Gene. 121 (1992) 305-311 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

305

0378-l 119/92/$05.00

06727

Cloning and characterization of the ADH.5 gene encoding dehydrogenase 5, formaldehyde dehydrogenase (Recombinant

DNA;

Man-Wook

Hur and Howard

ofBiochemistryand

Departments 5122,

pseudogene;

promoter;

CpG island;

housekeeping

gene; transcriptional

human alcohol

factors;

alcoholism)

J. Edenberg

Molecular Biology, and qf Medical and Molecular

Genetics, Indiana

University School

ofMedicine,Indianapolis,

IN 46202-

USA

Received

by J. Piatigorsky:

21 April 1992; Revised/Accepted:

12 June/l5

June 1992; Received

at publishers:

9 July 1992

SUMMARY

Human X-alcohol dehydrogenase (x-ADH) is a zinc-containing dimeric enzyme responsible for the oxidation of longchain alcohols and o-hydroxyfatty acids. Class-III ADHs, of which X-ADH is the prototype, are widely produced and well conserved during evolution. This suggests that they fulfill important housekeeping roles in cellular metabolism. Recent evidence suggests that class-III ADH and formaldehyde dehydrogenase (FDH) are the same enzyme. We have isolated and characterized two overlapping genomic clones that cover the entire ADH5 (FDH) gene. ADHS is composed of nine exons and eight introns. Two major transcription start points were identified by primer extension. The 5’ nontranslated region is unusual in that it contains two additional upstream ATG codons, which would encode peptides of 20 and 10 amino acids. Neither of the upstream ATGs is in a good context for translation initiation, whereas the ATG initiating &khgr;-ADH is in a favorable context. The 5’ region of ADHS is a CpG island; it is extremely G+C rich and has many CpG doublets. It does not contain either a TATA box or a CAAT box. This is consistent with ubiquitous expression, and contrasts with the promoters of all previously cloned ADH genes, which are expressed in a tissue-specific manner. The 5’ region of ADH.5 contains consensus binding sites for the transcriptional regulatory proteins, Spl, AP2, LF-Al, NF-1, NF-A2, and NF-El. A 1.5-kb upstream fragment from ADHS was able to drive the transcription of a cat reporter gene at high levels in monkey kidney cells (CV-1). Several processed pseudogenes were also isolated.

Mammalian alcohol dehydrogenases (ADH; EC 1.1.1.1) are zinc-containing dimeric enzymes that catalyze the reversible oxidation of a wide variety of alcohols, using

NAD + as the preferred coenzyme (Ehrig et al., 1990). They form a gene family divided into at least five distinct classes with about 60% aa identity in interclass comparisons (Strydom and Vallee, 1982; Pares et al., 1990; Edenberg, 1991; Yasunami et al., 1991). Human x alcohol dehydro-

Correspondence

monkey

INTRODUCTION

Molecular

to: Dr. H. J. Edenberg,

Biology, Indiana

Drive, Indianapolis,

University

IN 46202-5122,

Tel. (317) 274-2353;

Department

of Biochemistry

School of Medicine,

and

635 Barnhill

USA.

kidney

Abbreviations: acetyltransferase;

aa, amino acid(s);

ADH,

alcohol

dehydrogenase;

ADH; bp, base pair(s); cat, gene encoding Cm, chloramphenicol;

Denhardt

solution,

0.02’y0

Ficoll/

0.02%

lacZ, gene encoding

Fax (317) 274-4686.

ribonucleotide; gene encoding

cell line;

polyvinylpyrrolidone/ 0.02% bovine serum albumin; FDH, formaldehyde dehydrogenase; FDH , gene encoding FDH; kb, kilobase or 1000 bp;

cpm, count(s)

ADH,

CAT; CAT, Cm per minute; CV-1,

b-galactosidase; nt, nucleotide(s); oligo, oligodeoxyPipes, 1,4-piperazine-diethanesulfonic acid; SDS, sodium

dodecyl sulfate; SSC, 0.15 M NaCl/ 0.015 M Na,.citrate pH 7.6; SV40, simian virus 40; tsp,transcription start point(s); UTR, untranslated region.

