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Cloning and characterization of the gene encoding pyruvate phosphate dikinase from Giardia duodenalis
MOLlYC'ULAR ii&EMICAL PARit5IT0LOGY Molecular
and Biochemical
Parasitology
77 (1996) 225-333
Cloning and characterization of the gene encoding pyruvate phosphate dikinase from Giardia duodenalis’ Institute
Thomas
Bruderer,
of’ Parasitology.
Uniwrsity
Received
15 December
Claudia of Ziirich,
1995; revised
Wehrli,
Wititert/2urrrstrass~
5 March
Peter Kiihler* 266tr8057
1996; accepted
Ziirich.
7 March
Sn.it-_dard
1996
Abstract This paper reports the cloning and molecular characterization of the gene encoding pyruvate phosphate dikinase (PPDK) from Giardid. The ORF is 2652 nucleotide residues in length and not interrupted by introns. The gene appears to exist as a single copy in the genome and predicts a 97 629 Da protein containing 884 amino acid residues, Comparison of the deduced Giurdia PPDK sequence with those of homologous enzymes from other organisms revealed high sequence similarities and the presence of various conserved domains known to be essential for substrate binding and catalysis. Analysis of the ppdk gene and 19 other protein-coding genes from the protist revealed no typical TATA boxes, positioned at around - 30, but the presence of two novel consensus sequence motifs in the 5’ flanking regions. One is an AT-rich element immediately preceding the translation initiation codon and the other a 14-bp box centered at - 30. These shared consensus sequence patterns present in the 5’ flanking region of Giardio genes are suggested to play a role in the control of transcription initiation. Keywords: Giardia dwdewalis; Pyruvate
phosphate
dikinase;
1. Introduction Giurdia duodenalis nab)
is a binudear,
(syn: G. lumblia, G. intestiamitochondriate protozoon
Abbreciutiom: PCR, polymerase chain reaction; PPDK, pyruvate phosphate dikinase; ppfk, gene encoding PPDK; PP,, inorganic pyrophosphate. * Corresponding author. Tel: + 41 1 3651384; fax: + 41 1 3630478: e-mail: [email protected] ’ No/r: The nucleotide sequence information reported in this paper has been submitted to the EMBL Data Library with the accession No. 254168.
0166-6851~96~515.00 PII
S 166-685
‘i;) 1996 Elsevier
1(96)02605-9
Science
B.V. All rights
reserved
Promoters
of worldwide distribution that parasitizes the intestines of humans and other vertebrates. A remarkable feature of Giurdia and a variety of other protists, such as Entnmoebcl histolyticu. is that significant deviations occur in their metabolism from that of most other eukaryotes [1,2]. An important aspect is that some enzymes in these organisms rely on inorganic pyrophosphate (PP,) instead of adenine nucleotides as a donor of phosphate groups [3]. One of these is pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) that catalyzes the reversible conversion of phosphoenolpyruvate
226
T. Bruderer et al. f Molecular and Biochemical Parasitology 77 (1996) 225-233
to pyruvate. In certain parasitic protists and some prokaryotes, such as Clostridium syrnbiosurn (formerly called Bucteroides symbiosus), this enzyme appears to fulfil the glycolytic function assigned to pyruvate kinase of other organisms and involves the transfer of a high-energy bond of PP, to form ATP [3,4]. The PP, dependence of this metabolic step could thus be of energetic advantage for these organisms as compared to conventional glycolysis since it can increase the overall yield of cellular ATP production by utilizing the energy of a by-product of biosynthetic processes rather than wasting it by hydrolysis [3]. PPDK is also present in Propionibacterium shermanii, photosynthetic bacteria and plants where it primarily operates in the gluconeogenic direction catalyzing the synthesis of phosphoenolpyruvate from pyruvate [5]. However, the plant PPDK differs from the prokaryotic dikinases on the basis of its specific regulatory properties. Among protists, only the gene encoding PPDK from E. histolytica has been cloned and characterized and the relatively high extent of overall sequence similarity observed between the amebal, bacterial and plant PPDKs suggests that their genes have evolved from a common ancestral gene [6,7]. The presence of PPDK in Giardia and other pathogenic protozoa has chemotherapeutic and evolutionary implications [3,8]. As the enzyme is involved in an essential metabolic process and missing in higher animals, it may represent a suitable target for structure-based drug design. A second major current interest in PP,-dependent enzymes of protists is that they reflect features of the metabolism of early eukaryotes, and thus their analysis may provide new insights into ancestral molecular characteristics and the origins of nucleated cell evolution. In this respect Giardia spp. are of particular interest since these organisms have been recognized as one of the earliest diverging eukaryotic lineages [9]. In view of these considerations we have isolated and sequenced the gene encoding PPDK of Giardia and analyzed the deduced primary structure of this enzyme in comparison to previously identified PPDKs from other organisms. In addition, we have sequenced the genomic DNA from the ppdk gene upstream from the translation initiation site and analyzed
for the presence of potential consensus promoter elements. While this work was under way, the sequence of PPDK was established also for a human-derived Giardia isolate [lo].
2. Materials and methods 2.1. Source and cultivation of Giardia
trophozoites Trophozoites were collected from a cloned line of a sheep-derived Swiss Giardia isolate (02-4Al). The cells were cultured in modified TYI-S-33 medium and harvested as reported previously [11,12]. 2.2. Nucleic acid purification Total RNA was purified from trophozoite pellets by the single step method using acid guanidinium thiocyanate-phenol-chloroform extraction [I 31. Unshared genomic DNA was purified from trophozoites as described previously [ 141, except that the DNA solution was dialyzed in collodium bags (Sartorius) against 10 mM Tris-HCl containing 1 mM EDTA, pH 8.0, prior to RNAase A treatment. 2.3. Amp&cation
of a ppdk probe by PCR
Degenerate and inosine-containing oligonucleotides (P8, 5’-GCTCTAGATCGATGCCNGGNATGATGGAYAC_3’andP9,5’-CGGAATTCTGCAGCIACIACRTCYTCNCCYTGNGC-3’) corresponding to the highly conserved amino acid sequences (MPGMMDT and AQGEDWA, respectively) previously identified in various other PPDKs [6] were used as primers for amplification of a giardial PPDK gene-specific polymerase chain reaction (PCR) product. The PCR reaction mixture contained in a final volume of 100 ~1, 50 mM KCl, 2.5 mM MgCl,, 0.04% gelatin, 0.2 mM of dNTP, 250 ng genomic Giardia DNA and 0.5 ,uM each of the two primers. Samples were denatured (4 min at 96°C) and, following the addition of 2.5 U Taq-poly-
T. Bruderer
et al. / Molecular
and Biochemical
merase (Promega Corp.), subjected to 30 cycles of amplification (45 s at 95°C 30 s at 59°C 3 min at 72°C) in a Perkin Elmer Cetus thermal cycler. The PCR product was cloned into the phagemid Bluescript11 (Stratagene). For hybridization procedures, the cloned PCR fragment was labeled by random oligomer-priming with digoxigenine-1 ldUTP (Boehringer).
Parasitology
77 (1996) 225-233
221
strands were sequenced by the primer walking sequencing method using synthetic oligonucleotides (Microsynth). Sequence analysis was performed by using the programs available on the GCG software package of the University of Wisconsin [ 171.
3. Results and discussion 2.4. Screening 3.1. Cloning and sequencing of the ppdk gene Giardia chromosomal
DNA was subjected to partial cleavage with Sau3A, electrophoretically separated and fragments of 9-23 kbp size, isolated from a 0.6% agarose gel, were ligated into partially filled in XhoI sites of A-FIX11 arms (Stratagene) following the manufacturer’s instructions. Approximately 50000 plaques of the amplified library were transferred to a positively charged Nylon membrane (Boehringer) and screened by hybridization in 5 x SSC (1 x SSC contains 300 mM NaCl in 30 mM sodium citrate buffer, pH 7.0), 0.1% N-laurylsarcosine, 0.02% SDS, 1% blocking reagent (Boehringer) at 63°C. 2.5. Southern and Northern blotting Nucleic acids were fractionated electrophoretitally through agarose gels (5 pug restricted DNA) or in agarose-formaldehyde gels (5 fig total RNA) and transferred to positively charged Nylon membranes (Boehringer) by capillary blotting as described elsewhere [ 151. Hybridization to DNA was performed in 5 x SSC, 0.1 N-laurylsarcosine, 0.02% SDS, 1% blocking reagent (Boehringer) at 68°C. Hybridization to RNA was done in 5 x SSC, 50 mM sodium phosphate (pH 7.0), 50% formamide, 7% SDS, 2% blocking reagent (Boehringer) and 0.1% N-laurylsarcosine at 50°C. Membranes were washed under high stringency conditions (0.1 x SSC, O.l”/o SDS) at 68°C.
Amplification of genomic Giardia DNA using primer pair P8 and P9 yielded a major 579-bp product of which a 487-bp subfragment was subcloned using the PstI sites located within the PCR product and at the antisense primer region. The amino acid sequence encoded by the latter PCR fragment revealed substantial homology (4657%) to the corresponding region of previously identified ppdk genes. The non-radioactively labeled PCR fragment was used as a probe to screen a genomic DNA library derived from the cloned G. duodenalis isolate 02-4Al. Approximately 0.03% of the clones were positive. Two plaque purified clones of this library showed in their overlapping region an identical restriction pattern, and the ppdk could be located by Southern blotting and partial sequencing on a 5.2 kbp Hind111 fragment. Sequence analysis of this clone revealed an ORF of 2652 bp (Fig. I), encoding a polypeptide with a calculated molecular mass of 97 629 Da. The deduced sequence showed remarkable overall homology to the known PPDK sequences of C. symbiosum, E. histolytica and plants (Fig. 1). A 99.5% identity was found to the corresponding amino acid sequence established for a human-derived Giardia isolate reported in the accompanying paper [lo]. This small sequence deviation may reflect the genetic differences that were found to exist among the two G. duodenalis type organisms [ 181.
