A 6-phosphogluconate dehydrogenase gene from Trypanosoma brucei

Molecular and Biochemical Parasitology, 57 (1993) 89 100

89

(~) 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/93/$06.00 MOLBIO 01862

A 6-phosphogluconate dehydrogenase gene from Trypanosoma brucei M i c h a e l P. B a r r e t t a n d R i c h a r d W . F . Le P a g e University of Cambridge, Department of Pathology, Cambridge, UK (Received 7 May 1992; accepted 28 August 1992)

A Trypanosoma brucei gene encoding 6-phosphogluconate dehydrogenase (6-PGDH) (EC 1.1.1.44) was identified and cloned by functional complementation of Escherichia coli gnd mutants with genomic trypanosome DNA. The T. brucei gnd gene is present as a single copy. In Northern blot experiments a probe derived from the gene hybridises to 2 transcripts (2.9 kb and 3.1 kb) which are found in both bloodstream and procyclic form organisms; the larger transcript is more abundant in bloodstream form organisms. The derived amino acid sequence of the protein is 479 amino acids in length, with a molecular weight of 52 000. It is homologous to 6-PGDHs from bacterial and mammalian sources, but diverges significantly from these other enzymes. Key words: Trypanosoma brucei; Pentose phosphate pathway; 6-Phosphogluconate dehydrogenase gene; Complementation

Introduction

Much of our knowledge of the biochemistry of African trypanosomes has come from studies aimed at identifying new targets for selectively toxic chemotherapeutic agents [1]. The discoveries of the localization within organelles of the glycolytic pathway [2] and the use of a novel trypanosomatid specific cofactor N l ,N 8-bis(glutathionyl)spermidine (trypanothione) in redox reactions within the cell [3] represent major areas of interest in this regard. Until recently it was thought that the glycolytic pathway represented the sole route of glucose metabolism in these organisms. The pentose phosphate pathway (PPP) was beCorrespondence address." Michael P. Barrett, University of Cambridge, Dept. of Pathology, Tennis Court Road, Cambridge CB2 IQP, UK. Tel.: (0223) 333734; Fax: (0223) 333346. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number X65623.

Abbreviations." 6PGDH, 6-phosphogluconate dehydrogenase (EC I. 1.1.44): PPP, pentose phosphate pathway.

lieved to be absent because Ryley [4] was unable to detect 6-phosphogluconate dehydrogenase (6-PGDH) activity in trypanosome extracts. However, Cronin et al. [5] have demonstrated that all the enzymes required for a functional pentose phosphate pathway can be detected in procyclic organisms and that the oxidative branch of the pathway is present in bloodstream form organisms. The classical function of the pentose phosphate pathway is to convert hexoses to pentose sugars, which are essential for nucleotide biosynthesis. The pathway also serves as a vital source of N A D P H , which provides the reducing power required to maintain cellular redox potential and to protect against oxidative stress [6]. Owing to the key role of N A D P H we set out to identify the gene encoding one of the enzymes which regenerates this compound, namely 6-phosphogluconate dehydrogenase. Since heterologous D N A probes were unavailable, we adopted a strategy for direct detection of the gene by complementation of Escherichia coli gnd mutants. E. coli double mutants unable to grow on gluconate arise as a consequence of their lack of both 6-

90 phosphogluconate dehydrogenase and 6-phosphogluconate dehydratase activity [7]. These enzymes are encoded at the gnd and edd loci respectively. Expression of an introduced 6P G D H or 6-phosphogluconate dehydratase gene should restore the capacity of E. coli gnd,'edd to grow on gluconate as a carbon source. Complementation of E. coli mutants has been used to clone a number of eukaryotic genes, since the successful identification of the yeast imidazole glycerol-phosphate dehydratase gene [8] provided the first example of the usefulness of this approach. Subsequently, numerous eukaryotic genes have been similarly identified, including the glucose phosphate isomerase gene from Plasmodium falciparum [9]. Complementation provides a means to clone a gene without prior knowledge of its nucleotide sequence and in instances where the nucleotide sequence homology between genes encoding functionally similar proteins has diverged to a degree which renders heterologous gene probing unfeasible. Since the coding sequences of genes in most eukaryotes contain introns which E. coli cannot process the use of complementation for eukaryotic gene cloning has usually relied on the transformation of E. coli with expression plasmids carrying c D N A inserts. However, the structural genes of Trypanosoma brucei do not contain introns. Consequently it should be possible to screen genomic trypanosome D N A fragments in complementation tests, and to detect transcribed open reading frames directly. This approach has proved to be relatively simple and successful in this instance.

