Owen, Carolyn Ann; (1994) Characterisation of genes coding for a 235kDA rhoptry protein of plasmodium yoelii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The highly virulent rodent malaria parasite, Plasmodium yoelii strain YM is capable of invading both mature erythrocytes and reticulocytes throughout the course of the infection. YM had been reported to have arisen abruptly from the avirulent P. yoelii strain 17X which is reticulocyte-restricted for most of the course of infection. A genetic cross between YM and an avirulent parasite strain derived from 17X indicated that the two parasites differed only at a single genetic locus, which was considered to confer the virulent phenotype. Monoclonal antibodies directed at a complex of 235 kiloDalton (kDa) proteins, present in the rhoptries of the pre-invasive P. yoelii merozoites had been reported to convert a virulent YM infection into an avirulent, reticulocyte-restricted, 17X-like disease. This indicated that the rhoptry protein complex might mediate virulence. DNA clones encoding three different 235kDa rhoptry proteins had been isolated and sequenced. In this thesis electrophoretic karyotypes were produced for P. yoelii strains YM and 17X, and where possible individual chromosomes have been identified as homologues of those of other malaria species by their hybridisation with specific DNA probes. Four chromosomes of strain 17X were shown to migrate at appreciably different rates from their YM homologues, indicating that they differed in size. The genomic location of members of the 235kDa rhoptry protein gene family and the gene copy number have been examined in both strains. A two-dimensional Southern blot technique was used to visualise the restriction fragments containing members of the rhoptry protein gene family derived from resolved chromosomes. These blots permitted the localisation of the cloned rhoptry protein genes within individual chromosomes. In strain YM the gene family was demonstrated to be present on six chromosomes and to have an estimated minimum copy number of forty. On individual chromosomes, the gene family members are organised in tandem arrays which occur almost exclusively in sub-telomeric locations. Two chromosomes have rhoptry protein genes at each end. A series of subclones derived from strain YM, which exhibited different levels of virulence, were also examined. No differences were detected in the distribution of the major loci for the rhoptry protein gene family in these parasite clones but a difference was observed in the rhoptry complex proteins immunoprecipitated from one of the clones using anti-rhoptry protein monoclonal antibodies. The major rhoptry protein gene loci which had been identified in YM were also present in strain 17X, in additional two major loci were also detected in this parasite which do not occur in YM. One of these loci was present on a chromosome (13 or 14) which does not contain rhoptry protein genes in YM. Immunoprecipitation experiments indicated that there was differential expression of the rhoptry protein genes in strains YM and 17X. Hybridisation with rhoptry protein gene probes demonstrated the presence of homologous sequences on at least six chromosomes in another rodent malaria parasite, P. berghei. The extent and the organisation of the gene family appeared to be similar in P. yoelii and P. berghei. Initial experiments appeared to link a single genetic locus of the gene family with reticulocyte-restriction in both species. However analysis of the progeny of a genetic cross between virulent and avirulent parasites indicated that this was not the case. While ail of the data collected supported a very close relationship between P. yoelii strains YM and 17X, the fact that the strains had been demonstrated to differ at more than one genetic locus, and possessed four differently sized chromosomes indicated that YM could not have evolved directly from 17X as the result of an abrupt mutation. No evidence was found to support the hypothesis that the rhoptry protein complex is responsible for the virulent phenotype.

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