224

Biochimica et Biophysica Acta, 1171 (1992) 224-228 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

Short S e q u e n c e - P a p e r

BBAEXP 90427

Molecular characterization of a putative peroxidase gene of Drosophila melanogaster Shu Wing

N g 1, M i c h a e l

Wiedemann,

Roland

Kontermann

and Gabriele

Petersen

lnstitut fiir Molekulare Genetik, UniL'ersitatHeidelberg, Heidelberg (Germany) (Received 2 October 1992)

Key words: Peroxidase gene; (D. melanogaster) We have identified genomic clones and corresponding cDNAs that encode a putative peroxidase of Drosophila melanogaster. The gene (DmPO) appears as a single copy gene located on the third chromosome at position 89 D / E . It is interrupted by seven small introns and one unusually large 5' intron (about 11 kb). Sequence analysis of the cDNA showed an open reading frame of 690 amino acids resulting in a protein of 77 kDa. The deduced amino acid sequence reveals an overall homology to myelo-, eosinophil and thyroid peroxidase, a human superfamily of peroxidases.

P e r o x i d a s e - o n e of the t h r e e m a i n e n z y m e s r e s p o n sible for the b r e a k d o w n of free radicals g e n e r a t e d d u r i n g c e l l u l a r m e t a b o l i s m - carries out a variety o f b i o s y n t h e t i c a n d d e g r a d a t i v e functions r e l a t e d to the c o n s u m p t i o n of h y d r o g e n p e r o x i d e [1]. T h e y serve to p r o t e c t a e r o b i c o r g a n i s m s against h a r m f u l side reactions of oxygen m e t a b o l i s m : free radical a t t a c k a n d p e r o x i d a t i o n of cellular constituents, p a r t i c u l a r l y the u n s a t u r a t e d fatty acids o f o r g a n e l l e a n d p l a s m a m e m b r a n e s [2]. Two d i f f e r e n t families of p e r o x i d a s e s have b e e n d e s c r i b e d . T h e s m a l l e r e n z y m e s (19 to 35 k D a ) including h o r s e r a d i s h p e r o x i d a s e [3], g l u t a t h i o n e peroxidase [4], c y t o c h r o m e c p e r o x i d a s e [5], a n d m a n g a n e s e p e r o x i d a s e [6] s e e m to exist in all species, as distantly r e l a t e d as m i c r o o r g a n i s m s , plants, a n d m a m m a l s . Several p e r o x i d a s e s have b e e n i d e n t i f i e d in m a m m a l s that s e e m to b e l o n g to a d i f f e r e n t p e r o x i d a s e family: thyroid- ( T P O ) , myelo- ( M P O ) a n d e o s i n o p h i l p e r o x i d a s e ( E P O ) . H u m a n M P O a n d h u m a n E P O play an i m p o r tant role in the m i c r o b i c i d a l system of n e u t r o p h i l s ( m o n o c y t e s a n d granulocytes), the h u m a n d e f e n s e against m i c r o o r g a n i s m s [7-10]. In the p r e s e n c e of

Correspondence to: G. Petersen, Institut fiir Molekulare Genetik, Universifiit Heidelberg, Im Neuenheimer Feld 230, 6900 Heidelberg, Germany. Enzymes: peroxidase, EC 1.11.1.7. The sequence data reported in this paper have been deposited at the EMBL data library under the accession number X68131. i Present address: Department of Medicine, Haematology-Oncology Division, Beth Israel Hospital, Boston, MA, USA. Abbreviations: SSC, 0.15 M NaCI/0.015 M Na3.citrate (pH 7.6).

