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Proc. Nati. Acad. Sci. USAVol. 85, pp. 1782-1786, March
1988Biochemistry
Cloning and complete nucleotide sequence of a full-length
cDNAencoding a catalytically functional tumor-associatedaldehyde
dehydrogenase
(eukaryotic cDNA expression/antibody probe/hepatoma/inducible
isozyme)
DAVID E. JONES, JR.*, MARK D. BRENNAN*, JOHN HEMPELt, AND RONALD
LINDAHL***Biochemistry Program and Department of Biology,
University of Alabama, Tuscaloosa, AL 35487; and tDepartment of
Microbiology, Biochemistry andMolecular Biology, University of
Pittsburgh Medical School, Pittsburgh, PA 15261
Communicated by Sidney Weinhouse, November 23, 1987 (received
for review September 18, 1987)
ABSTRACT To study the mechanism(s) controllingexpression of the
tumor-associated aldehyde dehydrogenase(tumor ALDH), which appears
during rat hepatocarcinogen-esis, cDNAs encoding this isozyme were
cloned and identifiedwith an antibody probe. Poly(A)-containing RNA
from HTCrat hepatoma cells, which have been shown to possess
highlevels of tumor ALDH, was used as template to
synthesizedouble-stranded cDNA. The cDNA was methylated to
protectinternal sites. Two different synthetic DNA linkers were
addedsequentially to the cDNA to insure correct orientation
forexpression from the lac promoter of pUC8. A library of100,000
independent members carrying inserts >1 kilobasewas obtained.
From this library, two apparently identicaltumor ALDH clones,
differing only in size, were identifiedwith an indirect
immunological probe. The larger of the cDNAclones identified,
pTALDH, was chosen for further study.Interestingly, since tumor
ALDH is a dimeric enzyme,pTALDH directs synthesis of a functional
tumor ALDH in thebacterial cell. The cDNA sequence has been
confirmed bycomparison to the amino acid sequence of tumor
ALDHpurified from HTC cells.
Previous work has shown that a tumor-associated
aldehydedehydrogenase (tumor ALDH) can be induced by a numberof
different chemical carcinogens during rat hepatocarcino-genesis
(1-11). This phenotype is characterized by an in-crease in total
ALDH activity due to the appearance of acytosolic isozyme
undetectable in normal rat liver. Thisisozyme preferentially
oxidizes aromatic aldehyde sub-strates using NADP+ as coenzyme and
differs from normalliver ALDH isozymes in a number of physical and
functionalproperties (1, 4, 12, 13).The tumor ALDH isozyme has been
purified from a
variety of sources and its properties have been described (1,13,
14). The tumor ALDH isozyme has a Mr of 110,000 andis composed of
two apparently identical subunits. While thetumor ALDH isozyme
shares identical subunit size (Mr.54,000) with the normal liver
ALDH isozymes, it is adistinctly different enzymatic species based
on electropho-retic mobility, isoelectric point, sensitivity to
inhibitors,substrate and coenzyme preference, and immunologic
cross-activity (1, 4, 12-15).
In normal rat liver, ALDH activity is localized primarily tothe
mitochondrial and microsomal fractions (4, 12, 15, 16).At least
three distinct ALDH isozymes can be differentiatedon the basis of
substrate and coenzyme preference, substrateand coenzyme Ki,, and
sensitivity to inhibitors (4, 12, 15,16). Generally, the normal
liver isozymes are tetramers that
preferentially oxidize aliphatic aldehydes using NAD+
ascoenzyme.Another ALDH isozyme can be induced in normal liver
by
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (13, 17-19).Both the
tumor ALDH isozyme and the TCDD-inducibleisozyme have Mrs of
110,000 and preferentially oxidizearomatic aldehyde substrates
using NADP+ as coenzyme.By all criteria, these isozymes appear to
be identical (13-19).We have previously shown (2) that the
appearance of the
tumor ALDH isozyme during hepatocarcinogenesis is notdue to
derepression of a fetal ALDH gene. This gene productis also not
found in regenerating liver after partial hepatec-tomy (10).