306 genase (x-ADH) is the prototype of class-III ADHs (Strydom and Vallee, 1982); it is responsible for the oxidation of long-chain alcohols and o-hydroxyfatty acids (Pares

wise comparisons among human, rat, horse and mouse; Kaiser et al., 1988; 1989; Giri et al., 1989a: Sharma et al., 1989; Edenberg et al., 1990) suggests that all of the class-

and Vallee, 1981; Wagner et al., 1984; Giri et al., 1989b). X-ADH is expressed in all tissues tested, unlike other ADHs which are expressed in a tissue specific manner (Adinolfi et al., 1984; Duley et al., 1985; Edenberg, 1991). It is the only ADH present in brain, placenta, and testis (Pares

III ADHs and FDH conclusion: and FDH both been

et al., 1984; Beisswenger et al., 1985; Dafeldeker Vallee, 1986). The human gene encoding X-ADH

Molecular cloning of the ADH.5 (FDH) gene should provide information on the structure of this gene and on its evolutionary origin: did it diverge from the other ADHs or

named ADHS (Smith, 1986). Formaldehyde dehydrogenase

(FDH;

and was

EC 1.2.1.1) is im-

converge from another (FDH) gene? an important first step in studying expression, which differs from the ADH genes. In this communication

portant in the detoxification of formaldehyde (Pommotabbed et al., 1989). Formaldehyde can be formed in animal cells from several exogenous and endogenous precursors including methionine, choline, homoserine, methanol, and xenobiotics (Uotila and Koivusalo, 1987). FDH can also metabolize methylglyoxal and other a-ketoaldehydes. All mammalian cells studied so far contain FDH (Uotila and

It should also provide the regulation of its other, tissue-specific we report the cloning,

sequencing, and characterization of the human ADH.5 (FDH) gene, the first class-III ADH gene to be cloned, and demonstrate that its 5’ flanking sequence can drive the transcription of a reporter gene.

Koivusalo, 1974; Kahn et al., 1984). Recently, it was shown that the aa sequence of five tryptic peptides of rat FDH were identical to the sequences of the rat class-III ADH, ADH-2 (Koivusalo et al., 1989). The two activities copurify through numerous steps and have identical physiochemical properties and the same wide tissue distribution. Purified rat FDH has ADH activity

RESULTS

AND DISCUSSION

(a) Isolation and characterization of MM.5 genomic clones Initial screening was carried out with mouse Adh-B2 cDNA as the probe to avoid bias toward a single gene in case several genes were present. Clone 27-1, which contains the sequence from intron 4 to beyond the 3’ nontranslated region, was isolated along with several pseudogenes. No full-length clone was obtained. The second screening yielded clone AC-4, which overlaps with 17-l

with the characteristics of a class-III ADH (Koivusalo et al., 1989). Thus, rat class-III ADH and rat FDH appear to be the same enzyme. The high degree of aa conservation among the mammalian class-III ADHs (greater than 91% aa identity in pair0.95 n n

are also FDHs. The mapping of human ADHS to the same chromosomal region reinforces this ADHS (Carlock et al., 1985; Giri et al., 1989a) (Kahn et al., 1984; Hiroshige et al., 1985) have locahzed to chromosome 4q21-q25.

0.62 nn

3.0

B h7-1

C

K-4

D;

1 9

I

;

,

(f

5

E

I

I I

SP I

I"

8 I

I1

E

SH

K

S

H

H

E

I

II

I

I

I

I

I

10

I

12

I

I

I4

I

I

Fig. 1. Structure and restriction map ofADH5 (FDH). (A ) Gene structure. Exons arc represented signal is marked as (A)n. (B ) Sequencing strategy. (C ) I clones C-4 and 7-l. (D ) Restriction Methods: a human genomic

cDNA

fragment

tion (Denhardt, was isolated. Hybridization

library was prepared in the vector lGeml1 (Promega, Madison, (1.9-kb PstI-EcoRI fragment; Hur et al., 1992) as a probe. Hybridization

1966)/5%

SDS/O.02

M Na.phosphate/S%

dextran

16

I

I

18

I

I

E

20 kb

I

I

I

by open boxes, intron sizes are in kb. The polyadenylation map: E, EcoRI; H, HindIII; K, KpnI; Sp, SphI; S, SstI.