H 2 0 2 , h M P O a n d h E P O catalyze the p r o d u c t i o n of the p o t e n t m i c r o b i c i d a l agent h y p o c h l o r o u s acid. W h i l e s c r e e n i n g a g e n o m i c Drosophila melanogaster library for an R N A p o l y m e r a s e subunit g e n e two over-

E

E

I

I

(E') E

E

E

I I

I

I

(E)

E

E

I

I

I

Bgl

E H

E

I

I I

I

c~ E I I

DmPO22

DmPO24

H E

P

H

HH

K

H

E Bgl

I I I

l

I

II

I

I

I

K

I

DmPO2

I

I

I

I

I

I

B

B

B

B

B

B

~

cDmPO38

2 kb

Fig. l. Genomic organization of the DmPO gene region. The solid line represents the genomic DNA, the regions covered by ADmPO22, ADmPO24 and ADmPO2 are indicated above. The corresponding cDNA clone cDmPO38 is shown below. The coding regions of the cDNA are shown as solid boxes, introns are indicated as open triangles, 5' and 3' untranslated sequences are shown as open boxes. Restriction enzymes: B, BamHI; Bgl, Bglll; E, EcoRl; H, HindIII; K, KpnI; P, Pstl, note that BglII and PstI restriction sites have not been mapped for the entire phage clones but in subclones and fragments and that ADmPO22, ADmPO24 contain additional EcoRl restriction sites, phage derived EcoRI sites are in brackets. DNA was isolated and analyzed by standard techniques [16].

225 lapping genomic clones, ADmPO22 and ADmPO24 (Fig. 1) have been isolated by cross hybridization under low stringency conditions (5 × SSC, 58°C, washes with 1 × SSC, 58°C) with a 1.2 kb B a m H l fragment from the largest subunit of RNA polymerase II [11]. Prelimi-

nary sequence information of the cross-hybridizing 1.5 kb E c o R l fragment of ADmPO22 and ADmPO24 showed no homology to an RNA polymerase subunit but contained an interesting open reading frame which encouraged us to use this fragment to screen a

G~TTCGC~TTGCGCAcTCCAAATTGGTG~TTGTAAA/U~TACGGTGTTT~GTATCGC~GG~TATTCCTCCGCT~TCCCC~CTTCACC~TCCATTGGCA AT/UUUiGTGAAA~CTACATTGTATTCGC~C~CTGTT~TT~TCGTCTGGAG~G~TATTCGCTTC~GCTATC gtaagtacaaaaagtcgggtaa¢ gcaecttaategc 1 82

/ /

a~t

11

kb

/ /

aagtcaactttaagtattcatttatatacacttttcaattacaettttag

ATG ATA AGG GCA C ~ ~ T CTT CTG CTC TTG GCC CTC TTG G ~ TTC ATC TCT AGT GCA CTT GGC CTA ~ N I R A R D L L L L A L L G F I S S A L G L K V

GTT TCC TCT GGC S S G

27

TAT ~ C ATA GTC CAC ~ T C ~ CCG C ~ TCT TCT TTT CCC ~ C TAT CAT GGC TTT AGT TAC CTA C ~ GGA TCA GCT CCA TAT Y H I V H N Q P Q S S F P N Y H G F S Y L Q G S A P Y

54

1~

GTG ATT GG g t a c g t t t t t t g g a a t a t t t c t t c t t t t a t g c t c t t a t g t a a g c c a t t a a c t t t t a g G ~ C AOC CTG CCG kCk TCT CCG GCT CCC V I G N S L P T S P A P

256

C~ ~T Q N

337

GCT GCT CCT CCG GCA GTT TGC G ~ ~ A A P f l A V C E K

418

G ~ GTG GCT ~ T G V A N

513

ACT CCT ~ T P K

594

CTG GTG GCT TTC G ~ ~ ~ G ~ T GTA CCC ~ T CCG ~ G TTC ACG CTG CAC ~ C ATG CAG TGG GGC CAG ATC ATG ACC CAC L V A F G E Q D V P D P E F T L H N N Q W G Q I N T H 1 8 5

6~

~ T ATG AGC ATG CAG GCC GGT GGC ACT CAG TCC A D N S N Q A G G T Q S

769

~ K

CCG TTT TCA TCG CCT GCC AGT CCG CCG GTA TCA GCT TAT GGT TAC AGC TTT CCC ACT GCT GGC AGG GTG TCA TGT P F S S P A S P P V S A Y G Y S F P T A G R V S C

93

ACG GCC TAT CGC ACT TTG ~ T G ~ TCC TGC ~ T CAT TTG ~ G CAA CCG GGC TTA T A Y R T L D G S C N H L E Q P G L 1 2 0

TCC ~ g t a a t t t a t c t t a a a t a t t a t t e a g c t a t g c a a g t g t t t a t a t g a t t a t g t t c c t a t g t t a g S K

