Molecular and Biochemical Parasitology, 56 (1992) 169 176
169
© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01834
Analysis of the genes encoding immunodominant piroplasm surface proteins of Theileria sergenti and Theileria buffeli by nucleotide sequencing and polymerase chain reaction Shin-ichiro K a w a z u a, C h i h i r o S u g i m o t o b, T s u g i h i k o K a m i o a a n d K o z o Fujisaki a aNational Institute of Animal Health, Tsukuba, Ibaraki, Japan; and bFaculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan (Received 20 May 1992; accepted 28 July 1992)
The nucleotide sequences of the cDNAs encoding a 33-kDa piroplasm protein of Theileria sergenti (p33) and a similar protein of Theileria buffeli (p34) were determined. Both of the genes contained an open reading frame of 849 base pairs. The predicted amino acid sequence of p33 and p34, consisting of 283 residues, showed 82% similarity. A transmembrane hydrophobic domain and signal peptides were predicted. The polymerase chain reaction was used to amplify p33/34 genes from the piroplasm D N A of T. sergenti, T. buffeli and Theileria orientalis. Following amplification, p33 and p34 genes were clearly differentiated using the restriction enzymes sites that were not shared between them. These results indicated that p33 and p34 were conserved molecules among these Theileria species, and the genes that encode p33/34 proteins were suitable for discrimination of T. sergenti from T. buffeli/T, orientalis. Key words: Nucleotide sequence; Polymerase chain reaction; Piroplasm; Theileria buffeli; Theileria orientalis; Theileria
sergenti
Introduction
Theileria sergenti is a protozoan parasite that frequently causes anaemia as intraerythrocytic piroplasms in cattle in Japan. Several other theilerial species taxonomically related to T. sergenti are distributed in countries from the temperate zone to the subtropics [1]. Based on biochemical and serological examinations and transmission experiments using various species of tick vectors, we proposed that the benign Correspondence address: Shin-ichiro Kawazu, National Institute of Animal Health, Tsukuba, Ibaraki, 305 Japan. Fax: 0298-38-7880. Note." Nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL and GenBank T M data bases with the accession numbers Dl1046 (T. sergenti) and D11047 (T. buffeli). Abbreviations: PCR, polymerase chain reaction.
Theileria species from Japan, Australia and Britain, frequently referred to as the T. sergenti, T. buffeli and T. orientalis group of parasites, should be classified into two groups, i.e., T. sergenti and T. buffeli/T, orientalis [2-4]. Among piroplasm proteins of this parasite group, the major 33-kDa protein of T. sergenti (p33) and similar proteins of 34 kDa of T. buffeli (p34) and T. orientalis have been reported to be potential molecular markers to distinguish these Theileria species on twodimensional polyacrylamide gel electrophoresis (2D-PAGE) [2]. These proteins are exposed on the piroplasm surface and are the most immunodominant proteins recognized by infected cattle [3]. Antigenic comparisons among piroplasm lysates by enzyme-linked immunosorbent assay revealed that the serological dissimilarity between T. sergenti and T. buffeli/T, orientalis could be attributed to antigenic difference between p33 and p34 [3].
170
We have previously cloned genes of p33 and p34 from c D N A libraries constructed from T. sergenti and T. buffeli mRNA. Immunological analyses of recombinant p33 and p34 of T. sergenti and T. buffeli, suggested that these proteins contained both species-common and species-specific epitopes that were recognized by infected cattle [5]. In this study, we sequenced the cDNAs encoding p33 and p34, and compared their predicted amino acid sequences. In addition, p33/34 genes were amplified from the parasite genomes by PCR [6] and compared in regard to their internal restriction enzyme sites in order to determine whether they are suitable for the differentiation of T. sergenti from T. bu[feli/T, orientalis at the genetic level.
TBW 17 which contains a c D N A insert of 1000 bp was digested with EcoRI, and the resultant insert fragment was subcloned into the same plasmid vector.
Nucleotide sequence determination.
Sequence analysis was performed by the Sanger dideoxy chain termination method [12] using recombinant plasmid D N A as a template [13]. Synthetic oligonucleotides were used to prime subsequent sequencing reactions and the entire sequences were obtained on both strands. D N A sequences were analysed by genetic information processing programme (GENETYX; Software Development Co., Ltd., Japan) and deduced amino acid sequences were compared to the SWISSPROT protein sequence database (European Molecular Biology Laboratory).
Materials and Methods
Parasite stocks and preparation of piroplasm DNAs. T. sergenti (Ikeda stock) [7], T. buffeli (Warwick stock) [8] and T. orientalis (Essex stock) [9] were used in this study. Blood was collected from calves experimentally infected with each of the parasite stocks, and piroplasms were purified by the method described previously [10], using the Aeromonas hydrophila hemolysin. Genomic D N A of the parasites was prepared from purified piroplasms by treatment with SDS-proteinase K solution, and phenol extraction [11].
Subchming of cDNA inserts from phages expressing p33 and p34. Two phage clones, namely TSI 10, which expresses p33 of T.
sergenti, and TBW 17 which expresses p34 of T. buffeli were used. These clones were immunologically
selected
from
either
T.
sergenti or T. buffeli c D N A libraries constructed in 2gtll [5]. Phage D N A from the clone TSI 10 which contains a c D N A insert of 1030 bp was double digested with KpnI and SacI and fractionated by agarose gel electrophoresis. The resultant two insert-containing fragments, a 1.6-kb KpnI/SacI fragment and a 1.5kb Kpnl fragment were subcloned into the plasmid pUC 19. Phage D N A from the clone
Polymerase chain reaction amplijication. PCR amplification was performed in a 100 /tl reaction mixture containing 10 mM Tris-HC1, pH 8.3/50 m M KC1/1.5 mM MgCI2/0.001% gelatin/200 #M each of the four dNTPs/0.5 #M each of the oligonucleotide primers/2.5 units of Taq (Thermus aquaticus) DNA polymerase (Perkin Elmer Cetus, CT, U.S.A.). The primers used were: 5'-TATGTTGTCCAAGAGATCGT-Y; and 5 ' - T G A G A C T C A G T G C G CCTAGA-3' (Fig. l a). The template DNA added to the reactions was either 250 to 500 ng of piroplasm genomic D N A or 10 ng of recombinant phage DNA. The reaction mixture was overlaid with paraffin oil and subjected to 25 cycles of amplification in a programmable heating block (Perkin Elmer Cetus). The programme used was as follows; 94°C, 1 min; 58°C, 2 min and 72°C, 3 min. After amplification, I0 /~1 of each sample was subjected to agarose gel electrophoresis with or without restriction enzyme digestion. Results
Comparisons of p33 and p34 cDNAs. The nucleotide sequences of p33 and p34 cDNAs are shown in Fig. l a. A stop codon map
171
a) Ora TSI
10
I
CAGTT~TATAA~TTAAGTTTAATATTTAAATTATATT~TAAAC~ACACTT~TATACTTTAACTAGATAATTTGCTK~-~TGTCCAAGA~AACGTTCAACGTACTTTGCCTAG~ATACTTC 45
TSW 17
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& T G
. . . . . . . . . . . .
