DNA STRUCTURE AND GENE REGULATION

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The mechanism of gene regulation at the molecular level will be studied with special emphasis on the role of DNA properties and conformations as determinants of specificity. The properties of left-handed DNA will be investigated with restriction fragments of known sequence and recombinant plasmids. The influence of base sequence on the capacity of a region to form a left-handed helix will be studied with synthetic DNA polymers, plasmids containing simple repeating sequences (both of natural and synthetic origin) and with mutants of defined sequence in the structurally interesting segments. Conditions that promote the R to L transition (such as ionic environment, supercoiling, AAF modification of guanosine moieties, and methylation) will be investigated. Attempts will be made to determine the structure and chemical behavior of the junction between a left-handed Z-region and a right-handed B-segment. The biological significance of left-handed DNA will be evaluated in vitro and in vivo. Conformational properties will be determined by circular dichroism, proton and phosphorus NMR, and laser Raman spectroscopy as well as superhelix density determinations on topoisomers of the recombinant molecules. X-ray diffraction studies will be pursued on a collaboratilve basis. Gene cloning will be employed extensively to prepare chemically defined molecules with inserts with the desired sequences. Milligram quantities of the restriction fragment inserts will be purified by high pressure liquid chromatography on RPC-5 An understanding of the details of gene expression is fundamental to our ultimate comprehension of normal cellular growth and divison as well as disease syndromes characterized by unregulated growth such as cancer.