G TAT GGT CGC TTG CTG Y G R L L

131

TAT GCC ~ C GGT ATT TCG GCA CCC ACC AGA TCC GTG ACA GGT ~ T GAG CTG CCC AGT GCT CGT CTT GTT TCC Y A D G [ S A P T R S V T G D E L P S A R L V S 1 5 8

gtaagtagtacatctctagctagatatatccctggeteeagtcgcttgattetccaacag 196

~ G CAT CCC ACA CGT TGT TGC ACC GAC GAT GGT CGC CTG ATT GGC CTG GAC ACC GCC CAC ~ G ACC TGT TTC GCC ATT K H P T R C C T D D G R L | G L D T A H K T C F A | 2 2 3

~9

ATT GTG CCA CCT CAC ~ T CCG GCT TAC TCC C ~ GTG GGC ACT ~ G TGC CTC ~ C TTT GTG CGC ACT CTG ACG ~ T CGG ~ C ] V P P H D P A Y S Q V G T E C L N F V R T L T D R D 2 5 0

930

TCG ~ C TGT CAG TAC ~ G GGT G ~ CCG GCA ~ G CAG TTG ACG GTG GTC ACT TCG TAC TTG ~ T CTC TCG CTG GTA TAT GGC S N C Q Y Q G G P A E Q L T V V T S Y L D L S L V Y G 2 ~

1011 ~ C T C C A T T C ~ C A G ~ C A G C G A T A T C C G T ~ G T T C C ~ G ~ G G C C G G A T G A T T G T G ~ G ~ G C G T ~ C G ~ G C C ~ G T G G N S I Q Q N S D I R E F Q G G R N I V E E R N G A K W 3

0

2

1092

CTG C ~ CTC TCT C ~ ~ T GTG ACC GGC ~ T TGT ~ T GCC GTT ~ T GCC AGT GAG GTG TGC TAC CGT TCC G ~ ~ C GTG AGG L P L S R N V T G D C D A V D A S E V C Y R S G D V R

11~

GTC ~ T V N

~G ~T Q N

1254

CTG ~ T L N

C ~ CAC TAC ~ T ~ T CGC ACG TTG TTC CAG ~ G GCG C ~ ~ G ATC ~ C ATT GCC CAG TAT CAG CAG ATC AGT TAC P H Y D D R T L F Q E A R K I N I A Q Y Q Q I S Y ~ 5 8

1335

TAC ~ Y E

TGG CTG CCC ATC TTC TTG GGT GGC ~ G ~ C ATG CTG ~ G ~ C CGG CTG ATC TAC ~ G GCT CCA TCC G ~ AGC TAT ~ L P I F L G G E N N L K N R L I Y K A P S G S Y 4 1 2

1416

ATC ~ C ~ T TTC ~ T CCC ~ C ATT ~ T CCA TCG GTG CTG ~ T ~ G CAC GCG ACG GCC GCT TTC AGA TAC TTC CAT TCC CAG I N D F D P N I D P S V L N E H A T A A F R Y F H S Q 4 3 9

1497

ATT ~ ! E

1592

CGT C ~ GTT CTC GGC TCT CTG ACC CTC AGC ~ C TGG TTC ~ T CGT CCC GGT ATT ATT ~ G GTT G ~ ~ T ~ C TTT ~ T TCC R Q V L G S L T L S D W F N R P G | ] E V G D N F D S 4 ~

16~

CTG ~ C A ~ GGA CAC GCC ACG ~ G CCC G ~ ~ G CTA ACT ~ T ATC ~ T TTT GAC C ~ ~ G gtgggtacacaaataacaaaatgagac L T R G H A T Q P E E L T D I N F D R Q

I~0

cacctttctaacctctcaacctttcctggcttag

1~8

CTG ~ C ATT ~ G CGT ~ T CGC GAT CAC GGT CTG GCT TCC TAC AAT ~ C ATG CGG ~ TTC TGC GGA CTG A ~ CGT GCC CAC L D I Q R N R D H G L A S Y N D N R E F C G L R R A H 5 4 2

