Molecular characterization of the gene, mRNAS, precursor proteins, and mature subunits involved in the synthesis of the NADP-specific glutamate dehydrogenase isoenzymes in Chlorella sorokiniana

195 Pages·1994·3.7 MB·English

by  Miller, Philip

MOLECULAR CHARACTERIZATION OF THE GENE, mRNAS, PRECURSOR
PROTEINS, AND MATURE SUBUNITS INVOLVED IN THE SYNTHESIS OF
THE NADP-SPECIFIC GLUTAMATE DEHYDROGENASE ISOENZYMES IN
CHLORELLA SOROKINIANA
By
PHILIP W. MILLER
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994
.
This work is dedicated to my parents, Walter and Elaine
Miller, who taught me by example that success is only
achieved by dedication and hard-work, and that true success
can be measured in many ways
ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation to
his mentor, Dr. Robert R. Schmidt, for his support and
guidance during the course of this research. Thanks are due
to Dr. Phillip M. Achey, Dr. Richard P. Boyce, Dr. Francis C.
Davis, and Dr. William B. Gurley for their guidance while
serving on the advisory committee. The author would like to
acknowledge the initial training provided by Dr. Kyu Don Kim
and the continued interest and suggestions provided by Dr.
Mark Cock throughout this research. The author would also
like to thank Mrs. Phyllis Schmidt for her continued guidance
in meeting all the requirements for this degree.
Special thanks go to Dr. Mark Tamplin, Rendi Murphree,
and Victor Garrido for providing the materials, expertise,
and training for the production of the monoclonal antibodies
crucial to this research. The author would like to thank Dr.
Roy Jensen and lab, and Dr. L. 0. Ingram for the use of their
equipment that was important to this study.
The author wishes to extend special thanks to my lab
mates Richard Hutson, Brenda Russell, Jan Baer, and Dr. Mary
U. Connell for their friendship and participation during the
course of this project. To Ms. Waltraud Dunn, I extend my
sincere thanks for her friendship, patience, stimulating
in
conversations, and all the assistance she provided throughout
this study. To Julie Rogers, I offer my heartfelt thanks for
her unfailing support, confidence, and inspiration.
This research was supported in part by the USDA
Competitive Research Grants office (Grant 89-37262-4843).
The author was supported on a graduate research assistantship
funded by the Graduate School of the University of Florida.
IV
TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
LIST OF FIGURES vii
LIST OF TABLES X
LIST OF ABBREVIATIONS xi
INTRODUCTION
1
LITERATURE REVIEW 6
MATERIALS AND METHODS 25
Culture Conditions 25
Enzyme Assay 25
Isolation of RNA 26
Genomic DNA Isolation 28
NADP-GDH Protein Purification 29
Purification of the a-NADP-GDH holoenzyme 29
Partial purification of NADP-GDH isoenzymes 31
Anti-NADP-GDH Antibody Production and Purification 32
Monoclonal antibody production 32
Polyclonal antibody production 33
Western Blotting 34
Alkaline phosphatase conjugated antibody detection .... 34
125I-Protein A detection 35
Amino-Terminal Sequence Analysis of the NADP-GDH a-
Subunit and p-Subunit 37
DNA Probe Synthesis 37
Northern Blot Analysis 38
Southern Blot Analysis 39
NADP-GDH cDNA Cloning and Characterization 41
XgtlO library 41
*.ZAP II library 41
5 Race-PCR cloning 44
'
NADP-GDH cDNA characterization 46
Primer Extension Analysis 47
Genomic Allele-Specific PCR 48
Construction of NADP-GDH In vitro Transcription
Vectors 49
Comparison of the NADP-GDH mRNAs, Antigens, and
Activities in 29 mM Induced C. sorokiniana Cells 50
Culture conditions 50
RNase protection analysis 51
NADP-GDH antigen and activity analyses 52
RT-PCR analysis 52
RESULTS 54
NADP-GDH cDNA Cloning and Characterization 54
Restriction mapping and sequencing of XgtlO cDNA
clones 54
Isolation, restriction-mapping, and sequencing of
the A.ZAPII NADP-GDH cDNA clones 58
Primer extension analysis 61
RACE-PCR cloning of two NADP-GDH 5 termini 64
'
Analysis of the C. sorokiniana NADP-GDH cDNA
sequences 70
Determination of the Exon/Intron Boundaries of the
NADP-GDH Gene 75
Determination of the Number of NADP-GDH Genes in the
C. sorokiniana Genome 86
Southern blot analysis of the NADP-GDH gene 86
Allele-specific PCR analysis of the NADP-GDH gene 91
