The EMBO Journal vol.4 no. I I pp.2997 - 3003. 1985

Structure and regulated expression of genes encoding fructosebiphosphate aldolase in Trypanosoma brucei

Christine E.ClaytonThe Rockefeller University. 1230 York Avenue. New York. NY 10021. USA

Communicated by P.W.J.Rigby

Low stringency hybridisation with a rabbit aldolase cDNAwas used to select cDNA clones encoding fructose biphosphatealdolase in Trypanosoma brucei. A clone which is almost fulllength encodes a protein of 41 027 daltons which has 50%identity with rabbit aldolase A and slightly lower homologywith B-type aldolases. The homologous mRNA is at least6-fold more abundant in bloodstream trypomastigotes thanin procyclic forms, as expected from measurements of enzymeactivity. Genomic mapping results indicate that trypanosomeshave four copies of the aldolase gene arranged as two copiesof a tandem repeat. The protein has a short N-terminal ex-tension (relative to other known aldolases) which could beinvolved in the glycosomal localisation of the enzyme.Key words: Trypanosoma/aldolase/mRNA/glycosome

IntroductionThe African trypanosomes are parasites of wild and domesticanimals and of humans. Existing drugs are unsatisfactory andprospects for immunoprophylaxis are extremely poor. As try-panosomes in the mammalian bloodstream depend entirely onglycolysis for their energy supply the glycolytic pathway is anobvious target for chemotherapeutic attack (Fairlamb, 1982). Thehigh rate of glucose utilisation (Bowman and Flynn, 1976) maybe an intrinsic property of the enzymes involved, or it could bea consequence of their compartmentalization within unique micro-body-like organelles called glycosomes (Opperdoes and Borst,1977; Opperdoes et al., 1981). These membrane-bounded organ-elles contain 8-9% of the total protein in bloodstream formtrypanosomes, including the glycolytic enzymes (Opperdoes etal., 1984; Opperdoes and Borst, 1977) and enzymes involvedin pyrimidine (Hammond et al., 1981) and alkoxyphospholipid(Opperdoes and Cottem, 1982) biosynthesis and carbon dioxidefixation (Opperdoes, 1984). The glycolytic enzymes appear tobe very closely associated within the organelle (Oduro et al.,1980; Aman et al., 1985; Opperdoes et al., 1984), and tend toaggregate in vitro (Misset and Opperdoes, 1984). Until detailedstructural and kinetic information is available it will be imposs-ible to tell whether the rapid glycolysis is a consequence of indi-vidual enzyme characteristics, specific enzyme interactions orhigh local concentrations of glycolytic intermediates. Three ofthe glycosomal enzymes have been purified (Misset and Opper-does, 1984) but so far very little pertinent structural informationis available. The mechanism of glycosome assembly is unknown.The morphological and biochemical changes that occur when

bloodstream trypanosomes are ingested by Glossina, the insectvector, are a promising system for analysing the regulation ofgene expression. Long slender bloodstream trypanosomes lackcitric acid cycle enzymes and cytochromes; ATP is generatedonly by substrate-level phosphorylation (Bowman and Flynn,

1976), and mitochondrial structure is rudimentary (Brown et al.,1973). Glucose utilisation by the 'procyclic' forms growing inthe insect vector, Glossina, or in culture at 27°C, is much moreefficient: they have an elaborate mitochondrion with electrontransport chain-mediated respiration (Brown et al., 1973; Ghiottoet al., 1979; Bienen et al., 1981, 1983). Although the mor-phology of the glycosomes is unchanged during the transition,the activities of many of the glycolytic enzymes are halved. Some,such as hexokinase and fructose biphosphate aldolase, are reduced10- to 30-fold (Brown et al., 1973; Opperdoes et al., 1981; Hartet al., 1984).

Previous studies of nuclear transcription control in trypano-somes have mainly been restricted to the phenomenon of antigenicvariation: a process of sequential expression of genes encodingthe variant surface glycoproteins (VSGs) which often involve generearrangement (Bernards, 1984). When trypanosomes transforminto procyclics VSG expression is rapidly shut off (Overath etal., 1983). It is known that most or all trypanosome mRNAshave at their 5' end an independently coded (Goyaux et al., 1985)35-bp 'mini-exon' (Boothroyd and Cross, 1982; De Lange et al.,1984; Parsons et al., 1984) although housekeeping genes andVSGs appear to be transcribed by RNA polymerases of differ-ing a-amanitin sensitivity (Kooter and Borst, 1984). Transcrip-tional promoters have yet to be definitively identified. Clearlyit is necessary to investigate the control of transcription of genesdifferent from, and simpler than, those encoding VSGs in orderto distinguish general features of trypanosome transcription fromthose which are unique to VSG expression.The glycolytic enzyme whose activity is most strongly regu-

lated during procyclic differentiation is fructose biphosphatealdolase (Hart et al., 1984). I have cloned the cDNA and genesencoding this protein. Structural and sequence information areconsistent with trypanosome diploidy, indicate that trypanosomealdolase is most closely related to the A-type eukaryotic aldolases,and are suggestive of a processing event during import of theprotein into the glycosome.