1929

TCG TGG ~ G G ~ TAC GGT ~ T CTA ATA AGT CCA CCA ATT CTG ~ ~ CTT ~ G TCG CTA TAC CCG AGC CAC GAA ~ C GTG S W E G Y G D L I S P P I L E K L K S L Y P S H E D V 5 ~

2010

GAT CTG ACT GTG GGC GCC TCC TTG ~ G GCG CAT GTG GCC G ~ ACT TTG GCT GGA CCC ACC TTC CTG TGT ATC CTC ACG G ~ D L T V G A S L E A H V A G T L A G P T F L C I L T E 5 9 6

2091

C ~ TTC TAC AGA ACC A ~ GTG GGC ~ T CGC TTC TTC TTC G ~ ~ C G ~ GAC ~ G CTC ACT GGA TTT ACT CCT G gtaaaaag Q F Y R T R V G D R F F F E N G D K L T G F T P

21~

aacatttctcctctgactatttteaatattaatttatttatgtctaatttcgtag

2265

TTG CTG TGC ~ C ~ T GGC ~ C CAC ATT TCA TCC ATG CAG CCC ~ GCT TTC CGG ACT GTA TCC CAT TC L L C D N G N H I S S N Q P E A F R T V S H S

2347

attaaattggttatttggaaagtaactgagctctgaaattacag

2437

~ K

2525

~4

CCG G ~ CTG GCC ATT CTC CAG ACG ATC CTG CTG CGC G ~ CAC ~ T CGC ATT GCG GAC GCC CTG TCG GCA P G L A I L Q T I L L R E H N R I A D A L S A 3 3 1

GGT CGT TTG ~ g t e a g t g c c t a t t t a c t a t t a t g a g t t a a g a a c a t t t t a t t a a t t t t a a a a t t t t t g t g c a g T CTA CTG TCC ~ G CTG G R L D L L S E L

450

497

ATC ~ G CAC TTC CTG TTC A ~ A ~ ~ C ATG CCC TTC GGT TCC ~ T CTG CGT TCC I K H F L F R R N H P F G S D L R S 5 1 5

AT C ~ CTG ~ G ~ G CTG AGG ~ G GCC AGT ATG GCT CGT D Q L E E L R K A S H A R

620 633

gtaagtttaaettg 656

G ~ T CCG ATT ATA CCG TGC TCA ~ T ATT CCA C ~ GTC GAC CTC ACC N P ] I P C S N I P Q V D L T 6 7 1

TGG ATT GAT CAA ~ G CCA TAT GCC ACT GTA ~ T CCG TCT CAC TAC GGA ~ G ~ G T ~ TCCGCTGCTGTGG~TCTATGCTT~CT M I O Q K P Y A T V D P S H Y G K K * 690

AGTTGAATACAAATACATTTAGCTTAATCAATAACATAAACGTAAATGCAAAGCCCCTCCCCCCCCCCCCTTGTATGATAATGTCCCAAACTGATGTACAGAAATAAA

Fig. 2. DNA sequence and deduced protein sequence of DrnPO. Shown is the genomic DNA sequence and the deduced amino acid sequence except for the large 5' intron, where only the border sequences are given. The sequence starts with the 5' end of the cDNA clone cDmPO38 and ends with a putative polyadenylation signal. Numbering of the D N A sequence begins with the translation start codon. Intron sequences are given in lowercase letters. Single stranded templates were sequenced according to Sanger et al. [17] using [35S]dATP (Amersham) and T7 sequencing kits (Pharmacia). Reaction products were run on buffer gradient polyacrylamide gels [18]. Sequence analysis was done using P C / G E N E and H U S A R and the EMBL and SWlSS-PROT data libraries. The sequence data have been deposited at the EMBL data library under the accession number X68131.