2.6. DNA sequencing and sequence analysis 3.2. Transcript and copy number analysis Nucleotide sequences of plasmid or Ml3 DNA were determined by dideoxy nucleotide chain termination analysis [16] using either [3SS]dATP or a Pharmacia A.L.F. DNA sequencer. Both DNA
When total RNA isolated from the 02-4Al Giardia clone was analyzed in Northern blots, the ppdk-specific probe hybridized to a single tran-
228
T. Bruderer et al. i Molecular and Biochemical Parasitolog)) 77 (1996) 225-233
F 2 C G E
MMSSLSVEGMLLKSARESC..LPARVNQRRNGDLRRLNHHRQSSFVRCLTPARVSRPELRSSGLTPP~VLNPVSFPVTTAKKRVFTFGKGRSE..GNRD MAASVSRAICVQKPGSKCTRDREATSFRRRSVAAPRPPKAK....ARRRHPLRLRRGTGPHCS..PLRAWDA..APIQTTKKRVFHFGKGKSE..GNKT .. . . . . . . .. . . . . .. . . . .. . . . . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . .. . .. . . . .. . . ..~~YKFE....E..GNAS . . . . . . .. . . . .. . . . . . . .. . . . .. . . . .. . . . . . .. . .. . . . .. .. . . . .. . . . . .. . . .. . .. . . . .. . . . .. . ..MST~VYFFG~TPENQPANSE . . .. . . . . . .. . . . . . .. . . . .. . . .. . . ... . . .. . . . .. . . . .. . . . ... . . .. .. . .. . . . .. . . . .. . . .. . . . ...MQRVYAFEDGDGTN..... -----------_--------------------------------------------___---__---___----__---___-*__*____________
F 2 C G E
M.KSLLGGKGANLAEMSSIGLSVPPGLTISTEACEEYQQNGKSLPPGSWDEISEGLDYVQ~MSASLGDPSKPLLLSVRSG~ISMPG~DTVLNLGLND M.KELLGGKGANLAEMASIGLSVPPGFTVSTEACQQYQDAGCALPAGLWAEIVDGLQWVEEYMGATLGDPQRPLLLSVRSGAAVSMPGMMDTVLNLGLND M.RNLLGGKGCNLAEMTILGMPIPQGFTVTTEACTEYYNSGKQITQEIQDQIFEArTWLEELNGKKFGDTEDPLLVSVRS~~SMPG~DTILNLGLND LCRKVLGGKGISLAAMIKLGMPVPLGFTITCQTCVEYQKTA.SWPKGLKEEVASNLKLLEEKGKTFGDNTNPLLVSVRSGAAVSMPGMMDTILNLGLND ..KKLLGGKGAGLCTMTKIGLPVPQGFVITTEMCKQFIANGNKMPEGLMEEVKKEYQLVEKKSGKVFGGEENPLLVSVRSGAAMSMDTILNLGLND _____*****__*__*___*--- i-*_______*______________________-__--------*----***_***,-*--*~******_*******
195 189 113 120 112
F 2 C G E
EVVAGLAGKSGA.RFAYDSYRRFLDMFGNVVMGIPHSLFDEKLEQMKAEKGIHLDTDLTAADLKDLKDLVEKY~VYVEA.KGEKFPTDPKKQLELAVNAVFD EVAAGLAAKSGE.RFAYDSFRRFLDMFGNVVMDIPRSLFEEKLEHMKESKGLKNDTDLTASDLKELVGQYKEVYLSA.KGEPFPSDPKKQLELAVLAVFN VAVEGFAKKTGNPRFAYDSYRRFIQMYSDVVMEVPKSHFEKIIDAMKEEKGVHFDTDLTADDLKELAEKFKAVYKEAMNGEEFPQEPKDQLMGAVKAVFR ESVKGLAAVTGNARFAYDSYRR~QMFGDVCLGIDHDKFEHALDAVKTRYGRKTDPELTADELEEVCEAYRKICV~.TGKTFPQCPHEQLELAINAVFK KTWALAKLTNNERFAYDSYRRFVSLFGKIALNACDEVYDKTLENKKVEKGVKLDTELDANDMKELAQVFIKK.TEEFTKQPFPVDPYAQLEFAICAVFR ______*______******_***-----__--___----__-----*---*-_*__i_*______________-__-----**-_*_-**__+__***_
293 287 213 219 211
F Z C G E
SWDSPRRNKYRSINQITGLK..GTAVNIQSMVFGNMGNTSGTGVLFTRNPSTGEK..KLYGEFLINAQGEDWAGIRTPEDLGTMETCMP...EAYKELV SWESPRAXKYRSINQITGLR..GTAVNVQCMVFGNMGNTSGTGVLFTRNPNTGEK..KLYGEFLVNAQGEDWAGIRTPEDLDAMKNLMP...QAYDELV SWDNPRAIVYRRMNDIPGDW..GTAVNVQTMVFGNKGETSGTGVAFTRNPSTGEK..GIYGEYLINAQGEDWAGVRTPQPITQLENDMP...DCYKQFM SWTNPRAQAYRTLNKLDHNM..GTAVNVQSMVFGNTGDDSGTGVGFTRCPKTGEKFSYLYGEFLQNAQGEDWAGIRTPVNLKEMPTINASWKACYDELS SWMGKRAVDYRREFKITPEQADGTAVSWSMVYGNMGNMGNDSATGVCFTRDPGTGENM..FFGEYLKNAQGEDWAGIRTPQIISKMAEDRD.LPGCYEQLL i*___**__**_________-_*~**--__*+_**_*__*_***-*~*-*-***--____ **_*_+*********_***_--_--___-_____-*____
386 380 306 317 308
F 2 C G E
ENCEILERHYKDMMDIEFTVQENRLWMLQCRTGKRTGKGAVRIAVDMVNEGLIDTRTAIKRVETQHLDQLLHPQFEDPSAYKSHVVATGLPASPGAAVGQ ENCNILESHYKEMQDIEFTVQENRLWMLQCRTGKRTGKSAVKIAVDMVNEGLVEPRSAIKMVEPGHLDQLLHPQFENPSAYKDQVIATGLPASPGAAVGQ DLAMKLEKHFRDMQDMEFTIEEGKLYFLQTRNGKRTAPAALQIACDLVDEGMITEEEAWRIEAKSLDQLLHPTFNPIlALKAGEVIGSALPASPGAAAGK LIYAKLEGYYNDMVDLEFTVENGKLWMLQARAGKRTGFAMVRIAIDMCKEGMLTEEEALLRIDANKINEFLFKRFDPSV..KPWLGKGIPASPGILAVGV DIRKKLEGYFHEVQDFEFTIERKKLYMLQTRNGI
486 480 406 415 406
F Z C G E
VCFSAEDAETWHAQGKSAILVRTETSPEDVGGMHAAAGILDDMKIFTIGDRVIKEGDWLSLNGTTGEVI WFTAEDAEAWHSQGKAAILVRAETSPEDVGGMHAAVGILTERGGMTSHAAWARWWGKCCVSGCSGIRVNDAEKLVTIGSHVLREGEWLSLNGSTGEVI VYFTADEAKARHEKGERVILVRLETSPEDIEGMHAAEGILTVRGGMTSHAAWARGMGTCCVSGCGEIKINEEAKTFELGGHTFAEGDYISLDGSTGKIY ICFCPMRTCELAEQGKKVILTRIETSPEDILGMDRAVGILTARGGQTSHAAWARGMGKCCVAGADCCQINYATKTLVIGDRKFKEGDFISINGTTGEIY WFDADDAVE.QAKGKKVLLLREETKPEDIHGFFVAEGILTCRGGKTSHAKKIAKIGSLEVHEGDILTIDGSTGCVY __*___________*____ *_*_**_***__*___*_****_tiC_**~-*********-_~_-**_*_--____---*_---*-----~~__-___*_**___ -A__
586 580 506 515 505
F 2 C G E
LGKQLLAPPAM.SNDLEIFMSWADQARRLKVMANADTPNDALTARNNGAQGIGLCRTEHMFFASDERIKAVRKMIMAVTPEQRKVALDLLLPYQRSDFEG LGKQPLSPPAL.SGDLGTFMAWVDDVRKLKVLANADTPDDALTARNNGAQGIGLCRTEHMFFASDERIKAVRQMIMAPTLELRQQALDRLLTYQRSDFEG KGDIETQERSV.SGSFERIMVWADKFRTLKVRTNADTPEDTLNAVKLGAEGIGLCRTEHMFFEAD.RIMKIR~ILSDSVEAREEALNELIPFQKGDF~ NGAVQTIEPGI.TDDLQTIMDWSDKYRVLKIRTNADTPHDAAVARKFGAEGIGLCRTEHMFF~D.RI~EMILSDDEGARRTALNKLLPFQREDFIG KGEVPLEEPQVGSGYFGTILKWANEIKKIGVFAAGDLPSAAKKALEFGAEGIGLCRTERM.FNAVERLPIWKMILSNTLEERKKYLNELMPLQKQDFIG _*__________________-*-_----__-____*-*--__-*---**-~******~_*-*___-*--__--**-__----*_-_~_-*-__*--**__ -8__
685 679 604 613 604
F Z C G E
IFRAMDGLPVTIRLLDPPLHEFLPEGDL...EHIVNELAVDTG..MSADEIYSKIENLSEVNPMLGFRGCRLGISYPELTEMQV~IFQ~VS~TNQ.GV IFRAMDGLPVTIRLLDHPSYEFLPEGNI...EDIVSELCAETG..ANQEDALARIEKLSEVNPMLGFRGCRLGISYPELTEMQA~IFE~I~TNQ.GV MYKALEGRPMTVRYLDPPLHEFVPHTEE. ..EQ..AELAKNMG..LTLAEVKAKVDELHE~PMMGHRGCRLAVTYPEIA~QT~~E~IE~EETGI IFKAMDGKGVNIRLLDPPLHEFLPHTR..... DLQKKLAEDMN..KKHRHIHERVEDLHEVNPMLGFRGVRLGIVYPEISEMQVRAILEAACIVSRE.GV LLKTMNGLPVTVRLLDPPLHEFLPTLEELMREIFEMKLSGKTEGLAEKEWLK~EL~~PMIGHRGIRLGTTNPEIYEMQI~FLEATREVIKE.GI +_*_***_*_**_f*____**---**_~*-_-*--__----*______,----_*-**_*__**-*____-------__*---_____-______--_-
779 773 697 705 703
F Z C G G
TVIPEIMVPLVGTPQEL.RHQISVIRGVAANVFAEMGVTLEY~GTMIEIP~LIAEEIGKEADFFSFGTNDLTQMTFGYSRDDVG.KFLQIY~QGIL QVFPEIMVPLVGTPQEL.GHQVTLIRQVAEKVFANVGKTIGY~GTMIEIP~LVADEIAEQ~FFSFGTNDLTQMTFGYSRDDVG.KFIPVHLAQGIL DIVPEIMIPLVGEKKEL.KFVKDVWEVAEQVKKEKGSDMQYHIGTMIEIPRAALTADG.KFLDSYYKAKIY TVKPEIMIPVLFSENEM.EIMHALVNRVAASVFKEHGTTVDYEVGTMIELPRACVMADKIAQTAQYFSFGTNDLTQTTFGISRDDAG.KFIPKYIDRGIF NDHREIMIPNVTEVNELINLRKNVLEPVHEEVEKKYGIKVPFSYGTMVECVRAALTADKIATEASFFSFGTNDLTQGTFSYSREDSENKFIPKYVELKIL ____**~_*______*____--__---~__-*____*---_---***_*__**---,__*___*__****~~****_~*--,*-*___**-___----*_
877 871 795 803 803
F Z C G E
QHDPFEVIDQKGVGQLI~TEKGRAANPSLKVGICGEHGGEPSSVAFFDGVGLDYVSCSPFRVPIARLAAAQVIV...... QHDPFEVLDQRGVGELVKFATERGRKARPNLKVGICGEHGGEPSSVAFFAKAGLDFVSCSPFRVPIARLAAAQVLV...... ESDPFARLDQTGVGQLVEMAVKKGRQTRPGLKCGICGEHGGDPSSVEFCHKVGLNYVSCSPFRVPIARLAAAQAALNNK.... KVDPFVTLDQQGVGALMKIEGGRSTRTDMKIGICGEQTD.PASILFLHKIGLNYVSCSPYRVPVARVAAAIAAIKARTNQ PANPFEILDRPGVGEVMRIAVTKGRQTRPELLVGICGEHGGEPSSIEWCHMIGLNYVSCSSYRIPVARIAAAQAQIRHPREN ___i*___*__***_____*-_-~*__-__---,,***---_*-*-__----~*--~*~~--*_*-**-*~*----_____-C-
96 90
14 21 14
953 947 874 884 885
Fig. 1. Alignment of deduced amino acid sequences of PPDK from Giurdia and various other organisms. F. Flaueriu rrinrrria (EMBL Data Library accession No. X57141 [22]); Z. Zea map (JO3401 [21]); C, Clostridium symbiosum (M60920 [19], corrected sequence in [20]); G. Giardia dzroderzafis(254168); E, Etztanzoeba hystolytica (X74596 [6]). Identical amino acid residues are marked by asterisk. The underlined sequence stretches denote putative functional domains required for phosphorylation (A). catalysis of the phosphoenolpyruvate/pyruvate partial reaction (P-loop, B) and substrate binding and/or catalysis (C).
