Propagacion De Bacterias

IDENTIFICATION OF PROTEINS INVOLVED IN
CHLOROPLAST DNA REPLICATION

by
Matthew Gordon Lassen

A thesis submitted to the faculty of
Brigham Young University
in partial fulfillment of the requirements for the degree of

Master of Science

Department of Microbiology & Molecular Biology
Brigham Young University
December 2004

BRIGHAM YOUNG UNIVERSITY

GRADUATE COMMITTEE APPROVALof a thesis submitted by
Matthew G. Lassen
This thesis has been read by each member of the following graduate committee and by
majority vote has been found to be satisfactory.

Date

Brent L. Nielsen, Chair

Date

William R. McCleary

Date

Craig E. Coleman

BRIGHAM YOUNG UNIVERSITY

As chair of the candidate’s graduate committee, I have read the thesis of Matthew G.
Lassenin its final form and have found that (1) its format, citations, and bibliographical
style are consistent and acceptable and fulfill university and department style
requirements; (2) its illustrative materials including figures and tables are in place; and
(3) the final manuscript is satisfactory to the graduate committee and is ready for
submission to the university library.

Date

BrentL. Nielsen
Chair, Graduate Committee

Accepted for the Department
Alan R. Harker
Chair, Department of Microbiology and
Molecular Biology

Accepted for the College
R. Kent Crookston
Dean, College of Biology and Agriculture

ABSTRACT

IDENTIFICATION OF PROTEINS INVOLVED IN
CHLOROPLAST DNA REPLICATION
Matthew Gordon Lassen
Department of Microbiology & Molecular Biology
Master ofScience

Chapter 1
Chloroplast nucleoids (ct-nucleoids) are DNA/protein complexes involved in
compacting the chloroplast genome, and may play a role in regulating DNA replication.
Ct-nucleoids were isolated from young soybean plants and separated by 2-D gel
electrophoresis. Gel spots were excised and analyzed by MALDI-ToF mass
spectrometry, resulting in several protein identifications. Theproteins identified all have
functions unrelated to DNA replication. While some of these proteins may be due to
contamination, it is possible that some of these proteins are dual-functional, playing
direct roles in the regulation of DNA replication.
Chapter 2
A 28 kDa soybean protein was isolated by sequence specific DNA affinity
chromatography from total chloroplast protein isolations. Massspectrometry analysis
revealed that the 28 kDa protein contains some homology within an ssb domain of an

Arabidopsis mitochondrial-targeted SSB (mtSSB) of approximately 21 kDa. N-terminal
sequencing revealed that the 28 kDa soy protein is identical to a 36 amino acid region at
the N-terminus of the Arabidopsis mtSSB. Protein fractions containing the 28 kDa
protein shift oriA inelectrophoretic mobility shift assays (EMSAs). Arabidopsis mtSSB
fails to shift oriA in EMSAs run under identical conditions. Arabidopsis mtSSB causes a
shift of ssDNA in EMSAs, while the ability of the 28 kDa soy protein to bind ssDNA is
still unclear. Importantly, the 28 kDa soy protein was identified from total protein
extracts obtained from intact chloroplasts, while in-vitro targeting experimentssuggest
that the Arabidopsis mtSSB localizes only to mitochondria and not to chloroplasts.
BLAST searches of the available soybean genomic and EST databases do not produce
any significant homologies to the 36 amino acid N-terminal sequence.

ACKNOWLEDGEMENTS

Many people have been crucial in completing this thesis. First of all, I would like
to thank Dr. Brent Nielsen for the opportunity hegave me to work in his lab, as well as
for all of his patience and good advice. Drs. Bill McCleary and Craig Coleman have also
provided important advice and guidance throughout the project. Dr. Craig Thulin and
Katie Southwick assisted with the 2-D gels and mass spectrometry. Melisa Wall
performed the targeting experiments. Andrew Edmondson provided valuable information
and data concerning...