Distribution and characterisation of the 235 kDa rhoptry multigene family within the genomes of virulent and avirulent lines of Plasmodium yoelii

In the rodent malaria species, Plasmodium yoelii, a multi-gene family (Py235) encodes a 235 kDa rhoptry protein. This protein is believed to be involved in merozoite attachment and invasion of red blood cells. Only two members of Py235 have been sequenced so far. Using genomic DNA from the virulent P. yoelii YM line we have PCR amplified additional members of this gene family. These >8 kb full length clones have been cloned and sequenced. Based on differences within the tri-amino acid repeat structure at the C-terminal end of the Py235 protein, it has been possible to divide the multi-gene family into subtypes. The protein translations of five full-length genes (representing four different subtypes) were compared. While there was a high level of amino acid identity at the C-terminal end of these proteins, the N-terminal region revealed a great deal of sequence diversity. Critically, certain residues appeared to be conserved notably seven out of eight cysteines. Comparison of two full-length genes of a particular sub-type shows >99% amino acid identity at the protein level, implying that very closely related genes exist within the parasite genome. We have used this new sequence information to compare the distribution of Py235 in the virulent YM and avirulent 17X lines of P. yoelii. Our results indicate that while the overall distribution of Py235 genes is broadly conserved between the two lines, significant differences exist when individual subtypes are compared.