WI) and screened without amplification using a mouse Adh-B, was at 42°C in 40”/, formamide/S x SSCjl x Denhardt solu-

sulfate, and final washing

was at 5O”C, 90 min in 1 x SSC/O.l%

SDS. Then 17-l

A second library was constructed and screened with a human X-ADH cDNA fragment (220-bp EcoRI-DdeI fragment; Giri et al., 1989a). was at 50°C in 6 x SSCj5 x Denhardt solution/O.l% SDS/O.O2% Na.phosphate; the final wash was at 55°C 20 min in 1 x SSCjO. 1%

SDS. After stripping

off the probe, the filters were hybridized

with a 190-bp fragment

from intron 4 (excised from 17-I);

the final wash was at 55”C,

20 min in 0.1 x SSC / 0.1% SDS. Probes were labeled with [c(-32P]dATP and [a-32P]dCTP either by nick translation (Tabor and Struhl, 1987) or by using random hexamers (Feinberg and Vogelstein, 1984). DNAs from the positive phage clones were characterized by restriction endonuclease mapping and hybridization analysis (Southern, 1975).

307 and extends from the 5’ upstream region to intron 6. In total, ten positive bacteriophages were isolated from ap-

polypeptide

prox. lo6 plaques. The ;i DNAs were subjected to restriction mapping and sequencing. Most clones were processed pseudogenes related to ADH.5. These explain some of the multiple bands that were reported to hybridize with the X-ADH cDNA (Sharma et al., 1989). Another ADHS

as a strong translational initiation codon (Kozak, 1987). It is therefore likely that the two upstream ATGs are bypassed by the 40s ribosomal subunit most of the time; their presence might, however, modulate the translation efficiency of the X-ADH mRNA.

-related pseudogene has been reported (Matsuo and Yokoyama, 1990). ADHS is composed of nine exons and eight introns, spanning 15 kb (Fig. 1). The intron-exon boundaries were

Giri et al. (1989a) reported an apparent in-frame ATG 51 nt upstream from the main ATG in their cDNA se-

established by comparison to the nt sequence of human X-ADH cDNA (Giri et al., 1989a; Sharma et al., 1989). All intron-exon junctions follow the GT/AG rule (Breathnach and Chambon, 1981). The introns of ADH5 interrupt the coding sequence at the same positions as found for the class-I and class-II ADH genes (Edenberg, 1991). The identical intron locations, as well as the overall homology ofADH5 to the class-I and class-II genes, demonstrate that the ADHS (FDH) gene is clearly a member of the ADH multigene family. The sizes of the introns differ from those of the class I and class II ADHs , and there was no apparent sequence homology of the introns or of the 5’ and 3’ untranslated regions with genes of these other classes. The sequence of ADHS is shown in Fig. 2. The largest exon, exon 5, is 261 bp; the introns ranged from 13 1 bp (intron 8) to 4.3 kb (intron 4). There are several discrepancies among the protein, cDNA, and genomic sequences. ADHS encodes Asp167 and Phe246, as in the reported protein sequence (Kaiser et al., 1988) and in the cDNA described by Sharma et al. (1989); the cDNA of Giri et al. (1989a) differed in 2 nt that would result in Tyr’67 and Leu246. There are also some minor differences in the 3’ nontranslated region, as noted in Fig. 2. (b) Identification of the fsp and upstream ATGs The 5’ ends of the mRNA were determined by primer extension (Fig. 3). The major tsp were a doublet at 78 bp and 80 bp upstream from the ATG codon, and a weaker one was noted 162 bp upstream (Figs. 2 and 3). Both the longer and shorter 5’ untranslated regions have two ATG codons upstream from the ATG codon that initiates translation of the X-ADH polypeptide (Figs. 2 and 4). This is rare in eukaryotic genes (Kozak, 1987). The upstream ATGs are out of frame with the main polypeptide, but in frame with each other (Fig. 4). If they were to be translated, they would make a 20-aa and a lo-aa polypeptide, respectively; both would terminate just before the ATG that initiates the X-ADH polypeptide. The sequences around both upstream ATGs have Cs at the -3 position and pyrimidines at the + 4 position (Fig. 4); thus, both are expected to be weak for translational initiation (Kozak, 1987). The ATG codon that initiates the X-ADH