Materials and Methods

Chemical,; and enzymes. All chemicals used were of the highest grade available and purchased from Sigma. Restriction enzymes and D N A modifying enzymes were purchased from Boehringer Mannheim except T7 polymerase (Sequenase) which was purchased from the US Biochemical Corporation. Radioactive isotypes, Hybond-N nylon membranes and

autoradiography film were all purchased from Amersham International.

Trypanosomes. T. brucei strain TRUC427 [10] was grown either as bloodstream forms in adult Wistar rats or as cultured procyclic forms at 28°C in SDM-79 medium supplemented with 10% foetal calf serum using established procedures [11]. Vectors. The pUC series of vectors (pUC8, pUC8-1, pUC8-2, pUC9, pUC9-1, pUC9-2) has been constructed to contain the polylinkers of pUC 8 and pUC 9 in all three reading frames and both orientations relative to the laeZ promoter [12]. E. coli strains. E. coli strain DF1070 [7] (tonA22 ompF627 edd-l gnd-1 relAl SpoT1 pit-lO T2R) was obtained from B. Bachmann at the E. coli genetic stock centre in Yale. The mutations of the gnd and edd loci in this strain were induced by ethyl methane sulphonate mutagenesis [7]. E. coli strain GB23152 (W3110 trpR lacZ (Am) trpA9605 kdgR ~ A (edd-zwJ)22 A (sbcB-his-gnd-rJb) recA rpsL20 hsdR) [13] was a gift from R. Wolf Jr. All routine DNA procedures were carried out in E. coli strain DH5~(endA 1 hsdRl7 ,~k mk+ ) supE44 thi-I recA1 gyrAl Nal relA1 A(lacZYA-argF)ul(,9 (d?8OlacZAM15)). E. coil strain DFI070 was maintained on 1.5% agar plates including minimal medium containing 6% K2HPO4/0.2% KH2PO4/0.1% (NH4)SO4/ 0.025% trisodium citrate/0.01% MgSO4 pH 7.2 plus 0.2% glucose. In experiments designed to select transformed organisms the glucose was replaced by gluconate, and ampicillin was added to 100/~g m1-1. E. coli strain GB23152 was maintained on identical medium with the addition of 0.01% histidine and 0.01% tryptophan. Nucleic acid preparations. DNA was prepared from procyclic trypanosomes as described [14]. RNA was prepared from bloodstream form trypanosomes and from procyclic forms as described [15]. Large and small-scale preparations of plasmid DNA from E. coli

91

were made using the standard procedures [16]. A library of T. brucei genomic D N A was prepared by partially digesting total trypanosome D N A with the restriction endonuclease Sau3a and purifying fragments in the 2-10-kb size range from a 0.8% agarose gel by electroelution [16]. Fragments were ligated into the BamH1 site of a set of pUC vectors [12] which had been pooled in equal concentrations to give equal chances of ligating fragments in each of 3 reading frames. The ligation mix was transformed into E. coli strain DH5~ using a method modified from [17]. After the heat shock stage of the procedure, the organisms were grown for 1 h at 37°C in SOB medium (2% bacto-tryptone/0.5% bactoyeast extract/0.05% NaC1). A 1/100th aliquot was plated onto an SOB/amp plate containing 0.2% 5-bromo-4-chloro-3-indolyl-fl-D-galactopyranoside (X-gal). The remainder of the organisms were inoculated into 1 1 of SOB medium containing 100 #g m l - ~ ampicillin and were grown overnight in an orbital incubator at 37°C. A large-scale plasmid preparation from this overnight culture yielded an amplified library containing supercoiled D N A with an E. coli K-12 modification pattern. Nucleic acid analysis. Restriction endonucleases were used according to manufacturers' specifications. Electrophoresis of D N A was in 0.8% agarose gel in TAE buffer (0.04 M Trisacetate/0.001 M EDTA, pH 8.0). Electrophoresis of R N A was in 2.4% agarose gels in 10 mM NazHPO4 buffer which was circulated during the run. R N A was heated to 50°C and treated with glyoxal (4/~1 of RNA were mixed with 7.2 #1 of a mixture containing 105 ~1 dimethyl sulphoxide/30/A glyoxal/4.2 #l 0.5 M Na2HPO4) prior to running. Southern and Northern hybridiSation analyses were carried out according to standard procedures [16]. DNA sequencing. Randomly sheared fragments of the E. coli gnd complementing fragments within plasmid pTgGND3.2 were cloned into M13 and sequenced using the Sequenase procedure according to the manufacturer's specifications. Sequencing gels were

