AU606042B2

AU606042B2 - Asexual blood stage antigens of plasmodium falciparum

Info

Publication number: AU606042B2
Application number: AU66877/86A
Authority: AU
Inventors: Robin Fredric Anders; Graham Vallancey Brown; Ross Leon Coppel; David James Kemp
Original assignee: Walter and Eliza Hall Institute of Medical Research
Current assignee: Saramane Pty Ltd
Priority date: 1985-12-24
Filing date: 1986-12-19
Publication date: 1991-01-31
Anticipated expiration: 2006-12-18
Also published as: AU6687786A

Description

To: THE COMMISSIONER OF PATENTS (a member of the firm of DAVIES COLLISON for and on behalf of the Applicant).

IL,,QIGr AT SUB-OFF1ICE I 24DEC 19*5 Davies Coilisofl, Melbourne and Canberra.

I 60)60 bV.1142 C 0 4 M 0N WE A L TH OF A U PATENTS ACT 1952 0O 3 MPLETE SPECIFICATION (original) FOR OFFICE USE 3 T RA LIA -1 a1TImfl Class Int. .Class Application Numberz Lodged: 6 &0-7-7 /I-6.

Complete Specification Lodged: Accepted: Published: Prioritj 0e 0 000 @0 0 000 0* S 0 00 0* 0 0 0000 0 @0 0 0 *000 0@ 0 0 0000 @0 @0 0 *0 *0 o 0 0 @0 0 @0000 0 0S 0 0 0@ 0@*@00 0 4" Related Art: u-c'

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~.1 Name of Applicant: Address of Applicant: Actual Inventor(s): Address for Service: THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCH.

Royal Parade, Parkville, Victoria, 3052, Australia.

David James KEMP; Robin Fredric ANDERS; Ross Leon COPPEL, and Graham Vallancey BROWN.

DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete specification for the invention entitled: '.ASEXUAL BLOOD STAGE ANTIGENS OF PLASMODIUM FALCIPARUM"1 The following statement i& a full description of this including the best method of performing it known to invention, us it 4 44 '4

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'00 la "ASEXUAL BLOOD STAGE ANTIGENS OF PLASMODIUM FALCIPARUM" This invention relates to synthetic peptides and polypeptides which have antigenicity suitable for providing protective immunity against Plasmodium falciparum infections, and to methods for the production 5 thereof.

The human malaria parasite Plasmodium falciFarum encodes many polypeptides that elicit an immune response in man. Recently, molecular cloning techniques have 10 facilitated the analysis of individual polypeptide antigens that are present in this complex mixture Many cDNA clones encoding these antigens have been isolated by screening Escherichia coli colonies that express the cloned sequences with human antibodies. The 15 production and screening of these clones is described in detail in International Patent Specification No.

PCT/AU84/00016.

The present invention is based upon the identification and characterisation of further asexual blood-stage antigens of P.falciparum.

According to the present invention, there is provided a DNA molecule comprising a nucleotide sequence i 4 1 0O 0 subrtantially corresponding to all or a portion of a base sequence coding for one of the antigens of P.falciparum described in detail hereinafter. In particular, there is provided a DNA molecule comprising a nucleotide sequence characterized by at least a portion thereof comprising all or a portion of a base sequence shown in the accompanying Figures. Such a nucleotide sequence codes for a polypeptide comprising at least a portion which corresponds to a portion of the amino acid sequence of an antigen of P.falciparum as described herein.

The present invention also extends to synthetic peptides or polypeptides displaying the antigenicity of 15 all or a portion of an antigen of P.falciparum as described herein, as well as to compositions for stimulating immune responses against such an antigen in a mammal, which compositions comprise at least one synthetic polypeptide displaying the antigenicity of all or a portion of the antigen, together with a pharmaceutically acceptable carrier therefor. The synthetic peptides or polypeptides according to this aspect of the invention may be prepared by expression in a host cell containing a recombinant DNA molecule which 25 comprises a nucleotide sequence as broadly described above operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. The synthetic peptide or polypeptide so expressed may be a fusion polypeptide comprising in addition to a portion displaying the antigenicity of all or a portion of the antigen, an additional polypeptide coded for by the DNA of the recombinant DNA molecule. Alternatively, the synthetic peptides or polypeptides may be produced by see* so 0 5

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A Rhoptry Protein of P.falciparum Intraerythrocytic asexual parasites of Plasmodium falciparum are responsible for the morbidity and mortality of this serious protozoal infection of man.

Propagation of the asexual parasite occurs when mature schizonts rupture and release merozoites which invade fresh erythrocytes. Invasion begins when the merozoite abuts an erythrocyte and re-orientates so that the apex of the merozoite is in contact with the erythrocyte membrane. Paired apical organelles called rhoptries discharge their contents prior to perturbation of the S 15 erythrocyte membrane and subsequent entry of the merozoite. Rhoptry proteins have been implicated as I potential protective immunogens in several systems (2, A cDNA clone encoding a portion of a Mr 105,000 rhoptry protein of P.falciparum has now been identified S* 20 and characterised. A rhoptry protein of this molecular weight is present in several isolates of P.falciparum from widely separated geographical areas.

Several previously isolated cDNA clones expressing 25 P.falciparum antigens contained regions of tandemly repeated peptides. It has previously been shown that these repeat regions are frequently highly antigenic and are the immunodominant regions of the molecule recognized during natural infection. The clone Ag44 expressing part of the Mr 107,000 rhoptry protein is an example where a naturally antigenic determinant is encoded by non-repeat sequence. It is not known whether other portions of this molecule contain repeat regions.

The identification of this clone allows the preparation 'i 4 of monospecific reagents against this rhoptry protein which will enable tests of its function and potential as a protective immunogen.

Further details of the isolation and characterization of this protein will be apparent from the following detailed description, and from the accompanying Figures. In the Figures: Figure 1 shows indirect immunofluorescence of the FC27 isolate of P.falciparum asexual blood stages reacted with human antibodies against Ag44. Fluorescein I staining of A, a late trophozoite and early schizont exhibiting weak fluorescence excluded over the 4 nuclei; B, a mature schizont with the predominant pattern of punctate fluorescence (left) and a multiply-infected erythrocyte exhibiting both punctate and lattice patterns of fluorescence (right); C, punctate fluorescence associated with extracellular merozoites. Paired spots of fluorescent staining occur 20 within a single merozoite (arrowed). Inset is of a mature schizont from an adjacent field of view.

Figure 2 shows immunoelectron microscopy, using 4 the protein A-gold technique, of a schizont of the FC27 25 isolate of P.falciparum reacted with human antibodies against Ag44. The pear-shaped rhoptries containing antigen reacting with antibodies are arrowed.

(Magnified x 79,000..) Figure 3 shows immunoblots using human antibodies r affinity-purified on the fusion protein of clone Ag44.

