I. Selected applications in molecular biology of chemically synthesized deoxyribonucleotides. II. Structural determination of the ileR gene locus of Escherichia coli K-12
Abstract
Several deoxyribonucleotides were synthesized either in liquid phase using the phosphotriester method or on a solid support with phosphoramidite chemistry. These molecules were subsequently purified, and then analyzed as to their purity, base composition and base sequence. The oligomers were synthesized for several purposes, including: site-specific mutagenesis, NMR structural studies to show possible DNA protein interaction sites, and use in a novel cDNA cloning scheme. An improved method for generating cDNAs for cloning was investigated. This scheme consists of several enzymatic manipulations of poly A$\sp+$ mRNA and a synthetic deoxyribonucleotide containing a poly T region. To achieve the desired result of generating full length duplex DNA with "sticky ends" several studies were conducted independently. These included determining the rate of tailing for deoxynucleotidyl terminal transferase and the rate of Exonuclease III digestion under our reaction conditions. A study was also conducted as to the relative stability of RNA versus DNA in an alkaline environment. The scheme was then tested using rabbit globin mRNA in a background of mRNA from several different sources, and the $\lambda$gt10 cloning system. The nucleotide sequence of ileR, the structural gene for Ile repressor has been determined. The predicted gene product, a protein of 100 amino acids, has primary structural features similar to other known double stranded DNA-binding regulatory proteins. There are two promoters upstream from the ileR gene as determined by S1 Nuclease mapping. Within the ileR promoter region lie two 18 base pair segments of DNA bearing significant homology to putative operator targets also found within the thr and ilv promoters. A second open reading frame capable of specifying a protein of 83 amino acids, designated orf83, is transcribed on the other strand in an opposite orientation.
Degree
Ph.D.
Advisors
Weith, Purdue University.
Subject Area
Biochemistry
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