T ........
T ....................
I~ TSI
10
TBW 17
Dra
I
CTTATcGTCTCTGcTACCGCCGCAGAGG~AAAAAAAGATGCAAAGGCTGAAGAGAAGAAGGACTTAACTCTCGAAGTTAACGCCACCGCAGCCGAA~ATTTTAAAGTCGACGCCTCAAA~165 • .C...TG ......
A.
A.AG...A .....
. . . .
G..CCA . . . . . . . . . . . . . . . . . . . .
A..T...G
. . . . G. . . . . . . . . . . . . . .
CAG. G T . . . A . . . . . .
C.... A.T..AA.C..T
EcoR V TSI
10
TBW 17
•CCAA••ACGTCGTTTTTACTGCCGAAGA•GGATACCGCATCAAGACA•TCAAGGTC••AGATAAGAACCTGTATAC•GTA•ATACTTCCAAGTTCACC•CAACTGTCGCCCA•AGACTG ........................
~7524 TSI
10
TBW 17
TCG. ,T . . . . .
I
Hind
T, ,TT . . . . . . .
T..T .....
T ........
A.CTT . . . . . . . . . .
T .....
A .....
A .....
T .....
111
Hal
C..T ......... I
AAGCATG~TGACGACCT~TTCTTCAAGCTCAACCTGTCCCACG~AAAGCCATTGCT~TTCAA~AAGAAGACT~ACAAGGATTGGGTTCAATTCAG~TTCGCCCAGTACCTCGATGAA~TT 405 .......
G...T.C.T .............
TG . . . . T . . . . .
T..C ......
C.CT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G..T.A...T.G
.........
I TSI
10
285 A.T
T..C...T.. Ava
II
GTATGGAAGGAGAAGAAGGAAGTAAAAGACCTCGACGCATCCAAGTTCGCAGACGCAGGTCTTTTCGCCGCTGAGGCTTTCGGT•CCGGAAAGCTGTACAAcTTCATTGGAAACTTCAAG 525
TBW ~7
...........
TSI
GTCAAGAAGGTCATGTTC•AGGAGAAGGACGTTGGAGATTCAAACAAGGCCAAATACACCGCTGTCAAAGTT•ACGTCGGTTCCGATGAGAAGAAAGTC•TAAGACTCGACTACTTCTAC 645
10
TBW 17
• .T . . . .
A .........
GT. T . A . . . T
C.C..G..TA.A..T
.....
...........
T..A.TG .....
T .....
T..CC.T..A..T
G. . . . . . . . . . .
...........
T..A ........ H i nd
TSI
10
T..A .....
TA.A ........
A .....
T..T...G.T..TG
T...A .......
T ........
. . . . TG. C . . . C C . . . . . . .
A ........
T ............
Ill
ACT~GT~AT~A~AGATTCAA~GAGGTTTACTTCAAATTGGTAGA~GGAAAATGGAA~AAG~TT~A~CA~A~C~A~GCAAACAAGGATTTGCACGCCAT~AACA~T~CTTGG~CTTCGGAC
T ...........
A...C
..................................
T
765
TBW 17
• .A.C ........................................
TSf
TACAA~CCTCTTGTCGACAAGTTCTCACCACTTGCCGTTCTCAGC~CGGTTCTCATCGCCTCCCTCGCAGTATT~TATTATCTCT`~`mGCGCACTGAGTCTCACATATTATCGACGTTTAA 885
.......
A . . . . . . . . . . .
T
. . . .
Dra 10 TSW 17 TSI
10
TBW 17
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TT.C . . . . . . . . . . . .
.
T.T ..........
TC . . . . . .
TAG . . . . . . . . . . . . . . .
I
A.A ...............
AATAACAATTGATAATTTGTACAAGACTGAAAAAAAAAAAAAAAAAAAAAAA
937
.............................
939
T.
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AAA
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b) TSI-IO cDNA
D _j_
D
N
I
I
K
D
I
1_. polyA
ORF TBW47 cDNA
EV
H
BI
A
I
I
I
I
P K H
D
It"
J-.
I
,200 bp
Fig. 1. (a) The nucleotide sequences of p33 and p34 cDNAs. The TSI l0 (p33 cDNA) and TBW 17 (p34 cDNA) nucleotide sequences are aligned. For the TBW 17 sequence only those nucleotides that differ from TSI 10 are shown. Dots indicate identical nucleotides. The initiation (ATG) and termination (TAG) codons are underlined and are in bold. The nucleotides indicated by arrows at the 5' and 3' end of the genes denote the sequence of the primers used in the PCR. The restriction enzymes sites shown in the restriction maps (b) are underlined and indicated here above the sequences. (b) Restriction maps of p33 and p34 cDNAs. Restriction enzymes sites are designated as follows: DraI, D; Nsp7524I, N; Kpnl, K; EcoRV, EV; HindIII, H; Ball, Bl; Avail, A; Pst|, P. The open reading frame (ORF) and poly(A) tail are denoted by thick lines in the maps.
analysis revealed one open reading frame of sufficient size to encode a protein of approximately 30 kDa within each of the sequences. The open reading frames of the p33 and p34 genes each consist of 849 bp with 84% identity. Unique restriction enzyme sites observed within either of the sequences are shown in the restriction maps (Fig. lb). Southern blot analyses were consistent with the genes being present in one copy per genome (data not shown). The predicted proteins consist of 283 amino acid residues with 82% identity and 92% homology. The maximum homology was calculated by the DNASIS programme (Hi-
tachi Software Engineering Co., Ltd., Japan). The polypeptides of predicted size 32 kDa each contain a putative signal peptide at the Nterminus and a hydrophobic region at the Cterminus which may be an anchor sequence (Fig. 2). Potential cleavage sites for the Nterminal signal peptides were predicted to be after Ala 23 of p33 and after Ala 22 of p34, respectively (Fig. 2) by using a computer programme, SIGSEQ 2 [14], based on a statistic weight-matrix method [15]. An obvious difference observed between p33 and p34 was in the numbers and positions of predicted Asn-linked glycosylation sites, 2 in p33 and 3
172
TSI
MLSKRTF NVLCLGYFL IVSATAAEEKKDAKAEEKKDLTL EVNAIAAEHFKVDASNANDVV
I 0
TBW 1 7
.....