A. Identification of the corresponding parasite antigen in different life-cycle stages of FC27: uninfected cells rings trophozoites schizonts merozoites B. Identification of the corresponding parasite antigen in 4 different isolates of P.falciparum grown in asynchronous culture: NF7 K1 FC27 and Vi Figure 4 is a partial nucleotide and amino acid sequence of Ag44. At the 5' end of the insert the first 2 bases derived from P.falciparum put the sequence in frame with p-galactosidase but are not translated here because the corresponding hybrid codon would not be found in *P.falciparum sequence. Figure 4A shows the full amino acid sequence of the Rhoptry Protein, Ag44. Amino acid residues Nos. 763 to 895 in Figure 4A correspond to residues Nos. 2 15 to 134 in Figure 4. Figure 4B (3 sheets) shows the full nucleotide sequence. Nucleotide base Nos.

3710 to 4111 in Figure 4B correspond to base Nos. 6 to 407 in Figure 4.

20 Figure 5 shows the hybridization of Ag44 cDNA to restriction fragments of P.falciparum DNA. DNA from isolates FC27 K1 and NF7 was cleaved with EcoRI and Hind 3 fractionated by electrophoresis on a 1% 32 Sagarose gel, blotted to nitrocellulose, hybridized with P- Ag44 cDNA and autoradiographed.

Materials and Methods (see later).

RESULTS

Identification of a cDNA Encoding a Rhoptry Protein.

cDNA derived from the Papua New Guinea isolate- FCQ27/PNG (FC27) isolate of P.falciparum was prepared as described and inserted into the expression vector ygtll-Amp3 A large number of clones expressing P.falciparum sequences were screened with human antibodies affinity purified against the FC27 isolate and seventy-eight antigen ^RAI positive clones were w"! 1 I, i' t U I O' 0 0* 0 0 0~ to k 0 0*00 6* 0 06 0 0 0 0 0 identified One such clone, Ag44, was shown to encode part of a rhoptry protein as follows. E.coli lysogenic for XAg44 were grown in liquid culture, heat-induced, lysed and coupled to CNBr-activated Sepharose. Human antibodies specific for the Ag44 fused polypeptide were affinity purified on this absorbent, and used to identify the P.falciparum protein corresponding to Ag44 by immunofluorescence and immunoblot assays.

Asynchronous cultures of the FC27 isolate were fixed to glass slides and examined by direct immunofluorescence. Proteins reactive with anti-Ag44 antibodies were localised to mature schizonts, in paired 15 organelles within merozoites, a pattern characteristic of rhoptry proteins (Fig.la). Little reactivity was seen with ring forms. Several different isolate of P.falciparum: K1 from Thailand, NF7 from Ghana and VI from Vietnam, all showed identical patterns of fluorescence.

Immunoelectron microscopy confirmed the rhoptry location of the antigen recognised by anti-Ag44 antibodies. There was heavy labelling of the 25 pear-shaped organelles when sections of schizonts were incubated first with affinity purified human anti-Ag44 antibodies and then protein A-gold (Fig.2).

Immunoblot analysis of lysates of synchronized P.falciparum infected cells showed that the anti-Ag44 antibodies recognised 3 closely-spaced bands of Mr 107,000, 105,000 and 103,000 (Fig.3a). The higher molecular weight forms were more prominent in immature forms, and this may suggest a precursor product i 1

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0 S relationship. A similar set of bands was recognised when lysates of several different P.falciparum isolates were probed with anti-Ag44 antibodies (Fig.3b).

Nucleotide Sequence of Ag44 DNA was purified from phage expressing Ag44. Only 1 insert was present and this was subcloned into the pUC and M13 vectors. The nucleotide sequence of the 494 bp R1 fragment was determined by the dideoxy method (Fig.4). There was a long open reading frame present which extended up to nucleotide 404 and was in frame with a-galactosidase, accounting for the large fused polypeptide synthesized by XAg44 The predicted amino acid sequence is displayed (Fig.4). There are no tandemly repeated peptide elements as are commonly found in other P.falciparum antigens. The termination codon at nucleotides 405-407 presumably represents the 3' end of the coding region. This is consistent with the presence of deoxyadenosine bases present at the extreme 3' end of the DNA sequence which correspond to the poly(A) tail of the mRNA. This sequence predicted here would encode approximately 16% of the entire molecule.

Genomic Context of Ag44 DNA from three P.falciparum isolates FC27, K1 and NF7 was cleaved with EcoRI or AhaIII; size fractionated and blotted to nitrocellulose. The purified 570 bp R1 fragment of Ag44 was nick-translated and hybridized to the nitrocellulose filter. All isolates showed a common 30 band of 1800 bp in EcoRl digests and 5000 bp in Hind 3 digests S S

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a j: si i ii a 8 (II) An Acidic Basic Repeat Antigen (ABRA) of P.falciparum A Mr 102,000 antigen of P.falciparum, predominantly of schizonts, has been identified and characterized.

Sequencing studies on 4 cDNA clones encoding parts of this antigen revealed blocks of hydrophilic dipeptide and tripeptide repeates and so the antigen has been designated the Acidic Basic Repeat Antigen (ABRA).

Further details of the isolation and characterisation of this antigen will be apparent from the detailed description hereunder, and from the accompanying Figures. In the Figures: Figure 6 shows indirect immunofluorescence of j P.falciparum asexual blood stages reacted with human antibodies to Agl96. Single fields of view for isolate Vl (panels A, B) and FC27 (panels C, D) examined by fluorescein C) and propidium D) fluorescence the erythrocytes shown contain trophozoites and schizor's Figure 7, shows immunoblots using human antibodies affinity-purified on the fusion protein of clone Agl96.

A. Identification of ABRA in 4 different isolates of P.falciparum grownin asynchronous culture: uninfected red cells NF7 K1 FC27 and Vl B. Detection of ABRA in different life-cycle stages of FC27: uninfected red cells rings trophozoites schizonts and merozoites i, I i: u

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9 C. Triton X-100 extracts of the same life-cycle stages as in (lane 1 to Triton-insoluble pellets were resolubilized in NaDodSO uninfected cells rings trophozoites schizonts and merozoites Molecular weight markers are myosin (200kD), (-c-lactosidase (116 kD), phosphorylase D (92 kD), .bovine serum albumin (66 kD) and ovalbumin (45 kD).

Fi Lre 8 is the nucleotide and amino acid sequence of Ag189. The start of Agl44, Agl96 and Ag 126 in relation to Agl89 are indicated by arrows and the adjacent clone number.

Figure 9 shows hydridization of Agl26 cDNA to a 15 restriction fragments of P.falciparum DNA. DNA from the 3 isolates of P.falciparum indicated was cleaved with EcoRI and Aha III fractionated by electrophoresis on a 1% agarose gel, blotted to V nitrocellulose, hydridized with 32P-Ag26 cDNA and 0660 20 autoradiographed. The P.falciparum isolates were: FC27 from Papua New Guinea; NF7 from Ghana and K1 from Thailand.

Materials and Methods 25 (See later) S* p

RESULTS

ADRA is located in the mature sohizont Indirect i;:.iunofluorescence was performed on t 30 acetone-fixed, asexual blood-stage parasites using human antibodies affinity-purified on an immunoadsorbent of Ag196. The antibodies reacted strongly with erythrocytes containing schizonts and gave predominantly a lattice pattern of fluorescence whi<h is particularly rr I well resolved in isolate VI (Fig.6A). Counterstained nuclei of the developing merozoites appeared within regions that excluded fluorescein staining (Fig.6B).

