Proc. Natl. Acad. Sci. USAVol. 78, No. 5, pp. 2717-2721, May 1981Biochemistry

Alcohol dehydrogenase gene of Drosophila melanogaster:Relationship of intervening sequences to functionaldomains in the protein

(recombinant DNA/gene isolation/pBR322/evolutionary homology)

CHEEPTIP BENYAJATI*, ALLEN R. PLACEt, DENNIS A. POWERS, AND WILLIAM SOFERt§Department of Biology and McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

Communicated by Hamilton 0. Smith, January 15, 1981

ABSTRACT The gene that codes for Drosophila alcohol de-hydrogenase (ADH; alcohol:NAD' oxidoreductase, EC 1.1.1.1)was identified in a bacteriophage A library of genomic DrosophilaDNA by using ADH cDNA cloned DNA as a probe. The DNAsequence of the protein encoding region was shown to be in agree-ment with the amino acid sequence of the ADH. Two interveningDNA sequences (introns) were identified within the protein en-coding region: one was 65 nucleotides and located between thecodons for amino acid residues 32 and 33, and one was 70 nucleo-tides and located between the codons for amino acid residues 167and 168. Both contained the 5' G-T and 3' A-G dinucleotides char-acteristic of intron boundaries of eukaryotic genes. On the basisof secondary structure predictions, the first 140 amino acid resi-dues of Drosophila ADH are in an alternating a8-sheet/a-helixarrangement which is characteristic of the coenzyme binding do-main ofdehydrogenases. The smaller ofthe two introns interruptsthe domain predicted to bind the adenine portion ofthe coenzyme.

The molecular architecture ofmany eukaryotic genes resemblespatchwork (2-7). Interspersed within the protein encoding se-quences are untranslated regions that are excised during mRNAprocessing (4, 6). The exact function of these intervening se-quences, called "introns," is unclear (2, 6). A intriguing hy-pothesis is that the introns divide the coding sequence into unitsthat represent functional domains ofa protein (2-3, 7). This ideais best illustrated in the immunoglobulin genes (8-10). Suchunits could be used again and again in various genes to generatenew protein structures (3).The recent cloning and determination of sequence of the al-

cohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase,EC 1.1. 1. 1) gene ofDrosophila melanogaster (11) provided theopportunity to test this hypothesis with a protein from a classof enzymes with well-demarcated functional domains, the de-hydrogenases. All the dehydrogenases studied to date consistof at least two domains (12): one binds the coenzyme, and theother binds the substrate. The latter domain also determinessubstrate specificity and provides many ofthe residues requiredfor catalysis. Although there is little amino acid sequence ho-mology among dehydrogenases in the coenzyme-binding re-gion, the secondary structure of this domain has been highlyconserved (12). This has given rise to the hypothesis that thedifferent dehydrogenases evolved by gene fusion ofan ancestralnucleotide sequence coding for the coenzyme-binding domainwith different nucleotide sequences coding for different, cata-lytic properties (13).We present here the entire nucleotide sequence of the cod-

ing region for the Drosophila ADHR gene. Two introns havebeen identified, one ofwhich is located in the midst ofthe-region

encoding the presumptive coenzyme-binding domain. Thesefindings are discussed in light of the proposed role of intronsin eukaryotic genes.

MATERIALS AND METHODSMaterials. Drosophila ADH cDNA (pADH.cDNA) was

cloned in pBR322 and propagated in Escherichia coli strainHB101 (11). The Drosophila bacteriophage Charon 4 A librarywas that constructed by Maniatis et al. (14).

Restriction Analysis and DNA Sequence Determination.Restriction fragments were analyzed by electrophoresis on agar-ose and acrylamide gels. 5'-Terminal labeling ofrestriction frag-ments and DNA sequence analysis were performed accordingto Maxam and Gilbert (15). Where possible, the sequence ofboth strands was determined. Each restriction site was con-firmed by analysis across the cleavage point. For stretches ofDNA in which only one strand could be sequenced, the ex-periments were repeated several times.

Containment. All recombinant DNA experiments were per-formed in P2 facilities in accordance with National Institutes ofHealth guidelines.

Peptide Sequencing. The isolation, purification, and se-quence determination procedures used to analyze the trypticpeptides of Drosophila ADH are published elsewhere (16, 17).

Secondary Structure Prediction. The computerized methodof Chou and Fasman (18) was used to predict the secondarystructure of the Drosophila ADH.

