Part I. Turnover of the Hemolymph Proteins of Drosophila melanogaster. Part II. A New Method for the Detection of Deoxyribonucleases and its Application to Studies of Drosophila melanogaster

Citation

Boyd, James Brown (1966) Part I. Turnover of the Hemolymph Proteins of Drosophila melanogaster. Part II. A New Method for the Detection of Deoxyribonucleases and its Application to Studies of Drosophila melanogaster. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/4ZS0-CV69. https://resolver.caltech.edu/CaltechETD:etd-09102002-133403

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. Part I A method has been developed for measuring the radioactivity of proteins labeled with[.....] following their separation by disc electrophoresis. The radioactivity is measured directly in acrylamide gel with scintillation techniques after the water in the gel has been replaced with a toluene-based scintillator solution. Seventy-four gel slices can be prepared with a minimum of handling in 9 to 18 hours depending upon the size of the slices. This technique has been used in combination with densitometry measurements of separately stained gels to study the turnover of the hemolymph proteins of [Drosophila melanogaster]. By injecting labeled homologous proteins into unlabeled animals, active turnover of most of the hemolymph proteins has been demonstrated in both larvae and pupae. One particular group of proteins begins to turn over rapidly after puparium formation with a half life of 13 hours. Another group of proteins has been shown to be relatively inert in pupae. A postulated mechanism for protein turnover in metamorphosing insects is discussed. Part II A method is described for detecting deoxyribonucleases which have been separated in acrylamide gel containing trapped DNA. After electrophoresis the enzymes are incubated at their final resting positions with the substrate found at those positions. The remaining DNA is stained and recorded with a densitometer. The assay is sensitive to less than 0. 25 nanograms of crystallized deoxyribonuclease I and is quantitatively reproducible. An investigation of the deoxyribonucleases of [Drosophila melanogaster] with this technique has revealed a large number of peaks of enzymatic activity which undergo marked changes during the course of development. By varying the substrate and the magnesium concentration used for incubation, it has been shown that this organism probably produces at least eight different enzymes which can degrade DNA. A preliminary determination of the properties of some of these enzymes has been made.

Thesis Files