Little or no reactivity was seen with ring and trophozoite stages of Vl.

More intense fluorescence was observed at a given antibody dilution with isolate FC27. Staining again occurred predominantly with erythrocytes containing schizonts, but there was diffuse staining in trophozoites to a greater extent than with VI (Fig.6C).

Fluorescence of the surface of infected erythrocytes was not seen when the assay was performed using unfixed cells or lightly glutaraldehyde-fixed and air-dried 15 monolayers Similar results with FC27 were obtained using mouse antisera against clones Ag196, Ag189, Ag126 *e and Ag203 belonging to the same serological family.

In immunoblots of asynchronous parasite 20 preparations, affinity-purified human antibodies against clone Ag196 detected a dominant band of Mr 102,000, which did not vary between the 3 isolates NF7, FC27 and V1 (Fig.7A). The corresponding protein is approximately Mr 2,000 smaller in isolate K1 from Thailand (Fig.7A).

a S In immunoblots of life-cycle stages (Fig.7B) the dominant Mr 102,000 band was present in schizonts was poorly represented or absent from other stages. A weak o band .of Mr 230,000 was also present in schizont S 30 preparations (Fig.7B). The target antigen was recovered in Triton extracts of infected erythrocytes and no additional material was detected by anti-Agl96 antibodies when pellets were resolubilized in NaDodSO 4 sample buffer (Fig.7C).

-r 11 Nucleotide and amino acid sequence The cDNA inserts of 4 members of the Ag196-family iere isolated. The insert of Ag189 was subcloned into the vector Ml3mp8 and its nucleo-i ,a sequence determined by the dideoxy procedure. Ag189 contains an insert of 965 bp, which has a single open reading frame extending through the whole cDNA. This frame is shown in Figure 8. All the other frames are interrupted by multiple stop codons. Agl89 is not in frame with -galactosidase and does not produce a large fused polypeptide. A number of other clones from similar expression libraries were out of phase with 6-galactosidase Hydrophilic dipeptide and tripeptide repeats predicted 0* 15 from the sequence of ABRA The sequence of Ag189 from position 1 to 834 s oencodes predominantly hydrophilic amino acids. At the 3' end starting at position 835 extends a highly charged region which consists of 10 dipeptide repeats (Glu-Lys) 20 and 6 interspersed tripeptide repeats (Glu-Glu-Lys).

The repeat-block is flanked on either side by three glutamic acids.

Three blocks of 12 nucleotides starting from 25 position 678 to 714 exhibits a high degree of homology.

These "cryptic" dodeca-nucleotide repeats only show a minor degree of similarity on the amino acid level.

oo0, Asparagine and isoleucine in position 3 and 4 in the first repeat appear again in the same position in the 30 third repeat and glutamines were found in position 1 and 2 of the second and the third cryptic repeat.

A dodecapeptide was synthesized comprising the amino acid sequence *1 U t iY; I 12 Glu-Lys-Glu-Glu-Lys-Glu-Lys-Glu-Glu-Lys-Glu-Lys and the binding of antibodies in malarial sera from PNG to this peptide was tested by a radioimmunoassay (RIA). The malarial sera gave no signal in the RIA. This result was surprising because synthetic peptides corresponding to six other repeating sequences that have been determined in other antigens of P.falciparum all gave positive results 8, In order to exclude sequencing errors the complete nucleotide sequence of Ag189 was again determined and an identical sequence and reading frame was obtained In addition, the inserts of 3 further clones were sequenced, namely Agl26, Agl44 and Ag196 coding for 15 segments of the same P.falciparum. These 3 clones are all in phase with 8-gal, produce large fused S. polypeptides and exhibit the same open reading frame as Ag189. Therefore there is certainty about the reading frame. The sequence of Agl26, 144 and 196 includes in Sa" 20 all 3 clones the region with the block of di- and tripeptide repeats. However differences among the 4 cDNA clones were also noted.

S* Ag144 which is 581 bp long commences at position 25 387 in relation to Agl89 and has deleted 6 bp in position 950 to 955, but contains 7 additional As at the S* 3' end of the cDNA, which codes for two more lysines.

Ag126 and Ag196 are 451 and 452 bp long respectively and both start at position 458 in relation to Agl89. The S• 30 Ag126- and Agl96-insert exhibit a deletion extending from position 901 to .955 in the sequence shown in Figure 8. It is believed that these deletions are artefacts of cloning in M13. Similar problems of maintaining cDNA *i 1 13 inserts in M13 have been observed with other malarial antigens Ag126 and Ag196 both differ from the sequence of Ag189 and Ag144 in two nucleotides. Ag126 and Ag196 contain at position 461 (in relation to Ag189) a "T" instead of an replacing tyrosine by phenylalanine and in position 806 a instead of which has no effect at the amino acid level. Agl26 and Agl96 have 3 and 4 additional As at the 3' end coding for 1 and 2 more lysines, respectively.

Genomic organisation of ABRA The insert of Ag126 was used in Southern blot 15 experiments to investigate the genomic organisation of 0o. ABRA. DNA from 3 geographical isolates of P.falciparum, the homologous strain FC27 from Papua New Guinea, NF7 from Ghana and K1 from Thailand were restricted with EcoRI and AhaIII, size-fractionated on 1% agarose gels, *20 blotted on nitrocellulose and probed with the 32 p-labelled insert of Ag126. As can be seen in Figure 9, the insert hybridized to a single 6.4 kb EcoRI fragment and a 1 kb AhaIII fragment in each isolate investigated. In addition, the DNAs of a further 3 25 isolates from Papua New Guinea (IMR143, IMR144 and MAD71) were probed with the 581 bp insert of Agl44 and showed identical fragment sizes in these isolates (data not shown).

:So* 30 III Other Antigens cloned in E.coli Several other antigens of P.falciparum, which are natural immunogens in man (and therefore potential vaccine candidates), have been identified with antibodies raised against or affinity purified on

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i 14 P.falciparum antigens expressed from cDNA sequences cloned in E.coli using the XAmp3 vector. The clones, and the apparent molecular weights and stage specifities (determined by immunofluorescent microscopy) of the corresponding parasite antigens, are listed in Table 1.

Figure 10 is the nucleotide sequence of clone Ag169; Figure 11 is the nucleotide sequence of clone Ag303; Figure 12 is the nucleotide sequence of clone Ag358; Figure 13 is the nucleotide sequence of clone Ag361; Figure 15 is the nucleotide sequence of clone 15 Ag394; and Figure 15 shows indirect immunofluorescence on acetone-methanol fixed bloodstages of P.falciparum reacted with antibodies directed against antigens produced by-Ag501 in bacteria T. trophozoite (minimal 20 reaction). S-schizont. G. gametocyte (no reaction seen).

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Corresponding P.falciparum Antigen Clone Apparent Molecular Predominant Location by Weight Immunofluorescence Ag169 N.A. N.A.

