Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase

Gene, 173 (1996) 1377145 0 1996 Elsevier Science B.V. All rights reserved.

137

037%1119/96/$15.00

GENE 09832

Cloning and characterisation of a gene from Plasmodium vivax and I? knowlesi: homology with valine-tRNA synthetase (Malaria; amino acid sequence; database search; cross-hybridisation; phylogenetic analysis; antigenicity)

conserved sequence blocks; codon bias; splice sites;

Valerie A. Snewina, Elizabeth Khouri”, Denise Matteib, Fredj Tekaia”, Marc Delarued, Kamini N. Mendis” and Peter H. David” aUnit d’lmmunoparasitologie, CNRS URAl960,

bUnitt de Parasitologie Experimentale, CNRS URA1960, “Unit6 Genetique Moltculaire des Levures,

*Units d’Immunologie Structurale, Institut Pasteur, 25-28 Rue du Dr. Roux, 75724 Paris, cedex 15, France. Tel. (33-l) 45688000; and “Malaria Research Unit, Department of Parasitology, Faculty of Medicine, University of Colomho, Colombo 8, Sri Lanka. Tel. (94-l) 699284 Received by P.F.G. Sims: 29 June 1995; Revised/Accepted:

21 August/2

September

1995; Received at publishers:

14 March

1996

SUMMARY

We have previously described a hgtll clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vioax transmission in the mosquito vector. An epitope recognised by Al2 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA crosshybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

INTRODUCTION

Plasmodium viuax (Pv) is one of four malarial parasites infecting humans. Although not responsible for the majority of the mortality attributed to this disease, the Correspondence Department

to:

St. Mary’s, Norfolk Fax (44-171)

Dr.

of Medical

V.

A.

Snewin,

Microbiology,

Place, London

at

Imperial

her

current

address:

College of Medicine

at

W2 lPG, UK. Tel. (44-171) 5943955;

2626299; e-mail: [email protected]

Abbreviations:

aa, amino

acid(s);

GCG, Genetics

Computer

Group

Ab, antibody(ies); (Madison,

bp, base pair(s);

WI, USA); kb, kilobase

or 1000 bp; mAb, monoclonal Ab; nt, nucleotide(s); oligo, oligodeoxynucleotide; ORF, open reading frame; PCR, polymerase chain reaction; P., Plasmodium; Pl; P. falciparum; Pk, P. knowlesi; Pv, Plasmodium vivax; R, adenosine (A) or guanosine (G); RT, reverse transcription; rRNA, ribosomal RNA; SDS, NaCl/O.OlS M Na,citrate encoding Val-tRS. PII SO378-1119(96)00235-l

sodium dodecyl pH 7.6; tRS, tRNA

sulfate; SSC, 0.15 M synthetase(s); valS, gene

symptoms it induces are severe. A large proportion of the world’s population is at risk from this parasite, leading to increased economic and social deprivation, yet Pv is relatively understudied, in part due to the lack of a routine culture system in the laboratory (reviewed in Snewin et al., 1991). One possible control measure would be the development of transmission-blocking vaccines (Kaslow, 1994; David et al., 1990). Such vaccines are based on immunity mediated by Ab against parasite sexual stages that block the development of the malaria parasite in the mosquito host. While not protecting the individual, subsequent transmission of the parasite would be interrupted. mAb with transmission-blocking activity have allowed the definition of specific targets of transmission-blocking immunity on the surface of Plasmodium sexual stages (Kaslow, 1994; Premawansa et al., 1990). We have previously described an epitope (Snewin et al.,

138 1995a), recognised by a transmission-blocking mAb (Udagama et al., 1987) which identifies one such antigen, termed GAMl (Carter et al,, 1993). This mAb, A12, is unusual among transmission-blocking mAb in recognising a linear epitope which appears to be present in both sexual (gamete surface) and asexual (trophozoite parasitoporous vacuole) stages of Pu (Udagama et al., 1987). The Al2 mAb was employed to isolate the 3’ region of a gene, clone 4713, through screening a Pv hgtll expression library (Snewin et al., 1995a). Here we describe an ORF corresponding to 146 kDa encompassing this fragment, in frame with that of the original hgtll expression clone. The aim of this study has been the characterisation of this gene and its homologue from the simian parasite P. knowlesi (Pk). These genes appear to encode putative parasite Val-tRS, by comparison with sequences from other organisms in the sequence databases; the resulting multi-alignment reveals blocks of aa residues predicted to be required for enzymatic activity. For this reason, this gene will be referred to as ualS and not, as previously, GAMl (Snewin et al., 1995b).