ResultsCloning of the trypanosome aldolase cDNAsIn preliminary transfer hybridisation experiments, rabbit andhuman aldolase clones and various fragments from them wereradioactively labelled and hybridised to trypanosome genomicDNA. An 800 bp PstI fragment from the coding region of rab-bit aldolase specifically detected two HindIll fragments of - 4 kband 8 kb (not shown). This probe was therefore used to screen8000 plaques from a T. brucei 427 bloodstream form cDNAlibrary. Six clones were obtained showing hybridisation stablein 2 x SSC at temperatures up to 55°C. Each of the clonedbacteriophage DNAs hybridised to the central portions of the rab-bit and, more weakly, the human aldolase cDNAs; very weakhybridisation was also detected with a fragment containing thelast 100 bp of the 3' end of the rabbit coding region. The clonedtrypanosome DNAs showed strong homology with each other,

(A IRL Press Limited, Oxford. England. 2997

C.E.Clayton

had similar restriction maps, and gave similar patterns of hybrid-isation to Northern and Southern blots of trypanosome DNA andRNA. The three largest inserts were - 1.9 kb; each was sub-cloned into a plasmid vector and one, paldl7, was arbitrarilychosen for sequence analysis.Regulation of aldolase mRNA levelsThere is up to 30 times more aldolase activity in bloodstreamform trypanosomes than in procyclics (Hart et al., 1984). Toassay for developmental regulation, the aldolase clones were usedas probes on Northern blots of equal quantities of T. brucei 427bloodstream form and procyclic TREU 667 poly(A)+ RNAs(Figure 1). A single mRNA was detected of - 1.9 kb (lanes Aand B) with mobility intermediate between the T brucei VSG117 mRNA and the tubulin mRNAs. The mobility of the excisedcDNA insert was very similar (not shown) suggesting that thethree 1.9-kb clones were essentially full length. The homologousRNA was more abundant in the bloodstream form (lane B) thanthe procyclic (lane A) trypanosomes. Re-hybridisation of the blotswith a tubulin probe indicated that there was approximately twiceas much tubulin mRNA in bloodstream forms (lane D) as in pro-cyclics (lane C), as observed by Van der Ploeg et al. (1985).Subsequent re-hybridisation with a sequence from downstreamof the aldolase genes which is constitutively expressed at a lowlevel (not shown) served as a further control.To confirm and quantify the regulation of aldolase mRNA

levels the abundance of the transcript was measured in slot blotsof total RNA from bloodstream and procyclic forms of two

A B C.- D

-i-95* i _ -1 W85

s w-1'7Is

Fig. 1. Regulated expression of aldolase mRNA. Transfer hybridisationanalysis of procyclic (A,C) and bloodstream (B,D) poly(A)+ RNA (5 ag).The blot was hybridised with 32P-labelled paldl7 (lanes A,B), then afterprobe removal re-hybridised with a tubulin probe (lanes C,D). Sizes markedare those of the tubulin mRNAs and VSG 117 mRNA.

kb 0

EA R A PsRRAR XIt I,I II I I I I

trypanosome populations: strain LUMP 1026 (Bienen et al.,1981) and a clone (BUT55) isolated immediately after tsetse trans-mission of strain TREU 667 (Hudson et al., 1980). The aldolasemRNA was - 6 times more abundant in bloodstream than in pro-cyclic trypanosomes. (The apparent greater difference in Figure1 is an artifact of reproduction.) The intensity of hybridisationto control slots containing the paldl7 insert in the lambda vectorindicated that the abundance of the aldolase transcript in the blood-stream mRNA was - 0.1 % of poly(A)+ RNA (data not shown).[From oligo(dT) binding, -5% of total RNA was poly(A)+.]When the lambda cDNA banks were re-screened with a paldl7probe, it hybridised to 0.3% of bloodstream cDNA clones and0.03% of procyclic cDNA plaques, in general agreement withthe slot hybridisation results.Sequence of the aldolase cDNAThe paldl7 cDNA has a single long open reading frame flankedby 65 bp of 5'-untranslated region and a remarkably long (640bp) 3 '-untranslated region terminated by 57 bp of poly(A)+ tail(Figures 2 and 3). The 5' end does not show any homology tothe mini-exon. Eight more independently isolated 1.9-kb aldolasecDNAs were analysed for the presence of the mini-exon bySouthem transfer hybridisation, with a genomic mini-exon probe.A cDNA (from the same library) which contains 22 bp of themini-exon (Cully et al., 1985) served as positive control. Mini-exons were not detected on the aldolase clones tested. Furtherexperimentation will be necessary to determine whether the mini-exon is present on the aldolase mRNA.The protein mol. wt. predicted from the sequence is 41 027,

close to the published value for aldolase of 40 000 (Misset andOpperdoes, 1984). The codon usage is similar to that of the tubu-lin genes (Kimmel et al., 1985) and markedly at variance withthose for VSGs and 'expression site-associated' genes (Cully etal., 1985). For example, the percentages of G+C at the thirdposition are 57% for aldolase and tubulins, 49% for VSGs and43% for expression site-associated genes. Notable are a strongpreference for the arginine codons CGT and CGC and the avoid-ance of TTA, TTG and CTA for leucine.The nucleotide sequence shows 55% homology with that of

rabbit aldolase A (Tolan et al., 1984) and lower homology withthe published B-type aldolase sequences [chicken (Burgess andPenhoet, 1985); human (Rottman et al., 1984); rat (Tsustsumiet al., 1985)] (Table I); no other significant nucleotide or aminoacid homologies were found in the Genbank or Dayhoff data-bases. The DNA homology starts 71 nucleotides into the coding

1;005

R AI I

1.5 1.9

ER R A R R Ps R AI I II , * if

b c

-4

.U4-

4- 4-

Fig. 2. Restriction map of, and sequencing strategy for, paId17. Coding region represented by filled area. Labelling points for chemical sequencing denotedby asterisks; a, b and c are M 13 clones used as hybridisation probes for genomic mapping. Enzymes are: Ps - PstI; A - AluI; R - RsaI; E - EcoRI;X - Xi?oI.

