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

Schistosoma mansoni p14 gene encodes an eggshell precursor that is expresse d only in vitelline cells of mature female worms in response to a male stim ulus. The upstream region of the p14 gene contains several potential cis-ac ting regulatory sequences. We used the upstream region of the p14 gene as b ait in a yeast-one-hybrid screen of a S. mansoni cDNA library to identify i nteracting proteins. We report the identification and characterization of a cDNA (S. mansoni PUR-alpha (SmPUR-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p 14 upstream region and activates transcription of the HIS3 reporter gene in yeast. Sm-PUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha. The DNA binding domain o f SmPUR-alpha is highly conserved. We show by gel shift assays that GST-SmP UR alpha binds to oligonucleotides comprising the p14 upstream region. SmPU R-alpha binds preferentially to single-stranded DNA and also binds RNA. Unl ike the mammalian homologue, SmPUR-alpha exhibits little specificity for th e PUR element GGn, but shows strong preference for a sequence containing al ternating pyrimidines. Our data support that SmPUR-alpha is a single-copy g ene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-alpha. is constitutively transcribed in many cell types and thus likely plays a role as a general transcription act ivator in schistosomes. (C) 2000 Elsevier Science B.V. All rights reserved.