script of about 2700 nt (Fig. 2A). The size of this band was found to be in good agreement with that predicted from the ppdk ORF. The nearly complete colinearity of the deduced Giurdia PPDK sequence with other PPDK sequences together with the observation that the giardial ppdk transcript was of similar size as that of the corresponding ORF is consistent with the absence of introns in this gene. Southern blots of total genomic DNA, probed with the subcloned ppdk PCR product, yielded a single hybridization band for all restriction enzyme digestions, indicating that ppdk exists as a single copy gene within the giardial genome (Fig. 2B). The locations of the restriction enzyme sites identified by mapping the two i-clones and their subclones are depicted in Fig. 2C. 3.3. Anulj~sis
of theppdk gene coding region
Sequence comparison of the PPDKs from Giardie as deduced from the nucleotide sequence of the corresponding gene with those of C. synbiosun~ ([ 191; corrected sequence in [20]), E. histolytica [6,7], maize [21] and Fluveriu trineraia [22] revealed an overall sequence identity of 54.1, 46.6, 51.1 and 52.6X, respectively (Fig. 1). For the alignment of the deduced sequences only a few small amino acid gaps had to be introduced, except for the large amino-terminal putative transit peptide of the plant PPDKs (76 residues for the F. trinerviu and 71 residues for the maize sequences) that is missing in the sequences of bacteria and protists. The size of the PPDK sequence from Giardiu (884 amino acid residues) was found to be very similar to that of the corresponding enzyme from C’. s~whiosun~ (874 residues), E. /listo/j*ticu (885 residues) and the mature forms from plant PPDKs (876 and 882 residues for maize and F. trinerviu, respectively). More detailed examination of various PPDK sequences revealed that the catalytic histidine that mediates the multiple phosphoryl transfer steps during catalysis is located within a highly conserved decapeptide arrangement (Fig. 1, domain A). indicating that the structures of the active site of these dikinases may be very similar. In plant PPDKs, the threonine residue positioned two residues upstream of the catalytic histidine functions as a regulatory site via a phosphorylationldephosphorylation
mechanism [19]. As this threonine is found within an identical motif (Fig. 1, domain A) in the enzyme from Giardiu and E. lzistolvticu it would be interesting to see whether the activity of protist PPDKs may be subject to a similar type of regulatory control. Other domain structures, previously demonstrated to be implicated in substrate binding and catalysis of C. synzhioszm PPDK [20,23] are also well conserved in the Giurdiu enzyme (Fig. 1. domains A and B) supporting the view that these PPDKs possess a uniform mechanism of catalysis.
Nucleotide sequences of 410 bp downstream and of 1087 bp upstream from the coding region were also sequenced and analyzed for conserved elements. A consensus sequence AGTGAAT, predicted as a putative polyadenylation site in Giurdiu, was found starting 2 bp upstream from the stop codon TGA. The sequence AGTPuAAPy has been proposed as a polyadenylation signal for Gimlicl separated by a 6-19 bp interval from the stop codon [3]. The DNA sequences upstream of the translation initiation codon of ppdk were searched for homology to typical eukaryotic transcriptional regulatory elements. Examination of this region from pprlhand several of the available other Giwdiu genes encoding a number of cytosolic enzymes [24-281, nuclear proteins [29,30] and heat shock proteins [31] revealed the presence of a 14-bp AT-rich consensus sequence centered at about 30 nucleotides upstream of the translational start site (Fig. 3). This element has a sequence and location that resembles the 1%bp putative consensus promoter motif recently deduced from analysis of seven Giurdiu cytoskeleton protein genes [32 -381. The strictly conserved pattern AA-N9-C located at nucleotide positions 3-14 within the 14-bp box is present in all of the sequences examined except in the adenylate kinase and vacuolar-type ATPase genes where the C at nucleotide position 14 is replaced by a T [27,28]. Recently. a second potential promoter consensus sequence was identified immediately upstream of the ATG initiation codon in cytoskeleon protein genes. The same consensus motif was also found to exist in ppdk and most other currently
T. Bruderer et al. / Molecular and Biochemical Parasitology 77 (1996) 225-233
230
A
B 12345676
nt
kb 23 9.4
7400
6.6
5300
4.3
2800
1900
2.3
1600
2.0
C K
C
XbH
P
Xb
E
E
Xb
E
PPB
C
C
E
C
B
C
NBEC
K
HB
Xb
P
C
lkb Probe
Fig. 2. Nothern and Southern blot analysis using the PsfI digested and digoxigenin-labeled PCR fragment as probe. Sizes of the nucleic acids are indicated on the left. (A) Northern blot, 5 pg of Giardia total RNA was subjected to formaldehyde agarose gel electrophoreses, blotted onto a positively charged Nylon membrane and hybridized as described in Methods. (B) Southern blot, 5 ,ug of Giordia genomic DNA were completely digested with restriction endonucleases and analyzed on a 0.8% agarose gel. Lane I, EcoRI; lane 2, EcoRI + NotI; lane 3, HindHI; lane 4, PstI; lane 5, Hind111 + PstI; lane 6, XbaI; lane 7, ClaI; lane 8, XbaI + ClaI. (C) Physical restriction map of Giardiu 02-4Al chromosomal DNA. The black box indicates the DNA region completely sequenced in this study. The hatched arrow denotes the ORF corresponding to ppdk. Restriction sites are: B, BarnHI; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; N, NotI; P, PstI; Xb, Xbal.
available protein-coding genes of Giardia (Fig. 3). The highly conserved motif shows a predominant A and T content and is 9 bp long [32]. Exceptions to this pattern exist in the adenylate kinase gene where a comparable sequence motif could not be identified and in the RAN-encoding gene that
contains a similar sequence 33 bp upstream of the ATG [27,29]. Interestingly, primer extension experiments with the latter gene revealed a termination signal at - 29, in addition to those at - 2 and - 4, which would be consistent with the putative initiation site being farer from the ATG than that
T. Bruderer er al. / Molecular and Biochemical Parasitology 77 (1996) 225-373 14
bp Box
Initiation site
al-giardin
-3yCAAAAAATACCGCC
-%ggTTTTAAAAAtg
X52485 [33]
ail-giardin
-34TAAAATATATTTTC
-12tacATTTAGAAAatg
M34550 [34]
P-giardin
-34CAAAAATTTGAGCC
-13agaAAATAAAAGgatg
X85958 [32]
y-giardin
-26TAAAAAACTAACCC
-13ctaAAACAAGAAaatg
X55287 [35]
Median body protein
-42CAAATGCTAAATTC
-'ktaAATTAAAAAt_g
X64517 [36]
a-tubulin
-33CAAATTCCAGAGTC
-%ggAAATAAAAAtg
JO4648 [37]
P-tubulin
-33CCAAATCCAGATTC
-%cgATTTAAAAAtg
X06748 [37]
Actin
-22TTAAATTTATTTGC
-"tgcAAAGTAAAAtg
L29032 [38]
PPDK (02-4Al) -4aTTAAATGAAATATC
-l%ctAATAAATAAttggatg
254168
PPDK (WB)
-40TTAAATGAAATATC
-lkctAATAAATAAttggatg
u43531 [lo]
GAP-l
-35AAAAAACTTAACAC
-'laaaAATCTAAAAtg
M88062
PPi-PFK
-31AAAAATTAGCTAGC
-13gctAATTTGAAAtg
U12337 [24]
TIM (WB)
-31CAAATTAGAAATTC
-14ccaGAAAATAAAtcatg
LO2120 [25]
TIM (GS)
-3gAAAAACCCTGATTC
-13tcaGAAAATAAAttatg
LO2116 [26]
NADP-GDH
+"GAAAAGCTGACCAC
-'lcagATTTTAAAAtg
M84604 [26]
AK
-36CAAAAGCATAATGT
mm-1
-46TAAATAAATCTGTT
-'kaaAATTTGAAAtg
RAN
-3QTAAAACTTTAACCC
-36cgaAATTAAAACNlsagtagcatgU02589 [29]
AR??
