Molecular Cloning and Expression of the Pyrimidine B Gene From Bacillus Subtilis

The aspartate transcarbamylase of Bacillus subtilis is an example of a "vegetative" gene product which ceases to be synthesized once the cells begin to sporulate. To study the regulation of expression of this enzyme, the gene which encodes it (pyrB) was cloned from B. subtilis. Chromosomal DNA from wild type B. subtilis was digested with BamHI and ligated into the BamHI site of pHV14, a chimeric plasmid bearing chloramphenicol resistance and capable of replicating in either E. coli or B. subtilis. The recombinant plasmid pBS205 was selected by its ability to complement an E. coli pyrB deletion mutant (TB2). pBS205 contained a 5.8 kb fragment of B. subtilis DNA. The aspartate transcarbamylase made by TB2 carrying pBS205 was immunochemically indistinguishable from B. subtilis aspartate transcarbamylase. The cloned fragment was capable of transforming various B. subtilis pyr mutants to uracil prototrophy. As a result of the transformation experiments and complementation experiments in E. coli pyrC and pyrD hosts, it was determined that the fragment also carried the intact pyrC gene but not the entire pyrD gene.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Vollmer, Amy Cheng

• Biology, Microbiology

Identifier

(UMI)AAI8410065

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/70532