THE JOURNAL OF BIOLOGICAL. CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biolom, Inc

Vol. 269, No. 21, Issue of May 27, pp. 15179-15185, 1994 Printed in U.S.A.

Dihydrofolate Reductase of DrosophiZa CLONING AND EXPRESSION OF A GENE WITH A RARE TRANSCRIPT*

(Received for publication, December 21, 1993)

Hong Hao, Michael G. Tyshenko, and Virginia K. Walker$ From the Department of Biology, Queen’s University, Kingston, Ontario K7L 3N6, Canada

Traditionally, dihydrofolate reductase (DHFR) has been isolated and the corresponding gene cloned from drug-resistant cell lines which have amplified DHFR genes after selection. ADhfr sequence has now been ob- tained by nested polymerase chain reaction (PCR) from Drosophila bearing a single gene copy. Using the PCR- amplified partial cDNA as a probe, Dhfr was cloned by screening a Drosophila genomic library. It consists of regulatory regions as well as a 599-nucleotide coding region with a single 50-base pair (bp) intron and encodes a protein of 182 amino acids. Previously we have shown that the enzyme has kinetic properties characteristic of both “prokaryotic” and “eukaryotic” DHFRs. Here we show that the organization of Drosophila Dhfr is strik- ingly different from its mammalian counterparts and most similar to that of mosquito. A 790-bp transcript was detected by Northern blot analysis, with a single tran- scription start site located 27 bp upstream ofATG codon. The Drosophila genome contains a single Dhfr copy at 89E and a selected cell line has not amplified the gene. Confirmation of the identity of this gene has been ob- tained by kinetic studies of recombinant DHFR over- expressed in Escherichia coli cells.

Dihydrofolate reductase (DHFR; EC 1.5.1.3)’ is an important enzyme involved in the de novo synthesis of purines, pyrimi- dines, and glycine. Its critical role in intermediary metabolism has made i t a target for anti-folate drugs and a focus for the study of resistance mechanisms and gene amplification. Since the first DHFR gene (Dhfr) was reported from an anti-folate- resistant murine cell line (11, other Dhfr loci have been cloned from a variety of species, including human (21, hamster (3), yeast (4, 5 ) , Escherichia coli (6), Leishmania (71, Lactobacillus casei (81, and mosquito (9). The initial cloning ofDhfi from many organisms has been facilitated by the generation of cell lines with amplified copies ofthis gene. For example, mouse Dhfr was isolated from a methotrexate (MTX)-resistant S-180 cell line with 200 copies of Dhfr (10); similarly the mosquito gene was isolated from a MTX-resistant cell line with 300 copies.

The overall structure and organization of cloned mammalian Dhfr loci shows extensive similarity (11). The mammalian genes (from human, mouse, and hamster) span approximately 30 kb and have five introns. Many transcripts of these genes

* This work was supported in part by a Queen’s Graduate Scholarship (to H. H.) and by an Operating Grant (to V. K. W.) from the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTMIEMBL Data Bank with accession number(s) U06861.

t To whom correspondence and reprint requests should be addressed. Tel.: 613-545-6123; Fax: 613-545-6806.

The abbreviations used are: DHFR, dihydrofolate reductase; “E, methotrexate; bp, base pair(s); kb, kilobase paids).

~~ ~

have been detected and result from multiple initiation sites andor multiple 3’-polyadenylation sites. The genes have similar intron positions and conserved exodintron boundaries but show great divergence in intron size, with the exception of the first, and sequence (12). Interestingly, the human, mouse, and Chi- nese hamster Dhfr loci have bidirectional promoters. One such mouse gene with a 4.0-kb transcript shows homology to the bac- terial DNA mismatch repair genes Hex A and Mut S (13).

The purification and characterization of an insect DHFR was reported only recently (14, 15) due to the low level of this protein in these MTX-sensitive organisms. The isolation of the corresponding gene from Drosophila is important not only for the wealth of genetic, cytological, and molecular information available for this insect but because of the opportunity to de- velop insect-specific antimetabolic agents. We anticipate that such agents, or indeed mutations in this vital gene, will lead to embryonic lethality. In order to study Drosophila Dhfr, MTX- resistant cells were selected over a 4-year period from a S3 cell line. It was expected that selection would result in an overam- plification of Dhfr as in other species. Surprisingly, our highly resistant cell line (200 p~ MTX) did not show overproduction of DHFR or its mRNA. Therefore, we had to clone Dhfr from a DHFR limited source (representing about 0.001% of the soluble protein; Ref. 14).