-3aCAAAAAATGAAGGC
-14ggcCATTAAAAAgaatg
M86513 [30]
HSP70
-4QTCAAAAGATCTGAC
-13ggaAAATAAAATcatg
U04874 [31]
ER-HSP70
-24CAAAAAAGGACGAC
‘I’cggAAATAAAAAtg
U04875 1311
CONSENSUS
131
U19901 [27] U18938 [28]
YAAAAWNNWAWNNC
AAWTAAAAA Fig. 3.Sequence comparision of putative polymerase II promoter elements of various genes from Giurdio consisting of a l4-bp box and an initiation site. EMBL Data Library accession numbers and references are listed in the right column. The numbering of the nucleotides in the sequences is relative to the zero positions assigned to the A of the ATC translation start codon. Boldface nucleotides represent major signals obtained from Sl protection or primer extension experiments. PPDK, pyruvate phosphate dikinase (from a sheep- and a human-derived Giardia isolate); GAP-l, glyceraldehyde-3-phosphate dehydrogenase; PP,-PFK, pyrophosphate-dependent phosphofructokinase; TIM, triosephosphate isomerase (from two different human-derived Giurdia isolates); NADP-GDH, NADP-dependent glutamate dehydrogenase: AK, adenylate kinase; VMA, vacuolar type ATPase; RAN, US-like nuclear protein; ARF, ADP-ribosylation factor; HSP70. 70-kDa heat shock protein; ER-HSP70, endoplasmatic reticulum HSP70 homolog.
in all other known Giardia genes [29]. In most of the studies where primer extension experiments were performed to identify transcriptional initiation the start site was found to begin with an A at a relative position of 5 or 6 within the 9-bp box. Recent analysis of protein-coding genes from another amitochondriate protozoan, Trichomonas vaginalis, has also revealed the pres-
ence of a strong consensus sequence immediately upstream of the initiation site [39]. However, this start site motif does not show any similarity to the highly conserved 9-bp consensus sequence immediately upstream of the putative initiation site of Giardia genes. Interestingly, the genes encoding the variant surface proteins of Giardia were not found to possess an upstream motif
737 .._
T. Bruderer et al. ! Molecular und Biochemical Parasitology 77 (I 996) 225-233
similar to that found for all other known genes of this parasite. A large number of RNA polymerase II promoters of higher eukaryotes contain a TATA box that is centered at around - 27 within a consensus sequence of TATAa/tAa/t. In the region corresponding to a similar location of the Giardia genes analyzed, there were no TATA boxes identified with any significant homology to other promoter sequences. Several potential TATA motifs have been localized in some genes of Giardia and other protists further upstream of the transcription start site. However, these sequences were not found to be conserved. appropriately spaced and homologous to the typical TATA consensus sequence commonly observed in class II genes of higher eukaryotes. It is interesting to note that in TATAless genes of higher eukaryotes the presence of an initiator sequence surrounding the transcription start site is sufficient to support transcriptional initiation by RNA polymerase II. The accurate biological activity associated with the consensus elements occurring upstream of Giurdiu proteincoding genes is at present unknown. Analysis of the role these elements may play in gene transcription have to await deletion and point mutation experiments and the establishment of an in vitro transcription assay. Such studies may then also help to elucidate the evolutionary origins of the structural components of eukaryotic promoters.
Acknowledgements
We thank Dr. Miklos Mtiller (The Rockefeller University, New York) for valuable comments on this manuscript and for sharing his results during the development of this work. This study was supported by grant 31-36198.92 from the Swiss National Science Foundation.
References [I] Mtiller, M. (1988) Energy metabolism of protozoa without mitochondria. Annu. Rev. Microbial. 42. 465-488. [2] Adam, R.D. (1991) The biology of Giardia spp. Microbiol. Rev. 55. 706--732.
Glycolysis I31 Mertens, E. (I 993) ATP versus pyrophosphate: revisited in parasitic protists. Parasitol. Today 9, 122126. [41 Milner, Y., Michaels, G. and Wood, H.G. (1978) Pyruvate, phosphate dikinase of Bacteroides symbiosus. J. Biol. Chem. 253, 8788881. [51 Wood, H.G., O’Brien. W.E. and Michaels, G. (1985) Pyruvate phosphate dikinase. Adv. Enzymol. 45. 108155. @I Bruchhaus, I. and Tannich, E. (1993) Primary structure of the pyruvate phosphate dikinase in Entamoeba hisfo/ytic,u. Mol. Biochem. Parasitol. 62, 1533156. E. and Perez-Montfort, R. (1994) Cloning [71 Saavedra-Lira, and sequence determination of the gene coding for the pyruvate phosphate dikinase of Entamoeha his~ol~~ticw. Gene 142, 2499251. E. (1993) Ginrdirr PI Hrdy, I., Mertens, E. and Nohynkova, imestitzalis: Detection and characterization of a pyruvate phosphate dikinase. Exp. Parasitol. 76, 438-441. [91 Sogin, M.L. (1991) Early evolution and the origin of eukaryotes. Curr. Opin. Gen. Develop. I. 457-463. L.. Hrdy, 1. and Miiller, M. (1996) The PO1 Nevalainen, primary structure of a Giardia lumhlia gene coding for the glycolytic enzyme, pyruvate. orthophosphate dikinase. Mol. Biochem. Parasitol. 77, 101-107. P., Castro. R. and Kiihler. [Ill Bruderer, T.. Papanastasiou, P. (1993) Variant cysteine-rich surface proteins of Giardio isolates from human and animal sources. Infect. Immun. 61, 393772944. [I21 Stranden, A.M., Eckert, J. and Kohler. P. (1990) Electrophoretic characterization of Giardiu isolated from humans, cattle, sheep, and a dog in Switzerland. J. Parasitol. 76, 660-668. P. and Sacchi, N. (1987) Single-step u31 Chomczynski. method of RNA isolation by acid guanidinium-phenolchlorform extraction. Anal. Biochem. 162, I56- 159. 1141 Ey, P.L.. Khanna, K.. Andrews, R.H.. Manning. P.A. and Mayrhofer, G. (1992) Distinct genetic groups of Giardio iutestinalis distinguished by restriction fragment length polymorphisms. J. Gen. Microbial. 138. 26292637. [I51 Sambrook, J., Fritsch, E.F. and Maniatis. T. (1989) Molecular cloning: a laboratory manual, 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. [16] Sanger. F., Nicklen, S. and Coulson. A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Nat1 Acad. Sci. USA 74. 5463-5467. [17] Devereux, J.. Haeberli, P. and Smithies, 0. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucl. Acid Res. 12, 387-395. [18] Ey, P.L.. Bruderer, T., Wehrli, C. and Kohler, P. (1996) Comparision of genetic groups determined by molecular and immunological analysis of Giardio isolates from animal and humans in Switzerland and Australia. Parasitol. Res. 82, 52260. [19] Pocalyko. D.J.. Carroll, L.J., Martin, M.B., Babbitt, P.C. and Dunaway-Mariano. D. (1990) Analysis of sequence
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[23]
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[24]
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[26]
[27]
[28]
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arld Biochrmical
homologies in plant and bacterial pyruvate phosphate dikinase, enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and other PEP-utilizing enzymes. Identification of potential catalytic and regulatory motifs. Biochemistry 29, 10757 - 10765. Xu. Y., Yankie. L., Shen, L.. Jung, Y.-S., Mariano, P. S., Dunaway-Mariano. D. and Martin. B.M. (1995) Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Uostridium synhiosum pyruvate phosphate dikinase. I. Identification of an essential cysteine by chemical modification with [l“Clbromopyruvate and site-directed mutagenesis. Biochemistry 34. 2181~7187. Matsuoka. M., Ozeki. Y.. Yamamoto, N.. Hirano, H., Kano-Murakami. Y. and Yanaka, Y. (1988) Primary structure of maize pyruvate. orthophosphate dikinase as deduced from cDNA sequence. J. Biol. Chem. 263, 11080~11083. Rosche. E. and Westhoff, P. (1990) Primary structure of pyruvate. orthophosphate dikinase in the dicotyledonous C, plant F(ccrerLi rrifrerriu. FEBS Lett. 273. 116- 121. Yankie. L.. Xu. Y. and Dunaway-Mariano. D. (1995) Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium .vwhiosum pyruvate phosphate dikinase. 2. Site directed mutagenesis of an essentiell arginine contained within an apparent P-loop. Biochemistry 34. 218882194 Rozario. C., Smith, M.W. and Miiller. M. (1995) Primary structure of a putative pyrophosphate-linked phosphofructokinaae gene of Giardia Iumhlia. Biochim. Biophys. Acta 1260. 2188222. Mowatt. M.R., Weinbach. E.C.. Howard. T.C. and Nash, T.E. (1994) Complementation of an Escheruhia coli glycolysis mutant by Girrrdia lumhlia triosephosphate isomerase. Exp. Parasitol. 78. 85-92. Yce. J. and Dennis. P.P. (1994) The NADP-dependent glutamate dehydrogenase of Giardiu lamhlia: A study of function. gene structure and expression. System. Appl. Microbial. 16, 7599767. Rozario. C. and Miiiier, M. (1995) Primary structure of a putative adenylate kinase gene of Giardiir lum6lia. Mol. Biochem. Parasitol. 71, 279-283. Hilario, E. and Gogarten, J.P. (1995) The V-ATPase A subunit gene (rrncl-I ) from Ginrdia lamblia. Biochim. Biophys. Acta 128. 94-98.
