Gibberellin treatment stimulates nuclear factor binding to the gibberellin response complex in a barley alpha-amylase promoter.

The promoters of a majority of cereal alpha-amylase genes contain three highly conserved sequences (gibberellin response element, box I, and pyrimidine box). Recent studies have demonstrated the functional importance of four regions that either coincide with or are immediately proximal to these three conserved elements as well as an upstream Opaque-2 binding sequence. In this study, we describe the characterization of nuclear protein factors from barley aleurone layers whose binding activity toward gibberellin response complex sequences from the barley low-pl alpha-amylase gene (Amy32b) promoter is stimulated by gibberellin A3 (GA3) treatment. Barley proteins isolated from crude nuclear extracts prepared from aleurone layers incubated with or without GA3 were fractionated by anion exchange fast protein liquid chromatography and studied using band shift assays, sequence-specific competitions, and DNase I footprinting. A GA3-dependent binding activity eluting at 210 mM KCl was shown to bind specifically to the gibberellin response element and the closely associated box I. DNase I footprinting with the proteins in this fraction indicated interactions with sequences in the gibberellin response element and box I. A second DNA binding activity eluting at 310 mM KCl was present constitutively in extracts prepared from tissues incubated both in the absence and in the presence of hormone. Proteins in this fraction were able to bind to many DNA sequences and, in general, were largely nonspecific. DNase I footprinting with the proteins in this fraction indicated a large area of protection with a single unoccupied region located at the 3' end of box I. The possible function of such an activity in hormone regulation of the alpha-amylase genes is discussed.     

Characterization of genes encoding two manganese peroxidases from the lignin-degrading fungus Dichomitus squalens(1).

Genes encoding two manganese peroxidases from the white-rot basidiomycete Dichomitus squalens were cloned and sequenced. The mnp1 and mnp2 genes encode mature proteins of 369 and 365 amino acids, respectively. The amino acids involved in peroxidase function, those forming the Mn(II) binding site, and those forming the five disulfide bonds in other Mn peroxidases are conserved in these sequences. Both predicted D. squalens proteins contain multiple acidic residues in their C-terminal sequences, which may be involved in additional metal binding. Both genes contain seven small introns, the locations of which align with each other. The promoters of both D. squalens genes contain putative AP-2 sites, which may be involved in their regulation by nutrient nitrogen. Southern blot analysis of genomic PCR fragments suggests that these sequences represent separate genes rather than allelic variants.     

Excess information at bacteriophage T7 genomic promoters detected by a random cloning technique.

In our previous analysis of the information at binding sites on nucleic acids, we found that most of the sites examined contain the amount of information expected from their frequency in the genome. The sequences at bacteriophage T7 promoters are an exception, because they are far more conserved (35 bits of information content) than should be necessary to distinguish them from the background of the Escherichia coli genome (17 bits). To determine the information actually used by the T7 RNA polymerase, promoters were chemically synthesized with many variations and those that function well in an in vivo assay were sequenced. Our analysis shows that the polymerase uses 18 bits of information, so the sequences at phage genomic promoters have significantly more information than the polymerase needs. The excess may represent the binding site of another protein.