MATERIALS AND METHODS Standard Techniques-Standard procedures were used to carry out

phage and plasmid DNA isolation, ligation and transformation, DNA blotting, and RNA blotting (16). DNA probes were radiolabeled by nick translation (Life Technologies, Inc. labeling kit) and purified as sug- gested by the manufacturer. DNA bands from low melt agarose gel electrophoresis used for subcloning were purified by Sephaglas Band- Prep kit (Pharmacia LKB Biotechnology Inc.).

DHFR Assay, Purification, and Internal Amino Acid Sequencing- DHFR enzyme activity in cell extracts and purified protein prepara- tions was determined as described previously (14). The procedures for purification of DHFR from Drosophila have been described previously (14). 5 pg of partially purified protein from 18 g of adult flies were separated on a 12% polyacrylamide gel and electroblotted overnight onto a nitrocellulose membrane at 35 V, 4 “C. The protein on the nitro- cellulose membrane was subjected to internal amino acid sequencing according to the procedures described by Aebersold et al. (17) at the Harvard Microchemistry Facility.

To purify recombinant DHFR, 100 ml of transformed E. coli were harvested by centrifugation and resuspended in 100 m~ potassium phosphate buffer, pH 7.9, 1 mM dithiothreitol, and 1 mM EDTA. Cells were lysed by egg white lysozyme (100 pg/ml final concentration) on ice for 15 min, followed by three successive freeze-thaw cycles. The enzyme was precipitated by (NH,),SO, fractionation (50-70% saturation) and dialyzed against 10 mM KCl, 1 mM EDTA, 2 mM dithiothreitol, and 100 mM Tris-HCI, pH 7.5, overnight. The dialysate was bound to Ml-Gel blue column (Bio-Rad) and eluted with 1 M KCI. The DHFR-containing fractions were dialyzed and 1 mM dithiothreitol and 0.1 mg/ml bovine serum albumin was added for kinetic studies. The K,,, values for the substrate and cofactor of recombinant Drosophila DHFR as well as the inhibition and dissociation constant for trimethoprim and [3H]MTX (Amersham Corp.) were determined as described previously (15). These values were determined for three times on at least two preparations of recombinant DHFR.

15179

15180 Drosophila DHFR Gene Nested Polymerase Chain Reaction Amplification-Oligonucleotide

primers were synthesized on an Applied Biosystems 360 A DNA Syn- thesizer (Core Facility, Insect Biotech Canada). First strand cDNA was produced by RNase H- reverse transcriptase (Moloney murine leukemia virus reverse transcriptase, Life Technologies, Inc.) following the manu- facturer’s protocol. After completion the volume was made up to 250 pl. 1 pl of first strand cDNA solution or 1 pg of Drosophila genomic DNA was used as a template for the first round of amplification in the pres- ence of 100 mM Tris, pH 9.0, 1.5 mM MgCl,, 500 mu KCI, 1% Triton X-100, 0.2 mM each deoxynucleotide triphosphate, and 50 pmol of each primer. A I-pl aliquot of the first amplification reaction was added to the nested PCR reaction, which was performed with the same components as the first PCR reaction except using the two inner primers.

Screening of Drosophila Genomic and cDNA Libraries-The 0.6-kb Drosophila Dhfr amplification product from nested PCR was labeled and hybridized to a Drosophila genomic library (Maniatis) (16). A Dro- sophila adult cDNA library (ClonTech) and a Drosophila adult P cDNA library (181 were screened with the PCR-amplified Dhfr cDNA using conditions described by manufacturers.

Southern and Northern Blot Analysis-Drosophila genomic DNA was isolated from adult flies (19). 6 pg of digested genomic DNA was sepa-

brane (Hybond-N, Amersham Corp.). The blot was hybridized with the rated by agarose gel electrophoresis and transferred to a nylon mem-

0.6-kb Drosophila Dhfr amplification product following the manufac- turer’s protocols.

Poly(A)’ RNA was purified by passage over oligo(dT)-cellulose using the QuickPrep Micro mRNAPurification Kit (Pharmacia), electrophore- sed on agarose gel after denaturation with glyoxal and dimethyl sulf- oxide, transferred to a nylon membrane, and hybridized to Droso~hila Dhfr cDNA according to the methods described by the manufacturer.