analysed using the programs of Staden [18] and the G C G package [19]. D N A fragments for subcloning were purified from agarose gels using Geneclean (Bio 101, La Jolla, CA) according to the manufacturer's specifications. Construction of deletion derivatives of plasmid pTGR-1 to locate the 6-phosphogluconate dehydrogenase gene. Plasmid pTgGND3.2 was derived from pTGR-1. Purified pTGR-1 was digested to completion with PstI, and the largest fragment thus formed (5.7 kb) was reisolated, self-ligated and transformed into E. coli DH5~. Plasmid pTgGNDR1 was derived from pTGR-1. Purified plasmid was digested to completion with EcoRI and the 3.2-kb fragment depicted in Fig. 1 was isolated. Six ligation reactions were set up (using each of the 6 pUC vectors, since it was unknown which one of these 6 plasmids was represented in pTGR-1). The ligation mixtures were transformed into E. coli DH5~. Following bacterial growth four colonies were taken as samples of the product of each ligation mixture, and plasmid mini-preps checked by EcoRV digestion to ensure that the inserted D N A fragment was orientated identically in each plasmid selected for the complementation tests. Finally a single colony was used as the source of each test plasmid. This procedure ensured that at least one of the 6 plasmids tested would contain the genomic D N A fragment in the same orientation and reading frame relative to the lacZ promoter in the vector as the original fragment in pTGRI. Plasmid pTgGND1.6 was derived from pTgGND3.2 by linearisation with PstI, followed by partial digestion with EcoRV. The reaction was allowed to run for 5 min at 37°C using 0.1 unit of enzyme per #g of plasmid. The fragments formed were then separated on a 1% agarose gel. A 4.3-kb fragment was purified from the gel, end repaired, self-ligated, and re-transformed into E. coli strain DH57. All the derived plasmids were restriction mapped to confirm the presence and orientation of the desired inserts, and used to transform E. coli strains DF1070 and GB23152. Transformants were selected on SOB/amp plates at 37°C overnight, then

92

replica plated onto minimal/gluconate plates with other supplements as necessary. Replica plated colonies were grown at 28°C and at 37°C for 3 days.

Results

Identification of a trypanosome 6-phosphogluconate dehydrogenase gene by complementation in E. coli strain DFI070. Gluconate-positive transformants of E. coli strain DFI070 were obtained using the pUC-borne library of T. brucei genomic DNA fragments previously amplified and modified in E. coli strain DH5~. After transformation half the cells were plated out and incubated at 28°C and half at 37°C. The total number of transformants obtained was estimated to be 2.5 × 105 by plating a 1/100th aliquot of the transformed organisms onto SOB/ampicillin agar plates. After 10 days' incubation, 6 gluconate-positive colonies were found growing on the 28~C plates; no such colonies were recovered at 37°C. A similar number of organisms (5 × 105) transformed with pUC plasmid alone, did not yield any gluconate positive colonies at either temperature. Plasmids (designated pTGRI-6) were purified from the 6 colonies and used to retransform E. coli strain DF1070. Transformants were selected on SOB/amp plates overnight and then replica plated onto minimal plates containing gluconate and ampicillin. Five of the six initial plasmids proved to be stable, and conferred a gluconate-positive phenotype on E. coli strain DF1070. However, the phenotypes were again temperature-sensitive, with

Enzyme assays. E. coli strain DF1070 and GB23152 transformed with recombinant plasraids carrying complementing T. brucei D N A fragments were grown at 28°C to late log phase in SOB medium containing 100 #g ml l ampicillin. 1.5-ml aliquots of the organisms were harvested, washed in t ml of 10 mM MgCI2 and resuspended in 350 #1 of 10 mM Tris-HCl pH 7.5/10 m M MgC12/1 m M DTT (TMD). The cells were frozen in liquid nitrogen for 1 rain, then thawed on ice. 30 #1 of 2 M KC1 was added to the cells along with 10 #1 of 30 mg ml ~ lysozyme. The cells were vortexed, left on ice for 30 rain and then frozen for a further 1 min in liquid nitrogen. 600 #1 of T M D was added to the cells. Insoluble debris was separated by centrifugation in a microfuge for 5 min. Total protein concentration within the soluble extract was determined using the method of Lowry [20]. 6-Phosphogluconate dehydrogenase and 6-phosphogluconate dehydratase assays were performed according to refs. 21 and 22, respectively.

complementation of E.coli gnd mutants RV RI RV RV

IIII

pTGRI

Pst

I

RV RI

ii

+

ORF

pUC

pT GND3.2

I I I

I

pTgGNDRI

Ill

l

l

l

+ II

I

pTgGNDI.6

I

Ikb

I

Fig. 1. Deletions of plasmid pTGR-I to locate the 6-phosphogluconate dehydrogenase gene. Constructs were prepared as described in Materials and Methods. Of the recombinant plasmids tested only pTgGND3.2 complemented E. colignd mutants DFI070 and GB23152 on gluconate-minimal medium. (The location of the gnd open reading frame determined by sequence analysis is indicated: ORF).

