- Email: [email protected]
The sequence of a Drosophila Cyp4e2 cytochrome P450-encoding cDNA
GENE J O U R N A L ON GENES AND GENOMES
AN INTERNATIONAL
ELSEVIER
Gene 179 (1996) 295-296
Short communication
The sequence of a
Drosophila Cyp4e2
cytochrome P450-encoding cDNA 1
Barry R. Pittendrigh, Giovanni Mocelin, Olga Andreev, Richard H. ffrench-Constant * Department of Entomology, University of Wisconsin, 237 Russell Laboratories, 1630 Linden Drive, Madison, WI 53706, USA Received 6 February 1996; accepted 20 February 1996
Abstract
A composite 1458-bp cDNA that encodes cytoehrome P450 (P450) Cyp4e2 has been constructed from clones isolated from two Drosophila embryonic cDNA libraries. The Drosophila eDNA open reading frame encodes a protein of 486 amino acids that is 40% identical and 61% similar to Cyp4dl from Drosophila. The predicted protein is unusual in that it appears to lack the hydrophobic N-terminus typical of microsomal P450s and also contains a small insertion at its C-terminus.
Keywords." Cytochrome P450; Insecticide resistance; Drosophila
Cytochrome P450 (P450) enzymes have been implicated in a wide range of metabolic activities in insects, including the metabolism of synthetic insecticides (Feyereisen, 1993). Certain P450s appear constitutively overexpressed in certain insecticide-resistant strains of Drosophila melanogaster (Waters et al., 1992) and the house fly Musea domestica (Carino et al., 1994). P450 gene fragments have been amplified from Drosophila rnelanogaster (Dunkov et al., 1996) using degenerate primers in polymerase chain reaction (PCR). We are interested in obtaining full length cDNAs for each of these genes in order to examine their range of metabolic activities following expression in heterologous systems. The sequence of the PCR-generated gene fragment corresponding to Cyp4e2 and the chromosomal localization of the gene have been reported elsewhere (Dunkov et al., 1996). A partial sequence of the 5'-end of the gene has also been reported by Amichot et al. in GenBank (accession no. X86076). Here we report the cloning and sequence of a composite cDNA coding for the complete ORF. A Drosophila embryonic cDNA library constructed in * Corresponding author. Fax + 1 608 2623322; e-mail: [email protected] 1 On request, the authors will supply detailed experimental evidence for the conclusions reached in this Short communication. Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; kb, kilobase(s) or 1000 bp; ORF, open reading frame; P450, cytochrome P450; P450, gene (DNA, RNA) encoding P450; UTR, untranslated region. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 1 1 1 9 ( 9 6 ) 0 0 3 7 8 - 2
the vector pNB40 (Brown and Kafatos, 1988) was screened with the gene fragment previously derived from degenerate PCR (Dunkov et al., 1996). 1 x 106 colonies were screened and three positive hybridizing clones were obtained. The longest of these was sequenced on both strands. Analysis of the sequence revealed that it was not full length but contained the Y-end of the ORF. In order to construct a eDNA coding for the complete ORF we amplified the Y-end of the gene from a different embryonic cDNA library (a kind gift of K. Thummel). PCR primers were derived from the partial sequence deposited in GenBank (forward: GCC GGA GAA GCT TGC TTA ACA AGT TCA ACG; and reverse: AAG GCC TGC ACA ATG GAA GAG CTG C G G TTT). The composite eDNA was assembled by ligating the PCR product onto the pNB40 clone using the NcoI site at the 5'-end of the latter. The composite Drosophila cDNA's ORF encodes a predicted protein of 486 aa. The nucleotide sequence is identical to the sequence of the PCR-generated gene fragment (Dunkov et al., 1996) in the region of overlap. The aa sequence is also 99.5% identical, in the region of overlap, to that predicted from the partial nucleotide sequence deposited in Genbank (the two amino acid replacements, both conserved, are K 2 1 5 > E and D 261>S). The predicted aa sequence of Cyp4e2 shows 40% identity and 61% similarity to Cyp4dl from D. melanogaster (Gandhi et al., 1992). The ORF of the composite eDNA and its deduced aa sequence are shown in Fig. 1. There are two unusual features of the predicted aa sequence in relation to other P450 protein sequences.
