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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

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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
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Miller, Philip
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xv, 177 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Amino acids ( jstor )
Complementary DNA ( jstor )
DNA ( jstor )
Exons ( jstor )
Gels ( jstor )
Genomics ( jstor )
Messenger RNA ( jstor )
Polymerase chain reaction ( jstor )
Quaternary ammonium compounds ( jstor )
RNA ( jstor )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 164-176).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Philip Miller.

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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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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


iii








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.
















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 .............
Culture Conditions..............
Enzyme Assay....................
Isolation of RNA................
Genomic DNA Isolation...........


NADP-GDH Protein Purification........................
Purification of the a-NADP-GDH holoenzyme .........
Partial purification of NADP-GDH isoenzymes .......
Anti-NADP-GDH Antibody Production and Purification...
Monoclonal antibody production ....................
Polyclonal antibody production ....................
Western Blotting .....................................
Alkaline phosphatase conjugated antibody detection
1251-Protein A detection ..........................
Amino-Terminal Sequence Analysis of the NADP-GDH a-
Subunit and p-Subunit .............................
DNA Probe Synthesis ..................................
Northern Blot Analysis...............................
Southern Blot Analysis...............................
NADP-GDH cDNA Cloning and Characterization...........
kgtl0 library .....................................
XZAP II library ...................................
5' Race-PCR cloning ...............................
NADP-GDH cDNA characterization ....................
Primer Extension Analysis............................
Genomic Allele-Specific PCR..........................
Construction of NADP-GDH In vitro Transcription
Vectors ...........................................
Comparison of the NADP-GDH mRNAs, Antigens, and
Activities in 29 mM Induced C. sorokiniana Cells ..
Culture conditions ................................
RNase protection analysis .........................


....31
.... 32
.... 32
..... 33
.... 34
.... 34
.... 35

.... 37
.... 37
.... 38
....39
.... 41
....41
....41
....44
.... 46
....47
.... 48

....49

.... 50
.... 50
....51


........................
........................
o.......................








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 kgtl0 cDNA
clones .............................................54
Isolation, restriction-mapping, and sequencing of
the kZAPII 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













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 genomic 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


vii








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
40C 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 p-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 240
min induction period in 29 mM ammonium medium........... 136


viii







27. Relative abundances of the NADP-GDH mRNAs
synthesized in synchronous C. sorokiniana cells
throughout a 240 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














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














LIST OF ABBREVIATIONS


AP ................................

ATCase............................


BSA...............................

ca............................... .

CaM...............................

CPSase......... ....................


DHOase............................

DTT ...............................

Dsx...............................

EDTA..............................


EGTA..............................



ELISA ............................


GS..................... ...........

GOGAT..............................

HAT ...... ..................... ..


HCR ........ ......................

ICBR............................. .


Alkaline phosphatase

Aspartate trans-
carbamylase

Bovine serum albumin

Calculated average

Calmodulin

Carbomylphosphate
synthase

Dihydroorotase

Dithiothreitol

Double-sex

Ethylene diamine-
tetraacetic acid

Ethylene glycol-bis (3-
aminoethyl ether) N, N,
N', N',-tetraacetic acid

Enzyme-linked immuno-
absorption assay

Glutamine synthetase

Glutamate synthase

Hypoxanthine-aminopterin-
thymidine

Highly conserved region

Interdisciplinary Center
for Biotechnology
Research








LANT6.............................


mA ................................

MAb...............................

NAD-GDH...........................



NADP-GDH..........................




NBT.................. ... .......

NEB....................... ........

NMN .. ............ ...o ......... .

nt...... ....... ................

NT........................... .....

ODU.............................. .

OTCase............................


PK...... .. ..... ..... .............

PME............ ................. .

RACE-PCR...........................


RPA... ........ ...................


RT-PCR............................

Rubisco................ ...........


SNAP-25...........................


snRNA ................. ........


snRNP.............................


Neurotensin lysine-
asparagine rich

Milliamperes

Monoclonal antibody

Nicotinamide adenine
dinucleotide-specific
glutamate dehydrogenase

Nicotinamide adenine
dinucleotide phosphate-
specific glutamate
dehydrogenase

Nitroblue tetrazolium

New England Biolabs

Neuromedian N

Nucleotides

Nerotensin

Optical density unit(s)

Ornithine trans-
carbamylase

Pyruvate kinase

Pectin methylesterase

Rapid amplification of
cDNA ends-PCR

Ribonuclease protection
assay

Reverse transcriptase PCR

Ribulose bisphosphate
carboxylase/oxygenase

Synaptosomal associated
protein 25 kD

Small nuclear ribonucleic
acid

Small nuclear ribonuclear
proteins


xii








SPP ..............................


SPT................... ..... ......


Sxl................ ...............

TBS......................... ......

tra........ .............. .........

TTBS.............. ... ............


UTR. ...................... ..........

VR.........oo.... ... ........... .

X-phosphate........................


Stromal processing
peptidase

Serine:pyruvate amino-
transferase

Sex-lethal

Tris-buffered saline

Transformer

Tween Tris-buffered
saline

Untranslated region

Variable region

5-bromo-4-chloro-3-indole
phosphate


xiii














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


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

December, 1994




Chairman: Dr. Robert R. Schmidt
Major Department: Microbiology and Cell Science


Chlorella sorokiniana possesses seven chloroplastic

NADP-glutamate dehydrogenases (NADP-GDHs) composed of varying

ratios of a- and P-subunits. Southern blot analysis and

allele-specific PCR demonstrated the C. sorokiniana genome

possesses a single NADP-GDH gene encoding both the a- and 3-

subunits. PCR analysis, cDNA cloning and sequencing, and

RNase protection analysis identified two NADP-GDH mRNAs that

are identical with the exception of a 42 nt insert located in

the 5' coding region of the longer mRNA. Deduced amino acid

sequence analysis revealed that the 42 nt insert encodes an

additional 14 amino acids. The absence or presence of the

insert does not affect the downstream reading frame. The +42

nt mRNA encodes a 53850 D precursor protein, whereas the -42


xiv








nt mRNA encodes a 52350 D precursor protein. The +42 nt and

-42 nt mRNAs are postulated to be derived via alternative

splicing of a pre-mRNA from the single 7.1 kbp NADP-GDH gene

that consists of 22 or 23 exons, respectively.

Western blot analysis of the a- and P-subunits showed

them to be antigenically similar and to be 53.5 and 52.3 kD

in size, respectively. Amino-terminal sequence analysis

revealed the a-subunit shares amino acid sequence identity

with the P-subunit; however, the a-subunit possesses a unique

11 amino acid a-helical domain that is lacking in the P-

subunit.

The induction patterns and relative abundances of the

NADP-GDH mRNAs, antigens, and activities were measured in

cells induced in 29 mM ammonium medium. The relative

abundance of the +42 nt mRNA correlated with the a-subunit

antigen, whereas the -42 nt mRNA correlated with the P-

subunit. The ratio of NADPH:NADP+-GDH activity was highest

when the P-subunit was prominent and lowest when the a-

subunit was prominent.

These results are consistent with a single nuclear gene

being transcribed into a pre-mRNA that is alternatively

processed to yield two mRNAs encoding two precursor proteins.

The precursor proteins are processed to either the a- or p-

subunit and assembled into isoenzymes with varying ammonium-

affinities.













INTRODUCTION

Inorganic nitrogen acquired by plants is ultimately

converted to ammonium before being assimilated in organic

nitrogen metabolism. One of the enzymes postulated to be

involved in the assimilatory process is GDH, a ubiquitous

enzyme found to be present in almost all organisms from

microbes to higher plants and animals (Srivastava and Singh,

1987). GDH catalyses the reversible conversion of x-

ketogluterate to glutamate via a reductive amination that

utilizes NADH or NADPH as a cofactor. The role of plant GDHs

in the assimilation of ammonium into amino acids has been

questioned since the discovery of the GS/GOGAT pathway that

is believed to be the favored pathway for ammonium

assimilation in higher plants (Miflin and Lea, 1976). The

primary objection to GDH playing a major role in nitrogen

metabolism is its low affinity for ammonium that would

require high intracellular ammonium concentrations to

function anabolically. Early evidence indicated that GDH is

a catabolic enzyme catalyzing the deamination of glutamate

with only a partial anabolic function in synthesizing

glutamate (Wallsgrove et al., 1987). However, more recent

studies reveal that Km values for ammonium and other

sustrates may be affected by various internal and external

factors and the previously reported in vitro Km values may







not reflect in vivo conditions. The physiological role of

large amounts of GDH present in various plant tissues and

organelles is still unclear, and possible conditions under

which GDH may play a significant role in carbon and nitrogen

metabolism have not been resolved.

The majority of plant GDHs characterized to date are

localized in the mitochondria; however, GDH species differing

in several properties (i.e. cofactor specificity) have been

characterized from chloroplasts (Srivastava and Singh, 1987).

Chlorella sorokiniana cells have been shown to possess a

constitutive, mitochondrial, tetrameric NAD-specific GDH

(Meredith et al., 1978), and seven ammonium-inducible,

chloroplast-localized, homo- and heterohexameric NADP-

specific GDH isoenzymes (Prunkard et al., 1986; Bascomb and

Schmidt, 1987). The seven chloroplastic NADP-GDH isoenzymes

were shown to have different electrophoretic mobilities

during native-PAGE, and presumably result from the formation

of homo- and heterohexamers composed of varying ratios of a-

and P-subunits (53.5 and 52.3 kD, respectively). Chlorella

cells cultured in 1 to 2 mM ammonium medium accumulate only

the a-homohexamer (Bascomb and Schmidt, 1987). The addition

of higher ammonium concentrations (3.4 to 29 mM) to nitrate-

cultured cells results in the accumulation of both a- and p-

subunits in NADP-GDH holoenzymes (Prunkard et al., 1986;

Bascomb and Schmidt, 1987; Bascomb et al., 1987). Prunkard

et al. (1986) demonstrated that the NADP-GDH subunit ratio

and isoenzyme pattern is influenced by both the carbon and








nitrogen source as well as the light conditions under which

cells are cultured.

The purified a- and p-homohexamers have strikingly

different ammonium Km values; however, the Km values for their

other substrates are very similar. The a-homohexamer is

allosterically regulated by NADPH and possesses an unusually

low Km for ammonium that ranges from 0.02 to 3.5 mM,

depending on the NADPH concentration (Bascomb and Schmidt,

1987). In contrast, the P-homohexamer is a non-allosteric

enzyme with an ammonium Km of approximately 75 mM. It is

postulated that the heterohexamers have varying degrees of

affinity for ammonium; however, no kinetic analyses have been

performed on purified heterohexamers. Pulse-chase

experiments, performed when homo- and heterohexamers of NADP-

GDH were accumulating during early induction in 29 mM

ammonium medium, revealed the a-subunit antigen was degraded

with a half-life of 50 min whereas the p-subunit antigen was

degraded more slowly with a half-life of 150 min (Bascomb et

al., 1986). After the removal of ammonium from the induced

cells, enhanced rates of degradation were observed for the a-

and p-subunit antigens, half-lifes of 5 and 13.5 min,

respectively.

Although the a- and p-subunits have distinct in vivo

turnover rates and the corresponding homohexamers have

remarkably different ammonium Km values, the a- and P-

subunits are derived from precursor proteins of nearly

identical size (ca 58,000 D) and were shown to have very








similar peptide maps (Prunkard et al., 1986; Bascomb and

Schmidt, 1987). Moreover, antibodies prepared against the 3-

homohexamer are capable of immunoprecipitating all of the

NADP-GDH isoenzymes (Yeung et al., 1981, Bascomb et al.,

1987), but do not crossreact with the mitochondrial NAD-GDH.

In addition, previous research in this laboratory provided

genomic cloning and southern blot evidence that indicated the

C. sorokiniana genome possesses a single NADP-GDH structural

gene (Cock et al., 1991).

Biochemical and immunochemical properties of the NADP-

GDH a- and p- subunits suggest that the two subunits share a

significant amount of protein sequence identity. Similar

kinetic, isoenzyme pattern, and immunological properties have

been shown for the mitochondrial GDH of grapevine (Loulakakis

and Roubelakis-Angelakis, 1991) and Arabidopsis (Cammaerts

and Jacobs, 1985). The understanding of the molecular

mechanisms regulating the C. sorokiniana NADP-GDH isoenzymes

is critical to further elucidate the metabolic significance

of GDH in carbon and nitrogen metabolism in Chlorella and

higher plants. Therefore, the purpose of this study is to

determine if the two NADP-GDH subunits arise from the (i)

differential processing of a precursor protein encoded by a

single nuclear gene and mRNA, (ii) specific processing of two

similar precursor proteins encoded by two mRNAs formed by

alternative splicing of a pre-mRNA derived from a single

nuclear gene, (iii) specific processing of two precursor





5


proteins encoded by two mRNAs transcribed from two closely

related nuclear genes.













LITERATURE REVIEW

Extensive research into the molecular and biochemical

mechanisms that control the physiology and potential fate of

a living cell has revealed a myriad of complexities in the

regulation of cellular processes. In prokaryotes, metabolic

processes have been shown to be temporally regulated at

transcription, translation, mRNA turnover and processing, and

post-translational modifications. The presence of organelles

in eukaryotes has provided another level of intricacy in

metabolic regulation by providing compartmentalization, that

provides for both temporal and spacial separation of cellular

events. The spatial separation of cellular processes

provides additional steps where regulation of transriptional,

post-transcriptional, translational, and post-translational

events can occur.

In response to the spatial and temporal separation of

metabolism, cells have evolved enzymes that share a similar

biological activity within an organism. These enzymes often

differ in their primary amino acid sequence (isoenzymes or

isoproteins), but may be capable of subunit exchange

(isozymes). Enzymatically similar isoforms may also exist as

a result of post-translational modifications

phosphorylationn, acetylation, methylation, etc.) to a single

protein. Theoretically, multiple isoenzymes have allowed








organisms to respond differentially in a refined way to a

broader range of developmental and environmental conditions.

Isoenzymes can arise via all the aforementioned cellular

processes and provide a useful tool to study the molecular

mechanisms involved in regulating biochemical processes.

The majority of isoenzymes characterized to date are

encoded by two or more genes within the genome of a organism.

Isoenzymes encoded by multiple genes are believed to have

arisen through gene duplication within an organism or via

gene exchange between organelles of eukaryotes (Gray and

Doolittle, 1982; Sun and Callis, 1993). The amount of

similarity conserved among isoenzymes derived from different

genes is influenced by how recent the duplication or exchange

has occurred (Pickersky et al., 1984), or may reflect a

strong selective pressure to maintain the primary amino acid

sequence (Moncreif et al., 1990).

Multiple isoenzymes of CaM, a 16 kD acidic Ca2+-binding,

signal transducing protein, have been identified in all

eukaryotes examined (Ling et al., 1991). Increased binding

of Ca2+ by CaM in response to increased intracellular Ca2+

levels triggers a conformational change in the protein.

Alteration of conformation in turn facilitates specific

interactions with Ca2+/CaM-dependent enzymes (O'Neil and

DeGrado, 1990). One notable feature of CaM isoenzymes is the

highly conserved primary structure; comparisons of amphibian,

avian, mammalian, and plant CaM isoproteins showed identities

of over 90 percent (Roberts et al., 1986). Analysis of CaM








amino acid sequences from a wide variety of organisms has

revealed that 47 of 148 amino acid residues are variant,

allowing a degree of latitude in the physiological

constraints that regulate the structure and function of each

CaM isoenzyme (Moncreif et al., 1990).

CaM multigene families have been characterized in rat

(Nojima, 1989), and human (Fischer et al., 1988). In both of

these species the CaM proteins encoded by different gene

families possess identical amino acid sequences; however,

their respective nucleotide sequences have diverged by

approximately 20 percent (Ling et al., 1991). More recently,

at least four CaM isoforms have been identified in

Arabidopsis thaliana that differ from one another by as much

as six amino acid substitutions (Gawienowski et al., 1993).

Arabiodopsis CaM isoenzymes are encoded by a multigene

family consisting of at least six different genes. Southern

blot analysis and genomic cloning determined the CaM proteins

were not allelic and that their coding regions had diverged

thirteen to twenty percent, and no significant identities

were retained in their mRNA 3'-untranslated regions. Most of

the amino acid changes between the CaM proteins appear to be

functionally conservative, and are clustered within the

fourth Ca2+-binding domain that is involved in high affinity

Ca2+ binding. It has yet to be determined if there exists

significant biochemical differences or tissue-specific

expression differences that would warrant these multiple CaM

proteins (Gawienowski et al., 1993). Multiple CaM isoforms,








each having a defined set of targets, may explain how a

common intracellular Ca2+ concentration signal can activate

different physiological responses.

In eukaryotes, multiple isoenzymes of CPSase and ATCase,

enzymes involved in the de novo pyrimidine biosynthetic

pathway have been identified (Jones, 1980; Ross, 1981).

Isoenzymes of these proteins have proven to be critical in

the biochemical regulation of eukaryotic pyrimidine and

arginine biosynthesis, both of which utilize

carbomylphosphate as an intermediate. Prokaryotes possess a

single CPSase and ATCase enzyme, both of which are

metabolically regulated by negative effectors to control flux

of carbomylphosphate between the two competing pathways

(Markoff and Radford, 1978).

Eukaryotes, other than plants, utilize two isoenzymes of

CPSase to commit separate pools of carbomylphosphate to the

pyrimidine and arginine pathways. An arginine-specific

CPSase is localized in the mitochondria with OTCase, the

enzyme that utlizes carbomylphosphate to synthesize

citrulline in the arginine pathway (Davis, 1986). A

pyrimidine-regulated CPSase activity exists on a

multifunctional protein that also exhibits ATCase activity

and is localized in the nucleus of yeast (Nagy et al., 1989),

or a cytosloic localized multifunctional protein that also

exhibits ATCase and DHOase activity observed in other

eukaryotes (Davidson et al., 1990). The different CPSase








activities have been shown to be encoded by separate genes in

these organisms.

The mechanisms of coordinately regulating the pyrimidine

and arginine pathways of plants are less understood.

Sequence analysis of partial cDNA clones from alfalfa has

provided evidence for the existence of arginine- and

pyrimidine-specific CPSases (Maley et al., 1992); however,

biochemical studies have only demonstrated a single

glutamine-dependent CPSase activity. Regardless of the

number of CPSases in plants, metabolic studies indicate that

the arginine and pyrimidine pathways share a common pool of

carbomylphosphate (Lovatt and Cheng, 1984). Localization of

CPSase, ATCase, and OTCase activities to the plant

chloroplast indicates that allocation of carbomylphosphate to

each pathway must be regulated (Shibata et al., 1986).

Williamson and Slocum (1994) utilized an ATCase

deficient mutant of Escherichia coli to clone by functional

complementation two different ATCases from pea plants.

Comparison of the deduced amino acid sequences of the clones

revealed an 85 percent identity and indicated they both

possessed a chloroplast targeting transit peptide. Southern

blot analysis revealed the two ATCase mRNAs are encoded by

two independent genes of the pea genome. Biochemical studies

are in progress to determine if these genes are

differentially regulated and if different ATCase subunits

which exist as homotrimers can also exist as heterotrimers

with unique kinetic properties.








Cell wall PME enzymes that de-esterify galactosyluronic

acid units of pectin, have been detected in all tissues of

higher plants analyzed. PME has been implicated in

functioning in a broad range of cellular processes including

fruit softening (Fischer and Bennett, 1991), plant response

to infection (Collomer and Keen, 1986), and cell growth

(Moustacos et al., 1991). Multiple isoenzymes of PME have

been detected in most plant species and tissues. It is

hypothesized that the multiple isoenzymes function in a

tissue-specific manner and have different modes of de-

esterification of pectins (Markovic and Kohn, 1984).

Harriman et al. (1991) and Recourt et al. (1992)

demonstrated that tomato and Phaseolus vulgaris genomes

possess multiple PME genes. The cDNAS isolated from tissue-

specific libraries indicated that there are multiple PMEs

with high sequence homologies encoded by separate genes. In

vivo expression of antisense RNA constructs, designed to

block specified PME isoforms, revealed regulation of PME

isoenzymes occurs by regulating transcription from different

genes in a developmental, tissue-specific manner (Gaffe et

al., 1994).

Multiple isoproteins do not exist in all cases where

multiple genes are detected. Multiple genes encoding (1-3,1-

4)-p-glucanendohydrolases have been identified in wheat

(Triticum aestivium). Isolation of P-glucanase cDNA clones

revealed two different cDNAs with 31 nucleotide

substitutions; however, the coding regions of the mRNAs were







identical and only a single P-glucanase protein was detected.

Further analysis revealed that the two mRNAs originated from

homeologous chromosomes in the wheat hexaploid genome (Lai et

al., 1993). Therefore, the potential exists to derive

multiple isoenzymes by combining genomes in polyploid

species. The level of conservation of similar proteins will

be determined by relatedness of the parental species, the

time lapse since the genomes were combined, and selective

pressures to maintain the primary structures of the proteins.

Multiple isoenzymes have been shown to be derived from a

single gene in an organism. Saccharomyces cerevisiae cells

possess both a cytosolic and secreted form of invertase.

Both isoenzymes of invertase are encoded by a single

structural gene, SUC2, which gives rise to two distinct mRNA

species (Perlman and Halvorson, 1981; Carlson and Botstein,

1982). The polypeptides encoded by the invertase mRNAs, when

translated in vitro, are 60 kD (p60) and 62 kD (p62). The

p60 mRNA is 1.8 kb and encodes the cytoplasmic invertase,

whereas the p62 mRNA is 1.9 kb and encodes the secreted

invertase (Carlson and Botstein, 1982). The p62 form has

been shown to be glycosylated in the Golgi to an 87 kD

protein, a process that targets the invertase for secretion.

The p62 protein is preferentially targeted to the Golgi

apparatus via a labile 19 amino acid amino-terminal signal

sequence that is cotranslationally cleaved upon import to the

Golgi (Perlman et al., 1982).








Amino acid analysis of the p60 and p62 proteins revealed

that, starting at amino acid residue 21, the secreted

invertase was identical to the cytoplasmic invertase.

Comparison of the 5' ends of the mRNA nucleotide sequences

and the SUC2 gene revealed the presence of two unique

promoters (Tassig and Carlson, 1983; Sarokin and Carlson,

1984, 1985). The promoter region for the secreted invertase

was located -140 bp upstream of the coding region, whereas

the intracellular invertase promoter was located -40 bp

upstream of its coding region. Deletion of the nucleotide

sequence from -650 to -418 bp removed the regulation of the

secreted form by glucose; however, deletion from -1900 to -80

bp had no influence on the cytoplasmic invertase (Sarokin and

Carlson, 1984).

Mammalian SPT is localized in two subcellular

organelles, the mitochondria and peroxisomes of the liver.

The peroxisomal SPTp and the mitochondrial SPTm isoenzymes

have very similar immunochemical (Oda et al., 1982),

catalytic and physical properties (Naguchi and Takada, 1978),

but their responses to hormones or other stimuli are quite

different. The SPTm is synthesized as a large precursor

which is specifically translocated into the mitochondria,

both in vivo and in vitro, and is processed into a mature

form similar in size to the mature SPTp. Two different SPT

mRNAs were detected by northern analysis using a SPTm probe.

The longer 1.9 kb mRNA was glucagon inducible and the smaller

1.7 kb mRNA was hormone insensitive indicating the larger







mRNA codes for the SPTm protein (45 kD) and the smaller mRNA

encodes the SPTp protein (43 kD) (Oda et al., 1993).

Cloning and sequence analysis of the two transcripts

provided evidence that the two mRNAs were identical except

for a longer 5' end possessed by the 1.9 kb SPTm mRNA. These

results were verified by S1 nuclease protection, and RNase

protection analysis. Utilizing SPTm and SPTp probes,

Southern blot analysis detected a single SPT gene.

Comparison of the cDNA and gene sequences revealed that the

two SPT mRNAs were generated by transcription from two unique

promoters located upstream of exon one. The SPTm mRNA

contains an amino terminal extension of 22 amino acids that

acts as a mitochondrial targeting signal, whereas the SPT

mRNA lacks the targeting signal due to initiation of

transcription from a different promoter downstream of the

mitochondrial start methionine codon in exon one. Transport

of SPTp to the peroxisome occurs if the SPT preprotein lacks

the mitochondrial targeting peptide (Oda et al., 1993).

There are many other examples of multliple isoproteins

generated from a single gene via the use of alternate

upstream promoters (Beltzer et al., 1988; Chatton et

al.,1988).

Alternative splicing of pre-mRNA transcribed from a

common promoter of single gene has emerged as a widespread

mechanism for regulating gene expression. In most cases,

alternative splicing gives rise to multiple protein isoforms

that share high identity, but vary in specific domains that








allow fine regulation of protein function (Smith et al.,

1989). Alternative splicing allows for protein isoform

switching without the need for permanent genetic change that

would be necessary with gene rearrangement. The number of

genes known to be alternatively spliced reported to date is so

vast; therefore, a limited number of representative cases

will be discussed.

Alternative splicing of transcripts has been shown to

regulate the localization of isoproteins. The immunoglogulin

heavy chain protein Igi is present as a membrane bound form

in early B lymphocytes. Upon maturation of the B-cell, after

antigen activiation, the membrane-bound IgR form decreases

and a concomitant increase in the IgR secreted pentamer form

is observed. The switch from membrane bound to secreted form

is acheived by the alternate use of 3' end exons that encode

the hydrophobic membrane-binding segment (Alt et al., 1980;

Rogers et al.,1980).

Gelosin, a protein which severs actin filaments, exists

as a plasma and cytosolic protein. Analysis of the two

isoenzymes indicated the proteins were identical except for

an additional 25 amino-terminal amino acids in the plasma

form. Analysis of the gelosin gene revealed the two

isoenzymes are expressed from the same gene via the use of

alternate promoters and subsequent alternative splicing of a

5' exon. The extra 25 amino acids of the mature plasma form

and an additional 27 amino acid signal peptide is encoded in

the extra exon. The 27 amino acid sequence targets the








plasma form to a secretion pathway and is cleaved from the

mature protein (Kwiatkowski et al., 1986).

Alternative pre-mRNA splicing can also function to

produce a functional and nonfunctional form of a protein that

acts as an on/off switch. The pathway of sexual

diffentiation in Drosophilia has revealed that an entire

sexual developmental cascade is regulated by alternative

splicing (Bingham et al., 1988). Briefly reviewed here, in

response to the X chromosome:autosome ratio this pathway is

regulated initially by the alternative splicing of three

primary genes:Sxl, tra, and dsx.

The Sxl gene, the first gene in the cascade, pre-mRNA is

alternatively spliced to yield male and female specific

transcripts. The splicing results in the inclusion of an

exon in male transcripts and in the truncation of the major

open reading frame after 48 codons. In females, the male

specific exon is spliced out and a complete 354 amino acid

RNA-binding protein is produced (Bell et al., 1988). The

functional female Sxl protein regulates the splicing out of a

248 bp intron in the tra gene mRNA which produces a female

function tra protein. The lack of the functional sxl protein

product in males leads to a default splicing pattern in the

male tra mRNA which produces a nonfunctional tra protein

(Boggs et al., 1987; Nagoshi et al., 1988). The functional

tra protein of females regulates the splicing of the dsx pre-

mRNA to a female-specific transcript that encodes a dsx

protein that represses male differentiation genes.








Alternative splicing of the dsx pre-mRNA produces functional

products in males and females; however, the male dsx mRNA

possesses a male specific exon that is spliced out of the

female mRNA. The male dsx protein represses the female

diferrentiation genes (Baker and Wolfner, 1988). Further

analysis revealed that the functional tra protein of females

acts by recruiting general splicing factors to a regulatory

element downstream of the female-specific 3' splice site of

the dsx mRNA (Tain and Maniatis, 1993).

Changes in the enzymatic activity of the enzyme PK can

be attributed to mRNA alternative splicing. The four

isoforms of PK, M1 and M2, L and R, each form homotetrameric

holoenzymes. The M1 and M2 isoenzymes differ by the presence

of an internal 45 amino acid segment that is encoded by a

pair of mutually exclusive exons (Noguchi et al., 1986).

This variable region is nearly identical to a similar region

found in the L and R isoforms. The M2, L, and R

homotetramers are all allosterically regulated and show

sigmoidal kinetics, whereas the M1 isoenzyme shows no

allosterism and has Michaelis-Menton kinetics (Imamura and

Tanaka, 1982). The M2, L, and R proteins reside in tissues

where allosteric regulation is critical to prevent futile

cycling, whereas the Ml form exists in muscle tissue in which

glycolysis is the dominant metabolic state. The

alternatively spliced exons of Ml and M2 encode a region that

is important in intrasubunit contacts; therefore, it is

postulated that interactions between subunits dictate the








regulatory properties of the individual isoenzymes (Noguchi

et al., 1986).

Alternative splicing also influences post-translational

modifications to isoproteins. The 25 kD synaptosomal

associated protein, SNAP-25, exists as two isoforms in

chicken. Cloning and characterization of the SNAP-25 cDNAs

and gene revealed that two exon fives exist in the gene.

Alternative splicing of the pre-mRNA mutually excluded one of

the two exons that resulted in two isoproteins that differed

in nine amino acid substitutions. The amino acid

substitutions result in the loss of a palmitoylation site,

thus influencing the ability of the isoforms to interact with

neuronal membranes (Bark, 1993).

The intracellular signaling by Ca2+ has been shown to be

finely regulated by alternative splicing in humans. The Ca2+

pump is a calmodulin-regulated P-type ATPase that is critical

in controlling intracellular Ca2+ concentrations. Analysis

of the pump gene structure indicated that alternative

splicing of the Ca2+ pump pre-mRNAs altered the calmodulin-

binding domain. Alteration of this domain either increases

or decreases pump affinity for calmodulin. The decreased

affinity for calmodulin causes an apparent lower affinity of

the pump for Ca2+, thus lowering the intracellular Ca2+

concentration (Enyedi et al., 1994).

Although alternative splicing appears to be a common

mechanism of altering gene function without permanently

changing gene structure, few examples of alternative mRNA








have been reported for plants. This absence of published

reports likely reflects a lack of detection of alternative

mRNA splicing rather than a lack of its existence. Rubisco

initiates the pathway of photosynthetic carbon reduction in

plants. This enzyme exhibits catalytic activity only after

activation by rubisco activase. Immunoblots utilizing anti-

activase antibodies detected two polypeptides in spinach (41

and 45 kD) and Arabidopsis (44 and 47 kD) indicating that the

two polypeptides were similar (Werneke, 1988). Genomic DNA

blots indicated that rubisco activase was encoded by a single

gene in spinach and Arabidopsis. Werneke et al. (1989)

demonstrated by amino- and carboxyl-terminal amino acid

analysis and cDNA cloning that the two activase isoenzymes

were derived by alternative splicing of activase pre-mRNA.

In spinach, two different 5' splice sites are utilized in

processing an intron in the 3' end of the primary transcript.

Use of the first 5' splice site introduced a termination

codon that results in formation of the 41 kD protein, whereas

selection of the downstream 5' splice junction omits the

temination codon and yields the 45 kD isoprotein. In

Arabidopsis, alternative splicing by a similar mechanism

results in the synthesis of the 44 kD and 47 kD polypeptides.

However, retention of the intron sequence does not introduce

a termination codon, but creates a frameshift that leads to

early termination of the protein. These results represent

the first case of alternative splicing reported for plants.







The P gene of Zea mays is postulated to

transcriptionally regulate flavonoid-derived pigment

biosynthesis in floral tissues. Two different P transcripts

have been detected, a 1.8 kb mRNA encoding a 43.7 kD protein

and a 0.945 kb mRNA encoding a 17.3 kD protein.

Characterization of the two P transcripts revealed they are

derived from a single gene and arise via alternative splicing

of the 3' end of a pre-mRNA (Grotewold et al., 1991). The

alternative splicing between exons two and three of the pre-

mRNA results in a frameshift that leads to early termination

of translation and yields the 17.3 kD polypeptide. The

truncated protein still possesses its DNA-binding domain;

therefore, it is hypothesized that the 17.3 kD protein may

act as a negative regulator and inhibits binding of the 43.7

kD functional transcriptional activator (Grotewold et al.,

1991).

Three cDNAs encoding RNA-binding proteins have been

isolated from Nicotiana (Nicotiana sylvestris) that encode

proteins with high affinities for polyuracil and polyguanine

motifs. Two of the cDNAs appear to be derived from a common

gene whose pre-mRNA undergoes alternative splicing in a

tissue specific manner. The alternative splicing occurs via

differential selection of two 5' splice junctions and results

in the formation of a functional and a truncated polypeptide.

The physiological significance of the isoproteins is unknown;

however, the functional polypeptide shows high homology with








the RNA-binding protein involved in the dsx alternative

splicing machinery of Drosophilia (Hirose et al., 1994).

Multiple isoenzymes can be derived by differential

proteolytic processing of a common precursor protein;

however, to date this mechanism has rarely been demonstrated.

NT and NMN, putative endocrine and neural signal prohormones,

are present in a 1:1 ratio within a common precursor

preprohormone polypeptide in mammals (Dobner et al., 1987).

Post-translational precursor processing has been shown to

occur in a tissue specific manner to yield both NT and NMN

prohormones in canines (Carraway and Mitra, 1990). Chicken

NT and LANT6 are derived via differential processing of a

common precursor prohormone (Carraway et al., 1993). Since

both NT and LANT6 have similar pharmacological activities and

bind similar receptors, it is postulated that the larger

slower degraded LANT6 may produce similar effects with a

different temporal pattern.

There are other translational and post-translational

mechanisms that could potentially yield multiple isoforms of

a protein. Such mechanisms include internal initiation of

translation (McBratney et al., 1993), protein splicing (Neff,

1993), protein methylation (Clark, 1993), protein acylation,

phosphorylation, and glycosylation (Blenis and Resh, 1993).

Although these mechanisms of regulation are undergoing

extensive research, the role and significance they play in

isoprotein formation is not well documented.








GDH is a ubiquitous enzyme detected in almost all

organisms from microbes to higher plants and animals

(Srivastava and Singh, 1987). This enzyme catalyzes the

reversible conversion of a-ketoglutarate to glutamate via a

reductive amination that utilizes NADH/NADPH as a cofactor.

A multitude of studies utilizing various techniques has

revealed that isoenzymes of GDH can be localized within the

mitochondria, chloroplast, and the cytoplasm depending on the

organism (Srivastava and Singh, 1987; LeJohn et al., 1994).

Multiple roles have been attributed to GDH including ammonia

assimilation (Yamaya and Oaks, 1987), maintaining the

glutamate/a-ketoglutarate ratio to regulate flux between

carbon and nitrogen metabolism (Munoz-Blanco and Cardenos,

1989), stress response (Miranda-Ham and Loyola-Vargus, 1988;

LeJohn et al., 1994), and function as a RNA-binding protein

(Preiss et al., 1993). Collectively, these findings

implicate GDH as playing a variety of cellular roles and

these various metabolic functions are regulated by the

differential use of GDH isoenzymes.

Chlorella sorokiniana has been shown to synthesize

multiple GDH isoenzymes: a constitutive, tetrameric,

mitochondrial NAD-GDH subunitt 45 kD), and seven ammonium-

inducible, hexameric, chloroplastic NADP-GDHs subunitt

53kD,P- and 55.5 kD,a-) (Prunkard et al., 1986; Bascomb and

Schmidt, 1987). The multiple chloroplastic isoenzymes have

different molecular weights and charges and presumably result

from the formation of homohexamers and heterohexamers due to








mixing of the a-subunits and P-subunits. The chloroplastic

isoenzyme pattern in C. sorkiniana has been shown to be

influenced by light conditions, carbon and nitrogen source,

and ammonium concentration (Isreal et al., 1977; Prunkard et

al., 1986; Bascomb and Schmidt, 1987).

Kinetic and physical characterization of the purified a-

homohexamer and P-homohexamer show them to have several

properties in common as well as striking differences (Bascomb

and Schmidt, 1987). The purified homohexamers have

remarkably different affinities for ammonia; however,

affinity values for the other substrates are quite similar.

The a-homohexamer is an allosteric enzyme with a low Km for

ammonia depending on the NADPH concentration, whereas the p-

homohexamer is nonallosteric and has a high Km for ammonia.

In addition, pulse-chase experiments demonstrated that the

two subunit types are synthesized and degraded at different

rates (Bascomb and Schmidt, 1987). Although the two subunit

types have distinct in vivo turnover rates, they appear to be

derived from precursor proteins of near identical size.

Antibodies derived against one subunit type are able to

immunprecipitate both a-subunits and 3-subunits (Yeung et

al., 1981), and peptide mapping of the subunits revealed that

the subunits have 36 of 40 peptides in common (Bascomb and

Schmidt, 1987). These results indicate that the two subunits

are very similar. Genomic cloning and Southern blot analysis

indicated a single NADP-GDH gene exists in the C. sorokiniana








genome that encodes both types of subunits (Cock et al.,

1991).

In summary, multiple isoenzymes have evolved as a

mechanism to allow an organism to respond to a broad range of

developmental and environmental conditions in a tissue-

specific manner. Isoenzymes and isoproteins can arise by a

variety of molecular and biochemical events including gene

duplication, alternative RNA splicing, transcriptional,

translational, and post-translational events. GDH, a

ubiquitous enzyme, exists as multiple isoenzymes with various

roles in most organisms studied. Considering the wealth of

information that exists on the metabolic roles and regulation

of GDH isoenzymes, further research into the biochemical

genetics of these isoenzymes may provide insight into

mechacanisms of molecular regulation in general.