Ag303(Ag331)** 125,000-130,000 Schizonts Ag358 Dominant bands are 210,000; 190,000 and 140,000 All stages Ag361 70,000 Mature stages Ag372 195,000; 140,000 and 80,000 Mature stages Ag394 140,000*** All stages including rhoptry locations.

Ag501 130,000 Mature stages N.A. Not available The apparent molecular weights (Mr) have been determined by Western blotting from 7.5% gels using antigens from the FC27 P.falciparum isolate. In some cases, the Mr can vary considerably in other isolates and of other gel conditions are employed.

Also in some cases numerous other weaker bands are seen, presumably reflecting breakdown products or cross-reactions.

It has been found that Ag303 and Ag331 correspond to fragments of the one coding sequence.

Cross-reactions with Ag23 and with bands of 105,000 and 102,000 were also observed.

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16 MATERIALS AND METHODS Parasites P.falciparum isolates FCQ27/PNG (FC27), IMR143, IMR144 and MAD71 were obtained through the Papua New Guinea Institute of Medical Research. NF7 from Ghana, and Kl from Thailand, were obtained from D.Walliker, Edinburgh University. VI from Vietnam was obtained from L.Miller, National Institute of Health, Bethesda, U.S.A.

Parasites were maintained in asynchronous in vitro culture in Group O human erythrocytes according to Trager and Jensen To obtain stage-specific life-cycle forms, parasite cultures were synchronised twice to within a six hour spread of maturation using sorbitol (10) and harvested at various time points of 15 the asexual cycle. Naturally released merozoites were obtained as described previously (11).

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*40* Sera Sera were obtained with informed consent from individuals living in the Madang region of Papua New Guinea. Some patients presented with acute malaria S* while in others, asymptomatic parasitaemia was detected in the course of routine surveys. Parasitaemic individuals were treated with chlorcquine and convalescent serum was collected one or two weeks later.

25 Parental consent was obtained before taking samples from children. In all cases, serum was separated and stored at -20 0 C for up to 12 months then held at -700C.

.Presence or absence of splenomegaly was documented for some subsets and parasiteaemia was assessed from a thick blood smear in. all cases.

~Aim Clones expressing P.falciparum antigens Methods for construction of the P.falciparum cDNA expression library and isolation of clones by antibody screening have been published Replicas of the antigen-positive clones were grown overnight at induced at 380, and lysed in situ as described (12).

Individual human sera were pretreated to remove anti-E.coli activity, reacted with the colonies at a final dilution of 1:500 in 3% bovine serum albumin/Tris saline, pH 9.6 albumin, and the colonies then reacted 125 with I protein A from Staphylococcus aureus and autoradiographed as described (12).

*s* Hybridization experiments '"15 DNA carrying inserts were purified by CsC1 centrifugation, digested with EcoRI, end-labelled with 32 32 P-dATP by the Klenow fragment of DNA polymerase I and size-fractionated on a 1% low-melting agarose-gel. The labelled inserts were recovered and hybridized to the bank of antigen-positive clones. In some cases the insert was first subcloned in the plasmid pUC-9 (13) S purified by gel electrophoresis and then nick Stranslated. Inserts which had been subcloned in this way were used in Southern blot experiments. For 25 Southern blots, two micrograms of parasite DNA was digested with restriction, endonuclease according to the manufacturer's instructions, electrophoresed in a 1% agarose gel and blotted to nitrocellulose filters which 6 were then hybridised with 10 cpm/ml of the various probes.

a 18 Nucleotide sequence determination The dideoxy chain termination method was employed for sequence determination. The inserts of the various antigen-expressing clones and fragments generated by digestion with appropriate restriction endonucleases were cloned onto M13mp8 and/or M13mp9 (13).

Affinity purification of human antibodies against cloned malaria antigens Induced 50ml cultures of antigen positive clones were prepared as described previously The S. pelleted bacteria were sonicated and soluble proteins 00 were conjugated to CNBr-activated Sepharose (Pharmacia, Sweden). Antibodies from a pool of human plasma were affinity-purified on the immobilised antigen as pa described o° lasynchronous cultures of P.falciparum were fixed in acetone/10% methanol and reacted with affinity-purified human antibodies. Sera from mice immunized with bacterial lysates of antigen-positive clones were also 25 examined (16) Fluorescein-conjugated sheep anti-human Ig or sheep anti-mouse Ig antisera were used as the 2 second antibody. Parasite nuclei wtee counterstained with propidium iodide and the slides were mounted in PBS containing p-phenylenediamine fori viewing under U.V. illumination.

i purified against the FC27 isolate and seventy-eight antigen positive clones were Lui 19 Immunoelectron Microscopy Parasitized erythrocytes were fixed with glutaraldehyde, sectioned after being embedded in L.R.White resin and incubated with appropriately diluted antibodies and protein A-gold using published procedures (11).

Immunoblotting Merozoites and infected erythocytes containing either stage-specific or asynchronous parasites were diluted in samp.e buffer containing 3% SDS, 62.5 mM Tris-HC1, f-mercaptoethanol, pH 6.8 and heated for 2 min S'at 100C. After centrifugation at 12,000g for 10 min., protein extracts were fractionated on 7.5% or 15 polyacrylamide/SDS gels and transferred 125 S electrophoretically to nitrocellulose. Filters were I blocked with 5% non-fat milk powder in phosphate-buffered saline (PBS) pH 7.4 and reacted with incubated in I-labelled protein A and autoradiographed.

In a separate experiment, parasitized cells and merozoites were first incubated in PBS containing 25 Triton X-100, 5 mM PMSF, 1 mM TPCK, 2.5 mM EDTA and 2 mM iodo-acetamine for 30 mins at room temperature and centrifuged at 12,000g for 10 min. Supernatants and Spellets were then individually diluted to equivalent t final volumes in sample buffer and treated as before.

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REFERENCES

1. Kemp, Coppel, Cowman, A,F., Saint, Brown, G.V. and Anders, R.F. (1983) Proc.Natl.Acad.Sci.USA 80, 3787-3791.

2. Holder, A.A. and Freeman, R.R. (1981) Science, 193, 673-676.

3. Perrin, Chizzolini, Lebon, Shaw, A., Merki, B. and Stocker, J. In "Proc.Asia Pacific Conference on Malaria", Honolulu, Hawaii, USA, ****April 21-27, 1985.

Anders, Coppel, Brown, Saint, Cowman, Lingelbach, Mitchell, G.F.andKemp (184)Molec.Biol.Med. 2, 177-191.

Perlmann, Berzins, Wahlgren, Carlsson, Bj~rkmann, Patarvoyo, M.E. and Perlman, P.

:(1984) J.ExiD.Med. 159, 1686-1704.

Dame, Williams, McCutchan, Weber, Wirtz, Hockmeyer, Sanders, G.S., Reddy, Maloy, Haynes, Schneider, S Roberts, Diggs, C.L. and Miller, L.H.

(1984) Science 225, 593-599.

Coppel, Cowman, Lingelbach, K.R., Brown, Saint, Kemp, D.J. and Anders, R.F. (1983) Nature (London) 306, 751-756.

21 8. Coppel, Cowman, Anders, Bianco, Saint, Lingelbach, Kemp, P.J. and Brown, G.V. (1984) Nature (London) (in press).