2998

ism

Trypanosome aldolase genes

II* 20 * ~ 3()*o*5 *

ATI (;AI CCA CATI TIAA T(CA AAC AAT'IAT ACC AAC AAG CCI; GAA AA(: ATA AAC: TCA ACT GCA

h* 7o* o*93 90* iu5 lo*ACT AAG ATC TCC AAG cc;r (TTr CAA CTTC CTC' cTr ACC CAA crc CCTr GCCC TAC ATC CGCC

Melt Ser Lys Arg Cal CIGICal L.eu Leu The CIn Le,i Pen Ala Tyr Asn Aeg

I0 30* 14()* 50* 160* 170(CTG AAG AC(; CCA rAT GAA CCC GAG CTG; ATT GAA ACT GCC AAA A.AG ATG ACC GCC CCCLeni Lys 'The Pro Tyvr GItin Ala hia Leu Ilie Gu Ther Ala Lys Lys Met Thr Ala ilro

A;)* 193* )3*20 in* 22(1* 230*GGlT AAC (;(T cTC crcC CC CcC GAT CAC TCC ACT CCT TCT TGCl TCC AAC CGC TTT GcCCGiv Lys Ctv Leni Leai Ala Ala Asp Ciu See Thr Gly Ser Cys See l.ys Arg Phe Ala

24* 253* 26(3 2 70* 280*GGC ATC llGT CTC AGC AAC ACrCGCATGA CAC CGC CG;T CAC TAC CGC GCT CTC ATC CTG;Iy Ile TIy leu See Assi Ther Ala Gtin Ais Arg Aeg Glsi Tyr Arg Ala Leu Met Leu

290* 33o* 31 3) 32(0* 33()* 340*i;AA TGC GAS CCTTTi"C CAT CAT TAC ATC AGC CCT GCT ATC CTG; CAC CAT CAT ACT GTGGILi Cvs Glu Tip Phe TIu Tin Tyvr IlIe Ser Gly Cal Ilie Leti His Asp Glsi Thr Cal

350* 3603* 370* 380* 390* 400*TAT TAT ASS CCT AAC AC CCGCGAA ACA TTC CCT CAT TAT CTC CGCC CGT CGT GGT GTGTvr TIn Lvs Ala Cys Thir Cly GIL Thr Phe Pro Ginl Tyr LCe* Arg Arg Arg Tip Cal

41(1* 420* 4303* 440* 450(*1;TG CCTI GGiC ATC AAA ACT TATr TGC GGC cTC CAT CCC CTC GTTG CAT GCT GCC AAG GCCCal Pro Gly Ilie Cys Thr Asp Cys Glv Lei* Ciu Prs Ceu Cal Cisj Glv Ala Cys Tiv

460* 470* 483* 490 53* 510*CAT CAT ATG ACT GCCT GGT CTC GCA GGT TAT ATC AAA CGC GCC ATG AAA TAT TAT GCTGiu Clia Met The Ala Tip L.1* Asp Cly Tyr Ilie Cys Arg Ala Lys Cys Tyr Tyr Ala

5211* 533)* 540* 55((* 560* 570*ATTG CCC TGC cGCC TTC TITC AAG TCC CCC ATC CTG TAT ATG ATC CAT ATC GCC ACT GTTGMet Tip Cys Arg Phe Cys Cys Trp Arg Ass Cal Tpr Lys Ilie Tin Ass Tiy The Pal

5A3* 590* 600* 61(3* 620*TcT GAA GCT GTTr GTT CCC TTC ATC GCT GAA ACA CTTT GCT CCC TAT GCT APTC CTTT TCCSee GTin Ala Val Cal Arg Phe Ass Ala lTis The Ce* Ala Aeg Tye Ala Ilie Leu See

633)* 6403* 650)* 6610* 6710* 680*CAA CTC TGTCCG TTCTTGT CCIG ATT GTG GAG CCT TAT GTT ATG; ATC CAT CCC ACT TATGtin Leni Cys Tip Len Pal Pen Ilie Val G;la its Tin Pal Met Ilie Asp Tip The His

6911* 700* 7113* 7 20* 730* 740*TAT ATT TAT ACT TGC CAA CCC TT TC TAT TAT GTTG TGG TCG CAT GTTG GTT TCT GCAAsp Ilie G;lu The Cys Tin Aeg Val See Tin His Cal Tep See Ciu Cal Cal Pee Ala

753* 760* 770* 78(0* 790* 9133*CTC TAT CGCCTAT CCC GTT GTA TGG TAT TTA TGCT cTTG CTTG OATG CCC OATC ATG GTTT GTTTCe*i His Arg His Tip Pal Cal Tep GTin Tip Cvs Ce* Ce* Cps Pe* Ass Met Pal Pai