Circular Dichroism (CD) Measurements. CD spectra weremeasured on a Cary 60 spectropolarimeter under-nitrogen flushat 250C in 0.1 M sodium phosphate buffer (pH 7.50). The in-strument had been calibrated with d-10-camphorsulfonic acidas specified in the operation handbook of the unit. The CD datawere expressed in terms of mean residue ellipticitydeg cm2-dmol-'. A mean residue weight of 107 was used. Pro-tein concentrations were determined by amino acid analysis andabsorbance at 280 nm. The percentage helix was determinedas described by Siegel et al. (19).

Sequence Comparisons. The computer programs ALIGN,RELATE, and SEARCH and the data base available at theNational Biomedical Research Foundation were used to deter-mine the relationship among the protein sequences of Dro-sophila, yeast, and horse liver ADHs. The scoring matrix used

Abbreviations: ADH, alcohol dehydrogenase; CD, circular dichroism;kb, kilobase(s).* Present address: Cancer Biology Program, Frederick Cancer Re-search Center, P. 0. Box B, Frederick, MD 21701.

t Present address: Department of Biology, University of Pennsylvania,Philadelphia, PA 19104.

* Present address: Waksman Institute, Rutgers University, PiscatawayNJ 08854.

§ To whom reprint requests should be addressed.

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

2717

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2718 Biochemistry: Benyajati et al. Proc. Natl. Acad. Sci. USA 78 (1981)

0 2 3 4 5

& x S a. xSS MOe x S

,,, .lII11 II(11( i' II(A

no 2

r-37la 0 ~~~U)

M.% a M CD.U)Z ~ Oa m

IItmew I6xllx w

0w

(B) L

(C)

(D)

444 85bpADH-cDNA (529 b)

2645770 b691HOOC _ uNH2 ADH Polypeptide (255ao)

.88oa 135oo 32oo

FIG. 1. Restriction map and strategy for determining the sequencearound the ADH' gene of D. melanogaster. DNA for sequencing wasderived from a 4.8-kb EcoRI fragment of a 15-kb insert of DrosophilaDNA cloned in the A vector Charon 4 (14). (A) Restriction map of frag-ment. (B) Restriction fragments used in the sequence determination.(C) Regions covered by the sequence determination, as indicated byarrows. The region of the genomic DNA corresponding to the cDNAclone [pADH.cDNA (11)] is shown as a striped bar. (D) Coding regionof two introns in the 4.8-kb EcoRI fragment.

was the mutation data matrix at 250 PAM (percentage of ac-cepted point mutations). In the ALIGN comparison, a bias of6 was used and gap penalities of 6 and 12 were imposed (1). For

the RELATE and SEARCH analyses, segment lengths of 25residues were used.

RESULTS AND DISCUSSIONIsolation and Sequence of the ADH Gene. The cDNA

(pADH.cDNA) clone used to screen the genomic DNA libraryof Drosophila has been shown to contain sequences coding forthe COOH-terminal half of the ADHE protein (see Fig. 2) (11).By using this DNA as a probe, a clone from the A library ofDrosophila genomic DNA (14) with a 15-kilobase (kb) insertcontaining the ADH gene was identified. EcoRI digestion of theA DNA gave a 4.8-kb fragment to which the probe hybridized(Fig. 1). The location and orientation of the ADH gene withinthe 4.8-kb fragment were established by hybridization of thecDNA clone and labeled message to the various restrictiondigests.

By using the strategy depicted in Fig. 1, the nucleotide se-quence of the ADHs gene and flanking regions were deter-mined. Almost 2 kb of DNA was sequenced. In the presentpaper only the coding region (Fig. 2) of the gene and the intronswill be discussed.