position

fits the Kozak consensus

sequence,

with A at

-3 and G at + 4 (Fig. 4) and should, therefore,

act

quence, and therefore suggested that there may be a leader polypeptide (Fig. 4). No leader polypeptide was found in the reported protein sequence (Kaiser et al., 1988). Our ADHS genomic sequence does not have the particular ATG believed by Giri et al. (1989a) to be responsible for the leader polypeptide; that ATG is part of an 8-bp region that diverges from the gene sequence (Fig. 4). The 5’ region of the cDNA is very G+C-rich; our sequencing, using 7-deaza-dGTP at elevated temperatures, showed an additional C in the cDNA inserted between the putative leader ATG and the ATG that initiates synthesis of the X-ADH polypeptide, altering the reading frame. This C is also found in the ADHS gene (Fig. 4). Thus, there is no evidence in the ADHS gene for a leader peptide. (c) Structure of the ADHS promoter The proximal 5’ region of ADHS is extremely rich in G+C: 73% in the first 200 bp upstream from the translation start codon and 61% in the 358-bp upstream region (Fig. 2). This region has the characteristics of a CpG island. The CpG doublet is rare in mammalian DNA, when compared either with the number expected based upon the C and G content or with the number of GpC doublets (Gardner-Garden and Frommer, 1987; Bird, 1986). CpG is not, however, underrepresented in CpG islands. For ADHS , CpGs are about equal in frequency to GpC: the ratio is approx. 0.95 in the 358 bp upstream from the translation start codon. CpGs are found approximately as often as expected from the base composition: there are 35 CpGs in the upstream 358 bp, where 31 are expected, and 24 within the upstream 200 bp where 24 are expected. These data indicate that the upstream region of ADHS is a CpG island. CpG islands with similar characteristics are found in the 5’ regions of virtually all housekeeping genes (Gardiner-Garden and Frommer, 1987). Indeed, the wide tissue distribution of X-ADH (FDH) enzyme activity and its evolutionary conservation suggests that it plays an important housekeeping role. The presence of processed pseudogenes (this report and Matsuo and Yokoyama, 1990) implies that the gene is expressed even in germ line cells. The proximal 5’ region of the human ADHS (FDH) gene contains no TATA box and no CCAAT box. The G+Crich promoter does contain consensus sequences for the

308 NF-PC6 NF-A2 TTTCALU$BBIIYIZITTCCCTCACCGTCCACATCTGT&_I NF-~6

NF-El L(SBBGT~CGATAACTGCTGTMAGTTACAC~

NF-1 TTCCCGACMUAAAKGAGTTCTGCAACMGTCCGJCGGATTTTAGCA4TGAUC

-162 .

LF-Al

GCACMCTTAGCGGCACGCACCACAGCTCGA&&&UX

CGGCGCCGTGCAGCCTCGCCGGCGAGTG

SPI

-316

-211

TTCCGGTTGGAGCCATTGCAAGCCCC

-106

-80 -45 l * G3T JAGGCGCTCGCCACGCCCA~GCCTCCGTCGCTGCGCGGCCCACCCCGGATGTCAGCCCCCCGCGCCGACCAGAATCCGTGMC

SPI CCCCACGCCCCGCCCCCCTCGC __Ap_2___

___Ap_2___

NF-I _-_Ap_2___

1 Met Ala Asn Glu ATG CCC MC GAG UAGGGCCCGTTG

-1

___AP_2___ 10 Vol Ile Lys Cys Lys Ala Ala Val Ala Trp Glu Ala Gly GTT ATC A4G TGC AAG GCT GCA GTT GCT TGG GAG GCT CGA

0.35 kb ivs-l TTTCCCCACTTC&

30 20 Lys Pro Leu Ser Ile Glu Glu Ile Glu Vol Aia Pro Pro Lys Ala HIS Glu Val Arg Ile Lys 3.0 kb AAC CCT CTC TCC ATA GAG GAG ATA GAG GTG GCA CCC CCA AAG GCT CAT GAA GTT CGA ATC AAG ZLy\ATGATACATT ivs-2

