Physical and Biological Studies of Crab Deoxyribonucleic Acid

Citation

Widholm, Jack Milton (1966) Physical and Biological Studies of Crab Deoxyribonucleic Acid. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/1276-DQ97. https://resolver.caltech.edu/CaltechETD:etd-09232002-114928

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. The native dAT components of C. antennarius and of C. borealis DNA have been separated in pure form by a new mercury binding technique. Two further DNA species, not heretofore described, are apparently also present in C. antennarius DNA. They have melting points and buoyant densities intermediate between those of dAT and of the major DNA component. A series of tests using optical density melting electrophoresis, buoyant density, electron microscopic, band sedimentation, exonuclease I susceptibility and actinomycin D inhibition techniques are employed to show that melted and renatured C. antennarius dAT remains partially denatured. After strand separation and recombination approximately 7% of the base pairs do not reform. These unpaired bases are predominantly guanine and cytosine (G and C) since exonuclease I hydrolysis assays and RNA synthesis inhibition by actinomycin D give the results which would be expected on this basis. The C. antennarius dAT contains 3.5% G and C bases. C. borealis dAT with 2.5% GC behaves similarly to C. antennarius dAT, except that the denatured structure is less evident, apparently because of the lower GC content. When the aforementioned tests are applied to synthetic dAT, which contains no G or C bases, the results indicate that this alternating dAT copolymer is fully hydrogen bonded both before and after melting. dAT is present to the extent of 10-11% in another Cancer species, C. anthonyi. dAT is found in all C. antennarius tissues and is localized in the cell nucleus. Studies with the E. coli RNA polymerase system show that dAT is copied at a rate 2.6 times as fast as is the main DNA component. dAT also has a higher affinity for the enzyme. Actinomycin D inhibits the transcription of native primers more than it does that of melted ones. Incorporation of GC into RNA is preferentially inhibited. Protamine, bistones Ib, IIb, III and IV, listed in order of decreasing effectiveness, inhibit the RNA synthesis supported by DNA. These basic proteins inhibit incorporation of A and U preferentially indicating preferential binding of the inhibitors to AT rich regions of the template. The RNA synthesized from the dAT template by E. coli RNA polymerase is double-stranded and melts at 67[degrees]C. This rAU has an S value of 5-6 after ribonuclease T1 treatment. This result is due to the low G content of the dAT, 1.8%. Actinomycin D inhibition studies with crab hepatopancreas tissue indicate that rAU is not synthesized at an appreciable rate in vivo if at all. The RNA from this tissue also contains no rAU as shown by melting and by T1 treatment. The rAU if present would melt sharply and be detectable and would show up as 5-6 S material in sucrose density gradient centrifugation of the hepatopancreas RNA after T1 treatment. Chromatin is less active as template for RNA synthesis than is whole crab DNA and the chromatin dAT component is particularly repressed.

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