ABSTRACT
The CCAAT sequence in the amdS promoter of Aspergillus nidulans is recognized by AnCF, a complex consisting of the three evolutionary conserved subunits HapB, HapC, and HapE. In this study we have investigated the effect of AnCF on the chromatin structure of the amdS gene. The AnCF complex and the CCAAT sequence were found to be necessary for the formation of a nucleosome-free, DNase I-hypersensitive region in the 5' region of the amdS gene. Deletion of the hapE gene results in loss of the DNase I-hypersensitive site, and the positioning of nucleosomes over the transcriptional start point is lost. Likewise, a point mutation in the CCAAT motif, as well as a 530-bp deletion which removes the CCAAT box, results in the loss of the DNase I-hypersensitive region. The DNase I-hypersensitive region and the nucleosome positioning can be restored by insertion of a 35-bp oligonucleotide carrying the CCAAT motif. A DNase I-hypersensitive region has been found in the CCAAT-containing fmdS gene and was also hapE dependent. These data indicate a critical role for the AnCF complex in establishing an open chromatin structure in A. nidulans.
Subject(s)
Amidohydrolases/genetics , Aspergillus nidulans/genetics , DNA, Fungal/metabolism , DNA-Binding Proteins/metabolism , Deoxyribonuclease I/metabolism , Promoter Regions, Genetic , Aspergillus nidulans/enzymology , CCAAT-Enhancer-Binding Proteins , Chromatin/ultrastructure , Fungal Proteins , Genes, Fungal , Nucleosomes , Repressor Proteins , TATA BoxABSTRACT
Poly(ADP-ribose) is routinely detected by the use of radioactive polymers formed from labeled substrates. In this report a simple and time-saving method for the biotinylation and the detection of poly(ADP-ribose) on blots is described. The polymer modified by light-induced reaction with photobiotin was colorimetrically detected and quantified, using streptavidine-alkaline phosphatase conjugates. The separation of poly(ADP-ribose) chains on polyacrylamide gels was not affected by the biotinylation of the polymers. When biotinylated poly(ADP-ribose) was used to detect the poly(ADP-ribose) binding capability of proteins in ligand blots, the results were comparable to those obtained with poly([32P]ADP-ribose). Experiments with histones and rat liver nuclear proteins demonstrate that in studies on poly(ADP-ribose)-protein interaction, this method is applicable to the detection of poly(ADP-ribose) binding proteins.