B.R. Pittendrigh et al./Gene 179 (1996) 295-296
296 1 l
151 51 301 I01 451 }r~l 601 201 751 251 9rll 301 1051 351 1201 401 1351 451
A TGGGAAACGCTCATC AAATGGC~AAGAATCCGTCAGAAA TA C T C G A C A C G G T T ~ M
G
N
A
H
Q
M
G
K
N
P
S
E
I
L
D
T
V
F
CCTC~3,TC~3CATCAGTATGGGAAAGACAACTTCGT S
W
W
H
Q
Y
G
K
D
N
F
CTTTTGGATTGGAACGTA~A~TCACCAG~C V
F
W
I
G
T
Y
S
N
V
L
V
C~ G T A C ~ T
S
S
K
Y
L
E
150 F
ATAC TCAGCAGTCAGACCCTGATCACAAAGTC C G A C A T T T A T C A A C T A A C A C A C C C " F T C ' C ~ T T A G G T C T C C T C A ~ A ~ A ~ A ~ A T ~ C A C ~ G A ~ A C C C C A ~ ~A~ C~CA~I~AG I L S S Q T b I T K S D I Y Q L T H P W b G b O b L T S T G S K W H K H R K M I T P A F H F N I L Q GACTTCCACGAGGTGATGAA~GAGAACT~ACCAAGTTTATCAAC4~ACTTGAAGACAGTT~C~CGGGGACAATATATTCGACTTCCAAGAACAGGC~CACTATTTGA~CCTG~ATGTCATTT~ACACA~~CA~ D F H E V M N E N S T K F I K H L K T V A A C, D N I F D F Q E Q A H Y L T L D V I C D T A M G V E I AATGCCATGGAAAAC CGCAGCTCTTCCATTGTGC AG~,CCTTCAAAGA CATGTGCTACAAC ATC AACATGAGAGCCTTC C ACC CGCTGAAC~CGCAATGAAC ~ C ~ T A T C G ~ C ~ A ~ A C C ~ A T A C ~ A ~ N A M E N R S S S I V Q A F K D M C Y N I N M R A F H P L K R N E L L Y R L A P D Y P A Y S R T L K A C A C T G C A G G A C ~ A C C A A C G A G ATCATCGCTAAGCC~ATTGAAGC CCACAAATCC, C ~ T G C A G ' g T T C G A C C ~ C ~ A G ~ C A C T ~ G ~ C ~ A G A C A C ~ C ~C C A C T A ~ A ~ T L Q D F T N E I I A F, R I E A H K S G A V S T N A G D E F T R K K M A F L D T L L S S T I D G [~ P CTGAAC TC C AAGGAACTGTAC GAAGAGGTCTCCACAT ~'fATGTTTGAAGGACATGATACTACCACTTCTGGA GT~TCG T T C G C T C ' T C T A ~ T A ~ CAT C ~ G A ~ C A ~ A T ~ ~ A ~ C ~ L N S K E b Y E E V S T F M F E G H D T T T S G V S F A V Y L L S R H Q D E Q R K L F K E Q R E V M GGT AATTC C GAACTGGGTCC~C GAT<]CC AC C TTCCAGGAAA TAT C C C A A A T G A A G T A C T T G G A T C T C T T T A T C A A f ~ G A G G C C C A C ~ G ~ A ~ C C A G ~ C ~ A ~ C ~ A C ~ T ~ACC~A~GA~A ~ ' T A T A ~ T G N S E L G R D A T F Q E I S Q M K Y L D L F I K E A Q R V Y P S V P F I G R F T E K D Y V I D G t~ CTCGTACCCAAGCGCACCACACTGAAC C T G C ~ C T C G T G A T G C T ~ A T A A C G A A A A G G ~ T T C A A A G A T C C CCACAAGTTCCGACC TGAGCGCTT TG'AGCTC~'AGAAA CCAC'GAC C ~ A ~ A ~ T A C C C~AG~ACCC L V P K G T T L N L G b V M b G Y N E K V F K D P H K F R P E R F E L E K P G P F E Y V P F S A ~ P CGAAACTC.CATT~GTCAGAAGTTC(]cCCTGCTGGAGATTAAGACTGTGGTATCCAAGATCATTAC~AACTTCGA~GTC~CTTCCT~C~A~AC~C~ T~AT~TATAT~ACCA CTA~ACTCCC~AG R N C I G Q K F A L L E I K T V V S K I I R N F E V L P A L D E L V S K D G Y I S T T I G L P D A E AGGAAGAAGCGCGATCCATACCGTCACAAATACGACCCTATTCTTTCCGCTGTGCTGACC CTGAAAT C Y ' G A A A A T C 4 3 T C T A T A C A T T C ~ - C T G ~ C ~ C < ~ 1458 R K K R D P Y R H K Y D P I L S A V L T L K S E N G L Y I R L K E R H * 486
50
300 I00 450 150 600 200 750 250 900 300 1050 350 1200 400 1350 450
Fig. 1. Nucleotide sequence of the Drosophila Cyp4e2 cytochrome P450-encoding cDNA ORF and the deduced aa sequence (GenBank accession no. U56957).