226

Drosophila melanogaster embryonic cDNA library under stringent conditions (5 × SSC, 65°C, washes with 0.2 × SSC, 68°C). One of the positive clones (cDmPO38, Fig. 1) carrying the largest insert (2.4 kb) was chosen for further characterization. DNA sequence analysis revealed an open reading frame of

2070 nucleotides coding for a protein of 690 amino acids and 5' and 3' untranslated sequences of 189 and 140 nucleotides, respectively. By comparison of cDNA and genomic sequences we identified seven small introns (around 60 nucleotides) throughout the coding region of the gene. Restriction mapping and Southern

DmPO hNPO hEPO hTPO

" IR r ~ R D L ~ F I SSAL ............. ~L KVS~G Y ........ H H G V P F F S S L R C M V D L G p C ~ A G G L T[A]ENK]L L LA LI~GIV LA I LAT pQ p SE GAAPAVLIG]E VD TiS~LV L S SN E E A K~ N H~I~P~VLAT L V L A Q p CE G T D P A S P I q A V E TiS]VL R D C I A E A K l NRALA~$VTILIVHACT EA F FP F I S R G K E l L ~ K P E E I S I R V S S V L E E S K R

29 71 45 48

DmPO hNPO hEPO hTPO

lp]H . . . . . . . . . ['N~QP ~ S . . . . . . . . . . . . ~ P N Y - H G F[~Y L Q G Sr~P Y V I G N~---qPT S P A P -[~N P F~S~'P~ S[~]P LIVIDKA - Y K E R R ESl K[(~RL R S G S A S P N E L L]SrfF KQ p V A A T R T AVP~A~D y L H VAILID L L E R K L R S L ~RRI~F N V T LIVIDA A - Y N ~ T O K S l ~ R l R S G SA S p N D L USIY F KO p V A A T R T VVRI~AD yN H v,AL~G L L E E K L ~ P O R[~GL~F N.~VT L]V~DT A N YA T H Q R ~ L K KR - - - G I L S P A Q L ~ S K L P E PT[~GV I A$~_.~AE % N E ~ I Q A M K R K V N L K T Q Q SQ HIP~T

77 141 115 116

DmPO hHPO hEPO hTPO

V S A Y G Y S F P T A G R V~-~-~A P--~P]A V ~ . . . . E ~ T A~Y'R'~LD~S~-~H L E Q r ~ E ~ v A ~ S K Y GF~L~T~KF~A~D'~I~'~A D V l T P A Q L N V L S K SISIGICAIY O D V G V YIClP - E O DIKI- -Iy R TII TIG~I~CI~N R R Sp~T]L G~ASJNIRA F V]RJ~I~PAE]YJEp G~FJS~L D V L T E P Q L R L L S Q AISIGICAlL R D Q A E RICI- - - S D[KJ- -IY R ~ i T]GJRICI~N K R R]I~LL~G]ASINJQA L AII~II.IP ~AEIYZEIOGJLI~L D A L S E O L L S I IANH~JGcL~PYNLr~PK]C]PNTCLAN YI~.RJPIT[G[CAL~NRDHL~R~ASIMITALAL~.I:~PLPJVL~E[DGiF~Q

141 209 181 187

~7

DmPO hMPO hEPO hTPO

; G IJ T P G V K R NIG]FP V ALIA RIAIVSIN E IIV]RIFIP T D Q L TLP,DIQJEIRS~IN FIN Q~ GOIL L DIH I~L D F T P E P A A R ALSJFV T G V N GWT P S~]R R NIG[F L]'~L-~RIAIV,..~N ~ IL~A[F]p N e R L T SIDIRG R AL.qNI~HSU G OIF I OlH DIL D F['~P E S P A e V A F T A G V D

DmPO hHPO hEPO hTPO

~

DmPO hHPO hEPO hTPO

T V V]'~Y~----D]L]"S~L['~'~N['~ I Q O N S D I~'~- - E F['Q']G~RH i ~'~EE ~ - - -[M'-~Ag I ~ ' ~ L S R , V T G O]"C]D. . . . . . AVDr~S E V N A LIT S~FV]D[AIS]N[Vy GIS E E P L A R N L]RINN S N~JL~L L AMN ~R]F QI~N G]RA L]L P[F O N l HD D p]C[L - - - LTNRSIAIR i P N A LIT SIF V]D]AIS]NIVY GIS E V S L S L R UR]N R T N Y LIGIL L A 1M I~R]F Q DIN GIRA LIL I~F D N L H D D PICIL - - - LTNRSL~R I P N G LT ~ F L~AL.sJT~V Y GISJ'~pA L E R ~ I.~Nl~ T S A EIG]L L R~HA~JL R D " ~ R A YJL I~F V P P R A P AA CA P E P G I P G E T R G P

:'I~""~'"L'"LOTA'~I~I~]

ETSCV~OPP . . . . . . . . E R['~CAQ L P P . . . . . . . . QML~JCENQHP . . . . . . . .

