mAbs was localised within the rhoptries of both YM and 17X merozoites from the late schizont stage, by immunoelectron microscopy, but could not be detected in
association with either the parasite or the infected erythrocyte post-invasion (Oka et a i 1984). When the parasite proteins affinity purified using the mAb 25.77 were electrophoresed on 5% polyacrylamide gels, the protein band could be seen to resolve into at least three distinct bands, of unequal intensity. These bands could represent the differentially processed products of a single gene, or the products of multiple genes. When western blots of size fractionated total parasite proteins were probed with the mAbs, the same multibanded pattern was produced, indicating that all, or several of the proteins present in the complex contain the antigenic determinant recognised by the protective antibodies (A. Holder, unpublished observation).
A clone containing a 145bp P. yoe//7 YM genomic DNA insert was subsequently isolated from a Xgtl 1 expression library screened with P. yoelii
hyperimmune serum (Keen etal. 1990). Antibodies affinity-selected using this clone recognised protein within the rhoptries in immunofluorescence studies. The insert from the clone (J7), hybridised with multiple bands on a Southern blot of P. yoe//7 YM
genomic DNA, restricted with Psal, indicating that multiple copies of this sequence existed in the parasite genome. Subsequently a Oral genomic DNA recombinant library was screened using J7, and four 500bp positive clones were identified. The inserts of these clones, S6, S7, S8 and S I 2 were sequenced, and found to represent the 3’ termini of three highly conserved but distinct genes. S12 was identical to S6. In each of the clones the sequence could be translated into a single open reading frame ending with in-frame stop codons followed by non-coding sequence. S6 was cloned into a bacterial expression vector and expressed as a fusion protein. Anti-serum raised against this protein in rabbits immunoprecipitated a 235kDa P. yoelii protein from solubilised parasite extracts, and reacted with rhoptries in immunofluorescence assay. S6, was used subsequently used to screen an EcoRI library and two large clones, E3 (6.8kbp), and E8 (5.6kbp) were identified (Holder et ai, 1990; Keen et a i 1990).
Clones E3 and E8 were characterised and completely sequenced by Jane Keen and Katharine Sinha while the work presented in this thesis was in progress. Both clones had open reading frames (ORFs) throughout, with no introns present. E3 was identified as comprising an almost entire coding sequence which overlapped with 88. E8 represents the majority of a corresponding gene overlapping with the 86
sequence (see Figure 1.4). E3/S8 contains an open reading frame throughout encoding a protein of 268kDa. There is a methionine residue encoded by the twenty- sixth codon of the translated E3 sequence, and this is followed by what appears to be a 19 amino acid signal sequence (Keen etal., 1994). This could constitute the start of the gene, but as no in-frame stop codons occur prior to the putative initiating
methionine residue it cannot be stated that this is the true start. If this methionine did represent the start of the gene, the predicted size of the protein would be 265kDa, 13% larger than the observed size of the rhoptry proteins present in the 235kDa complex. The proteins predicted by the sequences E3/S8and E8/S6, appear to contain little structure. Generally the proteins were predicted to be fairly hydrophilic, with the exception of a 16 amino acid putative transmembrane domain, present towards the C-terminus in both sequences. This region was followed in both predicted proteins by a 45amino-acid hydrophilic ‘cytoplasmic tail’ sequence. The E3/S8
sequence contained eleven encoded cysteine residues, while the E8/S6 sequence predicted only seven cysteines, all of which were conserved in E3/S8. The conserved cysteine residues were not tightly grouped, although the additional cysteines did contribute to a cluster of six residues in E3/S8. There are a large number of potential N-glycosylation sites in each clone (38 in E3/S8, 27 in E8/S6) but there is no evidence to suggest that any are used in the native rhoptry protein (Sinha, 1992). Almost immediately N-terminal of the transmembrane domain of each clone are very short reiterated sequences. In E8/S6 the sequence is longest, consisting of seven tripeptide repeats, five of the repeats have the sequence aspartate-isoluecine-asparagine, while the third and sixth copies are degenerate comprising aspartate-valine-isoluecine, and aspartate-threonine-isoluecine respectively. In E3/S8 the sequence is as in E8/S6, except that the third repeat has been deleted. 87, the partial copy of a third member of the gene family contains the shortest repeat sequence consisting of two aspartate- isoluecine-asparagine repeats separated by a glutamate-isoluecine-asparagine sequence.
Because no upstream sequences are available for the genes represented by E3/S8 and E8/S6, it is still unclear whether both represent the complete sequences of functional genes, and if so whether either or both of these genes is expressed.
As the work in this thesis progressed it became apparent that there were more copies of the gene-family than had been anticipated. In order to provide a minimum copy number for the gene-family, Martin Borre amplified, from P. yoelii YM genomic DNA a Ikbp central region of the gene, using primers based on sequence conserved in E3/S8 and E8/S6. This region of the gene was chosen because the amplified
sequence contained unique Hinf\ restriction sites in E3/S8 and E8/S6. The products, which formed a single band on agarose gels, were cloned and the clones grouped
according to the restriction patterns produced from their inserts. Seven different patterns were observed, providing a revised minimum copy number for the gene family. Among the seven restriction pattem types, three variants appeared most frequently: one was recognised as being E3-like, another was E8-like and the third clone, for which no cloned gene existed was termed 5. When clones within these groups were sequenced, variation was observed among members of each group, as well as between the different restriction pattem groupings. In total sixteen clones were sequenced, and of these eleven were demonstrated to be different. This result
appeared to indicate that the minimum copy number of the gene family was eleven.
Aims of this project
The aim of this project was to establish the genomic organisation of the gene family encoding the 235kDa rhoptry protein complex of P. yoelii virulent strain YM, in order to establish a minimum copy number for the gene family and to investigate whether the members of the gene family occur independently, or in association. A further aim was to investigate the distribution of the gene family in the avirulent parasite strain 17X from which YM was believed to have evolved as the result of a single mutation event. Comparison of the genomes of the two parasite would permit an assessment of whether a single mutation event could separate the two parasites. Initially this would require the production of an electrophoretic karyotype for the species, including determination of whether the chromosome number of this parasite was the same as other in other Plasmodia. It was hoped that this work would also permit the question whether chromosomal rearrangements or polymorphisms affecting the rhoptry protein gene family could form the basis of the differentially expressed virulent character.
CHAPTER 2 : MATERIALS AND METHODS