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[29] Chen, L.-M., Chern. Y., Ong, S.-J. and Tai. J.-H. (1994) Molecular cloning and characterization of a rrrs-related gene of ran itcJ/spii subfamily in Giardia lamhliu. J. Biol. Chem. 269. 17297-17304. [30] Murtagh, J.J., Mowatt, M.R., Lee, C.-M.. Lee, F.-J.S.. Mishima, K.. Nash. T.E., Moss. J. and Vaughan. M. (1992) Guanine nucleotide-binding proteins in the intestinal parasite Giardia kamhliu. J. Biol. Chem. 267. 9654 9662. [31] Gupta. R.S.. Aitken, K.. Falah, M. and Singh. B. ( 1994) Cloning of Giardia lamblia heat shock protein HSP70 homologs: Implications regarding origin of eukaryotic cells and of endoplasmic reticulum. Proc. Nat1 Acad. Sci. USA 91, 2895-2899. [32] Holberton. D.V. and Marshall. J. (1995) Analysis of consensus sequence patterns in Giurdia cytoskeleton gene promoters. Nucl. Acid Res. 15. 2945 2953. [33] Peattie, D.A.. Alonso. R.A.. Hein. A. and Caultield, J.P. (1989) Ultrastructural localization of giardins to the edges of disk microribbons of Giardiu lamblia atoll the nucleotide and deduced protein sequence of alpha giardin. J. Cell Biol. 109, 737332335. [34] Alonso. A. and Peattie, D.A. (1992) Nuclcotide sequence of a second alpha giardin gene and molecular analysis of the alpha giardin genes and transcripts in t&r& lumblia. Mol. Biochem. Parasitol. 50. 95-104. 1351 Nohria. A., Alonso. R. A. and Peattie D. A. (1992) Identification and characterization of ;x-giardin and the ;s-giardin gene from Gurdk lamhlia. Mol. Biochem. Parasitol. 56. 27.-38. [36] Marshall. J. and Holberton. D.V. (1993) Sequence and structure of a new coiled coil protein from a microtubule bundle in Giardia. J. Mol. Biol. 231. 521- 530. [37] Kirk-Mason, K.E., Turner. M.J. and Chakraborty, P.R. (1989) Evidence for unusually short tubulin mRNA lcaders and characterization of tubulin genes in Ciurdirl kmhlia. Mol. Biochem. Parasitol. 36. 877100. [38] Drouin, G., Desa. M.M. and Zuker. M. (1995) The Giardia lambliu actin gene and the phylogeny of eukaryotes. J. Mol. Evol. 41. 841 -848. [39] Quon. D.V.K.. Delgadillo, M.G.. Khachi. A.. Smale. S.T. and Johnson, P.J. (1994) Similarity between a ubiquitous promoter element in an ancient eukaryote and mamahan initiator elements. Proc. Nat1 Acad. Sci. USA 91. 457Y4583.
and Biochemical
Parasitology
77 (1996) 225-333
Cloning and characterization of the gene encoding pyruvate phosphate dikinase from Giardia duodenalis’ Institute
Thomas
Bruderer,
of’ Parasitology.
Uniwrsity
Received
15 December
Claudia of Ziirich,
1995; revised
Wehrli,
Wititert/2urrrstrass~
5 March
Peter Kiihler* 266tr8057
1996; accepted
Ziirich.
7 March
Sn.it-_dard
1996
Abstract This paper reports the cloning and molecular characterization of the gene encoding pyruvate phosphate dikinase (PPDK) from Giardid. The ORF is 2652 nucleotide residues in length and not interrupted by introns. The gene appears to exist as a single copy in the genome and predicts a 97 629 Da protein containing 884 amino acid residues, Comparison of the deduced Giurdia PPDK sequence with those of homologous enzymes from other organisms revealed high sequence similarities and the presence of various conserved domains known to be essential for substrate binding and catalysis. Analysis of the ppdk gene and 19 other protein-coding genes from the protist revealed no typical TATA boxes, positioned at around - 30, but the presence of two novel consensus sequence motifs in the 5’ flanking regions. One is an AT-rich element immediately preceding the translation initiation codon and the other a 14-bp box centered at - 30. These shared consensus sequence patterns present in the 5’ flanking region of Giardio genes are suggested to play a role in the control of transcription initiation. Keywords: Giardia dwdewalis; Pyruvate
phosphate
dikinase;
1. Introduction Giurdia duodenalis nab)
is a binudear,
(syn: G. lumblia, G. intestiamitochondriate protozoon
Abbreciutiom: PCR, polymerase chain reaction; PPDK, pyruvate phosphate dikinase; ppfk, gene encoding PPDK; PP,, inorganic pyrophosphate. * Corresponding author. Tel: + 41 1 3651384; fax: + 41 1 3630478: e-mail: [email protected] ’ No/r: The nucleotide sequence information reported in this paper has been submitted to the EMBL Data Library with the accession No. 254168.
0166-6851~96~515.00 PII
S 166-685
‘i;) 1996 Elsevier
1(96)02605-9
Science
B.V. All rights
reserved
Promoters
of worldwide distribution that parasitizes the intestines of humans and other vertebrates. A remarkable feature of Giurdia and a variety of other protists, such as Entnmoebcl histolyticu. is that significant deviations occur in their metabolism from that of most other eukaryotes [1,2]. An important aspect is that some enzymes in these organisms rely on inorganic pyrophosphate (PP,) instead of adenine nucleotides as a donor of phosphate groups [3]. One of these is pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) that catalyzes the reversible conversion of phosphoenolpyruvate
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T. Bruderer et al. f Molecular and Biochemical Parasitology 77 (1996) 225-233
to pyruvate. In certain parasitic protists and some prokaryotes, such as Clostridium syrnbiosurn (formerly called Bucteroides symbiosus), this enzyme appears to fulfil the glycolytic function assigned to pyruvate kinase of other organisms and involves the transfer of a high-energy bond of PP, to form ATP [3,4]. The PP, dependence of this metabolic step could thus be of energetic advantage for these organisms as compared to conventional glycolysis since it can increase the overall yield of cellular ATP production by utilizing the energy of a by-product of biosynthetic processes rather than wasting it by hydrolysis [3]. PPDK is also present in Propionibacterium shermanii, photosynthetic bacteria and plants where it primarily operates in the gluconeogenic direction catalyzing the synthesis of phosphoenolpyruvate from pyruvate [5]. However, the plant PPDK differs from the prokaryotic dikinases on the basis of its specific regulatory properties. Among protists, only the gene encoding PPDK from E. histolytica has been cloned and characterized and the relatively high extent of overall sequence similarity observed between the amebal, bacterial and plant PPDKs suggests that their genes have evolved from a common ancestral gene [6,7]. The presence of PPDK in Giardia and other pathogenic protozoa has chemotherapeutic and evolutionary implications [3,8]. As the enzyme is involved in an essential metabolic process and missing in higher animals, it may represent a suitable target for structure-based drug design. A second major current interest in PP,-dependent enzymes of protists is that they reflect features of the metabolism of early eukaryotes, and thus their analysis may provide new insights into ancestral molecular characteristics and the origins of nucleated cell evolution. In this respect Giardia spp. are of particular interest since these organisms have been recognized as one of the earliest diverging eukaryotic lineages [9]. In view of these considerations we have isolated and sequenced the gene encoding PPDK of Giardia and analyzed the deduced primary structure of this enzyme in comparison to previously identified PPDKs from other organisms. In addition, we have sequenced the genomic DNA from the ppdk gene upstream from the translation initiation site and analyzed
for the presence of potential consensus promoter elements. While this work was under way, the sequence of PPDK was established also for a human-derived Giardia isolate [lo].
2. Materials and methods 2.1. Source and cultivation of Giardia
trophozoites Trophozoites were collected from a cloned line of a sheep-derived Swiss Giardia isolate (02-4Al). The cells were cultured in modified TYI-S-33 medium and harvested as reported previously [11,12]. 2.2. Nucleic acid purification Total RNA was purified from trophozoite pellets by the single step method using acid guanidinium thiocyanate-phenol-chloroform extraction [I 31. Unshared genomic DNA was purified from trophozoites as described previously [ 141, except that the DNA solution was dialyzed in collodium bags (Sartorius) against 10 mM Tris-HCl containing 1 mM EDTA, pH 8.0, prior to RNAase A treatment. 2.3. Amp&cation
of a ppdk probe by PCR
Degenerate and inosine-containing oligonucleotides (P8, 5’-GCTCTAGATCGATGCCNGGNATGATGGAYAC_3’andP9,5’-CGGAATTCTGCAGCIACIACRTCYTCNCCYTGNGC-3’) corresponding to the highly conserved amino acid sequences (MPGMMDT and AQGEDWA, respectively) previously identified in various other PPDKs [6] were used as primers for amplification of a giardial PPDK gene-specific polymerase chain reaction (PCR) product. The PCR reaction mixture contained in a final volume of 100 ~1, 50 mM KCl, 2.5 mM MgCl,, 0.04% gelatin, 0.2 mM of dNTP, 250 ng genomic Giardia DNA and 0.5 ,uM each of the two primers. Samples were denatured (4 min at 96°C) and, following the addition of 2.5 U Taq-poly-
T. Bruderer
et al. / Molecular
and Biochemical
merase (Promega Corp.), subjected to 30 cycles of amplification (45 s at 95°C 30 s at 59°C 3 min at 72°C) in a Perkin Elmer Cetus thermal cycler. The PCR product was cloned into the phagemid Bluescript11 (Stratagene). For hybridization procedures, the cloned PCR fragment was labeled by random oligomer-priming with digoxigenine-1 ldUTP (Boehringer).
Parasitology
77 (1996) 225-233
221
strands were sequenced by the primer walking sequencing method using synthetic oligonucleotides (Microsynth). Sequence analysis was performed by using the programs available on the GCG software package of the University of Wisconsin [ 171.