RESULTS

E

F

b D D b

D D

AND DISCUSSION

(a) Southern hybridisation

fragment containing the hgtll clone 47/3 (Snewin et al., 1995a) was employed as a probe against genomic Southern blots, demonstrating the existence of a 6.5-kb EcoRI fragment within the genome of Pu Belem strain (Fig. 1, lane A, see legend). Cross-hybridisation was also observed with EcoRI-cleaved restricted genomic DNA from the closely related simian malaria parasites, Pk and P. fragile (Fig. 1, lanes B and C). In addition, hybridisation was obtained, under conditions of relatively high stringency, with both DNA from Bacillus subtilis and Saccharomyces cerevisiae (Fig. 1, lanes E and F). No hybridisation was observed with genomic DNA from P. falciparum Pf (Fig. 1D). This may be explained by the difference in %G + C content of the genomes of these two parasites (approximately 20% G+ C for Pf coding regions (Weber, 1988) compared to 36-55% for Pv (Fig. 3A) and 41% for the S. cerevisiae genome (Sharp et al., 1986). A DNA

(b) Sequence data and molecular characterisation

Fig. 2 illustrates the complete coding sequence of the PO valS gene. The cloning of the 3’ sequence (boxed), by

immune-screening of a hgt 11 library with a transmissionblocking mAb A12, has already been described in Snewin et al. (1995a). Screening a hgt WES Pv genomic DNA EcoRI library (Del Portillo et al., 1991), with the insert from clone 47/3, led to the isolation of a clone containing

Fig. 1. Southern obrained

blot analysis

of the valS gene. Genomic

from: Lanes: A, Pu Belem; B, Pk H-strain;

iae AB1.580. Arrowheads

indicate

HindHI-digested

h DNA size markers

of 23.1, 9.4, 6.6, 4.4, 2.3 and 2.0 kb (NE Biolabs,

Methods: After restriction electrophoresis

digestion

on a 0.8% agarose

membrane

(Amersham

The filter was hybridised

Beverly,

gel, DNA was transferred

International,

at 65°C overnight

Buckinghamshire,

DNA, employing (Amersham

0.2 x SSC/O.l%

UK).

generated

the oligos Pv24

Nick Translation

Washes were carried

SDS, and the film was exposed

and

to Hybond

with a probe

to Pv25 (see Fig. 2), after nt labelling by the manufacturer).

MA, USA).

of 3-6 ug DNA with EcoRI

from by PCR on Pv Belem genomic as described

was

E, Bacillus subtilis 21K; and F, Saccharomyces cerevis-

D, PfPalo-Alto;

Nylon

DNA

C, P. ,fragile S+;

kit,

out at 65°C at

to the filter at -80°C

for both 48 h and 7 days. In lane B, the result after 7 days is presented. No additional bands were observed in the other lanes when exposed over this period. out according

Standard

to Ausubel

molecular

biology

techniques

et al. (1994), unless otherwise

were carried stated.

a 6.5-kb insert, corresponding to the band identified in Fig. 1, lane A. This fragment was sub-cloned into pUC13, producing the clone SG26, the partial sequence of which is shown in Fig. 2. This clone showed no evidence of rearrangement when restriction fragments were compared to genomic digests (data not shown). Here we describe the cloning and characterisation of 5.3-kb of Pv genomic DNA, containing this original fragment within a 4029-bp ORF. Comparison of the Kozak (1984) consensus sequence,

139 -750 -600 -450 -300 -150 -1 150 300 450 600 750 900 1050 1200 1350 1500 1650 1800 1950 2100 2250 2400 2550 2700 2850 3000 3150 3300 3450

