The overall objectives of this proposal are to characterize cis-and trans-acting factors that regulate the transcriptional expression of heat shock (HS) genes of soybean. The two representative genes to be characterized in this study, Gmhsp 17.5-E and Gmhsp 26-A, encode proteins of 17.5 and 26 kDa respectively and are members of the low molecular weight family of HS proteins (HSPs). This class contains from 20to 30 proteins that predominate the heat shock response in soybean and other plants (1,2). Gmhsp 17.5-E appears to be a typical low molecular weight HSP with regard to promoter and protein structure and is expressed primarily in response to heat shock. In contrast, Gmhsp 26-A represents a highly divergent member of the group, and is expressed in unstressed tissues and at elevated levels in responses to a variety of physiological stress (3,4). The identification of transcriptional regulatory sequences will be approached by in vivo analysis of promoter alterations including 5', internal, and single base mutations. The mechanisms of promoter function (DNA-looping and protein-protein contact) will be probed by the strategic placement of a series of insertions constructed in half helical turn increments. Expression of mutated genes will be primarily assessed transgenically by S1 nuclease mapping of RNA from crown gall tumors utilizing reference gene vectors that contain """"""""test"""""""" and """"""""reference"""""""" copies of the gene under study. The precision obtained using S1 nuclease mapping of RNA pooled from 200 to 300 tumors for each mutation will facilitate a fine structure analysis of the promoter. Once the indentity and location of functional domains of the promoter are known, the model developed using tumor expression will be tested in whole plants utilizing regeneration vectors. Characterization of trans-acting factors will be performed by in vitro binding and footprinting assays, and by in vivo footprinting of intact nuclei using gemonic sequincing methods. DNA binding proteins will be purified from soybean nuclear extracts using heparin agarose, ion exchange and oligonucleotide affinity chromatography. The specific interactions of purified protein fractions with DNA will be characterized in vitro by DNAse I, DMS, and hydroxyl radical footprinting and by methylation/ethylation interference assays. In vitro reconstitution experiments will be used to look for cooperativity in DNA binding due to interactions between multiple trans -acting factors.