TSI
FTAEEGYRIKILKVGDKNLYTVDTSKFIPTVAHRLKHADDLFFKL~LSHAKPLLFKKKTD
10
S..L ........
C ....
........
E . K .--EP .........
~,
.QG.N * .T
...SD...F
TSI
10
KDWVQFSFAQYLDEVVWKEKKEVKDLDASKFADAGLFAAEAFGTGKLYNFIGNFKVKKVM
TB~
17
......
TSI
I0
FEEKDVGDSNKAKYTAVKVYVGSDEKKVVRLDYFYTGDERFKEVYFKLVDGKWKKVEQSE
F..::_._~=L...I
......
E ......
,.9,V.,.PKN
TSI
ANKDLHAMNSAWPSDYKPLVDKFSPLAVLSAVLIASLAVFYYL
TBW IT
.
.
.
.
.
.
.
.
.
N...L
~.D ...........
.................
A ....
F ....
120
D ..............
DT . . . . .
TBW ~7
I0
...........
1..G.A .....
*N T
TBW 17
N.G . . . . .
f ................
A.
60
V.D.V.P ....
=~. . . . . . . . . . . .
180
S.K 240
L ....
283
F...F..
Fig. 2. The predicted amino acid sequences of p33 and p34. The predicted amino acid sequence of T. sergenti p33 (TS110) and that of T. bu(feh p34 (TBWI7) are aligned in the single letter code. For the p34 sequence only those amino acids that differ from p33 are shown. Dots indicate identical amino acids. Features within the sequences are denoted by the following: the single lines at the N-terminal mark the putative signal sequences; the double lines at the C-terminal mark the hydrophobic stretches; the asterisks mark the sites of potential glycosylation; the broken lines mark putative erythrocyte binding motifs, Lys-Glu-Lys (KEK) and Lys-Glu-Leu (KEL), or Lys-Glu (KE) ion pair [181.
a)
b) gO
cO
~
~n
0
~rnO
~Orn 0
~0~00
~Orn 0
1 1
Fig. 3. (a) The p33 and p34 genes amplified from genomic DNA and recombinant phage DNA by polymerase chain reaction. Piroplasm D N A from T. sergenti (TS), T. buffeli (TB) and T. orientalis (TO), and recombinant phage DNA from TSI 10 and TBW17 were used as templates for amplification. The amplification reaction mixtures were applied to a 1.5% agarose gel and stained with ethidium bromide after electrophoretic separation. (b) Differentiation of the PCR-amplified p33 and p34 genes by restriction enzyme digestion. The amplification reaction mixtures obtained from piroplasm DNAs were then digested with the restriction enzymes, electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. The restriction enzymes used were Dral (D), Nsp75241 (N), HindIll (H) and Aval! (A). Sizes are given in base pairs (bp) on the left.
173
in p34. No significant peptide repeats, as generally found in the surface proteins of Plasmodium falciparum [16], were recognized within either sequence. The predicted amino acid sequences did not have significant homology with other known protein sequences stored in the SWISSPROT database.
Polymerase chain reaction amplification of p33/ 34 genes. Two PCR primers of 20-residue oligonucleotides were synthesized to amplify the p33/34 gene as indicated in Fig. la. When phage DNA containing cDNA of p33 or p34 was used, a single fragment of approximately 870 bp was amplified, in good accordance with the predicted size, 868 bp (Fig. 3a). These primers also amplified a single fragment of 870 bp from genomic DNA template of T. sergenti, T. buffeli or T. orientalis. No detectable amplifications from bovine and phage DNA took place under the same conditions (data not shown). Differentiation of the PCR-amplified p33/34 genes by restriction enzyme digestion. PCRamplified p33/34 genes from parasite genomes were digested with restriction enzymes, and their cleavage patterns were compared by agarose gel electrophoresis. The restriction enzymes which cut the p33 and p34 genes at the unique restriction sites shown in Fig. l b were used for this comparison. The results with PCR-amplified p33/34 genes from T. sergenti, 7". buffeli and T. orientalis genomes digested with four different restriction enzymes are shown in Fig. 3b. The cleavage patterns of PCR-amplified p33/34 genes from T. sergenti and T. buffeli with these four enzymes were consistent with their restriction maps. The restriction patterns of PCR-amplified genes from T. buffeli and T. orientalis were identical for any of the enzymes tested, and clearly distinguished from that of the PCR-amplified gene from T. sergenti. The other four restriction enzyme sites (KpnI, EcoRV, Bali and PstI) indicated in the restriction map of the p34 gene (Fig. lb) were also conserved in the PCRamplified gene from T. orientalis (data not shown).