However, we have recently shown that an enzy-matic species very
similar to the tumor ALDH isozyme isfound in normal rat urinary
bladder (20).To study the regulation of tumor ALDH gene
expression,
we elected to clone a tumor ALDH cDNA. We chose
thewell-established rat hepatoma cell line HTC as a source ofmRNA
because previous studies have shown that these cellspossess
constitutively high levels of tumor ALDH activity(14, 21),
accounting for "1% of the total soluble protein inthese cells
(unpublished data). Compared to other sources,such as primary
tumors, the extremely high tumor ALDHactivity should reflect
relatively high levels of tumor ALDHmRNA in HTC cells.To maximize
the chances of obtaining full-length cDNA
clones, the cDNA prepared from HTC mRNA was furthermanipulated.
The cDNA was methylated with EcoRI methyl-ase and Pst I methylase
to protect any internal restrictionendonuclease sites of these
enzymes. In addition, EcoRI andPst I linkers were added
sequentially to the cDNA to insurecorrect orientation for
expression from the lac promoter ofpUC8.The identification and
complete nucleotide sequence of a
cDNA encoding the tumor ALDH monomer are describedhere.§
Interestingly, the immunologically detected cDNAclone directs
synthesis of very high levels of functional tumorALDH.
EXPERIMENTAL PROCEDURESAntigen and Antibody Preparation. Tumor
ALDH was
purified from HTC cells grown in spinner culture by ammo-nium
sulfate precipitation and passage over a 5'-AMP Seph-arose column
as described (13, 14).High titer anti-tumor ALDH antisera were
raised in rab-
bits with Freund's adjuvant used to enhance immunogenic-
Abbreviations: ALDH, aldehyde dehydrogenase; tumor
ALDH,tumor-associated ALDH; TCDD,
2,3,7,8-tetrachlorodibenzo-p-dioxin.tTo whom reprint requests
should be addressed.§This sequence is being deposited in the
EMBL/GenBank data base(Bolt, Beranek, and Newman Laboratories,
Cambridge, MA, andEur. Mol. Biol. Lab., Heidelberg) (accession no.
J03637).
1782
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page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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Proc. Natl. Acad. Sci. USA 85 (1988) 1783
ity. The specificity of this antisera to tumor ALDH and thelack
of cross-reactivity between other ALDH isozymes wastested by
Ouchterlony double diffusion (22). The IgG frac-tion of the
anti-tumor ALDH antisera was purified by am-monium sulfate
precipitation followed by passage over acombined DEAE-/CM-cellulose
column (23, 24). The puri-fied IgG fraction was preadsorbed to an
Escherichia coliextract before screening the cDNA library (25).
Preparation of Poly(A)+ RNA and Synthesis of cDNA.Polysomal RNA
was isolated from HTC cells grown inspinner culture by the sucrose
block-gradient method (26,27). Poly(A)-containing RNA was purified
from total poly-somal RNA by two cycles of adsorption and elution
fromoligo(dT)-cellulose (28, 29). Total RNA, poly(A)-containingRNA,
and non-poly(A)-containing RNA, were translated ina rabbit
reticulocyte translation system (Bethesda ResearchLaboratories).
The presence of tumor ALDH in the transla-tion products was
confirmed by immunoprecipitation (30,31) using anti-tumor ALDH
antibodies and analyzed byNaDodSO4/polyacrylamide gel
electrophoresis (32).Double-stranded cDNA was synthesized by using
avian
myeloblastosis virus reverse transcriptase (Life Sciences,St.
Petersburg, FL) as described by Maniatis et al. (29). ThecDNA was
methylated to protect any internal sites usingEcoRI and Pst I
methylases (New England Biolabs) (33).EcoRI and Pst I linkers were
ligated to the cDNA sequen-tially to insure correct orientations
for expression from thelac promoter of pUC8 (29, 33, 34). The cDNA
was cleavedwith both EcoRI and Pst I restriction endonucleases
(NewEngland Biolabs). DNA >1 kilobase (kb) was prepared
byagarose gel electrophoresis and then ligated into EcoRI/PstI-cut
pUC8. Recombinant plasmids were used to transformE. coli strain
HB101 (Bethesda Research Laboratories).Antibody screening of the
cDNA library was performed
by using purified anti-tumor ALDH IgG and an
anti-rabbitIgG/horseradish peroxidase-conjugated second
antibody(Sigma). Indirect immunologic screening was performed
asdescribed (35) using 4-chloro-1-naphthol as substrate for
thehorseradish peroxidase reaction.
