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Genet Mol Biol. 2009 Jan;32(1):129-32. doi: 10.1590/S1415-47572009005000021. Epub 2009 Jan 30.

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene.

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  • 1Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG Brazil.

Abstract

Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.

KEYWORDS:

Penicillium griseoroseum; 5' upstream regulatory sequence; electrophoretic mobility shift assay; polygalacturonase

PMID:
21637657
[PubMed]
PMCID:
PMC3032954
Free PMC Article
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