93 TABLE I Activity of gluconate metabolising enzymes in wild-type E. coil and in strain GB23152 transformed with the pTGR series of plasmids 6-Phosphogluconate dehydrogenase activity (nmol m i n - n (rag protein)- n)

6-Phosphogluconate dehydratase activity (nmol m i n - 1 (mg protein)-- n)

GB23152 transformed with pTGR-1

11.1 _+ 1.3

<0.1

GB23152 transformed with pTGR-2

18.6 _+ 4.2

<0.1

GB23152 transformed with pTGR-3

<0.1

<0.1

GB23152 transformed with pUC

<0.1

<0.1

DH5:~ ( + v e control)

59.5 + 9.6

8.2 + 1.3

growth occurring at 28°C, and not at 37°C. Following purification, these 5 plasmids were digested with a number of restriction endonucleases, and also used to probe Southern blots of T. brucei genomic DNA digested with EcoRI. This procedure showed that the 5 stable plasmids contained closely related inserts of trypanosomal DNA which hybridised to fragments of identical molecular size in the Southern blots. The inserts differed only in length. Of the 2 classes of insert recovered the second was seen to comprise a simple extension of the first (data not shown). One member of the class of plasmids containing the smaller insert (pTGR1) was chosen for further analysis. A set of deletion derivatives of pTGR1 was made by digesting the plasmid with restriction enzymes found to cut the insert, and by subcloning the resulting fragments. From these experiments a 3.2-kb fragment which retained the ability to complement E. coli strain DF1070 was identified (Fig. 1). Complementation of an E. coli deletion mutant. E. coli strain DF1070 is a point mutant. The nature of the mutation in the gnd gene remains uncharacterised. To ensure that the gluconate-positive phenotype of the transformants resulted from complementation and not from extragenic suppression arising from within the trypanosome DNA inserts we used our recombinant plasmids to transform E. coli strain GB23152. Both the gnd and edd genes

R

Bst E 1 1

V

B

H

P Sm Sa

Kb 7 . 2 8 .5 _ _ 6.4-5.7 4.8-4.3-3.7--

gll N

2.3

qD

1.9 1.4

-

1.3 m

-

Fig. 2. Southern hybridization of DNA fragments from T. brucei strain 427 probed with the 3.2-kb fragment of DNA encompassing the gene encoding 6-phosphogluconate dehydrogenase. Total DNA from T. brucei strain 427 was digested to completion with a variety of restriction endonucleases: R, EcoRI; V, EcoRV; B, BamHI; H, HindllI; P, Pstl; Sm, Smal; Sa, Sall. Fragments were separated by electrophoresis in a 1.0% agarose gel then transferred to a Hybond filter. The 3.2-kb insert from plasmid pTgGND3.2 was labelled with 32p and used to screen the filter at high stringency.

94

have been deleted from this strain. All five plasmids which conferred a gluconate-positive phenotype on E. coli strain DF1070 conferred the same phenotype on E. coli strain GB23152. Once again complementation was functional at 28°C but not at 37°C. Determination of enzyme activity in transformed organisms. The gluconate positive phenotype of the transformants could in principle have arisen by complementation acting at either the gnd or at the edd locus, although 6-phosphogluconate dehydratase activity was reported absent in T. brucei [5]. To determine which enzyme activity accounted for the gluconate positive phenotype of our transformants bacteria transformed with plasmid T A T A A C C C T A C A C A A A G A T T G A A G A T A A G T TT T T A T A C & T A T A T T T A C T A C C A C A T A A A T

60

pTgGND3.2 were assayed for both 6-PGDH and 6-phosphogluconate dehydratase activity. Only 6-phosphogluconate dehydrogenase activity was detected (Table I).

Copy number and chromosomal location oJ" the gndgene. Plasmid pTgGND3.2 was used as a probe against total T. brucei D N A digested with a number of restriction endonucleases. The results obtained, when taken in conjunction with a restriction map of the plasmid, suggest that the T. brucei, gnd gene is present as a single copy (Fig. 2). When the same probe was hybridised to T. brucei chromosomes separated by pulsed field gel electrophoresis into megabase-sized, intermediate (200-2000 kb) and mini-chromosomes (50-150 kb) it N A P G I T Q S ¥ G ¥ T L K CAATGCACCCCIG TA T T A C T C A A T C C C C T G G A T A C A C TC T C A ~

K S P S G C AAAAC-CCCC AG T G G

324 1140

120

P E I K O L Y D S V C GCCCC~TTAAGCAGCTCTACGACTCTGTGTGCAT

A I I S C Y A Q TGCCATT&TC TCATGCT&CGCTCA

344 1200

M S M D A T A A A A A T A A C T G T TGT TAC T T T C A G G A A G G G G A A A A A G A C G G A A G A T C A TG T C A A T G G ~