'VI~I"~ .........

~SOV~TEFclc'~V~TLTOR~SI-"ICOY~'~'P^EplL

IC FIP L K lip PI.IDI. . . . . . . . . IPle XK -.OAOICll PIFIFIRISCPA C P G S . I T I - - - R . p [ l [C F"IP[~K lIP P]NLDj . . . . . . . . . II~e I K- N O R olcIi PIFJFII~sA P S C pQ~K N e V - " " RNpII [CF1P[I]QL[~EARPAAGTACL[~F]-~]R-SSAA[~GT'~'D'~'GALFGNLSTANP---RQ~M

DmPO hHPO hEPO hTPO

~ Y R S['~'[V[~V N Q N~'I'I~G ~ I]-L]Q~ I [ ' ~ ' ~

DmPO hHPO hEPO hTPO

GJEJNH LJ'~N R C Ij'Y]KAP SG S Y I ND F~]P['~ZJ'~SJV'IL~IE H ~ F J ' ~ $ O ~ I E - - - G J ~ L L S E L R Q V~G SJ'~Tf~D ~ P T A H R[K.JYL P T]Y]R . . . . . . . . S Y N DS~D PIRJ A]NIV F "ITIqA F R,_YJqI~TLlilO P F N FIR L DIN R Y 0 P 14e P N P R V ell SIR V

DmPO hHPo hEPO hTPO

I[~D~ S~N"~

Y]D']DR T~'L~F ~ ' ~

F L AIGOITII~ISS E NJI~IEILI~.S14HIllLLL~RE HN IqLJAJT.ILJKS{L. ~lt ~ G e , ~ .

N I ~Q Y~]Qf]Is~']Y E ~

F L AJGDITIRIS T E TIPllqL A]A~I~I]L F~R e H N q q A I T ~ q l l .K]LN ~ W N GO ~ Y ~ E A R K ~ , qqN ~ q l l l l ~ e DF F L AIG DIGIRIAS E VLpJSLI.~TALI.~HL~L~]L R E H N R I L L ~ ~ A ~ U S A D A V YIO E A R KIV V GL.AJLI~IL]JT L R D Y

~ARAReT LGHIY[R. . . . . . . . PJEJAe Q O Y V G I~Je . . . . . . . .

C R

Z6~

13131 303 318 323 397 369 387

394 468 440 459

462 530 GYCSlVdVlg.P[RIV]~q~[VF-[TLqAFeIFqI~T~LQeF.FIeLI~SOYeASAP~SHVPIL~SA~02 G YIDIST A NLPJTM~JV F qT A A F RJFGLI~AT~H e L V RIqL~JA S e G e H e bLImPG~I,I L I . ~ A 522

F A S U RVV L E GG llOlp i LIRGIL.A TlPlAKILI)IR0 N0 l AVD EIilRe XlL FLEO~IR lt~Lp LIP~4. qO ~ R O, a 4P ~[Y ~A

601

F S~W t L I. RIGIGG LLDJPE]IJR GJL L A RIPIAKILJQV q..plO L N ME E L t E RLL.~VL S MS S T DLLD~AL~IM

593

P

DmPO hHPo hEPO hTPO

~ ~

DmPO hHPO hEPO hTPO

T K[~'miO Q K['~y A T V O p S H y G K K AS[I~R EAS SAI~IR G T EA]I~RE T F[~ODDKCGFPESVENGD FVHCEESGRRVLVYSCRHGYELQGREOLTCTGEGWD

