Molecular cloning and characterization of the ABC transporter expressed in Trachea (ATET) gene fromDrosophila melanogaster

BIOCHIMICA ET BIOPHYSICA ACTA

ELSEVIER

BBI Biochimica et Biophysica Acta 1309 (1996) 47-52

Short s e q u e n c e - p a p e r

Molecular cloning and characterization of the ABC transporter expressed in Trachea(ATET) gene from Drosophilamelanogaster H i s a n a g a K u w a n a a,b, K e i k o S h i m i z u - N i s h i k a w a a,~, H i d e n o r i Iwahana b, D a i s u k e Y a m a m o t o a,c,, a Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan b Department of Applied Biological Sciences, Tokyo Unit:ersity of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183, Japan c Yamamoto BehaL,ior Genes Project, ERATO (Exploratory Research Jbr Advanced Technology), Research Development Corporation of Japan (JRDC) at Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan Received 2 May 1996; revised 9 July 1996; accepted 17 July 1996

Abstract A novel member of the ATP-binding cassette (ABC) transporter proteins has been cloned from Drosophila melanogaster. The gene is designated as ABC Transporter Expressed in Trachea (Atet), because the transcript was localized to the respiratory system by in situ hybridization analysis of whole-mount embryos using digoxigenin-labeled RNA probes. The hybridization signal was also observed in amnioserosa. Northern blot analysis identified a single 4.5 kb mRNA expressed in all developmental stages at a relatively constant level. The Atet gene mapped to 24E on the left arm of the second chromosome. The Atet protein shows extensive homology with the Drosophila white gene product, which is reported to form heterodimers with the brown or scarlet gene product to transport guanine or tryptophan into the pigment cells in the compound eye. By analogy, Atet is suggested to be involved in transporting a small molecule after dimerization with a partner protein. Keywords: Multi-drug resistance; White gene; Transmembrane protein; Embryogenesis

The ATP-binding cassette (ABC) transporters represent a conserved family of transmembrane proteins that have two ABC motifs, each of which contains two adenosine triphosphate (ATP)-binding sites. They

Abbreviations: ABC, ATP-binding cassette; MDR, multi-drug resistance; CFTR, cystic fibrosis transmembrane conductance regulator. *Corresponding author. Fax: +81 427 212850; e-marl: daichan @fly.erato.jrdc.go.jp. Present address: Central Institute for Experimental Animals, 1430 Nogawa, Miyamae-ku, Kawasaki, Kanagawa 216, Japan.

have been identified in a diverse range of organisms, functioning to import or export a variety of molecules across the cell membrane [1,2]. The best known examples of the ABC family are the products of the multi-drug resistance (MDR) genes [3-6] and the cystic fibrosis transmembrane conductance regulator (CFTR) [7]. These proteins are integrated into the membrane as monomers, which have twelve transmembrane segments and two ABC domains in their cytoplasmic extension. Another type of ABC transporters operates as heterodimers of two different polypeptides, both of which possess only six membrane-spanning regions and a

0167-4781/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S01 67-4781 (96)001 37-6

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H. Kuwana et al. / Biochimica et Biophysica Acta 1309 (1996) 47-52

single ABC domain. The products of the white (w) [8], scarlet (st) [9] and brown (bw) [10] genes of Drosophila melanogaster belong to this subtype. The w, st and bw genes were originally identified by their effects on eye color when mutated: the st and bw mutations prevent the pigment cells from taking up the pigment precursors tryptophan and guanine, respectively, while the cells mutant for w are unable to take up both of these substances. It is inferred that the S t / W heterodimer mediates tryptophan transportation and the B w / W heterodimer transports guanine [ 11 ]. We report here the cloning of a new gene, ABC Transporter Expressed in. Trachea (Atet), from Drosophila melanogaster. The gene product shows extensive homology with W and Bw proteins in the first to sixth membrane-spanning regions and in the ABC domain, but has a unique putative ligand-binding sequence in the extracellular loop. Isolation and sequencing of cDNA. We screened a Agt 11 Drosophila embryonic cDNA library using a [32p]-labeled genomic DNA fragment derived from the 91B region as a probe. Through three successive rounds of library screening, seven overlapping clones were isolated. The cDNAs were subcloned into pBluescripts (Stratagene) and restriction mapped. Sequencing of cDNA clones was performed by the chain termination method after preparing appropriate deletion clones [12]. Chromosome in situ hybridization. The 1.1 kb PstI-XhoI cDNA fragment was used as a probe. The probe was labeled with bio-1 ldUTP (Enzo Biochemicals) by nick translation. Polytene chromosomes were prepared from salivary glands of late 3rd instar larvae of the Canton-S strain. Signal detection was performed with streptavidin-conjugated alkaline phosphatase (BRL) followed by histochemical detection with nitroblue tetrazolium and BCIP. Isolation of RNA and Northern hybridization analysis. RNAs were extracted from 0-12 h and 12-24 h embryos, 3rd-instar larvae, pupae and adults, Extraction of total RNA was performed by the single-step method [13]. Poly (A)+RNAs were prepared by oligo (dT)-cellulose affinity chromatography using an mRNA purification kit (Pharmacia LBK). Aliquots of poly (A) + RNA (each 7.5 rag) were separated on 0.8% agarose gels containing formaldehyde as described in Ref. [14]. Following transfer onto Nitro-

