Date of Award

Spring 1989

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

Abstract

The inclusion of 1% casein in buffer used to reactivate enzymes subjected to SDS-polyacrylamide electrophoresis resulted in faster and more complete restoration of nuclease and B-galactosidase enzyme activities. Enzyme activities which were absent from gels during longer wash procedures were detectable with this technique. The threshold of detection of two-dimensionally separated Deoxyribonuclease I was 1 picogram, tenfold lower than for previously reported wash procedures. Addition of BSA at concentrations above 50 ug/ml to nuclease gels was found to result in less effective detergent removal during wash procedures and reduced recovery of enzyme activity.

General and specific nuclease expression patterns were observed in two-dimensional sodium dodecyl sulfate polyacrylamide gels of 64 competence-induced com and spo mutants of Bacillus subtilis. General patterns of nucleases expression were as follows: (1) Four different groups of nucleases called "clusters" were found to be co-expressed in greater than 95% of the mutants. (2) Some cluster members are probably produced by post-translational modification. (3) A 14 kd nuclease reported by Venema was detected in the gels after use of the casein wash procedure.

Specific patterns of expression are as follows: (1) Sigma factor H is not required for nuclease 2 expression. (2) spoOK is required for nuclease 2 production. (3) Nuclease 2 is regulated differently than nuclease clusters A, B, C, and D. (4) crsa$\sb{47}$ (sigma A) is required for competence.

Download

Share

COinS