ELECTROPHORETIC AND ENZYMATIC IDENTIFICATION OF PROTEINS ASSOCIATED WITH THE DEVELOPMENT OF COMPETENCE IN BACILLUS SUBTILIS
type: CreativeWork
description: 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 redu…
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 redu…
author: COUGHLIN, SUSAN ALICIA
keywords: Biology, Genetics
date published: 1983-01-01T08:00:00Z
date modified: 2026-07-24T05:23:15Z
provider: University of New Hampshire
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of New Hampshire