Graduate Thesis Or Dissertation
 

Materials associated with the DNA fraction of Bacillus subtilis W168

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  • The purpose of this research was to determine if a basic protein was associated with the DNA of Bacillus subtilis W168, and to determine if there was a difference in the amount and type of protein associated with the DNA at different times during the growth cycle of the organism. The amino acid compositions of the cellular proteins at these times were also determined for comparison with any protein found associated with the DNA. The plan of approach was to grow cells for 5, 8, and 11 hours, and to extract the DNA complexes from these cells according to a modification of the procedure described by Marmur. The "DNA/protein" ratios of the DNA complexes, based upon the Lowry and diphenylamine tests, were 1:0.33, 1:0.35, and 1:0.36 for the 5, 8, and 11 hour cultures respectively. Ultraviolet absorption spectra of these DNA complexes showed that, except for the five hour culture, some material was associated with the DNA. An amino acid analysis of the DNA complex isolated from the eight hour culture showed the amino acids glutamic acid, glycine, alanine, and methionine to be present in significant amounts. No basic amino acids were found in the sample. An unknown peak, possibly glucosamine, was also observed to be present. The DNA complexes were treated with 3 M NaC1 to dissociate any protein from the DNA. Ultraviolet absorption spectra of the DNA samples obtained after this treatment were typical of those of pure DNA, indicating that some material had been removed. The material obtained from the dissociation treatment was found to contain very little protein. This material was treated with 0.25 N HC1 according to Butler and Godson's method for separating acidic and basic fractions. Very little acidic material was present. The three basic fractions were found to be similar when analyzed for total nitrogen, organic phosphorus, and amino acids. An amino acid analysis of the basic fraction obtained from the five hour culture showed the presence of trace amounts of all of the amino acids except lysine, histidine, arginine, and 1/2 cystine. The amino acids, however, were present in such low amounts as to make it uncertain that their presence was not due to contamination. An unknown peak, corresponding to the peak found in the amino acid analysis of the DNA complex isolated from the eight hour culture, was also observed to be present in this analysis. The middle layers obtained during the extraction of the DNA complexes were separated into acidic and basic protein fractions using 0.25 N HC1. These fractions were then hydrolyzed and analyzed for the presence of amino acids by two -dimensional paper chromatography. The basic protein fractions contained the most material, by weight, but very few amino acids were identified in these fractions. Most of the material present in these basic fractions was considered to be poly-β-hydroxybutyric acid. The amino acids found in the acidic protein fractions were basically the same for all three cultures.
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