Characterization of the cDNA. The location of
restrictionendonuclease cleavage sites within the cDNA insert
wasdetermined by digesting the DNA with restriction enzymesunder
the conditions recommended by the manufacturer. Thesizes of the DNA
fragments were determined by agarose gelelectrophoresis (29).RNA
blot analysis was performed using poly(A)-contain-
ing RNA from HTC cells and normal rat liver (29).
RNApreparations were electrophoresed with 1.2% agarose
gelscontaining formaldehyde (29). RNA was then transferred toa
nitrocellulose filter and hybridized to a
nick-translated32P-labeled cDNA probe (29). The washed filters were
auto-radiographed with an intensifying screen at -80°C usingKodak
X-Omat AR x-ray film.
Preparation of Cell Extracts. E. coli strain HB101 contain-ing
pTALDH was grown overnight on a shaking incubator in1% (wt/vol)
Bacto-tryptone/1% (wt/vol) yeast extract/0.5%(wt/vol) sodium
chloride/50 ,tg of ampicillin per ml.
E. coli cells were harvested by centrifugation at 15,000 xg for
10 min and washed in 60 mM sodium phosphate buffer(pH 8.5)
containing 1 mM EDTA and 1 mM 2-mercaptoeth-anol. This and all
further manipulations were carried out at0°C-4°C. The cells were
sonicated in a minimal volume ofphosphate buffer and centrifuged at
48,000 x g. The super-natant containing the tumor ALDH isozyme was
recoveredfor further study.
Determination of the ALDH Phenotype. Total ALDH ac-tivity was
assayed at 25°C by monitoring the increase inabsorbance at 340 nm
caused by NADH or NADPH produc-tion during the oxidation of an
aldehyde substrate as de-scribed (2, 7). Activities were expressed
as milliunits per mg
of protein [1 milliunit = 1 nmol of NAD(P)H produced permin].
Protein concentrations were determined by the methodof Lowry et al.
(36) using bovine serum albumin as standard.Immunoblot analysis of
the tumor ALDH clone was
performed following NaDodSO4/polyacrylamide gel electro-phoresis
(37, 38). First and second antibodies were the sameas those used
for screening the cDNA library.ALDH activity following
polyacrylamide gel electropho-
resis was determined by using either propionaldehyde
orbenzaldehyde as substrate and NAD or NADP as coenzymein a
tetrazolium-linked staining system (2, 7).DNA Sequence Analysis.
Restriction endonuclease frag-
ments and exonuclease III-digested fragments were sub-cloned
into pTZ18R and pTZ19R plasmids (Pharmacia).DNA sequence was
determined by the Sanger et al. dideoxy-nucleotide
chain-termination method (39).Amino Acid Sequence Analysis. Amino
acid sequence an-
alysis of purified tumor ALDH was performed as described(40-43).
The purified salt-free enzyme was Iyophilized, re-duced with
dithiothreitol (in 6M guanidine-HCI/2mM EDTA,pH 8.1),
S-[14C]carboxymethylated, dialyzed, lyophilized,and cleaved with
CNBr in 70%o HCOOH. The resultingfragments were fractionated in 30%
acetic acid on a G-75Sephadex column (1.5 x 180 cm) and finally
purified byreverse-phase high-performance liquid
chromatography(HPLC) using gBondapak C18 columns and gradients
ofacetonitrile against aqueous 0.1% trifluoroacetic acid. Aminoacid
compositions were determined after acid hydrolysis (6MHCI, 0.5%
phenol in vacuo, 110°C, 24 hr) using a Beckmanmodel 6300 amino acid
analyzer. Liquid-phase Edman degra-dations were performed using a
Beckman model 890M seque-nator, and phenylthiohydantoin derivatives
were identified byHPLC (43).
RESULTSThe presence of the tumor ALDH in both primary tumorsand
in HTC cells should also be reflected in the mRNApopulations
expressed in these cells. Tumor ALDH transla-tion products from in
vitro translation were readily detect-able in the
poly(A)-containing RNA of HTC cells (Fig. 1).No tumor ALDH was
detectable in immunoprecipitates of invitro translations using
normal liver poly(A)-containing RNA(data not shown).