TAATGTC‘CTGAT&

50 40 Ile Ile Ala Thr Ala Val Cys His Thr Asp Ala Tyr Thr Leu Ser Gly Ala Asp Pro Glu ATC ATT CCC ACT GCG GTT TGC CAC ACC CAT CCC TAT ACC CTG AGT CGA GCT CAT CCT GAG

70 80 60 Gly Cys Phe Pro Val Ile Leu Gly His Glu Gly Ala Gly Ile Val Glu Ser Val Gly Glu Gly VIII Thr Lys Leu Lys GGT TGT TTT CCA GTG ATC TTG CGA CAT GM GGT GCT CGA ATT GTG GM ACT CTT GGT GAG CGA GTT ACT MC CTG AAG Alo G GCG G WGGAGMTA

620 bp 1vs-3

100 90 ly Asp Thr Vol Ile Pro Leu Tyr Ile Pro Gin Cys Gly Glu Cys Lys ATTTGTTTCCffi CT GAC ACT GTC ATC CCA CTT TAC ATC CCA CAG TGT CGA GM TGC AAA

110 Phe Cys Leu As" Pro Lys Thr Asn Leu Cys Gin Lys Ile Ar TTT TGT CTA MT CCT MA ACT MC CTT TGC CAG AAG ATA AG LITAGTATCTT

4.3 kb ivs-4 TTTTTCTTT&

g Vol Thr Gin Gly A GTC ACT CM CCC

130 140 120 Lys Gly Leu Met Pro Asp Gly Thr Ser Arg Phe Thr Cys Lys Gly Lys Thr Ile Leu His Tyr Met Gly Thr Ser Thr AAA CGA TTA ATG CCA CAT GGT ACC AGC AGA TTT ACT TGC AM GGA AAG ACA ATT TTG CAT TAC ATG CGA ACC AGC ACA 160 167 170 150 Phe Ser Glu Tyr Thr Val Vol Alo Asp Ile Ser Vol Ala Lys Ile Asp Pro Leu Ala Pro Leu Asp Lys 'Jo1 Cys Leu TTT TCT GAA TAC ACA GTT GTG GCT CAT ATC TCT GTT GCT AA4 ATA CAT CCT TTA GCA CCT TTG CAT AbA GTC TGC CTT 180 Leu Gly Cys Gly Ile Ser Thr Gly Tyr Gly Ala Ala Val Asn Thr Ala Lys CTA GGT TGT GGC ATT TCA ACC GGT TAT GGT GCT GCT GTG AK ACT CCC AAG UAAGAGACTGAC

151 bp IVS-5 TCTTTACTCCTAG

200 210 190 Leu Clu Pro Gly Ser Vol Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ala Val Ile Met Gly Cys Lys Vol Ala TTG GAG CCT CCC TCT GTT TGT KC GTC TTT GGT CTG CGA CGA GTC CGA TTG GCA GTT ATC ATG CCC TGT AM GTG GCT 240 220 230 Cly Ala Ser A,-g Ile Ile Gly Val Asp Ile Asn Lys Asp Lys Phe Ala Arg Ala Lys Glu Phe Gly Ala Thr Glu Cys GGT GCT TCC CCC ATC ATT GGT GTG GAC ATC MT AM CAT AM TTT GCA AGG CCC AAA GAG TTT CGA CCC ACT GAA TGT 250 260 246 Ile Asn Pro Gin Asp Phe Ser Lys Pro Ile Gin Glu Val Leu Ile Glu Met Thr Asp Gly Gly Vol Asp Tyr Ser Phe ATT AAC CCT CAG CAT TTT ACT AM CCC ATC CAG GAA GTG CTC ATT GAG ATG ACC CAT CGA CGA GTG GAC TAT TCC TTT 270 Glu Cys Ile Gly Asn Val Lys Vol Met GAA TGT ATT GGT AAT GTG MC GTC ATG LIGAGTATGGGCT