813* 820* 8301* 8401* 853*ccTT CCC GCTT CGAA TCCCCC CTC OATG GCCTTAT GCCC CGAGCTAT GTT CCC TAT TAT ACT TTTPeo Tip Ala GIu See Tip Len Cps Tip His Ala Clis Tin Cal Ala Tin Tyr Tile Cal

8613* 873* 8(3* 8990* 900* 910*AAA ACTTCcCGcc CGT GTT ATT CCC CCT GCGCCTC CCC GGT G;TG ACT TTC CTA TCA GTTLys The Leu Ala Arg Pal Ite Pro Pen Ala Le* Pea Tip Cal The Phe Leu Pee Tip

92o* 930* 940* 950* 9601* 970*GCCCTTT ACT CAT GTTT ATG CCC TCC TAT TAT CTC OATC GCT ATG OATC OATC TTC CCC TTATip ILeu See Tin Clal Met Ala See Glu Tpe Ce* Ass Ala Met Ass Ass Cys Pen Leu

99(o* 9901* 1MO(* 131(3* (102(0*CCA CCC CCA TGC AAA CTTG ACT TTT PTCA TAT CCC CCT GCA CTT TAT TCC AGCT CCC ATAPen Arg Pen Tep Cps Leu The Phe See Tyr Ala Arg Ala Lets Tin See See Ala Ilie

11130* 104(3* 1(1513* 106(0* 1(17(1* IL(80*OATG CCC TGG TTT GGA OATG GAATTTCTGGTGTC GAA GCT CCC CCC CGT GCCC TTC ATT TATLys Arg Trp Tip Tip Lys Tin lee Tip Pal TIin Ala Tip Arg Arg Ala Phe Met His

1(090* ii((0* 1113* 1123* 1130* 1140*CCC GCA OATG OTT AAC PTCA CT'C CCC CAA cTTT CCC OAT TAT OATC CGT GCTT TAT TAT TATArg Ala Lys Met Ass See Lesi Ala Tin LenTip CpLs Tpe Asn Aeg Ala Asp Asp Asp

11513* 1163* 117(3* 118(3* 1193*OATG TATC TCCAT TCT CCC TAT GTT GTT CCC OATC ACA PAT TAA OAT' AAP PAP GCC CAPLys Asp lee Tin Ser Ten Tyr Pal Ala Tip Assi Tile Tyr--

121(1* 1210* 122(1* 123(1* 1240* 125(1*PTA GP CTTGG T CcCCTTT GTC TCGTTCTTTPP cCCTT GGA OATG GTT CCC GGA CPA APTC1260* 127(1* 1280* 129(1* 131)0* 13110*PTGA TGG CAT AGCT ACT TAT GTG CGCCTPG CAT GTT GGT TAT GAA CCC CCC APTG APT TAA

13213* 1330* 1340* 1350* 13601* 1373*OATG OATG AOA APT CC TTGA PAT CTT APTG TTT OATG ACT ATA OAT OAT CPTG OATCCCCTTP

1380* 1390* 1403* 1413* 1420*PAT APT CTTP GCT TGGTTGPTCPT CGT TGG TGC APTG CCC OAT CTPC CTA GTT TGC TTT TTA

1430* 1440* 14511* 1460* 1470* 1480*GCA CCC CCC CPA CAP CAA TGG GTA TAPr ATC PTTG CTT, CCT CCC GCA GCT TAT CGT GCCC

1490* 1503* 1510* 1520* 1530* 1540*AGC PTGC CCC TAT CCC G;TT TCT CTA ACTCGT GTTG TAT CTT CCI; TCT TAT ACT TGT AAA

1550* 1560* 15713* 1580* 159(1*ITT PTA OAA ACT OAT ATA TAA PTA CTP TTT GAA OATG TGG TAA AAA ACT OAT ATA TCT

16(10* 1610* 1623* 163(3* 1640* 1653*TPT TTT TPT GTT PTA ATA CCC TCT TPTT T TGT APTG AGA GCA ATA OAT OATG TGT GTG1660* 1*70* 1680* 1690* 1700* 1710*

TGT GTG TGT GTG TGT GTT GTT GTTP GTT GAA AAA APTG OATG TAT ACT AAA GCCC CTC APP1720* 1730* 1740* 17513* 1763*

OATG GGT CCC CAT ACA CCA ACT ATA APT APTc TCGA AOA PAT TTT CAP GCA CCA OAT OATG177o* 17813* 179(1* 1830* 181(3* 1820*

PTGA ACA TAT PTGA TCGA OAT CAA AGA GTT GGCA GGA CTG ATA CCGG ACT TTT CTA TG CAT18313* 184(1* 1850*TGG TAA APP CAT TTG ACTTA)5

Fig. 3. Sequence of paldI7.