Starting with 10 ,ug of poly(A)-containing RNA from HTCcells, a
cDNA library of 100,000 independent memberscarrying inserts >1
kb was obtained. From this library, twotumor ALDH clones were
obtained with an indirect immu-nological probe. Three other clones
with similar restrictiondigest patterns were also identified with a
cDNA fragmentencoding the TCDD-inducible ALDH (generously
providedby T. Dunn, McArdle Laboratory for Cancer
Research,University of Wisconsin). The largest cDNA insert of
theimmunologically obtained clones, pTALDH, was chosen forfurther
study.A restriction map ofthe pTALDH clone demonstrates that
three Pst I sites were protected by the methylation step inthe
cDNA cloning (Fig. 2). This cDNA is -1.8 kb long andappears to
contain the entire tumor ALDH coding sequence.RNA blot analysis of
poly(A)-containing RNA from HTC
cells with pTALDH used as a probe reveals a species ofRNA
migrating at -1.8 kb. No band is detectable in
thepoly(A)-containing RNA of normal liver (data not
shown).Immunoblot analysis of E. coli HB101 extracts containing
pTALDH reveals a polypeptide that is cross-reactive
withanti-tumor ALDH antibodies and that comigrates with tumorALDH
from HTC cells (data not shown). Thus, the proteinsexpressed in HTC
cells and in E. coli have indistinguishablesubunit molecular
weights. Very little cross-reactivity is
Biochemistry: Jones et al.
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Proc. Natl. Acad. Sci. USA 85 (1988)
1 2 3 4 5 6
_ 66
- 45
J 29
FIG. 1. In vitro translation and immunoprecipitation of
poly-somal RNA from HTC cells. RNAs were translated in vitro,
im-munoprecipitated, and electrophoresed in a 12%
NaDodSO4/poly-acrylamide gel. One microgram ofeach RNA was
translated in a 50-,ulreaction mixture. Lane 1, total translation
products of poly(A)-containing RNA from HTC cells (10 ,ul of
reaction mixture); lanes2-6, indirect immunoprecipitated
translation products from 40 p1 ofeach reaction mixture using
anti-tumor ALDH. Lanes: 2, endogenoustranslation products; 3,
rabbit globin poly(A)-containing RNA; 4,total RNA from HTC cells;
5, non-poly(A)-containing RNA fromHTC cells; 6, poly(A)-containing
RNA from HTC cells. Arrowindicates migration of authentic tumor
ALDH from HTC cells.Numbers on right represent Mr x io-'.
seen in the extract of normal rat liver or in untransformed
E.coli strain HB101.
It is also noteworthy, since tumor ALDH functions as amultimer
(dimer) with relatively large (M, 50,000) subunits,that the pTALDH
cDNA encodes a functional ALDH thatpossesses many properties of
tumor ALDH (Table 1) (1-14).Like authentic tumor ALDH, the
pTALDH-encoded poly-peptide prefers aromatic aldehydes as substrate
and NADP+as coenzyme. Nondenaturing polyacrylamide gel
electro-phoresis followed by ALDH activity staining shows that
thepTALDH-encoded ALDH comigrates with tumor ALDHactivity from both
HTC cells and a hepatocellular carcinoma(data not shown).The
strategy for sequencing the pTALDH cDNA involves
the insertion of restriction fragments or exonuclease
IIIdeletion fragments from pTALDH into pTZ plasmids (Fig.2). The
length of the pTALDH cDNA is 1780 base pairs,including the two
linkers added during the cloning procedure
(Fig. 3). The pTALDH cDNA contains 173 nucleotides of
5'noncoding sequence and 247 nucleotides of 3' noncodingsequence.
The coding region of this cDNA begins with theinitiation codon
(ATG) at nucleotide 174 of the cDNA andextends through nucleotide
1532 (1359 bases). An opal(TGA) stop codon is present at nucleotide
1533 of the cDNA.The coding sequence encodes a polypeptide of 453
aminoacids of Mr 50,209. The ATG codons of the lacZ gene and
atnucleotide 104 of the cDNA were ruled out as start codonssince
stop codons would be encountered at nucleotides 23and 188 of the
cDNA, respectively. The pTALDH cDNAalso possesses a poly(A) tail of
50 nucleotides.The polypeptide encoded by the pTALDH cDNA has
been confirmed by primary structural analysis of tumorALDH
isolated from HTC cells (Fig. 3). Both authenticALDH isolated from
HTC cells and the enzyme expressedby the pTALDH-containing E. coli
HB101 were submitted toEdman degradation. The enzyme from HTC cells
was re-fractive to direct Edman degradation, indicating a blocked
Nterminus. In contrast, the results from 10 cycles of
Edmandegradation using the enzyme expressed in E. coli
areconsistent with the N terminus given (Fig. 3). Thus,
thebacterially produced ALDH polypeptide does not possessthe leader
peptide sequence of the lacZ gene product (34).