plus 2000 (Micron Separations Inc.), the filters were hybridized in a solution containing 4 × SSC, 5 × Denhardt's solution, 0.2% SDS, 50% formamide and the appropriate [32p]-labeled probe at 42°C. The filters were washed in 0.1 × SSC at 65°C and then exposed to an imaging plate for analysis using a Bas 2000 image analyzer. Whole mount in situ hybridization. In situ hybridization to whole mount embryos was performed by a modification of the method described in Ref. [15]. Collected embryos were fixed with a mixture of paraformaldehyde in PBS and heptane for 30 min after removing the chorion membranes in sodium hypochlorite solution. All incubations and washes were performed at room temperature. After three 5-min washes in wash buffer (PBS/0.3% Triton-X), embryos were digested with proteinase K (50 m g / m l proteinase K in the wash buffer) for 5 min. Following digestion, embryos were washed twice in 2 m g / m l glycine solution for 5 min and twice for 5 min each in the wash buffer, before being fixed for 30 min with paraformaldehyde in PBS. Following four 5-min washes in the wash buffer, embryos were prehybridized and then hybridized with the appropriate probes. Non-radioactive probes were made by using digoxigenin-dUTP and detected with a monoclonal antibody against digoxigenin coupled to alkaline phosphatase according to the manufacturer's instruction (Boehringer Mannheim). The Atet gene was isolated fortuitously by crosshybridization under high stringency conditions (6 X SSC at 65°C without formamide) with a genomic sequence derived from the 91 B region in the third chromosome in a search for full-length cDNA of the satori gene [16]. Subsequent mapping of the isolated clones onto polytene chromosomes by in situ hybridization indicated that the cDNA originates from band 24E on the second chromosome (Fig. 1). Partial sequencing of the genomic probe used for cDNA screening revealed no significant homology with the 24E cDNA sequence. The cytological division 24E is known to contain as many as twelve genes. However, nothing is known about the molecular nature of any of these loci [21]. A more detailed analysis of the Atet gene was therefore initiated. Sequence analysis of the cloned cDNA indicated an open reading flame of 2496 base pairs (bp) which can potentially encode a protein of 832 amino acids

H. Kuwana et a l . / Biochimica et Biophysica Acta 1309 (1996) 47-52

Fig. 1. Mapping of the Atet locus on polytene chromosomes. A positive hybridization signal was detected in region 24E on the left arm of the second chromosome.

1 61 121 181 241 301 361 421 481 541 601 661 731 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441