3. Results and discussion 2.4. Screening 3.1. Cloning and sequencing of the ppdk gene Giardia chromosomal
DNA was subjected to partial cleavage with Sau3A, electrophoretically separated and fragments of 9-23 kbp size, isolated from a 0.6% agarose gel, were ligated into partially filled in XhoI sites of A-FIX11 arms (Stratagene) following the manufacturer’s instructions. Approximately 50000 plaques of the amplified library were transferred to a positively charged Nylon membrane (Boehringer) and screened by hybridization in 5 x SSC (1 x SSC contains 300 mM NaCl in 30 mM sodium citrate buffer, pH 7.0), 0.1% N-laurylsarcosine, 0.02% SDS, 1% blocking reagent (Boehringer) at 63°C. 2.5. Southern and Northern blotting Nucleic acids were fractionated electrophoretitally through agarose gels (5 pug restricted DNA) or in agarose-formaldehyde gels (5 fig total RNA) and transferred to positively charged Nylon membranes (Boehringer) by capillary blotting as described elsewhere [ 151. Hybridization to DNA was performed in 5 x SSC, 0.1 N-laurylsarcosine, 0.02% SDS, 1% blocking reagent (Boehringer) at 68°C. Hybridization to RNA was done in 5 x SSC, 50 mM sodium phosphate (pH 7.0), 50% formamide, 7% SDS, 2% blocking reagent (Boehringer) and 0.1% N-laurylsarcosine at 50°C. Membranes were washed under high stringency conditions (0.1 x SSC, O.l”/o SDS) at 68°C.
Amplification of genomic Giardia DNA using primer pair P8 and P9 yielded a major 579-bp product of which a 487-bp subfragment was subcloned using the PstI sites located within the PCR product and at the antisense primer region. The amino acid sequence encoded by the latter PCR fragment revealed substantial homology (4657%) to the corresponding region of previously identified ppdk genes. The non-radioactively labeled PCR fragment was used as a probe to screen a genomic DNA library derived from the cloned G. duodenalis isolate 02-4Al. Approximately 0.03% of the clones were positive. Two plaque purified clones of this library showed in their overlapping region an identical restriction pattern, and the ppdk could be located by Southern blotting and partial sequencing on a 5.2 kbp Hind111 fragment. Sequence analysis of this clone revealed an ORF of 2652 bp (Fig. I), encoding a polypeptide with a calculated molecular mass of 97 629 Da. The deduced sequence showed remarkable overall homology to the known PPDK sequences of C. symbiosum, E. histolytica and plants (Fig. 1). A 99.5% identity was found to the corresponding amino acid sequence established for a human-derived Giardia isolate reported in the accompanying paper [lo]. This small sequence deviation may reflect the genetic differences that were found to exist among the two G. duodenalis type organisms [ 181.
2.6. DNA sequencing and sequence analysis 3.2. Transcript and copy number analysis Nucleotide sequences of plasmid or Ml3 DNA were determined by dideoxy nucleotide chain termination analysis [16] using either [3SS]dATP or a Pharmacia A.L.F. DNA sequencer. Both DNA
When total RNA isolated from the 02-4Al Giardia clone was analyzed in Northern blots, the ppdk-specific probe hybridized to a single tran-
228
T. Bruderer et al. i Molecular and Biochemical Parasitolog)) 77 (1996) 225-233
F 2 C G E
MMSSLSVEGMLLKSARESC..LPARVNQRRNGDLRRLNHHRQSSFVRCLTPARVSRPELRSSGLTPP~VLNPVSFPVTTAKKRVFTFGKGRSE..GNRD MAASVSRAICVQKPGSKCTRDREATSFRRRSVAAPRPPKAK....ARRRHPLRLRRGTGPHCS..PLRAWDA..APIQTTKKRVFHFGKGKSE..GNKT .. . . . . . . .. . . . . .. . . . .. . . . . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . . .. . . .. . .. . . . .. . . ..~~YKFE....E..GNAS . . . . . . .. . . . .. . . . . . . .. . . . .. . . . .. . . . . . .. . .. . . . .. .. . . . .. . . . . .. . . .. . .. . . . .. . . . .. . ..MST~VYFFG~TPENQPANSE . . .. . . . . . .. . . . . . .. . . . .. . . .. . . ... . . .. . . . .. . . . .. . . . ... . . .. .. . .. . . . .. . . . .. . . .. . . . ...MQRVYAFEDGDGTN..... -----------_--------------------------------------------___---__---___----__---___-*__*____________
F 2 C G E
M.KSLLGGKGANLAEMSSIGLSVPPGLTISTEACEEYQQNGKSLPPGSWDEISEGLDYVQ~MSASLGDPSKPLLLSVRSG~ISMPG~DTVLNLGLND M.KELLGGKGANLAEMASIGLSVPPGFTVSTEACQQYQDAGCALPAGLWAEIVDGLQWVEEYMGATLGDPQRPLLLSVRSGAAVSMPGMMDTVLNLGLND M.RNLLGGKGCNLAEMTILGMPIPQGFTVTTEACTEYYNSGKQITQEIQDQIFEArTWLEELNGKKFGDTEDPLLVSVRS~~SMPG~DTILNLGLND LCRKVLGGKGISLAAMIKLGMPVPLGFTITCQTCVEYQKTA.SWPKGLKEEVASNLKLLEEKGKTFGDNTNPLLVSVRSGAAVSMPGMMDTILNLGLND ..KKLLGGKGAGLCTMTKIGLPVPQGFVITTEMCKQFIANGNKMPEGLMEEVKKEYQLVEKKSGKVFGGEENPLLVSVRSGAAMSMDTILNLGLND _____*****__*__*___*--- i-*_______*______________________-__--------*----***_***,-*--*~******_*******
195 189 113 120 112
F 2 C G E
EVVAGLAGKSGA.RFAYDSYRRFLDMFGNVVMGIPHSLFDEKLEQMKAEKGIHLDTDLTAADLKDLKDLVEKY~VYVEA.KGEKFPTDPKKQLELAVNAVFD EVAAGLAAKSGE.RFAYDSFRRFLDMFGNVVMDIPRSLFEEKLEHMKESKGLKNDTDLTASDLKELVGQYKEVYLSA.KGEPFPSDPKKQLELAVLAVFN VAVEGFAKKTGNPRFAYDSYRRFIQMYSDVVMEVPKSHFEKIIDAMKEEKGVHFDTDLTADDLKELAEKFKAVYKEAMNGEEFPQEPKDQLMGAVKAVFR ESVKGLAAVTGNARFAYDSYRR~QMFGDVCLGIDHDKFEHALDAVKTRYGRKTDPELTADELEEVCEAYRKICV~.TGKTFPQCPHEQLELAINAVFK KTWALAKLTNNERFAYDSYRRFVSLFGKIALNACDEVYDKTLENKKVEKGVKLDTELDANDMKELAQVFIKK.TEEFTKQPFPVDPYAQLEFAICAVFR ______*______******_***-----__--___----__-----*---*-_*__i_*______________-__-----**-_*_-**__+__***_
293 287 213 219 211
F Z C G E
SWDSPRRNKYRSINQITGLK..GTAVNIQSMVFGNMGNTSGTGVLFTRNPSTGEK..KLYGEFLINAQGEDWAGIRTPEDLGTMETCMP...EAYKELV SWESPRAXKYRSINQITGLR..GTAVNVQCMVFGNMGNTSGTGVLFTRNPNTGEK..KLYGEFLVNAQGEDWAGIRTPEDLDAMKNLMP...QAYDELV SWDNPRAIVYRRMNDIPGDW..GTAVNVQTMVFGNKGETSGTGVAFTRNPSTGEK..GIYGEYLINAQGEDWAGVRTPQPITQLENDMP...DCYKQFM SWTNPRAQAYRTLNKLDHNM..GTAVNVQSMVFGNTGDDSGTGVGFTRCPKTGEKFSYLYGEFLQNAQGEDWAGIRTPVNLKEMPTINASWKACYDELS SWMGKRAVDYRREFKITPEQADGTAVSWSMVYGNMGNMGNDSATGVCFTRDPGTGENM..FFGEYLKNAQGEDWAGIRTPQIISKMAEDRD.LPGCYEQLL i*___**__**_________-_*~**--__*+_**_*__*_***-*~*-*-***--____ **_*_+*********_***_--_--___-_____-*____
386 380 306 317 308
F 2 C G E
ENCEILERHYKDMMDIEFTVQENRLWMLQCRTGKRTGKGAVRIAVDMVNEGLIDTRTAIKRVETQHLDQLLHPQFEDPSAYKSHVVATGLPASPGAAVGQ ENCNILESHYKEMQDIEFTVQENRLWMLQCRTGKRTGKSAVKIAVDMVNEGLVEPRSAIKMVEPGHLDQLLHPQFENPSAYKDQVIATGLPASPGAAVGQ DLAMKLEKHFRDMQDMEFTIEEGKLYFLQTRNGKRTAPAALQIACDLVDEGMITEEEAWRIEAKSLDQLLHPTFNPIlALKAGEVIGSALPASPGAAAGK LIYAKLEGYYNDMVDLEFTVENGKLWMLQARAGKRTGFAMVRIAIDMCKEGMLTEEEALLRIDANKINEFLFKRFDPSV..KPWLGKGIPASPGILAVGV DIRKKLEGYFHEVQDFEFTIERKKLYMLQTRNGI
486 480 406 415 406
F Z C G E
VCFSAEDAETWHAQGKSAILVRTETSPEDVGGMHAAAGILDDMKIFTIGDRVIKEGDWLSLNGTTGEVI WFTAEDAEAWHSQGKAAILVRAETSPEDVGGMHAAVGILTERGGMTSHAAWARWWGKCCVSGCSGIRVNDAEKLVTIGSHVLREGEWLSLNGSTGEVI VYFTADEAKARHEKGERVILVRLETSPEDIEGMHAAEGILTVRGGMTSHAAWARGMGTCCVSGCGEIKINEEAKTFELGGHTFAEGDYISLDGSTGKIY ICFCPMRTCELAEQGKKVILTRIETSPEDILGMDRAVGILTARGGQTSHAAWARGMGKCCVAGADCCQINYATKTLVIGDRKFKEGDFISINGTTGEIY WFDADDAVE.QAKGKKVLLLREETKPEDIHGFFVAEGILTCRGGKTSHAKKIAKIGSLEVHEGDILTIDGSTGCVY __*___________*____ *_*_**_***__*___*_****_tiC_**~-*********-_~_-**_*_--____---*_---*-----~~__-___*_**___ -A__
586 580 506 515 505
F 2 C G E
LGKQLLAPPAM.SNDLEIFMSWADQARRLKVMANADTPNDALTARNNGAQGIGLCRTEHMFFASDERIKAVRKMIMAVTPEQRKVALDLLLPYQRSDFEG LGKQPLSPPAL.SGDLGTFMAWVDDVRKLKVLANADTPDDALTARNNGAQGIGLCRTEHMFFASDERIKAVRQMIMAPTLELRQQALDRLLTYQRSDFEG KGDIETQERSV.SGSFERIMVWADKFRTLKVRTNADTPEDTLNAVKLGAEGIGLCRTEHMFFEAD.RIMKIR~ILSDSVEAREEALNELIPFQKGDF~ NGAVQTIEPGI.TDDLQTIMDWSDKYRVLKIRTNADTPHDAAVARKFGAEGIGLCRTEHMFF~D.RI~EMILSDDEGARRTALNKLLPFQREDFIG KGEVPLEEPQVGSGYFGTILKWANEIKKIGVFAAGDLPSAAKKALEFGAEGIGLCRTERM.FNAVERLPIWKMILSNTLEERKKYLNELMPLQKQDFIG _*__________________-*-_----__-____*-*--__-*---**-~******~_*-*___-*--__--**-__----*_-_~_-*-__*--**__ -8__
685 679 604 613 604
F Z C G E