DISCUSSIONA cDNA encoding the tumor ALDH of rat liver has
beencloned, isolated, and confirmed by a variety of
techniques.Interestingly, this clone, pTALDH, produces a
functionaldimeric tumor ALDH molecule as demonstrated by
tradi-tional assay procedures and nondenaturing gel
electrophore-sis. In addition, the protein produced by E. coli
containingpTALDH begins with the same sequence (although with afree
N terminus), has the same subunit molecular weight,and displays the
same preference for aromatic aldehydes andNADP+ as substrate and
coenzyme as authentic tumorALDH (1-15). Moreover, the polypeptide
encoded bypTALDH is immunologically cross-reactive with
anti-tumorALDH antibodies by immunoblot analysis.The cloning
strategy used here offers a high probability of
obtaining a full-length cDNA clone. Methylation protectsagainst
internal cleavage by restriction enzymes while pre-paring the
linker ends of the cDNA for ligation. The sequen-tial addition of
the two linkers insures that all clones will becorrectly oriented
for expression from the lac promoter of apUC-type plasmid. If a
full-length clone is desired, gelpurification of cDNAs at least as
large as that necessary to
pTALDH cDNAE pp p B H H p
- - 4 - 0 - --
'M b b 6
4- * *
I I~~~0 0
1 200 400 600 800 1000 1200 1400 1600 184N UCLEOTIDES
FIG. 2. Restriction map and sequencing strategy for the pTALDH
cDNA. Arrows indicate direction and extent of nucleotide
sequencedetermination from clones obtained from restriction
endonuclease cleavage fragments of the cDNA (rightward arrows,
coding strand; leftwardarrows, noncoding strand). Arrows with
asterisks indicate sequence clones obtained from exonuclease III
cleavage of the cDNA. E, EcoRI;P, Pst I; B, BamHI; H, HindIII.
1784 Biochemistry: Jones et al.
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Proc. Natl. Acad. Sci. USA 85 (1988) 1785
Table 1. ALDH activity from various sourcesSpecific activity
Propionaldehyde/ Benzaldehyde/Cell type NAD NADP
pTALDH in HB101* 273.3 1125.6HTC* 153.1 567.7Rat hepatocellular
84.8 ± 14.6 260.4 ± 60.2carcinomat (18.3-285.9)t (6.5-1016.2)t
Normal rat liver§ 20.3 ± 0.4 7.2 ± 0.3E. coli strain HB101* 2.1
1.3*Average milliunits of protein per mg from at least three
determi-nations of specific activity.tAverage milliunits of protein
per mg for 24 tumors induced bydietary
acetylaminofluorene/phenobarbital exposure (7).lNumbers in
parentheses indicate the range of specific activities.§Average
milliunits of protein per mg from 51 determinations ofspecific
activity of normal liver.1Note that this activity is due not to the
presence of tumor ALDHbut to a decreased ability of normal rat
liver ALDHs to utilize thissubstrate/enzyme combination.
contain the complete coding sequence of the desired poly-peptide
greatly reduces the number of clones to be screened.RNA blot
analysis of poly(A)-containing RNA from HTC
cells demonstrates a mRNA species migrating at =1.8 kb.This,
coupled with the fact that a functional ALDH proteinis produced,
indicates that the entire coding sequence of thetumor ALDH isozyme
is contained within the pTALDHcDNA insert (1780 base pairs).
Furthermore, since no hy-bridization is detectable in RNA blots of
poly(A)-containingRNA from normal liver, it appears that expression
of tumorALDH mRNA is limited in the liver to tumor cells and
thatregulation of the tumor ALDH gene in the liver occurs at a
pretranslational level, although very low level expression
ofthis gene may not be detectable by RNA blot analysis. Thisalso
suggests that transcripts encoding the tumor ALDH aredistinctly
different from those encoding the normal ALDHisozymes.Another cDNA
clone encoding the TCDD-inducible
ALDH isozyme has been separately isolated and confirmed(T. Dunn,
personal communication). Identical restrictionenzyme sites are
found within the TCDD-inducible ALDHcDNA and the tumor ALDH cDNA.
