Abstract

The study of the regulation of gene expression in complex genes can be facilitated by the use of operon fusions which place a well characterized lacZ gene under the control of a promoter of interest. An hsd::MudX128 operon fusion mutant of E. coli K-12 isolated by Prakash (1986) was observed to have a high β-galactosidase activity (1000 units), ten to twenty times higher than other hsd::MudX mutants. This β-galactosidase activity was suppressed to a very low level (25 units) by the introduction of an F’ plasmid, F'101, which carries the 98 min to 2 min region of the chromosome. The mutant 128 was further characterized in this study to give explanations for the observations described above. In P1 transduction experiments, Ampr transductants that were r-km-k were also expressing high β-galactosidase activity. Therefore the r-km-k phenotype and high β-galactosidase activity are closely linked. DNA hybridization studies showed that the high β-galactosidase activity of 128 is due not to the combined expression of multiple insertions fusions but due to a single insertion of MudX(lacZ).

Little is known about the regulation of hsd genes. In this thesis, the hsdM gene product seems to have a regulatory function on the expression of the hsd genes. The F’ conjugation experiments, using an F’ kit in which various members carry different coding regions of the E. coli K chromosome and also derivatives of F'101 plasmids which contain various hsd::lacZ fusion mutants, suggest that the hsdM gene product is responsible for the suppression phenomenon which causes a decrease from 1000 β-galactosidase units to 25. Thus a "hsdM repressor" model has been proposed. To identify the strong promoter presumed to be responsible for the high β-galactosidase activity, a 16 kb BamHI fragment containing the chromosomal MudX fusion site as well as the intact lacZ gene from the 128 fusion strain was cloned into λ.dash™ (Stratagene). The restriction map of the hsd genes in the 128 strain did not correspond to the maps previously published suggesting that a DNA rearrangement had occurred near the MudXlacZ insertion. Furthermore, a mutant stability test and a DNA hybridization study suggest another DNA rearrangement had occurred at the MudX junction site in the strain 128, at a frequency of 10-2. Further characterization of the mutant and future studies concerning the regulation of hsd genes are discussed.

LLU Discipline

Microbiology

Department

Microbiology

School

Graduate School

First Advisor

Jun-ichi Ryu

Second Advisor

Leonard R. Bullas

Third Advisor

George T. Javor

Fourth Advisor

Anthony J. Zuccarelli

Degree Name

Master of Science (MS)

Degree Level

M.S.

Year Degree Awarded

1990

Date (Title Page)

6-1990

Language

English

Library of Congress/MESH Subject Headings

Gene Expression Regulation, Bacterial; Escherichia Coli -- genetics; Mutation -- physiology; Galactosidases -- physiology

Type

Thesis

Page Count

2 vii; 100

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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