hTPO

ADGAHPPCHASARCRNTKGGFQCLCADPYELGDDGRTCVDSGRLPRATWI

hTPO

RMTRTGTKSTLP I SETGGGTPELRCGKHQAVGTSPQRAAAQDSEQESAGHEGRDTHRLPRAL

DmPO hHPO hEPO hTPO

I FJ~

A. K IIV,~. ~IIIITI~R D Y ~ L ~

280 25: ~

~l~.swe~Y~D,

I SPPxlq-EI'rLIKsEqPS"E"vlq'TV~'~S~A"V~T'AI~T

E

Fq'elx'T~I'~YmT

POPE TVGO LI~TVL R N L KILIA RIKUN EgffIG T P N N l n l WNIGIGV SIEIP L KR K G R rIG PIL L AICIL I G TIQ F]R K L s Q p R . L A Q L S R V L K . Q DLLJA~KFL.[['yle. T p O . :lUll ~ XI~]X ~qq, L L ~ A R ~G ~L L ~ L F E . l O ~ e ~ A I ~ L E T PAD L S T A i A S R S VADLI..~| L DIL ylK H p O N IL~V~LJ~G L lq_qN F L p RA R T[G p[L F ALCJLI G I ~ H KAL

VJ~D-'~F FJE"~GO K L T G~'~P DJ"~LE~]RI-I'I'I~AJ-~NAJi~L L ~ ' ~ ' ~ ' ] N H l S S H O P" E U ~ T V~H S N P i I P[C'~N ~ Q V D['L[ DIGDR Fp/~[ENIE G - - -VIF[.~,NOlOlRQAIL.IAOIIS~LPlRIl IICDNITIGII - - TTVSKNN IIFINSMISIYPRD FVNICS~T LIPIAL NIq DIGDRFI.I~QKRG---VIFTIKRIQIRKA~I.ISRIISILSIP.[I IICDNITIGIi -TTVSR-D~JF,.~ANIYPRGFVN[CS~RjJPIRLN]q DDG[ . ~ ; / ~ S H - - - V[F T]O A]Q[RR['E'~E~'I'I~HL~LS[R]V i[C D N]TLGJL - X R V P N- ADIA.~JQV G K F P E D F E SLy) S[l PIG H NLl.q

601 672 644 664 670 738 709 729 679

FQPPLCKDVNEC

SHSLAALL ! GGFAGLTSTVI C

734 70/, 800

871 933

Fig. 3. Amino acid alignment of D m P O with hTPO, b M P O and bEPO. The sequence data used in this alignment are taken from Morishita et al. for h M P O [19], Ten et al. for h E P O [20] and Kimura et al. for hTPO [21] which has an additional C-terminal domain responsible for membrane attachment of the enzyme. Spaces are introduced for optimal alignment. The signal sequences are overlined. The proposed proximal histidine residue replaced by a glutamine in D m P O is indicated by an arrow, The proximal histidine identified by [15] is marked by an asterisk, the distal histidine and arginine are marked by open triangles. Identical amino acids are boxed.

227 analysis indicated that the most 5' intron was quite large and that the first exon was not included in ADmPO22. To isolate a genomic clone covering the 5' end of the gene we rescreened the genomic library under stringent conditions with a 0.2 kb EcoRI-BglII fragment from cDmPO38 containing almost exclusively the 5' exon. This resulted in the isolation of ADmPO2 (see Fig. 1). Restriction mapping and Southern analysis showed that this clone lacks the 3' located 1.5 kb EcoRI fragment but contains the rest of the coding region, the 5' end of the gene and the entire first intron. The genomic organization of the gene region is shown in Fig. 1. To confirm the hybridization results, a 0.9 kb EcoRI-PstI fragment hybridizing with the 0.2 kb EcoRI-BglII fragment representing the most 5' cDNA was sequenced. The sequence is identical with the 5' end of cDmPO38 up to a consensus splice site. The 5' intron is extremely large (about 11 kb) and separates the second exon immediately adjacent to the putative translation start codon from the untranslated first exon.The genomic DNA sequence (except for the first intron which has only been partially sequenced) and the deduced amino acid sequence of cDmPO38 are shown in Fig. 2. The deduced protein consists of 690 amino acids with a calculated molecular weight of 77 kDa and an isoelectric point of 6.3. There is a potential asparagine linked glycosylation site at position 310 and the first 14 amino acids show all characteristics of a signal peptide sequence as found in other secreted proteins [12]. A computer search in the SWISS-PROT and EMBL data libraries revealed a striking homology of the gene product of cDmPO38 to several mammalian peroxidases. An amino acid alignment of the putative Drosophila peroxidase (DmPO) with hMPO, hTPO, and hEPO is shown in Fig. 3. The human peroxidases are well characterized and despite differences and dissimilarities regarding absorption spectra and physicochemical and physiological properties, they are probably derived from a common ancestral gene [13]. Biochemical analysis of Drosophila peroxidases has shown the occurrance of several isozymes which could also be due to the existence of a gene family. To this end, we hybridized genomic DNA under low stringency conditions with a 1.9 kb BglIIHindIII fragment from cDmPO38, covering almost all of the coding region except for the last 130 nucleotides. Hybridization under these conditions results in the expected pattern (Fig. 4) and does not detect any additional bands, except for a weak signal at 3.8 kb in the BamHI-HindIII digest which should only be present in the BamHI digest. However, this signal persists under stringent hybridization and washing conditions and is most likely due to incomplete digestion of the genomic DNA by HindIII. The expected banding pattern has also been obtained with a genomic 4.3 kb

