1. Mechanism of splicing of introns from pre-mRNAs. a. Now that it has been established that U2-RNP is required for splicing, we need to test our hypothesis that U2-RNA recognizes and binds to the branch point sequence and the terminal C/T-A-G of an intron by hydrogen-bonding. First we will determine if the complementary nucleotides in the region 40-52 in U2-RNA are single stranded in native U2-RNP by the method of Inoue and Cech, 1984. Then we will form a splicing complex by adding a synthetic intron with flanking exons to a HeLa nuclear extract so that we can determine what regions of the U2-RNA are protected against methylation by the Inoue and Cech method. b. We will also try to identify a U2-like RNA in yeast by using cleavage with RNase H and dTACTAAC and a U2-RNA in Neurospora using dTGCTGAC and RNase H. c. A procedure is urgently needed to assess quantitatively the effect of variations in intron boundary signals on the splicing rate in vivo. We will use the intron and flanking exons of the Drosophila heat shock gene 83. Two copies of different length giving identifiable transcripts will be put into a vector with a selectable marker and then we will transform Drosophila tissue culture cells with this plasmid. We will compare the initial rate of spliced hsp83 mRNA formation in heat shock from the two truncated genes in the same plasmid. Then we will determine the change in this ratio when the 3' boundary signals in one truncated copy are systematically mutated, one nucleotide at a time. 2. Regulation of expression of cytoplasmic actin genes. Since cytoplasmic actin genes of metazoans all have an intron in the 5' untranslated region, we feel that this intron may contain an element involved in regulation of expression of these genes. We will construct a gene lacking this intron and assess its regulated expression in transformed cells. The work will be done with Drosophila actin gene 5C and Schneider line 3. We will look at gene expression in response to ecdysterone which stimulates cytoplasmic actin gene expression in this line. We will then determine the elements in the 5' flanking region of this gene necessary for regulated expression using the standard deletion and linker scanning methods.
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