Firstly, when aligned to other P450s, Cyp4e2appears to lack the hydrophobic N-terminus typical of microsomal P450s. Although the previously reported 5'-UTR of Amichot et al. in GenBank (accession no. X86076) contains a second potential ATG 5' to the reported start site, we also confirmed that this was not in frame with the ORF. It therefore remains to be confirmed if the Cyp4e2 protein is membrane associated, or indeed is functional as a P450. Secondly, Cyp4e2 contains a short aa insertion at its C-terminus relative to other P450s. We cannot, however, confirm that this represents an unspliced intron in the cDNA, since this is a feature shared by the closely related Cyp4el.
Acknowledgement This work is part of an ongoing collaboration with the laboratory of Dr. R. Feyereisen to whom we are extremely grateful for his assistance with DNA probes
and sequence alignments. We thank T. Rocheleau for advice on cloning.
References Brown, N.H. and Kafatos, F.C. (1988) Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203, 425-437. Carino, F.A., Koener, J.F., Plapp, F.W. Jr. and Feyereisen, R. (1994) Constitutive overexpression of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides. Insect Biochem. Mol. Biol. 24, 411-418; Issn: 0965-1748. Dunkov, B.C., Rodriguez-Arnaiz, R., Pittendrigh, B., ffrench-Constant, R.H. and Feyereisen, R. (1996) Cytochrome P450 gene clusters in Drosophila melanogaster. Mol. General Gen. 251, 290-297. Feyereisen, R. (1993) In: Schenkman, J.B. and Greim, H. (Eds.), Cytochrome P450. Springer-Verlag, Heidelberg, pp. 311-324. Gandhi, R., Varak, E. and Goldberg, M.L. (1992) Molecular analysis of cytochrome P450 gene of family 4 on the Drosophila X chromosome. DNA Cell Biol. 11,397-404. Waters, L.C., Zelhof, A.C., Shaw, B.J. and Ch'ang, L.Y. (1992) Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila. Proc. Natl. Acad. Sci. USA 89, 4855-4859.
AN INTERNATIONAL
ELSEVIER
Gene 179 (1996) 295-296
Short communication
The sequence of a
Drosophila Cyp4e2
cytochrome P450-encoding cDNA 1
Barry R. Pittendrigh, Giovanni Mocelin, Olga Andreev, Richard H. ffrench-Constant * Department of Entomology, University of Wisconsin, 237 Russell Laboratories, 1630 Linden Drive, Madison, WI 53706, USA Received 6 February 1996; accepted 20 February 1996
Abstract
A composite 1458-bp cDNA that encodes cytoehrome P450 (P450) Cyp4e2 has been constructed from clones isolated from two Drosophila embryonic cDNA libraries. The Drosophila eDNA open reading frame encodes a protein of 486 amino acids that is 40% identical and 61% similar to Cyp4dl from Drosophila. The predicted protein is unusual in that it appears to lack the hydrophobic N-terminus typical of microsomal P450s and also contains a small insertion at its C-terminus.
