Construction of recombinant molecules containing RNA processing signals and binding of human snRNPs to some nucleic acids
Berget, Susan M.
Master of Arts
The restriction endonuclease Pst I cleavage site map of Ad2 was generated. Several recombinant molecules were formed, containing Pst I-generated fragments of Ad2 DNA, inserted into the E. coli vector pBR322. These molecules are designed to be useful for the study of RNA processing because they contain signals for splicing and polyadenylation. A filter binding assay was developed to measure the affinity of snRNPs for nucleic acids. The of U1 snRNPs was determined to be on the order of 1 to 11 M for RNA with or without a donor splice junction and for denatured DNA. U2 snRNP also bound RNA containing a donor splice junction with a KD of 1 M. The dissociation of U1 snRNP from denatured DNA was followed and the remainder of the snRNPs dissociated in less than one minute and fifty percent dissociated with a t1/2 of 2 minutes.