Fig. 2. Sequence accordingly,

of the Pv ualS gene. The hgtll

including

are shown in lower-case and a potential sub-cloning Sequences created

characters

polyadenylation

into rUC13

US Biochemicals,

positions

were aligned

(Pv8, 9, 14 and 15), described

and the intron splice sites are arrowed. signal sequence

using standard

Cleveland

clone 47/3 (Snewin et al., 1995a) is shown boxed. Oligo sequences

of the nested PCR primers

Sequencing

OH, USA), using a combination

using the CCC

GELASSEMBLY

was carried of synthetic

The DNA sequence

oligo primers

has been deposited

GCCRCCATGG, with the two putative start codons shown circled in Fig. 2, indicates that the true start codon may be the second of the two observed at nt 28. An ORF would therefore be predicted between nt 28-4029, terminating with a TGA stop codon, followed by a highly A + T-rich region and a potential polyadenylation signal, (AATAAA) (Proudfoot and Brownlee, 1976), shown in bold in Fig. 2. The nt sequence from the Pv gene is 54.7% G + C, within the coding region, with more highly A + Tfound in the 5’ and 3’ flanking regions. The %G+C content for a number of malaria

parasites, including Pv and Pk has been described (Gutteridge et al., 1971; McCutchan et al., 1984). These investigations identified distinct genome fractions, in both organisms, with %G+C contents of both 18%

and labelled

non-coding

regions

start codons are circled, the TGA stop codon is asterisked,

out on both strands

series of programmes,

to cover the 5.3-kb region. The Pv ualS DNA sequence

rich regions total DNA

The two putative

is shown in bold. Methods:

techniques.

are dotted-underlined

in Snewin et al. (199513). The predicted

and sub-cloning

and a database

hgtIVES clone insert was obtained

of the

with dideoxy

nucleotide

through

naturally

technology occurring

of more than 5%kb of overlapping

in the EMBL/GenBank

database

under accession

through

(Sequenase restriction sequence

kit, sites. was

No. X84734.

and ~30%. When calculated for the coding of the Pv genes shown in Fig. 3A, however

regions (GCG

COMPOSITION

varying

program),

a %G+C

content

from 50% to 36% is obtained. This indicates that in general, coding regions of Pv genes contain a higher %G + C than the non-coding flanking regions (also discussed in Barnwell

and Galinski,

1995). The Pv valS gene appears

to be a marked example of this, illustrated in Fig. 3B. Such a bias is in fact also present in Pfalthough in this case, coding sequences are 80% A+T and non-coding regions 90% A +T (Weber, 1988). The equivalent gene from Pk was obtained through screening a library (Hudson et al., 1988), with Pv valS DNA sequences and by PCR amplification of Pk genomic DNA (see the legend to Fig. 4). The Pk DNA sequence

140

A

,F

%GC

Gene

Reference

54.7 49.7 49.7 44.5

a! vals &CSP &RNA

z*; 3616 35.7

Ezz I!Ycys protease & Duffy p_vREP1

this study Amot et al., 1988 Waters et al., 1989 Cheng and Saul 1994 de1 Portillo et al., 1991 Rosenthal et al., 1994 Fang et al., 1991 Galinski et al., 1992

46.7 46.3 39.4 36.8

pk vals pkCSP &AMA1 gkEsPa

this study Ozaki et al., 1983 Waters et al., 1991 Adams et al., 1990

III

II

III

GcQ CM A L

eukaryote

Fig. 3. Codon

cone.

llllllllll

4ooo

II

I

II

II

C/AA0

gta/gagt

. . . . . . . . . . . . . . . . . . . . . . . . . . . ..(t/c)n

of the Pu and Pk dS

sequence

for the Pu ualS gene sequence.

through

gene sequences.

C: The RT-PCR

carried directly

to a first-strand

La Jolla, CA, USA). Amplification

of the resulting

obtained

II

A: Comparison

from genomic

of %G+C

products

out from Pu Belem isolate RNA. Total RNA was purified cDNA

synthesis,

according

cDNA by PCR was carried

DNA (460 bp, lane 3). Size markers

of oligos Pv43 to Pv41L and the identity

of resulting

content

in Pu and Pk coding

based on the method

to manufacturers out, final volume

N

B

G

from Pv first-strand

obtained

H

III III III G~A (UC CAC

III1

..-.....................nc/tag

amplification

Cetus (Norwalk, CT, USA), 9600 GeneAmp PCR System, employing the following 10 s, 59°C 25 s, 72°C 55 s and extension for 4 min at 72°C. 10 ~1 were subsequently consisted

I

N

QCAGACCAC GA

RT-PCR

with the product

I

5000 bp

GA

Y

content

was detected

III1

Sacchi (1987). 30 pg was then added

Primers

3ooo

TAc OgtaaggtgPttgaggggt~agacacacagcgggag.79bp.ttatttccccttttatcttcccttttatgtagaC

bias and intron

B: Plot of %G+C

Stratagene,

2ooo

loo0

A L Y QCQCTQ TAT ~aea*ggetoaoccrptgtg~ggcegtgaggcc.7Obp.tttcgcctaacccccttgccgctccccctcagOT

PwalS

intron

/

instructions

cDNA.

sequences.