Discussion
The nucleotide sequences of cDNAs which encode p33 of T. sergenti and p34 of T. buffeli were analyzed. It has previously been reported that p33 of T. sergenti and p34 of T. buffeli and T. orientalis were the most abundant proteins in piroplasms [2] and the most immunodominant proteins in infected cattle [3]. These proteins are localized on the surface of piroplasms as determined by immunoelectron microscopy [17] and surface protein labeling with biotin [3]. The nucleotide sequence of p33 and p34 cDNAs showed high similarity in this study. All these results indicate that these proteins are equivalent molecules in these Theileria species. Southern blot analysis indicated that the p33 or p34 gene was present as a single copy in the respective parasite genome. PCR amplification of the p33/34 gene from both parasite genomic DNA and cloned cDNA indicated that the coding sequence of each gene is on a single exon. The different excesses of the observed molecular weights of p33 and p34 over the predicted 30 kDa for the mature polypeptides may be accounted for by the different numbers of potential glycosylation sites. In Western blot analysis, authentic p33 and p34 showed species-specific reactivities against sera from cattle infected with either of T. sergenti, T. buffeli or T. orientalis [3], but the reactivities of recombinant p33 and p34 against those sera were equal [5]. Post-translational modifications, possibly glycosylation, may generate species-specific epitopes that are predominantly recognized by infected cattle. The possibility that there are post-translational modifications of p33/34 should be examined using eukaryotic expression systems. The functions of p33 and p34 have not been clarified. Interestingly, the predicted amino acid sequences of p33 and p34 contained LysGlu-Lys (KEK) and Lys-Glu-Leu (KEL) motifs which were identified in a surface protein of blood-stage parasites of P. falciparum as erythrocyte-binding peptide sequences [18]. These motifs are considered to be an important feature of certain malaria
174
proteins which interact with erythrocytes, including the putative ligand for red cell receptor, EBA-175 [19] and the precursor of the major merozoite surface antigen (PMMSA) [16]. Binding and invasion of T. sergenti piroplasms to bovine erythrocytes via parasites surface structures has been demonstrated in vitro [20]. The piroplasm surface proteins p33/34 might play important roles in interaction with host cells. The KE sequence within a synthetic polypeptide used as malarial vaccine (SPf 66) is reported to be the key motif against which protective antibodies are directed. The repetition of this motif in p33 and p34 might produce cross-reacting antibodies in infected cattle. The restriction enzyme site comparison of the PCR-amplified fragments clearly discriminated T. sergenti from T. buffeli and T. orientalis. This procedure therefore provides a positive/negative marker system based on the differences of the genes coding the major piroplasm proteins between T. sergenti, T. buffeli and T. orientalis. This marker system will form a useful supplement to the other methods currently available for stock characterization such as restriction fragment length polymorphism analysis of genomic DNA using genomic D N A probes [21]. Whether this system could be applied for characterization and differentiation of other stocks of the T. sergenti/T, buffeli/T, orientalis group parasites is being examined. The results obtained in this study are in accordance with our previous conclusions that T. sergenti should be separated from T. buffeli and T. orientalis on the basis of tick-transmissibility and other phenotypic characteristics. These observations also suggest that the latter two parasites, T. buffeli and T. orientalis are the closely related species. It is possible that T. buffeli was introduced into Australia by Theileria-infected cattle imported from Britain in the early days of European settlement [22], and has adapted to the indigenous ticks Haemaphysalis bancrofti and Haemaphysalis humerosa, principally parasites of marsupials [8]. Australian T. buffeli and British 7". orientalis might belong to one and the same
species and in that case the latter name might have priority over T. buffeli which was introduced as the name of the parasite in Asian water buffalo [1].
Acknowledgements We would like to thank Drs. Y. Tsuchiya, S. Yamada, and T. Sekizaki for useful comments during the preparation of this paper.
References 1 Uilenberg, G. (1981) Theilerial species of domestic livestock. In: Advances in the Control of Theileriosis (Irvin, A.D., Cunningham, M.P. and Young, A.S., eds.), pp. 4-37. Martinus Nijhoff, The Hague. 2 Sugimoto, C., Kawazu, S., Kamio, T. and Fujisaki, K. (1991) Protein analysis of Theileria sergenti/buffeli/ orientalis piroplasms by two-dimensional gel electrophoresis. Parasitology 102, 341-346. 3 Kawazu, S., Sugimoto, C., Kamio, T. and Fujisaki, K. (1992) Antigenic differences between Japanese Theileria sergenti and other benign Theileria species of cattle from Australia (T. buffeli) and Britain (T. orientalis). Parasitol. Res. 78, 13(~135. 4 Fujisaki, K., Kamio, T. and Kawazu, S. (1991) Theileria sergenti cannot be regarded as the same species as "1". buffeli and T. orientalis because of its transmissibility only by Kaiseriana ticks. In: Modern Acarology, Vol. 1, (Dusb~.bek, F. and Bukva, V., eds.), pp. 233-237. Academia, Prague. 5 Kawazu, S., Sugimoto, C., Kamio, T. and Fujisaki, K. (1992) Molecular cloning and immunological analysis of immunodominant piroplasm surface proteins of The# leria sergenti and T. buffeli. J. Vet. Med. Sci. 54, 305311. 6 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable D N A polymerase. Science 239, 487~491. 7 Fujisaki, K., Ito, Y., Kamio, T. and Kitaoka, S. (1985) The presence of Theileria sergenti in Haemaphysalis longicornis overwintering in pasture in Japan. Ann. Trop. Med. Parasitol. 79, 519-524. 8 Stewart, N.P., DeVos, A.J., Shiels, I. and McGregor, W. (1987) The experimental transmission of Theileria buffeli of cattle in Australia by Haernaphysalis humerosa. Aust. Vet. J. 64, 81-83. 9 Morzaria, S.P., Barnett, S.F. and Brocklesby, D.W. (1974) Isolation of Theileria mutans from cattle in Essex. Vet. Rec. 94, 256. 10 Sugimoto, C., Sato, M., Kawazu, S., Kamio, T. and Fujisaki, K. (1991) Purification of merozoites of Theileria sergenti from infected bovine erythrocytes. Parasitol. Res. 77, 129 131. 11 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. 2nd edn.