9. Trager, and Jensen, J.B. (1976) Science 193, 673-676.

Lambros, C. and Vanderberg, J.P. (1979) J.Parasitol 418.

11. Brown, Culvenor, Crewther, P.E., seeBiaco A..,Copel R..,Saint, RBStahl, H-D, Kemp, D.J. and Anders, R.F. (1985) J.Exp.Med 162, 774-779.

12. Stahl, Coppel, Brown, Saint, tj Lingelbach, Cowman, Anders, R.F and ::.Kemp, D.J. (1984) Proc.Nat.Acad.Sci.USA 81, 2456-2460.

13. Messing, J. and Vieira, J. (1982) Gene 19, 269-276.

*14. Sanger, Nicklen, S. and Coulson, A.R. (1977) 0* Proc.Nat.Acad.Sci.USA 74, 5463-5467.

Stahl, 11-D., Crewther, Anders, Brown, Coppel, Bianco, Mitchell, F.G.

and Kemp, D.J. (1985) Proc.Nat4Acad.Sci.USA 82, 543-547.

1 Coppel, Brown, Mitchell, Anders, R.F. and Kemp, D.J. (1984) EMBO J. 3, 403-407.

Claims

2. A recombinant DNA molecule according to claim 1, wherein said nucleotide sequence is characterised by at least a portion thereof comprising a base sequence substantially as shown in Figure 4. recIc bnacnt
3. ALDNA molecule comprising a nucleotide sequence capable of being expressed as at least one polypeptide displaying the antigenicity of the Rhoptry Protein of P.falciparum defined herein, and other antigens of P.falciparum cross-reactive therewith.
4. A recombinant DNA molecule comprising a nucleotide S* sequence according to any one of claims 1 to 3, operatively linked to an expression control sequence.
5. A recombinant DNA cloning vehicle or vector capable of expressing all or a portion of at least one polypeptide or protein of P.falciparum, and having Sinserted therein a nucleotide sequence according to any j one of claims 1 to 3, said sequence being operatively i* linked to an expression control sequence.
6. A recombinant DNA cloning vehicle or vector according to claim 5, characterised in that said nucleotide sequence and said expression control sequence are inserted into a bacteriophage. g
7. A host cell containing a recombinant DNA molecule according to claim 4, or a recombinant DNA cloning vehicle or vector according to claim A i 23
8. A synthetic peptide or polypeptide displaying the antigenicity of all or a portion of the Rhoptry Protein P.falciparum defined herein, and other antigens of P.falciparum cross-reactive therewith.
9. A fused polypeptide comprising a polypeptide sequence displaying the antigenicity of the Rhoptry Protein of P.falciparum defined herein, and other antigens of P.falciparum cross-reactive therewith as the C-terminal sequence, and an additional polypeptide as the N-terminal sequence fused thereto. A fused polypeptide according to claim 9, wherein the additional polypeptide is a polypeptide coded for by the DNA of a recombinant DNA cloning vehicle or vector.
11. A composition for stimulating immune responses against P.falciparum antigens in a mammal, comprising at least one polypeptide displaying the antigenicity of the Rhoptry Protein of P.falciparum defined herein, and other antigens of P.falciparum cross-reactive therewith, together with a pharmaceutically acceptable carrier therefor.
12. A method of stimulating immune responses against P.falciparum antigens in a mammal, which comprises administering a composition according to claim 11 to said mammal. j Dated this 27th day of August, 1990, THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCH By its Patent Attorneys, DAVIES COLLISON *O 4 I 0 00 .00. a 0 FIG.!/ AIG2 12 3 45 1 23 4
200- 93- 69- 0O 0@ S. S 5* S S 46- F/G.J S. S 555 55 S S 55 55 5e 1 23 12 3 50- 0- S *SS 5* S S 55 -A 4~4~ A B S S S S. S S S S S S S S S '3 55. S S *5 .;V Vat Asp ItI eLeuG tuG Lu LysThrLysAspGnAspLeuG tuILeGuLeuTyrLysTyr GCGTTG ACATATTAG AAGAA AA AAC [AAG GATCAAG ATTTiAGAAATAGAATTATAC AAATAT 20 30 40 50 MetGlIy Pro Leu Lys G Iu GtInSerLys SerT hrSe rAc cLQSe rTh rSe rAs pGIu LeuSer ATGGGA CCATTAAAAGAA CA ATCTAAAAGTA[AAGTGCTGiCAT[TAC TAG TGATG AATTATC 72 82 92 102 102 122 G IySe rG IuG IyPro SerTh rG tuSerTh rSerTh r GyAsnGIn G [yGluAsp Lys Thr Thr AGGTTCTGAA GGTCCATCTA [TGAATCTA[ AAGTACAGGAAAT[AAGGTGAAGATAAAACAA 134 144 154 164 174 184 AspAsnTh rTyrLys Gt[u M eGIu GtIu LeuG uG u A.Gtu GtIy ThrSerAsnLeuLys CAG ATAATAC ATACA AAG AAAT 6 GM 6 AATTA GAAGAAGCTGAAGG6AACTT[AAATCTTAAA 196 206 216 226 236 246 L ysG t y LeuG u Ph eTy rL y sS erS e rLeu L ysLeuAspGtnLeuAspL ysG u LysProLys AMAGGTTTAGAATTTTATAAAT[CTT[ TCTAAAA[TTGATlCAATTAGATAAAGAAAAACTAA 258 