Methods: An

of Chomczynski

(Stratascript

of 50 ~1 per reaction,

and

RT-PCR

in a Perkin

kit,

Elmer

program: 91°C 5 min, 56°C 2 min followed by 35 cycles at 91°C electrophoresed on a 1.5% agarose gel (lane 1) for comparison

(M) correspond

322-bp fragment

to $X174 DNA after HaeIII

was confirmed

by oligo probing

digestion

(NE Biolabs).

with Pv40 (data not shown).

Positions and sequences of these primers are shown in Fig. 2. Fragments were subsequently cloned (TA-cloning kit, Invitrogen, San Diego, CA, USA), prior to sequencing (Sequenase kit, USB) with universal oligo primers. Controls include a reaction without addition of the reverse transcriptase (Stratascript

RNase

surrounding

the intron

H-),

for first-strand

cDNA

in the PO Reticulocyte

synthesis

Binding

(lane 2). In addition

Protein

1 gene (nt 65-86

a reaction

was performed

and 651-670

according

of 403 bp from first-strand

cDNA (lane 4) or 605 bp from genomic

DNA (lane 5). D: Pu ualS intron

from the Pk uaLS sequence

and the vertebrate

(Mount,

is 75% identical predicted

coding

the codon bias COMPOSITION

consensus

to that of the equivalent region

(GCG

of 46.7% program).

GAP

sequence

Pv gene in the program),

with

G+ C content (GCG Although relatively few

complete Pk gene sequences have as yet been deposited in the sequence databases, the %G + C content of a selection of such genes has been calculated (Fig. 3A). The coding region of the Pk valS gene (Pk valS), is 46.7% G+C-rich, again higher than that reported for the genome overall by sedimentation analysis (Gutteridge et al., 1971) and that of the Pv gene. (c) Intron structure The presence of a 138-bp

intron

has been identified

within the Pv sequence, using RT-PCR on Belem mixed blood-stage parasite RNA (Fig. 3C). The intron donor and acceptor sequences, marked by arrows in Fig. 2,

with primers to Galinski

sequence

derived

from the sequence

et al., 1992), giving a fragment

splice sites compared

to those predicted

1982).

follow the consensus sequences described in Mount (1982). The PO exon-intron boundary sequences, compared to those predicted from the Pk sequence and the eukaryotic consensus sequence are shown in Fig. 3D. The existence of the intron in Pk appears to be confirmed by the breakdown in homology both at the DNA- and translated aa-level between the two sequences (Fig. 3D), and by the presence of conserved splice donor and acceptor sequences. A 147-bp intron would therefore be predicted in the Pk gene. Stop codons are present in the equivalent sequence from both parasites in all three reading frames. The intragenic region of Pv is however 59% G+C, yet the Pk predicted intervening sequence is only 48 % G + C, Pk therefore appearing more A + T-rich from these limited comparisons. These data support evidence already obtained from several Plasmodium genes, that intervening sequences follow the same general rules for

If****

1

*

** **

Al

t ,*

*

*

* l **x**
(I** ****

l

f*ltttt*f*.****t*f**I

tt I, ** *** * **

**** *** **/*fff**.****.*l***

Fig, 4. The translated of the PO intron

of Pu t&S and Pk ua2S. The Pu sequence,

aa sequences

and its predicted

region in Pk is arrowed.