MATERIALS AND METHODS


Culture Conditions


C. sorokiniana cells were cultured autotrophically as

previously described by Prunkard et al. (1986) in a modified

basal salts medium. The modified medium contained in mM

concentration: CaCl2, 0.34; K2S04, 6.0; KH2PO4, 18.4; MgCl2,

1.5; in [M concentration CoCl2, 0.189; CuC12, 0.352; EDTA,

72; FeCl3, 71.6; H3BO3, 38.8; MnCl2, 10.1; NH4VO4, 0.20;

(NH4)6MO7024, 4.19; NiC12, 0.19; SnC12, 0.19; ZnCl2, 0.734.
The medium was supplemented with 1 mM NH4Cl, 29 mM NH4C1, or

29 mM KNO3 as a nitrogen source depending on the experimental

conditions. The medium containing NH4Cl was adjusted to pH

7.4, and medium containing KNO3 was adjusted to pH 6.8 with

KOH after autoclaving. Cells were supplied with a 2%(v/v)

C02-air mixture and light intensity sufficient to allow cell

division into four progeny.


Enzyme Assay


The aminating and deaminiating activity of the NADP-GDH

was measured spectrophotometrically at 340 nm, by adding a 10

to 20 iL aliquot of enzyme preparation to 500 AL of assay

solution. The deaminating assay solution was composed of 44

mM Tris, 20.4 mM glutamate, and 1.02 mM NADP+ (Sigma), pH









8.8. The aminating assay solution was composed of 50 mM

Tris, 25 mM a-ketoglutarate, 0.357 mM NADPH (Sigma), and

0.356 M (NH4)2SO4, pH 7.4. One unit of enzyme activity was

the amount of NADP-GDH required to reduce or to oxidize 1.0

VM of NADP+ or NADPH per min at 38.50C.


Isolation of RNA


All labware used in total cellular RNA isolation was

sterilized by baking at 2200C for 8 h, and sterile

plasticware was utilized whenever possible. All solutions

were made with H20 treated with 0.1% diethylpyrocarbonate

(Sigma) overnight at 370C, and autoclaved according to

Sambrook et al. (1989).

On the day of the RNA isolation, a pellet of C.

sorokiniana cells stored at -700C was resuspended 1 to 10

(w/v) in RNA breakage buffer: 0.1M Tris (pH8.5), 0.4M LiCl,

10 mM EGTA, 5 mM EDTA, 100 units/mL sodium heparin (Sigma,

100 units/mg), and 1 mM aurintricarboxylic acid (Sigma). The

cell suspension was centrifuged at 7000g for 5 min at 40C and

the supernatant was discarded. The cell pellet was

resuspendeed 1 to 10 (w/v) in RNA breakage buffer and

ruptured by passage through a French pressure cell at 20,000

p.s.i.. The cell homogenate was collected in a disposable 50

mL conical tube containing 0.05 times volume 20% (w/v) SDS,

0.05 times volume 0.5 M EDTA (pH 8), 200 ug/mL proteinase K

(Sigma), and allowed to incubate at room temperature for 15

min. One-half volume of TE buffer (Tris 10mM:EDTA 1mM, pH








8.0) equilibrated phenol was added to the homogenate and

after a 3 min incubation a one-half volume of

chloroform:isoamylalcohol (24:1,v/v) was added and mixed for

10 min on a wrist action shaker (Burrel). The extracted

homogenate was transfer to a 30 mL siliconized (Sigmacote,

Sigma) corex tube and centrifuged at 10OOg for 10 min at 40C.

The upper aqueous phase was removed and repeatedly extracted

with an equal volume of chloroform:isoamylalcohol (24:1,

v/v), as described above, until the aqueous interface was

clear. After the final extraction, the aqueous phase was

combined with an equal volume of 2X LiCl-Urea buffer (4 M

LiCl, 4 M urea, 2 mM EDTA, 1 mM aurintricarboxylic acid) and

the RNA was precipitated on ice for 16 h at 40C. The RNA

precipitate was centrifuged at 4000g for 20 min at 40C and

the resulting pellet was rinsed once with IX LiCl-Urea buffer

and centrifuged again to pellet the RNA. The RNA pellet was

solublized in TE (pH 7.5) and an aliquot was quantified

spectrophotometrically at 260 nm. After quantitation, the

total RNA was precipitated with 0.3M sodium acetate (pH 5.2)

and 2.5 times volume of 100% ethanol and stored at -200C as a

precipitate. The mRNA fraction was isolated from total

cellular RNA using an oligo(dT) spin column kit (mRNA

SeparatorTM, Clontech) according to the supplier's

instructions.








Genomic DNA Isolation


Total cellular DNA was isolated from C. sorokiniana

cells using a modified procedure of Ausubel et al. (1989). A

6.5 g pellet of 29 mM KN03 cultured cells and a 6.0 g of 29

mM NH4Cl was harvested by centrifugation at 7000 rpm for 10

min at 40C. Each cell pellet was resuspended in 25 mL of DNA

extraction buffer (0.1 M Tris-HCl, pH 8; 0.1 M EDTA, pH 8;

0.25 M NaCl), mixed with 2.7 mL of 10% (w/v) sarkosyl, and

incubated for 2 h at 550C. The cell homogenate was combined

with 2.78 mL of 5M NaCl, 3.2 mL of 10% (w/v) CTAB, and

incubated at 650C for 20 min. An equal volume of

chloroform:isoamylalcohol (24:1, v/v) was added and extracted

by gentle shaking for 10 min followed by centifugation at

3000 rpm for 10 min at 40C. The supernatant was transferred

to a new tube and the nucleic acids were precipitated with

0.6 times volume of isopropol alcohol at -200C for 30 min.

The precipitate was pelleted by centrifugation at 4500 rpm

for 20 min at 40C. The pellet was resuspended in 9.5 ml of

TE (pH 8) and combined with 10 g of CsCl (BRL) and 0.5 mL of

10 mg/mL ethidium bromide. The CsCl:DNA mix was placed on

ice for 30 min and the contaminating RNA was removed by

centrifugation at 7500g for 10 min. The CsCl supernatant was

transferred to a 13 mL Quickseal tube (Beckman) and

centrifuged in a Vi65 Beckman rotor for 6 h at 55,000 rpm.

The high molecular weight DNA band was visualized with a

hand-held UV light, eluted from the tube, butanol extracted,

and dialysed 15 h against two changes of 1 L of TE (pH 8).








The dialyzed genomic DNA was quantified spectro-

photometrically at 260 nm and stored as an ethanol

precipitate until use.


NADP-GDH Protein Purification



Purification of the a-NADP-GDH holoenzyme


C. sorokiniana cells were cultured with continuous light

in 29 mM ammonium medium in a 30 L Plexiglas chamber as

previously described (Baker and Schmidt, 1963). Cells were

harvested at 4.0 0D640 by centrifugation at 30,000 rpm through

a Sharples centrifuge (Pennwalt) and washed two times in 10

mM Tris (pH 8.5 at 40C). Pelleted cells (130 g) were stored

at -200C in 250 mL centrifuge bottles until use.

Purification of NADP-GDH was accomplished using a modified

procedure of Yeung et al. (1981). Procedural modifications

involved the substitution of Sephadex G-200 gel (Pharmacia)

for G-150 gel in the gel-filtration column, and the addition

of NADP+ as a stabilizer to a final concentration of 0.1 mM

to the gel-filtration buffer and all subsequent storage

buffers. As a final modification, the NADP+ affinity resin

step was omitted and a preparative nondenaturing-PAGE step

was substituted (Miller et al., 1994a).

Sephadex G-200 column fractions possessing NADP-GDH

activity were pooled and concentrated via Diaflow (Amicon)

filtration. The soluble enzyme (68 mg) was protected from

oxidation by the addition of DTT to a final concentration of








10 mM, and dialyzed for 30 min against 28.8 mM Tris, 192 mM

glycine, 2 mM DTT (pH 8.4). The dialysate was clarified by

centrifugation at 20,000g for 10 min at 40C and was combined

with 3 mL of 40% (w/v) sucrose and 1 mL of 0.02% bromophenol

blue.

For preparative nondenaturing PAGE, a 3 cm tall 7%

acrylamide (w/v, 28 acrylamide: 0.735 bis-acrylamide, pH 8.8)

resolving gel, and a 2 cm tall 2% acrylamide (w/v, 1.6

acrylamide: 0.4 bis-acrylamide, pH 6.6) stacking gel were

cast in the 28 mm ID gel tube of the Model 491 Prep Cell

(Bio-Rad). All acrylamide stocks were pretreated with AG501-

X8 mixed bed resin (Bio-Rad) to remove any contaminating

acrylic acid residue to prevent in vitro N-acylation of

proteins during electrophoresis. The protein sample was

electrophoresed at 15 mA constant power for 20 min and then

for 3.5 h at a constant power of 30 mA. Six milliliter

fractions were collected and assayed for NADP-GDH deaminating

activity and GDH containing fractions were pooled. The

enzyme in the pooled fractions in 10 mM KPO4 (pH 6.2), 0.1 mM

NADP+ was concentrated by Diaflow filtration to 1 mg/mL as

determined by the method of Bradford (1976), using BSA as a

standard. The concentrated enzyme preparation was stored at

-200C. The purity of the preparation was determined by

silver-staining using the Silverstain Plus kit (Bio-Rad) to

visualize proteins resolved by 10% (w/v) Tris-Tricine SDS-

PAGE (Shagger and von Jagow, 1987).








Partial purification of NADP-GDH isoenzymes

To insure that both the a-subunit and 3-subunit were

represented, NADP-GDH isoenzymes were purified from a mixture

of cells cultured for 240 min in 1 mM ammonium medium (14 g),

90 min in 1 mM ammonium medium (6 g), and for 20, 40, 60, and

80 min in 29 mM ammonium medium (1 g/time point) according to

Bascomb and Schmidt (1987). GDH isoenzymes were partially

purified using a scaled down modified procedure of Yeung et

al. (1981). The DEAE sephacel ion exchange columns (pH

7.4,pH 6) were scaled down to a 40 mL bed volume and a 400 mL

linear KC1 gradient (0 to 0.4 M) was used to elute the

proteins in 3 mL fractions. The pH 6 DEAE ion-exchange

column fractions containing NADP-GDH were combined into two

pools; corresponding to the leading and trailing halves of

the NADP-GDH activity peak. The separate pooled fractions

were dialyzed against 10 mM KP04 (pH 6.2), 2 mM DTT for 16 h,

and affinity purified using Type 3 NADP+ affinity gel

(Pharmacia) as previuosly described (Bascomb and Schmidt,

1987). The NADP-GDH in the pooled fractions was concentrated

via Diaflow filtration to 2 mg/ml protein, as determined by

the method of Bradford (1976), and stored at 40C until

further use. After resolution of the proteins by 8% (w/v)

Tris-Tricine SDS-PAGE (Shagger and von Jagow, 1987), the

purity of the preparation was determined by silverstaining.








Anti-NADP-GDH Antibody Production and Purification


Monoclonal antibody production


Mouse anti-NADP-GDH MAbs were produced as described by

Tamplin et al. (1991). BALB/C mice were immunized by

intraperitoneal injection of 40 pg purified NADP-GDH a-

subunit in 0.25 mL PBS mixed 1:1 (v/v) with Freunds complete

adjuvant (Sigma). On day 24, the mice were injected

intraperitonealy with 25 ig of purified a-subunit in 0.25 mL

PBS mixed 1:1 (v/v) with Freunds incomplete adjuvant (Sigma).

The mice were injected on day 38 with 25 gg of purified a-

subunit in 0.5 mL PBS, and once again 2 days prior to cell

fusion. Tail bleeds were performed one week prior to cell

fusion and analysed by ELISA to select mice producing a high

anti-NADP-GDH titer. Splenocytes and myeloma SP2/0 were

fused by the protocol of Van Deusen and Whetstone (1981).

After selection with HAT medium, hybridoma supernatants were

screened by ELISA in 96 well EIA plates (Costar) against 1.25

pg NADP-GDH a-subunit in 50 gl 0.1 M Na2CO3 (pH 9.6) per

well. The ELISA procedure was performed as previously

described (Tamplin et al., 1991). ELISA positive clones were

selected and hybridoma supernatants were screened for

reactivity on Western blots. Selected high-titer hybridomas

were cloned by limiting dilution (Harlow and Lane, 1988), and

hybridoma supernatants were collected and frozen at -200C for

future use.








Polyclonal antibody production


Rabbit anti-NADP-GDH antibodies were produced

commercially by Hazelton Washington (Vienna, VA) in six New

Zealand white rabbits. Rabbits were given a primary

immunization of 0.3 mg of purified NADP-GDH a-holoenzyme per

animal; followed by mulitple 0.15 mg booster injections.

Preimmune sera, test bleeds, and production bleeds were

shipped on dry ice for future analyses. Anti-NADP-GDH IgG

titer was determined by measuring the ability of a series of

increasing amounts of antiserum or purified IgG to

immunoprecipitate 1 unit of NADP-GDH activity. After

incubating the immunoprecipitation mix for 35 min at 250C,

immunoprecipitates were removed by centifugation at 14,000

rpm for 2 min in an Eppendorf microfuge. The titer was

recorded as the percent of NADP-GDH activity remaining in the

supernatant relative to a preimmune serum control. Standard

reaction conditions utilized 10 RL of 25 mM imidazole (pH 6),

1 unit of NADP-GDH deaminating activity in 20 pL of 25 mM

imidazole (pH 6), anti-NADP-GDH antiserum or purified IgG in

a range from 0 to 25%, and rabbit preimmune serum was added

as a stabilizer to a final volume of 40 RL.

Anti-NADP-GDH IgG was purified from rabbit serum using a

MASSTM Protein A affinity membrane 50 mm disc device (Nygene

Corp.). Anti-NADP-GDH rabbit serum was diluted 1:4 (v/v) in

6 mL of PBS and passed through 0.45 im syringe filter

(Gelman). Filtered serum was bound to the Protein A disc at

40C and washed with PBS until the A280 of the eluate was








zero. Purified IgG was eluted with 10 mL of 0.1 M glycine

(pH 2.5) and 1 mL fractions were collected in microfuge

tubes, containing 0.1 mL of 1 M Tris (pH 8), and then gently

inverted to mix the contents. Fractions were analyzed

spectrophotometrically at 280 nm and fractions containing

greater than 1 mg/mL IgG were combined and frozen at -200C in

0.25 mL aliquots.

Western Blotting



Alkaline phosphatase conjugated antibody detection


For NADP-GDH antigen determinations, the proteins in

samples were resolved by 8% (w/v) Tris-Tricine SDS-PAGE using

a Mini-Protean II cell (Bio-Rad) or a 400 SE cell(Hoeffer).

The proteins were electroblotted to a 7 cm x 10 cm Immun-

LiteTM nylon membrane (Bio-Rad) at 20 V constant voltage for

16 to 20 h at 40C in a Mini-Transblot II cell (Bio-Rad).

Whatmann 3MM filter paper, scotch-brite pads, and Immun-LiteTM

membrane, utilized in the electrotransfer, were pre-

equilibrated for 10 min in 10 mM MES (pH 6), 0.01% (w/v) SDS

transfer buffer at room temperature. The immunodetection

procedure was performed at room temperature using the Immun-

LiteTM chemoluminescent detection kit (Bio-Rad). The nylon

membrane was incubated for 1 h in TBS blocking buffer (20 mM

Tris, 0.5 M NaCl, pH 7.5; 5% [w/v] nonfat dry milk). The

membrane was washed for 5 min with TTBS (TBS with 0.05% [v/v]

Tween 20) and incubated 2 to 16 h in primary antibody buffer








(TTBS, 1% [w/v] nonfat dry milk, rabbit anti-NADP-GDH IgG

[1.9 mg/mL IgG faction] diluted 1:200 or mouse anti-NADP-GDH

MAb hybridoma supernatant diluted 1:10). The nylon membrane

was washed for 15 min with three changes of 40 mL TTBS, and

incubated for 1 h in secondary antibody buffer(TTBS, 1% [w/v]

nonfat dry milk, 1:3,000 dilution of goat anti-rabbit IgG AP

conjugate [Bio-Rad] or 1:1,500 dilution of goat anti-mouse

IgG [whole molecule] AP conjugate [Sigma]). The immunoblot

was washed for 15 min with 3 changes of 40 mL TTBS, and 5 min

in 40 mL of TBS. The membrane was immersed for 5 min in

chemoluminescent substrate, sealed in a heat-sealable bag

(Seal-n-Save, Sears), and exposed to Kodak X-Omat AR film for

30 s to 5 min. After chemoluminescent detection, the

immonoblot was rinsed for 5 min in TBS, and incubated with a

color development substrate (10 mL 0.1 M Tris-HCl [pH 9.5],

0.1 M NaCl, 50 mM MgCl, 45 FiL NBT, 35 AL X-phosphate

[Boehringer Manniheim]). Color development was monitored

visually and stopped by the addition of 30 mL of TE (pH 8),

and molecular weight determinations were made relative to a

series of prestained protein markers (Midrange kit,

Diversified Biotech; Rainbow markers, Amersham).


125I-Protein A detection


The proteins in 12 pL aliquots of clarified cell

homogenates (20 mL cell culture concentrated in 3 mL of GDH

breakage buffer) were resolved by 8% (w/v) Tris-Tricine SDS-

PAGE in the 400 SE cell. The gel was electrophoresed at 10






mA constant current until the prestain 30 kD protein marker

(Rainbow markers, Amersham) reached the bottom of the gel.

The SDS gel, nitrocellulose (Bioblot-NC, Costar), and

Whatmann 3MM filter paper were equilibrated for 30 min in

24.8 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.4

transfer buffer (Towbin et al. 1979). The proteins were

transferred electrophoretically at 30 V constant voltage for

16 h at 40C in a Trans-Blot cell (Bio-Rad). Immunodetection

was performed using a modified procedure of Towbin and Gordon

(1984) and Johnson et al. (1984). After electroblotting, the

nitrocellulose was air-dried and blocked in 50 mL of 40 mM

Tris (pH 7.4), 150 mM NaCl, 5% (w/v) nonfat dry milk

(Carnation), and 0.01% (v/v) Antifoam A (Dow-Corning)

blocking buffer for 1 h. All incubations were performed at

room temperature in a heat-sealable bag on a Labquake shaker

(Lab Industries). The blocking solution was decanted and the

nitrocellulose was incubated in 30 mL of 40 mM Tris (pH 7.4),

150 mM NaCl, 5% (w/v) nonfat dry milk, 0.01% (v/v) Antifoam

A, 0.05% (v/v) Tween 20 (Batteiger et al., 1982), and rabbit

anti-NADP-GDH antibody (1.9 mg/mL, IgG fraction) diluted

1:300 for 3 h. The immunoblot washed for 1 h with three

changes of 200 mL of 40 mM Tris (pH 7.4), 150 mM NaCIl and

transferred to 25 mL of 40 mM Tris, (pH 7.4), 150 mM NaCI, 5%

(v/v) nonfat dry milk, 0.01% (v/v) Antifoam A, 0.05% (v/v)

Tween 20, and 0.25 iCi/lane of 125I-labelled Protein A (30

mCi/mg, Amersham) for 1 h in a new heat-sealable bag. The

nitrocellulose was washed for 1 h with three changes of 300








mL of 40 mM Tris (pH 7.4), 150 mM NaCl. The immunoblot was

allowed to air dry for 3 h on a stack of paper towels and

exposed to Fuji RX autoradiogrphy film at -700C.


Amino-Terminal Sequence Analysis of the NADP-GDH a-Subunit
and P-Subunit


An aliquot of a preparation of purified NADP-GDH a-

subunit (120 pmol) and a partially purified preparation of

NADP-GDH a-subunit (80 pmol) and p-subunit (50 pmol) were

resolved by 8% (w/v) Tris-Tricine SDS-PAGE and electroblotted

to a PVDF membrane (Immobilon-PSQ, Millipore) as described by

Plough et al. (1989). To prevent in vitro acylation of the

protein amino-terminal residues, all polyacrylamide solutions

used in PAGE were treated with AG501-X8 mixed bed resin to

remove contaminating acrylic acid. Protein sequence analysis

of the electroblotted proteins was provided by the ICBR

Protein Chemistry Core facility.


DNA Probe Synthesis


Specific cDNA restriction fragments were excised from

purified plasmids using the appropriate restriction

endonuclease (BRL). The plasmid restriction endonuclease

fragments were separated by electrophoresis in an alkaline

agarose gel (0.8% to 3% [w/v]) in TAE buffer (40 mM Tris-

acetate, 1 mM EDTA). The ethidium bromide stained fragment

of interest was cut out of the gel and mascerated in the

upper portion of a 0.45 Rm nylon membrane microfilterfuge







tube (Ranin). The mascerated agarose was frozen for 1 h at

-200C and then centrifuged at 3000 rpm in an Eppendorf

microfuge. The gel purified fragment in the supernatant was

precipitated with 0.3 M sodium acetate (pH 5.2) and 2.5 times

volume of 100% ethanol at -20oC.

Twenty-five nanograms of purified cDNA fragment were

diluted in 33 RL dH20 and denatured at 1000C and chilled

rapidly on ice. The denatured fragment was radiolabeled with
32P-dGTP (3000 Ci/mmol, Amersham) by the random primer method

of Feinberg and Vogelstein (1984). Unicorporated nucleotides

were removed from the 32P-labeled probe using a Sehadex G-50

(Pharmacia) spin column (Sambrook et al., 1989).


Northern Blot Analysis


Total or poly(A)+ RNA stored as an ethanol precipitate

was pelleted by centrifugation at 14000 rpm for 15 min in an

Eppendorf microfuge. The vacuum dried pellet was resuspended

in 20 VL of 75% (v/v) formamide (BRL), 8.28% (v/v)

formaldehyde, 3% (v/v) 10X MOPS (20 mM MOPS, 50 mM sodium

acetate, 10 mM EDTA, pH 7) and heated for 5 min at 800C then

chilled on ice. The denatured RNA sample was combined with 6

PL of formamide loading buffer (Sambrook et al., 1989) and 1

pL of 10 mg/mL ethidium bromide and resolved on a 2% (w/v)

formaldehyde-agarose gel (Ausubel et al., 1989). The RNA was

electrophoresed at 5 V/cm with constant circulation of the

electrophoresis buffer using magnetic stir bars in the buffer

reservoirs. After visualization with a UV transilluminator








(Fotodyne), the RNA was transferred by capillary blotting to

a Hybond-N nylon membrane (Amersham) with 20X SSPE (3.6 M

NaCI, 0.2 M KP04, pH 7.7; 20 mM EDTA). The nylon membrane

was rinsed once in 2X SSPE, air dried for 1 h, and UV

irradiated for 5 min on a transilluminator to covalently link

the RNA to the membrane.

The membrane was prehybridized for 2 h at 400C in 10 mL

of 50% (v/v) formamide, 25 mM KP04 (pH 7.7), 5X SSPE, 5X

Denhardt,s solution (0.1% [w/v] Ficoll, 0.1% [w/v]

polyvinylpyrrolidone, 0.1% [w/v] BSA), 0.1% (w/v) SDS, 100

Ag/mL sheared denatured salmon sperm DNA, 100 Rg/mL yeast

tRNA in a heat-sealable bag on a Labquake shaker. The

prehybrization solution was decanted and the membrane was

hybridized for 16 h at 420C on a Labquake shaker in 10 mL of

prehybridization buffer with 1 x 106 to 1 x 108 cpm of a heat

denatured 32P-labeled cDNA probe. The membrane was washed

three times in 0.1X SSPE, 0.1% (w/v) SDS for 20 min per wash

at 650C. The Northern blot was covered with laboratory

plastic wrap and exposed to Kodak X-Omat AR film with one

intensifying sceen at -700C.


Southern Blot Analysis


C. sorokiniana high molecular weight genomic DNA (6 pg)

was digested with a threefold excess of Pvu II, or Taq I

(BRL) for 4 h at the appropriate buffer and temperature

conditions. Three, 2 ig aliquots of Pvu II digested, Taq I

digested, and undigested genomic DNA were electrophoresed at








1.5 V/cm in a 0.8% (w/v) agarose gel in TAE buffer. Ethidium

bromide (0.5 Rg/mL) was added to the gel prior to

polymerization to allow visualization of the DNA during

electrophoresis. After electrophoresis, the gel was soaked

for 10 min in 0.1 N HC1, 35 min in 0.5 N NaOH, 1.5 M NaCI

denaturing solution, and 45 min in 0.5 M Tris, 3 M NaCl, pH 7

neutralizing solution. The DNA was transferred to a Hybond-N

nylon membrane by capillary action using 20X SSC (3 M NaCI,

0.3M sodium citrate) as described by Southern (1975). After

transfer, the membrane was rinsed in 2X SSC, air dried, and

the DNA was covalently linked to the membrane for 5 min on a

UV transilluminator. The nylon membrane was cut into three

strips each containing Pvu II digested, Taq I digested, and

uncut genomic DNA for analysis with different probes.

The nylon membranes were prehybridized in a HB-1D

hybridization oven (Techne) in 15 mL of 20% (v/v) formamide,

0.6 M NaCl, 0.6 M sodium citrate, 10 mM EDTA, 0.1% (w/v) SDS,

5X Denhardt's solution, 100 pg/mL denatured sheared salmon

sperm DNA for 2 h at 400C. The membranes were hybridized

independently for 16 h at 450C in 15 mL of 50% (v/v)

formamide, 10% (w/v) dextran sulfate (Sigma), 1X Denhardt's

solution, 4X SSC, 5 pg/mL denatured sheared salmon sperm DNA,

and 1 x 106 to 1 x 108 cpm denatured 32P-labeled CDNA probe.

The cDNA probes corresponded to the 5'-VR, HCR, or 3'-UTR of

the consensus C. sorokiniana NADP-GDH mRNAs. The membranes

were washed independently three times in 50 mL of 0.1X SSC,

0.1% (w/v) SDS for 30 min per wash at 650C. The nylon








membranes were covered with plastic wrap and exposed to Fuji

RX film with one intensifying screen at -70oC.


NADP-GDH cDNA Cloning and Characterization



qtl0O library


Small scale liquid lysates of previously isolated plaque

pure NADP-GDH kgtl0O clones were produced as described by

Sambrook et al. (1989). The kgtl0O clone DNA was isolated

using a rapid small scale liquid lysate k phage DNA isolation

procedure (Ausubel et al., 1989). The cDNA inserts were

excised from the purified phage DNA with Eco RI restriction

endonuclease, gel purified as described above, subcloned into

the multiple cloning site of pUC 18, and transformed by the

CaCl2 method (Ausubel et al., 1989) into Eschericia coli DH5a

for further characterization.


kZAP II library


Synchronous C. sorokiniana cells were cultured in a 3 L

chamber in 1 mM ammonium medium or 29 mM ammonium medium

according to conditions reported by Bascomb and Schmidt

(1987) to yield primarily the NADP-GDH a-holoenzyme or

predominately the p-holoenzyme, respectively. The level of

ammonia in the 1 mM ammonium medium induction was

periodically measured by the method of Hardwood and Kuhn

(1970). Cells were harvested by centrifugation at 8000g at

40C, and frozen at -700C for use in total RNA isolation. The








cells in 20 mL from each growth condition were concentrated

by filtration, resuspended in 3 mL of GDH breakage buffer,

and ruptured by passage through a French pressure cell at

20,000 p.s.i.. Aliquots of the various homogenates from

ammonium induced cells were resolved by 7.5% (w/v)

nondenaturing PAGE and the NADP-GDH isoenzyme pattern was

determined by use of a selective activity stain (Yeung et

al., 1981).

Total cellular RNA was isolated from 2 g cell pellets

from each growth condition as described above and the

poly(A)+ RNA fraction was purified by elution from an

oligo(dT) cellulose spin column (Clontech). The poly(A)+

selection was repeated two times on each RNA preparation to

insure complete removal of contaminating tRNAs, and rRNAs.

Both total RNA (20 pg) and poly(A)+ RNA (10 Rg) were analyzed

by 2% (w/v) formaldehyde-agarose gel electrophoresis, and

northern blot analysis, using a 242 bp HCR radiolabeled cDNA

probe, to verify the purity, intactness, and approximate

quantity of NADP-GDH mRNA represented in the a-induced and p-

induced RNA preparations. Poly(A)+ RNA (50 Rg) from each

preparation was combined and utilized for the commercial

production of a custom XUni-ZAP XR C. sorokiniana cDNA

library (Stratagene Cloning Systems, Palo Alto, CA).

The amplified XZAP library, containing 2 x 1010 pfu/mL,

was plated on twenty 150 mm petri plates at 50,000 pfu per

plate for a total of 1 x 106 pfu screened. The phage plaques

were absorbed to duplicate Hybond-N 132 mm circular membranes








and treated according to the plaque blotting protocol of

Amersham (1985). Membranes were prehybridized in a common

container in 200 mL of 2X PIPES (0.8 M NaCI, 20 mM PIPES, pH

6.5), 50% (w/v) formamide, 0.5% (w/v) SDS, 100 Rg/mL

denatured sheared salmon sperm DNA at 400C. Blocked

membranes were hybridized at 420C in ten heat-sealable bags

(four membranes/bag) in prehybridization buffer containing 1

x 106 cpm/membrane of a 32P-labeled NADP-GDH 242 bp HCR cDNA

probe on a lab rocker (Reliable Scientific). The membranes

were washed three times in 200 mL of 0.1X SSC, 0.1% (w/v) SDS

for 20 min per wash at 500C. Duplicate membranes were

wrapped in plastic wrap and exposed to Kodak X-Omat AR film

at -700C for 28 h. Putative NADP-GDH cDNA plagues, detected

on duplicate membranes, were cored from the plate and plaque

purified by secondary and tertiary screenings with the 242 bp

HCR probe. Putative NADP-GDH cDNA phage clones (167),

selected in the primary screening, were combined and screened

a second time with a 32P-labeled 130 bp Eco RI/Bgl II cDNA

fragment isolated from the 5' terminus of the most complete

5' end NADP-GDH cDNA clone (pGDc 42). Ten plaque pure NADP-

GDH clones were subcloned in pBluescript KS+ and transformed

into E. coli DH5a F' via an in vivo excision protocol

provided by Stratagene. All plasmid isolations were

performed as described by Kraft et al. (1988).







5' RACE-PCR cloning


The 5'-terminal NADP-GDH cDNA sequences were cloned

using a modified anchored PCR procedure for the rapid

amplification of cDNA ends (Frohman, 1990; Jain et al.,

1992). A mixture of poly(A)+ RNA, used in the synthesis of

the XZAP library, was utilized to clone the 5' end of the

NADP-GDH mRNA. One hundred nanograms of the mRNA mixture

were combined with 10 ng of a gene-specific primer (RRS 9;

Table 1), designed to hybridize to the HCR of NADP-GDH mRNAs,

heated for 5 min, and chilled on ice. First strand DNA

synthesis was performed using SuperscriptTM reverse

transcriptase (BRL) according to the supplier's protocol.

The terminated reverse transcription reaction was treated

with one unit of ribonuclease H for 20 min at 370C, 5 min at

950C, and extracted once with chloroform:isoamyl alcohol

(24:1, v/v). Excess primers and dNTPs were removed by

centrifugation at 2000 rpm through an Ultrafree-MC filterfuge

tube (30,000 MW cutoff, Millepore) and the retentate was

concentrated to 10 il on a Savant Speedvac. The first-strand

synthesis products were combined with 10 pL of tailing mix

(IX tailing buffer [Promega Corp.], 0.4 mM dATP, 10 units

terminal deoxytransferase) and incubated at 370C for 10 min.

The reaction mixture was heated to 950C for 5 min, diluted to

0.5 mL with TE (pH 8), and utilized as a cDNA pool. A

mixture of 5gL of the cDNA pool, 5 gL of VentTM polymerase 10X

buffer (NEB), 200 AM of each dNTP, 25 pmol of a gene specific



























Table 1. Synthetic oligonucleotide sequences


Oligomer
RRS5
RRS6
RRS7
RRS9
RRS11
RRS12
RRS13
RRS14
RRS15
RRS16
RRS17
RRS 18
RRS19
RRS24
RRS25


Nucleotide Sequence
GGGCTGCGCAGGCCGGGCGGCCACGATAGG
GGGTCGACATTCTAGACAGAATTCGTGGATCC(T)18
GGGTCGACATTCTAGACAGAA
CTCAAAGGCAAGGAACTTCATG
GGACGAGTACTGCACGC
GAGCAGATCTTCAAGAACAGC
TCTGCACGTAGCTGATGTGG
CCCAGCCAGGGCCCTCACC
CACAGTATCGCATTCCGGGC
GATCTCGGTCAGCAGCTG
CTTTCTGCTCGCCCTCTC
GCGGCGACATCGCGC
CGTGCGCCAGCTGCTGAC
CCTTGTTGTACTTGTGG
CCACAAGTACAACAAGG








primer (RRS 9), 5 pmol of the poly(dT) adaptor primer

(RRS6), 0.2 units PerfectmatchTM DNA polymerase enhancer

(Stratagene), and 1 unit of VentTM polymerase (NEB) in 50 RL

was amplified according to Jain et al. (1992). The PCR

products were purified away from the excess primers by

centrifugation at 2,000 rpm through an Ultrafree-MC unit.

The retentate was collected and subjected to two more rounds

of amplification using a new nested gene specific primer at

each step (RRS 11; RRS16, respectively) and an adaptor primer

(RRS 7). PCR amplifications were performed in a Model 480

thermocycler (Perkin-Elmer Cetus), and all custom

oligonucleotides were synthesized by the ICBR DNA synthesis

facility. The standard PCR reaction mixture consisted of 10

pIL of 10X VentTM polymerase buffer, 100 VM of each dNTP, 0.4

units of PerfectmatchTM, 50 pmol of each primer, 1 unit VentTM

DNA polymerase in a 100 pl reaction volume. The optimal PCR

cycling parameters were determined using OligoTM 4.0 primer

analysis software (National Biosciences Inc.). The 5' RACE-

PCR products were gel purified, subcloned into the Sma I site

of pUC 18, and transformed into E. coli DH5a for further

characterization.


NADP-GDH cDNA characterization


Purified NADP-GDH cDNA clone plasmids were digested for

1 to 2 h with specific restriction endonucleases using buffer

and temperature conditions deemed optimal by the supplier

(BRL). The resulting DNA fragments were resolved by








electrophoresis in a 1% (w/v) agarose minigel in TAE buffer.

DNA fragments less than 500 bp were resolved by 4% (w/v, 29

acrylamide:1 bis-acrylamide) PAGE in TBE buffer (0.13 M Tris,

45 mM Borate, 2.5 mM EDTA, pH 9) at 7 V/cm until the

bromophenol blue dye was 1.5 cm from the base of the gel.

Restriction fragment size was determined by comparison to

XDNA/Hind III and OX174/Hae III restriction fragment

standards (BRL).

NADP-GDH cDNA clones were sequenced by the dideoxy

method of Sanger et al. (1977) using modified T7 polymerse

(Tabor and Richardson, 1987) and the Sequenase 2.0 kit

protocol (United States Biochemical Corp.). The products of

the sequencing reactions were resolved on a 7 M urea, 5%

(w/v, 29 acrylamide:1 bis-acrylamide) sequencing gel. All

cDNA clones were partially sequenced from both ends.

Internal sequences were determined by subcloning select

restriction fragments into pUC 18 or by generation of a set

of nested deletions by timed digestion with exonuclease III

(Henikoff, 1984) using the Erase-a-base system (Promega

Corp.). Sequence data were analyzed using the Genetics

Computer Group programs (Devereux et al., 1984) on the ICBR

VAX computer.


Primer Extension Analysis


The 5' transcriptional start sites of the NADP-GDH mRNAs

were mapped by primer extension analysis as described by

Sambrook et al. (1989). A 20 Rg aliqout of a poly(A)+ RNA








mixture, isolated for use in the XZAP library synthesis, was

combined with 3.5 x 105 cpm of a 32P-labeled 30 nucleotide

oligomer (RRS 5) designed to hybridize to the 5' end of the

NADP-GDH mRNA. The primer:RNA mixture was denatured at 850C

for 10 min and allowed to anneal for 12 h at 300C. After

annealing, the RNA:primer complex was extended at 450C for 2

h with 5 units of SuperscriptTM reverse transcriptase, 0.5 mM

of each dNTP, according to the supplier's protocol. The

extension reaction products were treated with 5 ng of DNase

free RNase A (Sigma) for 30 min at 370C,

phenol:chloroform:isoamylalcohol (25:24:1, v/v) extracted,

and ethanol precipitated at 00C for 1 h. The pelleted

precipitate was resuspended in 6 iL of formamide loading

buffer and resolved on a 7 M urea, 5% (w/v) acrylamide

sequencing gel. The primer extension product was detected by

autoradiography on Kodak X-Omat AR film at -700C and the size

of the extension product was estimated by comparison to a

sequencing ladder.