4 180

M F O C L R E M D K V H N F G L N L P A AATGTTTCAGTGCCTGCGTGAGATGGACAAGGTGCATAACT TCGGAC TCAATCT TCCAGC

364 1260

V G V V G L G V M G A N L TGTCGGTG TTGTCC~CTCC~TGATC~TC-CGAACCTC~CT

L N I A E K TC,AACATTC-CGGAGAA

24 240

T I A T F R A G C I L Q G Y L L K P M TACCATTC-CAACTTTCCCICGCCGGTTGCATT T T G C A G G G C T A C C T T T T A ~ C C A T G A C

T

384 1320

G F K V A V F N R T Y $ K S E E F M K A A G G G T T T A A A G T T G C T G TGT T C R A C C G T A C G T A C TC T A A G A G C G A G G A A T TCA T G A A A G C

44 300

E A F E K N P N I S N L X C A F Q T E TGAG~CATTCGAAAAGAATCCCAACATTAGCAATCTCATGTGTGCATTCCAAACCGAGAT

I

404 1380

N A S A P F A G N L K A F E T M E A F A G A A T G C C T C TGC T C C A T T T G C G G G T A A T T T G A A G G C G T T T G A A A C T & T G G A G G C A T T T G C

64 360

R A G L 0 N Y R D X V A L I T S K L E V CAGGGCAGGAC TAC&GAATTACCGCGATATGGTGGCAC T TATCACA TCAAAGTTGGAAGT

424 1440

A S L K K P R K A L I L V Q A G A A T D A G C G T C A C T C A A G A A A C C T C G A A A G C ~ C T C A T C C T G G T G C A G G C GGC,C C ~ TA C G G ~

84 420

S I P V L S A S GTCCATTCCTGTGCTGTCAG~TCCC

444 1500

S T I g O L K K V F E K G D I L V D T G C T C/~ C A A T T G A A C A A C T T A / q G ~ G T~ T T T G A G A A G G ~ G A TA T C C TC G T T G A T A C C G G

104 480

K Y G Q L V 6 L Q R D C A A G TA TGGGC A A C T T G T G TCG T T G C & G C ~ T G T

N A H F K D Q G R R A O Q TAACGCGCA T TT TA A C ~ T C A G G G A C G C C G ~ C C A C ~ A ~

124 540

V D K D G R E GGT~TAAAGAC~C~GAATCAT

144 600

TGTTGATATCATTACGAAGTC~

TGT~TGCAAACAACAGG~CACCTGTG~GGC

A

L E A A G L R TGGAC-GCC,G C A G G TC T C C G

F L G M G I S G G E E G A R K G GT TTC T T C ~ A T C ~ A T A T C C G ~ G A G G A G G G T ( ~ C C ~ G G ~ C A G C C T T T T T P G G T L 8 V W E E I CCCTGC~ACGCTTAGTGTGTC~TACGACCAAT

R

P

I

P

A

F

F

V E A A A A TGTTC~AGG~GCCC~AGC

K A D D G R P C V T M N G S G G T A A G G C A G A T GA T C ~ C G G C C C TG T G TGACC~% T G ~ C G G C A C ~ G G C G ~ T

A

G

S C C A TG

164 660 184 720 204 780

D I L R A M G L N N D E TGACATCCTTCC~TGG~CTC~AACGATC~AAGTTGC

224 S40

V

A

A V L E D W T C ~ C G T T C T T G A A G A T TG

K S K N F L X S Y M L D I 8 ~ A A A R A GAAA TCA~GAACTTCT TC~GTCTTAT&TGCTCC~TATC TCAATTGCAG~GCGCGC~

244 900

K D K D G S Y L T E H V M D R I G $ K G A A A G G A T A A G G ~ T G G A A G TTA TCT T A C G G A G C A C G T G A T G G A T C G T & T T G G A T C G A A G G G

264 9&0

T G L W S A Q E & L E I G V P A P S L N C ACC G G C T TA TGG TCC GC C C A A G ~ C ~ T C T C G A G A T T C,C~G T C C C T G C C ~ C C A G TT T C ~

284 1020

M A V V S R (} F T M Y K T E I% Q /% N A S CA T C ~ T G T C G T & T C G C G G C A G T T C A C A A T G T A T A / ~ C T G A G C G T C A A G C G A A T G C C A G

