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The high-mobility-group (HMG) chromosomal protein wheat HMGa was purified to homogeneity and tested for its binding characteristics to double-stranded DNA. Wheat HMGa was able to bind to P268, an A/T-rich fragment derived from the pea plastocyanin gene promoter, producing a small mobility shift in gel retardation assays where the bound complex was sensitive to addition of proteinase K but resistant to heat treatment of the protein, consistent with the identity of wheat HMGa as a putative HMG-I/Y protein. Gel retardation assays and southwestern hybridization analysis revealed that wheat HMGa could selectively interact with the DNA polynucleotides poly(dA).poly(dT), poly(dAdT).poly(dAdT), and poly(dG).poly(dC), but not with poly(dGdC).poly(dGdC). Surface plasmon resonance analysis determined the kinetic and affinity constants of sensor chip-immobilized wheat HMGa for double-stranded DNA 10-mers, revealing a good affinity of the protein for various dinucleotide combinations, except that of alternating GC sequence. Thus contrary to prior reports of a selectivity of wheat HMGa for A/T-rich DNA, the protein appears to be able to interact with sequences containing guanine and cytosine residues as well, except where G/C residues alternate directly in the primary sequence.

Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.