The long term objective is to develop a simple visual assay for gene expression in all of the major tissues and organs of the maize plant. Sophisticated maize genetics has allowed recognition of diverse regulatory phenomena including the discovery of transposable elements; the proposed studies using this model system will address important questions such as the regulation of gene expression in homologous organs for which this organism is particularly well-suited. These fundamental studies should be applicable to all eukaryotes. Using recently developed electroporation methods for the transient assay of gene expression and selection of stable transformed cultures of maize, promoters and other regulatory sequences conferring different constitutive levels of expressiion, tissue specificity and induction during hypoxia will be characterized. The role of viral sequences as enhancers will be tested in these transformation protocols as will the possibility that autonomous replicating sequences isolated from maize can act as origins of replication in their normal host. The properties of the transposable element Mutator required for its excision will be tested by screening for excision from the 5' region of the lacZ gene; constructs will be electroporated into maize cells, plasmids will be recovered after several days and transformed into E. coli cells where the frequency of blue colony formation indicative of restoration of lacZ function can be readily scored. Mutation of Mutator such as deletion of the host sequence duplications, inverted repeat ends and reading frames will define which features are required for excision; a complementation assay for the reading frames may be possible by co-porating RNA transcripts derived from each reading frame. In parallel with these experiments new methods will be developed for either the regeneration of whole plants from transformed protoplasts or the transformation of intact tissue segments capable of regeneration. Genetic stocks in which all tissues and organs should be purple due to anthocyanin accumulation will be transformed with the one missing structural gene of the pathway; expression of this structural gene will be driven by various promoters and a simple visual assay of the plant will indicate the tissue-specificity conferred by each construct.
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