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 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-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast. SmPUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha. The DNA binding domain of SmPUR-alpha is highly conserved. We show by gel shift assays that GST-SmPUR-alpha binds to oligonucleotides comprising the p14 upstream region. SmPUR-alpha binds preferentially to single-stranded DNA and also binds RNA. Unlike the mammalian homologue, SmPUR-alpha exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines. Our data support that SmPUR-alpha is a single-copy gene 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 activator in schistosomes.
Fantappie
M R
MR
Department of Microbiology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, 14214, USA.
Osman
A
A
Niles
E G
EG
LoVerde
P T
PT
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D006224:Cricetinae; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D008297:Male; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014157:Transcription Factors; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D000096985:Calponins
D017362:Cyclic AMP Response Element-Binding Protein; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D015801:Helminth Proteins; D009124:Muscle Proteins; D014157:Transcription Factors; C425295:PUR-alpha protein, Schistosoma mansoni; C118619:calponin homolog protein, Schistosoma japonicum; D000096985:Calponins