Met residues are shown in bold and the putative e, are boxed and labelled is shown deleted

i. The aa sequence

in bold and boxed (iii), aligned regions

was obtained generated

are denoted through

the 3’ part of valS including

a Malaysian

sequence

signal sequence

(1.1 * ** f **. t * * * ::

corresponds

to align the sequences is underlined.

region in Pk. Identical

H-strain

previously

described

clone A hgtll

(5’) and Pv8-Pv14

homologous

1+**

(Snewin,

cDNA

library

of 146 kDa. The position lines. The two N-terminal

blocks, described

in Pv by the mAb A12,

are indicated

by asterisks.

clone 47/3; Pk12 encompasses

The

by dots. Methods: The Pk nt sequence

et al., 1988), with the 5’ and 3’ regions

(3’) (see Fig. 2). Two clones were obtained:

with the PO hgtll

in more detail in section

recognised

in both sequences

1995b) is underscored (Hudson

* .f********f*f

to a polypeptide

The conserved

aa residues

**t**

are shown as dashed

in Snewin et al. (1995a) is shown in box ii and the sequence

with the equivalent

oligos Pv47-Pv48

Il. * *et**

when translated,

Gaps introduced

hydrophobic

described

Al and A2; the A2 region

screening

by PCR between

*** * ******t*

Pk6 contains

of Pu ualS

a region corresponding

most of the 5’ region

to

of the gene (Pk clone

positions are indicated). Both clones appeared to in fact contain genomic DNA, however, with no poly(A) tail and the putative intron sequence present in Pk6. The junction between these clones was obtained by PCR amplification of Pk genomic DNA (H-strain), between oligo Pkx and Pky. In addition,

the equivalent

sequence

sequences follows oligo are as 5’-ACCTTCCATGTTTACCTGAAGCAG. accession

to that recognised 3’); (5’ to

by the Pu mAb Al2 was obtained by PCR from Pkz to a Pu oligo Pv25b (see Fig. 2). The Pkx: 5’-CGGTACATAAGGAGGCGC, Pky: T-CTGCAGAATGTCCTTCCC; Pkz:

The nt sequence

No. X87933, within which the location

obtained

for the Pk valS gene has been deposited

in the EMBL/GenBank

database

under

of these Pk oligos is described.

these parasitic protozoa as for other eukaryotes (reviewed in Weber, 1988). (d) Comparison of the Pv and Pk aa sequences

Fig. 4 shows a comparison of the deduced aa sequence of the valS gene from Pv and Pk. The sequences show 66% identity and 78% similarity (GCG GAP program). The Pv translated aa sequence (1295 aa; predicted molecular weight 146 310 Da), contains an elevated content of Arg (8%), Leu ( 11%) Gly (8%) and Glu (7%). A putative, though atypical, hydrophobic signal sequence from the second of the two in frame start codons, nt 28, is underlined in Fig. 4, although reference to known signal sequences (von Heijne, 1985), indicates that this protein is unlikely to be exported. The Pk sequence available corresponds to an ORF of 977 aa; 113 983 Da. However as the 5’ sequence has not been obtained, only a partial comparison with the Pv sequence may be carried out. In Fig. 4, the sequence previously described in Snewiq et al. (1995) is shown in box ii. The consensus motifs used to define class-1 type tRS enzymes, HIGH and KMSKS, and the sequences predicted to be involved in binding

the aa valine are labelled as boxes marked i. Sequences shown in theses boxes, (i), are discussed in section e. Within these regions, the Pv and Pk aa sequences are 78-93% identical, substitutions often being conservative. All the blocks are arranged in the same order in the Plasmodium sequences as in Val-tRS sequences from other organisms. The polymorphic region described in Snewin et al. (1995b) is indicated in Fig. 4, underscored by dots (82). It is interesting to note that another potential ‘deleted region’ is also present (Al), which could indicate an additional polymorphic region in this gene. Short degenerate motifs (indels) are repeated in the Al and A2 regions (Fig. 4). The Al2 mAb epitope sequence (Snewin et al., 1995a) is partially conserved between the Pv and Pk translated sequences, consisting of the sequences TWEVLH and TWEIIH, respectively (box iii, in bold in Fig. 4). Four of the six aa are thus identical and the remaining two are conservative substitutions (Val and Leu to Ile). The SwissProt database (release 31) was searched with the six aa sequences (BLITZ server). No perfect matches were obtained with either sequence and