Genomic Allele-Specific PCR


C. sorokiniana genomic DNA was analysed by allele-

specific PCR as described by Saiki et al. (1986). Genomic

DNA (1 Rg) and three NADP-GDH genomic clones in pUC 18 (0.1

ng; pGDg 8.4.4, 14.10.1, 15.2.2) were amplified using exon-

specific primer pairs that hybridized to exons one and three

(RRS17, RRS18), exons 10 and 11 (RRS12, RRS13), and exon 22

(RRS14, RRS15) of the NADP-GDH gene. The standard genomic








PCR reaction mixture was composed of IX VentTM polymerase

buffer, 200mM of each dNTP, 50 pmol of each primer, 0.4 units

PerfectmatchTM, and 1 unit VentTM polymerase. PCR cycles were

executed under cycling parameters deemed optimal for each

primer pair. The allele-specific PCR products were resolved

by 4% (w/v) PAGE and visualized by ethidium staining. The

size of the PCR products was estimated relative to a 123 bp

ladder (BRL).


Construction of NADP-GDH In vitro Transcription Vectors


PCR generated fragments corresponding to the 5'-VR of

the +42 nt and -42 nt mRNAs, HCR, and 3'-UTR were cloned

downstream of the SP6 promoter of the SP65 in vitro

transcription vector (Promega Corp.). Using the 5' RACE-PCR

+42 bp and -42 bp cDNA clones (5'-VR; RRS16, RRS17) or pGDc

23 (HCR7 RRS 12, RRS13: 3'-UTR; RRS14, RRS15) as templates,

the PCR fragments were amplified, gel purified, and cloned

into the SmaI site of pUC 18. Clones corresponding to each

of the mRNA regions were selected on the basis of their

orientation in pUC 18, excised by digestion with Sal I/Eco

RI, and directionally cloned in the antisense orientation

into the SP65 transcription vector. Constructs were verified

by sequence analysis and were purified by a large scale CsCl

plasmid isolation procedure (Ausubel et al., 1989). 32p_

labeled antisense RNA probes corresponding to the four

regions of the mRNA were transcribed using the RiboprobeTM in

vitro transcription system (Promega Corp.). Full length in








vitro transcription products were selected by gel

purification on a 7 M urea, 5% (w/v) acrylamide sequencing

gel according to Ausubel et al. (1989) and used as protecting

fragments for RPA.


Comparison of the NADP-GDH mRNAs, Antigens, and Activities in
29 mM Induced C. sorokiniana Cells



Culture conditions


C. sorokiniana cells were synchronized using three

alternating light:dark periods (9 h: 7 h) in 29 mM KNO3

medium. Cells were harvested by centrifugation, washed with

nitrogen-free medium, and resuspended in 4 L of pre-

equilibrated nitrogen-free medium in a 4 L Plexiglas chamber.

The cells were induced in 29 mM ammonium medium for 240 min

as described by Cock et al. (1991). Samples of 500 mL of

cell culture (2.87 x 106 cells/mL)were collected at TO, and

at 20 min intervals for the first 140 min, and a final sample

was harvested at 240 min. Samples for RNA isolation were

concentrated by centrifugation at 40C at 9,000 rpm and

stored at -700C. The cells in 20 mL of culture were

harvested by filtration, resuspended in 3 mL of GDH breakage

buffer, and stored at -200C for protein and GDH activity

analyses.








RNase protection analysis


Total cellular RNA was isolated from a 1 g pellet from

each time point as described above. The amount of poly(A)+

RNA in the various total RNA preparations was quantified

based on the formation of ribonuclease-resistant hybrids with

poly-3H-5,6-uridylate (2-10 Ci/mmol, New England Nuclear) as

described by Davis and Davis (1978) using purified p-globin

mRNA (BRL) as a standard.

The quanity and form of mRNA present at each time

interval was determined by RPA. A total of 210 ng of Poly

(A)+ RNA or 25 ng of control yeast tRNA was combined with 1 x

105 cpm of a 32P-labeled antisense RNA probe corresponding to

the +42 nt and -42 nt 5'-VR, HCR, or 3'-UTR of the NADP-GDH

mRNAs and hybridized for 16 h at 420C. RPAs were performed

using the GuardianTm RPA kit (Clontech) according to the

suppliers instructions. The ribonuclease resistant fragments

were resolved on a 7 M urea, 5% (w/v) sequencing gel and

sized by comparison to a sequencing ladder. The resistent

fragments were transferred from the gel to 3MM Whatmann

filter paper, dried at 800C, and exposed to a phosphorous

screen and then to Fuji RX film at -700C. The amount of

residual antisense RNA probe was quantitated on a

PhospholmagerTM (Molecular Dynamics) at the ICBR DNA Synthesis

Core facility.








NADP-GDH antigen and activity analyses


C. sorokiniana cell samples from the various time points

were disrupted by two passages through a French pressure cell

at 20,000 p.s.i.. Aliquots from each cell homogenate were

analyzed for both aminating and deaminating NADP-GDH

activity. Total soluble protein concentration in the cell

homogenates was determined by the method of Bradford (1976)

using BSA as a standard. The proteins in 12 pL aliquots from

each homogenate were resolved by 8% (w/v) Tris-Tricine SDS-

PAGE, transferred to nitrocellulose, and NADP-GDH antigen was

detected by 125I-Protein A Western blot analysis as described

above. The amount of NADP-GDH antigen present as the a-

subunit and p-subunit at each time interval was quantified on

a Visage 60 laser desitometer (Bio Image).


RT-PCR analysis


A modified RT-PCR (Kawaski, 1990) procedure was used to

identify and quantify the NADP-GDH mRNAs present in the total

RNA preparations from the various induction time-points. A

series of primer pairs were selected to yield ovelapping PCR

fragments spanning the entire length of the NADP-GDH mRNAs:

RRS17, RRS18; RRS17, RRS16; RRS19, RRS13; RRS12, RRS13;

RRS12, RRS24; RRS15, RRS25. An aliquot of total RNA from

each time-point containing 10 Rg of poly(A)+ RNA was combined

with 300 pmol of random hexameric oligonucleotides

(Pharmacia), incubated at 750C for 5 min, and chilled on ice.

The RNA:primer mixture in 6 IL was combined with 2 tL of 10X








VentTM polymerase buffer, 10 units RNASINTM (Promega Corp.), 1

mM of each dNTP, 1 mM DTT, 200 units SuperscriptTM reverse

transcriptase in a final volume of 20 iL, and incubated at

220C for 10 min, 420C for 70 min, 500C for 30 min, and 950C

for 5 min. After termination of the reaction, 120 pL of IX

VentTM polymerase buffer was added to each reaction tube. The

resulting mixture served as cDNA stocks for subsequent

amplifications. The standard RT-PCR amplification mixture

consisted of 8 RL 10X VentTM polymerase buffer, 200 RM of each

dNTP, 50 pmol of each primer, 0.2 units PerfectmatchTM, 20 pL

of a cDNA stock, and 4 units of VentTM (exo-) polymerase (NEB)

in 100 [tL final volume. The RT-PCR mixtures were cycled at

conditions determined to be optimal for each primer pair

using the OligoTM 4.0 primer analysis software. RT-PCR

products were resolved on 1% to 3% (w/v) agarose gels and

sized by comparison to a 123 bp ladder (BRL). The relative

intensities of the PCR fragments was quantified from

PolaroidTM Type 55 negatives of ethidium bromide stained gels

on a Visage 60 laser densitometer.













RESULTS


NADP-GDH cDNA Cloning and Characterization



Restriction mapping and sequencing of XqtlO cDNA clones


A kgtlO cDNA library was constructed from poly(A)+ RNA

isolated from C. sorokiniana cells induced in 29 mM ammonium

medium for 80 min. Cells induced under these conditions were

reported by Prunkard et al. (1986) and Bascomb et al. (1987)

to accumulate both the a- and P-subunits as NADP-GDH

holoenzymes between 40 and 120 min, and at 80 min the a- and

P-subunits each constituted approximately 50% of the total

NADP-GDH antigen. Approximately 2 x 106 pfu were screened

with a heterologous 1.2 kb Salmonella typhimurium gdhA gene

probe (Miller and Brenchley, 1984) and six putative NADP-GDH

cDNAs were isolated. The cDNAs ranged from 0.6 to 1.91 kb

and their restriction maps were identical in regions in which

they overlapped (Fig. IB; Cock et al., 1991). The cDNA

clones appeared to be truncated forms of the 1.91 kb pGDc 23

cDNA clone that lacked a complete 5' terminus. A lacZ-pGDc

23 translation fusion expressed in E. coli JM 109 accumulated

antigen which was recognized by antibodies raised to purified

C. sorokiniana NADP-GDH verifying the cDNA authenticity (Cock

et al., 1991).





























Figure 1. Restriction maps of 17 cDNAs isolated from a
C.sorokinana library prepared from RNA isolated from cells
induced for 80 min in 29 mM ammonium medium. A, The 2145 bp
consensus NADP-GDH map. Regions corresponding to the HCR and
3'-UTR are indicated. B, NADP-GDH cDNA clones isolated using
a heterologous 1.2 kb probe from the gdhA gene from S.
typhymurium. cDNA clones were sequenced in the regions
denoted by arrows. C, The cDNA clones were isolated using a
homologous 115 bp PstI fragment from the 5' end of the HCR of
pGDc 23.




























0 2 0a o s 1 1 2


254 b Consv gn
Pst I Pst I
s ConservTd PSegon frobt


Pv' II H1o II Pvu II

a Rn a tI |qi II Eqi. II
jst l| st I Pst I N( I Nor I -st I Uar I


.< to 1S 20


3'-Untranlt.ed
Roelol Probe
!r" I S..!


H.I II


!1r I N.r I


I


S I Teco I
I .iY consensus
3 GDH-cDNA


FN, P B.o E P N ? JNc N N






?Kc 3g F x


39 F I IF PN Hcq x
Eq N N NN'N No!


? aC 2a P


P q 3 ,, q P





st I ?st I
5 Probe

P ? e B*g f





? P1 Ig P
,qN c Bg P









P No Eag P


SP ? B, g P




N- P -. E N
Nq P0 Eq N


N Nf Pv e I








S N PvN N E N


N a. NN


N a Pv p






N N P7 f Ke 3












N ? N N K.eN S


I p P. B ? P, Ns P


Po 3q N P c Eq PN


pGDc 23


pGDc 2


pGDc 3


pGDc 6


pGDc 7


pGDc 10






pGDc 30


pGDc 31


pGDc 32


pGDc 33


pGDc 34


pGDc 35


pGDc 36


pGDc 38


pGDc 39


S.
1.2., 0


0.1.,


N.l~4



0.1, 4


pGDc 42


SpGc 44


. ..............


,l








In an attempt to select additional NADP-GDH cDNAs with

complete 5' termini, the kgtl0 library was rescreened with a

homologous 115 bp PstI restriction fragment probe (Fig. IC)

derived from the 5'-proximal end of pGDc 23. Approximately

one-half of the 115 bp probe sequence overlaps the 5-terminus

of the 354 bp NADP-GDH HCR (Fig. 1A) that is predicted to be

present in all NADP-GDH cDNAs. Eleven additional cDNA clones

were isolated, restriction-mapped, and their 3' and 5'

termini sequenced (Fig. 1C). The additional cDNA clones had

identical restriction maps and sequences in regions that

ovelapped with pGDC 23. NADP-GDH cDNA clones pGDC 31, 38,

and 42 were longer than pGDC 23 at their 5' termini; however,

none of the cDNAs possessed an ATG start methionine codon or

a 5'-untranslated region. The 5'-terminal EcoRI/PvuII

fragment (Fig. IC) of pGDC 42 was subcloned into pUC 18 and

both strands were sequenced. Sequence analysis of the pGDc

42 subclone revealed that pGDc 42 possesed an additional 256

bp of coding region sequence not found in pGDc 23.

Although the cDNA library was prepared using an oligo

(dT) primer, 10 of the 17 NADP-GDH cDNAs lacked a poly(A)+

tail and additional 3'-terminal sequences (Fig. 1). None of

the 17 cDNA clones were full-length at their 5' termini.

From the combined sequences of the cDNA clones, a 2145 bp

consensus NADP-GDH cDNA sequence and restriction map was

constructed (Fig.lA). Bascomb et al. (1986) and Cock et al.

(1991) showed the NADP-GDH mRNA to be 2.2 kb; therefore, the








consensus NADP-GDH clone was determined to be approximately

98% full-length.


Isolation, restriction-mapping, and sequencing of the kZAPII
NADP-GDH cDNA clones


Since all of the gtl0O library NADP-GDH cDNA clones

lacked complete 5' termini and many lacked complete 3'

termini, a second ammonium-induced unidirectional cDNA

library was constructed. Synchronous C. sorokiniana cells,

used for total RNA isolation, were cultured in 1 mM or 29 mM

ammonium medium according to conditions reported to yield

primarily the NADP-GDH a-holoenzyme or predominately the 3-

holoenzyme, respectively (Bascomb and Schmidt, 1987). The

NADP-GDH isoenzyme pattern from each culture condition was

verified by activity staining crude preparations resolved by

nondenaturing PAGE. Poly(A)+ RNA from each culture condition

was analyzed by northern analysis, using the 242 bp HCR and

the 378 bp 3'-UTR probes (Fig. 1A), to verify the approximate

level and intactness of NADP-GDH mRNA in each preparation

(Fig. 2). An equal quantity of poly(A)+ RNA from each

preparation was combined, to ensure representation of

multiple types of NADP-GDH mRNAs if they existed, and the RNA

mixture was utilized in the cDNA library construction. The

XZAPII unidirectional cloning system was utilized to ensure

the cloning of the complete 3' ends of the mRNAs.

Initial screening of 1 x 106 pfu with the homologous 242

bp HCR probe derived from pGDc 23 (Fig. 1A) yielded 167































Figure 2. Northern blot analysis of poly(A)+ RNA isolated
from C. sorokiniana cells induced for 3 h in 1 mM ammonium
medium (Lanes 1,3) or continuously in 29 mM ammonium medium
(Lanes 2,4). Formaldehyde-agarose gel resolved RNA
preparations immobilized on nylon were hybridized with the
242 bp HCR probe and 378 bp 3'-UTR probe. A single-sized 2.2
kb NADP-GDH mRNA was detected in both RNA preparations with
both probes.









HCR 3'-UTR
1 2"34
9.49-
7.46-
4.40-
2.37-
2.235-
1.35-


0.24-








putative NADP-GDH cDNA clones. Ten of the primary screening

plaques were selected at random and plaque purified by

secondary and tertiary screenings. Restriction-mapping and

sequencing of the ten NADP-GDH cDNAs revealed eight unique

overlapping clones ranging in size from 1.46 to 2.04 kb (Fig.

3). Two of the 10 clones proved to be identical to pBGDC 52

and 58 indicating a detection of redundant clones in the

amplified library. All eight of the unique NADP-GDH clones

possessed identical complete 3' termini; however, they all

lacked complete 5' termini (Fig. 3). The longest kZAP NADP-

GDH cDNA clone, pBGDc 53, was 103 bp shorter than the gtl0O

NADP-GDH consensus cDNA.

To detect any cDNA clones longer at the 5' terminus than

pBGDc 53, the 167 putative NADP-GDH plaques selected in the

primary screening were combined and rescreened. The second

screening utilized a homologous 130 bp EcoRI/BglII cDNA

fragment probe derived from the 5' terminus of pGDc 42 (Fig.

IC). No NADP-GDH clones longer than pBGDc 53 were isolated

from the secondary screening.


Primer extension analysis


Although all of the NADP-GDH cDNA clones isolated from

the kZAP library possessed complete 3' ends, none of the 25

cDNA clones isolated from either library possessed complete

5' ends. In order to determine the amount of sequence

remaining proximal to the 5' end of the consensus NADP-GDH

cDNA clone, primer extension analysis was performed on the











o0 0






(0 C: MO
Q a) (a


>iC 00 *



- 0 r.l
u 0 -v






m Q
e, 0 <
H 4)

'- O 0
14.4 .i Z)
SH Hn a








- H0 0 H
to ao



















S0) 0 ta
0 MOH -H
1-i l ) Q- >0


















O *) ( 0 o

04 -0 Q
ao a)











0 0 4-)
0 0 4
4-) 0 r-











0cm>
d0 a )
0a 3 h .
U) tvH 4 -)





U0) 1O 4

0 a)



m -H 0 U)
A1-4 c4 Qa
M 3 -4)UOi















62

n
0" N w
L Lo LO Lo a |L La



(t
621





A %n
S S S A.IA
PI n


o N
I25 ^ '& I '


0






M
0 H

m-
C -H
H --











M g--E-


iT


0

S.
S.
U'
0


- 1 -


j I


ty..
p.

U'
0>
S.








mixture of C. sorokiniana poly(A)+ RNA previously isolated

for the XZAP library construction. As determined by

comparison to a cDNA generated sequencing ladder, a single

primer extension product of 87 nt was detected (Fig. 4). The

87 nt product corresponds to 53 bp of sequence identified in

the 5' terminus of pGDc 42 (Fig. IC) and 34 bp of sequence

previously undetected. The additional 34 bp extension

predicted a mRNA of 2.179 kb that approximated the 2.2 kb

mRNA determined by northern blot analysis (Fig. 2). The

primer extension analysis was repeated with identical

results.


RACE-PCR cloning of two NADP-GDH 5' termini


To determine the 5'-terminal sequences of the NADP-GDH

mRNA(s), a modified anchored PCR procedure for the rapid

amplification of cDNA ends was performed (Jain et al., 1992).

To ensure any possible sequence differences that might reside

in the 5' region proximal to the HCR would not be missed, a

RNA mixture previously prepared by mixing equal quantities of

poly(A)+ RNA from cells synthesizing primarily the a- or 3-

holoenzyme was used for the RACE-PCR cloning.

Agarose gel elelctrophoresis of the products from the

second step of the RACE-PCR amplification revealed two DNA

fragments of approximately 390 and 450 bp in size.

Reamplification using a different nested gene-specific

primer, designed to hybridize closer to the 5'-termini of the

mRNAs, yielded two unique PCR products of approximately 330






























Figure 4. Primer extension analysis of NADP-GDH mRNA(s). A
poly(A)+ RNA mixture isolated from Chlorella cells,
synthesizing primarily the NADP-GDH a- or P-subunit, was
hybridized with a 32P-labelled 5' NADP-GDH-specific
oligonucleotide and extended with reverse transcriptase. A
single 87 nt primer extension product (PE) was detected after
resolution on a 5% sequenceing gel. The approximate size of
the extension product was determined by comparison to a NADP-
GDH cDNA clone sequencing ladder.


















AGCT PE


- -87 nt








and 370 bp in size as determined by agarose gel

electrophoresis (Fig. 5). Sequence analysis of the two

cloned final PCR products revealed the actual size of the

products, minus the anchor primer, to be 269 and 311 bp.

Comparison of the two RACE-PCR cDNA clone sequences showed

them to be identical except for the presence of an additional

42 bp in the 5' coding region of the longer PCR product. The

additional 42 bp sequence encodes 14 amino acids that were

not present in the 269 bp RACE-PCR clone (Fig. 5). The

absence of the 42 bp sequence in the 269 bp clone results in

the deletion of the 14 amino acids from the amino-terminus of

the polypeptide; however, the downstream reading frame

remained unchanged. Both RACE-PCR products possessed

identical putative 5'-UTRs, translation initiation sites, and

were identical for the first 12 codons (Fig. 5). Both the

+42 bp and -42 bp clones overlapped in frame with pGDC 42 at

their 3' ends. The 5' RACE products were 33 bp longer than

pGDC 42 at their 5'-termini which is in close agreement with

the 34 bp length predicted by primer extension analysis.

The NADP-GDH 5' RACE-PCR clones possessed identical 32

bp 5'-terminal pyrimidine rich (89%) sequences and

transcription initiation sequences indicative of eukaryotic

5'-UTRs and translational start sites, respectively (Kozak,

1984). Furthermore, additional 5'-terminal guanine residues

were detected (2 on the +42 bp and 1 on the -42 bp clone) in

both clones that could not be accounted for in the NADP-GDH

gene sequence. The presence of unique 5'-terminal guanine












0



-H 0 m
0 4-) -I

.O i0 0



4) 0

U C
0 P- ) H
00 "A





o -' H-H
0) P a (d
Pu d -W o




U 0 .I ri
MO 4 *o







O H ) 0

rC4 4-)
Sa)04
SV r 0
-4 ) OH




0 40 0 I
P z









0o0 () a
V Q U)-4430 a












a4 -HP
4-) 3 )o (
0 4 -4 U







U 0) r N
I r0 QO 0







Sa) 01 X

-1 0 Q H
P4 13 u 0)








S0 1 -H
H 0 U) 0









*-i 1) m


H P) 0 4 -)










44( CO U) W 4-) Q





69















< 00
S> >




> 1


-)J
; Ir




/ >






<< < <- 0
F- F-
2i








residues on 5' RACE-PCR products has proven to be a

definitive means of identifying 5' capping points and

transcription initation sites of eukaryotic mRNAs (Bahring et

al., 1994). The RACE-PCR procedure was repeated with

identical results. RT-PCR performed on the poly(A)+ RNA

mixture using two new gene-specific primers that flanked the

42 bp variable region also yielded two PCR products differing

in size by 42 bp.

Both 5' RACE-PCR products appear to be complete at their

5' termini and overlap in frame with the consensus NADP-GDH

cDNA identified earlier. These results are consistent with

the existence of two separate NADP-GDH mRNAs that share a

common transcriptional start site and are identical with the

exception of a 42 nt insert identified in the longer mRNA.

The +42 nt mRNA predicted size is 2185 nt, whereas the -42 nt

mRNA predicted size is 2143 assuming a mean poly(A) tail

length of 70 nt.


Analysis of the C. sorokiniana NADP-GDH cDNA sequences


Sequence analysis of the two consensus NADP-GDH cDNAs

(+42 bp and -42 bp) revealed both mRNAs possessed an

identical 32 nt 5'-UTR (Fig 6). The +42 bp cDNA possesses an

ORF from nt 33 to a TAA stop codon at nt 1611 that encodes a

precursor protein with a molecular mass of 57850 D. The -42

bp cDNA possesses an ORF from nt 33 to nt 1569 that encodes a

precursor protein of 56350 D. The 1500 D difference in

molecular mass observed in the two precursor proteins is due



























Figure 6. Nucleotide sequence of the consensus NADP-GDH
mRNAs derived from the cDNA and 5' RACE-PCR clone sequences.
Beginning at nucleotide 33, two ORFs were identified from two
mRNAs that differed by 42 nt in the 5'-VR (boxed). The +42
bp mRNA encodes a polypeptide of 57,850 D, whereas the -42 bp
mRNA encodes a 56,350 D polypeptide. The deduced amino acid
sequences of the C. sorokiniana precursor polypeptides (Cs)
are compared with those of E. coli (Ec) and N. crassa (Nc)
NADP-GDHs. Arrows denote the boundaries of the highly
conserved glutamate binding domain identified by Mattaj et
al. (1982). A consensus algal polyadenylation signal
(underlined) is located 17 bp upstream from the poly(A) tail
of the NADP-GDH mRNAs.










Cs CTCTTTCTGCTCGCCCTCTCCGTCCCGCCCATGCAGACC 40
M 0 T
GCCCTCGTCGCCAAGCCTATCGTGGCCGCCCCGCTGGCGGCACGCCCGCGCTGCCTCGCGCCGTGGCCGTGCGCGTGGGTCCGCTCCGCC 130
A L V A K P I V AIA P L A A R P R C L A P W P|C A W V R S A
AAGCGCGATGTCCGCGCCAAGGCCGTCTCGCTGGAGGAGCAGATCTCCGCGATGGACGCCACCACCGCCGACTTCACGGCGCTGCAGAAG 220
K A D V R A K A V S L E E Q I S A M D A T T G D F T A L Q K
GCGGTGAAGCAGATGGCCACCAAGGCGGGCACTGAGGGCCTGGTGCACGGCATCAAGAACCCCGAGCTGCGCCAGCTGCTGACCGAGATC 310
Cs A V K Q N A T K A G T E G L V H G I K N P E L R Q L L T E I
E M D Q T Y S L S F L N H V Q K
BE S
TTCATGAAGGACCCGGAGCAGCAGGAGTTCATGCAGGCGGTGCGCGAGGTGGCCGTCTCCCTGCAGCCCGTGTTCGAGAAGCGCCCCGAG 400
F M K D P E Q Q E F M Q A V R E V A V S L Q P V F E K R P E
R N P N Q T E F A Q.A V R E V M T T L W P F L E Q N P K Y R
N L P S E P E F E QGA Y K E L A Y T L E N S S L Q H .
CTGCTGCCCATCTTCAAGCAGATCGTTGAGCCTGAGCGCGTGATCACCTTCCGCGTGTCCTGGCTGGACGACGCCGGCAACCTGCAGGTC 490
L L P I F K Q I V E P E R V I T F R V S W L D D A G N L Q V
Q M S L L E R L V .V R N Q I
Y R T A L T V A S I .. .. V .E N V.
AACCGCGGCTTCCGCGTGCAGTACTCGTCCGCCATCGGCCCCTACAAGGGCGGCCTGCGCTTCCACCCCTCCGTGAACCTGTCCATCATG 580
N R G F R V 0 Y S S A I G P Y K GG L R F H P S V N L S I M
A W F N L
Y F N L L L
AAGTTCCTTGCCTTTGAGCAGATCTTCAAGAACAGCCTGACCACCCTGCCCATGGGCGGCGGCAAGGGCGGCTCCGACTTCGACCCCAAG 670
K F L A F E Q I F K N S L T T L P M G G G K G G S D F D P K
S G T A .
G A G S A .
GGCAAGAGCGACGCGGAGGTGATGCGCTTCTGCCAGTCCTTCATGACCGAGCTGCAGCGCCACATCAGCTACGTGCAGGACGTGCCCGCC 760
G K S D A E V M R F C Q S F N T E L Q R H I S Y V 0 D V P A
E G A L Y L G A D T .
I R C A A H K G A D T .
GGCGACATCGGCGTGGGCGCGCGCGAGATTGGCTACCTTTTCGGCCAGTACAAGCGCATCACCAAGAACTACACCGGCGTGCTGACCGGC 850
G D I G V G A R E I G Y L F G Q Y K R I T K N Y T G V L T G
G V F N A K L S N T A C F .
G A R K A A N R F E .
AAGGGCCAGGAGTATGGCGGCTCCGAGATCCGCCCCGAGGCCACCGGCTACGGCGCCGTGCTGTTTGTGGAGAACGTGCTGAAGGACAAG 940
K G Q E Y G G S E I R P E A T G Y G A V L F V E N V L K D K
L S F L L Y T A R H
L S W L L Y Y G H M E Y S
GGCGAGAGCCTCAAGGGCAAGCGCTGCCTGGTGTCTGGCGCGGGCAACGTGGCCCAGTACTGCGCGGAGCTGCTGCTGGAGAAGGGCGCC 1030
G E S L K G K R C L V S G A G N V A Q Y C A E L L L E K G A
MN G F E N V S S A I K A F.
.A G S YA V A L S A L K I L .
ATCGTGCTGTCGCTGTCCGACTCCCAGGGCTACGTGTACGAGCCCAACGGCTTCACGCGCGAGCAGCTGCAGGCGGTGCAGGACATGAAG 1120
1 V L S L S D S Q G Y V Y E P N G F T R E 0 L Q A V Q D M K
R .I T A S T V D E S K K A R L I E I .
T .V K .A L V A T G E S G I T V E D I N A V A
AAGAAGAACAACAGCGCCCGCATCTCCGAGTACAAGAGCGACACCGCCGTGTATGTGGGCGACCGCCGCAAGCCTTGGGAGCTGGACTGC 1210
K KN N S A R I S E Y K S D T A V Y V G D R R K P U E L D C
A S R D RD G V A D A K E FG L V Y L E G 0 0 S P -
I E Q L T S F 0 H GH LK I E G A R L H V G
CAGGTGGACATCGCCTTCCCCTGCGCCACCCAGAACGAGATCGATGAGCACGACGCCGAGCTGCTGATCAAGCACGGCTGCCAGTACGTG 1300
Q V D I A F P C A T 0 N E I D E H D A E LL I K H G C Q Y V
L L V D A H Q A N V K A .
K L V S K E E .G .L AA K F.
GTGGAGGGCGCCAACATGCCCTCCACCAACGAGGCCATCCACAAGTACAACAAGGCCGGCATCATCTACTGCCCCGGCAAGGCGGCCAAC 1390
V E G A N M P S T N E A I H K Y N K A G I I Y C P G K A A N
A T .I T E L F Q V L F A .
A S G C L E VFENNRKE K EAW A .
GCCGGCGGCGTGGCGGTCAGCGGCCTGGAGATGACCCAGAACCGCATGAGCCTGAACTGGACTCGCGAGGAGGTTCGCGACAAGCTGGAG 1480
A G G V A V S G L E M T Q N R M S L N W T R E E V R D K L E
T .. A .A A R G K A K .D A R H
C ...A S Q R A D E K
CGCATCATGAAGGACATCTACGACTCCGCCATGGGGCCGTCCCGCGAGTACAATGTTGACCTGGCTGCGGGCGCCAACATCGCGGGCTTC 1570
R I M K D I Y S A M G P S R E Y N V D L A A G A I A G F
H L H H A C V E H G G GE T N Y V .
D N A F F N G L N T A K T Y V E AAE GEL P S V S .
ACCAAGGTGGCTGATGCCGTCAAGGCCCAGGGCGCTGTTTAAGCTGCCCAGGCCCAAGCCACGGCTCACCGGCAATCCAACCCAACCAAC 1660
T K V A D A V K A Q G A V *
V .. M L V I *
V Q M H D D W S K N*
TCAACGGCCAGGACCTTTTCGGAAGCGGCGCCTTTTTCCCAGCCAGGGCCCTCACCTGCCCTTTCATAACCCTGCTATTGCCGCCGTGCC 1750
CCTGCAATTCCACCCCAAGAAGAACTAGCGGCACTTGACTGCATCAGGACGGCTATTTTTTTCGCGACGCGCGCTCACCCCGAGAGCCTC 1840
TCTCCCCCGAGCCCTAAGCGCTGACGTCCGCCCGACTTTGCCTCGCACATCGCTCGGTTTTGACCCCCTCCAGTCTACCCACCCTGTTGT 1930
GAAGCCTACCAGCTCAATTGCCTTTTAGTGTATGTGCGCCCCCTCCTGCCCCCGAATTTTCCTGCCATGAGACGTGCGGTTCCTAGCCTG 2020
GTGACCCCAAGTAGCAGTTAGTGTGCGTGCCTTGCCCTGCGCTGCCCGGGATGCGATACTGTGACCTGAGAGTGCTTGTGTAAACACGAC 2110
GAGTC (Poly A)70 2185








to the presence of the additional 14 amino acid residues in

the +42 bp cDNA (Fig. 6).

The sequence TGTAA located 17 nt upstream of the

polyadenylation site has been identified as a conserved

polyadenylation signal generally used in algal genomes (Fig.

6). The conserved TGTAA signal has been identified in the

same position in numerous Chlamydomonas, Chlorella, Volvox,

and Euglena cDNAs (Wolf et al., 1991).

The deduced amino acid sequences of the C. sorokiniana

NADP-GDHs are 50% and 50.3% identical with the NADP-specific

GDH of E. coli (McPherson and Wooton, 1983) and Neurospora

crassa (Kinnaird and Finchum, 1983), respectively (Fig. 6).

Comparison of the sequences of the highly conserved glutamate

binding domain (Mattaj et al., 1982) shows a strong identity

of 76.6% and 73.4%, respectively. Alignment of the C.

sorokiniana NADP-GDH polypeptide sequences with the bovine

mitochondrial NAD-dependent GDH (Julliard and Smith, 1979)

revealed a significantly lower 23% identity for the entire

protein and a 27.4% identity over the GDH conserved region.

Analysis of the codon preference of both NADP-GDH mRNAs

showed a strong bias for C and G at the first position, in

the case of arginine and leucine, and the third position of

the codon (Table 2, Table 3). Furthermore, an extreme

preference for G or C at the third codon position correlates

with the 63% GC content reported for the C. sorokiniana

genomic DNA (Cock et al., 1990).















Table 2.

TTT phe F
TTC phe F
TTA leu L
TTG leu L


leu L
leu L
leu L
leu L


Codon usaqe of


the -42 bo NADP-GDH mRNA.


TAT tyr Y
TAC tyr Y
TAA OCH Z
TAG AMB Z


CCT pro P
CCC pro P
CCA pro P
CCG pro P

ACT thr T
ACC thr T
ACA thr T
ACG thr T

GCT ala A
GCC ala A
GCA ala A
GCG ala A


his H
his H
gln Q
gln Q


AAT asn
AAC asn
AAA lys
AAG lys

GAT asp
GAC asp
GAA glu
GAG glu


TGT
TGC
TGA
TGG

CGT
CGC
CGA
CGG

AGT
AGC
AGA
AGG


arg R
arg R
arg R
arg R

ser S
ser S
arg R
arg R


GGT gly
GGC gly
GGA gly
GGG gly


Table 3. Codon usage of the +42 bp NADP-GDH mRNA.


TTT phe F
TTC phe F
TTA leu L
TTG leu L


leu L
leu L
leu L
leu L


CCT pro P
CCC pro P
CCA pro P
CCG pro P

ACT thr T
ACC thr T
ACA thr T
ACG thr T


4 GCT
8 GCC
- GCA
31 GCG


ala A
ala A
ala A
ala A


TAT tyr Y
TAC tyr Y
TAA OCH Z
TAG AMB Z


his H
his H
gln Q
gln Q


AAT asn
AAC asn
AAA lys
AAG lys

GAT asp
GAC asp
GAA glu
GAG glu


TGT cys C
TGC cys C
TGA OPA Z
TGG trp W

CGT arg R
CGC arg R
CGA arg R
CGG arg R


ser S
ser S
arg R
arg R








Both of the deduced NADP-GDH polypeptides possess amino-

terminal extensions that are rich in alanine, serine, and

threonine and contain few acidic residues. These amino acid

sequence motifs are indicative of chloroplast targeting

domains (Smeekens et al., 1990). The boundaries of the

transit peptide were delineated by amino terminal sequence

analysis as discussed later.

The secondary structures of the deduced polypeptide

sequences of the +42 nt and -42 nt GDH cDNAs were predicted

by the method of Garnier et al. (1978; Fig. 7, Fig. 8).

Alignment of the predicted secondary structures to homologous

regions of the Clostridium symbiosum NAD-GDH 1.96A resolution

crystal structure (Baker et al., 1992) indicates the

predicted structures are accurate representations.

Comparison of the chloroplast transit peptide regions of the

two predicted structures showed that the 14 amino acids

encoded by the additional 42 nt of the longer mRNA introduced

a random coil structure with multiple turns (Fig. 8) that

disrupts an a-helical domain observed in the -42 nt mRNA

transit peptide region (Fig. 7).


Determination of the Exon/Intron Boundaries
of the NADP-GDH Gene

A 9873 bp region of genomic DNA containing the NADP-GDH

gene previously sequenced by Cock et al. (1991) was compared

to the two consensus NADP-GDH cDNA sequences (Fig. 9). Both

the +42 bp and -42 bp NADP-GDH cDNAs span a 7178 bp region of













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r-4 -
00


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r O) 0 -
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0 C' 40 V




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d ~ I-












co 0
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-
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to ) o --I









(4 0 o +
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H 4 4) ()



0 0 0 I


o E0





C! U li 4O
UOlM C0







-d Cd Ua
0)0 +





0H 0 U






p Ei 0
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4- 40 *.C
>'0







WM- 0 *I

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0)C
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Figure 9. Nucleotide sequence of the C. sorokiniana NADP-GDH
gene containing 22 exons (+42 bp mRNA) or 23 exons (-42 bp
mRNA). The position of the exons are identified by their
corresponding deduced amino acid sequences. The 5'-VR amino
acid sequences are indicated separately in the 5' region of
the gene. The highly conserved glutamate binding region
(Mattaj et al., 1982) is distributed over six exons
encompassing 2.09 kb as indicated by the two arrows. The 5'-
VR 42 bp auxon is denoted by arrowheads. Underlined regions
indicate the 5'-UTR and 3'-UTR, respectively.