CG~FD3CC~GGTCGAC~FI'I~GT~CACGAA'DDGAGTC CAATCGCA AC GGATCGGATCGC G G ~ A A T A A A A A C A C ~ A C ~ T A C A C " ~"~"lqki-CG TAT~CCTC~ CTC~ACCATCTGGT~TC43TATI'I"C GA _CT CGCGTCGAA'/~TTGTC~TTTAAATA C~CCATAACACAGAATACA~F_AAAGAAGCCAAAGCGACGAGCACGAGT ATCTC~GTATCFIL'TC*AAAGTATCTAA~q~C GCGGGA~ACAG'fGAAT ACC~L~kI-I - I C ' f ~ G C C ~ TATATATAATC,C T ; ~ T A A A C C A A ~ C AC~'~"~"F ~'ATC~CA%~TO-~ %~-FFF ~ TAGGCCCCA~AATAGAAATC TACAATCAACATATAACG~AC~CAATGCAGATC-C CAATt'GC~ATTCAA~/TAAATAACAGCA~Y~CAAGA~T~TACGTATCACAAA AAATATAAAGCATTAATCAATI~AGCCAAATC~/TGATGCCATATGCCAAAAGCGA~T~CA ACAAAAACTAAACGCAT~CTAGAACTACACAGAAACTAGCGTCATC, AATC~ M N E L CAGGCCTCCAGTGTCCCC~GTGCTCCTCCCACC~TCCTGCCGGCG Q A S S V P L V D K C S S H A V I 5 P A GCCAAGAGTCCCAACAGAGTCTCCGATTCGGCAGCCACTACGGCACCTAATGCCG~'fCTC A K S P N R V S D S A A T T A P N A V L TACACAAC~CA%~'~f CCCTATCAACC~ CACC~GCC~TCCAAATCCA Y T S G ~ M S L S T L S T G S G N P N P AATCCC~ATC~GTACCAATCCCAGACAGGAGCCAGTGCCACTGCTCAGCCAAC'r~AAG N P N A S T N P R Q E P V P L L S Q L K CTGAC CAACGA~-~fC~AAAC.%GAACAGTCACGGGTC C C A G A A C A ~ T C ~ T G G ~ L T N D L K Q N S H G S Q N N L C N G G ~Fr~'TAACAACCATC, CC~CCCCAAGGTGGCC~ACAACAC~AGTCCG L G S N N H A L A P K V A ~ N S G G S P AATGGCCAGAAGAAGGGTACCA TCTACCCCAA C N G Q K K G T I A L S H L P Q R P P V D ATCGAG~TATATC CTACTC~TTACCGATAGTCATCC~~ E F C D ~ S Y S V T D S H R R' G F ~ T ATCCTGAAGAGCGTCTC TGGAGAGATCACAGCTATCATGGGACCC L K S V S G K F R N G E I T A I M G P ZAGGAGC~GAGTACGCTGATGAACA~ACAAAACTGCCCAACTC G K G K S T L M N I L A G Y K T A Q L ~C,C~CGTACTGATCAACAGC~C-CGAAAT~GGCC~GCTCTCC S G S V L I N S K E R N L R R F R K L S TC~ACATCATC~!AGGACGA~D~'~ q ~.-~L~TCGC C ~ A T C ~ C ~ G G C C A T G A ~ C Y I M Q D D V L I A ~L T V R E A M M GTGC,C C G C C A A C C T C A A C ~ T A T G A T C A C G T A C G C C A A G G ~ T C A A N L K L G K N M I T Y A K V V V V

1501 ~ C ~ G A ~ C , A ~ T ' r , C ~ G T C C~ACACC.C~GACCTC,C~TCr~ E E I L E T I G L K E S V N T L T C ~ L 1561 ~ ~ C T G T C C A ~ C T C C , AATTGGTCAACAATCCACCAGTG S G G Q R K R L S I A L E L V N N P P V 1621 A T ~ C G A A C C C A C C T C C G G A C ~ C A C ~ C C ~ A G C T G A T A T ~ G I M F F D ~ P T S G L D S S T C F Q 5 I S 1681 C ' D G C T A A G A ~ C A G A G G ~ C C ~ _ A C C A T A G T G T G T A C A A T C CATCA~C CGTC G, L L R S L A R G G R T I V C T ~ H Q p S 174] G C G C G A C ~ ' T T C G A T C A T C ' I ~ T A _GTGTG~AC A R n ~ E K F D H L Y L L A Q G Q C V Y 1801 ~ A ~ X r ~ A C r ~ T ~ C C G T A C C T A T C A ~ V _ ~ C C . ~ T ~ C C C C r ~ E G R V K G L V P Y L S S L G Y E C P S 1861 T A T C A C A A T C C ~ A T ~ C C - ~ u G A .GTACGGCGATGCTGTA Y H N P A D Y V t E V A S G E Y G D A V 1921 _GAAGTACGCGC.ACA~F~GATTAT P K L V D A V K S G A C K K Y A H K D Y 1981 G T A C ~ CAA~/~ACATTATCAAGGGC~GTGGCAG~ V L T L A Q K G C N N D I I K G ~ G S G 2041 ~ T G C A A T G G C C A ' ~ ' ~ - x - x uaCCCTGGAGGATGAAAAGCCGCCGCTGGAGGACAGG A E N A M A I L T L E D E K P P L E D R

