Isolation of the genes encoding the 51-kilodalton and 28-kilodalton subunits of Crithidia fasciculata replication protein A

Abstract

The genes encoding the 51-kilodalton subunit (p51) and the 28-kilodalton subunit (p28) of replication protein A (RP-A), designated CfaRPA1 and CfaRPA2 respectively, were cloned from the trypanosomatid Crithidia fasciculata by screening a genomic DNA library in the expression vector lambda gt11 with antibodies raised against purified C. fasciculata RP-A. CfaRPA1 has a single open reading frame encoding a polypeptide of 467 amino acids and a molecular mass of 52.0 kDa. The predicted p51 polypeptide has sequence similarity to the corresponding subunits from human, Xenopus laevis, and Saccharomyces cerevisiae, but is lacking a segment of approximately 20 kDa from its amino terminus, accounting for its smaller molecular weight when compared to the large subunits of RP-A from these other organisms. CfaRPA1 contains a zinc-finger motif that is also found in the RP-A large subunits from human, frog, and yeast. CfaRPA2 contains a single large open reading frame encoding a polypeptide of 258 amino acids and a molecular mass of 27.5 kDa. The predicted polypeptide has significant sequence similarity to the middle subunit of RP-A from human cells, mouse cells, and the budding yeast S. cerevisiae. Northern hybridization analysis of polyadenylated RNA from C. fasciculata indicates that both cloned genes are expressed as polyadenylated transcripts. CfaRPA1 hybridized with a 2.30-kb transcript and CfaRPA2 hybridized with a 1.44-kb transcript.