268 278 288 298 308 Lys L ysLysSe rLys Arg Lys LysL ysArgAs pSerSerS erAspA rg I IteLeuLeuG I uG tu AAAGAAA AAA T[ TAAAAGAAAAMA AG A GAGACAGTTCTA GTGAC A G A ATATTATTAG AAG 320 330 340 350 360 370 SerLy sTh r PheTh rSe rG u As nG[u Leux) AATCTAAAA[ [TTTA CTTCT CAAAATGMATTGTAAATTAAAAA1TTAAT[ [TACATGTAGAT 382 392 402 412 422 432 ITTA TTATAT TA [AT [ATG T AAT [ATATTA TAG AATTTAT TTTTAAGAA AAAAAAAAAAA 444 454 464 474 484 494 F1G. S S S S S S S S S S S S *S S *S S *5 S S. *S S SS* S S S .5 5* S S 55 5 5e S SS S S S S S S S S MRS K11L VT 1'F 110 NRSWVSEFLK 210 NLYSTVEDEQ 310 SDFTLIKPOS 410 KELSEINITDF 510 TRYTSLYIHK 610 SIISVLYINE -710 EIPVQLETSN 810 20 30 tirril'St'sTV KVWGKOVFAG 120 130 EYEEPFVNPV MKFLDKEQRL 220 230 RKQLLKVIIE NESTNDISVQ 320 330 VWNVPIFDIY KPKNYLDSVO 420 430 SFKEDNSEIQ CQNVRKSLDL 520 530 FFKSVTLLKG NFLYKNNKAI 620 630 YYYEQLSCFY PKEEELIKSR 720 730 DGIGYRKQOV LYETDKPQTM 820 830 SIOtNOGEI) K1'i',i)NTrYEM 40Q F'V'I'KKtKTLL 140 FEFTYNFGDVE 240 CPTYNTKLIIY 340 NLDTECE'KKL 440 EVOVETMKGI 540 RYHVLVVKPH 640 MIliPNIVDRI 740 DEASYEETVD 840 .E lYE AECTIS 50 60 I)CNFALYYNI" KGNGPDAGSP 150 160 PQGKYTYE'QL RNFTKYCIL? 250 260 TKECANSNNI LKCIDEFLRK 350 360 NSKNLIFLSF [100IPNNPYY 450 460 AARKLCKTIE KFILTKDDAO 550 560 YTFHPFYTEE IYIFLKH-LII 650 660 LKGIDNLMKS TRYDKMRTMY 750 760 EOAHIIVNEKQ HSAI]FLDAIA 850 860 70 LOFVDEI QF .170 PLIK'rNI OG 270 TCIIKKTE SE I 370 NVELQEIVKL 470 KPEKSDI[IRG 570 I LGLSML VS S 670 LDFESSDIFS 770 EKDILEEKTK 870 rKP KKESK R 80 90 YWFVEIIFIISV KFRVPEIILKI) 180 190 ESGELKYQL NKEEYKVFLS 280 290 PSADLCCEHLQ ELFESLENPY 380 390 STYTYSIFDK LYNFFE'VFKK 480 490 FRILCILIST IIVEAYNIVRQ 580 590 NPSSPEFOTI IEFLVTYYNK 680 690 REKVFTALYN FOSFIKTNEQ 780 790 DQDLEIELYK YMGPLKEQSK 880 890 KKKRO).S.I)R Tll..ESKTrFT 100* KNIIINFTPCL' 200 SVGSQMTAIK LDNFKKFMTN 400 SGAPISPVSV 500 LLNMESMISL 600 GSEKFVLYFI 700 LKNLEEIS 800 STSAASTSDE 900 SENEI, FIGURE 4A 00 00 i~f 203040060780910 21 2 30 2 40 25~0 2 60 270 280 290 100 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 800 610 620 630 640 650 660 670 680 690 700 710 720 -730 740 750 760 7 70 780 790 800 810 820 830 840 850 860 870 880 890 900 91t0 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 105-0 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1150 1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 1510 1520 1530 1540 1550 1560 1570 1580 i590 1600 FIGURE 4B 0% 0* 0 9 Ss. 'e 00: 4 vowS* 0. .0 :0 SS 1610 1620 1630 1640 1650 16G0 16710 16180 1690 1I00 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 1810 .1820 1830 1840 11150 1860 111,70 111110 10190 1900 1910 1920 1930 1940 1950 1960 1970 19810 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100 2110 2120 2130. 2140 2150 2160 21710 2180 2190 2200 2210 2220 2230 2240 2250 2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400 2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550 2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700 27110 -720 2.730 2'140 27 r,0 27-1G0( 2770 2711 2790 21100 2810 2820 2830 2840 2850 2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000 3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150 3160 3170 3180 3190 3200 FIGURE 4B (cont'd) Se se 00 *0 Sa see *so .0 so: a*. .0 00 a. .1 .0 0S e e 0 0.0 S Se we 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300 3510 3520 3530 3540 3550 3560 3570 3580 3590 3400 L GGT 3610 3620AAATTG 3630ATAA 3640CAA 3650ATGA 3660ATA 3670TAAT 3680TTT 3690ACTA 3700A 3410~ 3720 3730 3740 3750 3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900 3910 3920 3930 3940 3950 3960 3970 3980 3990 4700 4110 41 320 4130 4140 4150 4160 4170 4180 4190 4200 GCGGAAATG-UATATAAAAAAACCAAGTGATAAGATTTAAATCATTAACATATTATGGGAATTAATTAAGAAAAAAAAAAA 3210 3920 4230 4940 4250 4260 4270 3280 4290 4000 ATAAAAAAATGGATAGAATTAGAAAATTTTGTTTTTTTGATCTTTTAAATTTTThTGAAAATATTAAAC TTTTCAAT~TTAAT 4310 4320 4330 4340 4350 4360 4370 4380 4390 4400 AAAATATTATATGAATTTTTTTTTTATTAAATTGAGATTTTAAACI TATGTGACATGAT PAAOATCGGTTCTAT FIGURE 4B (cont'd) -99 -r.6 -99 -99 9L 91LL* -004 0 see AftS 991I 9 Hi syr~s~ysrg~s~lG~nG[u~sG~~eu~oG~ Pr CATTTAAGA ***AAA*TC *G*AAAGG**AC *GACC*AT*** CTAT** His~yrL y s~eL s Leuys ysGLeuL ys~sty eP o Pro LsLsyGIuA"s ATTAAAAG ATAAA[TCAAGAAAAAATATATTC AA[TAAG~A[AATAA 130 240 30o 460610 AsGtu tyrVtuGpu~uhLy s~lytefG u IsPfe-ofLyssALuLePhA Leu AATATGTTAGAGATA CAAAAGTATTTAGAATGTCAAATTATT