49

with a relative molecular mass of approx. 91 kDa. The predicted sequence carries an ABC domain, six putative asparagine glycosylation sites and three potential glycosaminoglycan binding sites. Hydrophobicity analysis of the Atet protein reveals 6 hydrophobic segments each of 21 amino acids, which could form transmembrane domains (Fig. 2). The absence of an identifiable signal sequence implies that the N terminus is located on the cytoplasmic face of the membrane. Comparison of the Atet protein sequence with known proteins using the PIR and Swiss-Prot databases demonstrated the highest homology with the Drosophila melanogaster pigment precursor transporters, W [17] and Bw [10]. Partial

2101 C A G C T G G A G C C C T C T A T T C C C G T C G A G G A T C ~ C G T G A A A C C A C C G A A G C T A G A G Q L E P S I P V E D P A D V K P P K L E 2161 A C G C A C ~ C C A G A A C T C C G A T T G C A G C G ' P G G T C A A T A T G C C C * A C T A A T G C C G T ~ C T Q Q S Q N S D C S V V N M P T N A V P 2221 G A C A ~ G C T ~ T A G C T C ~ C A C A A A A T G C T G T A ~ T C G D S C S F S S S K G T Q N A V G G S G S 2281 G G A G G A C C C A G T G C C G T I ~ T G A C ~ A T T C G C A C G A G A G C G T G G G P S A V V G C M T S L L D S H E S V 2341 GTCACAT?GCCC;/ACAAGACGGGATrCCCCACCAGCGGCTGGACACAATIL'TGGATCCTG V T L P ~ K T G F F T S G W T Q F W I L 2401 CTC_AAAC GCTCATTCCGCAC C A T C C T ~ T A A A A T G C T G A C CCACA TC~GACTCTTC L K R S F R T I L R D K M L T H M R L F 2461 T C G C A C G ' / G A T C G ~ ' ~ G G A G C C A T C A ~ F I ~ T G A T C T A C T A C G A ~ C G A G G C C S M V I V G A I ~ ~ M ! Y Y D V G N E A 2521 AGCAAGATCATC,A G C A A T G C G G G A T G C . A T C ~ G ' F ~ C ~ C C A C C T T C A C G S K I M S N A G C I F F V S L F T T F T 2581 GC CATGATGC CCAC CA~TCIC~AC C'FFTCCC-ACCGAAATGTCAG .GCAT A M M P T I L T F P T E M S V F V R E H 2641 C T C A A C T A C T G G T A C T C C ~ G G C C T T C T A C T T T G C C A A G A C C A T A G C G G A C A T G C C A L N Y W Y S L K A F Y F A K T 1 A p M P 2701 ~ ' I ' P C A G A % ~ F G ~ G C G T C T A T G T C C T G G TGGTGTACTAC CTAAC C~3%CAGC CA F Q I V F S S V Y V L V V Y Y L T S Q P 2761 A T G G A A T I ~ C ~ T ~ T C T G % ~ G T G C T A A A C ' P C A C T G G T G G C A M E L E R V S M F V L I ¢ V L N S 5 V A 2821 CAATCGCIV, G G A C T G C T G A T C C ~ T G A A C A ~ A C G G G C G T A T T C C T C G G C S L G L L 1 G A G M N I E T G V F L G 2881 C C CGTC,ACGAC CATAC C C A C G A T A % ~ C G G A ~ C ~ C A C CATT P V T T I P T I L F S G F F V N F D T 2941 C C G C - G C T A C C ' D G C A G ~ C C T A C ~ G C T A ~ T G C G C T A T C . C ~ G G T G C C P G Y L Q W v T Y V S Y V R Y G F E G A 3001 ATGGTAGCCATCTATC-GAATGGATAGGGC CAAGATC-CAG TGC.AATCAAATGTACT~CAC M V A I Y G M D R A K M Q C N Q M Y C H 3061 T A T C G A G ~ F_~%GATGTCCATC,G A T A A ~ ~ F F I ~ Y R V P K K F L E E M S M D N A L F W V 3121 G A C G C ~ T C G G A A ~ C ~ T C A T C G C C T A C T ' F I ~ D A V A L I G I F F A L R I ~ A Y F V 3181 C G A T G G A A A C T G C A C A T G A T T ~ A A G C G G A C A Q ~ _ ' I " I ~ ' ~ C ~ _ , A T T ~ A T A G T C T A G R W K L H M I R 3241 G A A T A T C A ~ T A T T A C C A T A C G T T A G A G A T C A C C ~ A C A T A G A A A G G G ~ C T A 3301 G T C ~ C ~ T C CCAGGAAAGC~FI~AGCTAGTTGTAC~ATCGCA 3361 T T C T A A A A C q ' r C T C C C G T C ~ A C G I q T A T A C A G A C A G A C A G A C A T A C A C A C A C 3421 C-CA~2GCA~U, C A T ~ T A G G T A T C ~ G A C GA%~'TA~TATATACC CA~GATAT 3481 ATATACAC C C G T A C A G T A C ~ T A C C A C A A A A ~ T G C , AAT~ACATAGAA 3541 CTATATATAGAG~AATACTAGGAGTACCCACGTAGAGCC C C A C T ~ C A C A C A C ~ 3601 ATTC~.GGTACATATAA'FLt~'I%//GACCA ~ AAGTAGG~ 3681 AAACATAACAAATTGTAGACAAGATC _ .CCGAATCC~G'FITA 3721 GGAC.a/-'GA~--~"1."~;.~CCCAACG'~ffx.-J.'.tL A C T C ~ C T A A ~ , ~ " ~ - * ~ ~ 3781 CTATAA%~'TI%IT aTa ~ G C A A T C G A ~ TCAAAC 3841 G G A ~ T r A G G G C G A G ~ x G ' r a A A G C G G A A A C G G A A A C A G A A A T C A T ~ T A G C 3901 ~ TCCACATACCGGA CCAACAAA 3961 T C C ~ C ~ C C T ~ C T G G T ~ T A T A A ~ J 3 C A A T T A C T A ' L FF ~-L~CGATGGGAT 4021 G C A A ~ ' F ~"~~ *~ I T A G ~ A ' F F I ~ ' F F A ~ T A C A ~ I ' I T A A T T T A G T A C A T G 4081 " ~ A G T I T A A ' F I ~ A T A C G T C T A C A T I ~ T ~ T A A A T G C A A T ~ T I ~ A A 4141 ACAGCA .CACC CAAAGTAAA~'~AT~TAATC~ATAC//~ 4201 G G A T G T A A A A C A C A T T G C . A T E G A T A C A C A A A C C A A G ~ A ~ C T A C A G A A A ~ L - ~ 4261 CATA .GGACAGTACAGCATTGTAATTATTTA~AAGCAA~'TG 4321 " F I ~ C C CCCC~TATI~AAGTAT~ L~'1~ FTGTATATTTCGGTAG'F~ 4381 TGTAGCCCAAT~ITACACTGTACA~TAACGTTAAAGAA~GGCCATTACTGTACACAAAAG 4441 C C A T A T T A A T T A C T ~ T C ~ A G A G T A iT ~'FI~ATAAAGAACCCAAAACATC