81



GATCAGCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAACCGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCG 90
CATGCTTGTGCTGGTGAGGCTGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTGTGTGCTGGGCTTGCA 180
TGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCACTTGCTGGACGTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAG 270
GGATTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTTGCCAGAATGATCGGTTCAGCTGTTAATCAATG 360
CCAGCAAGAGAAGGGGTCAAGTGCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCAAGTGTGTGCGAGCCA 450
GCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTCATCCAACTAACCATAGCTGATCAACACTGCAATCATCGGCGGCTGATGCA 540
AGCATCCTGCAAGACACATGCTGTGCGATGCTGCGCTGCTGCCTGCTGCGCACGCCGTTGAGTTGGCAGCAGCTCAGCCATGCACTGGAT 630
CAGGCTGGGCTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCTGGCTGCTTGCTGGGTTGCATGGCATCG 720
CACCATCAGCAGGAGCGCATGCGAAGGGACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGTCACCAGGCC 810
CGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGACGTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGC 900
ACAGGCAGCAGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCGGGGTCGCGCCCCAGCCAGCGGTGATGC 990
GCTGATCnnnCCAAACGAGTTCACATTCATTTGCAGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGGAATGCG 1080
GGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATGTTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCT 1170
GTTTCTTTCGTGTTGCAAAACGCGCCACGTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAATCGTGTGCACGCCGATCAGCTGA 1260
TTGCCCGGCTCGCGAAGTAGGCGCC CTCTTTCTGCTCGCCCTCTCTCCGTCCCGCC ATG CAG ACC GCC CTC GTC GTGAGCAG 1342
+42GDH M Q T A L V
-42 GDH M Q T A L V
CGCTTGGGTTGCCTTGCAGCGGTTGTTGCTGGATCGCGCCGCCGGCCGACCGGGGCTGGTTGCACGGCCCGCCGCGCCGCGCACACTGAC 1432
CGGCGGTCCTGTTTCTCCTCATTGCGACTGCAG GCC AAG CCT ATC GTG GCC GCC CCG CTG GCG GCA CGC CCG CGC 1507
A K P I V AAA P L A A R P R
A K P I V A
TGC CTC GCG CCG TGG CCG TGC GCG TGG GTAAGCGGCTCGGGTGGGGCCCGGGGATGGCACGCTGGGGTTAGGGTTGCGCGG 1588
C L A P W PAC A W
C A W
TGTGCGACGACAACGCCGCTCACGTCCAGCCTCAGCTGCTGGCGCCTCGCTGGCCCGCTGCCATTGCTCATGTGCAAGACAGGATGCTTG 1678
CTGGGTGATGGGCGGAGCACCAGGGCTGTTGGTGGTGGGCGGCGCGCACGCTGCCGCCGCCGCCAGCCGCCGCGCGCCTGCCTCTCGCAG 1768
TGGTGTGGCCCATCCTGCCTCCCTGCCAACAACCTCACCGCTCGCCCCGACCCGCAG GTC CGC TCC GCC AAG CGC GAT GTC 1849
V R S A K A D V
V R S A K A D V
CGC GCC AAG GCC GTC TCG GTGAGTGCTCTGCGTGCACCGCCAGCCCTGCAAGCACGCCCCCGCCGGCGCCAAACCTCCAACCGC 1933
R A K A V S
R A K A V S
CGCGGGGACCCCGCTGCCATGCATGCACCTGCCGGCACCTGCACCGCCTTCGTGCGCGCCGCTCCTGTGCAGCCCTCACCGTCACTGACC 2023
AATCCAAACACTTTTTCGCCACTGTTCTGCAG CTG GAG GAG CAG ATC TCC GCG ATG GAC GCC ACC ACC GGC GAC 2097
L E E 0 I S A M D A T T G D
TTC ACG GTGCGCCGCCACAGCCGTACTTATGCGCCCTGTTGGACTCGGGCAGCCACTGTACCGCCCCTTCATAGCGCCCGCCGTCCTG 2185
F T
CCTGACATGGGCTCAACGCAAGCCATGCCATGCCTTCAAACAGCATGCATTCATCCCTGTCCTGACTCATCAAGATCGCCCTGTGTCTTG 2275
ACCCTGCGCCGCCCCGCAACCGCCATCCCGCTTGTTTCCCGACCTGCCCTCTCCCCCCGCCCGCCCTCGTCCTCATGTGCCGCAG GCG 2363
A
CTG CAG AAG GCG GTG AAG CAG ATG GCC ACC AAG GCG GGC ACT GAG GGC CTG GTG CAC GGC ATC AAG AA 2431
L Q K A V K 0 M A T K A G T E G L V H G I K N
C CCC GAC GTG CGC CAG GCAAGTCTTTAGCCTGATTGGAATGGAATGTAAGCCTGCCTTGTGCGCATTCCTTGGGCATCAACAAT 2515
P D V R Q
CCTGAGCTGCGCCAGGTGAGGAATAACACACCGTTTTTGAGCACTTCTATCGTCCCCACCTGCTGGCGTTGCGGCTCGACCGGGCTGCTT 2605
AGAGCAGCCCCGATGAGAAGAAAGCCCACGTGCGCAGAGTGCCAAACGCTGTCTCCTTCCCCCGCCCTGTCATCCACCACAGCTGCTGAC 2695
CGAGATCTTCATGAAGGACCCCGTTCAGCAGGAGTTCATGCAGGCTCATCTACATGCATGCGTAACAATAACCTGCCTCTTTCCTCTTCC 2785
CACCACGCAG CTG CTG ACC GAG ATC TTC ATG AAG GAC CCG GAG CAG CAG GAG TTC ATG CAG GCG GTG CGC 2855
L L T E I F M K D P E Q Q E F M Q A V R
GAG GTG GCC GTC TCC CTG CAG CCC GTG TTC GAG AAG CGC CCC GAG CTG CTG CCC ATC TTC AAG CAG GC 2923
E V A V S L Q P V F E K R P E L L P I F K 0
AAGCGCGCCTGAGGGGGGCAGGGGTGGTGCAGGGCGGGTCAGAGGGCTGGTTATAACTAACTAGGGTGCGGTGGACACGGGCGTGCAGAA 3013
GCCTGGCTCATCCACCAGTGACAGCAGCATGCTGGGTTGGCGAGCAGCAAGACACCCATTCACCGCTCGGCGACTGGCCTGACTAGCTGC 3103
AAGTCTGCTCTGTGTTATTCGCCATCCGCAG ATC GTT GAGACCT GAG CGC GTG ATC ACC TTC CGC GTG TCC TGG 3176
I V E /P E R V I T F R V S W
CTG GAC GAC GCC GGC AAC CTG CAG GTACAGCAGGCAGGCTGGCGCCTTGGCTGGCTAGTGTTCCCTTGCAGAGAGAAGCAGC 3258
L D D A G N L Q
ACACCACGCACGCACACTCGTCCCTGCCCGCCGCCATATGGCATGCATGCGGCATCCCGTGCGCCGACAATTCCACTGTTGTGCACTCAG 3348
TTCAGCTTCATTCTCATGGCCCATTCATTCACTTCACTGTTTGCAG GTC AAC CGC GGC TTC CGC GTG CAG TAC TCG TCC 3427
V N R G F R V Q Y S S
GCC ATC GGC CCC TAC AAG GGC GGC CTG CGC TTC CAC CCC TCC G GTGCGTGCCTGCACTGGCTGTGCCTGCGCTGG 3502
A I G P Y K G G L R F H P S
CTGTGCCTGCGCTGGCCGTGCCTGCACCGGCTGTGCCTGGCTCAGCGGGTGGGGATGTGAGGCATGTCGGTGCACCAACCCGCCCGGCTT 3592
GCTCCGACGTCTACACCTGCAACACGGCTGCACAATGGACAGGGCAGGGCGGGGCAGGCACTTGCATCGGTGCCCGCCCCTCCAGCATGC 3682
ATGGGCGTGGCGAGCTGGGGGCGGGCCGGGCACCAACGGAGCAACTTGCAGTTCACCCTACTTTTCATGTGCCCCTGTCCAATGCCGCAG 3772
TG AAC CTG TCC ATC ATG AAG TTC CTT GTGAGTGCTGCCAAGCCTTGAAAGCGCTGTGCTAGCTGGTGAAATTGAGCAAGGA 3853
V N L S I M K F L
GCTGGGAAGAGTATAGCCGTGGGGGCAGGCCAGCCACTTTGCTGGCGCAAAGGTGGCCCTGCGATGCGCTGCGGCGACTGACACAGCGGC 3943
CCCTCCATCCCTTCACAACCATATGCAG GCC TTT GAG CAG ATC TTC AAG AAC AGC CTG ACC ACC CTG CCC ATG 4016
A F E Q I F K N S L T T L P M
GGC GGC GGC AAG GGC GGC TCC GAC TTC GAC CCC AAG G GTGCGCCTTCCTTGAGTTAGTCGGCGGCAAGCTGCACATT 4093
G G G K G G S D F D P K
AAATGCCTCCGTCGGTCGTGTTTCAAGGCCCGCCCTGGCCCATCATTGGCTGACGGTCCACTGCCTGCCACCCTGTGTCGCCACCTACCT 4183
GCATACCACCCACCCAACACTCCCGCCCCTCCTGCAACCCCTCCCTCCCCACTACCGCAG GC AAG AGC GAC GCG GAG GTG 4263
G K S D A E V
ATG CGC TTC TGC CAG TCC TTC ATG ACC GAG CTG CAG CGC CAC ATC AGC TAC GTG CAG GAC GTG CCC 4329
M R F C 0 S F M T E L 0 R H I S Y V Q D V P
GCC GGC GAC ATC GGC GTG G GTGAGCGAGCGAGCGAGCAGCGAGCGGGCGTGTTTTTGAAAATTGCAGGGAGGGTAGTCGGGTG 4412
A G D I G V
GGGCAAAGGAAACGCACACACTTGCATGCGTAGCCAGCAAGCTTTCGTTCTCCTCATTCGCCGCTCCATTAGCTCACTGCCTTTGCCCAC 4502
CTCTTGTTTACCAACAACACGCAG GC GCG CGCAGAG ATT GGC TAC CTT TTC GGC CAG TAC AAG CGC ATC ACC 4573
G A R E I G Y L F G 0 Y K R I T










AAG AAC TAC ACC GGC GTG CTG ACC GGC AAG GG GTGAGGCCCGCTTGCACTGACTGAGCTCGAGCCGGGAGCAACTGTAC 4652
K N Y T G V L T G K G
TTTGCATTCCTGCCGGTCTGTTTCGGGGCGGCTGATCGGCAAGGGGTAAGGACCAGTGCCCACAGGAGCTCTAACGCTTGCCTGCCACGT 4742
TTGGGTGAACTGGTGTTCTCCAGCAGCCAGAGTTTTCCATGTCCACCCGCCTGCAAGCTCCTGGCTGTTCATCGCTGTGCTCTGTGTCTC 4832

CCTGCCAACACAATCCATACCAACACAATCCTGCGCCCTGCAG C CAG GAG TAT GGC GGC TCC GAG ATC CGC CCC GAG 4909
Q E Y G G S E I R P E
GCC ACC GGC TAC GGC GCC GTG CTG TTT GTG GAG AAC GTG CTG AAG GAC AAG GGC GAG AGC CTC AAG GT 4977
A T G Y G A V L F V E N V L K D K G E S L K
GCAGCTATATGCCTTGGTTGTGCTGCCCTTGGCAGCAGTGAAGGCTGCGATGGTCTTTCACCTGAACTTTCAACGTACCAGCATGCGCAC 5067
ATGAGGTAGAGCACAGCCCAAACTGCTCAGAACGTCCGCCTGCCAAGTTCTTTCTTCCATCCACACCCCACACACCTGTGCAG GGC 5153
G
AAG CGC TGC CTG GTG TCT GGC GCG GGC AAC GTG GCC CAG TAC TGC GCG GAG CTG CTG CTG GAG AAG 5219
K R C L V S G A G N V A Q Y C A E L L L E K
GGC GCC ATC GTG CTG TCG CTG TCC GAC TCC CAG GGC TAC GTG TAC GAG GTGCGGTTGATACATCTGGGCCATTT 5293
G A I V L S L S D S Q G Y V Y E
CGGCTGTTGATTGTGCTCTGTGTTGTCTGTAGTGTCTGACTCCCTGGGCTCGTGCACGAGGTGCGGAAGGCTCAGGCAGCAGTTCGGAGC 5383
TCTGCCTGTCTGCTGCTCCTAGAGCTACCTAATGAAGCATAGCTCTGCTGTGCTGCCCCCTCGCGCCTGCTCACCCGTCAACCACCACCG 5473
CCCCTCCCCACCCCCTTTTCATTTTTCCCGCAG CCC AAC GGC TTC ACG CGC GAG CAG CTG CAG GCG GTG CAG GAC 5548
P N G F T R E 0 L Q A V 0 D
ATG AAG AAG AAG AAC AAC AGC GCC CGC ATC TCC GAG TAC AAG GACAGTGATGACCGGTCCAGGAAACAAGTTGCAC 5624
M K K K N N S A R I S E Y K
ATGTCGTCTAGAAGGTCCCTGCCGCCGACACAGCAGCCGCGCCTGGGCTGCCGCTGCTTCGATAGCACCACCCACCCCTGCCGCCCCATC 5714
TCCTGCCTGCACTGCACCTTCCCATTTTGCCCACTAGCCACTGCTCACTCGAGTTCTCAACTGTCACTTGCAATTTTCTCTCTGCTTGCA 5804
G AGC GAC ACC GCC GTG TAT GTG GGC GAC CGC CGC AAG CCT TGG GAG CTG GAC TGC CAG GTG GAC ATC 5871
S D T A V Y V G D R R K P W E L D C Q V D I
GCC TTC CCC TGC GCC ACC CAG GTGCGCAGCCAGACTGGCTTGCATGCAACGCATCAAATGTCTCAAGGTTTGCCTGCAAGTGC 5954
A F P C A T Q
TCTAAGCCCTGTCAGAACTTTTTACAAGCAGCATGGCAGTGGAGGTGGTGAGGGCGACGTCCTGCACCGTTTCCTCAATGCCGCCGTGCC 6044
CCGGCTCTCTTGCCCTGTATGCAG AAC GAG ATC GAT GAG CAC GAC GCC GAG CTG CTG ATC AAG CAC GGC TGC 6116
N E I D E H D A E L L I K H G C
CAG TAC GTG GTG GAG GGC GCC AAC ATG CCC TCC ACC AAC GAG GCC ATC CAC AAG TAC AAC AAG GTGGCG 6185
0 Y V V E G A N M P S T N E A I H K Y N K
CTGCCTATACGAAGAATGTATTCCACTTGATGTTCAATACAGGGCGGGTGTTCAGAAACTAGGCGTGCCGCGAGGCCGTCCACAAGTACA 6275
ACAAGTGGGCGGTGGCTGCGAAGTTAGTTCTTAATCAAGGGCTGGTATGCTGTGCTGCACCAACGAGGCCATCCACAAGTACAACAAGGT 6365
GGGCCTGTTTTGAGCTTGCTGACAAGCTAGCCTCCCGACAGCTCTCCGGGTTGCGAGTTCCCAGCTGCTGCCTTCCGCAGTCTTTGGGAC 6455
CACGTGCGCCACCCACCCACCCATGTTTCTCCCGCACACATACTGCTCAGTACACACTTGCAGCTCCATGCAACCCAGCCTCTTTGCTGC 6545
CCCACCCTTCCCTCTCCCTGCCTCCGCGTCGCGCAG GCC GGC ATC ATC TAC TGC CCC GGC AAG GCG GCC AAC GCC 6620
A G I I Y C P G K A A N A
GGC GGC GTG GCG GTC AGC GGC CTG GAG ATG ACC CAG AAC CGC ATG GTGAGCGTGGCATGATTTCCCTGCTTGTCA 6695
G G V A V S G L E M T Q N R M
GGGCTTGCAGTATAAGCTGAAGAAACGAAGTGGTCTGCAGTCAGCAGCCTGCAGATGACCCAGAGCCGCATGGTGAGGAGGGCAGGGGCT 6785
GTTAACTGGGAGCAGCCTCAGCGACGCCCAGTGCTGGTGCTTTGTTCCTCGTGCACCTCAGCTGCTGCAACTTTGTGAGCGCATCGCCCT 6875
GAACCGCCACAACTGCCTGCGCCTGCCCTGCCGCAG AGC CTG AAC TGG ACT CGC GAG GAG GTT CGC GAC AAG CTG 6950
S L N W T R E E V R D K L
GAG CGC ATC ATG AAG GTGAGGGCTGATTGTGCGGCTATCACAGTGCAACCACGCAAGCTGGAGCGCATCATGAAGGTGAGGGCTG 7035
E R I M K
ATTGTGCGGOCTATCACAGTGCAACCACGCTCGTCATGGGCCTTGCGCGCCTCGCCCGTCGCGACTCGGCTGAAGTCGCTGCGGAAGCCGC 7125
CTTCGAGGAGGAAAGCCTGCGCCTTCGTCACGGCTCGCACTGCTTCCTTTCCCTCCACAGGACATCTACGACTCGCCATGGGCGCCTTTT 7215
GCAGGACAACCCATTCCGTTCACAACACTCAGCAACCCTGCCCTCATTCTTCTTCATCCCCGCAG GAC ATC TAC GAC TCC GCC 7298
D I Y D S A
ATG GGG CCG TCC CGC GAG TAC AAT GTT GAC CTG GCT G GTGAGTGCCTGGCTGTGCAGACAGACACGACACTTGTAAA 7375
M G P S R E Y N V D L A
CTCAGTTTTTTCATTCTAGCCTGCCGCCGTTTCTGCCGGCCAGGATTGGCTTTGGATATCGCTCTGCCCTGAGTAGCTAGTAGCCAGTTG 7465
CCCGGCAGCTATTGCCCCCCTGCCTGCTGTAGCTGTCTGCTGCCTGCGGTGCTGGTGTGCATGGAGCACCCACCGCAAAGCTCAAACGCC 7555
TGCGGTTGGTGGGCGCATGCTGTGCTTGCGGTGCTGCCCATCCGCCCTTGCGTTGCCACCCTGCTCACCCTGCTCACCCTGCCCCGCCTG 7645
CCCCCTCCCCCCGTCCTCCCAATTCTACAG CG GGC GCC AAC ATC GCG G GTGAGTTGGATTGGGGGGAGTTGTGCACACTGCT 7727
A G A N I A
GAAACGTGCAACGAGCACTGCTGCCTGTGCACTGCTGGCGCTGTTTTGGCACGATATGCTGCATTGCTGGTTGCCCGTCCTCAACTGTTG 7817
CAAGAGAGTGGCAGCTTGAACCGCCAATGCAGCGAAATGGTCGCGCACCCGCCTATTTGTGGCTTACGTTGCATTCCTCTCTCCGCTGCC 7907
TGCAG GC TTC ACC AAG GTG GCT GAT GCC GTC AAG GCC CAG GGC GCT GTT TAA GCTGCCCAGGCCCAAGCCACG 7980
G F T K V A D A V K A Q G A V *
GCTCACCGGCAATCCAACCCAACCAACTCAACGGCCAGGACCTTTTCGGAAGCGGCGCCTTTTTCCCAGCCAGGGCCCTCACCTGCCCTT 8070
TCATAACCCTGCTATTGCCGCCGTGCCCCTGCAATTCCACCCCAAGAAGAACTAGCGGCACTTGACTGCATCAGGACGGCTATTTTTTTC 8160
GCGACGCGCGCTCACCCCGAGAGCCTCTCTCCCCCGAGCCCTAAGCGCTGACGTCCGCCCGACTTTGCCTCGCACATCGCTCGGTTTTGA 8250
CCCCCTCCAGTCTACCCACCCTGTTGTGAAGCCTACCAGCTCAATTGCCTTTTAGTGTATGTGCGCCCCCTCCTGCCCCCGAATTTTCCT 8340
GCCATGAGACGTGCGGTTCCTAGCCTGGTGACCCCAAGTAGCAGTTAGTGTGCGTGCCTTGCCCTGCGCTGCCCGGGATGCGATACTGTG 8430
ACCTGAGAGTGCTTGTGTAAACACGACGAGTC GATCACCCGGTGCTTGGTGCACAAGCAGGGCATTGGAGCAGGGCAGCGGATCTGGAC 8519
TCCAGACTGGAGACGGCGGCCGCCGCCAGGTCAGCAGCCGGAAAACGCACCCGGAAAACTAGATCCCGAGCGCCTGGGCCGCTGCGCGCC 8609
GCATTTACAGTTCCAGACCCAGTCAGATCACCCAGGGCATCCACCAGCCACTGCAAAGCGGTTGCACAGCGGCTCGGCTCGATGGCGCCG 8699
CAATGGCAGGCCCGCGCTACGAGCCCGCTGCCTGATCCTAGCTGCTGCCGTGGCTGTTTGCCGTGTGCCGCTGAAGGTGCCGACCGCACG 8789
CCCGGGCGAGTGCTGGGACACGTGACGCGCGAGCTCAAGGCCTCCGAGCTGCCGGGCAAGGTAAGCGGAGCGTGTAAAAGATGGCTGGTA 8879
CTGCTGTTGACCCGATCCGCCCTGCTCGCGCGGGCCAGCACCACCCCTGCGTGCCGCCAACCTCACCCGCGCCGTGCCGCTCTGCCCCTC 8969
CGATTTCTGCTGCAGGATATGGCCTGATCTTCTATGGAGACAGCATCACGGAGAGCCTGCGTGGCACAGACAAGTGCCGCGACGTCTGCC 9059
TGAAGAGTAAGACGCGGTCCTCCTGCAAAGGCATTCCTGAGGTGCGCGAGCACGAGGGCCGCCACTCCTGCCGAATGGTTGCTACACATT 9149
GCATCGGCAGGGGTGGATTGGTTCATGGGCAGCCACTTCTTTCAGCTTCATAACTTTGCAACCAGTTTCCATGATCGCCGTCGTGCCGCC 9239
GCCGCATCCGCCGCTCTCCTGCCCGCTTGCCGATACGCCTTTCTGGGCCCCCGCTTACCGCACTGTGACCGAAGGTCCTGCAAAAGTACT 9329
TTGGCGCCTACCGCCCGGGTGTGATGGGCATGTCCATGGATGAGTCGGCCCACCTGCTGTGGCGCCTGCAGAACGGCCAGATCCCCCGCG 9419
TCAACCAGGTGAGCGCGCACAGGCAGTGCAGCGCAGGCAGCACAGCGCACGACATAGTGCAGCAGCGGCAGACTGGACGGGCCAACTGTC 9509
TGCCTGCGGTCTGCACTGGTGGGGCCAACTGCGTCTTCTGCGCCATGCCTTCAGCCAGGAATAGCACATGCTCCTTCGCCCTGCCTGCAG 9599
CCCAAGACAGTTGTGCTGAACATCGGCACCAACGACCTCACCAACTGCCGTGGAGCGCGAAGAACGCCCAGAAGAAGCAGGCGGCCATCA 9689
ACAAAGAAATCCCGGGGATCGTGGGCCGGTGAGCTGGGCGGTGGGGCATTCGCATGAACATGCATATCCTGGCTGCCACAGCGTGCCGCA 9779
TGCTATGTTGGGTGGGTCCACGGCAGTTGGCCGCTCGGTGCCGCTGGTGCATGCTGTGTGGGAGGGGAAGCTGCCTCGGTGTGACCTGAA 9869
GGACTT 9873


Figure 9 continued







the C. sorokiniana genome and are divided into 22 exons and

23 exons, respectively (Fig. 10). The exons in this gene

exhibit a range from 9 bp (exon 3 of the -42 nt mRNA) to a

large exon of 550 bp at the 3' end of the gene. The 9, 16,

and 18 bp exons are smaller than the smallest exons

identified in higher-plant genes (Tischer et al., 1986).

The introns identified in the NADP-GDH gene range

between 42 and 402 bp with a mean length of 233 bp. The mean

intron length is similar to the mean intron length of 249 bp

calculated in a survey of higher-plant introns (Hawkins,
C A TC
1988). The consensus sequence AG/GTG GG was derived as
A C CG
the 5' intron donor site using the criteria suggested by

Cavener (1987). The 5' donor site closely matches the

general consensus, CAG/GTAAGT observed in higher-plants and

animals (Hawkins, 1988) with the exception of G at position

+3 of the C. sorokiniana intron. Three of the 21 conserved

5' intron splice junctions do not conform to the GT-AG rule

of Breathnach and Chambon (1981) due to a substitution of C
TTTTTTT T T
at position +2. A consensus sequence, C CcCtt C GCAG/,
CCCCCCC C C
was derived for the 3' intron splice junction of the NADP-GDH

gene. The strong preference for G at position -4 observed in

19 of 21 conserved intron splice junctions is typical of

plant introns (Brown, 1986). The 5' (GCC/GCC) and 3'

(CCG/TGC) splice site junctions of the 42 bp auxiliary exon

(termed auxon hereafter as per Werneke et al. [1989]) showed

no conservation in splice site branch points.











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/' e ,9 n721 UL)1&( $5< 81,9(56,7< 2) )/25,'$



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

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.
rv

TABLE OF CONTENTS
ACKNOWLEDGMENTS ÍÜ
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 (3-Subunit 37
DNA Probe Synthesis 37
Northern Blot Analysis 38
Southern Blot Analysis 39
NADP-GDH cDNA Cloning and Characterization 41
igtlO library 41
7.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
v

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 igtlO cDNA
clones 54
Isolation, restriction-mapping, and sequencing of
the iZAPII 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 genomic 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
Vll

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 240
min induction period in 2 9 mM ammonium medium 136
Vlll

27. Relative abundances of the NADP-GDH mRNAs
synthesized in synchronous C. sorokiniana cells
throughout a 240 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:f$-
subunits 119
x

LIST OF ABBREVIATIONS
AP Alkaline phosphatase
ATCase Aspartate trans-
carbamylase
BSA Bovine serum albumin
ca Calculated average
CaM Calmodulin
CPSase Carbomylphosphate
synthase
DHOase Dihydroorotase
DTT Dithiothreitol
Dsx Double-sex
EDTA Ethylene diamine-
tetraacetic acid
EGTA Ethylene glycol-bis ((3-
aminoethyl ether) N, N,
N', N',-tetraacetic acid
ELISA Enzyme-linked immuno-
absorption assay
GS Glutamine synthetase
GOGAT Glutamate synthase
HAT Hypoxanthine-aminopterin-
thymidine
HCR Highly conserved region
ICBR Interdisciplinary Center
for Biotechnology
Research
xi

LANT6 Neurotensin lysine-
asparagine rich
mA Milliamperes
MAb Monoclonal antibody
NAD-GDH Nicotinamide adenine
dinucleotide-specific
glutamate dehydrogenase
NADP-GDH Nicotinamide adenine
dinucleotide phosphate-
specific glutamate
dehydrogenase
NBT Nitroblue tetrazolium
NEB New England Biolabs
NMN Neuromedian N
nt Nucleotides
NT Nerotensin
ODU Optical density unit(s)
OTCase Ornithine trans-
carbamylase
PK Pyruvate kinase
PME Pectin methylesterase
RACE-PCR Rapid amplification of
cDNA ends-PCR
RPA Ribonuclease protection
assay
RT-PCR Reverse transcriptase PCR
Rubisco Ribulose bisphosphate
carboxylase/oxygenase
SNAP-25 Synaptosomal associated
protein 25 kD
snRNA Small nuclear ribonucleic
acid
snRNP Small nuclear ribonuclear
proteins
Xll

SPP Stromal processing
peptidase
SPT Serine:pyruvate amino¬
transferase
Sxl Sex-lethal
TBS Tris-buffered saline
tra Transformer
TTBS Tween Tris-buffered
saline
UTR Untranslated region
VR Variable region
X-phosphate 5-bromo-4-chloro-3-indole
phosphate
xiii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
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
December, 1994
Chairman: Dr. Robert R. Schmidt
Major Department: Microbiology and Cell Science
Chlorella sorokiniana possesses seven chloroplastic
NADP-glutamate dehydrogenases (NADP-GDHs) composed of varying
ratios of cx- and (3-subunits. Southern blot analysis and
allele-specific PCR demonstrated the C. sorokiniana genome
possesses a single NADP-GDH gene encoding both the a- and (3-
subunits. PCR analysis, cDNA cloning and sequencing, and
RNase protection analysis identified two NADP-GDH mRNAs that
are identical with the exception of a 42 nt insert located in
the 5' coding region of the longer mRNA. Deduced amino acid
sequence analysis revealed that the 42 nt insert encodes an
additional 14 amino acids. The absence or presence of the
insert does not affect the downstream reading frame. The +42
nt mRNA encodes a 53850 D precursor protein, whereas the -42
xiv

nt mRNA encodes a 52350 D precursor protein. The +42 nt and
-42 nt mRNAs are postulated to be derived via alternative
splicing of a pre-mRNA from the single 7.1 kbp NADP-GDH gene
that consists of 22 or 23 exons, respectively.
Western blot analysis of the a- and p-subunits showed
them to be antigenically similar and to be 53.5 and 52.3 kD
in size, respectively. Amino-terminal sequence analysis
revealed the a-subunit shares amino acid sequence identity
with the (3-subunit; however, the a-subunit possesses a unique
11 amino acid a-helical domain that is lacking in the (3-
subunit.
The induction patterns and relative abundances of the
NADP-GDH mRNAs, antigens, and activities were measured in
cells induced in 29 mM ammonium medium. The relative
abundance of the +42 nt mRNA correlated with the a-subunit
antigen, whereas the -42 nt mRNA correlated with the p-
subunit. The ratio of NADPH:NADP+-GDH activity was highest
when the p-subunit was prominent and lowest when the a-
subunit was prominent.
These results are consistent with a single nuclear gene
being transcribed into a pre-mRNA that is alternatively
processed to yield two mRNAs encoding two precursor proteins.
The precursor proteins are processed to either the a- or (3-
subunit and assembled into isoenzymes with varying ammonium-
affinities .
xv

INTRODUCTION
Inorganic nitrogen acquired by plants is ultimately
converted to ammonium before being assimilated in organic
nitrogen metabolism. One of the enzymes postulated to be
involved in the assimilatory process is GDH, a ubiquitous
enzyme found to be present in almost all organisms from
microbes to higher plants and animals (Srivastava and Singh,
1987). GDH catalyses the reversible conversion of a-
ketogluterate to glutamate via a reductive amination that
utilizes NADH or NADPH as a cofactor. The role of plant GDHs
in the assimilation of ammonium into amino acids has been
questioned since the discovery of the GS/GOGAT pathway that
is believed to be the favored pathway for ammonium
assimilation in higher plants (Miflin and Lea, 1976). The
primary objection to GDH playing a major role in nitrogen
metabolism is its low affinity for ammonium that would
require high intracellular ammonium concentrations to
function anabolically. Early evidence indicated that GDH is
a catabolic enzyme catalyzing the deamination of glutamate
with only a partialy anabolic function in synthesizing
glutamate (Wallsgrove et al., 1987). However, more recent
studies reveal that Km values for ammonium and other
sustrates may be affected by various internal and external
factors and the previously reported in vitro Km values may
1

2
not reflect in vivo conditions. The physiological role of
large amounts of GDH present in various plant tissues and
organelles is still unclear, and possible conditions under
which GDH may play a significant role in carbon and nitrogen
metabolism have not been resolved.
The majority of plant GDHs characterized to date are
localized in the mitochondria; however, GDH species differing
in several properties (i.e. cofactor specificity) have been
characterized from chloroplasts (Srivastava and Singh, 1987).
Chlorella sorokiniana cells have been shown to possess a
constitutive, mitochondrial, tetrameric NAD-specific GDH
(Meredith et al., 1978), and seven ammonium-inducible,
chloroplast-localized, homo- and heterohexameric NADP-
specific GDH isoenzymes (Prunkard et al., 1986; Bascomb and
Schmidt, 1987). The seven chloroplastic NADP-GDH isoenzymes
were shown to have different electrophoretic mobilities
during native-PAGE, and presumably result from the formation
of homo- and heterohexamers composed of varying ratios of a-
and p-subunits (53.5 and 52.3 kD, respectively). Chlorella
cells cultured in 1 to 2 mM ammonium medium accumulate only
the a-homohexamer (Bascomb and Schmidt, 1987). The addition
of higher ammonium concentrations (3.4 to 29 mM) to nitrate-
cultured cells results in the accumulation of both a- and p-
subunits in NADP-GDH holoenzymes (Prunkard et al., 1986;
Bascomb and Schmidt, 1987; Bascomb et al., 1987). Prunkard
et al. (1986) demonstrated that the NADP-GDH subunit ratio
and isoenzyme pattern is influenced by both the carbon and

3
nitrogen source as well as the light conditions under which
cells are cultured.
The purified a- and p-homohexamers have strikingly
different ammonium Km values; however, the Km values for their
other substrates are very similar. The a-homohexamer is
allosterically regulated by NADPH and possesses an unusually
low Km for ammonium that ranges from 0.02 to 3.5 mM,
depending on the NADPH concentration (Bascomb and Schmidt,
1987). In contrast, the p-homohexamer is a non-allosteric
enzyme with an ammonium Km of approximately 75 mM. It is
postulated that the heterohexamers have varying degrees of
affinity for ammonium; however, no kinetic analyses have been
performed on purified heterohexamers. Pulse-chase
experiments, performed when homo- and heterohexamers of NADP-
GDH were accumulating during early induction in 29 mM
ammonium medium, revealed the a-subunit antigen was degraded
with a half-life of 50 min whereas the p-subunit antigen was
degraded more slowly with a half-life of 150 min (Bascomb et
al., 1986). After the removal of ammonium from the induced
cells, enhanced rates of degradation were observed for the a-
and p-subunit antigens, half-lifes of 5 and 13.5 min,
respectively.
Although the a- and p-subunits have distinct in vivo
turnover rates and the corresponding homohexamers have
remarkably different ammonium Km values, the a- and p-
subunits are derived from precursor proteins of nearly
identical size (ca 58,000 D) and were shown to have very

4
similiar peptide maps (Prunkard et al., 1986; Bascomb and
Schmidt, 1987). Moreover, antibodies prepared against the [3-
homohexamer are capable of immunoprecipitating all of the
NADP-GDH isoenzymes (Yeung et al., 1981, Bascomb et al.,
1987), but do not crossreact with the mitochondrial NAD-GDH.
In addition, previous research in this laboratory provided
genomic cloning and southern blot evidence that indicated the
C. sorokiniana genome possesses a single NADP-GDH structural
gene (Cock et al., 1991).
Biochemical and immunochemical properties of the NADP-
GDH a- and (3- subunits suggest that the two subunits share a
significant amount of protein sequence identity. Similar
kinetic, isoenzyme pattern, and immunological properties have
been shown for the mitochondrial GDH of grapevine (Loulakakis
and Roubelakis-Angelakis, 1991) and Arabidopsis (Cammaerts
and Jacobs, 1985). The understanding of the molecular
mechanisms regulating the C. sorokiniana NADP-GDH isoenzymes
is critical to further elucidate the metabolic significance
of GDH in carbon and nitrogen metabolism in Chlorella and
higher plants. Therefore, the purpose of this study is to
determine if the two NADP-GDH subunits arise from the (i)
differential processing of a precursor protein encoded by a
single nuclear gene and mRNA, (ii) specific processing of two
similar precursor proteins encoded by two mRNAs formed by
alternative splicing of a pre-mRNA derived from a single
nuclear gene, (iii) specific processing of two precursor

5
proteins encoded by two mRNAs transcribed from two closely
related nuclear genes.

LITERATURE REVIEW
Extensive research into the molecular and biochemical
mechanisms that control the physiology and potential fate of
a living cell has revealed a myriad of complexities in the
regulation of cellular processes. In prokaryotes, metabolic
processes have been shown to be temporally regulated at
transcription, translation, mRNA turnover and processing, and
post-translational modifications. The presence of organelles
in eukaryotes has provided another level of intricacy in
metabolic regulation by providing compartmentalization, that
provides for both temporal and spacial separation of cellular
events. The spatial separation of cellular processes
provides additional steps where regulation of transriptional,
post-transcriptional, translational, and post-translational
events can occur.
In response to the spatial and temporal separation of
metabolism, cells have evolved enzymes that share a similar
biological activity within an organism. These enzymes often
differ in their primary amino acid sequence (isoenzymes or
isoproteins), but may be capable of subunit exchange
(isozymes). Enzymatically similar isoforms may also exist as
a result of post-translational modifications
(phosphorylation, acetylation, methylation, etc.) to a single
protein. Theoretically, multiple isoenzymes have allowed
6

7
organisms to respond differentially in a refined way to a
broader range of developmental and environmental conditions.
Isoenzymes can arise via all the aforementioned cellular
processes and provide a useful tool to study the molecular
mechanisms involved in regulating biochemical processes.
The majority of isoenzymes characterized to date are
encoded by two or more genes within the genome of a organism.
Isoenzymes encoded by multiple genes are believed to have
arisen through gene duplication within an organism or via
gene exchange between organelles of eukaryotes (Gray and
Doolittle, 1982; Sun and Callis, 1993). The amount of
similarity conserved among isoenzymes derived from different
genes is influenced by how recent the duplication or exchange
has occurred (Pickersky et al., 1984), or may reflect a
strong selective pressure to maintain the primary amino acid
sequence (Moncreif et al., 1990).
Multiple isoenzymes of CaM, a 16 kD acidic Ca2+-binding,
signal transducing protein, have been identified in all
eukaryotes examined (Ling et al., 1991). Increased binding
of Ca2+ by CaM in response to increased intracellular Ca2+
levels triggers a conformational change in the protein.
Alteration of conformation in turn facilitates specific
interactions with Ca2+/CaM-dependent enzymes (O'Neil and
DeGrado, 1990). One notable feature of CaM isoenzymes is the
highly conserved primary structure; comparisons of amphibian,
avian, mammalian, and plant CaM isoproteins showed identities
of over 90 percent (Roberts et al., 1986). Analysis of CaM

8
amino acid sequences from a wide variety of organisms has
revealed that 47 of 148 amino acid residues are variant,
allowing a degree of latitude in the physiological
constraints that regulate the structure and function of each
CaM isoenzyme (Moncreif et al., 1990).
CaM multigene families have been characterized in rat
(Nojima, 1989), and human (Fischer et al., 1988). In both of
these species the CaM proteins encoded by different gene
families possess identical amino acid sequences; however,
their respective nucleotide sequences have diverged by
approximately 20 percent (Ling et al., 1991). More recently,
at least four CaM isoforms have been identified in
Arabidopsis thaliana that differ from one another by as much
as six amino acid substitutions (Gawienowski et al., 1993).
Arabiodopsis CaM isoenzymes are encoded by a multigene
family consisting of at least six different genes. Southern
blot analysis and genomic cloning determined the CaM proteins
were not allelic and that their coding regions had diverged
thirteen to twenty percent, and no significant identities
were retained in their mRNA 3'-untranslated regions. Most of
the amino acid changes between the CaM proteins appear to be
functionally conservative, and are clustered within the
fourth Ca2+-binding domain that is involved in high affinity
Ca2+ binding. It has yet to be determined if there exists
significant biochemical differences or tissue-specific
expression differences that would warrant these multiple CaM
proteins (Gawienowski et al., 1993). Multiple CaM isoforms,

9
each having a defined set of targets, may explain how a
common intracellular Ca2+ concentration signal can activate
different physiological responses.
In eukaryotes, multiple isoenzymes of CPSase and ATCase,
enzymes involved in the de novo pyrimidine biosynthetic
pathway have been identified (Jones, 1980; Ross, 1981).
Isoenzymes of these proteins have proven to be critical in
the biochemical regulation of eukaryotic pyrimidine and
arginine biosynthesis, both of which utilize
carbomylphosphate as an intermediate. Prokaryotes possess a
single CPSase and ATCase enzyme, both of which are
metabolically regulated by negative effectors to control flux
of carbomylphosphate between the two competing pathways
(Markoff and Radford, 1978).
Eukaryotes, other than plants, utilize two isoenzymes of
CPSase to commit separate pools of carbomylphosphate to the
pyrimidine and arginine pathways. An arginine-specific
CPSase is localized in the mitochondria with OTCase, the
enzyme that utlizes carbomylphosphate to synthesize
citrulline in the arginine pathway (Davis, 1986). A
pyrimidine-regulated CPSase activity exists on a
multifunctional protein that also exhibits ATCase activity
and is localized in the nucleus of yeast (Nagy et al., 1989),
or a cytosloic localized multifunctional protein that also
exhibits ATCase and DHOase activity observed in other
eukaryotes (Davidson et al., 1990). The different CPSase

10
activities have been shown to be encoded by separate genes in
these organisms.
The mechanisms of coordinately regulating the pyrimidine
and arginine pathways of plants are less understood.
Sequence analysis of partial cDNA clones from alfalfa has
provided evidence for the existence of arginine- and
pyrimidine-specific CPSases (Maley et al., 1992); however,
biochemical studies have only demonstrated a single
glutamine-dependent CPSase activity. Regardless of the
number of CPSases in plants, metabolic studies indicate that
the arginine and pyrimidine pathways share a common pool of
carbomylphosphate (Lovatt and Cheng, 1984). Localization of
CPSase, ATCase, and OTCase activities to the plant
chloroplast indicates that allocation of carbomylphosphate to
each pathway must be regulated (Shibata et al., 1986).
Williamson and Slocum (1994) utilized an ATCase
deficient mutant of Escherichia coli to clone by functional
complementation two different ATCases from pea plants.
Comparison of the deduced amino acid sequences of the clones
revealed an 85 percent identity and indicated they both
possessed a chloroplast targeting transit peptide. Southern
blot analysis revealed the two ATCase mRNAs are encoded by
two independent genes of the pea genome. Biochemical studies
are in progress to determine if these genes are
differentially regulated and if different ATCase subunits
which exist as homotrimers can also exist as heterotrimers
with unique kinetic properties.

