CHARACTERIZATION OF NUCLEIC ACID STRUCTURES
Abstract
The study of living organisms at the biochemical level has led to the discovery that nucleic acids, functioning in the maintenance of heredity or in the process of gene expression, represent the material by which information is coded for storage and decoded for expression. Intrinsic to this process of nucleic acid function are the various structures or shapes that nucleic acids maintain in their functionally active state. This work will describe new or improved techniques for analyzing and manipulating these nucleic acid structures. As an improved approach for analyzing secondary structures of ribonucleic acids, a new uridine-specific modification has been developed. This method involves the chemical modification of the uridine heterocyclic ring with a water-soluble carbodiimide, and its selective reduction by sodium borohydride. This modified nucleotide was utilized as a stable enzymatic blocking group and as a site for specific RNA cleavage. Preliminary application of this procedure has been applied to the structure of 5S rRNA. The potential of this modification for the specific labeling and cleaving of RNA structures will be described. Sequence determination of DNA is vital for understanding genetic function since information is stored in the order of nucleotides in the DNA template. An improved procedure for labeling DNA restriction fragments with T4 polynucleotide kinase has been developed. This procedure, which is a prerequisite for DNA sequence analysis, investigates the parameters affecting phosphorylation by T4 polynucleotide kinase. Evidence will indicate that the labeling efficiency of this reaction was found to fluctuate primarily with the alkaline phosphatase concentration and only slightly with other substrates and enzymes. In addition, two novel labeling methods for efficiently labeling small amounts of 3'-protruding and blunt-ended DNA restriction fragments will be discussed. Characterization of natural genetic mutants may provide information concerning mechanisms of mutation generation. Application of the previously described labeling techniques has been utilized for DNA sequence analysis of various adenovirus mutants. These mutants were examined for mutation specificity and, in the case of H5in317, for the activity for potential RNA transcriptional and translational signals. Various deletion mutants were found to contain a degree of terminal specificity, in which a seventeen-long nucleotide sequence was identified at the endpoint of several different mutants. Additional insertion mutants, containing terminal sequence duplications and functional RNA process signals, will be compared to DNA transposable elements. Application of recombinant DNA technology to the investigation of genetic expression represents another procedure for the analysis of DNA primary structures. A unique adenovirus mutant has been isolated that allows an improved characterization of the viral gene products produced from two overlapping transcripts synthesized from a viral gene known to encode products essential for viral gene expression and the oncogenic transformation of cells. This mutant was generated by the specific insertion of an octanucleotide into the DNA sequences coding for only one viral transcript. This insert produced a frame-shift mutation that prematurely terminates RNA translation of only one of the two transcripts from this gene. This work will include information outlining the effect of this mutation on viral gene expression, viral DNA replication, and the oncogenic transformation of rodent cells.
Degree
Ph.D.
Subject Area
Biochemistry
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