Fig. 2. The nucleotide and predicted amino acid sequences of Atet. Underlined nucleotide sequences indicate putative polyadenylation signals. Underlined amino acid sequences represent predicted membrane-spanning domains. The ABC domain is boxed. Underlined amino acid sequences in the box correspond to the Walker A and B regions [26], which are nucleotide-binding sites. Potential N-glycosylation sites and glycosaminoglycan binding sites are indicated with (©) and (O), respectively.

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H. Kuwana et al. / Biochimica et Biophysica Acta 1309 (1996) 47-52

alyze ATP hydrolysis-dependent drug efflux like their mammalian counterparts, MDRs, conferring multidrug resistance to various inhibitors upon amplification [6,20]. Overall identity between the amino acid sequences of Atet and W is 32%. These two proteins showed marked similarity in the fifth transmembrane domain and its flanks (Fig. 3C), where the W protein appears to dimerize with Bw [11]. Conservation of the amino acid sequence in this region suggests that Atet has the potential to form a dimer with W or other unknown

homology was found with the MDR-related proteins of Saccharomyces(ADP1 [18], PDR5 [19] and SNQ2 [20]) and the mouse MDR proteins [6], and some other members of the ABC transporter family, particularly in the ABC domain where the ATP-binding sites lie (Fig. 3A,B). The ADP1 (ATP-dependent permease 1) shows significant homologies to a class of bacterial permeases which hydrolyze two molecules of ATP per mol of transported substrate [18]. PDR5 (pleiotropic drug resistance 5) and SNQ2 (sensitive to 4-NQO 2) cat-