Table I. Homology between trypanosome aldolase and mammalian aldolases

Animal DNA Protein

Rabbit 55.3% 49.9%Rat 52.7% 46.8%Human 52.6% 46.5%Chicken 51.9% 46.5%

Table II. Amino acid sequence similarity to rabbit aldolase of trypanosomeglycosome and rat aldolase subdivided according to rat gene exons

Rat Trypanosome Rat Commentsexon Equivalent No. of %I %I+C Gaps %I

region residues

Ila 0-24 24 4% 8% 0 69% N terminuslIb 25-47 23 65% 82% 0 83%III 48- 117 70 50% 64% 1(T) 58%IV 118- 136 19 42% 52% 1(R) 61%V 137-190 54 44% 55% 0 79% Substrate

bindingVI 191-218 28 57% 75% 0 82%VII 219-276 58 45% 56% 0 83% Active siteVIII 277-343 67 59% 69% 0 67%IX 344-372 29 38% 55% 2(T) 45% C terminus

R: rat. T: trypanosome. I: identical. C: conservative change [groups ofamino acids defined as by Von Heijne (1983) except acidic and basicresidues separated].

region and dies away at the termination codon. The degree ofprotein sequence homology varies in parallel with DNA sequencehomology. There are only four gaps (Table II) in the alignmentbetween the trypanosome and rabbit enzymes; the protein se-quence is clearly more conserved in some regions than in others.At the N terminus, the trypanosome aldolase is 10 amino acidslonger (excluding the initiation methionine) than the other aldo-lases. Strong homology starts at residue 24 in the rabbit sequence(Figure 7).Organization of aldolase genesThe genomic organization of the aldolase coding sequence wasinvestigated by restriction digestion and transfer hybridisation ofgenomic DNA and of a genomic clone selected from a TREU667 library. The aldolase genes are present on a single 12.5-kbfragment bounded by EcoRI and PvuII sites (Figures 4 and 5,lanes C-G). The number of gene copies was estimated byhybridisation of DNA from T brucei 427, variant 221, whichcontains only the 'basic' unexpressed copy of the VSG 117 gene(Bernards et al., 1985; Van der Ploeg et al., 1982), with VSG117 and aldolase probes of identical lengths and specific activities(Figure 5, lanes D,F; VSG genes denoted by asterisks). Densito-metric scanning of the autoradiogram suggests that, assuminga single copy of the 117 basic copy gene, there are 4-5 copiesof the aldolase gene in the trypanosome genome. The 117 VSGprobe shows weak homology to sequences in the TREU 667genome, apparent after prolonged autoradiographic exposure.Digests with Sall, EcoRI and HindIII (including double digests)reveal no differences between bloodstream and procyclic formsof T. brucei 427 in the genomic arrangement of the aldolase se-quences (data not shown).The map (Figure 4) of the aldolase genes represented by the

2999

0

Pv EB FV BI I i I I

H

10 20

E Pv B SB X SSB X X E PvI,I I l .,I , ,,I t I

I I f I I I I I I

H Ps Ps H Ps Ps H Ps Ps

Fig. 4. Restriction map of the aldolase genes. Boundaries of the genomic clone denoted by double arrows; transcribed regions represented by tlilled areas witharrows indicating the direction of transcription. Enzymes are: B - BamnHI; E - EcoRl; H - HinidIII; Ps - PstIl Pv - Pi'iII: S - Still- X - X/oI.Dotted lines indicate additional PstI sites in a postulated second copy of the repeat.

. ,.

24

-~~~~~-

,440

.2:0

0-@

48- *

Fig. 5. Organization of the aldolase genes. Restriction digested DNA fromT. brucei TREU 667 procyclics (A,B,C,E,G) and EATRO 427 (variant221) bloodstream forms (D,F) (2 /g per lane) blotted and hybridised withaldolase and VSG 117 probes. Asterisks indicate the 117 basic copy bands.Enzymes are: A - PstI; B - HindIII; C - EcoRI, PvuII; D,E - PvuII;F,G - EcoRI.

genomic clone shows two aldolase genes in a 4-kb tandem repeatdelineated by BamHI, Sall and HindIII (Figure 5, lane B). Thedirection of transcription was defined by re-hybridisation of theblots with M13 clone probes 'a' and 'c' derived from the 3' and5' ends of paldl7 (Figure 2). The genes have the same restric-tion map as the cloned cDNA, with no evidence for introns.Scrutiny of the genomic PstI digest (Figure 5, lane A), however,suggests a more complicated picture. The flanking sequences are

present as a faint band at 3.3 kb and two co-migrating fragmentsof 2.9 and 2.85 kb; the central 1.05 kb coding region fragment,colinear with the cDNA probe, hybridises more strongly as ex-

pected. Unexpected are the two smaller bands, absent in thegenomic clone, whose sizes (600 and 450 bp) and combined in-tensities add up to those of the 1.05-kb band. The 600-bp frag-ment is cut by XhoI and hybridises to the internal M 13 clone'b' which spans the XhoI site (Figure 2). In all other respectsthe results of genomic digests are entirely consistent with the mapof the genomic clone. The simplest interpretation of these resultsis that the trypanosome genome contains two copies of the tandemrepeat, making a total of four copies of the coding region. Inthe second copy of the repeat, both aldolase genes contain an

additional PstI site at the positions indicated (Figure 4).

T. brucei A L

Type A A Laldolases

Type B A Laldolases

H R H G V V W E G C L

S D H H I Y L E G T L(B) (V) (Q)

N D H H V Y L E G T L

L K P N M V V P G A E S G L K

L K P N M V T P G H A C T Q K(D) (A) (B) (Z)

L K P N M V T A G H A C T K K(S) (P)

Fig. 6. Comparison of active sites of T. brucei and other aldolases.(:) Indicates amino acid identity and (.) conservative changes (as in TableII).