Reliability of the Sequence. The sequence given in Fig. 2is based on three independent sets of data: DNA sequence ofthe pADH.cDNA clone; DNA sequence of a portion of the ge-nomic 4.8-kb EcoRI fragment; and amino acid sequence andcomposition of several purified tryptic peptides. Moreover, thecodon change (ACG to AAG) responsible for the amino acidreplacement threonine to lysine observed in the ADHF andADHs allelic variants (20) is found. And lastly, the translatedsequence agrees with a recently published amino acid sequence(21) except for our identification of a glutamic acid at position

TG TCG TTT ACT TTG ACC AAC AAG AAC GTG ATT TTC GTT GCC GGT CTG GGA GGC ATT GCT CTG GAC ACC AGC AAG GAG CTG CrC AAG CGCSer-Pha-Thr-Lau-Thr-Asn-Lya-Asn-Vat-Ite-Pho-VaL-A La-CLy-Lau-CLy-CLy-ILe-GLy-Lou-A p-rhr-.qr-L -Gt-u-L u-Lou-Lys-Arg444 444 444 444 444 444 444 7 -7 -7 -7 -71 10 20

CAT CTG AAG AAC CTG GTG ATC CTC GAC CGC ATT GAG AAC CCG GCT GCC ATr GCC GAG CTG AAG GCA ATC AAT CCA AAG GTG ACC GrC ACCAsp-Leu-Lye-A sn-Leu-VaL-ILe-Lau-A ap-A rg-Ite-GLu-A sn-Pro-A La-A ta-Ite-A La-GLu-Lou-Lye-A ta-Ite-Aan-Pro-Lya-VaL-Thr-VaL-Thr7 7 -7 -7 7 7 .7 -7 -7 7 -7 7 7 7 -7 -7 -7 -7 -7 7 77 -7 -7 7 -7 -7 77

30 40 50

TTC TAC CCC TAT GAT GTG ACC GTC CCC ATT GCC GAG ACC ACC AAG CTG CTG AAG ACC ATC TTC GCC CAG CTG AAG ACC GTC GAT GTC CTGPhe-Tyr-Pro-Tyr-Asp-VaL-Thr-VaL-Pro-Ile-Ala-GLu-Thr-Th'r-Lys-Lau-Lau-Lya-Thr-ILe-Ph.-A la-GLn-Lau-Lya-Thr-VaL-Aap-VaL-Lau-7 -7 -7 7 -7-7- 7-7-7-7 -7 -7- 7-,7- 7-77-7 _ 77 -7 _-760 70 80

ATC AAC GGA GCT CCT ATC CTC CAC CAT CAC CAG ATC GAG CGC ACC ATT GCC GTC AAC TAC ACT GGC CTG GTC AAC ACC ACG ACG GCC ATTIte-AAn-Cly-Ala-Cly-Ile-Lau-Aap-Asp-His-GLn-Ita-Glu-Arg-Thr-Ite-AZa-VaL-Asn-Tyr-Thr-Gly-Lau-Val-Aan-Tlhr-rhr-Thr-Ata-Its-7 -7 77 -7 -7 -77 -7 -7 -7 7 -7 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 44490 100 110

CTG GAC TTC TGG GAC AAG CGC AAG CGC GGT CCC GGT GGT ATC ATC TGC AAC ATT GGA TCC GTC ACT GGA rrC AAr GCC ATC rAC CAG GTGLau-Asp-Phe-Trp-Asp-Ly s-Arg-Lys-GZy-GLy-Pro-Gly-GZy-ILaZe-La-Cys-Asn-IZe-GZy-Ser-VaZ-Thr-GLy-Pha-Aann-ta-Its-Try-Gtn-VaL444 444 444 444 444 444_ 7--7 7 7 _ 7 444 444 444 444 444 444 444 444 444 444 444 444 444 444120 130 140

G rCCC GTC TAC TCC CGC ACC AAG CCC GCC GTG CTC AAC TTC ACC AGC TCC CTG GCG AAA CTG GCC CCC Arr ACC GGC GrG ACC GCT rAC ACCPro-VaZ-Tyr-Ser-GLy-Thr-Lye-A La-A La-VaL-VaL-Asn-Pha-Thr-Ser-Ser-Lou-A Za-Lys-Lau-A La-Pro-ILe-Thr-Gty-VaL-rhr-A La-Tyr-Thr444 444 444 444 444 444 444 -7-7---7-7-7-7 -7 -77_7-77160 160 170

CTC AAC CCC CCC ATC ACC CGC ACC ACC CTC CTC CACLtAtGjTTC AAC TCC TCC TTG CAT CTT GAG CCC CAC Grr Ccr GAG AAG CrC CrG cCTVaL-Asn-Pro-CZy-IZL-Thr-Arg-Thr-Thr-Lau-VaZ-His-Lys-Phe-Aan-Ser-Trp-Lou-Aap-VaL-Clu-Pro-CGn-VaL-Ata-GCu-Lya-Lou-Lau-ALa180 190 200