304 1080

L N Y V T A M F T P T L TCAATTACGTTAC TGCGATGTTTACG~CAACACT F G R H G Y E R GT T C G G T C C ~ A C GC,CT A C G A A A G

464 1560

F Q W P E L Q * T C C A A T G G C C T G A G T T G C ~ T A A C A T A C T TTGC C

479 1620

T C A T A T T C TCT T C G T C C T TTCC TCTGT TCTAT T A A C A G A A A T C T A C G A T G T G A C A T G G A G

1680

CGAT TGTGTGATACGC~TTGATCC~

1740

CGTG~CAAC

S

V

TG T A C A C G C G C G T A T T T T C A T A C A C A A T T TG T

C TC GT G G A A C A A C C ~ A / ~ G G A C ,

GGGGAACGGAA~TGTGCGTGTGGGCAAGGCGGA GAAG~GAGGAGGGTTG

V K M ¥ H N S G E Y A I L Q I W G E V F CG T G A A G A T G T A C C A C A A T T C G G G T G A A T A C G C C A T TT T G C A A A T C T G G G G T G A G G T T TT

I

N

AGAGAC A~

TG T G C A A A A G G TAGC T G A A T A G C G

T~G TT TAT T G T T A T A C T G G T G C A ~ G T

AGTGACACTC-~TTCTTTC

GG T G T C G A A G

TGGGAGGGAG~G

1800 1860 1920

TT TT TCT T TT T T C A A T G C T C T G A A G C A C C T T C U G C C T TC T

1980

CAC T T C G C C G A T G T C G T G C A C A T C A T A A A C C C C T T T T G A G T A A TTAACT T T T T & T T T TGC

2040

CGTGGGAAAG& TCTGTGTG~

TGATT T T T C T T T T & C C A T T A C A A T TTAT T A T & T T A T T

2100

TG/qATT T T C T A A C G A C A T G TT T G T T T A G T T TGTC T TT TTC

2160

T T G / ~ G C C T G G

CAACACTATTTGGAGTACTT TGTTTA

2180

Fig. 3. Nucleic acid sequence of the 6-phosphogluconate dehydrogenase gene of T. brucei and its derived amino-acid sequence. 2.2 kb of contiguous sequence from plasmid pTgGND3.2 was analysed using the programmes of Staden [18]. An open reading frame of 1437 nucleotides was found and computer-translated. Amino acids are written above the central nucleotide in their corresponding codon, using the standard single letter nomenclature. The stop codon is highlighted with an asterisk above it.

95

bound only to the megabase-sized chromosomes, remaining in a compression zone 4 . 4 ~ (results not shown).

Bsf Pcf

Bsf Pcf

Nucleotide sequence of the gnd coding region in pTgGND3.2. The 3.2-kb insert of pTgGND- 2.4-- I~-*

3.2 was randomly sheared by sonication and the fragments sub-cloned into the bacteriophage vector M 13mp8. One hundred M 13 clones were sequenced and a 1437-bp open reading frame was found within 2.2 kb of contiguous sequence (Fig. 3). All of the open reading frame was sequenced on both strands. Two inframe A T G codons were found at the 5' end of the sequence. The first of these provides a translated sequence with best homology to the amino-termini of other known 6-PGDHs. The nucleotide positioned at - 3 relative to this codon is an A residue which is found at the - 3 position of most initiation codons in T. brucei [23]. The codon usage and GC content of the proposed gnd gene are typical of other genes analysed to date from T. brucei [24]. The O R F comprises 49% AT base pairs, while the remainder of the sequence cloned into pTgGND3.2 is 60% AT-rich, a value common to non-coding sequences flanking genes in T. brucei [25].

Northern hybridisation

analysis. A 0.4-kb fragment derived from within the open reading frame encoding gnd was labelled and used to probe poly(A) + enriched R N A isolated from both bloodstream form and procyclic trypanosomes (Fig. 4). The probe hybridised to 2 transcripts of 3.1 kb and 2.9 kb. One of the transcripts appeared to be more abundant in bloodstream form organisms, as judged by the relative intensity of hybridisation of the D N A probe to m R N A prepared from the two cell types. The larger transcript may be a precursor to the smaller, or both may be the products of a common precursor which is differentially trans-spliced or 3'-processed to yield 2 distinct m R N A species. The size of these transcripts is large relative to that of the gene (3.1 kb and 2.9 kb compared with 1.44 kb), although it is not known whether the additional size corresponds

| .4 m

Tubulin probe

gnd probe R

v,l

0.4kb probe

Fig. 4. Northern hybridisation of R N A from bloodstream and procyclic form T. brucei probed with a fragment within the gnd gene. Poly(A)+-enriched m R N A s from l09 bloodstream form (Bsf) and 109 procyclic form (Pcf) organisms were separated by agarose gel electrophoresis. Duplicate samples of each type of R N A were run and transferred to nylon filters. Blots were probed at high stringency with an c~/fl tubulin probe and with a probe derived from within the gnd gene. Autoradiography was carried out for 36 h.

to 5' or 3' flanking sequences.

