STUDIES ON THE SYNTHESIS AND SEQUENCE OF NUCLEIC ACIDS
Abstract
Techniques and methods for the chemical and enzymatic synthesis of oligodeoxynucleotides were developed. Protected deoxyribonucleoside-3' aryl phosphodiesters were synthesized and used in the synthesis of several oligonucleotides ranging in chain length from five to twenty residues. A new series of condensing reagents, arylsulfonyl 4-nitroimidazoles, were made and characterized. These reagents gave consistently high yields (60-90%) and clean products in condensation reactions. Also, diarylphosphorochloridates were used as condensing reagents by forming a tetrasubstituted pyrophosphate intermediate with the phosphodiesters. A scheme utilizing these reagents was developed in which required purification and workup procedures were minimal. Finally, a method for phosphorylating the 5'-termini of chemically synthesized oligomers was developed. This involved transferring a phosphate from a modified phosphorylated uridine existing in a 5' to 5' linkage with the oligomer. The enzymatic studies involved the characterization of riboterminated oligodeoxynucleotides as substrates for RNA ligase. Conditions were developed in which relatively large oligomers could be joined together by RNA ligase in 50 to 90% yields. Preferential use by the enzyme of ribo-terminated acceptor molecules showed the order rC>>rA>rU>rG. The conditons of acceptor excess over donor were found to enhance reaction much more so than donor excess conditions. A region of 1041 base pairs of the Escherichia coli genome was sequenced. This region contained the gene for the tryptophan repressor protein. The coding region was located resulting in the prediction of the Trp repressor amino acid sequence. Trp repressor is predicted to be 88 amino acids in length with a molecular weight of 10,250 daltons. A twenty-two base pair region preceding the trpR gene was found to have extensive homology with the trp operator DNA sequence. This finding suggests the possibility of autogenous regulation for the trpR gene.
Degree
Ph.D.
Subject Area
Biology
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