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12 13 14 15 16 17
18
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Hattori, M. and Sakaki, Y. (1986) Dideoxy sequencing method using denatured plasmid templates. Anal. Biochem. 152, 232-238. Folz, R.J. and Gordon, J.I. (1987) Computer-assisted predictions of signal peptidase processing sites. Biochem. Biophys. Res. Commun. 146, 870-877. von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683-4690. Kemp, D.J. and Cowman, A.F. (1990) Genetic diversity in Plasrnodiumfalciparum. Adv. Parasitol. 29, 75-149. Shirakata, S., Onuma, M., Kirisawa, R., Takahashi, K. and Kawakami, Y. (1989) Localization of surface antigens on Theileria sergenti merozoite by monoclonal antibodies. Jpn. J. Vet. Sci. 51, 831 833. Molano, A., Segura, C., Guzman, F., Lozada, D. and Patarroyo, M.E. (1992) In human malaria protective
antibodies are directed mainly against the Lys-Glu ion pair within the Lys-Glu-Lys motif of the synthetic vaccine SPf 66. Parasite Immunol. 14, 111 124. 19 Sim, B.K.L., Orlandi, P.A., Haynes, J.D., Klotz, F.W., Carter, J.M., Camus, D., Zegans, M.E. and Chulay, J.D. (1990) Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion. J. Cell Biol. 111, 1877 1884. 20 Kawamoto, S., Takahashi, K., Onuma, M., Kurosawa, T. and Sonoda, M. (1990) Invasion of bovine erythrocytes by Theileria sergenti piroplasms in vitro. Jpn. J. Vet. Sci. 52, 1261-1263. 21 Matsuba, T., Kawakami, Y., lwai, H. and Onuma, M. (1992) Genomic analysis of Theileria sergenti stocks in Japan with DNA probes. Vet. Parasitol. 41, 35-43. 22 Payne, W.J.A. (1970) The origin of tropical and subtropical breeds. In: Cattle Production in the Tropics, Vol. 1, (Payne, W.J.A., ed.), pp. 31-61. Longman, London.
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© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01834
Analysis of the genes encoding immunodominant piroplasm surface proteins of Theileria sergenti and Theileria buffeli by nucleotide sequencing and polymerase chain reaction Shin-ichiro K a w a z u a, C h i h i r o S u g i m o t o b, T s u g i h i k o K a m i o a a n d K o z o Fujisaki a aNational Institute of Animal Health, Tsukuba, Ibaraki, Japan; and bFaculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan (Received 20 May 1992; accepted 28 July 1992)
The nucleotide sequences of the cDNAs encoding a 33-kDa piroplasm protein of Theileria sergenti (p33) and a similar protein of Theileria buffeli (p34) were determined. Both of the genes contained an open reading frame of 849 base pairs. The predicted amino acid sequence of p33 and p34, consisting of 283 residues, showed 82% similarity. A transmembrane hydrophobic domain and signal peptides were predicted. The polymerase chain reaction was used to amplify p33/34 genes from the piroplasm D N A of T. sergenti, T. buffeli and Theileria orientalis. Following amplification, p33 and p34 genes were clearly differentiated using the restriction enzymes sites that were not shared between them. These results indicated that p33 and p34 were conserved molecules among these Theileria species, and the genes that encode p33/34 proteins were suitable for discrimination of T. sergenti from T. buffeli/T, orientalis. Key words: Nucleotide sequence; Polymerase chain reaction; Piroplasm; Theileria buffeli; Theileria orientalis; Theileria
sergenti
Introduction
Theileria sergenti is a protozoan parasite that frequently causes anaemia as intraerythrocytic piroplasms in cattle in Japan. Several other theilerial species taxonomically related to T. sergenti are distributed in countries from the temperate zone to the subtropics [1]. Based on biochemical and serological examinations and transmission experiments using various species of tick vectors, we proposed that the benign Correspondence address: Shin-ichiro Kawazu, National Institute of Animal Health, Tsukuba, Ibaraki, 305 Japan. Fax: 0298-38-7880. Note." Nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL and GenBank T M data bases with the accession numbers Dl1046 (T. sergenti) and D11047 (T. buffeli). Abbreviations: PCR, polymerase chain reaction.
Theileria species from Japan, Australia and Britain, frequently referred to as the T. sergenti, T. buffeli and T. orientalis group of parasites, should be classified into two groups, i.e., T. sergenti and T. buffeli/T, orientalis [2-4]. Among piroplasm proteins of this parasite group, the major 33-kDa protein of T. sergenti (p33) and similar proteins of 34 kDa of T. buffeli (p34) and T. orientalis have been reported to be potential molecular markers to distinguish these Theileria species on twodimensional polyacrylamide gel electrophoresis (2D-PAGE) [2]. These proteins are exposed on the piroplasm surface and are the most immunodominant proteins recognized by infected cattle [3]. Antigenic comparisons among piroplasm lysates by enzyme-linked immunosorbent assay revealed that the serological dissimilarity between T. sergenti and T. buffeli/T, orientalis could be attributed to antigenic difference between p33 and p34 [3].
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We have previously cloned genes of p33 and p34 from c D N A libraries constructed from T. sergenti and T. buffeli mRNA. Immunological analyses of recombinant p33 and p34 of T. sergenti and T. buffeli, suggested that these proteins contained both species-common and species-specific epitopes that were recognized by infected cattle [5]. In this study, we sequenced the cDNAs encoding p33 and p34, and compared their predicted amino acid sequences. In addition, p33/34 genes were amplified from the parasite genomes by PCR [6] and compared in regard to their internal restriction enzyme sites in order to determine whether they are suitable for the differentiation of T. sergenti from T. bu[feli/T, orientalis at the genetic level.
TBW 17 which contains a c D N A insert of 1000 bp was digested with EcoRI, and the resultant insert fragment was subcloned into the same plasmid vector.
Nucleotide sequence determination.
Sequence analysis was performed by the Sanger dideoxy chain termination method [12] using recombinant plasmid D N A as a template [13]. Synthetic oligonucleotides were used to prime subsequent sequencing reactions and the entire sequences were obtained on both strands. D N A sequences were analysed by genetic information processing programme (GENETYX; Software Development Co., Ltd., Japan) and deduced amino acid sequences were compared to the SWISSPROT protein sequence database (European Molecular Biology Laboratory).
Materials and Methods
Parasite stocks and preparation of piroplasm DNAs. T. sergenti (Ikeda stock) [7], T. buffeli (Warwick stock) [8] and T. orientalis (Essex stock) [9] were used in this study. Blood was collected from calves experimentally infected with each of the parasite stocks, and piroplasms were purified by the method described previously [10], using the Aeromonas hydrophila hemolysin. Genomic D N A of the parasites was prepared from purified piroplasms by treatment with SDS-proteinase K solution, and phenol extraction [11].
