Study of gene regulation and genome organization of rice (Oryza sativa L.)

The promoter of the constitutively expressed rice (Oryza sativa) actin1 gene (Act1) is highly active in transformed rice plants. I have constructed a series of Act1 promoter deletions fused to a bacterial {dollar}beta{dollar}-glucuronidase reporter sequence (Gus). Transient expression assays in transformed rice protoplasts, as well as transformed maize cells and tissues, identified two distinct cis-acting regulatory elements in the Act1 promoter. A 40 bp poly(dA-dT) region was found to be a positive regulator of Act1 promoter activity. By gel retardation and MPE footprinting, I have identified a ubiquitous rice protein which specifically recognizes this poly(dA-dT) element in the constitutively active Act1 promoter. A CCCAA pentamer repeat containing region was found to be a negative regulator of the Act1 promoter in transformed rice protoplasts. Transient expression assays in different maize cells and tissues using the Act1 deletion constructs suggested that the CCCAA pentamer repeat region functions in a complex tissue-specific manner. A CCCAA-binding protein was detected only in root extracts. The results suggest that the Act1 promoter contains multiple cis-acting elements, each of which interacts with either ubiquitous or tissue-specific trans-acting factors to confer the observed constitutive pattern of Act 1 promoter activity.; The development of methods for cleavage of DNA at specific site(s) that are widely spaced would facilitate physical mapping of large genomes. I have developed a new method to specifically cleave intact chromosomal DNA by using {dollar}lambda{dollar}-terminase. A plasmid containing two specific cleavage sites (cohesive-end sites) for {dollar}lambda{dollar}-terminase was specifically introduced into the E. coli genome and into chromosome V of S. cerevisiae. Chromosomal DNA was prepared from the resulting strains, and then cleaved with {dollar}lambda{dollar}-terminase. The results showed that the 4.7 megabase pair (Mb) circular E. coli chromosome and the 0.58 Mb linear yeast chromosome V were specifically cleaved at the desired sites with very high efficiencies. To test this method in large genomes, I introduced the cos site(s) into Arabidopsis thaliana (A. thaliana) and rice genomes. Transgenic plants containing the cos sites have been obtained. I then used {dollar}lambda{dollar}-terminase to cleave the plant chromosomal DNA and analyzed the cleavage products by either conventional or pulsed-field gel electrophoreses (PFGE). The results suggested that the {dollar}lambda{dollar}-terminase mediated cleavage reaction is a simple one-step procedure with a high specificity. Thus, it is particularly suitable for mapping very large genomes of eukaryotes.; I have also examined genome localization of several rice repetitive sequences. These repetitive elements differ from each other in their organization and abundance in the rice genome. PFGE analysis using rice repetitive sequences as well as A. thaliana telomere DNA as probes revealed that certain members of several families of the repetitive elements are closely linked to each other and are located near the telomeric regions of rice chromosomes.