RNA blot analysis ofRNA from livers of rats exposed to TCDD also
shows amRNA species migrating at :1L8 kb. No hybridization usingthe
TCDD-inducible ALDH cDNA is seen with mRNA fromnormal rat liver.
Therefore, it appears that these two inducibleALDH isozymes are
encoded by the same gene.Sequence analysis of the pTALDH cDNA
identifies an
open reading frame encoding a polypeptide with a molecularweight
comparable to the tumor ALDH present in vivo (13,14). The two ATG
codons upstream from the translationstart codon (i.e., the ATG
codons of the lacZ gene and theATG at nucleotide 104 of the cDNA;
see Fig. 3) were ruledout as initiator codons since they encounter
stop codons atnucleotides 23 and 188, respectively. The amino acid
com-position of a blocked tryptic peptide is also consistent withan
N-terminal tryptic fragment that would be generated iftranslation
started at nucleotide 174 (unpublished data).Interestingly, E. coli
transformed with pTALDH expressesan active protein with a free N
terminus that does not includeany lacZ residues. Rather, the
sequence obtained corre-sponds exactly to that expected for HTC
ALDH, but with-out the N-terminal blocking group. Whether this is a
result ofa difference in sequence recognition specificity of the
hostacetylation system is not known.
10 20 30 40 50 60 70 80 90 100 110 120
130GAATTCCTGCTGCACCCATATTTGAATATTGTTTTATCCTCATCACGCGAACTTCCTGGGGAAGGACTCCAAGGTCCAAGTGGGCGGGAAGGAGGGTCCTTAAATGTGCCCTCTTTCAGCTCTTGCTCAT
140 150 160 170 200
230TCCAGGAGTCCCAGCTGCTGGAGAAGGCTGTGTAGGAGTTGCA ATG AGC AGT ATC AGT
GAC ACC GTG AAA CGG GCC AGG GAG GCC TTT AAC TCC GGC AAG ACT CGA
Met Ser Ser Ile Ser Asp Thr Val Lys Arg Ala Arg Glu Ala Phe Asn
Ser Gly Lys Thr Arg260 290 320
TCG CTG CAG TTC CGA ATC CAG CAG TTG GAG GCG CTG CAG CGC ATG ATC
AAT GAG AAC CTG AAA AGC ATC TCT GGG GCG CTG GCC TCT GAC CTG GGCSer
Leu Gln Phe Arg Ile Gln Gln Leu Glu Ala Leu Gln Arg Met Ile Asn Glu
Asn Leu Lys Ser Ile Ser Gly Ala Leu Ala Ser Asp Leu Gly
350 380 410AAG AAC GAA TGG ACC TCC TAA TAT GAG GAA GTG GCT CAC
GTA CTG GAG GAG CTT GAT ACC ACA ATT AAG GAG CTC CCT GAT TGG GCT GAG
GAT GAGLys Asn Glu Trp Thr Ser Tyr Tyr Glu Glu Val Ala His Val Leu
Glu-Glu Leu Asp Thr Thr Ile Lys Glu Leu Pro Asp Trp Ala Glu Asp
Glu
440 470 500CCT GTG GCC AAG ACT CGC CAG ACC CAG CAG GAT GAC CTC
TAC ATC CAC TCG GAG CCC CTG GGT GTG GTC CTT GTC ATA GGT GCT TGG AAC
TAC CCCPro Val Ala Lys Thr Arg Gln Thr Gln Gln Asp Asp Leu Tyr Ie
His Ser Giu Pro Leu Gly Val Val Leu Val le Gly Ala TrP Asn Tyr
Pro
530 560 590 620TTC AAC CTC ACC ATC CAG CCC ATG GTG GGT GCC GTT
GCT GCA GGA AAC GCA GTG ATC CTC AAG CCC TCA GAA GTG AGC GGG CAC ATG
GCA GAC CTGPhe Asn Leu Thr Ile Gln Pro Met Val Gly Ala Val Ala Ala
Gly Asn Ala Vai Ile Leu Lys Pro Ser Giu Val Ser Gly His Met Ala Asp
Leu
650 680 710CTA GCG ACA CTC ATC CCT CAG TAT ATG GAC CAG AAT CTG
TAC CTA GTG GTC AAA GGG GGT GTC CCT GAA ACC ACG GAG CTG CTC AAA GAG