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Fig. 4. Genomic Southern blot hybridized under low stringency conditions with a 1.9 kb BgllI-Hindlll cDNA fragment. Genomic DNA was isolated according to Ashburner [22], digested with restriction enzymes as indicated, electrophoresed on 1% agarose gels (5/zg per lane), transferred to Hybond N ÷ (Amersham) and hybridized under low stringency (5 x SSC, 30% formamide, 42°C, washes with 2 x SSC, 42°C) following standard protocols [16]. DNA was radioactively labelled with [32p]dCTP by the random-primed method using multiprime labelling kits (Amersham).

EcoRI fragment covering parts of the first large intron and most of the coding region from ADmPO2 (data not shown). These results indicate that DmPO is a single copy gene and probably does not belong to a gene superfamily. Using the complete DmPO cDNA as a probe on Northern blots a single band of about 3.2 kb is detected in the poly(A)+-RNA fraction (data not shown). In situ hybridization under stringent conditions of the 4.3 kb EcoRI fragment on polytene chromosomes gives a single band on the right arm of the third chromosome at position 89 D / E (Fig. 5). The overall amino acid sequence homology of DmPO with the mammalian peroxidases is about 35% and in regions which are supposed to harbor the functional domains, the similarity increases to about 70%. These regions include the putative sites for the proximal and the distal ligands to the heme iron atom. In DmPO, however, the putative proximal histidine is replaced by a glutamine (see Fig. 3). This is in line with the recent observation that in hMPO substitution of His-416 by alanine has no effect on the properties of the enzyme [14]. The authors suggest that His-502 (His-437 in DmPO) constitutes the proximal ligand since its substitution by alanine leads to the loss or inappropriate configuration of the heme together with the loss of

228 working conditions. We also wish to thank V. Pirotta and B. Hovemann for the generous gift of D N A libraries. S.W.N. was supported by a fellowship of the Deutscher Akademischer Austauschdienst.

References

Fig. 5. In situ localization of DrnPO on polytene chromosomes. Salivary glands of third instar larvae of Drosophila melanogaster were squashed and hybridized following the protocol of Ashburner (1989). DNA was labelled with biotin-16-UTP by nick-translation (ENZO) and detection of hybridization was performed using the ENZO detection system. The chromosomes were hybridized with a 4.3 kb genomic EcoRl fragment (see Fig. 1). A single band can be detected on the third chromosome at position 89 D / E . The bar represents 50/zm.

peroxidasic activity. By X-ray crystal structure analysis of canine MPO Zeng and Fenna [15] clearly identified His-502 as the proximal ligand to the heme iron. In addition they have evidence that on the distal side of the heme His-261 and Arg-331 participate directly in the catalytic mechanism analogous to the non-homologous cytochrome c peroxidase. Both these amino acids are well conserved among the human peroxidase family and also in DmPO (see Fig. 3). The cloning of a putative peroxidase gene provides an excellent tool for further biochemical characterization of peroxidase function in Drosophila. We are especially grateful to Prof. E.K.F. Bautz for his encouragement, constant support and excellent

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