Subchming of cDNA inserts from phages expressing p33 and p34. Two phage clones, namely TSI 10, which expresses p33 of T.
sergenti, and TBW 17 which expresses p34 of T. buffeli were used. These clones were immunologically
selected
from
either
T.
sergenti or T. buffeli c D N A libraries constructed in 2gtll [5]. Phage D N A from the clone TSI 10 which contains a c D N A insert of 1030 bp was double digested with KpnI and SacI and fractionated by agarose gel electrophoresis. The resultant two insert-containing fragments, a 1.6-kb KpnI/SacI fragment and a 1.5kb Kpnl fragment were subcloned into the plasmid pUC 19. Phage D N A from the clone
Polymerase chain reaction amplijication. PCR amplification was performed in a 100 /tl reaction mixture containing 10 mM Tris-HC1, pH 8.3/50 m M KC1/1.5 mM MgCI2/0.001% gelatin/200 #M each of the four dNTPs/0.5 #M each of the oligonucleotide primers/2.5 units of Taq (Thermus aquaticus) DNA polymerase (Perkin Elmer Cetus, CT, U.S.A.). The primers used were: 5'-TATGTTGTCCAAGAGATCGT-Y; and 5 ' - T G A G A C T C A G T G C G CCTAGA-3' (Fig. l a). The template DNA added to the reactions was either 250 to 500 ng of piroplasm genomic D N A or 10 ng of recombinant phage DNA. The reaction mixture was overlaid with paraffin oil and subjected to 25 cycles of amplification in a programmable heating block (Perkin Elmer Cetus). The programme used was as follows; 94°C, 1 min; 58°C, 2 min and 72°C, 3 min. After amplification, I0 /~1 of each sample was subjected to agarose gel electrophoresis with or without restriction enzyme digestion. Results
Comparisons of p33 and p34 cDNAs. The nucleotide sequences of p33 and p34 cDNAs are shown in Fig. l a. A stop codon map
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a) Ora TSI
10
I
CAGTT~TATAA~TTAAGTTTAATATTTAAATTATATT~TAAAC~ACACTT~TATACTTTAACTAGATAATTTGCTK~-~TGTCCAAGA~AACGTTCAACGTACTTTGCCTAG~ATACTTC 45
TSW 17
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& T G
. . . . . . . . . . . .
T ........
T ....................
I~ TSI
10
TBW 17
Dra
I
CTTATcGTCTCTGcTACCGCCGCAGAGG~AAAAAAAGATGCAAAGGCTGAAGAGAAGAAGGACTTAACTCTCGAAGTTAACGCCACCGCAGCCGAA~ATTTTAAAGTCGACGCCTCAAA~165 • .C...TG ......
A.
A.AG...A .....
. . . .
G..CCA . . . . . . . . . . . . . . . . . . . .
A..T...G
. . . . G. . . . . . . . . . . . . . .
CAG. G T . . . A . . . . . .
C.... A.T..AA.C..T
EcoR V TSI
10
TBW 17
•CCAA••ACGTCGTTTTTACTGCCGAAGA•GGATACCGCATCAAGACA•TCAAGGTC••AGATAAGAACCTGTATAC•GTA•ATACTTCCAAGTTCACC•CAACTGTCGCCCA•AGACTG ........................
~7524 TSI
10
TBW 17
TCG. ,T . . . . .
I
Hind
T, ,TT . . . . . . .
T..T .....
T ........
A.CTT . . . . . . . . . .
T .....
A .....
A .....
T .....
111
Hal
C..T ......... I
AAGCATG~TGACGACCT~TTCTTCAAGCTCAACCTGTCCCACG~AAAGCCATTGCT~TTCAA~AAGAAGACT~ACAAGGATTGGGTTCAATTCAG~TTCGCCCAGTACCTCGATGAA~TT 405 .......
G...T.C.T .............
TG . . . . T . . . . .
T..C ......
C.CT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G..T.A...T.G
.........
I TSI
10
285 A.T
T..C...T.. Ava
II
GTATGGAAGGAGAAGAAGGAAGTAAAAGACCTCGACGCATCCAAGTTCGCAGACGCAGGTCTTTTCGCCGCTGAGGCTTTCGGT•CCGGAAAGCTGTACAAcTTCATTGGAAACTTCAAG 525
TBW ~7
...........
TSI
GTCAAGAAGGTCATGTTC•AGGAGAAGGACGTTGGAGATTCAAACAAGGCCAAATACACCGCTGTCAAAGTT•ACGTCGGTTCCGATGAGAAGAAAGTC•TAAGACTCGACTACTTCTAC 645
10
TBW 17
• .T . . . .
A .........
GT. T . A . . . T
C.C..G..TA.A..T
.....
...........
T..A.TG .....
T .....
T..CC.T..A..T
G. . . . . . . . . . .
...........
T..A ........ H i nd
TSI
10
T..A .....
TA.A ........
A .....
T..T...G.T..TG
T...A .......
T ........
. . . . TG. C . . . C C . . . . . . .
A ........
T ............
Ill
ACT~GT~AT~A~AGATTCAA~GAGGTTTACTTCAAATTGGTAGA~GGAAAATGGAA~AAG~TT~A~CA~A~C~A~GCAAACAAGGATTTGCACGCCAT~AACA~T~CTTGG~CTTCGGAC
T ...........
A...C
..................................
T
765
TBW 17
• .A.C ........................................
TSf
TACAA~CCTCTTGTCGACAAGTTCTCACCACTTGCCGTTCTCAGC~CGGTTCTCATCGCCTCCCTCGCAGTATT~TATTATCTCT`~`mGCGCACTGAGTCTCACATATTATCGACGTTTAA 885
.......
A . . . . . . . . . . .
T
. . . .
Dra 10 TSW 17 TSI
10
TBW 17
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TT.C . . . . . . . . . . . .
.
T.T ..........
TC . . . . . .
TAG . . . . . . . . . . . . . . .
I
A.A ...............
AATAACAATTGATAATTTGTACAAGACTGAAAAAAAAAAAAAAAAAAAAAAA
937
.............................
939
T.
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AAA
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b) TSI-IO cDNA
D _j_
D
N
I
I
K
D
I
1_. polyA
ORF TBW47 cDNA
EV
H
BI
A
I
I
I
I
P K H
D
It"
J-.