Primer Extension Analysis-A 31-bp oligonucleotide primer corre- sponding to the inverse complement of bp 21-52 was end-labeled using [y-32PlATP by T4 polynucleotide kinase in a forward exchange reaction. The labeled primer was annealed to either 10 pg of poly(AP RNA or 50 pg of total RNA at 58 “C for 20 min and cooled slowly to room tempera- ture. After treating the reactions with avian myeoloblastosis virus re- verse transcriptase at 42 “C for 30 min according to the manufacturer’s protocol (Promega), extension products were electrophoresed on a 8% denatured polyacrylamide gel and visualized after exposing to Kodak X-AR film at room temperature for 36 h.

In Situ Hybridization to Polytene Chromosomes-Squashes of poly- tene chromosomes were prepared from salivary glands that were dis- sected from third instar Iarvae (20, 21). Drosophila Dhfr (D2 phage insert) was labeled by b i o t i n - 2 l - d ~ P (ClonTech~ using the nick trans- lation labeling kit (Life Technologies, Inc.). Hybridization of the biotin- labeled DNA probes to chromosome squashes was carried out by the method of Mariana Wolfner.’

DNA Sequencing-cDNA and genomic DNA fragments to be se- quenced were subcloned into Bluescript SK’ vector. Automated DNA sequencing was carried out by Applied Biosystems 373A fluorescent sequencer using DyeDeoxy terminators in sequencing reactions. Addi- tional genomic DNA sequences were determined by synthesizing spe- cifrc 17-bp oligonucleotides that were used as primers in the sequencing reaction.

Analysis of DNA sequence was done using PC-Gene programs, in-

Dhfr (SIGNAL) and manipulations of DNA sequences (SEQIN, NMA- cluding the search for regulatory elements in the 5”flanking region of

NIP, TRANSL, ASSEMGEL, NALIGN). The comparison and alignment of the DNAsequence and its deduced protein sequence with those in the data base were done using Fast Pairwise Comparison of Sequences program (release 5.41.

Western Blot Analysis-TOP10 cells bearing the expressed recombi- nant Drosophila DHFR were lysed by three cycles of freeze-thaw (In- vitrogen Corp.). After centrifugation, the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (Bio-Rad manual) and trans- ferred to a nitrocellulose membrane. Western blot analysis was per- formed (22) using DHFR-specific antibody. The ~rosophila DHFR an- tibody was produced by three subcutaneous injections of rabbits with 20-50 pg of purified DHFR (either directly from the Mi-Gel blue col- umn or after SDS-polyacrylamide gel electrophoresis) in RIBI adjuvant (RIBI Immunochem Research Inc.).

Expression of Drosophila DHFR in E. coli Cells-The wild type Dro- sophila Dhfr cDNA was amplified by PCR using Vent DNA polymerase with 5‘ proofreading ability (New England Biolabs) and two primers designed from the amino- and c a r b o x y l - ~ ~ i n a l sequence of Dhfr and bearing BamHI or EcoRI restriction sites on each end. The amplified ~- ’ M. Wolfner, personal communication.

product was digested with BamHI and EcoRI and ligated into the BamHUEcoRI restriction sites of the expression vector pTrcHis A (In- vitrogen Corp.) to produce proper in-frame recombinant Dhfr. This re- combinant expression vector, pTrcHis-Dhfr, was transformed into E. coli strain TOPI0 and induced by treatment with isopropy~-l-thio-~-~ galactop~anoside (1 mM final concentration). Cell extracts were pre- pared for Western blot analysis and DHFR assays.

RESULTS

Cloning of Drosophila Dhfr and Northern Analysis-A num- ber of strategies were employed in the attempt to clone Dhfr from wild type Drosophi~a. Degenerate oligonucleotide probes based on the a m i n o - ~ ~ i n a l sequence of Drosophila DHFR (14) were hybridized to both Drosophila genomic and cDNA libraries and used to prime first strand cDNA for the rapid amplification of cDNA ends (RACE-PCR). The Drosophila li- braries were also hybridized with heterologous Dhfr cDNAs from mouse and mosquito under low stringency conditions. Additionally, a Dros~ph i~a cDNA expression library was screened with two Drosophila DHFR-specific antibodies (from purified native DHFR and denatured DHFR). The analysis of these results are presented under “Discussion.”

The failure of these standard methods led us to employ an alternative way to clone Dhfr. DHFR preparations which were eluted from an AfE-Gel blue column (14) were electroblotted onto a nitrocellulose membrane followed by in situ protease digestion. The resulting peptide fragments were separated by reverse-phase high performance liquid chromatography and subjected to sequence analysis. Four degenerate oligonucle- otide primers were synthesized according to the internal and confirmed amino terminal sequence of DHFR (Fig. 1B). PCR was performed using first strand cDNA as template for ampli- fication by primers 1 and 4 (Fig. lA, lane f ); an aliquot of this reaction was then reamplified with one of two combinations involving nested primers (primers 1 and 3, lane 2; primers 2 and 3, lane 3) . In both cases, a unique amplified band with the expected molecular weight of about 500 bp was obtained (Fig. LA, lane 2 and 3). Analysis of the sequenced amplification prod- uct revealed homology with other Dhfrs.