Keywords." Cytochrome P450; Insecticide resistance; Drosophila
Cytochrome P450 (P450) enzymes have been implicated in a wide range of metabolic activities in insects, including the metabolism of synthetic insecticides (Feyereisen, 1993). Certain P450s appear constitutively overexpressed in certain insecticide-resistant strains of Drosophila melanogaster (Waters et al., 1992) and the house fly Musea domestica (Carino et al., 1994). P450 gene fragments have been amplified from Drosophila rnelanogaster (Dunkov et al., 1996) using degenerate primers in polymerase chain reaction (PCR). We are interested in obtaining full length cDNAs for each of these genes in order to examine their range of metabolic activities following expression in heterologous systems. The sequence of the PCR-generated gene fragment corresponding to Cyp4e2 and the chromosomal localization of the gene have been reported elsewhere (Dunkov et al., 1996). A partial sequence of the 5'-end of the gene has also been reported by Amichot et al. in GenBank (accession no. X86076). Here we report the cloning and sequence of a composite cDNA coding for the complete ORF. A Drosophila embryonic cDNA library constructed in * Corresponding author. Fax + 1 608 2623322; e-mail: [email protected] 1 On request, the authors will supply detailed experimental evidence for the conclusions reached in this Short communication. Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; kb, kilobase(s) or 1000 bp; ORF, open reading frame; P450, cytochrome P450; P450, gene (DNA, RNA) encoding P450; UTR, untranslated region. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 1 1 1 9 ( 9 6 ) 0 0 3 7 8 - 2
the vector pNB40 (Brown and Kafatos, 1988) was screened with the gene fragment previously derived from degenerate PCR (Dunkov et al., 1996). 1 x 106 colonies were screened and three positive hybridizing clones were obtained. The longest of these was sequenced on both strands. Analysis of the sequence revealed that it was not full length but contained the Y-end of the ORF. In order to construct a eDNA coding for the complete ORF we amplified the Y-end of the gene from a different embryonic cDNA library (a kind gift of K. Thummel). PCR primers were derived from the partial sequence deposited in GenBank (forward: GCC GGA GAA GCT TGC TTA ACA AGT TCA ACG; and reverse: AAG GCC TGC ACA ATG GAA GAG CTG C G G TTT). The composite eDNA was assembled by ligating the PCR product onto the pNB40 clone using the NcoI site at the 5'-end of the latter. The composite Drosophila cDNA's ORF encodes a predicted protein of 486 aa. The nucleotide sequence is identical to the sequence of the PCR-generated gene fragment (Dunkov et al., 1996) in the region of overlap. The aa sequence is also 99.5% identical, in the region of overlap, to that predicted from the partial nucleotide sequence deposited in Genbank (the two amino acid replacements, both conserved, are K 2 1 5 > E and D 261>S). The predicted aa sequence of Cyp4e2 shows 40% identity and 61% similarity to Cyp4dl from D. melanogaster (Gandhi et al., 1992). The ORF of the composite eDNA and its deduced aa sequence are shown in Fig. 1. There are two unusual features of the predicted aa sequence in relation to other P450 protein sequences.
B.R. Pittendrigh et al./Gene 179 (1996) 295-296
296 1 l
151 51 301 I01 451 }r~l 601 201 751 251 9rll 301 1051 351 1201 401 1351 451
A TGGGAAACGCTCATC AAATGGC~AAGAATCCGTCAGAAA TA C T C G A C A C G G T T ~ M
G
N
A
H
Q
M
G
K
N
P
S
E
I
L
D
T
V
F
CCTC~3,TC~3CATCAGTATGGGAAAGACAACTTCGT S
W
W
H
Q
Y
G
K
D
N
F
CTTTTGGATTGGAACGTA~A~TCACCAG~C V
F
W
I
G
T
Y
S
N
V
L
V
C~ G T A C ~ T
S
S
K
Y
L
E
150 F