142 none of the potential matches were present as repeated motifs. (e) Conserved sequence blocks for Val-tRS Using a BLASTx search (Altschul et al., 1990) of the GenBank (Genpept program) database (release 87) significant homology was detected between both deduced aa sequences and Val-tRS enzymes from bacteria, yeast and higher eukaryotes. Although only a low level of homology was detected over the majority of the deduced aa sequence, certain sequences (shown schematically in Fig. 5) typical of class-I type aminoacyl-tRS were identified, in addition to the sequences predicted to confer specificity for valine. It should be noted that no such homology was observed when the translated aa sequence from the hgtll clone 47/3 was analysed in this way (Snewin et al., 1995a). Aminoacyl-tRS constitute an essential part of the translation apparatus, responsible for addition of each aa to its corresponding tRNA molecule. These enzymes have been grouped into two classes (ten in each). Class-I tRS (Arg, Cys, Gln, Glu, Ile, Leu, Met, Tyr, Trp and Val), are

defined by HIGH and KMSKS signature sequences, diagnostic for the presence of a structural domain that binds ATP, known as the Rossman fold (Eriani et al., 1990; Land&s et al., 1995; Delarue, 1995). Sequences conserved between tRS from different organisms have been predicted to give specificity for each aa. Two such sequences, conferring specificity for valine, are also found in the translated Pv valS and Pk valS gene sequences. Five putative Pf tRS have been detected by expressed sequence tag analysis (Chakrabarti et al., 1994) including Met and Ile class-1 type tRS enzymes. In addition, nine putative tRNA genes have been characterised on the 35-kb circular DNA of Pf(Gardner et al., 1994). (f ) Multi-alignment and phylogenetic analysis

Two types of analysis were employed on the multiple sequence alignments. Fig. 6A is a representation of the similarity of the sequences, presented as a hierarchy. This indicates that the two Plasmodium sequences group together, yet with aa sequences highly divergent from ValtRS sequences from other organisms. Direct comparison (GCG GAP analysis) indicates only 21% overall aa iden-

Fv Fk Bt Bs LC

EC SC NC

/-is

*

***

**

*

*

**

****

Fig. 5. Comparison of Pv dS and Pk ualS translated sequences with aa sequence blocks from Val-tRS. Val-tRS sequences were obtained from the GenBank database for Homo sapiens (Hs), Neurospora crassa (NC), Saccharomyces cerevisiae (SC), Escherichia coli (EC), Lactobacilus casei (Lc), Bacillus subtifis (Bs) and B. stearothermophilus (Bt), with accession Nos. X59303, P28350, 502719, X05891, S58666, X77239 and M16318, respectively. The aa sequences were aligned, using the CLUSTALW programme (Thompson et al., 1994), with manual adjustments, to those of the ualS gene sequence from Pu and Pk. Only those regions predicted to be involved in enzymatic activity (the HIGH and KMSKS signature sequences), and those regions predicted to be involved in binding to the aa valine, are illustrated. Identical residues are highlighted against a black background and marked with an asterisk,

whereas

conservative

changes

are shown in grey.

143 when compared to the human enzyme, the sequences being 45% similar yet again only 21% identical. When the aa sequence of the human enzyme is compared to that of B. subtilis, they are 61% similar, 41% identical. The Pv valS and Pk valS translated sequences however show 66% aa identity and 78% similarity between the two sequences. This disparity is also observed when the predicted antigenicity was calculated (GCG PEPSCAN program), the deduced aa sequences from the Pv ualS gene appears highly antigenic in comparison to that of the sequence of the B. subtilis Val-tRS gene (data not shown). The difference is especially marked at the C-terminal end of the predicted protein, within which we have previously mapped the epitope of the Al2 transmission-blocking mAb. The rooted phylogenetic tree obtained from Parsimony, however (Fig. 6B), indicates that the Plasmodium sequences do nevertheless, in evolutionary terms, group with Val-tRS from other organisms.

H. sapiens S. cerevisiae N. crassa E. coli L. casei B. subtilis I

B. sterothermophilus

B

1 I

rw 1oo

(g) Conclusions (I) Since only those

E. coli

L. casei

100

B.

100

100

B. sterothermophilus

P. vivax

P. knowlesi

-

outgroup

Fig. 6. Multi-alignment

ualS translated aa sequences described

and phylogenetic

aa sequences were aligned

in the legend

dendrogram

from other

over their entire lengths to Fig. 5, with manual

shown has been generated

duced by CLUSTALW

of Pu u&S and Pk

analysis

with Val-tRS

sequence

organisms.

(CLUSTALW),

scores pro-

This represents

the sim-

ilarity of the sequences at the aa level, displayed in graphical Branch lengths are proportional to the similarity scores obtained the multi-alignment. B: Two programs Neighbour version

Joining

The scale bar represents were then applied: (NEIGHBOR),

3.5~. Consensus

majority-rule

criteria

a similarity

Parsimony

trees

were

and

package,

(CONSENSE),

subsequently

form. from

score of 0.10.