AGG TTTLeu Ala Thr Leu Ile Pro Gln Tyr Met Asp Gln Asn Leu Tyr Leu
Val Val Lys Gly Gly Val Pro Glu Thr Thr Glu Leu Leu Lys Glu Arg
Phe
740 770 800GAC CAC ATC ATG TAC ACT GGG AGC ACA GCC GTA GGG AAG
ATT GTT ATG GCC GCT GCC GCC AAG CAT CTG ACT CCT GTC ACC CTG GAG CTT
GGA GGGAsp His Ile Met Tyr Thr Gly Ser Thr Ala Val Gly Lys Ile Val
Met Ala Ala Ala Ala Lys His Leu Thr Pro Val Thr Leu Glu Leu G1y
Gly
830 860 890AAA AGC CCT TGT TAC GTG GAC AAG GAC TGT GAT CTA GAT
GTG GCT TGC AGG CGT ATC GCC TGG GGG AAA TTT ATG AAC AGT GGC CAG ACC
TGT GTGLys Ser Pro Cys Tyr Val Asp Lys Asp Cys Asp Leu Asp Val Ala
Cys Ara Arg Ile Ala Trp Gly Lys Phe Met Asn Ser Gly Gln Thr Cys
Val
920 950 980GCC CCA GAC TAC ATC CTC TGT GAC CCC TGC ATT CAG AAC
CAA ATC GTG GAG AAG CTC AAG AAG TCA CTC AAA GAT TTC TAT GGG GAA GAT
GCT AAGAla Pro Asp Tyr Ile Leu Cys Asp Pro Ser Ile Gln Asn Gln Ile
Val Glu LYS Leu Lys Lys Ser Leu Lys Asp Phe Tyr Gly Glu Asp Ala
Lys
1010 1040 1070 1100CAG TCC CGT GAT TAT GGG AGG ATC ATC AAT GAC
CGT CAC TTC CAG CGG GTC AAA GGC CTG ATT GAC AAC CAG AAA GTA GCC CAT
GGA GGC ACT TGGGln Ser Arg Asp Tyr Gly Arg Ile Ile Asn Asp Arg His
Phe Gln Arg Val Lys Gly Leu Ile Asp Asn Gin Lys Val Ala His Gly Gly
Thr Trp
1130 1160 1190GAC CAG TCC TCA CGA TAC ATA GCT CCA ACC ATC CTG
GTG GAT GTG GAC CCC CAG TCC CCA GTG ATG CAG GAG GAG ATC TTT GGG CCG
GTG ATG CCCAsp Gln Ser Ser Arg Tyr Ile Ala Pro Thr Ile Leu Val Asp
Val Asp Pro Gln Ser Pro Val Met Gln Glu Glu Ile Phe Gly Pro Val Met
Pro
1220 1250 1280ATT GTG TGT GTT CGA AGC TTG GAG GAG GCC ATT CAA
TTT ATC AAC CAG CGT GAG AAG CCC CTG GCA CTC TAT GTG TTC TCC AAC AAT
GAG AAG GTGIle Val Cys Val Arg Ser Leu Glu Giu Ala Ile Gln Phe Ile
Asn Gln Arg Glu Lys Pro Leu Ala Leu Tyr Val Phe Ser Asn Asn Glu Lys
Val
1310 1340 1370ATC AAG AAA ATG ATC GCA GAG ACA TCC AGC GGT GGA
GTG ACA GCC AAT GAC GTC ATT GTT CAC ATC ACC GTG CCC ACT TTG CCC TTT
GGT GGT GTGIle Lys Lys Met Ile Ala Glu Thr Ser Ser Gly Gly Val Thr
Ala Asn Asp Val Ile Val His Ile Thr Val Pro Thr Leu Pro Phe Gly Gly
Val
1400 1430 1460GGG AAC AGC GGC ATG GGG GCC TAC CAT GGC AAG AAA
AGC TTC GAG ACC TTC TCC CAC CGC CGC TCT TGC CTG GTG AAG TCT CTG TTG
AAT GAA GAAGly Asn Ser Gly Met Gly Ala Tyr His Gly Lys Lys Ser Phe
Glu Thr Phe Ser His Arg Arg Ser Cys Leu Val Lys Ser Leu Leu Asn Glu
Glu
1490 1520 1540 1550 1560 1570 1580 1590GCT CAC AAG GCC AGG TAT
CCC CCA AGC CCA GCC AAG ATG CCC CGG CAC
TGAGAGAGTGCTGCTGCACCTGACACATGTTTTGCTGGCTGTCTTGTCCTTGAGGAGTTGCTCATGAla
His Lys Ala Arg Tyr Pro Pro Ser Pro Ala LYS Met Pro Arg His *op1600
1610 1620 1630 1640 1650 1660 1670 1680 1690 1700 1710
1720GAGCCTCATCCTGGCTTATTCCCACCTGCCGTCCTGTGCTTAACTCCCCGAAGGACTCTCACCTCACTCCTCCAAATTCCACTGTTTGCTGGGCACAGAAATCAATAAAAGCCTCTGAGGAAAAGTCAAA1730
1740 1750 1760 1770 1780AAAAAAAAAAAAAAAAA
aAAAA1A~AAA1AAAAAAAAGCTGCAG
FIG. 3. Complete nucleotide sequence and deduced amino acid
sequence of the pTALDH cDNA. Solid underlining denotes