11
Cell wall PME enzymes that de-esterify galactosyluronic
acid units of pectin, have been detected in all tissues of
higher plants analyzed. PME has been implicated in
functioning in a broad range of cellular processes including
fruit softening (Fischer and Bennett, 1991), plant response
to infection (Collomer and Keen, 1986), and cell growth
(Moustacos et al., 1991). Multiple isoenzymes of PME have
been detected in most plant species and tissues. It is
hypothesized that the multiple isoenzymes function in a
tissue-specific manner and have different modes of de¬
esterification of pectins (Markovic and Kohn, 1984).
Harriman et al. (1991) and Recourt et al. (1992)
demonstrated that tomato and Phaseolus vulgaris genomes
possess multiple PME genes. The cDNAS isolated from tissue-
specific libraries indicated that there are multiple PMEs
with high sequence homologies encoded by separate genes. In
vivo expression of antisense RNA constructs, designed to
block specified PME isoforms, revealed regulation of PME
isoenzymes occurs by regulating transcription from different
genes in a developmental, tissue-specific manner (Gaffe et
al., 1994).
Multiple isoproteins do not exist in all cases where
multiple genes are detected. Multiple genes encoding (1-3,1-
4J-p-glucanendohydrolases have been identified in wheat
(Triticum aestivium). Isolation of p-glucanase cDNA clones
revealed two different cDNAs with 31 nucleotide
substitutions; however, the coding regions of the mRNAs were

12
identical and only a single p-glucanase protein was detected.
Further analysis revealed that the two mRNAs originated from
homeologous chromosomes in the wheat hexaploid genome (Lai et
al., 1993). Therefore, the potential exists to derive
multiple isoenzymes by combining genomes in polyploid
species. The level of conservation of similar proteins will
be determined by relatedness of the parental species, the
time lapse since the genomes were combined, and selective
pressures to maintain the primary structures of the proteins.
Multiple isoenzymes have been shown to be derived from a
single gene in an organism. Saccharomyces cerevisiae cells
possess both a cytosolic and secreted form of invertase.
Both isoenzymes of invertase are encoded by a single
structural gene, SUC2, which gives rise to two distinct mRNA
species (Perlman and Halvorson, 1981; Carlson and Botstein,
1982). The polypeptides encoded by the invertase mRNAs, when
translated in vitro, are 60 kD (p60) and 62 kD (p62). The
p60 mRNA is 1.8 kb and encodes the cytoplasmic invertase,
whereas the p62 mRNA is 1.9 kb and encodes the secreted
invertase (Carlson and Botstein, 1982). The p62 form has
been shown to be glycosylated in the Golgi to an 87 kD
protein, a process that targets the invertase for secretion.
The p62 protein is preferentially targeted to the Golgi
apparatus via a labile 19 amino acid amino-terminal signal
sequence that is cotranslationally cleaved upon import to the
Golgi (Perlman et al., 1982).

13
Amino acid analysis of the p60 and p62 proteins revealed
that, starting at amino acid residue 21, the secreted
invertase was identical to the cytoplasmic invertase.
Comparison of the 5' ends of the mRNA nucleotide sequences
and the SUC2 gene revealed the presence of two unique
promoters (Tassig and Carlson, 1983; Sarokin and Carlson,
1984, 1985). The promoter region for the secreted invertase
was located -140 bp upstream of the coding region, whereas
the intracellular invertase promoter was located -40 bp
upstream of its coding region. Deletion of the nucleotide
sequence from -650 to -418 bp removed the regulation of the
secreted form by glucose; however, deletion from -1900 to -80
bp had no influence on the cytoplasmic invertase (Sarokin and
Carlson, 1984) .
Mammalian SPT is localized in two subcellular
organelles, the mitochondria and peroxisomes of the liver.
The peroxisomal SPTp and the mitochondrial SPTm isoenzymes
have very similar immunochemical (Oda et al., 1982),
catalytic and physical properties (Naguchi and Takada, 1978),
but their responses to hormones or other stimuli are quite
different. The SPTm is synthesized as a large precursor
which is specifically translocated into the mitochondria,
both in vivo and in vitro, and is processed into a mature
form similar in size to the mature SPTp. Two different SPT
mRNAs were detected by northern analysis using a SPTm probe.
The longer 1.9 kb mRNA was glucagon inducible and the smaller
1.7 kb mRNA was hormone insensitive indicating the larger

14
mRNA codes for the SPTm protein (45 kD) and the smaller mRNA
encodes the SPTp protein (43 kD) (Oda et al., 1993).
Cloning and sequence analysis of the two transcripts
provided evidence that the two mRNAs were identical except
for a longer 5' end possessed by the 1.9 kb SPTm mRNA. These
results were verified by Si nuclease protection, and RNase
protection analysis. Utilizing SPTm and SPTp probes,
Southern blot analysis detected a single SPT gene.
Comparison of the cDNA and gene sequences revealed that the
two SPT mRNAs were generated by transcription from two unique
promoters located upstream of exon one. The SPTm mRNA
contains an amino terminal extension of 22 amino acids that
acts as a mitochondrial targeting signal, whereas the SPT
mRNA lacks the targeting signal due to initiation of
transcription from a different promoter downstream of the
mitochondrial start methionine codon in exon one. Transport
of SPTp to the peroxisome occurs if the SPT preprotein lacks
the mitochondrial targeting peptide (Oda et al., 1993).
There are many other examples of multliple isoproteins
generated from a single gene via the use of alternate
upstream promoters (Beltzer et al., 1988; Chatton et
al.,1988).
Alternative splicing of pre-mRNA transcribed from a
common promoter of single gene has emerged as a widespread
mechanism for regulating gene expression. In most cases,
alternative splicing gives rise to multiple protein isoforms
that share high identity, but vary in specific domains that

15
allow fine regulation of protein function (Smith et al.,
1989). Alternative splicing allows for protein isoform
switching without the need for permanent genetic change that
would be necessary with gene rearrangement. The number of
genes known to be alternativly spliced reported to date is so
vast; therefore, a limited number of representative cases
will be discussed.
Alternative splicing of transcripts has been shown to
regulate the localization of isoproteins. The immunoglogulin
heavy chain protein Ign is present as a membrane bound form
in early B lymphocytes. Upon maturation of the B-cell, after
antigen activiation, the membrane-bound Igjx form decreases
and a concomitant increase in the Ig(n secreted pentamer form
is observed. The switch from membrane bound to secreted form
is acheived by the alternate use of 3' end exons that encode
the hydrophobic membrane-binding segment (Alt et al., 1980;
Rogers et al.,1980).
Gelosin, a protein which severs actin filaments, exists
as a plasma and cytosolic protein. Analysis of the two
isoenzymes indicated the proteins were identical except for
an additional 25 amino-terminal amino acids in the plasma
form. Analysis of the gelosin gene revealed the two
isoenzymes are expressed from the same gene via the use of
alternate promoters and subsequent alternative splicing of a
5' exon. The extra 25 amino acids of the mature plasma form
and an additional 27 amino acid signal peptide is encoded in
the extra exon. The 27 amino acid sequence targets the

16
plasma form to a secretion pathway and is cleaved from the
mature protein (Kwiatkowski et al., 1986).
Alternative pre-mRNA splicing can also function to
produce a functional and nonfunctional form of a protein that
acts as an on/off switch. The pathway of sexual
diffentiation in Drosophilia has revealed that an entire
sexual developmental cascade is regulated by alternative
splicing (Bingham et al., 1988). Briefly reviewed here, in
response to the X chromosome:autosome ratio this pathway is
regulated initially by the alternative splicing of three
primary geneszSxl, tra, and dsx.
The Sxl gene, the first gene in the cascade, pre-mRNA is
alternatively spliced to yield male and female specific
transcripts. The splicing results in the inclusion of an
exon in male transcripts and in the truncation of the major
open reading frame after 48 codons. In females, the male
specific exon is spliced out and a complete 354 amino acid
RNA-binding protein is produced (Bell et al., 1988). The
functional female Sxl protein regulates the splicing out of a
248 bp intron in the tra gene mRNA which produces a female
function tra protein. The lack of the functional sxl protein
product in males leads to a default splicing pattern in the
male tra mRNA which produces a nonfunctional tra protein
(Boggs et al., 1987; Nagoshi et al., 1988). The functional
tra protein of females regulates the splicing of the dsx pre-
mRNA to a female-specific transcript that encodes a dsx
protein that represses male differentiation genes.

17
Alternative splicing of the dsx pre-mRNA produces functional
products in males and females; however, the male dsx mRNA
possesses a male specific exon that is spliced out of the
female mRNA. The male dsx protein represses the female
diferrentiation genes (Baker and Wolfner, 1988). Further
analysis revealed that the functional tra protein of females
acts by recruiting general splicing factors to a regulatory
element downstream of the female-specific 3' splice site of
the dsx mRNA (Tain and Maniatis, 1993).
Changes in the enzymatic activity of the enzyme PK can
be attributed to mRNA alternative splicing. The four
isoforms of PK, Ml and M2, L and R, each form homotetrameric
holoenzymes. The Ml and M2 isoenzymes differ by the presence
of an internal 45 amino acid segment that is encoded by a
pair of mutually exclusive exons (Noguchi et al., 1986).
This variable region is nearly identical to a similar region
found in the L and R isoforms. The M2, L, and R
homotetramers are all allosterically regulated and show
sigmoidal kinetics, whereas the Ml isoenzyme shows no
allosterism and has Michaelis-Menton kinetics (Imamura and
Tanaka, 1982). The M2, L, and R proteins reside in tissues
where allosteric regulation is critical to prevent futile
cycling, whereas the Ml form exists in muscle tissue in which
glycolysis is the dominant metabolic state. The
alternatively spliced exons of Ml and M2 encode a region that
is important in intrasubunit contacts; therefore, it is
postulated that interactions between subunits dictate the

18
regulatory properties of the individual isoenzymes (Noguchi
et al., 1986 ) .
Alternative splicing also influences post-translational
modifications to isoproteins. The 25 kD synaptosomal
associated protein, SNAP-25, exists as two isoforms in
chicken. Cloning and characterization of the SNAP-25 cDNAs
and gene revealed that two exon fives exist in the gene.
Alternative splicing of the pre-mRNA mutually excluded one of
the two exons that resulted in two isoproteins that differed
in nine amino acid substitutions. The amino acid
substitutions result in the loss of a palmitoylation site,
thus influencing the ability of the isoforms to interact with
neuronal membranes (Bark, 1993).
The intracellular signaling by Ca2+ has been shown to be
finely regulated by alternative splicing in humans. The Ca2+
pump is a calmodulin-regulated P-type ATPase that is critical
in controlling intracellular Ca2+ concentrations. Analysis
of the pump gene structure indicated that alternative
splicing of the Ca2+ pump pre-mRNAs altered the calmodulin¬
binding domain. Alteration of this domain either increases
or decreases pump affinity for calmodulin. The decreased
affinity for calmodulin causes an apparent lower affinity of
the pump for Ca2+, thus lowering the intracellular Ca2+
concentration (Enyedi et al., 1994).
Although alternative splicing appears to be a common
mechanism of altering gene function without permanently
changing gene structure, few examples of alternative mRNA

19
have been reported for plants. This absence of published
reports likely reflects a lack of detection of alternative
mRNA splicing rather than a lack of its existence. Rubisco
initiates the pathway of photosynthetic carbon reduction in
plants. This enzyme exhibits catalytic activity only after
activation by rubisco activase. Immunoblots utilizing anti¬
activase antibodies detected two polypeptides in spinach (41
and 45 kD) and Arabidopsis (44 and 47 kD) indicating that the
two polypeptides were similar (Werneke, 1988). Genomic DNA
blots indicated that rubisco activase was encoded by a single
gene in spinach and Arabidopsis. Werneke et al. (1989)
demonstrated by amino- and carboxyl-terminal amino acid
analysis and cDNA cloning that the two activase isoenzymes
were derived by alternative splicing of activase pre-mRNA.
In spinach, two different 5' splice sites are utilized in
processing an intron in the 3' end of the primary transcript.
Use of the first 5' splice site introduced a termination
codon that results in formation of the 41 kD protein, whereas
selection of the downstream 5' splice junction omits the
temination codon and yields the 45 kD isoprotein. In
Arabidopsis, alternative splicing by a similar mechanism
results in the synthesis of the 44 kD and 47 kD polypeptides.
However, retention of the intron sequence does not introduce
a termination codon, but creates a frameshift that leads to
early termination of the protein. These results represent
the first case of alternative splicing reported for plants.

20
The P gene of Zea mays is postulated to
transcriptionally regulate flavonoid-derived pigment
biosynthesis in floral tissues. Two different P transcripts
have been detected, a 1.8 kb mRNA encoding a 43.7 kD protein
and a 0.945 kb mRNA encoding a 17.3 kD protein.
Characterization of the two P transcripts revealed they are
derived from a single gene and arise via alternative splicing
of the 3' end of a pre-mRNA (Grotewold et al., 1991). The
altenative splicing between exons two and three of the pre-
mRNA results in a frameshift that leads to early termination
of translation and yields the 17.3 kD polypeptide. The
truncated protein still possesses its DNA-binding domain;
therefore, it is hypothesized that the 17.3 kD protein may
act as a negative regulator and inhibits binding of the 43.7
kD functional transcriptional activator (Grotewold et al.,
1991).
Three cDNAs encoding RNA-binding proteins have been
isolated from Nicotiana (Nicotiana sylvestris) that encode
proteins with high affinities for polyuracil and polyguanine
motifs. Two of the cDNAs appear to be derived from a common
gene whose pre-mRNA undergoes alternative splicing in a
tissue specific manner. The alternative splicing occurs via
differential selection of two 5' splice junctions and results
in the formation of a functional and a truncated polypeptide.
The physiological significance of the isoproteins is unknown;
however, the functional polypeptide shows high homology with

21
the RNA-binding protein involved in the dsx alternative
splicing machinery of Drosophilia (Hirose et al., 1994).
Multiple isoenzymes can be derived by differential
proteolytic processing of a common precursor protein;
however, to date this mechanism has rarely been demonstrated.
NT and NMN, putative endocrine and neural signal prohormones,
are present in a 1:1 ratio within a common precursor
preprohormone polypeptide in mammals (Dobner et al., 1987).
Post-translational precursor processing has been shown to
occur in a tissue specific manner to yield both NT and NMN
prohormones in canines (Carraway and Mitra, 1990). Chicken
NT and LANT6 are derived via differential processing of a
common precursor prohormone (Carraway et al., 1993). Since
both NT and LANT6 have similar pharmacological activities and
bind similar receptors, it is postulated that the larger
slower degraded LANT6 may produce similar effects with a
different temporal pattern.
There are other translational and post-translational
mechanisms that could potentially yield multiple isoforms of
a protein. Such mechanisms include internal initiation of
translation (McBratney et al., 1993), protein splicing (Neff,
1993), protein methylation (Clark, 1993), protein acylation,
phosphorylation, and glycosylation (Blenis and Resh, 1993).
Although these mechanisms of regulation are undergoing
extensive research, the role and significance they play in
isoprotein formation is not well documented.

22
GDH is a ubiquitous enzyme detected in almost all
organisms from microbes to higher plants and animals
(Srivastava and Singh, 1987). This enzyme catalyzes the
reversible conversion of a-ketoglutarate to glutamate via a
reductive amination that utilizes NADH/NADPH as a cofactor.
A multitude of studies utilizing various techniques has
revealed that isoenzymes of GDH can be localized within the
mitochondria, chloroplast, and the cytoplasm depending on the
organism (Srivastava and Singh, 1987; LeJohn et al., 1994).
Multiple roles have been attributed to GDH including ammonia
assimilation (Yamaya and Oaks, 1987), maintaining the
glutamate/a-ketoglutarate ratio to regulate flux between
carbon and nitrogen metabolism (Munoz-Bianco and Cárdenos,
1989), stress response (Miranda-Ham and Loyola-Vargus, 1988;
LeJohn et al., 1994), and function as a RNA-binding protein
(Preiss et al., 1993). Collectively, these findings
implicate GDH as playing a variety of cellular roles and
these various metabolic funtions are regulated by the
differential use of GDH isoenzymes.
Chlorella sorokiniana has been shown to synthesize
multiple GDH isoenzymes: a constitutive, tetrameric,
mitochondrial NAD-GDH (subunit 45 kD), and seven ammonium-
inducible, hexameric, chloroplastic NADP-GDHs (subunit
53kD,(3- and 55.5 kD,cx-) (Prunkard et al., 1986; Bascomb and
Schmidt, 1987). The multiple chloroplastic isoenzymes have
different molecular weights and charges and presumably result
from the formation of homohexamers and heterohexamers due to

23
mixing of the a-subunits and (3-subunits. The chloroplastic
isoenzyme pattern in C. sorkiniana has been shown to be
influenced by light conditions, carbon and nitrogen source,
and ammonium concentration (Isreal et al., 1977; Prunkard et
al., 1986; Bascomb and Schmidt, 1987).
Kinetic and physical characterization of the purified a-
homohexamer and (3-homo hex ame r show them to have several
properties in common as well as striking differences (Bascomb
and Schmidt, 1987). The purified homohexamers have
remarkably different affinities for ammonia; however,
affinity values for the other substrates are quite similar.
The a-homohexamer is an allosteric enzyme with a low Km for
ammonia depending on the NADPH concentration, whereas the (3-
homohexamer is nonallosteric and has a high Km for ammonia.
In addition, pulse-chase experiments demonstrated that the
two subunit types are synthesized and degraded at different
rates (Bascomb and Schmidt, 1987). Although the two subunit
types have distinct in vivo turnover rates, they appear to be
derived from precursor proteins of near identical size.
Antibodies derived against one subunit type are able to
immunprecipitate both a-subunits and |3-subunits (Yeung et
al., 1981), and peptide mapping of the subunits revealed that
the subunits have 36 of 40 peptides in common (Bascomb and
Schmidt, 1987). These results indicate that the two subunits
are very similar. Genomic cloning and Southern blot analysis
indicated a single NADP-GDH gene exists in the C. sorokiniana

24
genome that encodes both types of subunits (Cock et al.,
1991) .
In summary, multiple isoenzymes have evolved as a
mechanism to allow an organism to respond to a broad range of
developmental and environmental conditions in a tissue-
specific manner. Isoenzymes and isoproteins can arise by a
variety of molecular and biochemical events including gene
duplication, alternative RNA splicing, transcriptional,
translational, and post-translational events. GDH, a
ubiquitous enzyme, exists as multiple isoenzymes with various
roles in most organisms studied. Considering the wealth of
information that exists on the metabolic roles and regulation
of GDH isoenzymes, further research into the biochemical
genetics of these isoenzymes may provide insight into
mechacanisms of molecular regulation in general.

MATERIALS AND METHODS
Culture Conditions
C. sorokiniana cells were cultured autotrophically as
previously described by Prunkard et al. (1986) in a modified
basal salts medium. The modified medium contained in mM
concentration: CaCl2, 0.34; K2S04f 6.0; KH2PO4, 18.4; MgCl2,
1.5; in (.iM concentration C0CI2, 0.189; CuCl2, 0.352; EDTA,
72; FeCl3, 71.6; H3BO3, 38.8; MnCl2, 10.1; NH4V04, 0.20;
(NH4)6MO7O24, 4.19; NÍCI2, 0.19; SnCl2, 0.19; ZnCl2, 0.734.
The medium was supplemented with 1 mM NH4CI, 29 mM NH4CI, or
29 mM KNO3 as a nitrogen source depending on the experimental
conditions. The medium containing NH4CI was adjusted to pH
7.4, and medium containing KNO3 was adjusted to pH 6.8 with
KOH after autoclaving. Cells were supplied with a 2%(v/v)
C02-air mixture and light intensity sufficient to allow cell
division into four progeny.
Enzyme Assay
The aminating and deaminiating activity of the NADP-GDH
was measured spectrophotometrically at 340 nm, by adding a 10
to 20 f.iL aliquot of enzyme preparation to 500 (.iL of assay
solution. The deaminating assay solution was composed of 44
mM Tris, 20.4 mM glutamate, and 1.02 mM NADP+ (Sigma), pH
25

26
8.8. The aminating assay solution was composed of 50 mM
Tris, 25 mM a-ketoglutarate, 0.357 mM NADPH (Sigma), and
0.356 M (NH4)2SO4, pH 7.4. One unit of enzyme activity was
the amount of NADP-GDH required to reduce or to oxidize 1.0
^M of NADP+ or NADPH per min at 38.5°C.
Isolation of RNA
All labware used in total cellular RNA isolation was
sterilized by baking at 220°C for 8 h, and sterile
plasticware was utilized whenever possible. All solutions
were made with H2O treated with 0.1% diethylpyrocarbonate
(Sigma) overnight at 37°C, and autoclaved according to
Sambrook et al. (1989).
On the day of the RNA isolation, a pellet of C.
sorokiniana cells stored at -70°C was resuspended 1 to 10
(w/v) in RNA breakage buffer: 0.1M Tris (pH8.5), 0.4M LiCl,
10 mM EGTA, 5 mM EDTA, 100 units/mL sodium heparin (Sigma,
100 units/mg), and 1 mM aurintricarboxylic acid (Sigma). The
cell suspension was centrifuged at 7000g for 5 min at 4°C and
the supernatant was discarded. The cell pellet was
resuspendeed 1 to 10 (w/v) in RNA breakage buffer and
ruptured by passage through a French pressure cell at 20,000
p.s.i.. The cell homogenate was collected in a disposable 50
mL conical tube containing 0.05 times volume 20% (w/v) SDS,
0.05 times volume 0.5 M EDTA (pH 8), 200 ug/mL proteinase K
(Sigma), and allowed to incubate at room temperature for 15
min. One-half volume of TE buffer (Tris 1OmM:EDTA ImM, pH

27
8.0) equilibrated phenol was added to the homogenate and
after a 3 min incubation a one-half volume of
chloroform:isoamylalcohol (24:l,v/v) was added and mixed for
10 min on a wrist action shaker (Burrel). The extracted
homogenate was transferd to a 30 mL siliconized (Sigmacote,
Sigma) corex tube and centrifuged at lOOOg for 10 min at 4°C.
The upper aqueous phase was removed and repeatedly extracted
with an equal volume of chloroform:isoamylalcohol (24:1,
v/v), as described above, until the aqueous interface was
clear. After the final extraction, the aqueous phase was
combined with an equal volume of 2X LiCl-Urea buffer (4 M
LiCl, 4 M urea, 2 mM EDTA, 1 mM aurintricarboxylic acid) and
the RNA was precipitated on ice for 16 h at 4°C. The RNA
precipitate was centrifuged at 4000g for 20 min at 4°C and
the resulting pellet was rinsed once with IX LiCl-Urea buffer
and centrifuged again to pellet the RNA. The RNA pellet was
solublized in TE (pH 7.5) and an aliquot was quantified
spectrophotometrically at 260 nm. After quantitation, the
total RNA was precipitated with 0.3M sodium acetate (pH 5.2)
and 2.5 times volume of 100% ethanol and stored at -20°C as a
precipitate. The mRNA fraction was isolated from total
cellular RNA using an oligo(dT) spin column kit (mRNA
Separatorâ„¢, Clontech) according to the supplier's
instructions.

28
Genomic DNA Isolation
Total cellular DNA was isolated from C. sorokiniana
cells using a modified procedure of Ausubel et al. (1989). A
6.5 g pellet of 29 mM KNO3 cultured cells and a 6.0 g of 29
mM NH4CI was harvested by centrifugation at 7000 rpm for 10
min at 4°C. Each cell pellet was resuspended in 25 mL of DNA
extraction buffer (0.1 M Tris-HCl, pH 8; 0.1 M EDTA, pH 8;
0.25 M NaCl), mixed with 2.7 mL of 10% (w/v) sarkosyl, and
incubated for 2 h at 55°C. The cell homogenate was combined
with 2.78 mL of 5M NaCl, 3.2 mL of 10% (w/v) CTAB, and
incubated at 65°C for 20 min. An equal volume of
chloroform:isoamylalcohol (24:1, v/v) was added and extracted
by gentle shaking for 10 min followed by centifugation at
3000 rpm for 10 min at 4°C. The supernatant was transferred
to a new tube and the nucleic acids were precipitated with
0.6 times volume of isopropol alcohol at -20°C for 30 min.
The precipitate was pelleted by centrifugation at 4500 rpm
for 20 min at 4°C. The pellet was resuspended in 9.5 ml of
TE (pH 8) and combined with 10 g of CsCl (BRL) and 0.5 mL of
10 mg/mL ethidium bromide. The CsCl:DNA mix was placed on
ice for 30 min and the contaminating RNA was removed by
centrifugation at 7500g for 10 min. The CsCl supernatant was
transferred to a 13 mL Quickseal tube (Beckman) and
centrifuged in a Vi65 Beckman rotor for 6 h at 55,000 rpm.
The high molecular weight DNA band was visualized with a
hand-held UV light, eluted from the tube, butanol extracted,
and dialysed 15 h against two changes of 1 L of TE (pH 8).

29
The dialyzed genomic DNA was quantified spectro-
photometrically at 260 nm and stored as an ethanol
precipitate until use.
NADP-GDH Protein Purification
Purification of the oc-NADP-GDH holoenzyme
C. sorokiniana cells were cultured with continous light
in 29 mM ammonium medium in a 30 L Plexiglas chamber as
previously described (Baker and Schmidt, 1963). Cells were
harvested at 4.0 OD640 by centrifugation at 30,000 rpm through
a Sharpies centrifuge (Pennwalt) and washed two times in 10
mM Tris (pH 8.5 at 4°C). Pelleted cells (130 g) were stored
at -20°C in 250 mL centrifuge bottles until use.
Purification of NADP-GDH was accomplished using a modified
procedure of Yeung et al. (1981). Procedural modifications
involved the substitution of Sephadex G-200 gel (Pharmacia)
for G-150 gel in the gel-filtration column, and the addition
of NADP+ as a stabilizer to a final concentration of 0.1 mM
to the gel-filtration buffer and all subsequent storage
buffers. As a final modification, the NADP+ affinity resin
step was omitted and a preparative nondenaturing-PAGE step
was substituted (Miller et al., 1994a).
Sephadex G-200 column fractions possessing NADP-GDH
activity were pooled and concentrated via Diaflow (Amicon)
filtration. The soluble enzyme (68 mg) was protected from
oxidation by the addition of DTT to a final concentration of

30
10 inM, and dialyzed for 30 min against 28.8 mM Tris, 192 mM
glycine, 2 mM DTT (pH 8.4). The dialysate was clarified by
centrifugation at 20,000g for 10 min at 4°C and was combined
with 3 mL of 40% (w/v) sucrose and 1 mL of 0.02% bromophenol
blue.
For preparative nondenaturing PAGE, a 3 cm tall 7%
acrylamide (w/v, 28 acrylamide: 0.735 bis-acrylamide, pH 8.8)
resolving gel, and a 2 cm tall 2% acrylamide (w/v, 1.6
acrylamide: 0.4 bis-acrylamide, pH 6.6) stacking gel were
cast in the 28 mm ID gel tube of the Model 491 Prep Cell
(Bio-Rad). All acrylamide stocks were pretreated with AG501-
X8 mixed bed resin (Bio-Rad) to remove any contaminating
acrylic acid residue to prevent in vitro N-acylation of
proteins during electrophoresis. The protein sample was
electrophoresed at 15 mA constant power for 20 min and then
for 3.5 h at a constant power of 30 mA. Six milliliter
fractions were collected and assayed for NADP-GDH deaminating
activity and GDH containing fractions were pooled. The
enzyme in the pooled fractions in 10 mM KPO4 (pH 6.2), 0.1 mM
NADP+ was concentrated by Diaflow filtration to 1 mg/mL as
determined by the method of Bradford (1976), using BSA as a
standard. The concentrated enzyme preparation was stored at
-20°C. The purity of the preparation was determined by
silver-staining using the Silverstain Plus kit (Bio-Rad) to
visualize proteins resolved by 10% (w/v) Tris-Tricine SDS-
PAGE (Shagger and von Jagow, 1987).

31
Partial purification of NADP-GDH isoenzymes
To insure that both the a-subunit and p-subunit were
represented, NADP-GDH isoenzymes were purified from a mixture
of cells cultured for 240 min in 1 mM ammonium medium (14 g),
90 min in 1 mM ammonium medium (6 g), and for 20, 40, 60, and
80 min in 29 mM ammonium medium (1 g/time point) according to
Bascomb and Schmidt (1987). GDH isoenzymes were partially
purified using a scaled down modified procedure of Yeung et
al. (1981). The DEAE sephacel ion exchange columns (pH
7.4,pH 6) were scaled down to a 40 mL bed volume and a 400 mL
linear KC1 gradient (0 to 0.4 M) was used to elute the
proteins in 3 mL fractions. The pH 6 DEAE ion-exchange
column fractions containing NADP-GDH were combined into two
pools; corresponding to the leading and trailing halves of
the NADP-GDH activity peak. The separate pooled fractions
were dialyzed against 10 mM KPO4 (pH 6.2), 2 mM DTT for 16 h,
and affinity purified using Type 3 NADP+ affinity gel
(Pharmacia) as previuosly described (Bascomb and Schmidt,
1987). The NADP-GDH in the pooled fractions was concentrated
via Diaflow filtration to 2 mg/ml protein, as determined by
the method of Bradford (1976), and stored at 4°C until
further use. After resolution of the proteins by 8% (w/v)
Tris-Tricine SDS-PAGE (Shagger and von Jagow, 1987), the
purity of the preparation was determined by silverstaining.

32
Anti-NADP-GDH Antibody Production and Purification
Monoclonal antibody production
Mouse anti-NADP-GDH MAbs were produced as described by
Tamplin et al. (1991). BALB/C mice were immunized by
intraperitoneal injection of 40 |ig purified NADP-GDH cx-
subunit in 0.25 mL PBS mixed 1:1 (v/v) with Freunds complete
adjuvant (Sigma). On day 24, the mice were injected
intraperitonealy with 25 [xg of purified a-subunit in 0.25 mL
PBS mixed 1:1 (v/v) with Freunds incomplete adjuvant (Sigma).
The mice were injected on day 38 with 25 |.ig of purified a-
subunit in 0.5 mL PBS, and once again 2 days prior to cell
fusion. Tail bleeds were performed one week prior to cell
fusion and analysed by ELISA to select mice producing a high
anti-NADP-GDH titer. Splenocytes and myeloma SP2/0 were
fused by the protocol of Van Deusen and Whetstone (1981).
After selection with HAT medium, hybridoma supernatants were
screened by ELISA in 96 well EIA plates (Costar) against 1.25
|xg NADP-GDH a-subunit in 50 [.il 0.1 M Na2C03 (pH 9.6) per
well. The ELISA procedure was performed as previously
described (Tamplin et al., 1991). ELISA positive clones were
selected and hybridoma supernatants were screened for
reactivity on Western blots. Selected high-titer hybridomas
were cloned by limiting dilution (Harlow and Lane, 1988), and
hybridoma supernatants were collected and frozen at -20°C for
future use.