To clone full-length Dhfr, the PCR amplified Dhfr cDNA was used as a probe to screen Drosophila genomic and cDNA librar- ies. No positive cDNA recombinant phage were observed; how- ever, genomic library hybridization yielded two phage (D2, D3). A3.5-kb Sal1 fragment from DZ (Fig. 2 A ) hybridized to the Dhfr cDNA probe and was subcloned into pBluescript SK” vector and sequenced using the strategy shown in Fig. 2 B .

To determine the copy number of Drosophila Dhfr, DNAfrom adult flies was digested to completion with a variety of restric- tion enzymes (Fig. 3) and hybridized with PCR-amplified Dhfr. A unique labeled band was observed in each lane of the South- ern blot. A single hybridization band was localized to the right arm of the third chromosome a t position 89E in polytene chro- mosomes after hybridizing with biotin-labeled Dhfr genomic DNA from the phage D2 insert (Fig. 4).

Northern analysis of Drosophila Dhfr was performed using 20 pg of poly(A)” RNA from adult flies and hybridized with PCR-amplified Dhfr cDNA. A 790-bp transcript was detected (Fig. 5), sufficient to encode DHFR. Primer extension analysis using 10 pg of poly(A)’ RNA resulted in a 78-bp extension product (Fig, 6), whereas 50 pg of total RNA was not sufficient to yield a detectable extension product signal. No minor bands were observed even after longer exposure, indicating the ab- sence of minor transcription start sites.

Organization of Drosophila Dhfr-Both strands of the 3.5-kb genomic Sal1 fragment were sequenced (Fig. 2B), as well as the cDNA amplified by nested PCR. The coding region of Dro- sophila Dhfr spans 599 nucleotides and encodes a 182-amino acid protein (Fig. 7). Comparison of the genomic Dhfr sequence

Drosophila DHFR Gene 15181

A L

1 2 3 4 5 6 5 - 4.36

- 2.32 - 2.03

- 0.56

B N'-terminal Sequence

MLRFNLIVAVSENFGIGIX'GDLP

I \ I

5' CCGTCGACAATTTT GTATCGGrAT 3' A A A \

' C T C

Internal Sequence V A ~ D S D ~ l P L G V O ~ ~ ~ G l ~ F ~ S ~

G C 5' .

DIIFR cDNA

" * 3'

+T FIG. 1. A, nested PCR amplification of a Dhfr cDNA fragment. First

strand cDNA from adults was amplified using the two outer primers (primers 1 and 4) at 40 cycles of 94 "C, 1 min; 46 "C, 1 min; and 72 "C, 1 min (lane 1 ). A 1-pl aliquot of the PCR reaction was reamplified with primers 1 and 3 (lane 2 ), primers 2 and 3 (lane 3 ), primer 1 alone (lane 4 ) , primer 2 alone (lane 5), and primer 3 alone (lane 6) , in cycles of 94 "C, 1 min; 56 "C, 1 min; and 72 "C, 1 min, repeated 30 times. HindIII- digested A DNA marker is shown on the right. B, nested PCR primer design. Four degenerate oligonucleotide primers were synthesized ac- cording to the amino-terminal and internal amino acid sequences of Drosophila DHFR. I represents inosine. C, nested PCR strategy. Primer positions are shown schematically; the PCR products amplified by the two outer primers ( I and 4 ) were reamplified by the two inner primers (2 and 3 ).

B. I K b - t

" t

4" "b 4" t c -b

FIG. 2.A, restriction endonuclease map of the 30-kb genomic insert of phage D2 and 3.5-kb SalI subclone. Restriction enzyme abbreviations are: S, SalI; K, KpnI; H, HindIII; Xh, XhoI; Xb, XbaI; Sm, SmaI; P, PvuI. Solid bars show the region hybridizing with the 0.6-kb Dhfr probe. B, DNA sequencing strategy. The arrows indicate the direction and extent of each sequencing run.

