Date of Award
Doctor of Philosophy
Zoology and Genetics
John E. Mayfield
Brucella abortus is a gram negative bacteria that can infect and cause serious disease in many mammals including humans. Any organism, including Brucella, that can survive as an intracellular parasite must be able to survive the stress of a foreign and often hostile environment of the host. Stress usually results in a dramatic change in gene expression including an elevated synthesis of heat shock proteins;We have subcloned, sequenced and expressed the Brucella HSP70 and the upstream sequences. The gene was put under the control of the strong "tac" promoter. However, when expressed, we saw a high accumulation of a 23 kDa polypeptide. Upon sequencing, this peptide was shown to be from the N-terminus of the protein;Upstream of the initiation codon of the Brucella HSP70 gene is a small ORF complete with its own initiation and termination codon and a good ribosome binding site. Various subclones were made and western blots from these showed that if the Brucella HSP70 gene is expressed without the ORF, the 23 kDa polypeptide accumulates. If the ORF is present, the protein accumulates intact. So, we concluded that this ORF is an important upstream regulatory element. Such a system has not been reported before and seems to be unique to Brucella. However, Brucella is an unusual parasite in that it can survive and multiply in the macrophage--the cell type of the immune system designed to kill it. It's unknown whether this form of regulation has any relationship to survival as an intracellular parasite;To facilitate expression we also constructed seven expression vectors, pJE1-pJE7. All these plasmids were derived from pKK223-3. They all carry the M13 origin of replication to facilitate production of single stranded DNA and an expanded polycloning site to facilitate cloning. pJE1-6 are in vivo constitutive expression vectors with modified promoters such that there is a graded expression with pJE1 carrying the strongest promoter. pJE7 is designed for in vitro regulated expression using the "tac" promoter. This plasmid carries the lac I[superscript]q gene to prevent over expression from the "tac" promoter.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Emily C. L. Chin
Chin, Emily C. L., "Cloning, sequencing, and expression of Brucella abortus heat shock 70 gene " (1993). Retrospective Theses and Dissertations. 10803.