ACACAGTTT 790 800 910 100 110 240 LysVGLysG euro~euGeu Assp eurAs n Iyetsl~erLys nysnLtAsp AGAGGTTAAAGCATTTAAAAAATTAAAAAAATATT[[[ CATATAATAAAAGGAT 132 50 16 7020 170 100 AGtyl. I.ueh~~Asp~h~e~~stuerul e tAsn~snA snrAsnVct[etLsP GAAATTGATTTC TTTG AAAA AGAATTAATC G AAAATAATAATA CA C [TAATGTTG TA C CA 310 320 330 340 350 360 FI .8A. S Pro ThrGLnSerLysLysLysAsnLysAsnGuThrV cL eGL ltA pS~nh CCAACTCAATAAAAAAAAAAAACA ATGAAACTGTATCTGTATGGATGAAAATTT 370 380 390 A144 400 410 420 Asp AsnnHi s Pr o G I u AsnnTyrrPheeLy s GI G I u Tyr Tyyr Tyyr AsppG LuuAsnoAsp As p Met GATAATCATCCTGAAAATTATTTTAAAGAAGAATATTTTAGTAAGTAA-G 430 440 450 4640 Ag126/ 470 4du Ag 196 G L u V ci L y sV a L y s Ly s I I e G L y Va I T h r L e u L y s L y s P h e G L u P r o L e u L y s As n G I y GAAGTAAAAG TAAAAAAATAGG TG TCACATTAAAAAAATTTG AA CC TTAAAAATGGA 490 500 510 520 530 540 AsprVa I Sr G Lu Ty r I L e Lys Leu I L e Hi s LeuG yAn Ly sAspL ys LysH i s I L ee AATTTAGTG AAACCATTAAATTGATTCATTTAGAAATAAAGATAAAAAACACATTGAA 550 560 570 580 590 600 A L a I L e AsnA s nAs p I I e G L I eLy n G L u eu G I n A I a I L e Tyr Asn G L u Le GCTATAACAACGATATCAAAT TATTAAACAAGAATT T TATGATATTAT6ATGAACTT 610 440 630 640 650 660 Me tA s n T y r T h r A s n G I y As n L y s As n I L e G L n G L n 1 e P h e G L n G L n A s n 1 L e L eu G L u ATGAATTATTAAGGAAACAAAATT[AATTATATTCA[ AATATGGAA 490 500 690 700 710 720 f 8B. AsnVt~er~u~r~teys~e~teis~e6Iysn~y~sp~s~y~isie~t i I C a C C C C C C C C C CC CC C C*C CCC C C C C C C. CC C C C C C C C C C C C C C C AsnAspValLeuAsnG I nGtuThrGuG~uG [uMetGluLysG tnVQ[G~uA I aI [eThr MATGATGTTCTTAATCAAGAAA 730 740 750 760 770 780 Lys sGlnlteGlu At ci3tuVal.AspALQaLeuAl aProLys AsflLysGuGtuGIuGu Lys AAGCAAATAG AAGCTGAAGTGGATGC CCTC GCACCAAAAAATAAGGAAGAAGAAGAAAMA 790 800 810 820 830 840 Glu Lys GLuLysGuLy [uLysGuG~u Lystu Lys G I uGuLysG LysGuLys GAAAAAGAM AA 3AA G1AAAM6GAAGAAAM GAAAAG AAG AAMAAGAAAAAG3AAAAA 850 860 870 880 890 900 G[uGtuLysGluLysGtIuGlu Lys GluLys GuGuLysGGuLysGtu Glu Glu Lys AAAAAG AAAAAG AAGAAAAAGAAAGAA GAAAAAGAAGA AAAAGAAGAAGAAAMA 910 920 930 940 950 960 L ysAs n AAAAA f 1G.8C A FC-27 r4F-7 K-i 4j -6.4 kb 1 21 2 12 FIG. 9 0* F1/G. S. *S* Se C. CS C C S C S C C C C C S C S 20 30 40 50 60 701 TT[CTCTT[TTC[TCTT[TTCTTCTT[[TTTCTTCTTCTTTTCTTCTTCATCTTCTT[TA[TTCTTCTI 810 820 830 840 850 860 87 01 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Li 90 100 T[ATCTTCTTGTAC CTCTTTAGATT[TT[T 280 290 300. TTCTT[AA[TTTTGATTTATC CATTAATGT 480 490 500 CTTTTTTTGC TTTTTTATGTTTTCCTTTAT 680 690 700 GTTACt3T[TGT[AATGGA[TT6G TAATGGT. 880 890 900 S 5 *5 S S S. S S S S S S S S S S S 110 120 130 14-0 150 160 170 TGTACCT[TTTAIGATTCTTCTTTTTTGT[TTCTTCCTTATCAACATGTTTCTGTTTTTGAAGTTCAG[TT 310 320 330 340 350 360 370 TAATTGTTCTGfTTCATCTATTAGTTTA[ATTGTG6TTGGCTTAGTGGTCTACTTT[TACTGTTATGTGT 510 520 530 540 550 560 570 710 720 730 740 750 760 770 A[CATAATTT[ATTGTTGGTTT[TT[A[TTGTTTGAT[TGGTTGTTf3CATTGTTTCTT[ATTTTCCTTAT 910 920 930 940 950 960 970 I~ 180 190 200 W[-TCTAT[TT[CCCATTTTTTTCTTATGAT 380 390 400 iTGTT[ TTCA[ATh [CT[TTTATCTT[TAAG L1 580 590 600 JTTCTTCTTTGTGTTC-TT[ TT[TTTGTGTT[ 780 790 80,0 C ATG TTTTATTAATATATGATTAATTAAAA FIG. /0B. YOU y J v I uV'u ft ft ft e ft ft ft C ft ft ft.. ft ft ft. ft.. ft.. ft ft ft ft S. SC ft ft a C ft S ft S ft 20 GAATTCAG AGCTCAMAGT 82 92 GATTCCCCTA CTGTTAAACC 154 164 GTATATATTA AGGAGAATAC 30 40 50 60 TCTAt3TTCA[ AGTTCTAGTT CAAGTTCAGA AAGTIZTT[[T GCTAATGGA[ 102 112 122 132 142 G[CAAGAAAT TTAtZAAAATA TATGTGAAA[ TGGAMAAAAC TTCAAGTTCC 174 184 194 204 214 ATTAATA[TT AMATGGAAAG TATAWGGAGA AACAA&4AGAA TACTACTGAA 226 TAACAMAGTT G 236 246 256 266 276 286 FIG. AATAAT-CAAAACAATAATGGMAAGTGTAAA[ATATATAATAATCAAAG [GATTTAATAA ATAA[TATTTTAATAATAATAATAATAATAATAATM[CAATATG [CAAATATATT[TAGT r3AATATGTA[A-GM[CATATTATAACA[ACATATGAAT[ATTCATAATGATAATAAGGAA TTATCATATTGATGATTCAAAAAGAAT-'fTTAATTAT FIG./2 gar a C S a a S S S a. S e~c S *a .4 S 4 P 4 1 ama a r a a a a a S a S S 20 30 40 .50 60 701 GAATTCTTTAATGGTAAAGAACCAAATAGAi3GTATAAATCCT'iATGAAGCTGTTGCTTATGGTG[TG[TAI 140 150 160 170 180 190 200 CATTAACTTTAGGTATAGAAACTGTGGGTGGTATTATGA[ACAATTAATTAAAAGAAATACTGT[AT[CCC 270 280 290 300 310 320 330 TTTTGAA6GAGAAAGAGCATTAACCAAAGATAAT[A[[TTTTAGGAAAGTTTGAATTATCTG6TATTC[A 400 410 420 430 440 450 460 TTACATGTTGAAG[TGAAGA [AAAGGTACAGGTAAAAGTAA3GGTATACTATTA[TAATGACAGTA3 530 54-0 550 