Studies on the DNA of Bacteriophage T7

Author: Studier, Frederick William

Year: 1963

Degree: Dissertation (Ph.D.)

Advisor: Bonner, James Frederick

Committee Member: Unknown, Unknown

Option: Biology; Chemistry

DOI: 10.7907/GV9J-HV27

Abstract

The molecular weight of T7 DNA was determined to be 15-17 x 10^6 by the method of sedimentation equilibrium in a CsCl density gradient. Possible sources of error in the optical reproduction of the concentration distribution in the cell were considered and evaluated. Comparison of the above molecular weight with the weight of the total DNA content of T7 phage determined by Davison and Freifelder (22) suggests: 1) that the entire DNA content of T7 bacteriophage can be released into solution as a single molecule and 2) that the density gradient method for determining molecular weights of homogeneous DNA samples is accurate within 10-20%.

The denaturation and renaturation of T7 DNA was studied using formamide as the denaturing agent. The complete cycle of denaturation and renaturation can thus be studied at room temperature without degradation of the primary structure of DNA.

Kinetics of denaturation were found to be essentially independent of concentration but were not first order, the rate falling off with time. Irreversible denaturation does not commence until the DNA has reached 80-90% of its full hyperchromicity. Samples of DNA isolated from denaturing conditions appear to be mixtures of fully denatured molecules and molecules which exhibit native characteristics to a high degree. Stirring solutions of DNA under denaturing conditions markedly accelerates the rate of denaturation. It seems possible that the molecular configuration of DNA molecules in intermediate states of denaturation may be a factor in stability to denaturation.

Optimally renatured T7 DNA has 85-90% of the native hyperchromic effect and bands at a slightly denser position than native DNA in a CsCl density gradient. Preliminary studies indicate second order kinetics for the renaturation reaction.

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