title: Site-specific mutagenesis of the nodule-infected cell expression (NICE) element and the AT-rich element ATRE-BS2* of the Sesbania rostrata leghemoglobin glb3 promoter.
abstract: Sesbania rostrata leghemoglobin glb3 (Srglb3) promoter sequences responsible for expression in infected cells of transgenic Lotus corniculatus nodules were delimited to a 78-bp Dral-Hinfl fragment. This region, which is located between coordinates -194 to -116 relative to the start codon of the Srglb3 gene, was named the nodule-infected cell expression (NICE) element. Insertion of the NICE element into the truncated nopaline synthase promoter was found to confer a nodule-specific expression pattern on this normally root-enhanced promoter. Within the NICE element, three distinct motifs ([A]AAAGAT, TTGTCTCTT, and CACCC[T]) were identified; they are highly conserved in the promoter regions of a variety of plant (leg)hemoglobin genes. The NICE element and the adjacent AT-rich element (ATRE-BS2*) were subjected to site-directed mutagenesis. The expression patterns of nine selected Srglb3 promoter fragments carrying mutations in ATRE-BS2* and 19 with mutations in the NICE element were examined. Mutations in ATRE-BS2* had varying effects on Srglb3 promoter activity, ranging from a two- to threefold reduction to a slight stimulation of activity. Mutations in the highly conserved (A)AAAGAT motif of the NICE element reduced Srglb3 promoter activity two- to fourfold, whereas mutations in the TCTT portion of the TTGTCTCTT motif virtually abolished promoter activity, demonstrating the essential nature of these motifs for Srglb3 gene expression. An A-to-T substitution in the CACCC(T) motif of the NICE element also abolished Srglb3 promoter activity, while a C-to-T mutation at position 4 resulted in a threefold reduction of promoter strength. The latter phenotypes resemble the effect of similar mutations in the conserved CACCC motif located in the promoter region of mammalian beta-globin genes. The possible analogies between these two systems will be discussed.
authors.affiliation: Department of Energy Plant Research Laboratory, Michigan State University, East Lansing 48824-1312.
authors.forename: K
authors.initials: K
authors.lastname: Szczyglowski
authors.forename: L
authors.initials: L
authors.lastname: Szabados
authors.forename: S Y
authors.initials: SY
authors.lastname: Fujimoto
authors.forename: D
authors.initials: D
authors.lastname: Silver
authors.forename: F J
authors.initials: FJ
authors.lastname: de Bruijn
mesh_terms: D001483:Base Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D007887:Fabaceae / Q000235:genetics* / Q000382:microbiology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005966:Glucuronidase; D007874:Leghemoglobin / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000235:genetics; D030821:Plants, Genetically Modified; D010946:Plants, Medicinal; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012231:Rhizobium; D012689:Sequence Homology, Nucleic Acid; D013559:Symbiosis / Q000235:genetics
chemical_list: D007874:Leghemoglobin; D005966:Glucuronidase