560 570 580 590 AACTTAAGAGAAAAGTTGAA6G[CCAAAATAACCTGATAATTATATA[AGAGTATGAAAG[AA[TGTTGAA 660 670 680 690 700 710 720 TGTTAAAGATGTTGAAGATTGUGTTAAATAATAACTCGAATGTTG ATT[T6AAG [ATTAAAA[AAAAATTA 790 800 810 820 830 840 850 CTT[AC CAC'AAC[TAGTGGAGA[GAAGATGTAGATAGTGACGAATTATAAAAT[TT[A[ATTTTATGAAT 920 930 940 950 9 60 970 9801 TTTAAA[AAATTAAAAAAAATAACATATATATGTATATATATATATATATATATATATATATATGTATAT FIG- /JA. 90 100 110 120 130 T[[AAt3CAGGTATTATTTTAtGTGAAAATTA[AAGACGTTGTTTTATTAGATGTTA[TC 210 220 230 240 250 260 AN? [AAAAAATtIA[AAAC CTTTT CAACATATCAAGATAAC[CACTGT[TTIAATTCAGT 340 350 360 370 380 390 rCACA[AAAGAGGAGTA[[[AAAATTGAAGTTACICTTTACCGTAGACLAAAAATGGTAT[ 470 480 490 500 510 520 ATTATCGAAAGAA[AAT[GAAAAAATGATGAATGATGCAGAAAATTCG[AGTTGAGATAA 600 610 620 630 640 650 S 730 740 750 760 770 780 AGAT[TTGAAGCTGTATG[[AA[[AATCATGTTAAATTATATGGTCAA[CAGGAGGA[ 860 870 880 890 900 910 ATATTATTATTTAATGTATAATTATGATATATTATGAATTA[[TT[TTTTT ~AGTTAAATGTATATATAAAAAAAAAACGGAATT[ 0010014 FIG. /38. C A S S S. *.S S S. S S S S AATTCCGTA[ 130 TAGATTTGGA 250 AATC[AAT 66 370 TAAAA6AAAA 490 ATAC[ATCT 66 61 0 TAT CTTATTT 20 TAATGTTGTA 140 TTT6 6TT6AA 2 60 TTCAAGAAMA 30 A[A[[ACTTA 150 6 GATT6TT6T 270 A6TA 6TAATA 40 50 601 T[ATA[AA6[ ACAATCA6TA ATG[ACT[TAI 160 170 180 GTIZ6TAAAAA TGAATTGCCZA TATTT6AAAA 280 290 300 AAMAATA TAATGAATCT GATAAAAGAG, 380 390 400 410 AG[TAAAACA CTTGGGTTAA GTATTAT[6T ATTT6ATAAT 500 AACAT[ T666 620 ACCC 6TAT[CT 510 AAAC[[AAAG 630 CATATATATG 520 530 6 T6TTAT6TT M6GCAATA66 640 650 AAA6 66TTAT ITTTTTTCATT 420 ATGAAIAG3A 5401 AATTT6 TATA 660 G[TTTGTTTT 730 740 750 760 770 780 ATTCAAAA6C TGAAATTATA TTA66A6TAC CCAM6GTTTT TAATA6AAT6 TATGCAiAZTA 850 860 870 880- 890 9001 ATTTA[6TMA AGGTAAAAAT AAT66AAATT TCA6TAAA'3T TGTTGAAG6T ATTA[TAATA 970 980 990 1000 1010 1020 G6AAATTATCT[[AG466TT 6CT6A66GTT TAAGT6TTCLT ATTAAATGTTAAGTATTATC FIGA/A. S .5 a S a a. a a a a S a a a a a t AATTTAGTAT 190 AGC TGATAAT 310 AAGA[ATTAG 80 AGATATAATT 200 TTTAGATAAT 320 TTTGTGTG CC 430 440 ATAAMATAGC [AATGTTACT 550 560 *ATGGTGTAAT A[CT[[ATGT 670 680 TGGGTGTAAA GATAAATATA 790 800 TTATGACG6AA AATAAATAAT 910 920 TATCAAGAAA AATAAAAGAT 1030 1040 AAGG ATATGG TTTAAfIGGMA 90 Afl GATATAT 210 C TAACTAAGC 330 TTAGAATG TG 450 GTTCAAAAC6 570 GATTGTAATA 690 TGGAGTAGAG 810 TTATCACGT 930 AAGATAAA[C 1 00 TAMATAATAC 220 GTAGTGMAAT 340 ATAAGGAAAA 460 AAGATCCTAA 580 TAATAAAGAA 700 ATATAAAA1T 820 GTMAGAAGTG 940 CTAATATGGA 110 AAAATTAGMA 230 GAAGATAGMA 350 AATAGAAA4G 470 TTTTATTGCC 590 ATAT [CT TA 710 TTTGAATACA 830 GATAGCAAMA 950 TGTTATCTTA 120 TGGTTGTG]T 240 AATGAAGAMA 360 ATTAATTCAT 480 TCTATTGTGT 600 ACAACACATT 720 GACATATG TA 840 CAGGCTATAA 960 AATG6N3GGTG 1050 1060 1070 1080 TCTACGGGTC C[ATATTTTTACAAGATGTAGATGACTGTA fl-G.148. 0 .0. 0 S 0 *0 0 **0 0* 00 0 0 0 0 S 0 0 0 C S 1090 1100 ACA[T6AAAG TAT666A6TA 6C 1110 1120 130 f f T6TTT[T[ [TA6TA[4A ATA[AAA6TA A6M[CATG66 121 0 T6TTTA6 T66 1330 GAT[AAA 666 1220 ATA[TTT TTA 1340 TTT66TTAAA 1230 6AAAA66MAT 1350 TTATCTCM6G 1 240 [TA CA6MCA 1360 6T6AATATAT 1250 T6[TTT [AC6 1370 AGAAACTGAA 14H0 1460 1470 1480 1490 AT66ACCATT 666AATTATA T[T6T66A[A AA[ATAAATT ATTTACATTT 1570 AMACATT A 1690 666 ACA[TA[ 1810 6CA[66 6T/A3 1930 AAAGT66AAG 1580 T 6AT[CT AITT 1700 AMACTA [CIT 1820 TAT 6AATA AT 1940 TAM6GATG AT 1590 TAT6TT6ATT 171 0 ACTCCMACAT 1 830 6 6TMM6AT6 1950 ATAMAAA6TG 1600 AT6TAAAf366 1720 TAAMAATAAG 1840 6M AAATC 1960 GAAGTAAAGt\ 161 0 MMAAT6AT6 1730 M6GATTC[MT 1850 T6ATAT TAMA 1970 TCATATAA 1260' MT6AT66TT 1380 AT6ATAAAT A 1500 TTAAAATG 16201 GAAMATA 1740' 6TAT TTAM6G 1860 66T66AAEVA, 1980 C66MIT FIG. 14C. 4. a *Z. S S a V S V V V V eq. a 9. 59 5 V S. .5 -1 V S V S V S S a 5 V V S 1150 AAATTTATAA 66CTA( 1160 IAGAT 1170 1180 1190 ACTATACCAA M66A6GAATT 6ITAATTA 1270 AlTITAA A C 1390 ATTTATATTC 1280 G6GA6ATATT 1400 [CAMTC CCT 1290 6 TACAlTA 1410 'TTT'iAAATT 1300 1310 AAAT6GG TTCTMTACA 1420 1430 TITTGTGTTC ATATGGT6AT 1200 AGT6iATTCTA 1320 TITTTIAGATA 1440 6ATTCTAT6 6 1510 1520 1530 1540 1550 1560 ATIAATATG TI AAA6ACAACT 6 6I6TA6ATG A GAMMIATI TICA6AAAMA ITAATI6ATG 1630 1640 1650 1660 1670 1680 AMAMCTAA TTTAAATA6A TACAAT6TTA TIAAIGACAI ATACTIAACT TCCAMACCAI 1750 1760 AT11111CIII ITTITATAGAT 1770 GAAGTAMM 1690 A6IAAAGATG 1780 1790 AGAAATAI6A A6AAAAAIIA 1900 1910 AIATAAAAAG I66M61AAA 1800 AGI66MAT 1920 6 CI6AATAA 1870 AAGAI6 AIAT 1880 AAAAAGTGGA 1990 2000 201 0 202 0 2030 2040 FIG./4D. IF A