A Atet H.white D.white

brown scarlet ADPI PDR5 SNQ2 MDR3

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Fig. 3. Alignment of conserved amino acid sequences. The Walker A (A) and B (B) [26] regions in Atet compared with those of Human white homolog, White, Brown, Scarlet, ADP1, PDR5, SNQ2 and mouse MDR3. In (C), the sequences in the fifth putative transmembrane domain are compared between Atet, Human white homolog, Scarlet, and White. The alignment was performed using the Multiple Sequence Program of DNASIS. White letters on the black background represent identical amino acids.

H. Kuwana et al. / Biochimica et Biophysica Acta 1309 (1996) 47-52

12345

4.5kb

Dras2 Fig. 4. Developmental Northern blotting of Drosophila Canton-S mRNA. Each lane contained 7.5 mg of poly (A) + RNA. Lane l, Early stage embryos (0-12 h); lane2, Late stage embryos (12-24 h); lane 3, Third instar larvae; lane 4, Pupae; lane 5, Adults. The Drosophila ras2 cDNA was used as a control probe.

partner proteins. A remarkable similarity is found between ATET and so-called 'Human white homolog', giving rise to the possibility that the latter protein is actually a human homolog of ATET rather than White. The amino acid identity is 63% between these two proteins in the conserved region spanning a.a. 134-a.a 423 (the residue number for ATET). To determine the size, abundance and complexity of the A t e t gene message, we performed developmental Northern analysis. As shown in Fig. 4, a

51

single 4.5 kb mRNA was detected at all stages analyzed, and the amount of the transcript was practically constant throughout development. The spatial pattern of expression of the A t e t gene was examined by in situ hybridization of whole-mount embryos using digoxigenin-labeled RNA probes (Fig. 5). In stage 5 embryos before cellularization, no signal was detected. At stages 8 and 9, the A t e t mRNA was detected in amnioserosa. At stages 10 to 14, strong signals appeared in the amnioserosa and the respiratory organs, i.e., the posterior spiracles and the tracheae. In fact, the A t e t transcript may serve as a useful marker to trace development of these structures. The w and b w mutations are characterized phenotypically by their altered pigmentation of the adult compound eye and larval Malpighian tubules, and no defect has been reported in these mutants in amnioserosa or the embryonic respiratory system [2224]. It is even unclear whether the w and b w transcripts are present in the developing embryo. It is possible that Atet forms a heterodimer with W so as to produce a functional transporter in the developing embryonic cells, provided these proteins are coexpressed.

1

iiiiiiiii!!

iii ~¸ ~!iiiiiiiiiil]!ii~iiiiiiii~{iii

Fig. 5. The expression patterns of the Atet transcript revealed by in situ hybridization on whole-mount embryos. Lateral (A, C and E) and dorsal (B, D and F) views of embryos at stage 5 (A), stage 11 (C and D) and stage 13 (E and F) hybridized with the antisense RNA probe. Stage 13 embryos hybridized with the sense probe (B) served as controls.

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H. Kuwana et al. / Biochimica et Biophysica Acta 1309 (1996) 47-52

Alternatively, the Atet protein might function independently of W, transporting a molecule required for tracheal development in early embryogenesis. In this context, it is particularly interesting to note that the embryos homozygous for the deficiency, Df12L)dph25, that may delete the Atet locus, reveal 'tracheae disconnected phenotype' [25]. We are in the process of generating transformant fly lines, which carry a hsp70-Atet + construct to examine whether its overexpression can rescue the defect of the Df(2L)dp-h25 embryos. We are also pursuing the possibility of tagging the Atet gene by means of 'local jumping' of a P-element inserted at 24E1.2. Thus, the identification, characterization, mapping, and expression analysis of the Atet gene represent the foundation on which a more comprehensive genetic investigation into the biological function of the W-like ABC transporter can be built and its possible role in early development determined. We thank Hiroshi Miyamoto for his technical guidance in whole mount in situ hybridization, Kai Zinn for the cDNA library, R. Dorn for the deficiency fly lines, Kuniaki Takahashi and Eric Nilsson for their comments on the manuscript, and June Takahashi for secretarial assistance.

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