Understanding how genes are differentially regulated during the development of an organism is a fundamental problem in biology. Genetic studies of maize have provided many examples of programmed changes in gene expression during development and have identified regulatory phenomena such as paramutation and the effects of controlling elements. One such genetically characterized locus in maize is the B locus which regulates the expression of anthocyanin pigments in the plant. Variation in the tissue-specific synthesis of anthocyanins is determined by which allele is present at B. Examining different B alleles at the molecular level should reveal nucleotide sequences responsible for regulating the quantity, timing and tissue-specificity of pigment production. Certain B alleles undergo or promote paramutation: a heritable alteration in gene expression promoted by the presence of two specific alleles in the same plant. Many models have been proposed to explain paramutation, including alteration in chromatin structure, gene conversion, or the interaction or transposition of DNA elements. A molecular description of this allelic interaction should reveal general principles on cell heritable regulation of gene expression as it occurs during development. These principles should be applicable to other organisms where a combined genetic and molecular approach is not yet feasible. The long term goals of this proposal are to examine the molecular mechanisms controlling the expression of the B gene in maize.
The specific aim of this proposal is to clone the B genomic sequences using the transposable element Robertson's Mutator to mark the locus. Multiple independent insertion mutations have been isolated in order to increase the probability of obtaining the entire gene. The gene will then be recovered by taking advantage of the homology between the element inserted into the B gene and the cloned transposable element. The mutants and the cloned sequences generated in this study, will provide tools with which to begin a molecular analysis of paramutation and gene expression at B.
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