Identification and functional characterization of a member of the PUR-α family from Schistosoma mansoni

Schistosoma mansoni p14 gene encodes an eggshell precursor that is expressed only in vitelline cells of mature female worms in response to a male stimulus. The upstream region of the p14 gene contains several potential cis-acting regulatory sequences. We used the upstream region of the p14 gene as bait in a yeast-one-hybrid screen of a S. mansoni cDNA library to identify interacting proteins. We report the identification and characterization of a cDNA (S. mansoni PUR-alpha (SmPUR-α)) encoding a protein homologous to single-stranded DNA transcription activator PUR-α, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast. SmPUR-α has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-α. The DNA binding domain of SmPUR-α is highly conserved. We show by gel shift assays that GST-SmPUR-α binds to oligonucleotides comprising the p14 upstream region. SmPUR-α binds preferentially to single-stranded DNA and also binds RNA. Unlike the mammalian homologue, SmPUR-α exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines. Our data support that SmPUR-α is a single-copy gene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-α is constitutively transcribed in many cell types and thus likely plays a role as a general transcription activator in schistosomes. (C) 2000 Elsevier Science B.V.