C GCAT CCC ACC CAG CCA TCC TTC CCC TCC CCC GAG AAC rTC CTC AAC CCT ATC CAA CTC AAC CAC AAC GGA CCC ATC rCC AAA CTC CAC CTCHis-Pro-Thr-GLn-Pro-Ser-Lau-A la-Cys-A La-GZu-Aan-Pha-VaZ-Lye-A ta-Ita-CGu-Lau-Aan-CZn-Aan-CLy-A La-IZa-rrp-Lya-Lau-Asp-Lou

V 7_II ___J -- 7 __ _71 P 7_ 7_ Jr --- _ _7' 7__ 7_ 7 7 7_ 7W 7_ 7_ -7 7

210 220 230

CCC ACC CTC GAG CCC ATC CAC TCC ACC AAC CAC TCC CAC TCC GCC ATC TAACGy -Th r-Lou-Clu -A La-It e-CL n-Trp-Thr-Lye-Hi -Trp-Ap-Se r-Cty-Ite240 250

FIG. 2. DNA sequence for the Adhs gene ofD. melanogaster. The initiation and termination codons are indicated by boxes. The translated proteinsequence is indicated below the nucleotide sequence. , Residues observed in tryptic peptides analyzed by automatic Edman degradation (16,17); ****, tryptic peptide whose composition agrees with that predicted from the DNA; solid vertical arrow, location ofthe two introns; open verticalarrow, 5' terminus ofthe cDNA clone (pADH.cDNA) derived from AdhF mRNA. The dashed line encloses the second-base codon change responsiblefor the threonine-to-lysine change observed between ADHF and ADH9 (20). Six silent third-base substitutions are observed between the ADHs andthe COOH-terminal segment of ADHF.

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Proc. Natl. Acad. Sci. USA 78 (1981) 2719

Table 1. Codon usage in the Drosophila ADH geneAmino

No. Codon acidAmino

No. Codon acidAmino

No. Codon acidAmino

No. Codon acidSer 1 TATSer 5 TAOSer 1 TAASer 0 TAG

LeuLeuLeuLeu

IleIlefle

Met

0 CCT8 0C02 CCA1 CCG

3 ACT22 ACC0 ACA2 ACG

Val 6 GCTVal 15 GCCVal 1 GCAVal 1 GCG

Pro 1 CATPro 3 CACPro 0 CAAPro 7 CAG

ThrThrThrThr

AlaAlaAlaAla

2 AAT14 AAC2 AAA

16 AAG

5 GAT7 GAC1 GAA9 GAG

His 0 CGTHis 5 CGCGln 0 CGAGln 0 CGG

Asn

AsnLysLys

AspAspGluGlu

0 AGT2 AGC0 AGA0 AGG

6 GGT8 GGC5 GGA0 GGG

25 and an additional tryptophan at position 251. We are con-

fident in our assignment at these positions because we haveisolated tryptic peptides whose sequences agree exactly withthose predicted from the DNA at these positions (see Fig. 2).Codon Usage. There is a distinct bias in the use ofdegenerate

codons for many genes (22, 23). In mammals, for example, Cand G are often used as the third codon base whereas A and Uare preferred in viruses (23). The codon frequencies in the trans-lation of the Drosophila ADHs gene (Table 1) show preferentialuse of codons with C or G as the third base. A similar bias isobserved with the major heat-shock protein from Drosophila(24). From the small amount of data available, it appears thatDrosophila may show choices among synonymous codons sim-ilar to what is observed with mammals (25).

Location of the Introns. We found two untranslated regionsin the protein encoding portion of the ADHs gene (Fig. 2). Thefirst, 65 bases, interrupts the protein sequence at amino acidresidue 32, whereas the longer (70 bases) second intron inter-rupts the protein sequence at amino acid residue 167. Two in-trons have also been observed by Goldberg (26) using S1 nu-

clease mapping experiments. The location of intron 2 issubstantiated by its absence from the nucleotide sequence ofthe pADH.cDNA clone. Moreover, both introns contain the 5'G-T and 3' A-G dinucleotides (27, 28) and the consensus se-

quence (29) (Fig. 3) found at the coding-intron junction ofmanyeukaryotic genes.

