Ainu Prakash

Abstract

The DNA restriction and modification of (R-M) genes in bacteria play a defensive role in the organism, offering protection to the host from invaders such as bacteriophages. The expression of these genes in E. coli K-12 has no previously been studied due to the complexity of EcoK R-M enzymes. The use of lacZ gene fusion techniques allowed studies of the expression of the hsdK genes for the first time. Operon fusions of Mu(lac) to the hsdK genes show that the two hsdK promoters, Pres and Pmod, are weak (50 Miller units) and that both operate at the same level. Growth studies indicate that transcription from Pres occurs approximately 1 h later than Pmod. An unusual feature of these genes is that they can be transferred to new bacterial strains which contain nonmodified DNA A control mechanism must exist to allow transfer of the hsdK genes without "killing" the recipient by expression of the donor restriction endonuclease. Indeed, the restriction activity was expressed later than the modification activity after conjugal transfer. What is the basis for this control? A mutant has been described previously that is "killed" upon conjugal transfer of the hsdK genes. This mutant supports the hypothesis that a control gene ("C") exists which regulates the restriction function encoded by the incoming hsdK genes. This work demonstrates that the hsdK genes are regulated by a gene in the recipient cell that is not part of the hsdK system. The location of the postulated control gene was indicated by gene replacement experiments in which various Hfr donors, wild type with respect to the "C" phenotype, were conjugated with the "C" mutant. The appearance of "C+t' recombinants suggested that the "C" gene is downstream from the HfrH origin of transfer. The "C" gene was subsequently cloned from E. coli C in pBR322 and three independent clones expressing the control function contained a common 1.2 kb EcoRI-Pstl fragment. Further experiments suggested that this gene may work at a post-translational level. These studies have opened the door to a new and exciting branch in the field of restriction enzymes.

LLU Discipline

Microbiology

Department

Microbiology

School

Graduate School

First Advisor

Jun-ichi Ryu

Second Advisor

Leonard R. Bullas

Third Advisor

John W. Patrickson

Fourth Advisor

W. Bart Rippon

Fifth Advisor

Anthony J. Zuccarelli

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1992

Date (Title Page)

3-1992

Language

English

Library of Congress/MESH Subject Headings

Escherichia Coli -- genetics; DNA Restriction-Modification Enzymes -- analysis; Gene Expression Regulation, Bacterial; Lac Operon -- genetics

Type

Dissertation

Page Count

x; 172; 2

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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