Comparison of the primary protein sequence oJ the enzyme with other 6-phosphogluconate dehydrogenases. The predicted amino acid sequence of the protein derived from the nucleotide sequence of the complementing TABLE II Net charge and isoelectric points of 6-phosphogluconate dehydrogenase from various sources Species

Charge"

pl b

Trypanosoma brucei

+2 + 1 - 11 - 12 - 14 - 18

7.98 7.65 5.01 4.91 4.94 4.79

Sheep

Salmonella typhimurium Synechecoccus sp. Escheriehia coli Bacillus subtilis

~The net charge of each of the enzymes was calculated based on the charged residue composition (each arginine and lysine residue count as + 1, each glutamate and each aspartate as - 1). bThe isoelectric points for each of the enzymes were calculated using the ISOELECTRIC programme of the G C G package [19].

96 T A B L E Ill Percentage amino-acid identity between 6-phosphogluconate dehydrogenases from various sources

E. coli S. typhimurium B. subtilis Svnechecoccus sp. Sheep T. hrucei

E. coli

S. typhimurium

B. subtilis

Synechecoccus sp.

Sheep

* 96 55 56 52 37

* 56 56 51 37

* 48 48 35

* 51 35

* 33

T. hrucei

*

Sequences were aligned with a gap penalty o f l0 using the SIP program o f Staden [18].

D N A fragment consists of 479 amino acids and gives rise to a protein with a predicted molecular weight of 52 000. The predicted pl of the protein is 7.98, with an overall charge of + 2 (Table II). The amino acid sequence was compared with those of 6-phosphogluconate dehydrogenase enzymes derived from: sheep [26], E. eoli [27], Bacillus subtilis [28], Salmonella typhimurium [29], and Synechococeus sp. [30]. The percentage of identical amino acids shared between these proteins is given in Table III. The trypanosomal enzyme is less closely related to the mammalian and bacterial enzymes than any of the bacterial and mammalian enzymes are to one another. The T. brucei enzyme possesses two internal clusters of amino acids (50-PheAla-Gly-52 and 332-Asp-Ser-Val-Cys-335) not present in the other enzymes. A C-terminal extension present on the mammalian enzyme is absent from the T. brucei 6 - P G D H although the trypanosome enzyme does have a very small extension (2 amino acids) at its Cterminus relative to the enzyme derived from bacterial species.

Discussion

The two principal points of interest reported here are the extent to which the nucleotide sequence of the T. brucei gnd gene diverges from that of all previously reported sequences for genes which encode this enzyme, and the finding that it has been possible to identify the T. brucei gene by complementation of E. coli gnd mutants with genomic trypanosome DNA.

The nucleotide (and corresponding amino acid sequence) divergences characteristic of the T. brucei 6 - P G D H raise the possibility that the trypanosome enzyme possesses structural features which would make it a suitable target for rational drug design. Mutations in 6 - P G D H in yeast [31] and drosophila [32] are lethal, a result which has been attributed to the toxicity of accumulated 6-phosphogluconate. The successful use of a complementation strategy to identify the gnd gene in T. brucei implies that complementation of mutant genes in other microbial species (or in eukaryotic cells) may represent a generally useful approach to the cloning of other trypanosome genes. A full discussion of the significance of the differences between the primary structures of the trypanosome and mammalian 6 - P G D H s must await the outcome of crystallographic studies which are now under way. Inspection of the primary sequence of the trypanosome enzyme reveals that a number of regions of the enzyme are particularly well conserved, as they are in the 6-PGDHs t¥om all other species studied to date. For example, in the sheep liver enzyme the coenzyme binding site has been shown to involve the motif 10-GIy-X-AIa-X-XGly [33]. In the corresponding sequence from T. brucei the ala-12 is replaced by a glycine. However, the coenzyme binding site nevertheless conforms to the fingerprint motif (GIyX-Gly-X-X-Gly) for NADPH-binding enzymes proposed in [34]. In the sheep liver enzyme, Arg-446 and His-452, and Tyr-191 and Lys-260 appear to be involved in binding substrate [33]. All of these residues are conserved in all species for which amino acid