DiscussionConservation of the aldolase sequence

Genomic and cDNA clones have been selected from trypanosomelibraries by virtue of their homology to a rabbit-aldolase-A-encoding cDNA. The homology with mammalian aldolase se-

quences, although lower than between trypanosome tubulins(84%) (Kimmel et al., 1985) and calmodulin (89%) (Tschudiet al., 1985) and the corresponding proteins from highereukaryotes, is unlikely to be fortuitous: the trypanosome sequence

shows no homology with any non-aldolase protein or DNA se-

quences in the Dayhoff and Genbank databases, and, most import-ant, the active site lysine (residues 229 in the rabbit sequence

and 240 in the trypanosome sequence) three amino acids im-plicated in substrate binding (Arg 55, Lys 146, and Arg 148 inthe rabbit sequence) and the C-terminal tyrosine whose removaldecreases aldolase activity (Horecker et al., 1972) are all con-

served. Tsutsumi et al. (1985) compared the rat aldolase B se-

quence with human aldolase B and rabbit aldolase A aftersubdividing the protein into portions corresponding to the ratgenomic exons. Although, for the most part, residues that are

least conserved between the vertebrate aldolases are also differentin trypanosome aldolase, the pattern of conservation of exon

equivalents is not parallel to that observed for the mammalianenzymes (Table II). Some amino acids in the active site regionwhich are conserved in all higher eukaryotic aldolases (Rottmanet al., 1984) are altered in the trypanosome enzyme (Figure 6).

B-type aldolases hydrolyse fructose-1-phosphate and fructose-1,6-diphosphate with equal efficiencies, whereas for A-type en-

zymes the ratio of specific activities with the two substrates is- 50:1 (Horecker et al., 1972). The trypanosome enzyme shows

greater sequence homology to rabbit aldolase A than to human,rat and chick B type aldolases. This is consistent with the availablekinetic information: Misset and Opperdoes (1984) reported thatthe trypanosome enzyme was seven times more active with thebiphosphate than with the monophosphate substrate. The trypano-some sequence should be of considerable utility in attempts to

define the elements of primary and secondary structure respon-sible for particular aspects of the enzyme mechanism.Developmental regulationThe activity of fructose biphosphate aldolase is 20-30 times

3000

C.E.Clayton

30 k b

Ps

B

H

Trypanosome aldolase genes

Spinachchloroplast SSYADELVKTA-KTVASPG

>< . .. .. .: . ..:Trypanosome MSKRVEVLLTQLPAYNRLKTPYEAELIETAKKMTA-PGK

Rabbit PHSHPALTPEQKKELSDIAHRIVA-PGK1* 14* 24*

Fig. 7. N-terminal alignment of trypanosome. spinach chloroplast and rabbitaldolases. Symbols as in Figure 6. Arrows indicate the putative processingpoint.

higher in bloodstream than in procyclic forms (Misset and Opper-does, 1984), but the difference in steady-state mRNA levelsobserved in this study was only 6-fold. Hence it is possible thatthere is some form of translational control. However, the blood-stream trypanosomes were ice-cooled before mRNA extraction.It is now known that cooling causes a rapid shut-off of VSG tran-scription (Bernards et al., 1985) and also influences the expres-sion of heat shock-like sequences (Van der Ploeg et al., 1985).The effect of the use of citrate, which may be a promoter of pro-cyclic transformation (Brun and Schoenenberger, 1981) as anti-coagulant is also uncertain. It will therefore be necessary toemploy revised methodology to assess accurately the degree oftranscriptional regulation.Genomic structure and ploidyThe aldolase genes are arranged as a direct tandem repeat; anorganization similar to those of glyceraldehyde phosphate dehy-drogenase and phosphoglycerate kinase (Gibson et al., 1985).Trypanosome calmodulin (Tschudi et al., 1985) and tubulin(Thomashow et al., 1983) are encoded by multiple tandemrepeats; the only exception found so far to this type of house-keeping gene organization is the single-copy triosephospate iso-merase gene (Gibson et al., 1985). Restriction mapping resultsindicate that there are two copies of the aldolase repeat in thetrypanosome genome, differing in the fine structure of the codingregion. It is tempting to speculate that these two copies representallelic variants of the aldolase gene. Observations of restrictionsite polymorphisms in the three housekeeping genes examinedby Gibson et al. (1985) and of isoenzyme distribution patterns(Gibson et al., 1980; Kilgour, 1980), together with measurementsof DNA content and complexity (Borst et al., 1980, 1982) indi-cated that trypanosomes are diploid and undergo some form ofgenetic recombination (Tait, 1983). However, the karyotypeobserved by pulse-field-gradient electrophoresis (Sloof et al.,1983) and the behaviour of VSG genes (Bernards, 1984) sug-gest that not all trypanosome chromosomes are present ashomologous pairs.Glycosome biogenesisThe most remarkable aspect of the trypanosome aldolase sequencepresented here is its dramatic divergence from other publishedsequences at the N terminus (Figure 7). [Careful inspection ofthe trypanosome sequence does not reveal alternative, morehomologous, reading frames in this region. Also, DNA and RNAtransfer hybridisation data, using as probe both paldl7 and the5' end M13 subclone 'a' (Figure 2), indicate that the cDNA iscolinear with a single mRNA. A cloning artifact therefore is im-probable.] The amino acid and nucleotide sequence homologybetween the rabbit and trypanosome enzymes starts abruptly ata position corresponding to residue 24 (rabbit) and residue 35(trypanosome) although very weak correspondence begins atresidues 14 and 25. Comparison with the N terminus of spinachchloroplast aldolase, a nuclear-encoded enzyme which may beprocessed during uptake into the plastid (Lebherz et al., 1984)

