Nan Sook Lee

Abstract

Two possible type I restriction-modification (R-M) systems, KpnAI and KpnBl, were recognized in Klebsiella pneumoniae strains M5al and GM236, respectively. In this project, four hsdRKpnAI clones (pNLA1 to pNLA4) were obtained by using an endonuclease expression-selection method. The hsdRKpnAI gene was identified on a 3.6 kb Sau3Al fragment. To map the gene, the hsdRKpnAI clone was hybridized to a membrane which contained pulsed-field gel electrophoresed fragments of the chromosome of the strain M5al. The hsdRKpnAI clone was hybridized to a 19 kb Xbal fragment and a 420 kb Blnl fragment, located at about 16 to 20 min on the recently constructed physical map of the M5al chromosome.

Similarly, the hsdRKpnBI clone was hybridized to a 40 kb Xbal fragment and a 330 kb Blnl fragment of the GM236 chromosome. Among five subclones (pNLB1 to pNLB5), the 3.5 kb BamHI-EcoRl fragment of pNLB2 was the smallest DNA fragment to retain restriction activity through complementation testing. The DNA sequence of the 3.5 kb fragment was determined. An open reading frame (ORF) of 3,039 bp was concluded to be the coding region for the HsdRkpnBI polypeptide. The 1,013 amino acid product (111,430 daltons) deduced from the nucleotide sequence is in excellent agreement with the 116 kD size estimated from the electrophoretic mobility of the putative HsdRkpnBI protein in SDS polyacrylamide gels. The nucleotide sequence of the hsdRkpnBI gene showed no significant similarity to any other sequence in the GenBank database. However, a homology search of the amino acid sequence scored highly with EcoR124/3I, a type IC restriction enzyme. After alignment of the two proteins, two ATP binding domains, a DNA binding domain and seven helicase motifs were found. Eight small ORFs were identified within a few kb on either side of the hsdRKpnBI gene. When subcloned, none of these ORFs showed any modification activity. However, complementation with r-KpnBI m-KpnBI mutant suggests that one downstream ORF is a positive effector for expression of the KpnBI modification activity.

LLU Discipline

Microbiology

Department

Microbiology

School

Graduate School

First Advisor

Junichi Ryu

Second Advisor

Leonard R. Bullas

Third Advisor

Kelvin A. W. Hill

Fourth Advisor

John J. Rossi

Fifth Advisor

Anthony J. Zuccarelli

Degree Name

Doctor of Philosophy (Medical Science)

Degree Level

Ph.D.

Year Degree Awarded

1994

Date (Title Page)

8-1994

Language

English

Library of Congress/MESH Subject Headings

Klebsiella pneumoniae -- genetics; Restriction Mapping Cloning; Molecular

Type

Dissertation

Page Count

xii: 202

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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Microbiology Commons

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