A partial characterization of the deoxyribonucleic acid found in the thoracic muscles of the tobacco hornworm moth (Manducta sexta) Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/2514nq30d

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  • The DNAs (deoxyribonucleic acids) of the thoracic tissue of the tobacco hornworm, Manducta sexta, were studied by physical and chemical means. The adult moth was considered to be fully formed two days after emergence from the pupal case, as indicated by its ability to fly and the integrity of the dorsal longitudinal flight muscle as seen in electron micrographs. The dorsal longitudinal flight muscle development was followed by assaying for total RNA (ribonucleic acid), DNA and protein during the period of time four days prior to emergence and two days after, α-glycerolphosphate dehydrogenase and cytochrome C oxidase were used as mitochondrial marker enzymes, and to follow the development of the mitochondria over a similar period of time. The enzyme assays also showed that the majority of activity was in the mitochondrial fraction. Once it was established that the insect tissue was developing normally and that an adult moth could be considered fully developed at the end of two days the DNA from the thoracic muscles was isolated and characterized. The material isolated from the nuclear (and debris) fraction was identified as DNA and characterized by its buoyant density in CsC1 gradients, purine and pyrimidine composition, melting temperature (hyperchromicity), spectral analysis and molecular weight. The genome size was estimated from renaturation kinetics and the amount of rapidly reassociating material (repeated sequences) was determined. DNA was isolated from the mitochondrial fraction in impure form but was identified on analytical CsC1 gradients and its buoyant density determined. The number and presence of super coils was estimated by reversible denaturation experiments (snap back). The molecular weight of the DNA was determined by two different methods; 1) from its band width in CsC1 gradients and 2) from contour lengths, measured on electron micrographs, of the circular and super coil forms. Circles and super coils were readily identified in the electron micrographs. The molecular weight found by band width analysis is 10.4 x 10⁶ daltons and the molecular weight range as determined from the electron micrographic studies is 7.9 x 10⁶ to 16.8 x 10⁶ daltons. The molecular circumference of the mitochond rial DNA, on formvar coated grids, is 4.0μ.
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