Isolation, sequence and differential expression of the p58 gene family of Babesia bigemina

Abstract

Four copies of the gene encoding the merozoite surface protein p58 from the protozoan hemoparasite Babesia bigemina were amplified from genomic DNA by polymerase chain reaction (PCR) techniques, molecularly cloned and subjected to DNA sequence analysis. The amplified DNA (Bbg7, Bbg9, Bbg13, Bbg14) could be placed into 2 classes with respect to its size and the length of the open reading frame (ORF). With the exception of a single base substitution, the sequence of Bbg13 is identical to the cDNA sequence published earlier [1]. The Bbg7 and Bbg14 copies of p58 diverged from Bbg13 sequence at regions towards the 3' and 5' ends, respectively. In contrast, Bbg9 has incorporated both regions of divergence within its sequence. Using a cloned strain of B. bigemina, RNA-PCR and Northern blot analyses demonstrate the in vivo transcription of 3 of the 4 copies, although one of the 3 expressed copies is present in very low abundance. The relative abundance and size of the two p58 mRNA species detected are consistent with the 58- and 55-kDa proteins detected by in vitro translation of B. bigemina poly(A)+ mRNA by immunoprecipitation with an anti-p58 monospecific antibodies. These results indicate that the gene encoding p58 exists as a multigene family that appears to be differentially expressed in the blood stage of the parasite's life cycle.