1.2 kb lvs-6 CTCTGTGCCTGW

280 Arg Ala Ala Leu Glu Ala Cys HLS AGA GCA GCA CTT GAG GCA TGT CAC

290 300 Lys Gly Trp Gly Val Ser Val Val Val Gly Vol Ala Ala Ser Gly Glu Glu Ile Alo Thr Arg Pro Phe Gin Leu Val AAG CCC TGG CCC GTC AGC GTC GTG GTT CGA GTA CCT GCT TCA GGT GM GAA ATT CCC ACT CGT CCA TTC CAG CTG GTA 320 310 Thr Gly Arg Thr Trp Lys Gly Thr Ala Phe Gly G ACA GGT CCC ACA TGG A4A CCC ACT CCC TTT CGA G WTTCCATGG

2.2 kb 1vs-7

GATTTCTTTT&

ly Trp Lys Ser Val Glu GA TGG AAG ACT GTA GAA

340 350 330 Ser Vol Pro Lys Leu Val Ser Glu Tyr Met Ser Lys Lys Ile Lys Val Asp Glu Phe Vol Thr HIS Asn Leu Ser Phe ACT GTC CCA AAG TTG GTG TCT GM TAT ATG TCC AA4 AAG ATA &!A GTT CAT GM TTT GTG ACT CAC MT CTG TCT TTT 360 Asp Glu Ile Asn Lys Ala Phe Glu Leu Met His SW Gly Lys Se CAT GAA ATC MC AAA CCC TTT GM CTG ATG CAT TCT CGA AAG AG UAGGCTTTCT

370 131 bp P Ile Arg Thr ~VS-8 TTCCTTTTACffi C ATT CGA ACT

374 Vol Vol Lys 1le l ** 1 GTT GTA MG ATT TM TTCAAnnGAGAAAAAT~TGTCCATCCTGTCGTGATGTGATAGGAGCAGCTTMCAGGCAGGGAG~GCGCCTCCMCCTCACA

