Organization and functional analysis of the Schistosoma mansoni cathepsin D-like aspartic protease gene promoter.
We have cloned a 969-bp fragment of genomic DNA that spans 821 bp of the 5' untranslated region, exon 1, a short intron, and part of exon 2 of the Schistosoma mansoni cathepsin D gene by inverse PCR. Inspection of this sequence revealed the presence of two TATA-box motifs, two inverted CCAAT-box (inverted NF-Y) motifs and sequences with homology to binding sites for the transcription factors, AP-1 and NF-Y. This sequence and deletion variants were cloned into reporter gene constructs, in order to examine the ability of these putative regulatory sequences to drive heterologous reporter gene activity. PCR products were cloned into the luciferase reporter vector pXP2. These reporter gene constructs were used to transform HeLa cells which were cultured and examined for luciferase activity. Additionally, HeLa cells transiently transfected with an EGFP reporter plasmid driven by the putative promoter from the S. mansoni cathepsin D gene were examined for EGFP transcripts and fluorescence. The 5' untranslated region of the S. mansoni cathepsin D gene, from position -772 to +40 (translation start ATG), included functional regulatory sequences capable of driving luciferase and EGFP expression, whereas shorter fragments from position -264 or -185 to +40 were insufficient to drive reporter activities.
Schulmeister
Alexandra
A
Department of Molecular Parasitology, Institute of Biology, Humboldt University Berlin, Philippstrasse 13, 10115 Berlin, Germany.
Heyers
Oliver
O
Morales
Maria E
ME
Brindley
Paul J
PJ
Lucius
Richard
R
Meusel
Grit
G
Kalinna
Bernd H
BH
D024506:5' Flanking Region; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002402:Cathepsin D / Q000235:genetics*; D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000032:analysis; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000235:genetics*; D016385:TATA Box; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014162:Transfection
D014157:Transcription Factors; D008156:Luciferases; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D