I
,200 bp
Fig. 1. (a) The nucleotide sequences of p33 and p34 cDNAs. The TSI l0 (p33 cDNA) and TBW 17 (p34 cDNA) nucleotide sequences are aligned. For the TBW 17 sequence only those nucleotides that differ from TSI 10 are shown. Dots indicate identical nucleotides. The initiation (ATG) and termination (TAG) codons are underlined and are in bold. The nucleotides indicated by arrows at the 5' and 3' end of the genes denote the sequence of the primers used in the PCR. The restriction enzymes sites shown in the restriction maps (b) are underlined and indicated here above the sequences. (b) Restriction maps of p33 and p34 cDNAs. Restriction enzymes sites are designated as follows: DraI, D; Nsp7524I, N; Kpnl, K; EcoRV, EV; HindIII, H; Ball, Bl; Avail, A; Pst|, P. The open reading frame (ORF) and poly(A) tail are denoted by thick lines in the maps.
analysis revealed one open reading frame of sufficient size to encode a protein of approximately 30 kDa within each of the sequences. The open reading frames of the p33 and p34 genes each consist of 849 bp with 84% identity. Unique restriction enzyme sites observed within either of the sequences are shown in the restriction maps (Fig. lb). Southern blot analyses were consistent with the genes being present in one copy per genome (data not shown). The predicted proteins consist of 283 amino acid residues with 82% identity and 92% homology. The maximum homology was calculated by the DNASIS programme (Hi-
tachi Software Engineering Co., Ltd., Japan). The polypeptides of predicted size 32 kDa each contain a putative signal peptide at the Nterminus and a hydrophobic region at the Cterminus which may be an anchor sequence (Fig. 2). Potential cleavage sites for the Nterminal signal peptides were predicted to be after Ala 23 of p33 and after Ala 22 of p34, respectively (Fig. 2) by using a computer programme, SIGSEQ 2 [14], based on a statistic weight-matrix method [15]. An obvious difference observed between p33 and p34 was in the numbers and positions of predicted Asn-linked glycosylation sites, 2 in p33 and 3
172
TSI
MLSKRTF NVLCLGYFL IVSATAAEEKKDAKAEEKKDLTL EVNAIAAEHFKVDASNANDVV
I 0
TBW 1 7
.....
TSI
FTAEEGYRIKILKVGDKNLYTVDTSKFIPTVAHRLKHADDLFFKL~LSHAKPLLFKKKTD
10
S..L ........
C ....
........
E . K .--EP .........
~,
.QG.N * .T
...SD...F
TSI
10
KDWVQFSFAQYLDEVVWKEKKEVKDLDASKFADAGLFAAEAFGTGKLYNFIGNFKVKKVM
TB~
17
......
TSI
I0
FEEKDVGDSNKAKYTAVKVYVGSDEKKVVRLDYFYTGDERFKEVYFKLVDGKWKKVEQSE
F..::_._~=L...I
......
E ......
,.9,V.,.PKN
TSI
ANKDLHAMNSAWPSDYKPLVDKFSPLAVLSAVLIASLAVFYYL
TBW IT
.
.
.
.
.
.
.
.
.
N...L
~.D ...........
.................
A ....
F ....
120
D ..............
DT . . . . .
TBW ~7
I0
...........
1..G.A .....
*N T
TBW 17
N.G . . . . .
f ................
A.
60
V.D.V.P ....
=~. . . . . . . . . . . .
180
S.K 240
L ....
283
F...F..
Fig. 2. The predicted amino acid sequences of p33 and p34. The predicted amino acid sequence of T. sergenti p33 (TS110) and that of T. bu(feh p34 (TBWI7) are aligned in the single letter code. For the p34 sequence only those amino acids that differ from p33 are shown. Dots indicate identical amino acids. Features within the sequences are denoted by the following: the single lines at the N-terminal mark the putative signal sequences; the double lines at the C-terminal mark the hydrophobic stretches; the asterisks mark the sites of potential glycosylation; the broken lines mark putative erythrocyte binding motifs, Lys-Glu-Lys (KEK) and Lys-Glu-Leu (KEL), or Lys-Glu (KE) ion pair [181.
a)
b) gO
cO
~
~n
0
~rnO
~Orn 0
~0~00
~Orn 0
1 1
Fig. 3. (a) The p33 and p34 genes amplified from genomic DNA and recombinant phage DNA by polymerase chain reaction. Piroplasm D N A from T. sergenti (TS), T. buffeli (TB) and T. orientalis (TO), and recombinant phage DNA from TSI 10 and TBW17 were used as templates for amplification. The amplification reaction mixtures were applied to a 1.5% agarose gel and stained with ethidium bromide after electrophoretic separation. (b) Differentiation of the PCR-amplified p33 and p34 genes by restriction enzyme digestion. The amplification reaction mixtures obtained from piroplasm DNAs were then digested with the restriction enzymes, electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. The restriction enzymes used were Dral (D), Nsp75241 (N), HindIll (H) and Aval! (A). Sizes are given in base pairs (bp) on the left.
173
in p34. No significant peptide repeats, as generally found in the surface proteins of Plasmodium falciparum [16], were recognized within either sequence. The predicted amino acid sequences did not have significant homology with other known protein sequences stored in the SWISSPROT database.
Polymerase chain reaction amplification of p33/ 34 genes. Two PCR primers of 20-residue oligonucleotides were synthesized to amplify the p33/34 gene as indicated in Fig. la. When phage DNA containing cDNA of p33 or p34 was used, a single fragment of approximately 870 bp was amplified, in good accordance with the predicted size, 868 bp (Fig. 3a). These primers also amplified a single fragment of 870 bp from genomic DNA template of T. sergenti, T. buffeli or T. orientalis. No detectable amplifications from bovine and phage DNA took place under the same conditions (data not shown). Differentiation of the PCR-amplified p33/34 genes by restriction enzyme digestion. PCRamplified p33/34 genes from parasite genomes were digested with restriction enzymes, and their cleavage patterns were compared by agarose gel electrophoresis. The restriction enzymes which cut the p33 and p34 genes at the unique restriction sites shown in Fig. l b were used for this comparison. The results with PCR-amplified p33/34 genes from T. sergenti, 7". buffeli and T. orientalis genomes digested with four different restriction enzymes are shown in Fig. 3b. The cleavage patterns of PCR-amplified p33/34 genes from T. sergenti and T. buffeli with these four enzymes were consistent with their restriction maps. The restriction patterns of PCR-amplified genes from T. buffeli and T. orientalis were identical for any of the enzymes tested, and clearly distinguished from that of the PCR-amplified gene from T. sergenti. The other four restriction enzyme sites (KpnI, EcoRV, Bali and PstI) indicated in the restriction map of the p34 gene (Fig. lb) were also conserved in the PCRamplified gene from T. orientalis (data not shown).