33
Polyclonal antibody production
Rabbit anti-NADP-GDH antibodies were produced
commercially by Hazelton Washington (Vienna, VA) in six New
Zealand white rabbits. Rabbits were given a primary
immunization of 0.3 mg of purified NADP-GDH a-holoenzyme per
animal; followed by mulitple 0.15 mg booster injections.
Preimmune sera, test bleeds, and production bleeds were
shipped on dry ice for future analyses. Anti-NADP-GDH igG
titer was determined by measuring the ability of a series of
increasing amounts of antiserum or purified IgG to
immunoprecipitate 1 unit of NADP-GDH activity. After
incubating the immunoprecipitation mix for 35 min at 25°C,
immunoprecipitates were removed by centifugation at 14,000
rpm for 2 min in an Eppendorf microfuge. The titer was
recorded as the percent of NADP-GDH activity remaining in the
supernatant relative to a preimmune serum control. Standard
reaction conditions utilized 10 ¡.iL of 25 mM imidazole (pH 6),
1 unit of NADP-GDH deaminating activity in 2 0 (iL of 25 mM
imidazole (pH 6), anti-NADP-GDH antiserum or purified IgG in
a range from 0 to 25%, and rabbit preimmune serum was added
as a stabilizer to a final volume of 40 fiL.
Anti-NADP-GDH IgG was purified from rabbit serum using a
MASSâ„¢ Protein A affinity membrane 50 mm disc device (Nygene
Corp.). Anti-NADP-GDH rabbit serum was diluted 1:4 (v/v) in
6 mL of PBS and passed through 0.45 |xm syringe filter
(Gelman). Filtered serum was bound to the Protein A disc at
4°C and washed with PBS until the A280 of the eluate was

34
zero. Purified IgG was eluted with 10 mL of 0.1 M glycine
(pH 2.5) and 1 mL fractions were collected in microfuge
tubes, containing 0.1 mL of 1 H Tris (pH 8), and then gently
inverted to mix the contents. Fractions were analyzed
spectrophotometrically at 280 nm and fractions containing
greater than 1 mg/mL IgG were combined and frozen at -20°C in
0.25 mL aliquots.
Western Blotting
Alkaline phosphatase conjugated antibody detection
For NADP-GDH antigen determinations, the proteins in
samples were resolved by 8% (w/v) Tris-Tricine SDS-PAGE using
a Mini-Protean II cell (Bio-Rad) or a 400 SE cell(Hoeffer).
The proteins were electroblotted to a 7 cm x 10 cm Immun-
Liteâ„¢ nylon membrane (Bio-Rad) at 20 V constant voltage for
16 to 20 h at 4°C in a Mini-Transblot II cell (Bio-Rad).
Whatmann 3MM filter paper, scotch-brite pads, and Immun-Liteâ„¢
membrane, utilized in the electrotransfer, were pre¬
equilibrated for 10 min in 10 mM MES (pH 6), 0.01% (w/v) SDS
transfer buffer at room temperature. The immunodetection
procedure was performed at room temperature using the Immun-
Liteâ„¢ chemoluminescent detection kit (Bio-Rad). The nylon
membrane was incubated for 1 h in TBS blocking buffer (20 mM
Tris, 0.5 M NaCl, pH 7.5; 5% [w/v] nonfat dry milk). The
membrane was washed for 5 min with TTBS (TBS with 0.05% [v/v]
Tween 20) and incubated 2 to 16 h in primary antibody buffer

35
(TTBS, 1% [w/v] nonfat dry milk, rabbit anti-NADP-GDH IgG
[1.9 mg/mL IgG faction] diluted 1:200 or mouse anti-NADP-GDH
MAb hybridoma supernatant diluted 1:10). The nylon membrane
was washed for 15 min with three changes of 40 mL TTBS, and
incubated for 1 h in secondary antibody buffer(TTBS, 1% [w/v]
nonfat dry milk, 1:3,000 dilution of goat anti-rabbit IgG AP
conjugate [Bio-Rad] or 1:1,500 dilution of goat anti-mouse
IgG [whole molecule] AP conjugate [Sigma]). The immunoblot
was washed for 15 min with 3 changes of 40 mL TTBS, and 5 min
in 40 mL of TBS. The membrane was immersed for 5 min in
chemoluminescent substrate, sealed in a heat-sealable bag
(Seal-n-Save, Sears), and exposed to Kodak X-Omat AR film for
30 s to 5 min. After chemoluminescent detection, the
immonoblot was rinsed for 5 min in TBS, and incubated with a
color development substrate (10 mL 0.1 M Tris-HCl [pH 9.5],
0.1 M NaCl, 50 mM MgCl, 45 |LL NBT, 35 ^iL X-phosphate
[Boehringer Manniheim]). Color development was monitored
visually and stopped by the addition of 30 mL of TE (pH 8),
and molecular weight determinations were made relative to a
series of prestained protein markers (Midrange kit,
Diversified Biotech; Rainbow markers, Amersham).
125i_protein A detection
The proteins in 12 [iL aliquots of clarified cell
homogenates (20 mL cell culture concentrated in 3 mL of GDH
breakage buffer) were resolved by 8% (w/v) Tris-Tricine SDS-
PAGE in the 400 SE cell. The gel was electrophoresed at 10

niA constant current until the prestain 30 kD protein marker
(Rainbow markers, Amersham) reached the bottom of the gel.
The SDS gel, nitrocellulose (Bioblot-NC, Costar), and
Whatmann 3MM filter paper were equilibrated for 30 min in
24.8 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.4
transfer buffer (Towbin et al. 1979). The proteins were
transferred electrophoretically at 30 V constant voltage for
16 h at 4°C in a Trans-Blot cell (Bio-Rad). Immunodetection
was performed using a modified procedure of Towbin and Gordon
(1984) and Johnson et al. (1984). After electroblotting, the
nitrocellulose was air-dried and blocked in 50 mL of 40 mM
Tris (pH 7.4), 150 mM NaCl, 5% (w/v) nonfat dry milk
(Carnation), and 0.01% (v/v) Antifoam A (Dow-Corning)
blocking buffer for 1 h. All incubations were performed at
room temperature in a heat-sealable bag on a Labquake shaker
(Lab Industries). The blocking solution was decanted and the
nitrocellulose was incubated in 30 mL of 40 mM Tris (pH 7.4),
150 mM NaCl, 5% (w/v) nonfat dry milk, 0.01% (v/v) Antifoam
A, 0.05% (v/v) Tween 20 (Batteiger et al., 1982), and rabbit
anti-NADP-GDH antibody (1.9 mg/mL, IgG fraction) diluted
1:300 for 3 h. The immunoblot washed for 1 h with three
changes of 200 mL of 40 mM Tris (pH 7.4), 150 mM NaCl and
transferred to 25 mL of 40 mM Tris, (pH 7.4), 150 mM NaCl, 5%
(v/v) nonfat dry milk, 0.01% (v/v) Antifoam A, 0.05% (v/v)
Tween 20, and 0.25 ¡.iCi/lane of 125I_iaj;)elled Protein A (30
mCi/mg, Amersham) for 1 h in a new heat-sealable bag. The
nitrocellulose was washed for 1 h with three changes of 300

37
mL of 40 mM Tris (pH 7.4), 150 mM NaCl. The immunoblot was
allowed to air dry for 3 h on a stack of paper towels and
exposed to Fuji RX autoradiogrphy film at -70°C.
Amino-Terminal Sequence Analysis of the NADP-GDH «-Subunit
and 3-Subunit
An aliquot of a preparation of purified NADP-GDH a-
subunit (120 pmol) and a partially purified preparation of
NADP-GDH a-subunit (80 pmol) and p-subunit (50 pmol) were
resolved by 8% (w/v) Tris-Tricine SDS-PAGE and electroblotted
to a PVDF membrane (Immobilon-PsQ, Millipore) as described by
Plough et al. (1989). To prevent in vitro acylation of the
protein amino-terminal residues, all polyacrylamide solutions
used in PAGE were treated with AG501-X8 mixed bed resin to
remove contaminating acrylic acid. Protein sequence analysis
of the electroblotted proteins was provided by the ICBR
Protein Chemistry Core facility.
DNA Probe Synthesis
Specific cDNA restriction fragments were excised from
purified plasmids using the appropriate restriction
endonuclease (BRL). The plasmid restriction endonuclease
fragments were separated by electrophoresis in an alkaline
agarose gel (0.8% to 3% [w/v]) in TAE buffer (40 mM Tris-
acetate, 1 mM EDTA). The ethidium bromide stained fragment
of interest was cut out of the gel and mascerated in the
upper portion of a 0.45 |xm nylon membrane microfilterfuge

38
tube (Ranin). The mascerated agarose was frozen for 1 h at
-20°C and then centrifuged at 3000 rpm in an Eppendorf
microfuge. The gel purified fragment in the supernatant was
precipitated with 0.3 M sodium acetate (pH 5.2) and 2.5 times
volume of 100% ethanol at -20°C.
Twenty-five nanograms of purified cDNA fragment were
diluted in 33 ^iL dH20 and denatured at 100°C and chilled
rapidly on ice. The denatured fragment was radiolabeled with
32P-dGTP (3000 Ci/mmol, Amersham) by the random primer method
of Feinberg and Vogelstein (1984). Unicorporated nucleotides
were removed from the 32P-labeled probe using a Sehadex G-50
(Pharmacia) spin column (Sambrook et al., 1989).
Northern Blot Analysis
Total or poly(A)+ RNA stored as an ethanol precipitate
was pelleted by centrifugation at 14000 rpm for 15 min in an
Eppendorf microfuge. The vacuum dried pellet was resuspended
in 20 [iL of 75% (v/v) formamide (BRL), 8.28% (v/v)
formaldehyde, 3% (v/v) 10X MOPS (20 mM MOPS, 50 mM sodium
acetate, 10 mM EDTA, pH 7) and heated for 5 min at 80°C then
chilled on ice. The denatured RNA sample was combined with 6
|xL of formamide loading buffer (Sambrook et al., 1989) and 1
(.iL of 10 mg/mL ethidium bromide and resolved on a 2% (w/v)
formaldehyde-agarose gel (Ausubel et al., 1989). The RNA was
electrophoresed at 5 V/cm with constant circulation of the
electrophoresis buffer using magnetic stir bars in the buffer
reservoirs. After visualization with a UV transilluminator

39
(Fotodyne), the RNA was transferred by capillary blotting to
a Hybond-N nylon membrane (Amersham) with 20X SSPE (3.6 M
NaCl, 0.2 M KPO4r pH 7.7; 20 mM EDTA). The nylon membrane
was rinsed once in 2X SSPE, air dried for 1 h, and UV
irradiated for 5 min on a transilluminator to covalently link
the RNA to the membrane.
The membrane was prehybridized for 2 h at 40°C in 10 mL
of 50% (v/v) formamide, 25 mM KPO4 (pH 7.7), 5X SSPE, 5X
Denhardt,s solution (0.1% [w/v] Ficoll, 0.1% [w/v]
polyvinylpyrrolidone, 0.1% [w/v] BSA), 0.1% (w/v) SDS, 100
¡rg/mL sheared denatured salmon sperm DNA, 100 |.ig/mL yeast
tRNA in a heat-sealable bag on a Labquake shaker. The
prehybrization solution was decanted and the membrane was
hybridized for 16 h at 42°C on a Labquake shaker in 10 mL of
prehybridization buffer with 1 x 108 to 1 x 108 cpm of a heat
denatured 82P-labeled cDNA probe. The membrane was washed
three times in 0.1X SSPE, 0.1% (w/v) SDS for 20 min per wash
at 65°C. The Northern blot was covered with laboratory
plastic wrap and exposed to Kodak X-Omat AR film with one
intensifying sceen at -70°C.
Southern Blot Analysis
C. sorokiniana high molecular weight genomic DNA (6 ng)
was digested with a threefold excess of Pvu II, or Taq I
(BRL) for 4 h at the appropriate buffer and temperature
conditions. Three, 2 (itg aliquots of Pvu II digested, Taq I
digested, and undigested genomic DNA were electrophoresed at

40
1.5 V/cm in a 0.8% (w/v) agarose gel in TAE buffer. Ethidium
bromide (0.5 ng/mL) was added to the gel prior to
polymerization to allow visualization of the DNA during
electrophoresis. After electrophoresis, the gel was soaked
for 10 min in 0.1 N HC1, 35 min in 0.5 N NaOH, 1.5 M NaCl
denaturing solution, and 45 min in 0.5 M Tris, 3 M NaCl, pH 7
neutralizing solution. The DNA was transferred to a Hybond-N
nylon membrane by capillary action using 20X SSC (3 M NaCl,
0.3M sodium citrate) as described by Southern (1975). After
transfer, the membrane was rinsed in 2X SSC, air dried, and
the DNA was covalently linked to the membrane for 5 min on a
UV transilluminator. The nylon membrane was cut into three
strips each containing Pvu II digested, Taq I digested, and
uncut genomic DNA for analysis with different probes.
The nylon membranes were prehybridized in a HB-1D
hybridization oven (Techne) in 15 mL of 20% (v/v) formamide,
0.6 M NaCl, 0.6 M sodium citrate, 10 mM EDTA, 0.1% (w/v) SDS,
5X Denhardt's solution, 100 ng/mL denatured sheared salmon
sperm DNA for 2 h at 40°C. The membranes were hybridized
independently for 16 h at 45°C in 15 mL of 50% (v/v)
formamide, 10% (w/v) dextran sulfate (Sigma), IX Denhardt's
solution, 4X SSC, 5 [.ig/mL denatured sheared salmon sperm DNA,
and Ixl06tolxl08 cpm denatured 82P-labeled cDNA probe.
The cDNA probes corresponded to the 5'-VR, HCR, or 3'-UTR of
the concensus C. sorokiniana NADP-GDH mRNAs. The membranes
were washed independently three times in 50 mL of 0.1X SSC,
0.1% (w/v) SDS for 30 min per wash at 65°C. The nylon

41
membranes were covered with plastic wrap and exposed to Fuji
RX film with one intensifying screen at -70°C.
NADP-GDH cDNA Cloning and Characterization
^qtlO library
Small scale liquid lysates of previously isolated plaque
pure NADP-GDH ^gtlO clones were produced as described by
Sambrook et al. (1989). The kgtlO clone DNA was isolated
using a rapid small scale liquid lysate X phage DNA isolation
procedure (Ausubel et al., 1989). The cDNA inserts were
excised from the purified phage DNA with Eco RI restriction
endonuclease, gel purified as described above, subcloned into
the multiple cloning site of pUC 18, and transformed by the
CaCl2 method (Ausubel et al., 1989) into Eschericia coli DH5a
for further characterization.
XZAP II library
Synchronous C. sorokiniana cells were cultured in a 3 L
chamber in 1 mM ammonium medium or 29 mM ammonium medium
according to conditions reported by Bascomb and Schmidt
(1987) to yield primarily the NADP-GDH a-holoenzyme or
predominately the p-holoenzyme, respectively. The level of
ammonia in the 1 mM ammonium medium induction was
periodically measured by the method of Hardwood and Kuhn
(1970). Cells were harvested by centrifugation at 8000g at
4°C, and frozen at -70°C for use in total RNA isolation.
The

42
cells in 20 mL from each growth condition were concentrated
by filtration, resuspended in 3 mL of GDH breakage buffer,
and ruptured by passage through a French pressure cell at
20,000 p.s.i.. Aliquots of the various homogenates from
ammonium induced cells were resolved by 7.5% (w/v)
nondenaturing PAGE and the NADP-GDH isoenzyme pattern was
determined by use of a selective activity stain (Yeung et
al., 1981).
Total cellular RNA was isolated from 2 g cell pellets
from each growth condition as described above and the
poly(A)+ RNA fraction was purified by elution from an
oligo(dT) cellulose spin column (Clontech). The poly(A)+
selection was repeated two times on each RNA preparation to
insure complete removal of contaminating tRNAs, and rRNAs.
Both total RNA (20 fig) and poly(A)+ RNA (10 (xg) were analyzed
by 2% (w/v) formaldehyde-agarose gel electrophoresis, and
northern blot analysis, using a 242 bp HCR radiolabeled cDNA
probe, to verify the purity, intactness, and approximate
quantity of NADP-GDH mRNA represented in the a-induced and |3-
induced RNA preparations. Poly (A)+ RNA (50 |iig) from each
preparation was combined and utilized for the commercial
production of a custom XUni-ZAP XR C. sorokiniana cDNA
library (Stratagene Cloning Systems, Palo Alto, CA).
The amplified kZAP library, containing 2 x 1010 pfu/mL,
was plated on twenty 150 mm petri plates at 50,000 pfu per
plate for a total of 1 x 106 pfu screened. The phage plaques
were absorbed to duplicate Hybond-N 132 mm circular membranes

43
and treated according to the plaque blotting protocol of
Amersham (1985). Membranes were prehybridized in a common
container in 200 mL of 2X PIPES (0.8 M NaCl, 20 mM PIPES, pH
6.5), 50% (w/v) formamide, 0.5% (w/v) SDS, 100 ¡.ig/rnL
denatured sheared salmon sperm DNA at 40°C. Blocked
membranes were hybridized at 42°C in ten heat-sealable bags
(four membranes/bag) in prehybridization buffer containing 1
x 10^ cpm/membrane of a 32p_iabeled NADP-GDH 242 bp HCR cDNA
probe on a lab rocker (Reliable Scientific). The membranes
were washed three times in 200 mL of 0.1X SSC, 0.1% (w/v) SDS
for 20 min per wash at 50°C. Duplicate membranes were
wrapped in plastic wrap and exposed to Kodak X-Omat AR film
at -70°C for 2 8 h. Putative NADP-GDH cDNA plagues, detected
on duplicate membranes, were cored from the plate and plaque
purified by secondary and tertiary screenings with the 242 bp
HCR probe. Putative NADP-GDH cDNA phage clones (167),
selected in the primary screening, were combined and screened
a second time with a 32p_iabeled 130 bp Eco RI/Bgl II cDNA
fragment isolated from the 5' terminus of the most complete
5' end NADP-GDH cDNA clone (pGDc 42). Ten plaque pure NADP-
GDH clones were subcloned in pBluescript KS+ and transformed
into E. coli DH5a F' via an in vivo excision protocol
provided by Stratagene. All plasmid isolations were
performed as described by Kraft et al. (1988).

44
5 ' RACE-PCR cloning
The 5'-terminal NADP-GDH cDNA sequences were cloned
using a modified anchored PCR procedure for the rapid
amplification of cDNA ends (Frohman, 1990; Jain et al.,
1992). A mixture of poly(A)+ RNA, used in the synthesis of
the X.ZAP library, was utilized to clone the 5' end of the
NADP-GDH mRNA. One hundred nanograms of the mRNA mixture
were combined with 10 ng of a gene-specific primer (RRS 9;
Table 1), designed to hybridize to the HCR of NADP-GDH mRNAs,
heated for 5 min, and chilled on ice. First strand DNA
synthesis was performed using Superscriptâ„¢ reverse
transcriptase (BRL) according to the supplier's protocol.
The terminated reverse transcription reaction was treated
with one unit of ribonuclease H for 20 min at 37°C, 5 min at
95°C, and extracted once with chloroform:isoamyl alcohol
(24:1, v/v). Excess primers and dNTPs were removed by
centrifugation at 2000 rpm through an Ultrafree-MC filterfuge
tube (30,000 MW cutoff, Millepore) and the reténtate was
concentrated to 10 ^il on a Savant Speedvac. The first-strand
synthesis products were combined with 10 fxL of tailing mix
(IX tailing buffer [Promega Corp.], 0.4 mM dATP, 10 units
terminal deoxytransferase) and incubated at 37°C for 10 min.
The reaction mixture was heated to 95°C for 5 min, diluted to
0.5 mL with TE (pH 8), and utilized as a cDNA pool. A
mixture of 5¡.iL of the cDNA pool, 5 [iL of Vent™ polymerase 10X
buffer (NEB), 200 ¡.iM of each dNTP, 25 pmol of a gene specific

45
Table 1. Synthetic oligonucleotide sequences
Oligomer
Nucleotide Sequence
RRS5
RRS6
RRS7
RRS9
RRS11
RRS12
RRS13
RRS14
RRS15
RRS16
RRS17
RRS18
RRS19
RRS24
RRS25
GGGCTGCGCAGGCCGGGCGGCCACGATAGG
GGGTCGACATTCTAGACAGAATTCGTGGATCC(T)i8
GGGTCGACATTCTAGACAGAA
CTCAAAGGCAAGGAACTTCATG
GGACGAGTACTGCACGC
GAGCAGATCTTCAAGAACAGC
TCTGCACGTAGCTGATGTGG
CCCAGCCAGGGCCCTCACC
CACAGTATCGCATTCCGGGC
GATCTCGGTCAGCAGCTG
CTTTCTGCTCGCCCTCTC
GCGGCGACATCGCGC
CGTGCGCCAGCTGCTGAC
CCTTGTTGTACTTGTGG
CCACAAGTACAACAAGG

46
primer (RRS 9), 5 pmol of the poly(dT) adaptor primer
(RRS6), 0.2 units Perfectmatchâ„¢ DNA polymerase enhancer
(Stratagene), and 1 unit of Ventâ„¢ polymerase (NEB) in 50 (.iL
was amplified according to Jain et al. (1992). The PCR
products were purified away from the excess primers by
centrifugation at 2,000 rpm through an Ultrafree-MC unit.
The reténtate was collected and subjected to two more rounds
of amplification using a new nested gene specific primer at
each step (RRS 11; RRS16, respectively) and an adaptor primer
(RRS 7). PCR amplifications were performed in a Model 480
thermocycler (Perkin-Elmer Cetus), and all custom
oligonucleotides were synthesized by the ICBR DNA synthesis
facility. The standard PCR reaction mixture consisted of 10
(.iL of 1 OX Ventâ„¢ polymerase buffer, 100 of each dNTP, 0.4
units of Perfectmatchâ„¢, 50 pmol of each primer, 1 unit Ventâ„¢
DNA polymerase in a 100 ¡.il reaction volume. The optimal PCR
cycling parameters were determined using Oligoâ„¢ 4.0 primer
analysis software (National Biosciences Inc.). The 5' RACE-
PCR products were gel purified, subcloned into the Sma I site
of pUC 18, and transformed into E. coli DH5a for further
characterization.
NADP-GDH cDNA characterization
Purified NADP-GDH cDNA clone plasmids were digested for
1 to 2 h with specific restriction endonucleases using buffer
and temperature conditions deemed optimal by the supplier
(BRL). The resulting DNA fragments were resolved by

47
electrophoresis in a 1% (w/v) agarose minigel in TAE buffer.
DNA fragments less than 500 bp were resolved by 4% (w/v, 29
acrylamide:l bis-acrylamide) PAGE in TBE buffer (0.13 M Tris,
45 mM Borate, 2.5 mM EDTA, pH 9) at 7 V/cm until the
bromophenol blue dye was 1.5 cm from the base of the gel.
Restriction fragment size was determined by comparison to
XDNA/Hind III and standards (BRL).
NADP-GDH cDNA clones were sequenced by the dideoxy
method of Sanger et al. (1977) using modified T7 polymerse
(Tabor and Richardson, 1987) and the Sequenase 2.0 kit
protocol (United States Biochemical Corp.). The products of
the sequencing reactions were resolved on a 7 M urea, 5%
(w/v, 29 acrylamide:1 bis-acrylamide) sequencing gel. All
cDNA clones were partially sequenced from both ends.
Internal sequences were determined by subcloning select
restriction fragments into pUC 18 or by generation of a set
of nested deletions by timed digestion with exonuclease III
(Henikoff, 1984) using the Erase-a-base system (Promega
Corp.). Sequence data were analyzed using the Genetics
Computer Group programs (Devereux et al., 1984) on the ICBR
VAX computer.
Primer Extension Analysis
The 5' transcriptional start sites of the NADP-GDH mRNAs
were mapped by primer extension analysis as described by
Sambrook et al. ( 1989). A 20 [xg aliqout of a poly(A)+ RNA

48
mixture, isolated for use in the kZAP library synthesis, was
combined with 3.5 x 105 cpm of a 32P-labeled 30 nucleotide
oligomer (RRS 5) designed to hybridize to the 5' end of the
NADP-GDH mRNA. The primer:RNA mixture was denatured at 85°C
for 10 min and allowed to anneal for 12 h at 30°C. After
annealing, the RNA:primer complex was extended at 45°C for 2
h with 5 units of Superscriptâ„¢ reverse transcriptase, 0.5 mM
of each dNTP, according to the supplier's protocol. The
extension reaction products were treated with 5 ng of DNase
free RNase A (Sigma) for 30 min at 37°C,
phenol:chloroform:isoamylalcohol (25:24:1, v/v) extracted,
and ethanol precipitated at 0°C for 1 h. The pelleted
precipitate was resuspended in 6 of formamide loading
buffer and resolved on a 7 M urea, 5% (w/v) acrylamide
sequencing gel. The primer extension product was detected by
autoradiography on Kodak X-Omat AR film at -70°C and the size
of the extension product was estimated by comparison to a
sequencing ladder.
Genomic Allele-Specific PCR
C. sorokiniana genomic DNA was analysed by allele-
specific PCR as described by Saiki et al. (1986). Genomic
DNA (1 ^ig) and three NADP-GDH genomic clones in pUC 18 (0.1
ng; pGDg 8.4.4, 14.10.1, 15.2.2) were amplified using exon-
specific primer pairs that hybridized to exons one and three
(RRS17, RRS18), exons 10 and 11 (RRS12, RRS13), and exon 22
(RRS14, RRS15) of the NADP-GDH gene. The standard genomic

49
PCR reaction mixture was composed of IX Ventâ„¢ polymerase
buffer, 200mM of each dNTP, 50 pmol of each primer, 0.4 units
Perfectmatchâ„¢, and 1 unit Ventâ„¢ polymerase. PCR cycles were
executed under cycling parameters deemed optimal for each
primer pair. The allele-specific PCR products were resolved
by 4% (w/v) PAGE and visualized by ethidium staining. The
size of the PCR products was estimated relative to a 123 bp
ladder (BRL).
Construction of NADP-GDH In vitro Transcription Vectors
PCR generated fragments corresponding to the 5'-VR of
the +42 nt and -42 nt mRNAs, HCR, and 3'-UTR were cloned
downstream of the SP6 promoter of the SP65 in vitro
transcription vector (Promega Corp.). Using the 5' RACE-PCR
+42 bp and -42 bp cDNA clones (5'-VR; RRS16, RRS17) or pGDc
23 (HCR; RRS 12, RRS13: 3'-UTR; RRS14, RRS15) as templates,
the PCR fragments were amplified, gel purified, and cloned
into the Smal site of pUC 18. Clones corresponding to each
of the mRNA regions were selected on the basis of their
orientation in pUC 18, excised by digestion with Sal I/Eco
RI, and directionally cloned in the antisense orientation
into the SP65 transcription vector. Constructs were verified
by sequence analysis and were purified by a large scale CsCl
plasmid isolation procedure (Ausubel et al., 1989). 32p_
labeled antisense RNA probes corresponding to the four
regions of the mRNA were transcribed using the Riboprobeâ„¢ in
vitro transcription system (Promega Corp.). Full length in

50
vitro transcription products were selected by gel
purification on a 7 M urea, 5% (w/v) acrylamide sequencing
gel according to Ausubel et al. (1989) and used as protecting
fragments for RPA.
Comparison of the NADP-GDH mRNAs, Antigens, and Activities in
29 mM Induced C. sorokiniana Cells
Culture conditions
C. sorokiniana cells were synchronized using three
alternating light:dark periods (9h: 7h) in 29 mM KNO3
medium. Cells were harvested by centrifugation, washed with
nitrogen-free medium, and resuspended in 4 L of pre¬
equilibrated nitrogen-free medium in a 4 L Plexiglas chamber.
The cells were induced in 29 mM ammonium medium for 240 min
as described by Cock et al. (1991). Samples of 500 mL of
cell culture (2.87 x 10^ cells/mL)were collected at To, and
at 20 min intervals for the first 140 min, and a final sample
was harvested at 240 min. Samples for RNA isolation were
concentrated by centrifugation at 4°C at 9,000 rpm and
stored at -70°C. The cells in 20 mL of culture were
harvested by filtration, resuspended in 3 mL of GDH breakage
buffer, and stored at -20°C for protein and GDH activity
analyses.

51
RNase protection analysis
Total cellular RNA was isolated from a 1 g pellet from
each time point as described above. The amount of poly(A)+
RNA in the various total RNA preparations was quantified
based on the formation of ribonuclease-resistant hybrids with
poly-^H-5,6-uridylate (2-10 Ci/mmol, New England Nuclear) as
described by Davis and Davis (1978) using purified (3-globin
mRNA (BRL) as a standard.
The quanity and form of mRNA present at each time
interval was determined by RPA. A total of 210 ng of Poly
(A)+ RNA or 25 ng of control yeast tRNA was combined with 1 x
105 cpm of a 32P-labeled antisense RNA probe corresponding to
the +42 nt and -42 nt 5'-VR, HCR, or 3'-UTR of the NADP-GDH
mRNAs and hybridized for 16 h at 42°C. RPAs were performed
using the Guardianâ„¢ RPA kit (Clontech) according to the
suppliers instructions. The ribonuclease resistant fragments
were resolved on a 7 M urea, 5% (w/v) sequencing gel and
sized by comparison to a sequencing ladder. The resistent
fragments were transferred from the gel to 3MM Whatmann
filter paper, dried at 80°C, and exposed to a phosphorous
screen and then to Fuji RX film at -70°C. The amount of
residual antisense RNA probe was quantitated on a
Phospholmagerâ„¢ (Molecular Dynamics) at the ICBR DNA Synthesis
Core facility.

52
NADP-GDH antigen and activity analyses
C. sorokiniana cell samples from the various time points
were disrupted by two passages through a French pressure cell
at 20,000 p.s.i.. Aliquots from each cell homogenate were
analyzed for both aminating and deaminating NADP-GDH
activity. Total soluble protein concentration in the cell
homogenates was determined by the method of Bradford (1976)
using BSA as a standard. The proteins in 12 ¡xL aliquots from
each homogenate were resolved by 8% (w/v) Tris-Tricine SDS-
PAGE, transferred to nitrocellulose, and NADP-GDH antigen was
detected by 125I-Protein A Western blot analysis as described
above. The amount of NADP-GDH antigen present as the a-
subunit and (5-subunit at each time interval was quantified on
a Visage 60 laser desitometer (Bio Image).
RT-PCR analysis
A modified RT-PCR (Kawaski, 1990) procedure was used to
identify and quantify the NADP-GDH mRNAs present in the total
RNA preparations from the various induction time-points. A
series of primer pairs were selected to yield ovelapping PCR
fragments spanning the entire length of the NADP-GDH mRNAs:
RRS17, RRS18; RRS17, RRS16; RRS19, RRS13; RRS12, RRS13;
RRS12, RRS24; RRS15, RRS25. An aliquot of total RNA from
each time-point containing 10 ¡Lig of poly (A)+ RNA was combined
with 300 pmol of random hexameric oligonucleotides
(Pharmacia), incubated at 75°C for 5 min, and chilled on ice.
The RNA:primer mixture in 6 ^iL was combined with 2 ^iL of 10X

53
Ventâ„¢ polymerase buffer, 10 units RNASINâ„¢ (Promega Corp.)/ 1
mM of each dNTP, 1 mM DTT, 200 units Superscriptâ„¢ reverse
transcriptase in a final volume of 20 |xL, and incubated at
22°C for 10 min, 42°C for 70 min, 50°C for 30 min, and 95°C
for 5 min. After termination of the reaction, 120 ^iL of IX
Ventâ„¢ polymerase buffer was added to each reaction tube. The
resulting mixture served as cDNA stocks for subsequent
amplifications. The standard RT-PCR amplification mixture
consisted of 8 fiL 10X Ventâ„¢ polymerase buffer, 200 fiM of each
dNTP, 50 pmol of each primer, 0.2 units Perfectmatchâ„¢' 2 0 (.iL
of a cDNA stock, and 4 units of Ventâ„¢ (exo-) polymerase (NEB)
in 100 |xL final volume. The RT-PCR mixtures were cycled at
conditions determined to be optimal for each primer pair
using the Oligoâ„¢ 4.0 primer analysis software. RT-PCR
products were resolved on 1% to 3% (w/v) agarose gels and
sized by comparison to a 123 bp ladder (BRL). The relative
intensities of the PCR fragments was quantified from
Polaroidâ„¢ Type 55 negatives of ethidium bromide stained gels
on a Visage 60 laser densitometer.

RESULTS
NADP-GDH cDNA Cloning and Characterization
Restriction mapping and sequencing of XqtlO cDNA clones
A kgtlO cDNA library was constructed from poly(A)+ RNA
isolated from C. sorokiniana cells induced in 29 mM ammonium
medium for 80 min. Cells induced under these conditions were
reported by Prunkard et al. (1986) and Bascomb et al. (1987)
to accumulate both the a- and p-subunits as NADP-GDH
holoenzymes between 40 and 120 min, and at 80 min the a- and
p-subunits each constituted approximately 50% of the total
NADP-GDH antigen. Approximately 2 x 10^ pfu were screened
with a heterologous 1.2 kb Salmonella typhimurium gdhA gene
probe (Miller and Brenchley, 1984) and six putative NADP-GDH
cDNAs were isolated. The cDNAs ranged from 0.6 to 1.91 kb
and their restriction maps were identical in regions in which
they overlapped (Fig. IB; Cock et al., 1991). The cDNA
clones appeared to be truncated forms of the 1.91 kb pGDc 23
cDNA clone that lacked a complete 5' terminus. A lacZ-pGDc
23 translation fusion expressed in E. coli JM 109 accumulated
antigen which was recognized by antibodies raised to purified
C. sorokiniana NADP-GDH verifying the cDNA authenticity (Cock
et al., 1991) .
54

Figure 1. Restriction maps of 17 cDNAs isolated from a
C.sorokinana library prepared from RNA isolated from cells
induced for 80 min in 29 mM ammonium medium. A, The 2145 bp
consensus NADP-GDH map. Regions corresponding to the HCR and
3'-UTR are indicated. B, NADP-GDH cDNA clones isolated using
a heterologous 1.2 kb probe from the gdhA gene from S.
typhymurium. cDNA clones were sequenced in the regions
denoted by arrows. C, The cDNA clones were isolated using a
homologous 115 bp PstI fragment from the 5' end of the HCR of
pGDc 2 3.

56
A
Ico RI
Sgl II ¡Bgl II
|Pst I'| I
3gl n
Pst I a*r I air I Pst I air I
|air I air I
S»i I Eco
I T«lv «I
5‘
TAÁ
3
B
J kb
Coas#rv«d R»gioiv
Pst I
â– =i Conserved P*giorv Prob*
3' -Urxtrinslitid
JUgioa Prob*
^ 1 >7, M S7 1
?YU II Kmc II
c
Pv Bg ? ? He 3g P
—
5 IT Pv P
3g
5 5 Pv P
? He 3g
5 H P
^
Py 3g ? ? He 3g
a a p* ?
pv Bg P ? â– 
\i
5 a tr ?
115 bp
P ? He Bg
5' Prob*
P
Pv 3g ? ? He Bg
a a Pv P
a a ?v p
a a Pv p
a a fv p
? P He Bg ? a a Pv ?
? ? He Bg P a a Py P
Pv 3g ? ? He Bg ?
a a ft p
? ? He Bg P
a a p* ?
3g p ?v Bg ? ? He 3g ? 5 a PvJ
Pv 3g P ? He Bg P
a a fv p
consensus
7 GOH-cDNA
pGDc 23
pGDc 2
pGDc 3
pGDc 6
pGDc 7
pGDc 10
pGDc 30
pGDc 31
pGDc 32
pGDc 33
pGDc 34
pGDc 35
pGDc 36
pGDc 38
pGDc 39
pGDc 42
pGDc 44

57
In an attempt to select additional NADP-GDH cDNAs with
complete 5' termini, the >^gtlO library was rescreened with a
homologous 115 bp PstI restriction fragment probe (Fig. 1C)
derived from the 5'-proximal end of pGDc 23. Approximately
one-half of the 115 bp probe sequence overlaps the 5-terminus
of the 354 bp NADP-GDH HCR (Fig. 1A) that is predicted to be
present in all NADP-GDH cDNAs. Eleven additional cDNA clones
were isolated, restriction-mapped, and their 3' and 5'
termini sequenced (Fig. 1C). The additional cDNA clones had
identical restriction maps and sequences in regions that
ovelapped with pGDC 23. NADP-GDH cDNA clones pGDC 31, 38,
and 42 were longer than pGDC 23 at their 5' termini; however,
none of the cDNAs possessed an ATG start methionine codon or
a 5'-untranslated region. The 5'-terminal FcoRI/Pvull
fragment (Fig. 1C) of pGDC 42 was subcloned into pUC 18 and
both strands were sequenced. Sequence analysis of the pGDc
42 subclone revealed that pGDc 42 possesed an additional 256
bp of coding region sequence not found in pGDc 23.
Although the cDNA library was prepared using an oligo
(dT) primer, 10 of the 17 NADP-GDH cDNAs lacked a poly(A)+
tail and additional 3'-terminal sequences (Fig. 1). None of
the 17 cDNA clones were full-length at their 5' termini.
From the combined sequences of the cDNA clones, a 2145 bp
consensus NADP-GDH cDNA sequence and restricition map was
constructed (Fig.lA). Bascomb et al. (1986) and Cock et al.
(1991) showed the NADP-GDH mRNA to be 2.2 kb; therefore, the

58
consensus NADP-GDH clone was determined to be approximately
98% full-length.
Isolation, restriction-mapping, and sequencing of the Á.ZAPII
NADP-GDH CDNA clones
Since all of the XgtlO library NADP-GDH cDNA clones
lacked complete 5' termini and many lacked complete 3'
termini, a second ammonium-induced unidirectional cDNA
library was constructed. Synchronous C. sorokiniana cells,
used for total RNA isolation, were cultured in 1 mM or 29 mM
ammonium medium according to conditions reported to yield
primarily the NADP-GDH a-holoenzyme or predominately the f$-
holoenzyme, respectively (Bascomb and Schmidt, 1987). The
NADP-GDH isoenzyme pattern from each culture condition was
verified by activity staining crude preparations resolved by
nondenaturing PAGE. Poly(A)+ RNA from each culture condition
was analyzed by northern analysis, using the 242 bp HCR and
the 378 bp 3'-UTR probes (Fig. 1A), to verify the approximate
level and intactness of NADP-GDH mRNA in each preparation
(Fig. 2). An equal quantity of poly(A)+ RNA from each
preparation was combined, to ensure representation of
multiple types of NADP-GDH mRNAs if they existed, and the RNA
mixture was utilized in the cDNA library construction. The
kZAPll unidirectional cloning system was utilized to ensure
the cloning of the complete 3' ends of the mRNAs.
Initial screening of 1 x 10^ pfu with the homologous 242
bp HCR probe derived from pGDc 23 (Fig. 1A) yielded 167

Figure 2. Northern blot analysis of poly(A)+ RNA isolated
from C. sorokiniana cells induced for 3 h in 1 mM ammonium
medium (Lanes 1,3) or continuously in 29 mM ammonium medium
(Lanes 2,4). Formaldehyde-agarose gel resolved RNA
preparations immobilized on nylon were hybridized with the
242 bp HCR probe and 378 bp 3'-UTR probe. A single-sized 2.2
kb NADP-GDH mRNA was detected in both RNA preparations with
both probes.

60
HCR 3'-UTR
1 2" 3 4 '
1.35-
0.24-

61
putative NADP-GDH cDNA clones. Ten of the primary screening
plaques were selected at random and plaque purified by
secondary and tertiary screenings. Restriction-mapping and
sequencing of the ten NADP-GDH cDNAs revealed eight unique
overlapping clones ranging in size from 1.46 to 2.04 kb (Fig.
3). Two of the 10 clones proved to be identical to pBGDC 52
and 58 indicating a detection of redundant clones in the
amplified library. All eight of the unique NADP-GDH clones
possessed identical complete 3' termini; however, they all
lacked complete 5' termini (Fig. 3). The longest kZAP NADP-
GDH cDNA clone, pBGDc 53, was 103 bp shorter than the XgtlO
NADP-GDH consensus cDNA.
To detect any cDNA clones longer at the 5' terminus than
pBGDc 53, the 167 putative NADP-GDH plaques selected in the
primary screening were combined and rescreened. The second
screening utilized a homologous 130 bp EcoRI/Bglll cDNA
fragment probe derived from the 5' terminus of pGDc 42 (Fig.
1C). No NADP-GDH clones longer than pBGDc 53 were isolated
from the secondary screening.
Primer extension analysis
Although all of the NADP-GDH cDNA clones isolated from
the X.ZAP library possessed complete 3' ends, none of the 25
cDNA clones isolated from either library possessed complete
5' ends. In order to determine the amount of sequence
remaining proximal to the 5' end of the consensus NADP-GDH
cDNA clone, primer extension analysis was performed on the

Figure 3. Restriction maps of eight cDNAs isolated from a C. sorokiniana library prepared
from a RNA mixture containing equal amounts of poly(A)+ RNA from cells induced in 1 mM
ammonium and 29 mM ammonium medium. NADP-GDH clones were isolated using a homologous 242 bp
HCR probe derived from pGDC 23 (Fig. 1). All clones possess a complete 3' terminus and no
clones were longer than the previously determined consensus NADP-GDH cDNA.