Because the x-ray crystallographic structure is not known forthe Drosophila ADH, the significance of the introns' locationswithin the gene is not immediately evident. The existence andlocation of functional domains must be based either on aminoacid sequence homology with other dehydrogenases whose pri-

CODING

5'GAT CT

Asp Le

5'TCCSer

rG AAG

eu Lys

mary and tertiary structures are known or on a search for a rec-

ognizable pattern of secondary structural elements predictedfrom the amino acid sequence.

Sequence Comparison. To assess homology among the al-cohol dehydrogenases, we compared the yeast, horse liver, andDrosophila ADH protein sequences. The yeast and horse liversequences were highly related. The significance of such com-

parisons can be assessed from an alignment score. The align-ment score is the number of standard deviations by which themaximal score for the sequences compared exceeds the averagemaximal score for random permutations of these sequences. Analignment score of3 is usually taken as an indication ofprobablerelatedness. For the yeast and horse liver sequences, a score

of 15.1 was found. The alignment obtained by this analysis wasessentially that proposed by Jornvall (30); we found 78 of the83 amino acid identities he observed. When the Drosophilasequence was aligned with either the yeast or the horse liversequence, the alignment scores were 1.13 and -1.01,respectively.A similar analysis using the computer program RELATE,

which makes an exhaustive comparison ofall segments ofa givenlength (25 residues in this case) from one sequence with thosefrom the other, again found little similarity between DrosophilaADH and the other two proteins. Moreover, when the Dro-sophila sequence was compared to itself, no duplicated se-

quences were found. Finally, to test whether the Drosophilaenzyme might be related to some other protein, 25-residue seg-ments of the Drosophila sequence were compared with all the25-residue segments of proteins or peptides currently in thedata bank. At the time the analysis was done, more than 1400sequences were available. In only one case was a significant

INTERVENING SEQUENCE CODING

65 nucleotides 3

GTAACTATGCGATGCC............ GTGTATTCAATCCTAGIAAC CTG GTG

Asn Leu Val

70 nucleotides 3'CTG GCGIGTAAGTTGATCAAAGG............ TTTATAACACCTTTAGIAAA CTG GCC

Leu Aiaj ILys Leu AlaI I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

FIG. 3. The DNA at theexon-intron boundaries of theDrosophila ADH gene. Verticalbroken lines show where the ex-

cision-ligation events must occur

to give mature mRNA. Theunderlined nucleotides are theconsensus sequence found at thecoding-intron junction of manyeukaryotic genes (29).

1 TIT8 TTC0 TTA3 ¶ITG

PhePheLeuLeu

o TCT5 TCCo TCA2 TCG

TyrTyrOCAM

o TGT2 TGCo TGA5 TGG

CysCysOPTrp

o CTT3 CTCo CTA

21 CTG

9 ATT14 ATCo ATA1 ATG

3 GTT9 GTCo GTA

10 GTG

ArgArgArgArg

SerSerArgArg

GlyGlyGlyGly

Biochemistry: Benyajati et al.

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2720 Biochemistry: Benyajati et al.

similarity found, involving residues 231-255 of the Drosophila near the boundary of the coenzyme-binding domain. It appearsADH and residues 344-368 of rabbit phosphorylase. The reason to divide the coenzyme-binding domain from what we assumefor this is unclear. to be the catalytic domain. The smaller intron, however, is in

Secondary Structure Prediction. Although there is little se- a /-sheet structure in what we predict is the adenine mononu-quence homology among the dehydrogenases, they all possess cleotide binding site. In fact, it separates the two invariant gly-a coenzyme-binding domain of extremely similar structure (12). cines in this unit from the invariant aspartate. Thus, whereasThe domain consists oftwo roughly identical units with the main the location ofone intron supports the idea that exons representelements being six strands of parallel sheet (PA, PB, P3C, /3D, functional units in a protein, the location of the other intron/3E, and 3F) and four helices (aB, aC, aE, and alF). There raises serious doubts about the hypothesis.are two helices on each side of the sheet. A similar situation has been observed in the chicken ovo-

To search for such a structure in the protein sequence of mucoid gene (35). Three ofthe introns delineate the three func-Drosophila ADH, the secondary structure of the enzyme was tional trypsin-binding domains ofthe secreted ovomucoid poly-predicted by the Chou-Fasman procedure (18). Overall, nine peptide as well as a separate domain that contains the signala-helical regions, 10 ,8-sheet regions, and 15 ,8-turn structures peptide. The domains delineated by these introns are similarare predicted (Fig. 4). The percentage helix present in this to those initially suggested from the protein sequence (36). Astructure (47.3%) is in good agreement with that calculated from second set of introns, however, divide these domains. To ex-the CD spectrum of the protein (51.7%). plain this finding, it is assumed that the primordial gene con-

