Date of Award

Fall 1983

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

Abstract

A two-dimensional zymogram procedure for the analysis of nucleases is described. Using purified deoxyribonuclease I (bovine pancreas), as little as 10 pg of nuclease can be detected. Isoelectric focusing (IEF) and non-equilibrium pH gradient electrophoresis (NEPHGE) were compared as first dimensions, in combination with SDS electrophoresis as the second dimension, in analyzing nucleases in lysates of Bacillus subtilis. All renaturable nuclease activities detected following SDS electrophoresis alone were resolved in NEPHGE-SDS electrophoresis gels whereas, in IEF-SDS gels, most were either at the basic end or were not present in the second dimensional gel. This method of analysis has revealed a complexity in nuclease species in B. subtilis not previously recognized.

Eighty-three discrete nuclease activities have been detected in B. subtilis lysates. These nucleases have been characterized with respect to monomeric molecular weights, cation-activation requirements, and preference for single or double stranded substrate. Substrate and cation analysis reveals a minimum of 16 classes of activities. The nucleases identified in this study are compared to those previously reported in B. subtilis.

The total protein composition and nuclease composition of cellular lysates from competent and non-competent B. subtilis strain SB25 and several transformation-defective mutants of SB25 has been examined by two-dimensional electrophoretic and zymogram analysis. Thirty-six competence-associated polypeptides have been identified. Nuclease analysis has revealed that the development of competence is accompanied by the induction of 4 nucleases and the suppression of 6 nucleases. Three of the competence-associated nucleases (monomeric MW; 17,500, 18,000, and 18,500) are primarily activated by Mn('2+) and are most active on denatured DNA. The fourth (monomeric MW; 17,000) is also Mn('2+)-activated but is distinguished by its preference for native DNA. The nuclease activities which are reduced in competent cells are all primarily Mn('2+)-activated. Three of these nucleases (monomeric MW; 18,000, 21,000, and 24,000) show equal activity on native and denatured DNA. The remaining three (monomeric MW; 17,000, 18,500, and 19,250), show a preference for native DNA.

Transformation-defective mutants have been identified which have a reduced level of the 17,000 MW competence-associated nuclease. These mutants are deficient in uptake and the ability to inactivate exogenous DNA. In addition to the suppression of the 17,000 MW nuclease, these mutants fail to induce a large number of additional competence-associated polypeptides.

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