0.4
L
Oí
1.4
L
1 8
2.0
2.2
J
35d bp
Conserved Region
Fst I Pst I
— ( Conserved Region Probe
3'-lfntranslated
Region Probe
1 378 bp S7 1
Pvu II Hiñe II
Pvu II
Eco RI
V
Hiñe II
II
Bgl II
Bgl II
I Pst I
Pit I
Pst I War I War I
Pit I War I
/ \
War I War I
!â–  I
i
TM
Sma I Eco RI
I >olv fl| consensus
GDU-cDHA
3'
P He Bg P
i
W W Pv P
l l
He W
L J
Sm
^l!-y ,R, pBGDC 50
P P He Bg
J.X i
Bg
W W Pv P
l i X—
He W
â– â– â– â–  i 1
N Pv P
-J'ti—
He N N
—i—i i
Sm
,oly " pBGDc 51
Sm
f01* " pBGDc 52
o>
GO
Bg p Pv Bg P P He Bg
-.1, J L V
W Pv P
l ii
He W
i i
Sm
joly R, pBGDc 53
w W Pv P H
l i li i
He W
Sm
,°'Y R. pBGDc 51
W Pv P
j ixL
He W
. â– â–  1.1
Sm
â–  pBGDc 55
P P He Bg
J.Y.i
W Pv P
I
He N
..ml l
Sm
|oly ", pBGDc 56
Pv Bg P P He Bg
L L Y i
W W Pv P w
-I 1 L.
He W
Sm
*°ly B pBGDc 50

64
mixture of C. sorokiniana poly(A)+ RNA previously isolated
for the XZAP library construction. As determined by
comparison to a cDNA generated sequencing ladder, a single
primer extension product of 87 nt was detected (Fig. 4). The
87 nt product corresponds to 53 bp of sequence identified in
the 5’ terminus of pGDc 42 (Fig. 1C) and 34 bp of sequence
previously undetected. The additional 34 bp extension
predicted a mRNA of 2.179 kb that approximated the 2.2 kb
mRNA determined by northern blot analysis (Fig. 2). The
primer extension analysis was repeated with identical
results.
RACE-PCR cloning of two NADP-GDH 5' termini
To determine the 5'-terminal sequences of the NADP-GDH
mRNA(s), a modified anchored PCR procedure for the rapid
amplification of cDNA ends was performed (Jain et al., 1992).
To ensure any possible sequence differences that might reside
in the 5’ region proximal to the HCR would not be missed, a
RNA mixture previously prepared by mixing equal quantities of
poly(A)+ RNA from cells synthesizing primarily the a- or p-
holoenzyme was used for the RACE-PCR cloning.
Agarose gel elelctrophoresis of the products from the
second step of the RACE-PCR amplification revealed two DNA
fragments of approximately 390 and 450 bp in size.
Reamplification using a different nested gene-specific
primer, designed to hybridize closer to the 5'-termini of the
mRNAs, yielded two unique PCR products of approximately 330

Figure 4. Primer extension analysis of NADP-GDH mRNA(s). A
poly(A)+ RNA mixture isolated from Chlorella cells,
synthesizing primarily the NADP-GDH a- or p-subunit, was
hybridized with a 32P-labelled 5' NADP-GDH-specific
oligonucleotide and extended with reverse transcriptase. A
single 87 nt primer extension product (PE) was detected after
resolution on a 5% sequenceing gel. The approximate size of
the extension product was determined by comparison to a NADP-
GDH cDNA clone sequencing ladder.

66

67
and 370 bp in size as determined by agarose gel
electrophoresis (Fig. 5). Sequence analysis of the two
cloned final PCR products revealed the actual size of the
products, minus the anchor primer, to be 269 and 311 bp.
Comparison of the two RACE-PCR cDNA clone sequences showed
them to be identical except for the presence of an additional
42 bp in the 5' coding region of the longer PCR product. The
additional 42 bp sequence encodes 14 amino acids that were
not present in the 269 bp RACE-PCR clone (Fig. 5). The
absence of the 42 bp sequence in the 269 bp clone results in
the deletion of the 14 amino acids from the amino-terminus of
the polypeptide; however, the downstream reading frame
remained unchanged. Both RACE-PCR products possessed
identical putative 5'-UTRs, translation initiation sites, and
were identical for the first 12 codons (Fig. 5). Both the
+42 bp and -42 bp clones overlapped in frame with pGDC 42 at
their 3' ends. The 5' RACE products were 33 bp longer than
pGDC 42 at their 5'-termini which is in close agreement with
the 34 bp length predicted by primer extension analysis.
The NADP-GDH 5' RACE-PCR clones possessed identical 32
bp 5'-terminal pyrimidine rich (89%) sequences and
transcription initiation sequences indicative of eukaryotic
5'-UTRs and translational start sites, respectively (Kozak,
1984). Furthermore, additional 5'-terminal guanine residues
were detected (2 on the +42 bp and 1 on the -42 bp clone) in
both clones that could not be accounted for in the NADP-GDH
gene sequence. The presence of unique 5'-terminal guanine

Figure 5. 5' RACE-PCR generated NADP-GDH 5'-terminus clones. Two PCR products,
approximately 370 and 330 bp, were detected after resolution on a 3% agarose gel (inset).
Sequence analysis revealed the two 5'-end clones differed by 42 bp in their 5'-coding
regions. The deduced amino acid sequences of the two NADP-GDH mRNAs showed the two mRNAs
to encode identical polypeptides with the exception of an additional 14 amino acids encoded
by the 42 nt insert in the longer mRNA.

ATG
i
AAAAAA
ATG
. +42 â– 
• I
t 4-
/
I
I
I
I
I
I
I
AAAAAA
vo
MQTALVAKPIVAAPLAARPRCLAPWPCAWVRSAKADVRA
MQTALVAKPIVA - ; CAWVRSAKADVRA

70
residues on 5' RACE-PCR products has proven to be a
definitive means of identifying 5' capping points and
transcription initation sites of eukaryotic mRNAs (Bahring et
al., 1994). The RACE-PCR procedure was repeated with
identical results. RT-PCR performed on the poly(A)+ RNA
mixture using two new gene-specific primers that flanked the
42 bp variable region also yielded two PCR products differing
in size by 42 bp.
Both 5' RACE-PCR products appear to be complete at their
5' termini and overlap in frame with the consensus NADP-GDH
cDNA identified earlier. These results are consistent with
the existence of two separate NADP-GDH mRNAs that share a
common transcriptional start site and are identical with the
exception of a 42 nt insert identified in the longer mRNA.
The +42 nt mRNA predicted size is 2185 nt, whereas the -42 nt
mRNA predicted size is 2143 assuming a mean poly(A) tail
length of 70 nt.
Analysis of the C. sorokiniana NADP-GDH cDNA sequences
Sequence analysis of the two consensus NADP-GDH cDNAs
(+42 bp and -42 bp) revealed both mRNAs possessed an
identical 32 nt 5'-UTR (Fig 6). The +42 bp cDNA possesses an
ORF from nt 33 to a TAA stop codon at nt 1611 that encodes a
precursor protein with a molecular mass of 57850 D. The -42
bp cDNA possesses an ORF from nt 33 to nt 1569 that encodes a
precursor protein of 56350 D. The 1500 D difference in
molecular mass observed in the two precursor proteins is due

Figure 6. Nucleotide sequence of the consensus NADP-GDH
mRNAs derived from the cDNA and 5' RACE-PCR clone sequences.
Beginning at nucleotide 33, two ORFs were identified from two
mRNAs that differed by 42 nt in the 5'-VR (boxed). The +42
bp mRNA encodes a polypeptide of 57,850 D, whereas the -42 bp
mRNA encodes a 56,350 D polypeptide. The deduced amino acid
sequences of the C. sorokiniana precursor polypeptides (Cs)
are compared with those of E. coli (Ec) and N. crassa (Nc)
NADP-GDHs. Arrows denote the boundaries of the highly
conserved glutamate binding domain identified by Mattaj et
al. (1982). A consensus algal polyadenylation signal
(underlined) is located 17 bp upstream from the poly(A) tail
of the NADP-GDH mRNAs.

72
Cs
Cs
Ec
He
CTCTTTCTGCTCGCCCTCTCCGTCCCGCCCATGCAGACC AO
HOT
GCCCTCGTCGCCAAGCCTATCGTGGCCGCCCCGCTGGCGGCACGCCCGCGCTGCCTCGCGCCGTGGCCGTGCGCGTGGGTCCGCTCCGCC 130
A L V A K P I V A | A P L A A R P R C L A P U P] C A U V R S A
AAGCGCGATGTCCGCGCCAAGGCCGTCTCGCTGGAGGAGCAGATCTCCGCGATGGACGCCACCACCGCCGACTTCACGGCGCTGCAGAAG 220
KADVRAKAVSLEEQISAHDATTGDFTALQK
GCGGTGAAGCAGATGGCCACCAAGGCGGGCACTGAGGGCCTGGTGCACGGCATCAAGAACCCCGAGCTGCGCCAGCTGCTGACCGAGATC 310
AVKQMATKAGTEGLVHGIKNPELRQLLTE I
MDQTYSL.SFLNHVQK
S
TTCATGAAGGACCCGGAGCAGCAGGAGTTCATGCAGGCGGTGCGCGAGGTGGCCGTCTCCCTGCAGCCCGTGTTCGAGAAGCGCCCCGAG A00
FMKDPEQQEFHQAVREVAVSLQPVFEKRPE
RNPMQTEFA Q A VREVHTTLUPFLEQNPKYR
N L P S E P E F E QjA Y K E L A Y T L E N S S L . Q . H . .
CTGCTGCCCATCTTCAAGCAGATCGTTGAGCCTGAGCGCGTGATCACCTTCCGCGTGTCCTGGCTGGACGACGCCGGCAACCTGCAGGTC A90
LLP I FKQIVEPERVI TFRVSULDDAGNLQV
QMSLLERL Q...V.V..RNQ1..
YRTALTVASI O...V.E..N..V..
AACCGCGGCTTCCGCGTGCAGTACTCGTCCGCCATCGGCCCCTACAAGGGCGGCCTGCGCTTCCACCCCTCCGTGAACCTGTCCATCATG 580
NRGFRVQYSSAIGPYKGGLRFHPSVNLSIM
. .AU...F H L
...Y...FN..L L L
AAGTTCCTTGCCTTTGAGCAGATCTTCAAGAACAGCCTGACCACCCTGCCCATGGGCGGCGGCAAGGGCGGCTCCGACTTCGACCCCAAG 670
KFLAFEQI FKNSLTTLPHGGGKGGSDFDPK
â–  â– â– G***Tia*Atai((i(a(aaa*aaaia
. . .G A..G.S A
GGCAAGAGCGACGCGGAGGTGATGCGCTTCTGCCAGTCCTTCATGACCGAGCTGCAGCGCCACATCAGCTACGTGCAGGACGTGCCCGCC 760
GKSDAEVMRFCQSFHTELQRHISYVQDVPA
. . . EG...Q...AL....Y..LGADT....
I R|. . . C A . . A . . H < . . G A D T . . . .
GGCGACATCGGCGTGGGCGCGCGCGAGATTGGCTACCTTTTCGGCCAGTACAAGCGCATCACCAAGAACTACACCGGCGTGCTGACCGGC 850
GDIGVGAREIGYLFGQYKRITKMYTGVLTG
G..V.FHA.MM.KLSN.TAC.F..
G M..A.RKAANRFE
AAGGGCCAGGAGTATGGCGGCTCCGAGATCCGCCCCGAGGCCACCGGCTACGGCGCCGTGCTGTTTGTGGAGAACGTGCTGAAGGACAAG 9A0
KGQEYGGSE 1 RPEATGYGAVLFVENVLKDK
. .LSF...L L.Y.T.AM..RH
..LSU...L L.YY.GHM.EYS
GGCGAGAGCCTCAAGGGCAAGCGCTGCCTGGTGTCTGGCGCGGGCAACGTGGCCCAGTACTGCGCGGAGCTGCTGCTGGAGAAGGGCGCC 1030
GESLKGKRCLVSGAGNVAQYCAELLLEKGA
a MGFE.M.VS. a .S A I a K A M . F a a
. AGSYA...VAL..S A.LK.I.L..
ATCGTGCTGTCGCTGTCCGACTCCCAGGGCTACGTGTACGAGCCCAACGGCTTCACGCGCGAGCAGCTGCAGGCGGTGCAGGACATGAAG 1120
IVLSLSDS9GYVYEPNGFTREQLQAVQDHK
R. I T A a a a S a T a V D E S a a a K a K a A R L I E I
T . V K.ALVATGESGITVEDIHAV.A
AAGAAGAACAACAGCGCCCGCATCTCCGAGTACAAGAGCGACACCGCCGTGTATGTGGGCGACCGCCGCAAGCCTTGGGAGCTGGACTGC 1210
KKNNSAR I SEYKSDTAVYVGDRRKPUE LDC
ASRORDG.VAD.AKEFGLVY-LEGOQ..S.P-
I . E- - . .Q.LTSFQH.GHLKUIEGAR . L H V G
CAGGTGGACATCGCCTTCCCCTGCGCCACCCAGAACGAGATCGATGAGCACGACGCCGAGCTGCTGATCAAGCACGGCTGCCAGTACGTG 1300
QVDIAFPCATQNE1DEHDAELLIKHGCOYV
- L L . V D A . H Q . .AN . V K A
K....L VSKEE..G.LAA..KF.
GTGGAGGGCGCCAACATGCCCTCCACCAACGAGGCCATCCACAAGTACAACAAGGCCGGCATCATCTACTGCCCCGGCAAGGCGGCCAAC 1390
VEGANHPSTNEAI HKYMKAGI IYCPGKAAN
A T.I..TELFQO..VLFA
A..S..GC.L...E VFENNRKE . K . EAVW .A
GCCGGCGGCGTGGCGGTCAGCGGCCTGGAGATGACCCAGAACCGCATGAGCCTGAACTGGACTCGCGAGGAGGTTCGCGACAAGCTGGAG 1480
AGGVAVSGLEHTONRHSLRWTREEVRDKLE
T A..AAR.G.KA.K.DAR.H
C '..A..SQR....QA..DE..K
CGCATCATGAAGGACATCTACGACTCCGCCATGGGGCCGTCCCGCGAGTACAATGTTGACCTGGCTGCGGGCGCCAACATCGCGGGCTTC 1570
RIHKDIYDSAMGPSREYNVDLAAGAMIAGF
H . . L. .HHACVEHGG.GEQTNYVQ
D . . . NAFFNGLNTAKTYVEAAEGELP S . V . . S
ACCAAGGTGGCTGATGCCGTCAAGGCCCAGGGCGCTGTTTAAGCTGCCCAGGCCCAAGCCACGGCTCACCGGCAATCCAACCCAACCAAC 1660
TKVADAVKAQGAV*
V Ml v i *
v! ! !q!mhd! duuskn*
TCAACGGCCAGGACCTTTTCGGAAGCGGCGCCTTTTTCCCAGCCAGGGCCCTCACCTGCCCTTTCATAACCCTGCTATTGCCGCCGTGCC 1750
CCTGCAATTCCACCCCAAGAAGAACTAGCGGCACTTGACTGCATCAGGACGGCTATTTTTTTCGCGACGCGCGCTCACCCCGAGAGCCTC 1840
TCTCCCCCGAGCCCTAAGCGCTGACGTCCGCCCGACTTTGCCTCGCACATCGCTCGGTTTTGACCCCCTCCAGTCTACCCACCCTGTTGT 1930
GAAGCCTACCAGCTCAATTGCCTTTTAGTGTATGTGCGCCCCCTCCTGCCCCCGAATTTTCCTGCCATGAGACGTGCGGTTCCTAGCCTG 2020
GTGACCCCAAGTAGCAGTTAGTGTGCGTGCCTTGCCCTGCGCTGCCCGGGATGCGATACTGTGACCTGAGAGTGCTTGTGTAAACACGAC 2110
GAGTC (Poly A)70 2185

73
to the presence of the additional 14 amino acid residues in
the +42 bp cDNA (Fig. 6).
The sequence TGTAA located 17 nt upstream of the
polyadenylation site has been identified as a conserved
polyadenylation signal generally used in algal genomes (Fig.
6). The conserved TGTAA signal has been identified in the
same position in numerous Chlamydomonas, Chlorella, Volvox,
and Euglena cDNAs (Wolf et al., 1991).
The deduced amino acid sequences of the C. sorokiniana
NADP-GDHs are 50% and 50.3% identical with the NADP-specific
GDH of E. coli (McPherson and Wooton, 1983) and Neurospora
crassa (Kinnaird and Finchum, 1983), respectively (Fig. 6).
Comparison of the sequences of the highly conserved glutamate
binding domain (Mattaj et al., 1982) shows a strong identity
of 76.6% and 73.4%, respectively. Alignment of the C.
sorokiniana NADP-GDH polypeptide sequences with the bovine
mitochondrial NAD-dependent GDH (Julliard and Smith, 1979)
revealed a significantly lower 23% identity for the entire
protein and a 27.4% identity over the GDH conserved region.
Analysis of the codon preference of both NADP-GDH mRNAs
showed a strong bias for C and G at the first position, in
the case of arginine and leucine, and the third position of
the codon (Table 2, Table 3). Furthermore, an extreme
preference for G or C at the third codon position correlates
with the 63% GC content reported for the C. sorokiniana
genomic DNA (Cock et al., 1990).

74
Table 2. Codon usaqe of
TTT
phe
F
2
TCT
ser
s
1
TTC
phe
F
17
TCC
ser
s
16
TTA
leu
L
-
TCA
ser
s
-
TTG
leu
L
-
TCG
ser
s
3
CTT
leu
L
2
CCT
pro
p
3
CTC
leu
L
2
CCC
pro
p
14
CTA
leu
L
-
CCA
pro
p
-
CTG
leu
L
32
CCG
pro
p
3
ATT
ile
I
1
ACT
thr
T
2
ATC
ile
I
25
ACC
thr
T
18
ATA
ile
I
-
ACA
thr
T
-
ATG
met
M
15
ACG
thr
T
2
GTT
val
V
4
GCT
ala
A
3
GTC
val
V
8
GCC
ala
A
31
GTA
val
V
-
GCA
ala
A
—
GTG
val
V
31
GCG
ala
A
15
Table 3. Codon usage of
TTT
phe
F
2
TCT
ser
s
1
TTC
phe
F
17
TCC
ser
s
16
TTA
leu
L
-
TCA
ser
s
_
TTG
leu
L
-
TCG
ser
s
3
CTT
leu
L
2
CCT
pro
p
3
CTC
leu
L
3
CCC
pro
p
14
CTA
leu
L
-
CCA
pro
p
_
CTG
leu
L
33
CCG
pro
p
7
ATT
ile
I
1
ACT
thr
T
2
ATC
ile
I
25
ACC
thr
T
18
ATA
ile
I
-
ACA
thr
T
ATG
met
M
15
ACG
thr
T
2
GTT
val
V
4
GCT
ala
A
3
GTC
val
V
8
GCC
ala
A
32
GTA
val
V
-
GCA
ala
A
1
GTG
val
V
31
GCG
ala
A
17
the
-4
2
bD
NADP
-GDH
mRNA
TAT
tyr
Y
2
TGT
cys
C
TAC
tyr
Y
16
TGC
cys
C
8
TAA
OCH
Z
1
TGA
OPA
z
—
TAG
AMB
Z
-
TGG
trp
w
4
CAT
his
H
-
CGT
arg
R
_
CAC
his
H
6
CGC
arg
R
26
CAA
gln
Q
-
CGA
arg
R
-
CAG
gln
Q
28
CGG
arg
R
-
AAT
asn
N
1
AGT
ser
S
_
AAC
asn
N
19
AGC
ser
s
8
AAA
lys
K
-
AGA
arg
R
1
AAG
lys
K
37
AGG
arg
R
-
GAT
asp
D
3
GGT
gly
G
_
GAC
asp
D
23
GGC
giy
G
44
GAA
glu
E
-
GGA
gly
G
-
GAG
glu
E
35
GGG
gly
G
1
+42 bp NADP-GDH mRNA.
TAT
tyr
Y
2
TGT
cys
c
_
TAC
tyr
Y
16
TGC
cys
C
9
TAA
OCH
Z
1
TGA
OPA
z
_
TAG
AMB
Z
-
TGG
trp
w
5
CAT
his
H
_
CGT
arg
R
CAC
his
H
6
CGC
arg
R
28
CAA
gln
Q
-
CGA
arg
R
_
CAG
gln
Q
28
CGG
arg
R
-
AAT
asn
N
1
AGT
ser
S
AAC
asn
N
19
AGC
ser
s
8
AAA
lys
K
-
AGA
arg
R
1
AAG
lys
K
37
AGG
arg
R
-
GAT
asp
D
3
GGT
gly
G
__
GAC
asp
D
23
GGC
gly
G
44
GAA
glu
E
-
GGA
gly
G
_
GAG
glu
E
35
GGG
gly
G
1

75
Both of the deduced NADP-GDH polypeptides possess amino-
terminal extensions that are rich in alanine, serine, and
threonine and contain few acidic residues. These amino acid
sequence motifs are indicative of chloroplast targeting
domains (Smeekens et al., 1990). The boundaries of the
transit peptide were delineated by amino terminal sequence
analysis as discussed later.
The secondary structures of the deduced polypeptide
sequences of the +42 nt and -42 nt GDH cDNAs were predicted
by the method of Gamier et al. (1978; Fig. 7, Fig. 8).
Alignment of the predicted secondary structures to homologous
regions of the Clostridium symbiosum NAD-GDH 1.96Á resolution
crystal structure (Baker et al., 1992) indicates the
predicted structures are accurate representations.
Comparison of the chloroplast transit peptide regions of the
two predicted structures showed that the 14 amino acids
encoded by the additional 42 nt of the longer mRNA introduced
a random coil structure with multiple turns (Fig. 8) that
disrupts an «-helical domain observed in the -42 nt mRNA
transit peptide region (Fig. 7).
Determination of the Exon/Intron Boundaries
of the NADP-GDH Gene
A 9873 bp region of genomic DNA containing the NADP-GDH
gene previously sequenced by Cock et al. (1991) was compared
to the two consensus NADP-GDH cDNA sequences (Fig. 9). Both
the +42 bp and -42 bp NADP-GDH cDNAs span a 7178 bp region of

Figure 7. Secondary structure prediction of the C. sorokiniana -42 bp NADP-GDH mRNA
precursor polypeptide. Structural predictions were made according to Gamier et al. (1978)
and were verified by comparison to the secondary structures determined from the three-
dimensional crystal structure of the C. symbiosum NAD-GDH. Arrow denotes region of a-
helicies that is disrupted by the additional 14 amino acids in the +42 bp mRNA precursor
polypeptide (Fig. 8).

NH2
UO
t
HOOC
100
350
a-helix
p-sheet
Turn

Figure 8. Secondary structure prediction of the C. sorokiniana +42 bp NADP-GDH mRNA
precursor polypeptide. Structural predictions were made according to Gamier et al. (197 8)
and were verified by comparison to the secondary structures determined from the three-
dimensional crystal structure of the C. symbiosum NAD-GDH. Arrows denote borders of region
of secondary structure introduced by the additional 14 amino acid residues unique to the
+42 bp NADP-GDH mRNA.

NH2

Figure 9. Nucleotide sequence of the C. sorokiniana NADP-GDH
gene containing 22 exons (+42 bp mRNA) or 23 exons (-42 bp
mRNA). The position of the exons are identified by their
corresponding deduced amino acid sequences. The 5'-VR amino
acid sequences are indicated separately in the 5' region of
the gene. The highly conserved glutamate binding region
(Mattaj et al., 1982) is distributed over six exons
encompassing 2.09 kb as indicated by the two arrows. The 5'-
VR 42 bp auxon is denoted by arrowheads. Underlined regions
indicate the 5'-UTR and 3’-UTR, respectively.

81
GATCAGCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAACCGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCG 90
CATGCTTGTGCTGGTGAGGCTGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTGTGTGCTGGGCTTGCA 180
TGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCACTTGCTGGACGTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAG 270
GGATTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTTGCCAGAATGATCGGTTCAGCTGTTAATCAATG 360
CCAGCAAGAGAAGGGGTCAAGTGCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCAAGTGTGTGCGAGCCA 450
GCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTCATCCAACTAACCATAGCTGATCAACACTGCAATCATCGGCGGCTGATGCA 540
AGCATCCTGCAAGACACATGCTGTGCGATGCTGCGCTGCTGCCTGCTGCGCACGCCGTTGAGTTGGCAGCAGCTCAGCCATGCACTGGAT 630
CAGGCTGGGCTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCTGGCTGCTTGCTGGGTTGCATGGCATCG 720
CACCATCAGCAGGAGCGCATGCGAAGGGACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGTCACCAGGCC 810
CGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGACGTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGC 900
ACAGGCAGCAGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCGGGGTCGCGCCCCAGCCAGCGGTGATGC 990
GCTGATCnnnCCAAACGAGTTCACATTCATTTGCAGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGGAATGCG 1080
GGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATGTTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCT 1170
GTTTCTTTCGTGTTGCAAAACGCGCCACGTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAATCGTGTGCACGCCGATCAGCTGA 1260
TTGCCCGGCTCGCGAAGTAGGCGCC CTCTTTCTGCTCGCCCTCTCTCCGTCCCGCC ATG CAG ACC GCC CTC GTC GTGAGCAG 1342
+42 GDH M Q T A L V
-42 GDH M Q T A L V
CGCTTGGGTTGCCTTGCAGCGGTTGTTGCTGGATCGCGCCGCCGGCCGACCGGGGCTGGTTGCACGGCCCGCCGCGCCGCGCACACTGAC 1432
CGGCGGTCCTGTTTCTCCTCATTGCGACTGCAG GCC AAG CCT ATC GTG GCC.GCC CCG CTG GCG GCA CGC CCG CGC 1507
akpivaAaplaarpr
A K P I V A
TGC CTC GCG CCG TGG CCG.TGC GCG TGG GTAAGCGGCTCGGGTGGGGCCCGGGGATGGCACGCTGGGGTTAGGGTTGCGCGG 1588
clapwpAcaw
CAW
TGTGCGACGACAACGCCGCTCACGTCCAGCCTCAGCTGCTGGCGCCTCGCTGGCCCGCTGCCATTGCTCATGTGCAAGACAGGATGCTTG 1678
CTGGGTGATGGGCGGAGCACCAGGGCTGTTGGTGGTGGGCGGCGCGCACGCTGCCGCCGCCGCCAGCCGCCGCGCGCCTGCCTCTCGCAG 1768
TGGTGTGGCCCATCCTGCCTCCCTGCCAACAACCTCACCGCTCGCCCCGACCCGCAG GTC CGC TCC GCC AAG CGC GAT GTC 1849
VRSAKADV
VRSAKADV
CGC GCC AAG GCC GTC TCG GTGAGTGCTCTGCGTGCACCGCCAGCCCTGCAAGCACGCCCCCGCCGGCGCCAAACCTCCAACCGC 1933
R A K A V S
R A K A V S
CGCGGGGACCCCGCTGCCATGCATGCACCTGCCGGCACCTGCACCGCCTTCGTGCGCGCCGCTCCTGTGCAGCCCTCACCGTCACTGACC 2023
AATCCAAACACTTTTTCGCCACTGTTCTGCAG CTG GAG GAG CAG ATC TCC GCG ATG GAC GCC ACC ACC GGC GAC 2097
LEEQI SAMDATTGD
TTC ACG GTGCGCCGCCACAGCCGTACTTATGCGCCCTGTTGGACTCGGGCAGCCACTGTACCGCCCCTTCATAGCGCCCGCCGTCCTG 2185
F T
CCTGACATGGGCTCAACGCAAGCCATGCCATGCCTTCAAACAGCATGCATTCATCCCTGTCCTGACTCATCAAGATCGCCCTGTGTCTTG 2275
ACCCTGCGCCGCCCCGCAACCGCCATCCCGCTTGTTTCCCGACCTGCCCTCTCCCCCCGCCCGCCCTCGTCCTCATGTGCCGCAG GCG 2363
A
CTG CAG AAG GCG GTG AAG CAG ATG GCC ACC AAG GCG GGC ACT GAG GGC CTG GTG CAC GGC ATC AAG AA 2431
LQKAVKQMATKAGTEGLVHG I K N
C CCC GAC GTG CGC CAG GCAAGTCTTTAGCCTGATTGGAATGGAATGTAAGCCTGCCTTGTGCGCATTCCTTGGGCATCAACAAT 2515
P D V R Q
CCTGAGCTGCGCCAGGTGAGGAATAACACACCGTTTTTGAGCACTTCTATCGTCCCCACCTGCTGGCGTTGCGGCTCGACCGGGCTGCTT 2605
AGAGCAGCCCCGATGAGAAGAAAGCCCACGTGCGCAGAGTGCCAAACGCTGTCTCCTTCCCCCGCCCTGTCATCCACCACAGCTGCTGAC 2695
CGAGATCTTCATGAAGGACCCCGTTCAGCAGGAGTTCATGCAGGCTCATCTACATGCATGCGTAACAATAACCTGCCTCTTTCCTCTTCC 2785
CACCACGCAG CTG CTG ACC GAG ATC TTC ATG AAG GAC CCG GAG CAG CAG GAG TTC ATG CAG GCG GTG CGC 2855
LITEIFMKDPEQQEFMQAVR
GAG GTG GCC GTC TCC CTG CAG CCC GTG TTC GAG AAG CGC CCC GAG CTG CTG CCC ATC TTC AAG CAG GC 2923
EVAVSLOPVFEKRPELLPI F K Q
AAGCGCGCCTGAGGGGGGCAGGGGTGGTGCAGGGCGGGTCAGAGGGCTGGTTATAACTAACTAGGGTGCGGTGGACACGGGCGTGCAGAA 3013
GCCTGGCTCATCCACCAGTGACAGCAGCATGCTGGGTTGGCGAGCAGCAAGACACCCATTCACCGCTCGGCGACTGGCCTGACTAGCTGC 3103
AAGTCTGCTCTGTGTTATTCGCCATCCGCAG ATC GTT GAGiCCT GAG CGC GTG ATC ACC TTC CGC GTG TCC TGG 3176
IVEÍPERVITFRVSW
CTG GAC GAC GCC GGC AAC CTG CAG GTACAGCAGGCAGGCTGGCGCCTTGGCTGGCTAGTGTTCCCTTGCAGAGAGAAGCAGC 3258
LDDAGNLQ
ACACCACGCACGCACACTCGTCCCTGCCCGCCGCCATATGGCATGCATGCGGCATCCCGTGCGCCGACAATTCCACTGTTGTGCACTCAG 3348
TTCAGCTTCATTCTCATGGCCCATTCATTCACTTCACTGTTTGCAG GTC AAC CGC GGC TTC CGC GTG CAG TAC TCG TCC 3427
VNRGFRVQYSS
GCC ATC GGC CCC TAC AAG GGC GGC CTG CGC TTC CAC CCC TCC G GTGCGTGCCTGCACTGGCTGTGCCTGCGCTGG 3502
AIGPYKGGLRFHPS
CTGTGCCTGCGCTGGCCGTGCCTGCACCGGCTGTGCCTGGCTCAGCGGGTGGGGATGTGAGGCATGTCGGTGCACCAACCCGCCCGGCTT 3592
GCTCCGACGTCTACACCTGCAACACGGCTGCACAATGGACAGGGCAGGGCGGGGCAGGCACTTGCATCGGTGCCCGCCCCTCCAGCATGC 3682
ATGGGCGTGGCGAGCTGGGGGCGGGCCGGGCACCAACGGAGCAACTTGCAGTTCACCCTACTTTTCATGTGCCCCTGTCCAATGCCGCAG 3772
TG AAC CTG TCC ATC ATG AAG TTC CTT GTGAGTGCTGCCAAGCCTTGAAAGCGCTGTGCTAGCTGGTGAAATTGAGCAAGGA 3853
VNLSIMKFL
GCTGGGAAGAGTATAGCCGTGGGGGCAGGCCAGCCACTTTGCTGGCGCAAAGGTGGCCCTGCGATGCGCTGCGGCGACTGACACAGCGGC 3943
CCCTCCATCCCTTCACAACCATATGCAG GCC TTT GAG CAG ATC TTC AAG AAC AGC CTG ACC ACC CTG CCC ATG 4016
AFEQI FKNSLTTLPM
GGC GGC GGC AAG GGC GGC TCC GAC TTC GAC CCC AAG G GTGCGCCTTCCTTGAGTTAGTCGGCGGCAAGCTGCACATT 4093
GGGKGGSDFDPK
AAATGCCTCCGTCGGTCGTGTTTCAAGGCCCGCCCTGGCCCATCATTGGCTGACGGTCCACTGCCTGCCACCCTGTGTCGCCACCTACCT 4183
GCATACCACCCACCCAACACTCCCGCCCCTCCTGCAACCCCTCCCTCCCCACTACCGCAG GC AAG AGC GAC GCG GAG GTG 4263
G K S D A E V
ATG CGC TTC TGC CAG TCC TTC ATG ACC GAG CTG CAG CGC CAC ATC AGC TAC GTG CAG GAC GTG CCC 4329
MRFCQSFMTELQRHISYVQDVP
GCC GGC GAC ATC GGC GTG G GTGAGCGAGCGAGCGAGCAGCGAGCGGGCGTGTTTTTGAAAATTGCAGGGAGGGTAGTCGGGTG 4412
A G 0 I G V
GGGCAAAGGAAACGCACACACTTGCATGCGTAGCCAGCAAGCTTTCGTTCTCCTCATTCGCCGCTCCATTAGCTCACTGCCTTTGCCCAC 4502
CTCTTGTTTACCAACAACACGCAG GC GCG CGCiGAG ATT GGC TAC CTT TTC GGC CAG TAC AAG CGC ATC ACC 4573
garTeigylfgqykrit