IFRAMDGLPVTIRLLDPPLHEFLPEGDL...EHIVNELAVDTG..MSADEIYSKIENLSEVNPMLGFRGCRLGISYPELTEMQV~IFQ~VS~TNQ.GV IFRAMDGLPVTIRLLDHPSYEFLPEGNI...EDIVSELCAETG..ANQEDALARIEKLSEVNPMLGFRGCRLGISYPELTEMQA~IFE~I~TNQ.GV MYKALEGRPMTVRYLDPPLHEFVPHTEE. ..EQ..AELAKNMG..LTLAEVKAKVDELHE~PMMGHRGCRLAVTYPEIA~QT~~E~IE~EETGI IFKAMDGKGVNIRLLDPPLHEFLPHTR..... DLQKKLAEDMN..KKHRHIHERVEDLHEVNPMLGFRGVRLGIVYPEISEMQVRAILEAACIVSRE.GV LLKTMNGLPVTVRLLDPPLHEFLPTLEELMREIFEMKLSGKTEGLAEKEWLK~EL~~PMIGHRGIRLGTTNPEIYEMQI~FLEATREVIKE.GI +_*_***_*_**_f*____**---**_~*-_-*--__----*______,----_*-**_*__**-*____-------__*---_____-______--_-
779 773 697 705 703
F Z C G G
TVIPEIMVPLVGTPQEL.RHQISVIRGVAANVFAEMGVTLEY~GTMIEIP~LIAEEIGKEADFFSFGTNDLTQMTFGYSRDDVG.KFLQIY~QGIL QVFPEIMVPLVGTPQEL.GHQVTLIRQVAEKVFANVGKTIGY~GTMIEIP~LVADEIAEQ~FFSFGTNDLTQMTFGYSRDDVG.KFIPVHLAQGIL DIVPEIMIPLVGEKKEL.KFVKDVWEVAEQVKKEKGSDMQYHIGTMIEIPRAALTADG.KFLDSYYKAKIY TVKPEIMIPVLFSENEM.EIMHALVNRVAASVFKEHGTTVDYEVGTMIELPRACVMADKIAQTAQYFSFGTNDLTQTTFGISRDDAG.KFIPKYIDRGIF NDHREIMIPNVTEVNELINLRKNVLEPVHEEVEKKYGIKVPFSYGTMVECVRAALTADKIATEASFFSFGTNDLTQGTFSYSREDSENKFIPKYVELKIL ____**~_*______*____--__---~__-*____*---_---***_*__**---,__*___*__****~~****_~*--,*-*___**-___----*_
877 871 795 803 803
F Z C G E
QHDPFEVIDQKGVGQLI~TEKGRAANPSLKVGICGEHGGEPSSVAFFDGVGLDYVSCSPFRVPIARLAAAQVIV...... QHDPFEVLDQRGVGELVKFATERGRKARPNLKVGICGEHGGEPSSVAFFAKAGLDFVSCSPFRVPIARLAAAQVLV...... ESDPFARLDQTGVGQLVEMAVKKGRQTRPGLKCGICGEHGGDPSSVEFCHKVGLNYVSCSPFRVPIARLAAAQAALNNK.... KVDPFVTLDQQGVGALMKIEGGRSTRTDMKIGICGEQTD.PASILFLHKIGLNYVSCSPYRVPVARVAAAIAAIKARTNQ PANPFEILDRPGVGEVMRIAVTKGRQTRPELLVGICGEHGGEPSSIEWCHMIGLNYVSCSSYRIPVARIAAAQAQIRHPREN ___i*___*__***_____*-_-~*__-__---,,***---_*-*-__----~*--~*~~--*_*-**-*~*----_____-C-
96 90
14 21 14
953 947 874 884 885
Fig. 1. Alignment of deduced amino acid sequences of PPDK from Giurdia and various other organisms. F. Flaueriu rrinrrria (EMBL Data Library accession No. X57141 [22]); Z. Zea map (JO3401 [21]); C, Clostridium symbiosum (M60920 [19], corrected sequence in [20]); G. Giardia dzroderzafis(254168); E, Etztanzoeba hystolytica (X74596 [6]). Identical amino acid residues are marked by asterisk. The underlined sequence stretches denote putative functional domains required for phosphorylation (A). catalysis of the phosphoenolpyruvate/pyruvate partial reaction (P-loop, B) and substrate binding and/or catalysis (C).
script of about 2700 nt (Fig. 2A). The size of this band was found to be in good agreement with that predicted from the ppdk ORF. The nearly complete colinearity of the deduced Giurdia PPDK sequence with other PPDK sequences together with the observation that the giardial ppdk transcript was of similar size as that of the corresponding ORF is consistent with the absence of introns in this gene. Southern blots of total genomic DNA, probed with the subcloned ppdk PCR product, yielded a single hybridization band for all restriction enzyme digestions, indicating that ppdk exists as a single copy gene within the giardial genome (Fig. 2B). The locations of the restriction enzyme sites identified by mapping the two i-clones and their subclones are depicted in Fig. 2C. 3.3. Anulj~sis
of theppdk gene coding region
Sequence comparison of the PPDKs from Giardie as deduced from the nucleotide sequence of the corresponding gene with those of C. synbiosun~ ([ 191; corrected sequence in [20]), E. histolytica [6,7], maize [21] and Fluveriu trineraia [22] revealed an overall sequence identity of 54.1, 46.6, 51.1 and 52.6X, respectively (Fig. 1). For the alignment of the deduced sequences only a few small amino acid gaps had to be introduced, except for the large amino-terminal putative transit peptide of the plant PPDKs (76 residues for the F. trinerviu and 71 residues for the maize sequences) that is missing in the sequences of bacteria and protists. The size of the PPDK sequence from Giardiu (884 amino acid residues) was found to be very similar to that of the corresponding enzyme from C’. s~whiosun~ (874 residues), E. /listo/j*ticu (885 residues) and the mature forms from plant PPDKs (876 and 882 residues for maize and F. trinerviu, respectively). More detailed examination of various PPDK sequences revealed that the catalytic histidine that mediates the multiple phosphoryl transfer steps during catalysis is located within a highly conserved decapeptide arrangement (Fig. 1, domain A). indicating that the structures of the active site of these dikinases may be very similar. In plant PPDKs, the threonine residue positioned two residues upstream of the catalytic histidine functions as a regulatory site via a phosphorylationldephosphorylation
mechanism [19]. As this threonine is found within an identical motif (Fig. 1, domain A) in the enzyme from Giardiu and E. lzistolvticu it would be interesting to see whether the activity of protist PPDKs may be subject to a similar type of regulatory control. Other domain structures, previously demonstrated to be implicated in substrate binding and catalysis of C. synzhioszm PPDK [20,23] are also well conserved in the Giurdiu enzyme (Fig. 1. domains A and B) supporting the view that these PPDKs possess a uniform mechanism of catalysis.
Nucleotide sequences of 410 bp downstream and of 1087 bp upstream from the coding region were also sequenced and analyzed for conserved elements. A consensus sequence AGTGAAT, predicted as a putative polyadenylation site in Giurdiu, was found starting 2 bp upstream from the stop codon TGA. The sequence AGTPuAAPy has been proposed as a polyadenylation signal for Gimlicl separated by a 6-19 bp interval from the stop codon [3]. The DNA sequences upstream of the translation initiation codon of ppdk were searched for homology to typical eukaryotic transcriptional regulatory elements. Examination of this region from pprlhand several of the available other Giwdiu genes encoding a number of cytosolic enzymes [24-281, nuclear proteins [29,30] and heat shock proteins [31] revealed the presence of a 14-bp AT-rich consensus sequence centered at about 30 nucleotides upstream of the translational start site (Fig. 3). This element has a sequence and location that resembles the 1%bp putative consensus promoter motif recently deduced from analysis of seven Giurdiu cytoskeleton protein genes [32 -381. The strictly conserved pattern AA-N9-C located at nucleotide positions 3-14 within the 14-bp box is present in all of the sequences examined except in the adenylate kinase and vacuolar-type ATPase genes where the C at nucleotide position 14 is replaced by a T [27,28]. Recently. a second potential promoter consensus sequence was identified immediately upstream of the ATG initiation codon in cytoskeleon protein genes. The same consensus motif was also found to exist in ppdk and most other currently
T. Bruderer et al. / Molecular and Biochemical Parasitology 77 (1996) 225-233
230
A
B 12345676
nt
kb 23 9.4
7400
6.6
5300
4.3
2800
1900
2.3
1600
2.0
C K
C
XbH
P
Xb
E
E
Xb
E
PPB
C
C
E
C
B
C
NBEC
K
HB
Xb
P
C
lkb Probe
Fig. 2. Nothern and Southern blot analysis using the PsfI digested and digoxigenin-labeled PCR fragment as probe. Sizes of the nucleic acids are indicated on the left. (A) Northern blot, 5 pg of Giardia total RNA was subjected to formaldehyde agarose gel electrophoreses, blotted onto a positively charged Nylon membrane and hybridized as described in Methods. (B) Southern blot, 5 ,ug of Giordia genomic DNA were completely digested with restriction endonucleases and analyzed on a 0.8% agarose gel. Lane I, EcoRI; lane 2, EcoRI + NotI; lane 3, HindHI; lane 4, PstI; lane 5, Hind111 + PstI; lane 6, XbaI; lane 7, ClaI; lane 8, XbaI + ClaI. (C) Physical restriction map of Giardiu 02-4Al chromosomal DNA. The black box indicates the DNA region completely sequenced in this study. The hatched arrow denotes the ORF corresponding to ppdk. Restriction sites are: B, BarnHI; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; N, NotI; P, PstI; Xb, Xbal.