Discussion
The nucleotide sequences of cDNAs which encode p33 of T. sergenti and p34 of T. buffeli were analyzed. It has previously been reported that p33 of T. sergenti and p34 of T. buffeli and T. orientalis were the most abundant proteins in piroplasms [2] and the most immunodominant proteins in infected cattle [3]. These proteins are localized on the surface of piroplasms as determined by immunoelectron microscopy [17] and surface protein labeling with biotin [3]. The nucleotide sequence of p33 and p34 cDNAs showed high similarity in this study. All these results indicate that these proteins are equivalent molecules in these Theileria species. Southern blot analysis indicated that the p33 or p34 gene was present as a single copy in the respective parasite genome. PCR amplification of the p33/34 gene from both parasite genomic DNA and cloned cDNA indicated that the coding sequence of each gene is on a single exon. The different excesses of the observed molecular weights of p33 and p34 over the predicted 30 kDa for the mature polypeptides may be accounted for by the different numbers of potential glycosylation sites. In Western blot analysis, authentic p33 and p34 showed species-specific reactivities against sera from cattle infected with either of T. sergenti, T. buffeli or T. orientalis [3], but the reactivities of recombinant p33 and p34 against those sera were equal [5]. Post-translational modifications, possibly glycosylation, may generate species-specific epitopes that are predominantly recognized by infected cattle. The possibility that there are post-translational modifications of p33/34 should be examined using eukaryotic expression systems. The functions of p33 and p34 have not been clarified. Interestingly, the predicted amino acid sequences of p33 and p34 contained LysGlu-Lys (KEK) and Lys-Glu-Leu (KEL) motifs which were identified in a surface protein of blood-stage parasites of P. falciparum as erythrocyte-binding peptide sequences [18]. These motifs are considered to be an important feature of certain malaria
174
proteins which interact with erythrocytes, including the putative ligand for red cell receptor, EBA-175 [19] and the precursor of the major merozoite surface antigen (PMMSA) [16]. Binding and invasion of T. sergenti piroplasms to bovine erythrocytes via parasites surface structures has been demonstrated in vitro [20]. The piroplasm surface proteins p33/34 might play important roles in interaction with host cells. The KE sequence within a synthetic polypeptide used as malarial vaccine (SPf 66) is reported to be the key motif against which protective antibodies are directed. The repetition of this motif in p33 and p34 might produce cross-reacting antibodies in infected cattle. The restriction enzyme site comparison of the PCR-amplified fragments clearly discriminated T. sergenti from T. buffeli and T. orientalis. This procedure therefore provides a positive/negative marker system based on the differences of the genes coding the major piroplasm proteins between T. sergenti, T. buffeli and T. orientalis. This marker system will form a useful supplement to the other methods currently available for stock characterization such as restriction fragment length polymorphism analysis of genomic DNA using genomic D N A probes [21]. Whether this system could be applied for characterization and differentiation of other stocks of the T. sergenti/T, buffeli/T, orientalis group parasites is being examined. The results obtained in this study are in accordance with our previous conclusions that T. sergenti should be separated from T. buffeli and T. orientalis on the basis of tick-transmissibility and other phenotypic characteristics. These observations also suggest that the latter two parasites, T. buffeli and T. orientalis are the closely related species. It is possible that T. buffeli was introduced into Australia by Theileria-infected cattle imported from Britain in the early days of European settlement [22], and has adapted to the indigenous ticks Haemaphysalis bancrofti and Haemaphysalis humerosa, principally parasites of marsupials [8]. Australian T. buffeli and British 7". orientalis might belong to one and the same
species and in that case the latter name might have priority over T. buffeli which was introduced as the name of the parasite in Asian water buffalo [1].
Acknowledgements We would like to thank Drs. Y. Tsuchiya, S. Yamada, and T. Sekizaki for useful comments during the preparation of this paper.
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Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Hattori, M. and Sakaki, Y. (1986) Dideoxy sequencing method using denatured plasmid templates. Anal. Biochem. 152, 232-238. Folz, R.J. and Gordon, J.I. (1987) Computer-assisted predictions of signal peptidase processing sites. Biochem. Biophys. Res. Commun. 146, 870-877. von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683-4690. Kemp, D.J. and Cowman, A.F. (1990) Genetic diversity in Plasrnodiumfalciparum. Adv. Parasitol. 29, 75-149. Shirakata, S., Onuma, M., Kirisawa, R., Takahashi, K. and Kawakami, Y. (1989) Localization of surface antigens on Theileria sergenti merozoite by monoclonal antibodies. Jpn. J. Vet. Sci. 51, 831 833. Molano, A., Segura, C., Guzman, F., Lozada, D. and Patarroyo, M.E. (1992) In human malaria protective
antibodies are directed mainly against the Lys-Glu ion pair within the Lys-Glu-Lys motif of the synthetic vaccine SPf 66. Parasite Immunol. 14, 111 124. 19 Sim, B.K.L., Orlandi, P.A., Haynes, J.D., Klotz, F.W., Carter, J.M., Camus, D., Zegans, M.E. and Chulay, J.D. (1990) Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion. J. Cell Biol. 111, 1877 1884. 20 Kawamoto, S., Takahashi, K., Onuma, M., Kurosawa, T. and Sonoda, M. (1990) Invasion of bovine erythrocytes by Theileria sergenti piroplasms in vitro. Jpn. J. Vet. Sci. 52, 1261-1263. 21 Matsuba, T., Kawakami, Y., lwai, H. and Onuma, M. (1992) Genomic analysis of Theileria sergenti stocks in Japan with DNA probes. Vet. Parasitol. 41, 35-43. 22 Payne, W.J.A. (1970) The origin of tropical and subtropical breeds. In: Cattle Production in the Tropics, Vol. 1, (Payne, W.J.A., ed.), pp. 31-61. Longman, London.