23.1 - - 0

9.41 - 0

6.56 -

4.36 -

2.32 - 2.03 -

FIG. 3. Southern blot analysis of the Drosophila Dhfr. 6 pg of Drosophila genomic DNA digested by the restriction enzymes indicated on each lane was electrophoresed on 0.7% agarose gel, transferred to a

Dhfr. Hybond-N membrane, and hybridized to "P-labeled PCR-amplified

with its cDNA revealed a single 50-bp intron which interrupts the Lys codon at amino acid position 27. The location of the intron is conserved compared with the single mosquito intron and with the first intron in mammals. The splice junctions with a GT dinucleotide in the 5' junction sequence and an AG dinucleotide in the 3' junction sequence conform to the "Cham- bon" rule (23). The putative branch-point sequence CTAAA in the intron is consistent with the consensus branch-point se- quence CTAAT in Drosophila genes (24).

Potential promoter elements were identified within the 933 bp upstream of the ATG translation start site. Primer extension revealed that the single transcription start site was located a t 27 bp upstream of the ATG start codon, which is 23 bp down- stream of a TATA sequence (Fig. 7). Although several putative TATA boxes were detected at further upstream regions: posi- tions -218 to -223, -233 to -237 and -362 to -367, there was no experimental evidence of their function. The 5'-flanking region of Drosophila Dhfr appears to lack the 48-bp GC box which is conserved in the promoter region of mammalian Dhfr loci. A classic AATAAA polyadenylation signal was not found in the 3'-flanking sequence, but like murine Dhfr (10) polyadeny- lation, may occur at an alternate sequence, TAAAAT, which is

15182 Drosophila DHFR Gene ! pm *

2 4 * 0 +I - c z

118 - 110-

82 -

66 -

FIG. 4. Chromosome location of Drosophila Dhfr. Polytene chro- mosome squashes from third instar larvae were hybridized to biotin- labeled D2 phage insert which contains full-length Dhfr. The arrow points to the hybridization signal a t band 89E.

h

a 0 -

9.49 - 7.46 - 4-40 -

2.37 - 1.35 -

w%'

0.24 -

FIG. 5. Northern analysis. A single 790-bp transcript from Dhfr was detected. 20 pg of poly(AY RNA isolated from adult Canton S flies was electrophoresed, transferred to a Hybond-N membrane, and hybridized to a 32P-labeled Dhfr cDNA probe. The Northern blot was exposed for 7 days a t -70 "C.

FIG. 6. lkanscription initiation sites analysis by primer exten- sion. An inverse complement primer (corresponding to bp 21-52) was annealed to 10 pg of poly(AY RNA and 50 pg of total RNA a t 58 "C for 20 min, cooled slowly to room temperature, and extended using avian myeoloblastosis virus reverse transcriptase a t 42 "C for 30 min. The

ide gels with end-labeled X 174 HinfI markers. Gels were exposed to extension products were electrophoresed on 8% denatured polyacrylam-

Kodak X-AFt film a t room temperature for 36 h.

total of 24 residues consewed between Drosophila and other species. Of these, it is thought that 9 residues are involved in NADPH binding, 5 residues are involved in MTX binding, and 3 residues are involved in both NADPH and MTX binding.

Expression of Drosophila DHFR in E. coli-In order to pro- duce large quantities of DHFR for structural studies and ki- netic characterization as well as to confirm the identity of the gene, Drosophila Dhfr cDNA was cloned into a p'lYcHis expres- sion vector and transformed into TOP10 E. coli cells. After isopropyl-1-thio-P-D-galactopyranoside induction an increas- ingly intense band with a molecular mass of 27 kDa was ob- served on Western blots using Drosophila DHFR-specific anti- sera (Fig. 9A). A few lower molecular weight bands were also detected and were probably due to site-specific protease degra- dation of DHFR. The higher molecular weight of the expressed recombinant DHFR is due to the polyhistidine leader peptide fused to the amino terminus of Drosophila DHFR. Increased DHFR activity in cell lysates at various times after induction was seen compared with a control (TOP10 cells transformed wit,h exprefision vector alnne) (Fig. 9B), and this incrcasc cor= related well with the more intense protein band on the Western blot. DHFR specific activity was 3800 times greater in trans- formed E. coli cells as compared with crude homogenates of wild type flies.