83

GCCTCGTAGAGCTTCACAGCTACTCCAG~TAGGGTTATGTGTGTCATTCATG~TCTCTATMTCMGGACMGGATPTTCAGTCATGMCCTGTTTTCT

187

GGATGCTCCTCCACATAAATMTTGCTAGTTI/\T~GG~TATTTIMCATBBZBBBI\GTPTT~CTACATT~GTGTG~~TTGTCTTGTTT~TGCT~TC

290

ATCATTGTCACGGTTTGTCTGCCCATTATCTTCATTCTGCMGGG~GGG~GGMGCAGGGCAGTGGTGGGTGTCTGAAACCTCAGAAACAT~CGTTGM

334

CTTTTMGGGTCTCAGTCCCCGTTGATTAAAGMCAGATCCTCCATCAGTGACAnnGTTMTCAGGACCCMGTCTGCTTCTGTGATATTATCTT~MGGGA

438

GGTACTGTGCCTTGTTCATACCTGTACCCC~TTCCTAGGATG~CATCTGCCTTCAGGGGGCACT~TGTATTATTGAAACAGCATTCTGGGCTTAAATAG

602

GTGTATGTATGTGTTGGTTGTGACTGTACTATTICTAGTATAGTGMCTACATACTGAATATCCMGTTCTCAGCACCTACTTTTGTCAAATCTTMCATTTTG

706

CCACTTCGAGATCACATTGCCATTCCTCCCCTCC~GAGGTMC~TTATCCACMTTTGATGTTTATCATTCCTGTGTTGTTGTACTTTCACTGTGTATMCC

810

TAAACCATCTACTCTTTAGTACTGTTTTATATATTTTTAAGCCTCATACTTGCTCATTCTACAGCTTTTTTCACTCATTATTGTATAATTATATCTGAAGCTCT

914

CGTTCATTMTTTTAGTCCTGTGTAGCAGMTTCMTTACGGGMCTACCATMTTTATCTGTTCTCCAGTTGMGGCATGMGTTGTTGCCAGTTTCTGTATT

1018

AT~CACTGTAGTGG~CATTCTTCTGCATTGGGCTLACTGCGTGTTACCTMGACGTATCACAGBACATTTAGCCTTATAGACATTGCCAAATTGC

1121

TCTTCAMCTAAATGTGACTTTTTGTGMTTACATGAGTATGGMT *

Fig. 2. Nucleotide

sequence

of the ADH5 (FDH) gene: exons, the boundaries

of introns,

and the 5’ and 3’ flanking

regions

are shown.

The 5’ flanking

region is numbered at the right, counting from the ATG start codon. Potential binding sites (based upon consensus sequences) for the transcriptional activators AP-2, LF-Al, NF-1, NF-El, NF-AZ, NF-KB, and Spl are indicated, as is an inverted G, T-related site (Carr and Edenberg, 1990). The tsp arc shown by single asterisks and the stop codon by three asterisks. The deduced aa sequence is numbered above. Asp’“’ and Phe246, in bold, match the cDNA of Sharma et al. (1989) but differ from that of Giri et al. (1989a). Two potential polyadenylation signals, AATAAA, are bold and underlined. The nt where polyadenylate was added in the cDNA of Sharma et al. (1989) is indicated by A Those nt underlined in the 3’ UTR were not found in

309 transcriptional regulatory protein Spl (Kadonaga et al., 1986) as might be expected from its base composition. It

TGCA

also has potential binding sites for several transcriptional regulators: AP-2, liver-specific transcription factor LF-Al, and nuclear factors NF-1, NF-El, NF-A2, and NF-KB (Faisst

and Meyer,

1992).

(d) Function of the ADHS

promoter

To test the ability of the 5’ flanking region of ADH.5 to drive transcription, a 1.5-kb fragment containing the region upstream from the coding sequences was subcloned into pCAT-Basic (Promega) to create pCAT-ADHS. This construct was transfected into CV- 1 cells for transient expression assays. Plasmid pCAT-ADHS showed strong promoter activity in CV-1 cells (Fig. 5), nearly comparable to that of the SV40 promoter + enhancer contained in the pCAT-Control plasmid (Promega). (e) Structure of the 3’ untranslated region of ADH.5 ADHS has two potential polyadenylation signals (AATAAA), 243 bp and 1092 bp downstream from the stop codon TAA. Giri et al. (1989a) found that the major X-ADH mRNA in human liver, spleen, and in several human cell lines was about 1.7 kb, the size expected for use of the first polyadenylation signal; there was also a weak

Fig. 3. Primer extension Methods: A human

of human liver RNA. Arrows

X-ADH cDNA fragment

) was isolated, dephosphorylated, nucleotide

with HaeIII. The 73-bp antisense

from a 6% polyactylamide/‘l

for primer extension. lo6 cpm)

were

formaldehyde/40

Total human

dissolved

fragment

kinased with [ Y-~*P]ATP and T4 poly-

kinase, and digested

was isolated

three tsp.

indicate

(98-bp EcoRI-PstI

liver RNA (50 ng) and primer (0.1 x

in 30 pl of hybridization

mM Pipes/O.4

strand

M urea gel, purified, and used

M NaCl/l

solution

mM EDTA).

(75%

After denatur-

ation at 90°C for 5 min, the mixture was slowly cooled to 36°C (2) or 42°C

(1) and hybridized

precipitation,

at this temperature

the primer extension

reaction

overnight.

band of about 2.6 kb. Sharma et al. (1989) isolated a cDNA of about 2.3 kb from a placental cDNA library, consistent with use of the second polyadenylation signal. There is 67% sequence identity between the proximal portion of the 3’ untranslated region of ADHS and the entire 3’ UTR region of mouse Adh-B, cDNA (Hur et al., 1992). This region extends from 67 bp to 250 bp downstream from the stop codon, ending just beyond the first AATAAA of the human gene. The sequence from 197 bp to 247 bp downstream from the stop codon, surrounding the first polyadenylation signal, is 87% identical. Thus, there is evolutionary conservation of the region containing