Starting with residue 8, the predicted structure ofDrosophila sisted only of the 5' subdomain and that the intron was createdADH resembles the alternating /3-sheet/a helix arrangement in the course of the addition of the 3' subdomain (35).found in the coenzyme-binding domain ofdehydrogenases (12). We are therefore left with the problem ofdefining what con-This is seen more clearly in Fig. 5 where the predicted /3 sheet stitutes a domain in a protein. Such a problem was apparent withstructures of the Drosophila ADH are aligned with the /3 sheets the globins until it was noted that the amino acid sequence en-in the coenzyme binding domain of horse liver ADH. Perhaps coded in the central exon contained all the necessary contactmore importantly, the four amino acid residues found in all residues that determine the heme-binding site (3). In fact, thiscoenzyme binding domains (12, 13) are also present in Dro- region of the globin polypeptide can bind heme tightly and spe-sophila ADH. In all dehydrogenases, the last residue in /3A is cifically (37). It has been suggested that this "miniglobin" en-invariably a glycine; any larger residue would interfere with coded in the central exon might be a remnant of an early hemethe adenine ribose position (12). An invariant aspartate forms carrier (3). Perhaps a similar case can be made for the 5' sub-a hydrogen bond to the 2'-OH of the same ribose. A second domains of ovomucoid. However, if we accept the x-ray crys-invariant glycine in aB is close to the AMP phosphate. The third tallographic data which suggest that this protein family ofNAD-invariant glycine assumes the same relative position at the nic- dependent dehydrogenases is very old (12) and that the pri-otinamide ribose as does the first one at adenosine. Each one mordial dehydrogenase contained a mononucleotide bindingof these residues can be found at the same position in Dro- domain represented by the alternating /Ba/3 structure observedsophila ADH. in extant dehydrogenases (38), then the location of the intron

Based on these observations, we believe that the first 140 within this domain ofthe DrosophilaADH is not consistent withresidues of the Drosophila ADH are involved in binding of the this exon being a remnant of a nucleotide binding protein.coenzyme. This differs considerably from the horse liver en- These first 32 amino acid residues ofDrosophila ADH do notzyme, in which the first 170 residues include the Zn-binding appear to possess any unique features that would require suchresidues (31). The Drosophila enzyme does not have any bound a division from the rest of the protein sequence. Although themetals (32). 3-sheet structure of residues 9-13 has the highest nucleation

In support of our assignment of the coenzyme binding do- probability of all the 3-sheets, it is only speculative that themain to the NH2-terminal half of the enzyme is our observation nucleation site for the coenzyme binding fold resides in thisthat ADH from the ethyl methanesulfonate-generated mutant region. It should be noted, however, that the predicted struc-Adhn~l cannot bind coenzyme (33). Except for a charge differ- ture for this region bears a striking resemblance to the second-ence, ADH from Adhn~l resembles the wild-type enzyme in ary structure of a synthetic 34-residue polypeptide designed toevery physical property examined (33). The enzyme from this bind nucleotides that was shown to possess strong interactionmutant has been shown to have a glycine-to-aspartate change with cytidine phosphates and single-stranded DNA (39).at position 14 (34), the position where replacement by any larger It is possible that the smaller intron is a recent insertion intoresidue would interfere with adenine binding. the Drosophila gene and therefore may not represent the an-

If we now reexamine the location of the introns in the Dro- cestral genetic unit. However, from the work available on thesophila ADH gene, we find that the longer of the two is found globin (40), ovomucoid (35), and insulin genes (41), the acqui-

+55

H eli x ooo 63 + +

0l-sheet A/\A 10 - +++

coi 138/

183.FIG. 4. Secondary structure prediction for>211 Drosophila ADH by the computerized method of

214 - Chou and Fasman (18). a-Helix, 47.3%; f-sheet,- ++ - 231 24.2%; random coil, 28.5%. Percentage a-helix254

calculated from the CD spectrum of DrosophilaADH is 51.8 ± 2.6% (mean ± SD).