Kinetic Characterizations of Recombinant Drosophila DHFR--To further characterize the recombinant Drosophila DHFR, kinetic studies were carried out using purified enzyme preparations. The K,,, values for NADPH and dihydrofolate were determined from primary and secondary Hanes plots and shown to be 11.28 and 4.71 p ~ , respectively. The Ki value for trimethoprim, a competitive inhibitor of DHFR, was deter- mined to be 95.95 UM. The dissociation constant for MTX was

adjacent to the translation stop codon (Fig. 7). measured by equil'ibrium dialysis of the enzyme preparation

Species-The DHFR sequence from Drosophila was compared with the DHFR from human, mouse, chicken, yeast, mosquito, DISCUSSION and E. coli (Fig. 8). Drosophila DHFR has 49% identity with Our interest in cloning Dhfr from Drosophila was to study mammalian DHFR, 27% identity with E. coli DHFR, 34% iden- this important "housekeeping" gene in a model insect, to un- tity with yeast DHFR, and 58% identity with mosquito DHFR. derstand the molecular genetic basis of MTX resistance in a Examining the consewed residues in the alignment reveals a Drosophila cell line, and further to use this as a model for the

Drosophila DHFR Is Homologous to the DHFRs from Other against [3H]MTX, the Kd value was 0.30 nM.

15183

-933 -813 -693

-453 -573

-333 -213

-93

28

148

26%

388

508

628 748 868 988

TGiiATATCZATGACAGWGG TCCAACAACAWAGGATGC CATGCAGGCGG'EACCACCT TAAMMAGTGTlTPCGGCA TXGGACGAAGAGATCATPXC CCCGlTlQTlTACAAAG'JXE A G A C C " X f l l l ! T A C ATGGC%'GXR!CGGGTGCTCC TcA!ETGAATCCCCTGCATG CCGAACACGCCTGCGATlTG A A G G G T G A W A A G A A GGTCAAAGCCCATGCTCTTC CCGGGGTGGCCATACGGGTG GGCAmAACTCGGGACCCGT CGTCGCCGGCGTGGTXGGA TGAAGGTTCCTCGATACTGT m G A C A C m A A CACCGCCTCGCGMTGGAGA GCAGCAGCGATCCCFGGSiTG ATCCAGCTGlYXAM3ATAC TGCGCXA&-GG IWGCTATAAGGT.PGMGCG CGGGGATIToTcRAGGTCAii CGCCAAOOOCWLWLTGGGGCGAGATGGAGA CCTA-GGGA CCAGAGTAGAACTGGAGACT ~~A G G A A T ~ C C A ~ ~ A ~ T A T ~ ~ T A T ~ G A ~ ~ ~ G T MCC-TAATCAG GCTCGAAAkCCTCZTATATC ~ ~ A T C T A A T M ~ T ~ A A T A G m ~ C ~ C Z T mAATCCCCTGTAAAGTATA ~ A T C A ~ A T ~ A MFPC!A&GACACAlTSTATC TA-TA!W@TTXTCGTGG GAGTGGCGCGMACCAACCT GAMTCTGACTAAGAWTTC ATMGCGTGTFXGATAATA GGTMGTAATCGAAW2TATC

- w G G T T C C A a c m c m m w m m m m g G C m m G A T A A m W A A A ~ A T G C ~ T C A ~ M G G A ~ T ~ ~ C G A - ~ M T c G ~ G C A H L R F N L I V A

V C E N F G I G I R G D L P W R I K GTlTGCGAGAATlTCGGAAT CGGCATCAGAGGCGATCTAC CATGGCGCAlTAAgtaagtt ggtaacaaggattgaaccgc aqctaaagcttttgtaccat C a g A T C W A G C ~ G T A C T

S B L K Y P

TCAGCCGCACCACCAAGCGA ACAAGTGATCCCACCAAGCA AAATGCCGTGGTMTGGGCA " X T A C W G G A G T G CCGGAAAGCAAGAGGCCTCT TCCCGATCGGCTGAACATAG S R T T K R T S D P T K Q N A V V H G R K T Y F G V P E S K R P L P D R L N I V

~ a C C A ~ ~ A G - ~ T C T G C C C ~ G G G A G T A ~ ~ T G C C C C M T C T C ~ ~ C G G C C A ~ G A T C ~ ~ a G M C G A A G W ~ G A A C A T l T G G A ~ T G G L S T T L Q E S D L P X G V L L C P N L E T A M X I L B E Q N E V E N I W I V G

G S G V Y E E A H A S P R C E R L Y I T Q I H Q X F D C D T F F P A I P D S F R GCGGTAGTGGAGWTACGAG GAGGCCATGGCCTCGCCMG GFGWACCGGCTGTACAlTA CCCAAATAATGCAGAAGlTC GATWCGACACC!WRTCCC CGCGATCCCTGACAGClT'XC

GAGAGGTCGCGCCCGATTCC GACATGCXA- GGAGGAGAAWGCATPARAT TCGAGTACAAGATTTIGGAG AAACAC-TU TACACTGAXR!ATETGCCTP E V A P D S D H P L G V Q E E N G I K F E Y X I L E K M S

