Our long-term goal is to study the regulation of the tissue specificity of gene expression in well-defined genetic systems of maize. To do this we will require cloned genes and the ability to re-introduce modified genes into the plant. Our proposal addresses the 3 major issues in developing a transformation system for maize: isolation and characterization of maize genes, vector construction and transformation methods. We describe the use of 1) a novel gene cloning antibody screening method and 2) a new mutator of maize as general methods for the cloning and identification of maize genes for which the gene product is or is not known. We propose to clone the Bronze I gene of maize as a test of the application of these methods. Concurrently we will use the maize Adh I gene in transformation experiments with yeast and mammalian cells to define the components of plant gene structure required for vector replication and expression in heterologous systems. We have already characterized several maize sequences (ars) which can substitute for the yeast origin of replication on the YIp5 vector and plan to substitute additional maize sequences for other vector functions. We have recently perfected a micro-injection method for introducing vector DNA into the 300-500 free nuclear stage of triploid maize endosperm. We propose to use this tissue as a model system to screen vectors for replication and expression in maize. Simultaneously we are exploring methods for the direct transformation of maize embryos and meristems in order to introduce DNA into the germ line. As much of what we know about gene mutability and programmed changes in gene expression comes from classical maize genetics, the molecular dissection of these phenomena will provide information of general significance to our understanding of gene expression in higher eukaryotes.
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