(PROTPARS)

both from the PHYLIP

trees were constructed and

as

A: The

adjustments.

from the similarity

comparisons.

The

plotted

with with

TREETOOL. Only Parsimony routinely gave a robust tree, Neighbour Joining possibly being influenced to a greater degree by the divergent aa sequences

(Felsenstein,

100 bootstrapped number

1988). The consensus

samples

of times the group

right of each fork, occurred tree is shown, the outgroup

consisting among

when the E. coli Leu-tRS

from the

of the species which are to the

the trees (out of 100 trees). A rooted

sequence

tRS (a class-11 type tRS (Eriani obtained

tree was obtained

and at each node, the value indicates

illustrated

being the E. coli Pro-

et al., 1990). An identical (class-1 tRS) (GenBank

tree was accession

No. X06331) was employed.

tity between the Pv valS gene and the B. subtilis Val-tRSencoding gene, despite the fact that these sequences show the highest degree of homology according to the BLASTx GenBank database search. A similar figure is obtained

regions deemed essential for enzyme function are conserved, we would predict that the proteins encoded by the two genes described in this study are indeed Val-tRS. However, the plasmodial sequences appear to have significantly diverged from other Val-tRS at the aa sequence level (Fig. 6A); whilst, in phylogenetic terms they still group with them (Fig. 6B). (2) Immuno-localisation with the Al2 mAb has previously shown that, as expected for a molecule involved in transmission-blocking immunity, the GAMl antigen appears to be found on the gamete surface, reactivity also being observed with asexual parasites within the trophozoite parasitophorous vacuole (Udegama et al., 1987). These data indicate that Pv valS may have been cloned by fortuitous cross-reactivity of a sequence encoded by the fusion protein expressed from clone 47/3 and the transmission-blocking mAb A12. Such cross-reactive epitopes have previously been observed in Plasmodium (reviewed in Mattei and Scherf, 1991). (3) Our attempts to resolve this dilemma have included expression of Pv valS gene fragments in the pGEX bacterial expression system. The resulting fusion proteins, while immunogenic in mice, led to the production of Ab which failed to recognise the parasite on immunofluorescence or Western blot. tRS commonly manifest a lack of reactivity when analysed in this way with Ab raised against them. This is possibly due to low levels of expression, or instability and rapid degradation of protein or message (reviewed in Schimmel and Siill, 1979). In contrast, a circulating protective antigen of Brugia malayi has been predicted from sequence data to be a tRS enzyme (Eriani et al., 1990; Kazura et al., 1990). Moreover, numerous other examples exist of tRS capable

144

of inducing an immune response (see Bunn et al., 1986; Mathews et al., 1984; Mitra et al., 1994; Targoff, 1990; Targoff et al., 1993). (4) The absence of an in vitro culture system for Pv leaves remote the possibility of directly obtaining the sequence of the Pv polypeptides recognised by the Al2 mAb. The possibility therefore cannot be ruled out that a cross-reacting epitope, efficiently recognised by Al2 and capable of inducing transmission-blocking Ab, possibly acting as a ‘mimotope’, may have led to of a gene unrelated to the true transmission-blocking candidate molecule GAMl.

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Vaccines. Marcel Dekker, New York, 1990, pp. 531-544.

Del Portillo,

We wish to thank Profs. Michel Rabinovitch and Luiz Pereira da Silva for support. We acknowledge Dr. Shirley Longacre for early contributions to this study. Dr. JeanClaude Michel and his laboratory at the Pasteur Institute, French Guyana, are thanked for providing access to Pv parasites, as are Diana Hudson-Taylor and Dr. Louis Miller, NIH, for the Pk library. Drs. Serge Bonnefoy, Sam Gobin, Diana Hudson-Taylor, Odile Mercereau-Puijalon, Mario Sefiorale, Brian Sheehan and Alan Thomas are also thanked for kind gifts of genomic DNA. This work was supported by the United Nations Development Programme/World Bank/World Health Organization Special Programme for Research and Training in Tropical Disease (TDR), by the Rockefeller Foundation, the EEC programme for Life Science and Technologies in Developing Countries (STDS), the CNRS and the Pasteur Institute. V.A.S. completed this study while in receipt of a fellowship from the ‘Foundation pour la Recherche Medicale’.

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