is informative: the few residues conserved in the trypanosomeand rabbit N termini are also present in the plastid enzyme (Figure7). The mature N terminus of the plant enzyme, however, isshorter than that of the rabbit protein, whereas trypanosome aldo-lase has an N-terminal extension of 10 amino acids.The trypanosome glycolytic enzymes have distinctive physio-

chemical properties, including a tendency to aggregate (Missetand Opperdoes, 1984). They also presumably have some mechan-ism to ensure localisation within the glycosome. Mechanisms oforganellar assembly are varied. The lysosomal entry systemrecognises a phosphomannose modification on lysosomal enzymes(Sly and Fisher, 1982). There is no other evidence for carbo-hydrate addition to trypanosome aldolase and the mol. wt. pre-dicted from the cDNA sequence is similar to that measured bygel mobility (Misset and Opperdoes, 1984) so such a mechanismis improbable. Opperdoes (1984) has suggested that the presenceof the initial steps of alkoxyphospholipid biosynthesis in the glyco-some is indicative of a relationship to peroxisomes. Peroxisomalproteins are synthesised on free polysomes; unlike nuclear-encoded mitochondrial proteins (Schatz and Butow, 1983) theyappear to maintain constant mol. wt. during uptake (Fujiki andLazarow, 1985, and references therein): only one exception tothis has so far been found (Fujiki et al., 1985). Whether or notproteolytic processing takes place, it seems reasonable to assumethat, as with other organellar proteins, the arrival of the glyco-lytic enzymes at their organellar destination is dependent uponspecific aspects of primary structure (Ellis, 1985). The main bodyof the trypanosome sequence aligns almost exactly with the se-quence of rabbit aldolase, a cytoplasmic protein, so the N-terminal23 amino acids are strong candidates for a role in aggregation,glycosome assembly or both. This region has similarities to othereucaryotic signal sequences: when residues 1-50 are examinedby a computer programme (H.S.Ip, unpublished), based on prin-ciples outlined by Von Heijne (1983) which identify knowneucaryotic signal sequences with a success rate of 85 %, a signalsequence is identified at the N terminus with a predicted cleavagesite between Asn 16 and Arg 17. Removal of this peptide wouldreduce the protein mol. wt. from 41 027 to 39 159. Comparisonof the N-terminal sequence presented here with that of the matureprotein will determine whether such a processing event occurs.

Materials and methodsTrnpanosomesStrains used were T. brucei TREU 667 BUT55, a cloned first relapse populationafter tsetse transmission (Hudson et al., 1980), LUMP 1026 (Bienen et al., 1981)and 427 antigenic types 117 and 221 (Cross, 1975). Bloodstream trypomastigoteswere grown in rats and mice and isolated by ion exchange chromatography (Lan-ham and Godfrey, 1970). Procyclic trypomastigotes were cultured in semi-definedmedium (Brun and Schonenberger. 1979).Plasinid clonesThe rabbit (pRM223) (Tolan et al., 1984) and human (pHL4 13) (Rottman et al.,1984) aldolase cDNA clones were a gift from Dr D.Tolan (University of California,Berkeley). Clones containing T. brucei a- and f-tubulin genes and the entire 117VSG cDNA (the latter a fusion of clones described in Boothroyd et al., 1982)were provided by P.Hevezi (then at Rockefeller University). A plasmid contain-ing a 700-bp mini-exon-containing genomic PvuII fragment from T. brucei (DeLange et al., 1983) was made and provided by Dr D.F.Cully (Rockefeller Uni-versity).

Nucleic acid preparation and analvsisTrypanosome DNA was prepared by SDS lysis, proteinase K digestion and equi-librium centrifugation in caesium chloride (Cully et al., 1985). RNA was purifiedfrom exponentially growing organisms by lysis in guanidinium thiocyanate(Chirgwin et al., 1979) followed by centrifugation through a caesium chloridecushion. Established procedures with occasional minor modifications wereemployed for plasmid (Birnboim and Doly, 1979) and bacteriophage (Maniatis