GCGCATCFRZACTCCCPE GGCGCCAGCATCAGCTCGTA T A ~ C T A C ~ ~ G T CFECAACAYlTCTACAGTC ~ G T C A A ~ ~ G C C CAWTCTCCCITG- CCCGAATCCCTAGTWXTGG @3XilTSTGGACGCATATCG CTGCAAWGAAGATFECTT AGTATGGCGCCTZETGTFE ATTACGTAA?UC!FI!ACGGCC CACWTGTCATAAAGAGCm TDCCTCCATGGGATSGCACT TGAGGTG'XGCCGGMCTCG TCCCDcACATAGmATCGCC CAGAGCACGA&G"f2CGCTG GAAGTCCTACGGAGTACAGA AGXR!AG~TGAAATGCANC AATITATAGWMTAGGGAAT TlTPCATCTTACCT

FIG. 7. Nucleotide sequence and deduced amino acid sequence of Drosophila DhF: The sequence shown was determined on both strands of the genomic and cDNA clones. The first base of the initiation codon is designated as nucleotide 1. The TATA box is ~nderZ~ned, and the transcription start site is indicated by an arrow. The amino acid initiation codon (ATG) and termination codon ( T U ) are boxed. The deduced protein

of the 50-bp intron. sequence is shown in single-letter code and aligned above the nucleotide sequence. The gap between the protein sequence indicates the presence

D m with other DHFRs. The se- FIG. 8. kilignment of €kosophila

quence of other DHFRs are from human (21, mouse (251, chicken (26), yeast (51, mosquito (91, and E. coli (6). The Dros- ophila DHFR sequence is shown in bold. Residues that are identical to R ~ s ~ p h i ~ u DHFR are hozed.

Chicken Mouse Euman Drosophila Mosquito Yeast E.Coli

Chicken Mouse

Drosophila Human

Mosquito Yeast E.Coli

Chicken Mouse Human Drosophila Mosquito

E.Coli Yeast

Chicken Mouse Human Drosophila Mosquito

E.Coli Yeast

eventual development of insect-specific antimetabolic agents. Cloning of Dhfr from other species has been greatly facilitated by cell lines in which this gene has undergone amplification. ~ t h o ~ g h the Drosophila cell lines were resistant to 2000-fold more MTX than the control lines, there was no evidence of increased DHFR activity and no Dhfr gene amplification (not shown). Thus, without amplified cell lines the isolation of such a meagerly expressed gene was extremely di~lcult. Initial at- tempts a t cloning Dhfr failed. In hindsight, the heterologous Whfr probes from mouse and mosquito show low overall se- quence homology with Drosophila. Second, the low abundance of Whfr transcripts results in these sequences being under- represented in cDNA libraries. Indeed two independently con- structed cDNA libraries made from adult flies were hybridized with Drosophila Dhfr, and both libraries failed to yield Dhfr cDNAs. For the same reason we failed to identify Dhfr se- quences when expression cDNA libraries were screened with antibodies made to native and denatured forms of DHFR.

Third, there were two discrepancies in the amino-terminal amino acids as determined by Edman degradation and nucle- otide sequencing, resulting in the synthesis o f some oligonucle- otide probes with mismatches.

Nested PCR is a sensitive technique especially useful for low copy number DNAs or mRNA templates (27). The sensitivity of nested PCR can be 1000 times higher than standard PCB with the same number of cycles, since two pairs of primers are re- quired to amplify the target sequence. This can be seen from Fig. LA; after the first round of amplification with the two outer primers, DNA of the expected size was barely observed on the gel (lane 1). However, a prominent 500-bp band was present after the second round of amplification using two inner primers (lane 3) . By determining an internal sequence as well as the amino-terminal sequence of DHFR and performing nested PCR with two pairs of Dhfr specific primers, we have cloned Dhfr from a low abundance DHFR source.

The organization of D r ~ ~ a p h i ~ a Whfr is similar to that of

Drosophila DHFR Gene 15184

A.

kDa

43.0 - 29&

18.4 - 14.3 - 6.2 -

B.