regionsconfirmed by Edman degradation; dashed underlining indicates
residue assignments supported at the protein level by amino acid
compositions.
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Proc. Natl. Acad. Sci. USA 85 (1988)
For pTALDH, the 5' consensus sequence (CCACC) re-ported for
eukaryotic mRNAs (44) is not present at nucleotidepositions - 5
through - 1 relative to the ATG start codon atposition 174 (1TGCA).
The consensus guanine at position + 4(44) relative to the ATG start
codon is also not present. Incontrast, the 3' nontranslated
sequence of the pTALDHcDNA insert does contain the AATAAA consensus
polya-denylylation signal (nucleotides 1700-1705) 19
nucleotidesfrom the poly(A) tail. The CACTG consensus sequence
noted(nucleotides 1676-1680) in class I genes (45) is also found
44nucleotides upstream from the poly(A) sequence.By alignment of
the peptide sequences of tumor ALDH
from HTC cells with the known human liver (NAD+ spe-cific) ALDHs
(40-42, 46), clear similarities are noted. Aminoacids 186-197 of
tumor ALDH are 58% similar to amino acids243-254 of the human E1 or
E2 sequences. Tumor ALDHamino acids 332-340 are 89o similar to
amino acids 397-405of E1 or E2. Weaker similarities (25-30%o) are
seen betweenamino acids 1-35 of tumor ALDH and amino acids 57-91
ofEl or E2 as well as for amino acids 341-377 of tumor ALDHand
amino acids 406-442 of E1 and E2. Interestingly, tumorALDH appears
to begin 57 residues downstream relative tothe start of the human
isozymes. Overall, a pattern of exten-sive variations within a
conserved basic framework appearsto characterize the HTC
NADP+-specific enzyme relative tothe human NAD + -specific enzymes.
A more detailed analysisof this conservation and divergence, with
functional implica-tions, will be the subject of a forthcoming
publication.The tumor ALDH cDNA obtained will be useful in
study-
ing the expression and the regulation of the tumor ALDHgene
during rat hepatocarcinogenesis. Studies using thisprobe to examine
the expression of the tumorALDH isozymein the resistant hepatocyte
model (47) will be helpful. On alarger scale, expression of this
isozyme in normal rat urinarybladder (20) and cornea (unpublished
results) as well as rathepatocarcinomas is being examined. Further
work directedtoward the isolation of cDNAs encoding the normal
liverALDH isozymes, genomic clones of ALDH isozymes, anddetermining
structure-function relationships between rat liverALDH isozymes
will also be useful.
We are grateful to Mr. Tracy Dunn for use of his cDNA
encodingthe TCDD-inducible ALDH isozyme. We would also like to
thankMs. June Reese for preparation of this manuscript. The work
issupported by National Cancer Institute Grant CA-21103 to
R.L.,University of Alabama funds to M.D.B., and National Institute
onAlcohol Abuse and Alcoholism Grant AA06985 to J.H.
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