Purification of the NADP-GDH Isoenzymes 94
Determination of the Stability of the NADP-GDH a-
Holoenzyme in the Presence of NADP+ 98
Production of Anti-NADP-GDH Polyclonal and Monoclonal
Antibodies 100
Analysis of the a- and p-Subunit Similarity with Mouse
Anti-NADP-GDH MAbs 103
Determination of the Molecular Mass of the NADP-GDH
Subunits 104
Comparison of the Induction Patterns of the NADP-GDH
Antigens, Activities, and mRNAs in 29 mM Ammonium
Medium 114
RT-PCR Analysis of the NADP-GDH mRNAs 133
DISCUSSION 139
LIST OF REFERENCES 164
BIOGRAPHICAL SKETCH 177
VI
...
LIST OF FIGURES
Figure page
1. Restriction maps of 17 cDNAs isolated from a
C.sorokinana cDNA library prepared from RNA isolated
from cells induced for 80 min in 29 mM ammonium
medium 56
2. Northern blot analysis of poly(A)+ RNA isolated
from C. sorokiniana cells induced for 3 h in 1 mM
ammonium medium or continuously in 29 mM ammonium
medium 60
3 Restriction maps of eight cDNAs isolated from a C.
sorokiniana cDNA library 63
4. Primer extension analysis of NADP-GDH mRNA(s) 66
5. 5' RACE-PCR generated NADP-GDH 5 '-terminus clones... 69
6. Nucleotide sequence of the consensus NADP-GDH
mRNAs derived from the cDNA and 5 RACE-PCR clone
'
sequences 72
7 Secondary structure prediction of the C.
sorokiniana -42 nt NADP-GDH mRNA precursor
polypeptide 77
8 Secondary structure prediction of the C.
sorokiniana +42 nt NADP-GDH mRNA precursor
polypeptide 79
9. Nucleotide sequence of the C. sorokiniana NADP-GDH
gene 81
10. Restriction maps and exon domains of four NADP-
GDH qenomic clones spanning 21.9 kbp of the
C. sorokiniana genome 85
11. Transcriptional initiation site for the C.
sorokiniana NADP-GDH nuclear gene and the upstream
region in the genomic DNA 88
vn
12. Southern blot analysis of undigested genomic DNA
and restriction fragments 90
13. Polyacrylamide gel electrophoresis of the PCR
products amplified from C. sorokiniana genomic DNA and
three NADP-GDH genomic clones 93
14. Analytical SDS-PAGE of the NADP-GDH a-holoenzyme
purified by preparative nondenaturing gel 97
15. Stability of purified NADP-GDH a-holoenzyme at
4°C in the presence of 0.1 mM NADP+ 102
16. Mouse anti-NADP-GDH monoclonal antibody
immunoblot analysis of the NADP-GDH 106
17. Estimation of the molecular weights of the C.
sorokiniana NADP-GDH a- and (3-subunits 108
18. Alignment of the C. sorokiniana NADP-GDH a- and
p-subunit deduced amino acid sequences 112
19. Increase in culture turbidity of Chlorella cells
cultured for 240 min 116
20. Pattern of the total soluble protein in
synchronized daughter cells 118
21. Patterns of accumulation of NADP-GDH antigens in
illuminated cells cultured in 29 mM ammonium medium 121
22. Patterns of accumulation of NADP-GDH antigens in
cells cultured in 29 mM ammonium medium for 240 min 123
23. Pattern of NADP-GDH activities in homogenates of
synchronous C. sorokiniana cells cultured in 29 mM
ammonium medium 125
24. Ribonuclease protection analysis of the NADP-GDH
mRNAs synthesized in synchronous C. sorokiniana cells
throughout a 240 min induction period in 29 mM
ammonium medium 128
25. Relative abundance patterns of NADP-GDH mRNA in
cells induced in 29 mM ammonium medium 132
26. RT-PCR analysis of the NADP-GDH mRNAs synthesized
in synchronous C. sorokiniana cells throughout a 24
min induction period in 29 mM ammonium medium 136
Vlll
27. Relative abundances of the NADP-GDH mRNAs
synthesized in synchronous C. sorokiniana cells
throughout a 24 min induction period in 29 mM
ammonium medium 138
28. Model for the regulation of the processing of the
two NADP-GDH precursor proteins 147
29. Helical wheel projections of the unique
C.sorokiniana NADP-GDH amino-terminal helical domains... 152
30. Diagramatic representation of the assembled
hexameric NADP-GDH 154
31. Model for the regulation of the C. sorokiniana
chloroplastic NADP-specific GDH isoenzymes 163
IX
..
LIST OF TABLES
Table page
1 Synthetic oligonucleotide sequences 45
2 Codon usage of the -42 nt NADP-GDH mRNA 74
3. Codon usage of the +42 nt NADP-GDH mRNA 74
4. Steps for the purification of the NADP-GDH a-
holoenzyme 95
5. Steps for the partial purification of NADP-GDH
isoenzymes 99
6. Ratios of NADP-GDH :NADP+ activities and a:p-
subunits 119