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et al., 1982) DNA isolation and for gel electrophoresis of denatured glyoxylatedRNA (McMaster and Carmichael, 1977) and of native or denatured DNA (Maniatiset al., 1982) followed by blotting onto nitrocellulose (Southern, 1975) or APTpaper (Alwine et al., 1977; Seed, 1982) and hybridisation with nick-translated32P-labelled probes (Rigby et al., 1977). DNA was routinely depurinated (Wahlet al., 1979) before transfer to ensure efficient detection of large fragments. Hybrid-isation of trypanosome probes to trypanosome nucleic acids was performed for15 h at 42°C in 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M Na citrate),5 x Denhardt's solution, 50 mM sodium phosphate (pH 6.8), 0.1% SDS and250yIg/ml sonicated denatured salmon sperm DNA (Wahl et al., 1979); the finalwash was in 0.2 x SSC and 0.1 % SDS at 65°C. For cross-species hybridis-ations, the temperature was 37°C, pre-incubation was overnight, hybridisationfor 2 days, the formamide concentration was reduced to 20% and wheat germtRNA was used as carrier; washes were of 2 or 3 h duration in 2 x SSC, 0.1 %SDS at progressively higher temperatures; these latter conditions were also usedfor mini-exon detection except that the hybridisation temperature was 22°CProbe removal from blots was achieved by washing for 1 h at 65°C in 50% for-mamide, 10 mM sodium phosphate pH 6.8, 0.1% SDS and 0.1 % sodium pyro-phosphate. Quantitation of RNA transcripts was performed by the slot hybridisationprocedure (Brown et al., 1983). Total RNA concentrations were estimated spectro-photometrically. The RNA was glyoxylated; equivalent serial dilutions of thevarious samples were absorbed to nitrocellulose using a slot apparatus, baked,then hybridised to experimental and control probes. For these and other experimentsinvolving quantitation of hybridisation, films were exposed for a range of timesat -700C with intensifying screens, developed and scanned with a densitometer.DNA sequencing was predominantly by the chain termination method (Sanger

et al., 1977) with [35S]ATP as label; buffer gradient (Biggin et al., 1983) andnon-gradient gels were used and dGTP was replaced by dITP to solve ambiguitiescaused by residual secondary structure in the migrating DNA. Templates werespecific restriction fragments cloned into M13mp8 and mp9 (Messing, 1983).Fragments which were unstable in or inhibited the growth of M 13 were clonedinto pEMBL vectors (Dente et al., 1983). Chemical sequencing (Maxam andGilbert, 1980) was employed for the 3' end [whose poly(A)+ tail disturbed poly-merase transcription], for an internal fragment which was very poorly copiedby the polymerase, and to confirm the structure of the 5' end.

Cloning proceduresA partial genomic library was made from TREU 667 procyclic DNA by partialSau3A digestion, ligation into the lambda vector EMBL 3 (Frischauf et al., 1983),and in vitro packaging (Hohn and Murray, 1977; Maniatis et al., 1982). cDNAlibraries were prepared using a combination of methods described by Huynh etal. (1985) and Gubler and Hoffman (1983) with accumulated modifications.Poly(A)-selected RNA was reverse transcribed and second strands synthesisedby DNA polymerase I and RNase H. The ends were repaired by T4 DNA polymer-ase and the products ligated to EcoRI adaptors (gift from W.Fulford, RockefellerUniversity) before size selection and cloning into the EcoRI site of lambda gtlO(Huynh et al., 1985). All desalting steps were performed using spin columns(Maniatis et al., 1982). Control experiments indicated that for mRNAs of - 2 kbat least 50% of the copies were full length immediately before cloning. The ef-ficiency with which full-length copies survive growth in the vector seems to besequence dependent.Recombinants were screened with radioactive probes (Benton and Davis, 1977)

and the desired inserts gel-purified (Holland and Wangh, 1983) and subclonedinto pAT153 (Twigg and Sherrat, 1980) or pUC12 (Messing, 1983) using standardprotocols (Grunstein and Hogness, 1975; Hanahan, 1983).

Computer analysisComputer analyses were performed on the Rockefeller University VAX 11/750computer using the Staden, ARP and Pustell software packages with local modifi-cations. Signal sequence analysis was done by Mr H.S.Ip (Rockefeller University)on an Apple II personal computer.

AcknowledgementsI thank Dr D.F.Cully, Mr P.Hevezi, Dr C.Gibbs and Dr S.Kidd (RockefellerUniversity) and Dr J.Jackson (Imperial College, London) for clones, strains, vec-tors and DNAs; Dr L.Simpson (UCLA) for trypanosome strains, Dr D.Tolan(University of California, Berkeley) for mammalian aldolase cDNA clones andMr W.Fulford for the EcoRl adaptors. I am grateful to Dr G.S.Lamont for helpwith trypanosome cultures, Dr T.Bargiello for cDNA cloning advice, and DrC.Gibbs, Dr D.F.Cully, Mr H.S.Ip and Ms L.O.C.Haas for sequencing tuition;also to Dr S.Kidd, Dr C.D.Wilde (University of Cambridge, UK), Dr S.Beverly(Harvard University) and Dr D.Peattie (Stanford University) for sequencing sug-gestions, and Mr H.S.Ip for computing assistance. I also wish to acknowledgethe technical help of Mr E.Davidowitz, in particular in characterising the genomicclones and elucidating the genomic structure. I thank all members of the Crosslaboratory for help and discussions, also, together with Dr P.W.J.Rigby (Im-perial College, London), for critical reading of the manuscript; Ms L.Curran

for typing, and Drs P.Borst (Netherlands Cancer- Institute) and L.R.Yarbrough(University of Kansas Medical Center) for communicating unpublished results.I am indebted to Professor G.A.M.Cross tor providing an excellent research envir-onment and for his advice. encouragemiient and support. This work was supportedin part by NIH grant no. PHS A12222901.

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Received on 29 July 1985

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