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

Krzysztof Szczyglowski, L. Szabados, Susan Y. Fujimoto, David Silver, Frans J. De Bruijn

Research output: Contribution to journalArticle

36 Citations (Scopus)

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 β-globin genes. The possible analogies between these two systems will be discussed.

Original languageEnglish
Pages (from-to)317-332
Number of pages16
JournalPlant Cell
Volume6
Issue number3
Publication statusPublished - Mar 1994

Fingerprint

Leghemoglobin
Sesbania
Sesbania rostrata
leghemoglobin
Mutagenesis
site-directed mutagenesis
Site-Directed Mutagenesis
promoter regions
Mutation
mutation
cells
Genes
Genetic Promoter Regions
Globins
Initiator Codon
Gene expression
nopaline
Hemoglobins
Substitution reactions
start codon

ASJC Scopus subject areas

  • Plant Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

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. / Szczyglowski, Krzysztof; Szabados, L.; Fujimoto, Susan Y.; Silver, David; De Bruijn, Frans J.

In: Plant Cell, Vol. 6, No. 3, 03.1994, p. 317-332.

Research output: Contribution to journalArticle

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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 β-globin genes. The possible analogies between these two systems will be discussed.",
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