After ethanol

mixture was added (1 unit/PI ng per ml of actinomycin

D/4

same primer was used in a sequencing

units per ~1 of Moloney

murine leukemia virus reverse transcriptase),

and

Perron

the extension

were done at 44°C

placental

RNase inhibitor/l reactions

with phenol/chloroform was analyzed

mM dNTPj50

and ethanol

by electrophoresis

either of the reported

cDNAs

for 70 min. After extraction

precipitation,

the extended

in 6% polyacrylamide/‘l

product

M urea gels. The

(Giri et al., 1989a; Sharma

et al., 1985) subclone

region of ADHS as template quencing

reaction

by comparison

et al., 1989). An A 11 bp downstream

reaction

that contains to provide

with the pUC18 (Yanisch-

exon

1 and the 5’ upstream

size markers.

Although

shown was not clear, we were able to determine

with cleaner

sequencing

the sethe tsp

ladders.

from the stop codon in both of the cDNAs

was not found

in ADH5. The G at 491 bp, A at 956 bp, and C at 1060 bp downstream from the stop codon in the cDNA of Sharma et al. (1989) were not found in ADHS. The TA 436 bp downstream from the stop codon in ADHS was CC in Giri et al. (1989a). Methods: the Sst I and Eco RI fragments of the i clones were subcloned

into pUC18

and sequenced

with universal

and reverse primers (BRL). The inserts were excised from the plasmid,

digested

with Sau3A1,

and subcloned into M13mp18 or mp19 (Yanisch-Perron et al., 1985) for sequencing by the dideoxynucleotide chain termination method (Sanger et al., 1977). Sequencing was in both directions, except for the 262 bp at the end of the 3’ untranslated region; these 262 bp match a published cDNA (Sharma et al., 1989). Sequences across the Sau 3AI restriction sites were confirmed either by sequencing the sites. The sizes of the introns were determined from their nt sequence, by restriction mapping, intron

1. These sequences

have been submitted

to GenBank

under accession

with oligo primers or by subcloning or by the polymerase chain reaction

Nos. M8 1112-M8 1118.

fragments that span using oligos flanking

310

Fig. 4. Sequence comparison of this cDNA,

of these are underlined. cDNAs

ofthe 5’ untranslated

using 7-deaza-dGTP.

The critical residues

is doubly underlined;

region ofthe ADHS (FDH) gene and cDNA: cDNA’

In the ADH5 sequence, (at -3,

it is embedded

the two upstream

+ 4) of the Kozak

in a short sequence

(1987) consensus

sequence

= our resequencing

polypeptides

initiated

for each ATG are bold. The additional

at either

ATG in the

that diverges from the gene (underlined).

the first human polyadenylation signal and the only polyadenylation site detected in the mouse Adh-B2 mRNA (Hur et al., 1992). (f) conclusions (I) This report describes

= Giri et al. (1989a); cDNA2

ATGs and the TGA that would terminate

ACKNOWLEDGEMENTS

We thank

Steve Fox for the preparation

of human

liver

RNA, Dr. Celeste J. Brown for the preparation and first screening of one human genomic library, and Dr. David the isolation

and characteriza-

tion of the human ADH5 (FL)H ) gene, the first ADH gene of its class to be isolated. The organization of this gene into nine exons interrupted with introns at the same positions as in the other members of the ADH multigene family firmly places it in the ADH family and suggests its FDH activity evolved from this basis. (2) The 5’ nontranslated region of the cDNA contains two ATG codons preceding the ATG that initiates X-ADH synthesis. Both upstream ATGs are in a poor translation initiation context, whereas the ATG that initiates &khgr;ADH is in a good context. (3) The promoter region is a CpG island with no TATA or CCAAT boxes. We have demonstrated that it is a strong promoter in CV-1 cells.

Goldman for the human X-ADII cDNA. This research was supported in part by grant RO 1 AA06460 from the National Institute on Alcohol Abuse and Alcoholism.

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