Dr. Fosket has isolated and sequenced two soybean beta-tubulin genes (SB1 and SB2) which exhibit different patterns of developmental and organ specific expression. However, both genes are expressed in elongating internodes and they are strongly and rapidly down regulated at the transcriptional level when etiolated seedlings are exposed to light. He will determine the nature of the cis-acting elements controlling the internodal expression of SB1 and SB2. Constructs in which a promoterless E. coli beta-glucosidase (GUS) reporter gene is driven by the SB1 and SB2 promoters have been made and transferred to tobacco by Agrobacterium mediated transformation, and the chimeric genes are expressed in the transgenic tobacco plants. The effect of deletions and mutations of the putative promoter regions of SB1 and SB2 on the expression of the GUS reporter gene in transgenic tobacco will be determined to identify the regulatory elements responsible both for their expression in elongating internodes and for their light induced down regulation. GUS enzymatic activity assays and S1 nuclease protection experiments will be used to verify that GUS enzymatic activity is a reflection of the transcriptional activity of the chimeric genes. The precise cellular localization of GUS activity in the transgenic tobacco plants will be determined histochemically, while the cell specific expression of the SB1 and SB2 genes in soybean internodes will be will be determined by means of in situ hybridizations, using labeled, gene specific probes. Additionally, SB1 and SB2 specific antibodies will be generated and used to ascertain which microtubule arrays contain these tubulins. The trans-acting factors regulating the transcription of the SB1 and SB2 tubulin genes in soybean internodal tissues will be identified by means of footprinting and gel retardation assays. These DNA binding regulatory proteins will be isolated by affinity chromatography and the nature of their interaction with SB1 and SB2 promoter elements will be studied. Antibodies raised to these DNA binding proteins will be used to isolate cDNAs encoding them from a lambda gt11 expression library. The cDNAs will be used as probes to examine the regulation of expression of the genes encoding these DNA binding proteins. %%% In plants microtubules are an important subcellular component involved in determining how cells expand and as a consequence, how tissues grow. This study will elucidate how the expression of a microtubule protein is expressed during plant growth and in response to light.