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Proc. Natl. Acad. Sci. USA 78 (1981) 2721

Adenine mononucleotide binding domain8B

Horse LADH SVF LGGVELSV MGCKAAG-AAR II GVEINKDK FAKAKEV-----GATECVN............ADHNvI:-:E,.G:ELDTS,.LKD-LK LERIENPAAEL.:,,..:.VFY

D m. ADH NVI FVAMLGGILDTSKELLKRD- LKNLR IENPAAIAELK'AINPKVTVTFYP

Nicotinamide mononucleotide binding domainflD PE

Horse LADH GGVD FS FEV RLDTMVTA LS CCOEAYGVSV IVGVPPDSONLSMNPMLL LSGRTW

D-.--- m.- ,Din. ADH 01LK TVDVL NEAG LDODH ERT AVNYTGL VNTTTA LDFWDKRKGGPGG I ICNI

sition of introns appears to be an early evolutionary event, per-

haps concomitant with the formation of a functional ancestralgene.

The final assignment of structural and functional domains forthe Drosophila ADH may have to await x-ray crystallographicstudy ofthe protein. However, the data presented here suggestthat the introns in the Drosophila ADH gene do not clearlyassort the nucleotide sequence into functional elements. Wemust await the isolation and analysis of other dehydrogenasegenes, especially those that code for proteins whose tertiarystructures are known, to see if the conclusions presented herecan be generalized.

We thank Peter Chou for performing the computerized secondarystructure prediction of the ADH protein. This work was supported inpart by National Institutes of Health Grants ES-01527 and GM-28791and Contract EY-76-S-02-2965 from the Department ofEnergy to W. S.;C. B. was a Postdoctoral Fellow ofthe Damon Runyon-Walter WinchellCancer Fund (DRG-175-F).

1. Dayhoff, M. 0. (1972) Atlas of Protein Sequence and Structure,5 (National Biomedical Research Foundation, Washington, DC),Suppls. 1-3, 1973, 1976, and 1978, Vol. 5.

2. Gilbert, W. (1978) Nature (London) 271, 501.3. Gilbert, W. (1979) in Eukaryotic Gene Regulation, ICN-UCLA

Symposium on Molecular and Cellular Biology, eds. Axel, R.,Maniatis, T. & Fox, C. F. (Academic, New York), Vol. 14, pp.1-12.

4. Darnell, J. E., Jr. (1978) Science 202, 1257-1260.5. Doolittle, W. F. (1978) Nature (London) 272, 581-582.6. Crick, F. (1979) Science 204, 264-271.7. Blake, C. C. F. (1979) Nature (London) 277, 598.8. Tonegawa, S., Maxam, A. M., Tizard, R., Bernard, 0. & Gilbert,

W. (1978) Proc. Natl. Acad. Sci. USA 75, 1485-1489.9. Sakano, H., Rogers, J. H., Hueppi, K., Brack, C., Traunecker,

A., Maki, R., Wall, R. & Tonegawa, S. (1979) Nature (London)277, 627-633.

10. Early, P. W., Davis, M. M., Kaback, D. B., Davidson, N. &Hood, L. (1979) Proc. Natl. Acad. Sci. USA 76, 857-861.

11. Benyajati, C., Wang, N., Reddy, A., Weinberg, E. & Sofer, W.(1980) Nucleic Acids Res., in press.

12. Rossmann, M. G., Liljas, A., Brad6n, C. I. & Banaszak, L. J.(1975) in The Enzymes, ed. Boyer, P. D. (Academic, New York),3rd Ed., Vol 11, pp. 61-102.

13. Eventoff, W. & Rossmann, M. G. (1975) in CRC Critical Reviewsin Biochemistry, ed. Fasman, G. D. (CRC Press, Cleveland,OH), pp. 111-140.

14. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell,C., Quon, D., Sim, G. K. & Efstratiadis, A. (1978) Cell 15,687-701.

15. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65,499-560.

I FIG. 5. Alignment of the /3-* sheet regions in the NAD+-binding

domain of horse LADH with theY D predicted 3sheets in residues 8-140

of Drosophila ADH (D. m. ADH).The adenine mononucleotide bind-ing fold (Upper) is aligned with thenicotinamide mononucleotidebinding fold (Lower) below. The

PF four invariant residues found in alldehydrogenases (12) are indicated

_KGA FG by white letters in black boxes. Thehelical section (aCD) connecting

*C and P3D in horse LADH (resi-G S VT G dues 250-258) is also found to beI @ helical in the Drosophila enzyme

(residues 68-83).

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