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Title:Studies on the Mechanism of Immunity for Colicin Ia
Author(s):Hsu, Chi-Hsin
Doctoral Committee Chair(s):Konisky, J.,
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
Abstract:The nucleotide sequences for the colicin Ia immunity gene and the 3$\sp\prime$ half of the colicin Ia structural gene were determined. The deduced amino acid sequences for colicin Ia and Ib were compared. The difference between these two proteins resides in the C-terminal 181 amino acids, however, the overall hydropathy profiles are conserved. Comparison of the deduced amino acid sequence for the colicin Ia immunity protein with that for the colicin Ib immunity protein revealed that these two proteins share no homology but do exhibit a conservation of overall hydrophobicity.
The colicin Ia immunity gene has been cloned into the HpaI site of the expression vector pKC30 bringing its expression under control of the phage lambda P$\sb{\rm L}$ promoter. The direction of transcription for the immunity gene was determined to be in the opposite but in a convergent way relative to the colicin Ia structural gene. Upon temperature induction of the P$\sb{\rm L}$-driven immunity gene, the immunity protein was found to accumulate to a level of 1-2% of the total cytoplasmic membrane proteins. The immunity protein was purified to apparent homogeneity by a combination of membrane extraction, isoelectric focusing and preparative SDS polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The immunity protein is not a processed membrane protein.
Possible interaction between colicin Ia and its immunity protein were examined. The immunity protein did not appear to form complex with colicin Ia in membrane vesicles or in detergent solutions. Nor did it modify the colicin. Purified Ia-immunity protein when reconstituted into liposomes did not prevent the liposomes from being depolarized by colicin Ia. It was found that the immunity protein could cause membrane leakage in a pH-dependent manner under certain conditions. The mechanism of immunity for colicin Ia is still largely unknown.
Issue Date:1987
Description:156 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.
Other Identifier(s):(UMI)AAI8803069
Date Available in IDEALS:2014-12-16
Date Deposited:1987

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