Abstract

Analysis of recently sequenced microbial genomes has revealed many DNA sequences that code for previously unknown restriction endonucleases and their corresponding methyltransferases. These findings show that numerous restriction enzymes abundant in bacteria have yet to be discovered. Traditionally, restriction enzymes have been discovered by the classical restriction and modification (R-M) phenomena of bacteriophages (type I and III enzymes), or by direct enzyme assays (type II enzymes). To avoid the limitations of these traditional approaches, a quantitative R-M test based on plasmid transformation efficiency (Plasmid R-M Test) was established using DNA fragments derived from the E. coli bacteriophage lambda. This test is similar to traditional “efficiency of plating” (EOP) assays but measures “efficiency of transformation” (EOT). To determine the feasibility of using plasmid transformation to detect restriction activity, five known R-M systems were tested, including: type I (EcoBI, EcoAl, Eco124I), type II (Hindlll), and type III (EcoP1I). To test the hypothesis that this methodology could be used to locate recognition sequences, we applied this methodology to determine the DNA recognition sequence for KpnAl, which was found to be GAA(6N)TGCC. For this, the computer program, RM Search was developed to analyze positive and negative DNA sequence data. In addition, a simple method was designed and used to identify the modification sites for the KpnAI methyltransferase. This method employs the concept of restriction enzyme sensitivity to the methylation status of double-stranded DNA. The recognition sequences for three previously characterized Salmonella R-M systems, StySEAI, StySENI, and StySGI were found to be ACA(6N)TYCA, CGA(6N)TACC, and TAAC(7N)RTCG, respectively. In addition, this project identified R-M systems in clinical E. coli strains EC826, EC851, and EC912. The recognition sequences for these systems respectively are GCA(6N)CTGA, GTCA(6N)TGAY, and CAC(5N)TGGC. Because plasmid transformation methods are available for many bacteria and enzyme purification is not required, this model system can be extended to further bacterial species to search for new R-M systems. Combined with RM Search, a newly developed computer program, this new test may become one of the standard methods used to find new restriction enzymes, and to predict their recognition sequences.

LLU Discipline

Microbiology and Molecular Genetics

Department

Microbiology

School

Graduate School

First Advisor

Junichi Ryu

Second Advisor

Istvan Fodor

Third Advisor

George T. Javor

Fourth Advisor

William H. R. Langridge

Fifth Advisor

Anthony J. Zuccarelli

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2003

Date (Title Page)

6-2003

Language

English

Library of Congress/MESH Subject Headings

Bacteriophages -- analysis; DNA Restriction Enzymes; Bacteria; Escherichia Coli.

Type

Dissertation

Page Count

xv; 176

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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