1999b) Metabolic deficiences in alcohol dehydrogenase Adh1, Adh3, and Adh4 null mutant mice J Biol Chem 274, 16796-16801
13 Liu, L., Hausladen, A., Zeng, M., Que, L., Heitman, J & Stamler, J.S (2001) A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans Nature 410, 490-494
14 Ang, H.L., Deltour, L., Hayamizu, T.F., Zgombic-Knight, M & Duester, G (1996) Retinoic acid synthesis in mouse embryos during gastrulation and craniofacial development linked to class
IV alcohol dehydrogenase gene expression J Biol Chem 271,
9526-9534
15 Ang, H.L., Deltour, L., Zgombic-Knight, M., Wagner, M.A & Duester, G (1996) Expression patterns of class I and class IV alcohol dehydrogenase genes in developing epithelia suggest a role for alcohol dehydrogenase in local retinoic synthesis Alcohol Clin Exp Res 20, 1050-1064
16 Deltour, L., Foglio, M.H & Duester, G (1999a) Impaired retinol utilization in Adh4 alcohol dehydrogenase mutant mice Dey Genet 25, 1-10
17 Burnett, K.G & Felder, M.R (1980) Ethanol metabolism in Peromyscus genetically deficient in alcohol dehydrogenase Biochem Pharmacol 29, 125-130.
Trang 918
19
20
21
22
23
24
25
26
27
28
29
30
31
Holmes, R.S., Albanese, R., Whitehead, F.D & Duley, J (1981)
Mouse alcohol dehydrogenase isozymes: products of closely
localized duplicated genes exhibiting divergent kinetic properties
J Exp Zool 217, 151-157
Peirce, J.L., Derr, R., Shendure, J., Kolata, T & Silver, L.M
(1998) A major influence of sex-specific loci on alcohol preference
in C57B1/6 and DBA/2 inbred mice Mamm Genome 9, 942-948
Balak, K.J., Keith, R.H & Felder, M.R (1982) Genetic and
developmental regulation of mouse liver alcohol dehydrogenase
J Biol Chem 257, 15000-15007
Felder, M.R., Watson, G., Huff, M.O & Ceci, J.D (1988)
Mechanism of induction of mouse kidney alcohol dehydrogenase
by androgen J Biol Chem 263, 14531-14537
Tussey, L & Felder, M.R (1989) Tissue-specific genetic variation
in the level of mouse alcohol dehydrogenase is controlled tran-
scriptionally in kidney and posttranscriptionally in liver Proc
Natl Acad Sci USA 86, 5903-5907
Dolney, D.E.A., Szalai, G., Duester, G & Felder, M.R (2001)
Molecular analysis of genetic differences among inbred mouse
strains controlling tissue expression pattern of alcohol dehydro-
genase 4 Gene 267, 145-156
Lin, Z., Edenberg, H.J & Carr, L (1993) A novel negative element
in the promoter of mouse liver alcohol dehydrogenase J Biol
Chem 268, 10260-10267
Carr, L.G., Zhang, K & Edenberg, H.J (1989) Protein-DNA
interaction in the 5’ region of the mouse alcohol dehydrogenase
gene Adh-1 Gene 78, 277-285
Xie, D., Narasimhan, P., Zheng, Y.-W., Dewey, J.J & Felder,
M.R (1996) Ten kilobases of 5’-flanking region confers proper
regulation of the mouse alcohol dehydrogenase-1 (Adh-]} gene in
kidney and adrenal of transgenic mice Gene 181, 173-178
Szalai, G., Ceci, J., Dewey, M & Felder, M.R (2001) Identifica-
tion and expression of cosmids with an allelic variant of class I
alcohol dehydrogenase in transgenic mice Chemico-Biol Interact
130-131, 481-490
Feinberg, A.P & Vogelstein, B (1983) A technique for radiola-
belling DNA restriction endonuclease fragments to high specific
activity Anal Biochem 132, 6-32
Pan, H.Q., Wang, Y.P., Chissoe, S.L., Bodenteich, A., Wang, Z.,
Iyer, K., Clifton, S.W., Crabtree, J.S & Roe, B.A (1994) The
complete nucleotide sequence of the SacBII domain of the Pl
pAD10-SacBII cloning vector and three cosmid cloning vectors:
pICF, svPHEP, and LAWRISTI6 Genet Anal Tech Appl 11,
181-186
Bodenteich, A., Chissoe, S Wang, Y.F & Roe, B.A (1993)
Shotgun cloning as the strategy of choice to generate templates for
high-throughput dideoxynucleotide sequencing In Automated
DNA _ Sequencing and Analysis Techniques (Venter, J.C., ed.),
pp 42-50 Academic Press, London, UK
Chissoe, S.L., Bodenteich, A., Wang, Y.F., Wang, Y.P., Burian,
D., Clifton, S.W., Crabtree, J., Freeman, A., Iyer, K., Jian, L.,
Ma, Y., McLaury, H.J., Pan, H.Q., Sharan, O., Toth, S., Wong,
Z., Zhang, G., Heisterkamp, N., Groffen, J & Roe, B.A (1995)
Sequence and analysis of the human ABL gene, the BCR gene, and
regions involved in the Philadelphia chromosomal translocation
Genomics 27, 67-82
32
33
34
35
36
37
38
39
40
4I
42
43
44
45
46
47
48
49
Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Ewing, B., Hillier, L., Wendl, M & Green, P (1998) Basecalling of automated sequencer traces using phred I Accuracy assessment Genome Res 8, 175-185
Ewing, B & Green, P (1998) Basecalling of automated sequencer traces using phred II Error probabilities Genome Res 8, 186-194 Gordon, D., Abajian, C & Green, P (1998) Consed: A graphical tool for sequence finishing Genome Res 8, 195-202
Genetics Computer Group (1994) Program Manual for the Wisconsin Package, Version 8, Genetics Computer Group, Madison, WI, USA
Altschul, S.F., Gish, W., Myers, E.W & Lipman, D.J (1990) Basic local alignment search tool J Mol Biol 215, 403-410 Worley, K.C., Wiese, B.A & Smith, R.F (1995) BEAUTY: an enhanced BLAST-based search tool that integrates multiple bio- logical information resources into sequence similarity searches Genome Res 5, 173-184
Henikoff, S & Henikoff, J.G (1994) Protein family classification based on searching a database of blocks Genomics 19, 97-107 Thompson, J.D., Higgins, D.G & Gibson, T.J (1994) CLU- STALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22,
4673-4680
Saitou, N & Nei, M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees Mol Biol Evol 4,
406-425
Nei, M & Kumar, S (2000) Molecular Evolution and Phylogenies Oxford University Press, New York
Felsenstein, J (1985) Confidence limits on phylogenies: an approach using the bootstrap Evolution 39, 783-791
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W & Lipman, D.J (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search programs Nucleic Acids Res 25, 3389-3402
Zgombic-Knight, M., Deltour, L., Haselbeck, R.J., Foglio, M.H
& Duester, G (1997) Gene structure and promoter for Adh3 encoding mouse class IV alcohol dehydrogenase (retinol dehy- drogenase) Genomics 41, 105-109
Zhang, K., Bosron, W.F & Edenberg, H.J (1987) Structure of the mouse Adh-] gene and identification of a deletion in a long alternating purine-pyrimidine sequence in the first intron of strains expressing low alcohol dehydrogenase activity Gene 57, 27-36 Stromberg, P & H6ég, J.-O (2000) Human class V alcohol dehy- drogenase (ADHS): a complex transcription unit generates C-ter- minal multiplicity Biochem Biophys Res Comm 278, 544-549 von Bahr-Landstrém, Jé6rnvall, H & H66g, J.-O (1991) Cloning and characterization of the human ADH# gene Gene 103,
269-274
Foglio, M.H & Duester, G (1996) Characterization of the func- tional gene encoding mouse class III alcohol dehydrogenase (glutathione-dependent formaldehyde dehydrogenase) and an unexpressed processed pseudogene with an intact open reading frame Eur J Biochem 277, 496-504.