ATAC TCAGCAGTCAGACCCTGATCACAAAGTC C G A C A T T T A T C A A C T A A C A C A C C C " F T C ' C ~ T T A G G T C T C C T C A ~ A ~ A ~ A ~ A T ~ C A C ~ G A ~ A C C C C A ~ ~A~ C~CA~I~AG I L S S Q T b I T K S D I Y Q L T H P W b G b O b L T S T G S K W H K H R K M I T P A F H F N I L Q GACTTCCACGAGGTGATGAA~GAGAACT~ACCAAGTTTATCAAC4~ACTTGAAGACAGTT~C~CGGGGACAATATATTCGACTTCCAAGAACAGGC~CACTATTTGA~CCTG~ATGTCATTT~ACACA~~CA~ D F H E V M N E N S T K F I K H L K T V A A C, D N I F D F Q E Q A H Y L T L D V I C D T A M G V E I AATGCCATGGAAAAC CGCAGCTCTTCCATTGTGC AG~,CCTTCAAAGA CATGTGCTACAAC ATC AACATGAGAGCCTTC C ACC CGCTGAAC~CGCAATGAAC ~ C ~ T A T C G ~ C ~ A ~ A C C ~ A T A C ~ A ~ N A M E N R S S S I V Q A F K D M C Y N I N M R A F H P L K R N E L L Y R L A P D Y P A Y S R T L K A C A C T G C A G G A C ~ A C C A A C G A G ATCATCGCTAAGCC~ATTGAAGC CCACAAATCC, C ~ T G C A G ' g T T C G A C C ~ C ~ A G ~ C A C T ~ G ~ C ~ A G A C A C ~ C ~C C A C T A ~ A ~ T L Q D F T N E I I A F, R I E A H K S G A V S T N A G D E F T R K K M A F L D T L L S S T I D G [~ P CTGAAC TC C AAGGAACTGTAC GAAGAGGTCTCCACAT ~'fATGTTTGAAGGACATGATACTACCACTTCTGGA GT~TCG T T C G C T C ' T C T A ~ T A ~ CAT C ~ G A ~ C A ~ A T ~ ~ A ~ C ~ L N S K E b Y E E V S T F M F E G H D T T T S G V S F A V Y L L S R H Q D E Q R K L F K E Q R E V M GGT AATTC C GAACTGGGTCC~C GAT<]CC AC C TTCCAGGAAA TAT C C C A A A T G A A G T A C T T G G A T C T C T T T A T C A A f ~ G A G G C C C A C ~ G ~ A ~ C C A G ~ C ~ A ~ C ~ A C ~ T ~ACC~A~GA~A ~ ' T A T A ~ T G N S E L G R D A T F Q E I S Q M K Y L D L F I K E A Q R V Y P S V P F I G R F T E K D Y V I D G t~ CTCGTACCCAAGCGCACCACACTGAAC C T G C ~ C T C G T G A T G C T ~ A T A A C G A A A A G G ~ T T C A A A G A T C C CCACAAGTTCCGACC TGAGCGCTT TG'AGCTC~'AGAAA CCAC'GAC C ~ A ~ A ~ T A C C C~AG~ACCC L V P K G T T L N L G b V M b G Y N E K V F K D P H K F R P E R F E L E K P G P F E Y V P F S A ~ P CGAAACTC.CATT~GTCAGAAGTTC(]cCCTGCTGGAGATTAAGACTGTGGTATCCAAGATCATTAC~AACTTCGA~GTC~CTTCCT~C~A~AC~C~ T~AT~TATAT~ACCA CTA~ACTCCC~AG R N C I G Q K F A L L E I K T V V S K I I R N F E V L P A L D E L V S K D G Y I S T T I G L P D A E AGGAAGAAGCGCGATCCATACCGTCACAAATACGACCCTATTCTTTCCGCTGTGCTGACC CTGAAAT C Y ' G A A A A T C 4 3 T C T A T A C A T T C ~ - C T G ~ C ~ C < ~ 1458 R K K R D P Y R H K Y D P I L S A V L T L K S E N G L Y I R L K E R H * 486
50
300 I00 450 150 600 200 750 250 900 300 1050 350 1200 400 1350 450
Fig. 1. Nucleotide sequence of the Drosophila Cyp4e2 cytochrome P450-encoding cDNA ORF and the deduced aa sequence (GenBank accession no. U56957).
Firstly, when aligned to other P450s, Cyp4e2appears to lack the hydrophobic N-terminus typical of microsomal P450s. Although the previously reported 5'-UTR of Amichot et al. in GenBank (accession no. X86076) contains a second potential ATG 5' to the reported start site, we also confirmed that this was not in frame with the ORF. It therefore remains to be confirmed if the Cyp4e2 protein is membrane associated, or indeed is functional as a P450. Secondly, Cyp4e2 contains a short aa insertion at its C-terminus relative to other P450s. We cannot, however, confirm that this represents an unspliced intron in the cDNA, since this is a feature shared by the closely related Cyp4el.
Acknowledgement This work is part of an ongoing collaboration with the laboratory of Dr. R. Feyereisen to whom we are extremely grateful for his assistance with DNA probes
and sequence alignments. We thank T. Rocheleau for advice on cloning.
References Brown, N.H. and Kafatos, F.C. (1988) Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203, 425-437. Carino, F.A., Koener, J.F., Plapp, F.W. Jr. and Feyereisen, R. (1994) Constitutive overexpression of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides. Insect Biochem. Mol. Biol. 24, 411-418; Issn: 0965-1748. Dunkov, B.C., Rodriguez-Arnaiz, R., Pittendrigh, B., ffrench-Constant, R.H. and Feyereisen, R. (1996) Cytochrome P450 gene clusters in Drosophila melanogaster. Mol. General Gen. 251, 290-297. Feyereisen, R. (1993) In: Schenkman, J.B. and Greim, H. (Eds.), Cytochrome P450. Springer-Verlag, Heidelberg, pp. 311-324. Gandhi, R., Varak, E. and Goldberg, M.L. (1992) Molecular analysis of cytochrome P450 gene of family 4 on the Drosophila X chromosome. DNA Cell Biol. 11,397-404. Waters, L.C., Zelhof, A.C., Shaw, B.J. and Ch'ang, L.Y. (1992) Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila. Proc. Natl. Acad. Sci. USA 89, 4855-4859.