title: Upstream regulatory elements controlling expression of the Entamoeba histolytica lectin.
abstract: Entamoeba histolytica genomic organization and putative promoter elements appear to be distinct from both metazoan and better characterized protozoan organisms. The recent development of DNA-mediated transfection for E. histolytica enabled characterization of cis-acting promoter elements required for gene expression. A deletion and replacement analysis was conducted on the promoter of an E. histolytica gene encoding the heavy subunit of the N-acetyl-beta-D-galactosamine-specific adhesin (hgl5). Deletion of the DNA from -1000 bases to -272 bases upstream from the start of transcription of hgl5 did not decrease reporter gene expression. Subsequent nested deletions and 10-bp replacement mutagenesis identified four positive upstream regulatory elements between bases -219 to -200, -189 to -160, -69 to -60, and -49 to -40. A negative upstream regulatory element between bases -89 to -80 was conserved upstream of three other E. histolytica genes. Mutation of the previously unidentified 'GAAC' element conserved within the putative core promoter decreased reporter gene expression by 75%. Site directed mutagenesis of the putative TATA element decreased reporter gene expression by greater than 50%, while mutation of the putative initiator element resulted in a more modest decrease. This analysis suggests that E. histolytica promoters are unlike other protozoan promoters, with AT-rich upstream regulatory elements, a non-consensus TATA element, the "GAAC' element, and an unusual initiator element.
authors.affiliation: Department of Medicine, University of Virginia, Charlottesville 22908, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Purdy
authors.forename: L T
authors.initials: LT
authors.lastname: Pho
authors.forename: B J
authors.initials: BJ
authors.lastname: Mann
authors.forename: W A
authors.initials: WA
authors.lastname: Petri
mesh_terms: D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004748:Entamoeba histolytica / Q000235:genetics*; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D005809:Genes, Regulator; D037102:Lectins / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D014162:Transfection
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D037102:Lectins; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; C052452:galactose inhibitable adherence protein, Entamoeba histolytica