97

sequence data are now available, including the trypanosome enzyme. Clearly, the trypanosome enzyme retains certain key features which have been detected in other 6-PGDHs, and the major differences in the primary sequence reported here relate to other regions of the enzyme. In addition to attempting to identify a trypanosome 6-PGDH gene we have also inquired into the possibility that T. brucei might possess additional 6-PGDH isoforms, for 2 reasons. Firstly, 6-PGDH isoenzymes have been reported in a number of species [31,35]. More interestingly, some of the 6PGDH activity in rat liver cells has been localised to peroxisomes [36]. These organelles are believed to represent analogues of the trypanosomal glycosomes [2,37]. Following the experiments reported here we therefore developed an improved complementation procedure. By using electroporation (instead of the Hanahan procedure) we isolated and screened more than 250 Gnd + E. coli transformants following complementation with genomic trypanosome DNA. All of the positive clones contained the same gene as that reported here, as judged by hybridisation to the trypanosome gnd gene. When used as a probe in Southern blots of restriction enzyme-digested genomic DNA this gene did not hybridise (even at low stringency) to any restriction fragments other than those detected at high stringency. Our evidence suggests either that T. brucei has only a single 6-PGDH gene, or that any other gene present cannot be detected by complementation or by hybridisation to the gene we have identified. Complementation of defined microbial mutant may represent a generally useful strategy for the identification of other trypanosomal genes from genomic DNA. We adopted this approach partly because of the technique's apparent simplicity and partly because complementation imposes a useful functional requirement on the cloned gene product (and will therefore distinguish genes from pseudogenes). Additionally, complementation can be used even when the structure of the target gene is entirely unknown, or when its

homology with cognate genes from other species is too low to permit its tentative identification by hybridisation with heterologous DNA probes. The absence of known introns in genomic trypanosomal DNA, and the success experienced by other workers in detecting other eukaryotic genes from cDNA sources [8,9,38] also encouraged the application of this method in this instance. Furthermore, complementation can occur without the need for efficient gene expression provided that slow-growing colonies such as those obtained in the present study are not out-grown by revertants or leaky mutants. If deletion mutants are available (as in the present instance) complementation can be distinguished directly from extragenic suppression, and potential cloning artefacts due to recombination between the complementing fragment and the wild type gene can be avoided. Certain features of the cloning strategy we adopted appeared to be critical, whilst other precautions taken by us appeared unexpectedly irrelevant. In order to ensure transcription of the complementing gene or gene fragment the genomic DNA library was prepared so that the DNA fragments were ligated downstream of the E. coli lacZ gene regulatory elements. This was done using an entire family of vectors (pUC 8, pUC8-1, pUC8-2, pUC9, pUC9-1 and pUC9-2), to ensure that the ligated DNA fragments would be introduced in both orientations and in all 3 reading frames relative to the adjacent regulatory elements. Nevertheless, in common with several other reported experiments where eukaryotic genes have been identified by complementation in E. coli [38,39], transcription of the ligated DNA fragments arose independently of the presence of the promoter in the vector, and complementation was detected regardless of the orientation of the DNA fragment relative to the lacZ promoter. This was apparent from the finding that re-cloning the insert from pTgGND3.2 into each of the pUC vectors independently resulted in identical rates of growth of complemented colonies (data not shown). In apparent contrast to the ease with which

98

we obtained a sufficient level of gene expression to result in functional complementation are the recent findings that a previously cloned 7". brucei phosphoglycerate kinase gene could only complement P g k - E. coli cells selected on glucose after a functional E. coli ribosome binding site was created by site-specific mutagenesis of the trypanosome D N A [40]. We have noted that the D N A sequence 5' to the open reading frame of the T. brucei gnd gene described in this paper has a G G A trinucleotide motif (which is a central feature of the Shine-Dalgarno sequence [41]) located 7 bp upstream of the A T G initiation codon, and in view of the results of Alexander et al. [40] the presence of this motif may have been significant. In developing the approach used here for trypanosome gene cloning by complementation in E. coli we also took into account the conditions in which the bacterial cells were incubated. The incubation period was prolonged so that relatively inefficient gene expression might still give rise to complementation and be detected. Additionally the bacterial cells were incubated at both 28°C and 37°C after transformation, to allow for the possibility that a eukaryotic gene product might not fold correctly, and therefore aggregate or degrade in E. coli at temperatures above 30°C [42]. This was a particularly critical precaution, since the complementation we have observed is consistently temperature sensitive. Recent temperature shift experiments imply that this effect is due to rapid degradation of the trypanosome protein in E. coli at temperatures over 30°C We conclude that complementation can be used successfully to clone trypanosome genes, providing that careful attention is paid to both the molecular genetics and to the physiology of the mutant and complemented phenotypes.

Acknowledgements We thank the Wellcome Trust for financial support, Dr. B. Bachmann for E. coli strain DF1070, Dr. R. Wolf Jr. for E. coli strain GB23152, and Dr. R. Brousseau for providing

the pUC series of vectors. We also thank Dr. H. Gardner and Dr. H.P. Voorheis for advice and discussion.

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