available protein-coding genes of Giardia (Fig. 3). The highly conserved motif shows a predominant A and T content and is 9 bp long [32]. Exceptions to this pattern exist in the adenylate kinase gene where a comparable sequence motif could not be identified and in the RAN-encoding gene that
contains a similar sequence 33 bp upstream of the ATG [27,29]. Interestingly, primer extension experiments with the latter gene revealed a termination signal at - 29, in addition to those at - 2 and - 4, which would be consistent with the putative initiation site being farer from the ATG than that
T. Bruderer er al. / Molecular and Biochemical Parasitology 77 (1996) 225-373 14
bp Box
Initiation site
al-giardin
-3yCAAAAAATACCGCC
-%ggTTTTAAAAAtg
X52485 [33]
ail-giardin
-34TAAAATATATTTTC
-12tacATTTAGAAAatg
M34550 [34]
P-giardin
-34CAAAAATTTGAGCC
-13agaAAATAAAAGgatg
X85958 [32]
y-giardin
-26TAAAAAACTAACCC
-13ctaAAACAAGAAaatg
X55287 [35]
Median body protein
-42CAAATGCTAAATTC
-'ktaAATTAAAAAt_g
X64517 [36]
a-tubulin
-33CAAATTCCAGAGTC
-%ggAAATAAAAAtg
JO4648 [37]
P-tubulin
-33CCAAATCCAGATTC
-%cgATTTAAAAAtg
X06748 [37]
Actin
-22TTAAATTTATTTGC
-"tgcAAAGTAAAAtg
L29032 [38]
PPDK (02-4Al) -4aTTAAATGAAATATC
-l%ctAATAAATAAttggatg
254168
PPDK (WB)
-40TTAAATGAAATATC
-lkctAATAAATAAttggatg
u43531 [lo]
GAP-l
-35AAAAAACTTAACAC
-'laaaAATCTAAAAtg
M88062
PPi-PFK
-31AAAAATTAGCTAGC
-13gctAATTTGAAAtg
U12337 [24]
TIM (WB)
-31CAAATTAGAAATTC
-14ccaGAAAATAAAtcatg
LO2120 [25]
TIM (GS)
-3gAAAAACCCTGATTC
-13tcaGAAAATAAAttatg
LO2116 [26]
NADP-GDH
+"GAAAAGCTGACCAC
-'lcagATTTTAAAAtg
M84604 [26]
AK
-36CAAAAGCATAATGT
mm-1
-46TAAATAAATCTGTT
-'kaaAATTTGAAAtg
RAN
-3QTAAAACTTTAACCC
-36cgaAATTAAAACNlsagtagcatgU02589 [29]
AR??
-3aCAAAAAATGAAGGC
-14ggcCATTAAAAAgaatg
M86513 [30]
HSP70
-4QTCAAAAGATCTGAC
-13ggaAAATAAAATcatg
U04874 [31]
ER-HSP70
-24CAAAAAAGGACGAC
‘I’cggAAATAAAAAtg
U04875 1311
CONSENSUS
131
U19901 [27] U18938 [28]
YAAAAWNNWAWNNC
AAWTAAAAA Fig. 3.Sequence comparision of putative polymerase II promoter elements of various genes from Giurdio consisting of a l4-bp box and an initiation site. EMBL Data Library accession numbers and references are listed in the right column. The numbering of the nucleotides in the sequences is relative to the zero positions assigned to the A of the ATC translation start codon. Boldface nucleotides represent major signals obtained from Sl protection or primer extension experiments. PPDK, pyruvate phosphate dikinase (from a sheep- and a human-derived Giardia isolate); GAP-l, glyceraldehyde-3-phosphate dehydrogenase; PP,-PFK, pyrophosphate-dependent phosphofructokinase; TIM, triosephosphate isomerase (from two different human-derived Giurdia isolates); NADP-GDH, NADP-dependent glutamate dehydrogenase: AK, adenylate kinase; VMA, vacuolar type ATPase; RAN, US-like nuclear protein; ARF, ADP-ribosylation factor; HSP70. 70-kDa heat shock protein; ER-HSP70, endoplasmatic reticulum HSP70 homolog.
in all other known Giardia genes [29]. In most of the studies where primer extension experiments were performed to identify transcriptional initiation the start site was found to begin with an A at a relative position of 5 or 6 within the 9-bp box. Recent analysis of protein-coding genes from another amitochondriate protozoan, Trichomonas vaginalis, has also revealed the pres-
ence of a strong consensus sequence immediately upstream of the initiation site [39]. However, this start site motif does not show any similarity to the highly conserved 9-bp consensus sequence immediately upstream of the putative initiation site of Giardia genes. Interestingly, the genes encoding the variant surface proteins of Giardia were not found to possess an upstream motif
737 .._
T. Bruderer et al. ! Molecular und Biochemical Parasitology 77 (I 996) 225-233
similar to that found for all other known genes of this parasite. A large number of RNA polymerase II promoters of higher eukaryotes contain a TATA box that is centered at around - 27 within a consensus sequence of TATAa/tAa/t. In the region corresponding to a similar location of the Giardia genes analyzed, there were no TATA boxes identified with any significant homology to other promoter sequences. Several potential TATA motifs have been localized in some genes of Giardia and other protists further upstream of the transcription start site. However, these sequences were not found to be conserved. appropriately spaced and homologous to the typical TATA consensus sequence commonly observed in class II genes of higher eukaryotes. It is interesting to note that in TATAless genes of higher eukaryotes the presence of an initiator sequence surrounding the transcription start site is sufficient to support transcriptional initiation by RNA polymerase II. The accurate biological activity associated with the consensus elements occurring upstream of Giurdiu proteincoding genes is at present unknown. Analysis of the role these elements may play in gene transcription have to await deletion and point mutation experiments and the establishment of an in vitro transcription assay. Such studies may then also help to elucidate the evolutionary origins of the structural components of eukaryotic promoters.
Acknowledgements
We thank Dr. Miklos Mtiller (The Rockefeller University, New York) for valuable comments on this manuscript and for sharing his results during the development of this work. This study was supported by grant 31-36198.92 from the Swiss National Science Foundation.
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[29] Chen, L.-M., Chern. Y., Ong, S.-J. and Tai. J.-H. (1994) Molecular cloning and characterization of a rrrs-related gene of ran itcJ/spii subfamily in Giardia lamhliu. J. Biol. Chem. 269. 17297-17304. [30] Murtagh, J.J., Mowatt, M.R., Lee, C.-M.. Lee, F.-J.S.. Mishima, K.. Nash. T.E., Moss. J. and Vaughan. M. (1992) Guanine nucleotide-binding proteins in the intestinal parasite Giardia kamhliu. J. Biol. Chem. 267. 9654 9662. [31] Gupta. R.S.. Aitken, K.. Falah, M. and Singh. B. ( 1994) Cloning of Giardia lamblia heat shock protein HSP70 homologs: Implications regarding origin of eukaryotic cells and of endoplasmic reticulum. Proc. Nat1 Acad. Sci. USA 91, 2895-2899. [32] Holberton. D.V. and Marshall. J. (1995) Analysis of consensus sequence patterns in Giurdia cytoskeleton gene promoters. Nucl. Acid Res. 15. 2945 2953. [33] Peattie, D.A.. Alonso. R.A.. Hein. A. and Caultield, J.P. (1989) Ultrastructural localization of giardins to the edges of disk microribbons of Giardiu lamblia atoll the nucleotide and deduced protein sequence of alpha giardin. J. Cell Biol. 109, 737332335. [34] Alonso. A. and Peattie, D.A. (1992) Nuclcotide sequence of a second alpha giardin gene and molecular analysis of the alpha giardin genes and transcripts in t&r& lumblia. Mol. Biochem. Parasitol. 50. 95-104. 1351 Nohria. A., Alonso. R. A. and Peattie D. A. (1992) Identification and characterization of ;x-giardin and the ;s-giardin gene from Gurdk lamhlia. Mol. Biochem. Parasitol. 56. 27.-38. [36] Marshall. J. and Holberton. D.V. (1993) Sequence and structure of a new coiled coil protein from a microtubule bundle in Giardia. J. Mol. Biol. 231. 521- 530. [37] Kirk-Mason, K.E., Turner. M.J. and Chakraborty, P.R. (1989) Evidence for unusually short tubulin mRNA lcaders and characterization of tubulin genes in Ciurdirl kmhlia. Mol. Biochem. Parasitol. 36. 877100. [38] Drouin, G., Desa. M.M. and Zuker. M. (1995) The Giardia lambliu actin gene and the phylogeny of eukaryotes. J. Mol. Evol. 41. 841 -848. [39] Quon. D.V.K.. Delgadillo, M.G.. Khachi. A.. Smale. S.T. and Johnson, P.J. (1994) Similarity between a ubiquitous promoter element in an ancient eukaryote and mamahan initiator elements. Proc. Nat1 Acad. Sci. USA 91. 457Y4583.