0.30 -

0.25 - E = 0.20 -

0.00 ’ I I I 0 1 2 3 4

Time (hour) FIG. 9. Expression of Drosophila DHFR in E. coli. A, Western

blot analysis shows the expressed Drosophila DHFR fusion protein. Extracts from cells containing the pTrcHis-Dhfr vector collected at the indicated time course (lanes la), and cells containing pTrcHis vector alone (Control) were electroblotted to nitrocellulose and incubated with Drosophila DHFR antibody. The arrow points to the full-length fusion DHFR. B , DHFR activity from expressed fusion protein. 10 pl of soluble protein from expressed E. coli cells at different time points were tested for DHFR activity.

mosquito. Both insect Dhfrs are about 1 kb long and contain a single intron, compared with mammalian Dhfis which are about 30 kb in length and include five introns. Dhfi has an intron of 50 bp, one of the smallest Drosophila introns found to date (24). In contrast to mammalian Dhfr with introns which show virtually no sequence homology except around splice junc- tions (2, 12), the Drosophila Dhfr intron has 58% sequence homology with the mosquito intron, equal to the identity shared by the exons of these two Dhfrs. Thus, it seems that the exons and intron of Dhfr in these two insects are evolving at the same rate from an ancestral dipteran Dhfr.

A striking difference between the Drosophila and mamma- lian Dhfr is at the 5’ promoter region of the gene. TATA-like sequences have been identified in Drosophila Dhfr, but mam- malian Dhfrs lack a TATA box, replacing it with 48 bp of tan- dem repeated GC boxes (2,28,29). These GC boxes have bind- ing sites for transcription factor Spl and function as bidirectional promoter elements. Only a short divergent open reading frame located from -356 to -727 (372 bp long) was uncovered by computer analysis of the Drosophila Dhfr, and this putative transcript has no significant homology with other sequences in GenBank (data not shown). Mammalian Dhfis usually have multiple mRNA species due to heterogeneity at both the 5’- and 3’-nontranslational regions of mRNAs (2, 29- 31). Mosquito Dhfr also shows multiple transcription initiation sites at the 5’ end, but has a single polyadenylation site (32).

Unlike its mammalian and mosquito counterparts, Drosophila Dhfr has only one detectable transcription initiation site lo- cated 27 bp upstream of the ATG codon. This transcription start site lies 23 bp downstream of a TATA sequence. Based on Northern and primer extension analysis, the 3’ end of the Dro- sophila Dhfr transcript is about 210 bp long. DHFR assays and Western analysis confirmed the identity of the recombinant DHFR. There were some differences in the kinetic data for the substrate, cofactor, and inhibitors of the recombinant Dro- sophila enzyme; the K, for NADPH and DHF was 2- and 16- fold higher, respectively, than Drosophila DHFR, and the Kd for MTX was %fold lower than the fly enzyme. We attribute these differences to the extra 36 amino acid residues on the relatively hydrophobic amino terminus of DHFR. These additional amino acids may affect normal post-translational modifications or folding of the enzyme and result in the altered kinetic proper- ties.

A comparison of the deduced amino acid sequences of DHFRs from several species (Fig. 7) using a CLUSTAL program of PC-Gene (IntelliGenetics) showed, not surprisingly, that Dro- sophila DHFR was most closely related to the mosquito en- zyme. The hypothetical tree (not shown) also indicates that the insect DHFRs are more similar to those of vertebrates than to those from yeast and E. coli. Curiously, however, Drosophila DHFR showed some characteristics which were typical of non- vertebrate enzymes (15). In addition, insect DHFRs appear to be less sensitive to MTX than other DHFRs. The MTX disso- ciation constant (K,) of Drosophila DHFR is 34-6615 times higher than that determined for mammalian DHFRs (15). All 17 residues involved in MTX binding (4) are conserved between Drosophila and mosquito DHFR, but only 12 residues are con- served between insect and mammalian DHFRs. Therefore, the other 5 residues in mammals (equivalent to Leu-30, Lys-31, Ser-34, Gly-58, and Val-59 of Drosophila DHFR) may interact more strongly with MTX. The identification of these important residues should facilitate future molecular modeling studies and contribute to the understanding of the structure-function relations of this crucial enzyme.

Drosophila DHFR has both typically vertebrate and prokary- otic physical and kinetic characteristics (15). This dual feature of Drosophila DHFR has now also been demonstrated in its gene structure. As in many Drosophila genes, Dhfr is compact like the lower eukaryotic Dhfr but has more homology with the vertebrate DHFR sequence than that from prokaryotes or lower eukaryotes. Despite these differences between DHFRs from different species, most of the residues involved in the interaction of the protein with its substrates and/or inhibitors are conserved, which reflects the strong evolutionary con- straints in the enzyme’s structure and function.

Acknowledgments-We are grateful to Tina Schaefer (Core Facility, Insect Biotech Canada) for performing automated DNA sequencing. We thank M. Wolfner and A. Hilliker for their advice on in situ hybridiza- tion and acknowledge all the help and encouragement given by our laboratory colleagues.

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