82
AAG AAC TAC ACC GGC GTG CTG ACC GGC AAG GG GTGAGGCCCGCTTGCACTGACTGAGCTCGAGCCGGGAGCAACTGTAC 4652
KNYTGVLTGKG
TTTGCATTCCTGCCGGTCTGTTTCGGGGCGGCTGATCGGCAAGGGGTAAGGACCAGTGCCCACAGGAGCTCTAACGCTTGCCTGCCACGT 4742
TTGGGTGAACTGGTGTTCTCCAGCAGCCAGAGTTTTCCATGTCCACCCGCCTGCAAGCTCCTGGCTGTTCATCGCTGTGCTCTGTGTCTC 4832
CCTGCCAACACAATCCATACCAACACAATCCTGCGCCCTGCAG C CAG GAG TAT GGC GGC TCC GAG ATC CGC CCC GAG 4909
QEYGGSE I R P E
GCC ACC GGC TAC GGC GCC GTG CTG TTT GTG GAG AAC GTG CTG AAG GAC AAG GGC GAG AGC CTC AAG GT 4977
ATGYGAVIFVENVIKDKGESLK
GCAGCTATATGCCTTGGTTGTGCTGCCCTTGGCAGCAGTGAAGGCTGCGATGGTCTTTCACCTGAACTTTCAACGTACCAGCATGCGCAC 5067
ATGAGGTAGAGCACAGCCCAAACTGCTCAGAACGTCCGCCTGCCAAGTTCTTTCTTCCATCCACACCCCACACACCTGTGCAG GGC 5153
G
AAG CGC TGC CTG GTG TCT GGC GCG GGC AAC GTG GCC CAG TAC TGC GCG GAG CTG CTG CTG GAG AAG 5219
KRCLVSGAGNVAQYCAELLLEK
GGC GCC ATC GTG CTG TCG CTG TCC GAC TCC CAG GGC TAC GTG TAC GAG GTGCGGTTGATACATCTGGGCCATTT 5293
GA I VLSLSOSQGYVYE
CGGCTGTTGATTGTGCTCTGTGTTGTCTGTAGTGTCTGACTCCCTGGGCTCGTGCACGAGGTGCGGAAGGCTCAGGCAGCAGTTCGGAGC 5383
TCTGCCTGTCTGCTGCTCCTAGAGCTACCTAATGAAGCATAGCTCTGCTGTGCTGCCCCCTCGCGCCTGCTCACCCGTCAACCACCACCG 5473
CCCCTCCCCACCCCCTTTTCATTTTTCCCGCAG CCC AAC GGC TTC ACG CGC GAG CAG CTG CAG GCG GTG CAG GAC 5548
PNGFTREQIQAVQO
ATG AAG AAG AAG AAC AAC AGC GCC CGC ATC TCC GAG TAC AAG GACAGTGATGACCGGTCCAGGAAACAAGTTGCAC 5624
MKKKNNSARI SEY<
ATGTCGTCTAGAAGGTCCCTGCCGCCGACACAGCAGCCGCGCCTGGGCTGCCGCTGCTTCGATAGCACCACCCACCCCTGCCGCCCCATC 5714
TCCTGCCTGCACTGCACCTTCCCATTTTGCCCACTAGCCACTGCTCACTCGAGTTCTCAACTGTCACTTGCAATTTTCTCTCTGCTTGCA 5804
G AGC GAC ACC GCC GTG TAT GTG GGC GAC CGC CGC AAG CCT TGG GAG CTG GAC TGC CAG GTG GAC ATC 5871
SDTAVYVGDRRKPWELDCQVDI
GCC TTC CCC TGC GCC ACC CAG GTGCGCAGCCAGACTGGCTTGCATGCAACGCATCAAATGTCTCAAGGTTTGCCTGCAAGTGC 5954
A F P C A T Q
TCTAAGCCCTGTCAGAACTTTTTACAAGCAGCATGGCAGTGGAGGTGGTGAGGGCGACGTCCTGCACCGTTTCCTCAATGCCGCCGTGCC 6044
CCGGCTCTCTTGCCCTGTATGCAG AAC GAG ATC GAT GAG CAC GAC GCC GAG CTG CTG ATC AAG CAC GGC TGC 6116
NEIDEHDAELLIKHGC
CAG TAC GTG GTG GAG GGC GCC AAC ATG CCC TCC ACC AAC GAG GCC ATC CAC AAG TAC AAC AAG GTGGCG 6185
QYVVEGANMPSTNEAIHKYNK
CTGCCTATACGAAGAATGTATTCCACTTGATGTTCAATACAGGGCGGGTGTTCAGAAACTAGGCGTGCCGCGAGGCCGTCCACAAGTACA 6275
ACAAGTGGGCGGTGGCTGCGAAGTTAGTTCTTAATCAAGGGCTGGTATGCTGTGCTGCACCAACGAGGCCATCCACAAGTACAACAAGGT 6365
GGGCCTGTTTTGAGCTTGCTGACAAGCTAGCCTCCCGACAGCTCTCCGGGTTGCGAGTTCCCAGCTGCTGCCTTCCGCAGTCTTTGGGAC 6455
CACGTGCGCCACCCACCCACCCATGTTTCTCCCGCACACATACTGCTCAGTACACACTTGCAGCTCCATGCAACCCAGCCTCTTTGCTGC 6545
CCCACCCTTCCCTCTCCCTGCCTCCGCGTCGCGCAG GCC GGC ATC ATC TAC TGC CCC GGC AAG GCG GCC AAC GCC 6620
AGI IYCPG GGC GGC GTG GCG GTC AGC GGC CTG GAG ATG ACC CAG AAC CGC ATG GTGAGCGTGGCATGATTTCCCTGCTTGTCA 6695
GGVAVSGLEMTONRM
GGGCTTGCAGTATAAGCTGAAGAAACGAAGTGGTCTGCAGTCAGCAGCCTGCAGATGACCCAGAGCCGCATGGTGAGGAGGGCAGGGGCT 6785
GTTAACTGGGAGCAGCCTCAGCGACGCCCAGTGCTGGTGCTTTGTTCCTCGTGCACCTCAGCTGCTGCAACTTTGTGAGCGCATCGCCCT 6875
GAACCGCCACAACTGCCTGCGCCTGCCCTGCCGCAG AGC CTG AAC TGG ACT CGC GAG GAG GTT CGC GAC AAG CTG 6950
SLNUTREEVRDKL
GAG CGC ATC ATG AAG GTGAGGGCTGATTGTGCGGCTATCACAGTGCAACCACGCAAGCTGGAGCGCATCATGAAGGTGAGGGCTG 7035
E R I M <
ATTGTGCGGCTATCACAGTGCAACCACGCTCGTCATGGGCCTTGCGCGCCTCGCCCGTCGCGACTCGGCTGAAGTCGCTGCGGAAGCCGC 7125
CTTCGAGGAGGAAAGCCTGCGCCTTCGTCACGGCTCGCACTGCTTCCTTTCCCTCCACAGGACATCTACGACTCGCCATGGGCGCCTTTT 7215
GCAGGACAACCCATTCCGTTCACAACACTCAGCAACCCTGCCCTCATTCTTCTTCATCCCCGCAG GAC ATC TAC GAC TCC GCC 7298
D I Y D S A
ATG GGG CCG TCC CGC GAG TAC AAT GTT GAC CTG GCT G GTGAGTGCCTGGCTGTGCAGACAGACACGACACTTGTAAA 7375
MGPSREYNVDLA
CTCAGTTTTTTCATTCTAGCCTGCCGCCGTTTCTGCCGGCCAGGATTGGCTTTGATGATCGCTCTGCCCTGAGTAGCTAGTAGCCAGTTG 7465
CCCGGCAGCTATTGCCCCCCTGCCTGCTGTAGCTGTCTGCTGCCTGCGGTGCTGGTGTGCATGGAGCACCCACCGCAAAGCTCAAACGCC 7555
TGCGGTTGGTGGGCGCATGCTGTGCTTGCGGTGCTGCCCATCCGCCCTTGCGTTGCCACCCTGCTCACCCTGCTCACCCTGCCCCGCCTG 7645
CCCCCTCCCCCCGTCCTCCCAATTCTACAG CG GGC GCC AAC ATC GCG G GTGAGTTGGATTGGGGGGAGTTGTGCACACTGCT 7727
A G A N I A
GAAACGTGCAACGAGCACTGCTGCCTGTGCACTGCTGGCGCTGTTTTGGCACGATATGCTGCATTGCTGGTTGCCCGTCCTCAACTGTTG 7817
CAAGAGAGTGGCAGCTTGAACCGCCAATGCAGCGAAATGGTCGCGCACCCGCCTATTTGTGGCTTACGTTGCATTCCTCTCTCCGCTGCC 7907
TGCAG GC TTC ACC AAG GTG GCT GAT GCC GTC AAG GCC CAG GGC GCT GTT TAA GCTGCCCAGGCCCAAGCCACG 7980
GFTKVADAVKAQGAV*
GCTCACCGGCAATCCAACCCAACCAACTCAACGGCCAGGACCTTTTCGGAAGCGGCGCCTTTTTCCCAGCCAGGGCCCTCACCTGCCCTT 8070
TCATAACCCTGCTATTGCCGCCGTGCCCCTGCAATTCCACCCCAAGAAGAACTAGCGGCACTTGACTGCATCAGGACGGCTATTTTTTTC 8160
GCGACGCGCGCTCACCCCGAGAGCCTCTCTCCCCCGAGCCCTAAGCGCTGACGTCCGCCCGACTTTGCCTCGCACATCGCTCGGTTTTGA 8250
CCCCCTCCAGTCTACCCACCCTGTTGTGAAGCCTACCAGCTCAATTGCCTTTTAGTGTATGTGCGCCCCCTCCTGCCCCCGAATTTTCCT 8340
GCCATGAGACGTGCGGTTCCTAGCCTGGTGACCCCAAGTAGCAGTTAGTGTGCGTGCCTTGCCCTGCGCTGCCCGGGATGCGATACTGTG 8430
ACCTGAGAGTGCTTGTGTAAACACGACGAGTC GATCACCCGGTGCTTGGTGCACAAGCAGGGCATTGGAGCAGGGCAGCGGATCTGGAC 8519
TCCAGACTGGAGACGGCGGCCGCCGCCAGGTCAGCAGCCGGAAAACGCACCCGGAAAACTAGATCCCGAGCGCCTGGGCCGCTGCGCGCC 8609
GCATTTACAGTTCCAGACCCAGTCAGATCACCCAGGGCATCCACCAGCCACTGCAAAGCGGTTGCACAGCGGCTCGGCTCGATGGCGCCG 8699
CAATGGCAGGCCCGCGCTACGAGCCCGCTGCCTGATCCTAGCTGCTGCCGTGGCTGTTTGCCGTGTGCCGCTGAAGGTGCCGACCGCACG 8789
CCCGGGCGAGTGCTGGGACACGTGACGCGCGAGCTCAAGGCCTCCGAGCTGCCGGGCAAGGTAAGCGGAGCGTGTAAAAGATGGCTGGTA 8879
CTGCTGTTGACCCGATCCGCCCTGCTCGCGCGGGCCAGCACCACCCCTGCGTGCCGCCAACCTCACCCGCGCCGTGCCGCTCTGCCCCTC 8969
CGATTTCTGCTGCAGGATATGGCCTGATCTTCTATGGAGACAGCATCACGGAGAGCCTGCGTGGCACAGACAAGTGCCGCGACGTCTGCC 9059
TGAAGAGTAAGACGCGGTCCTCCTGCAAAGGCATTCCTGAGGTGCGCGAGCACGAGGGCCGCCACTCCTGCCGAATGGTTGCTACACATT 9149
GCATCGGCAGGGGTGGATTGGTTCATGGGCAGCCACTTCTTTCAGCTTCATAACTTTGCAACCAGTTTCCATGATCGCCGTCGTGCCGCC 9239
GCCGCATCCGCCGCTCTCCTGCCCGCTTGCCGATACGCCTTTCTGGGCCCCCGCTTACCGCACTGTGACCGAAGGTCCTGCAAAAGTACT 9329
TTGGCGCCTACCGCCCGGGTGTGATGGGCATGTCCATGGATGAGTCGGCCCACCTGCTGTGGCGCCTGCAGAACGGCCAGATCCCCCGCG 9419
TCAACCAGGTGAGCGCGCACAGGCAGTGCAGCGCAGGCAGCACAGCGCACGACATAGTGCAGCAGCGGCAGACTGGACGGGCCAACTGTC 9509
TGCCTGCGGTCTGCACTGGTGGGGCCAACTGCGTCTTCTGCGCCATGCCTTCAGCCAGGAATAGCACATGCTCCTTCGCCCTGCCTGCAG 9599
CCCAAGACAGTTGTGCTGAACATCGGCACCAACGACCTCACCAACTGCCGTGGAGCGCGAAGAACGCCCAGAAGAAGCAGGCGGCCATCA 9689
ACAAAGAAATCCCGGGGATCGTGGGCCGGTGAGCTGGGCGGTGGGGCATTCGCATGAACATGCATATCCTGGCTGCCACAGCGTGCCGCA 9779
TGCTATGTTGGGTGGGTCCACGGCAGTTGGCCGCTCGGTGCCGCTGGTGCATGCTGTGTGGGAGGGGAAGCTGCCTCGGTGTGACCTGAA 9869
GGACTT 9873
Figure 9 continued

83
the C. sorokiniana genome and are divided into 22 exons and
23 exons, respectively (Fig. 10). The exons in this gene
exhibit a range from 9 bp (exon 3 of the -42 nt mRNA) to a
large exon of 550 bp at the 3' end of the gene. The 9, 16,
and 18 bp exons are smaller than the smallest exons
identified in higher-plant genes (Tischer et al., 1986).
The introns identified in the NADP-GDH gene range
between 42 and 402 bp with a mean length of 233 bp. The mean
intron length is similar to the mean intron length of 249 bp
calculated in a survey of higher-plant introns (Hawkins,
C A TC
1988). The consensus sequence AG/GTG GG , was derived as
A C CG
the 5' intron donor site using the criteria suggested by
Cavener (1987). The 5' donor site closely matches the
general consensus, CAG/GTAAGT observed in higher-plants and
animals (Hawkins, 1988) with the exception of G at position
+3 of the C. sorokiniana intron. Three of the 21 conserved
5' intron splice junctions do not conform to the GT-AG rule
of Breathnach and Chambón (1981) due to a substitution of C
TTTTTTT *1* T
at position +2. A consensus sequence, C CcCtt C GCAG/,
CCCCCCC C C
was derived for the 3' intron splice junction of the NADP-GDH
gene. The strong preference for G at position -4 observed in
19 of 21 conserved intron splice junctions is typical of
plant introns (Brown, 1986). The 5' (GCC/GCC) and 3'
(CCG/TGC) splice site junctions of the 42 bp auxiliary exon
(termed auxon hereafter as per Werneke et al. [1989]) showed
no conservation in splice site branch points.

Figure 10. Restriction maps and exon domains of four NADP-GDH genomic clones spanning 21.9
kbp of the C.sorokiniana genome. The exons (black boxes) are interupted by introns with
consensus exon/intron junction splice sites with the exception of the alternatively-spliced
region denoted in exon two (arrow). Heavy black lines denote regions in genomic clones
that have been sequenced on both strands. Restriction maps were generated using AccI (Ac),
Bamñl (B), BglII (Bg), EcoRI (R), HindiII (H), Kpnl (K), Smal (Sm), Xbal (Xb), and Xhol
(Xh). Primary genomic cloning and NADP-GDH gene sequencing performed by Drs. Mark Cock and
Kyu Don Kim with revisions provided by the author (Cock et al., 1991).

O 1
I L
2 3 4
J I L
5 6
J L
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
â– J 1 1 1 1 1 1 1 1 1 1 I I I I i
H K
J L
lílll 111 IIINIIU II I I ■
H H Sm Bg Bg Bg Ac Bg H Xh Xb Xh
J 1 I. I Y I I v v
H H
I I
Sm Bg Bg Bg Ac Bg H Xh
.. í.-ü.-V-
■í—L- m
Bg Bg Ac Bg H Xh xb Xh
> I i V 2lfl_
H H
J L
Sm Bg
1 1 .
Ac
Ac Sm Sm Sm Sm H B
Ll 1 l Li L
Ac Sm Sm Sm Sm H B
u r i ir i
NADP-GOH
EXON DOMAINS
pGDg 14 10 I
pGOg 0 4 4
R
J , pGDg 14 4 |
pGDg 15 2 1
00
Ln

86
Analysis of the 1285 bp sequence upstream from the
transcriptional start site revealed no consensus cis-
regulatory elements common with those published for other
eukaryotic organisms (Fig. 11).
Determination of the Number of NADP-GDH Genes in
the C. sorokiniana Genome
Southern blot analysis of the NADP-GDH gene
Using cDNA probes corresponding to the HCR and 3'-UTR,
Cock et al. (1991) provided evidence that indicated the C.
sorokiniana genome possessed a single NADP-GDH gene.
However, the possiblity remained that two recently diverged
GDH genes differing only in their 5' ends might exist in the
C. sorokiniana genome. C. sorokiniana genomic DNA was
digested with PvuII or TaqI and the resulting restriction
fragments were subjected to Southern blot analysis using cDNA
probes that hybridized to the 5'-VR (exons 1 to 3), HCR
(exons 10 and 11), and the 3'-UTR (exon 22) of the NADP-GDH
gene. The respective restriction enzyme and predicted
hybridizing fragment sizes were as follows: 5'-VR PvuII 634,
429, 367 bp Tagl 1465 bp; HCR PvuII 2646 bp TagI 590 bp, 1159
bp; 3'-UTR PvuII 4300 bp Tagl 1334 bp.
The 5'-VR and 3’-UTR probes yielded a hybridization
pattern that was predicted from the sequenced NADP-GDH gene
(Fig. 12). The HCR probe gave the predicted hybridization
pattern; however, an additional weakly hybridizing fragment

Figure 11. Transcriptional initiation site for the C.
sorokiniana NADP-GDH nuclear gene and the upstream region in
the genomic DNA that contains the putative NADP-GDH promoter.
Also shown is the 5'-UTR and the first seven amino acid
codons identified in the two NADP-GDH mRNAs. The entire
region of genomic DNA was derived from genomic clone 14.10.1
(Fig. 10). The identification of the transcription
initiation site, 5'-UTR, start codon, and other amino acid
codons was acheived by computer alignment with the 5’ RACE-
PCR generated cDNA fragments with the NADP-GDH gene sequence.
In the 1285 sequence upstream of the transcriptional start
site, no strong consensus cis-regulatory elements common with
those published for other eukaryotic organisms were
identified (Miller et al., 1994b).

-1250
-1200
GATCAGCCGCCTGCAACGCAAGGGCAGCCACAGCCGCTCCCACCCGCCGCTGAACCGACACGTGCTTGGGCGCCTGCCGCCTGCCTGCCG
-1150
• ••••••••
CATGCTTGTGCTGGTGAGGCTGGGCAGTGCTGCCATGCTGATTGAGGCTTGGTTCATCGGGTGGAAGCTTATGTGTGTGCTGGGCTTGCA
-1100 -1050
• ••••••••
TGCCGGGCAATGCGCATGGTGGCAAGAGGGCGGCAGCACTTGCTGGACGTGCCGCGGTGCCTCCAGGTGGTTCAATCGCGGCAGCCAGAG
-1000 -950
• ••••••••
GGATTTCAGATGATCGCGCGTACAGGTTGAGCAGCAGTGTCAGCAAAGGTAGCAGTTTGCCAGAATGATCGGTTCAGCTGTTAATCAATG
-900 -850
• ••••••••
CCAGCAAGAGAAGGGGTCAAGTGCAAACACGGGCATGCCACAGCACGGGCACCGGGGAGTGGAATGGCACCACCAAGTGTGTGCGAGCCA
-800 -750
• ••••••••
GCATCGCCGCCTGGCTGTTTCAGCTACAACGGCAGGAGTCATCCAACTAACCATAGCTGATCAACACTGCAATCATCGGCGGCTGATGCA
-700
• ••••••••
AGCATCCTGCAAGACACATGCTGTGCGATGCTGCGCTGCTGCCTGCTGCGCACGCCGTTGAGTTGGCAGCAGCTCAGCCATGCACTGGAT
-650 -600
• ••••••••
CAGGCTGGGCTGCCACTGCAATGTGGTGGATAGGATGCAAGTGGAGCGAATACCAAACCCTCTGGCTGCTTGCTGGGTTGCATGGCATCG
-550 -500
• ••••••••
CACCATCAGCAGGAGCGCATGCGAAGGGACTGGCCCCATGCACGCCATGCCAAACCGGAGCGCACCGAGTGTCCACACTGTCACCAGGCC
-450 -400
• ••••••••
CGCAAGCTTTGCAGAACCATGCTCATGGACGCATGTAGCGCTGACGTCCCTTGACGGCGCTCCTCTCGGGTGTGGGAAACGCAATGCAGC
-350
-300
ACAGGCAGCAGAGGCGGCGGCAGCAGAGCGGCGGCAGCAGCGGCGGGGGCCACCCTTCTTGCGGGGTCGCGCCCCAGCCAGCGGTGATGC
-290 -280 -270 -260 -250 -240 -230 -220 -210
• ••••••••
GCTGATCnnnCCAAACGAGTTCACATTCATTTGCAGCCTGGAGAAGCGAGGCTGGGGCCTTTGGGCTGGTGCAGCCCGCAATGGAATGCG
-200 -190 -180 -170 -160 -150 -140 -130 -120
• ••••••••
GGACCGCCAGGCTAGCAGCAAAGGCGCCTCCCCTACTCCGCATCGATGTTCCATAGTGCATTGGACTGCATTTGGGTGGGGCGGCCGGCT
GTTTCTTTCGTGTTGCAAAACGCGCCACGTCAGCAACCTGTCCCGTGGGTCCCCCGTGCCGATGAAATCGTGTGCACGCCGATCAGCTGA
-20 -10 *1 *10 *20 *30 *40 -»50
• • •••• • •
TTGCCCGGCTCGCGAAGTAGGCGCC CTCCTTTCTGCTCGCCCTCTCTCCGTCCCGCC ATG CAG ACC GCC CTC GTC GCC
5'-Untranslated Region M Q T A L V A
00
00

Figure 12. Southern blot analysis of undigested genomic DNA
(U) and restriction fragments obtained by digestion of C.
sorokiniana genomic DNA with PvxzII (P), and TagI (T). The
blots were hybridized under stringent conditions to cDNA
probes derived from the 5'-VR, HCR, and the 3'-UTR of the
NADP-GDH cDNAs. The stringency of the washes was O.lx SSC,
0.1% SDS at 65°C.

90
5-VR HCR 3-UTR
PTUPTUPTU
kbp
23.13- V W
9.42-
6.56-
4.36- W
2.32-
2.03-
1.35-
1.08-
.872-
.603-
.310-

91
was detected in both the PvuII and TagI digest that could not
be explained by incomplete restriction digestion (Fig. 12).
Since the 155 bp NADP-GDH HCR probe is derived from the
highest region of conservation in the glutamate binding
domain, the faintly hybridizing band most likely corresponds
to the C. sorokiniana mitochondrial NAD-GDH gene (Meredith
and Schmidt, 1991). These results provide evidence for the
existence of a single NADP-GDH gene in the C. sorokiniana
genome.
Allele-specific PCR analysis of the NADP-GDH gene
Since the Southern blot analysis is limited by the
resolution capability of agarose gel electrophoresis, the
remote possibility remained that two NADP-GDH genes exist
that only differ in their 5' ends by the 42 bp detected in
the NADP-GDH +42 nt mRNA. In order to test this possibility,
C. sorokiniana genomic DNA was analyzed by allele-specific
PCR (Saiki et al., 1986). Genomic DNA and three NADP-GDH
genomic clones spanning various regions of the NADP-GDH gene
(pGDg 8.4.4, 14.10.1, 15.2.2; Fig. 10) were amplified using a
series of exon specific primers. The primers were designed
to anneal to the 5'-VR (exons 1 and 3), HCR (exons 10 and
11), and 3'-UTR (exon 22) of the NADP-GDH gene. The
resulting PCR products were resolved by high-resolution PAGE
and analyzed for DNA polymorphisms (Fig. 13).
Allele-specific PCR of C. sorokiniana genomic DNA
yielded a single PCR fragment corresponding to the 5'-VR (564

Figure 13. Polyacrylamide gel electrophoresis of the PCR
products amplified from C. sorokiniana genomic DNA and three
NADP-GDH genomic clones, lacking or possessing the regions of
interest, using exon-specific primer pairs. Primer pairs
hybridized to exons 1 and 3 (5'-VR), exons 10 and 11 (HCR),
and the last 3' exon of the NADP-GDH gene (3'-UTR). Lanes:
1, pGDg 15.2.1; 2, pGDg 8.4.4; 3, pGDg 14.10.1; 4, C.
sorokiniana genomic DNA; 5, pUC 18 control template. A
single PCR fragment of the predicted size was amplified from
the genomic DNA template with each set of exon specific
primers. PCR products for a given template were combined
before separation by PAGE. Arrow denotes (lower figure)
location of the 42 nt auxon.

93
fHI III III! Ill M II I I
246
â—„"3'-UTR
â—„-HCR

94
bp), HCR (344 bp), and 3'-UTR (387 bp). The products
generated from the genomic clones template DNA yielded
identical products to the total genomic DNA (Fig. 13) and
were identical in size to the sizes predicted from the NADP-
GDH gene sequence. These results provide strong evidence
that the two NADP-GDH mRNAs are derived from a single NADP-
GDH gene rather than two closely related genes.
Purification of the NADP-GDH Isoenzymes
The NADP-GDH a-holoenzyme was purified from a 130 g
pellet of C. sorokiniana cells induced continuously in 29 mM
ammonium medium according to Yeung et al. (1981). The final
step of purification was modified from that of Yeung et al.
(1981) by substituting a nondenaturing preparative PAGE step
(Miller et al., 1994) for a NADP-hexane-agarose affinity
chromatography step. This procedural alteration was required
due to the lack of availability of the affinty agarose.
Approximately 11.5 mg of pure NADP-GDH a-holoenzyme with a
specific activity of 254 units/mg protein was obtained from
the 130 g of cells with only a 17% loss of the initial
activity (Table 4). The nondenaturing PAGE step effectively
purified the the enzyme an additional 8.5-fold over the
Sephadex G-200 step with less than a 2% loss of activity. A
single protein band of 53.5 kD corresponding to the NADP-GDH
«-subunit was detected by silver-staining after resolution by
Tris-Tricine SDS-PAGE (Fig. 14).

95
Table 4. Steps for the purification of the NADP-GDH u-
holoenzyme
Total
Sp.
%
Fold
Step
Units3
Activity*3
Recovery
Purification0
Supernatant
frozen/thawed
cell homogenate
Ammonium sulfate
3546
0.678
100
0
fractionation
(35%- 70% )
3526
1.22
99
1.8
DEAE-Sephacel
pH 7.4
3411
5.16
96
7.6
DEAE-Sephacel
pH 6.0
3060
23
86
33.9
Sephadex G-200
Model 491
3011
30
85
44.2
preparative
native PAGE
2938
254
83
375
aOne Unit is defined as the amount of enzyme necessary to
reduce lumol of NADP+/min at 38.5°C
bSpecific activity is defined as units/mg protein
cFold purification is defined as the specific activity of
step/specific activity of supernatant cell homogenate

Figure 14. Analytical SDS-PAGE of the NADP-GDH cx-holoenzyme
purified by preparative nondenaturing gel electrophoresis.
Proteins were detected by rapid silver-staining. Lanes: 1,
Molecular mass standards; 2, Sephadex G-200 NADP-GDH
fraction; 3, Nondenaturing preparative gel purified NADP-GDH
a-subunit (53.5 kD).

97
12 3
kD

98
A mixture of NADP-GDH isoenzymes containing both the a- and
P-subunit were functionally purified from a 24 g pellet of a
mixture of C. sorokiniana cells cultured under various time
regimes and ammonium concentrations. The NADP-GDH isoenzymes
were purified using a modified procedure of Yeung et al.
(1981). In this procedure, all column volumes were decreased
ten-fold and the Sephadex G-200 column was omitted (Table 5).
The isoenzyme preparation was purified 116-fold to a
specific activity of 45 units/mg protein with a loss of 37.5%
of the initial activity. The greater loss of activity
observed in the isoenzyme mixture during the purification
procedure may be in part due to the presence of a more labile
heterohexameric and/or p-homohexameric form. Silver-stained
Tris-Tricine SDS gels of the partially purified enzyme
preparation revealed that the preparation still contained
several contaminating proteins. However, the NADP-GDH a- and
P-subunits were sufficiently resolved from the contaminants
to allow further protein analyses.
Determination of the Stability of the NADP-GDH cx-Holoenzyme
in the Presence of NADP+
Purified NADP-GDH was reported to undergo polymerization
and inactivation during storage at -20°C (Yeung et al.,
1981). Since many studies require the longterm use of
catalytically active NADP-GDH, a means of storage without
enzyme activity loss was developed. Several researchers in
this laboratory observed a stabilization of NADP-GDH activity

99
Table 5. Steps for the partial purification of NADP-GDH
isoenzymes
Total
Sp.
%
Fold
Step
Units3
Activity*3
Recovery
Purification0
Supernatant
frozen/thawed
cell homogenate
Ammonium sulfate
320
0.387
100
0
fractionation
(35%- 70% )
310
0.812
97
2.1
DEAE-Sephacel
pH 7.4
272
2.67
88
6.9
DEAE-Sephacel
pH 6.0
183
71
9.28
79
24
NADP+ Type 3
Affinity Resin
132
68
45
62.5
116
aOne Unit is defined as the amount of enzyme necessary to
reduce l^mol of NADP+/min at 38.5°C
^Specific activity is defined as units/mg protein
cFold purification is defined as the specific activity of
step/specific activity of supernatant cell homogenate

100
in the presence of one of the cofactors, NADP+. To test the
ability of NADP+ to stabilize the GDH enzyme activity during
storage at 4°C, 205 [xg (52 units) of purified a-holoenzyme in
10 mL of 10 mM KP04 (pH 6.2), 2 mM DTT, and 0.1 mM NADP+ was
placed at 4°C and periodically tested for NADP-GDH
deaminating activity. The purified NADP-GDH retained 90% of
the original activity after 10 weeks of storage and 50% of
the activity was lost after 20 weeks storage (Fig. 15).
Thus, the NADP-GDH a-holoenzyme can be stored in the presence
of NADP+ at 4°C for extended periods of time without a rapid
loss of enzyme activity.
Production of Anti-NADP-GDH Polyclonal and Monoclonal
Antibodies
Polyclonal rabbit anti-NADP-GDH antibodies were produced
against the a-holoenzyme in six rabbits. Preimmune sera and
production bleed anti-sera were titered by their ability to
immunoprecipitate one unit of NADP-GDH at 22°C. All six
anti-sera possessed the ability to immunoprecipitate the
NADP-GDH activity to varying degrees in a dilution range from
1 x 10-4 to 2%. The preimmune sera lacked the ability to
precipitate the GDH activity and was shown to stabilize the
GDH activity at 22°C when compared to a control that lacked
serum. The anti-serum possessing the highest titer was
selected for use in western blot analyses.
Monoclonal antibodies were produced from splenocytes of
female Balb/C mice immunized with the NADP-GDH a-subunit

Figure 15. Stability of purified NADP-GDH a-holoenzyme at
4°C in the presence of 0.1 mM NADP+. A sample of purified a-
holoenzyme was stored at 4°C in 10 mM KP04, 2 mM DTT, and 0.1
mM NADP+ and periodically analyzed to determine the amount of
NADP-GDH deaminating activity that remained. NADP-GDH
deaminating activity was measured at 38.5°C by a
spectrophotometric analysis.

% Activity Remaining
fV) CD 00 O
o o o o o o
102

103
(Tamplin et al., 1991). Splenocyte-myeloma fusions produced
more than 1000 hybridomas. A total of 12 hybridomas
initially tested ELISA positive for producing anti-NADP-GDH
antibodies; however, only nine of the hybridoma lines were
stable. Results of western blot tests of hybridoma
supernatants showed that all nine hybridomas were producing
antibodies capable of detecting the denatured cx-subunit
immobilized on membranes. Hybridoma supernatants were
collected and stored at -20°C.
Analysis of the «- and P-subunit similarity with Mouse Anti-
NADP-GDH MAbs
Yeung et al. (1981) demonstrated that rabbit polyclonal
antibodies derived against the NADP-GDH p-holoenzyme were
able to immunoprecipitate both the a- and p-subunits of the
NADP-GDH. Futhermore, Bascomb and Schmidt (1987) revealed by
peptide mapping that the two subunits had 36 of 40 peptides
in common. To test further the hypothesis that the a- and p-
subunits are nearly identical, a protein preparation
possessing both subunit types was analyzed for cross¬
reactivity with MAbs derived against the a-subunit. Proteins
in a partially purified mixture of NADP-GDH isoenzymes were
resolved by Tris-Tricine SDS PAGE and transferred to a nylon
membrane. The blot was cut into strips and each strip was
incubated individually with one of five mouse anti-NADP-GDH
MAbs. All five (MAb: GDH 1, 3, 4,8, 9) of the anti-NADP-GDH
cx-subunit MAbs detected both the a- and p- subunit on western

104
blots (Fig. 16; MAb GDH 4,8 not shown). These results
provide further support for the high degree of similarity
between the two subunit types.
Determination of the Molecular Mass of the NADP-GDH Subunits
Prunkard et al. (1986) determined the molecular mass of
the NADP-GDH subunits, based on their mobilities in Tris-
glycine SDS-PAGE, to be 55.5 kD and 53 kD for the a-subunit
and (3-subunit, respectively. In this study, it was
determined that the earlier molecular mass estimations were
in error. To determine the molecular masses of the NADP-GDH
subunits, an aliquot of a partially purified protein
preparation was resolved by Tris-Tricine SDS-PAGE and the
position of each subunit was determined by immunodetection
with mouse anti-NADP-GDH antibodies. The mobilities of the
subunits were compared to the moblilities of molecular mass
standards ranging from 69 to 30 kD. The relative mobilities
of the standards were plotted against the molecular mass and
the slope of the line was statistically computed (Fig. 17).
The a-subunit and p-subunit sizes were calculated from the
slope to be 53.48 kD and 52.27 kD, respectively. These
values are in close agreement with the molecular mass
predicted for the subunits from the deduced amino acid
sequences (53.5 and 52.3 kD) of the mature subunits discussed
later.

Figure 16. Mouse anti-NADP-GDH monoclonal antibody
immunoblot analysis of the NADP-GDH a- and (3-subunit
similarity. MAbs developed against purified NADP-GDH a-
subunit were tested for their ability to immunoreact with
both the a- and (3-subunits. Lanes: 1, MAb GDH 1; 2, MAb GDH
3; 3, MAb GDH 9. MAb:NADP-GDH subunit complexes were
detected with alkaline-phosphate conjugated goat anti-mouse
antibodies. All MAbs tested immunoreacted with both subunit
types.

106
43 —

Figure 17. Estimation of the molecular weights of the C.
sorokiniana NADP-GDH a- and (3-subunits. NADP-GDH subunits
synthesized in vivo were partially purified and
electrophoresed on a 8% SDS polyacrylamide gel and detected
by immunoblotting. The molecular weight of the NADP-GDH a-
and (3-subunit was determined to be 53.48 and 52.27 kD,
respectively.

108
Relative Mobility

109
Determination of the Amino-terminal Amino Acid Residues of
the «- and (3-Subunits
In order to identify the amino-terminal sequences of the
mature NADP-GDH a- and (3-subunits and to locate the transit
peptide cleavage sites, the amino-terminus of both subunits
was sequenced by automated Edman degradation. Purified a-
subunit, and a second functionally pure mixture of a- and (3-
subunits were separated by Tris-Tricine SDS-PAGE and
transferred to a PVDF membrane. The amino acid composition
of the amino-terminal sequences were determined according to
Plough et al. (1989).
A total of 20 amino-terminal amino acid residues of the
a-subunit were identified: AVSLEEQISAMDATTGDFTA. Ten of the
amino-terminal residues of the (3-subunit were identified:
DATTGDFTAL. In order to verify that the higher molecular
weight band (53.5 kD) in the partially purified NADP-GDH
mixture was in fact the same subunit sequenced from the
purified a-subunit preparation, five amino-terminal residues
were analyzed a second time. All five residues agreed with
the aforementioned a-subunit amino-terminal residues.
Comparison of the a-subunit and (3-subunit amino-terminal
sequences revealed that the (3-subunit lacked 11 amino-
terminal amino acid residues identified in the a-subunit.
Furthermore, comparison of the amino acid residues of the
mature subunits to the deduced amino acid sequences of the
NADP-GDH cDNAs predicted the a-subunit to be 53,501 D and the
(3-subunit to be 52,342 D. The 1159 D difference in the two

110
subunits was due to the additional amino-terminal residues in
the a-subunit. The deduced molecular mass of the two
subunits are extremely close to those predicted by SDS gel
analysis.
The transit peptide SPP cleavage sites were identified:
MOTALVAKPIVAAPLAARPRCLAPWPCAWVRSAKRDVRAK AVSLEEQISAM DATTGDFT
VR amino acids A(3
Since both SPP cleavage sites were located downstream of the
14 amino acid variable region, it was not possible to
determine by comparison if one or both of the two precursor
proteins are processed to yield the a- or (3-subunits,
respectively.
All the protein data presented thus far support the
strong physical similarities of the a- and |3-subunits. In an
attempt to explain the kinetic and biochemical differences
observed between the a- and (3-homohexameric isoenzymes
(Bascomb and Schmidt, 1987), a comparison of the structural
predictions of the two subunits was performed. Structural
predictions were made according to Gamier et al. (1978) and
by comparison to the published 1.96Á crystal structure of the
C. symbiosum NAD-GDH (Baker et al., 1992).
The 486 amino acid residues comprising the a-subunit are
organised into two domains. Domain one consists of residues
42 to 238 and residues 460 to 486 (Fig. 18). The second
domain spans residues 239 to 406. The two helicies ai5 and
«16 (residues 407-459) serve as a link between the two
domains. Domain one contains the glutamate-binding domain

Figure 18. Alignment of the C. sorokiniana NADP-GDH a- and
(3-subunit deduced amino acid sequences (Cso) with the C.
symbiosum NAD-GDH amino acid sequence (Cs). The secondary
structural elements of the NADP-GDH subunits are shown above
the aligned sequences, with helicies represented as cylinders
and strands shown as arrows. Amino acid residues are
numbered in relation to the NADP-GDH a-subunit. The amino-
terminal amino acid residues of the a- and (3-subunits are
denoted by small arrows and residues highlighted by asterisks
(*) are conserved in the majority of GDHs sequenced to date.
The GDH structural elements are labelled in reference to the
three-dimensional structural elements identified in the C.
symbiosum NAD-GDH (Britton et al., 1992). Structural
elements a'i_2 (residues 1 to 41) are unique to the C.
sorokiniana NADP-GDHs. Amino acid identities are denoted by
vertical lines, high degrees of similarity are denoted by
colons, and low degrees of similarity by periods.

112
a
IK
a'
B
a.
B-
C. ♦ ♦ . . . I . : : . I : . I
Cs. a P MSKYVDRVIAEVEKKY
PEQQEFMQAVREVAVSLQPVFEKRPEL LPIFKQIVEPERVITFRVSWLDDAGNLQVNRG 116
: : : . I I : I . I II I I . I I . : : I I . I I I II - I I I . I I I . I . : : I I I
ADEPEFVQTVEEVLSSLGPWDAHPEYEEVALLERMVIPERVIEFRVPWEDDNGKVHVNTG
X.
O.,
I
* ***** * ** * * * * * *
FRVQYSSAIGPYKGGLRFHPSVNLSIMKFLAFEQIFKNSLTTLPMGGGKGGSDFDPKGKSD 177
: I I I I I I I I I I II I I 11111111111:111 I I:I I II II I I I :I I II II I I . I I I I
YRVQFNGAIGPYKGGLRFAPSVNLSIMKFLGFEQAFKDSLTTLPMGGAKGGSDFDPNGKSD
{ «7, ((°7bI)~CZ^> C
a
8
BK>
* * * * *
AEVMRFCQSFMTELQRHISYVQDVPAGDIGVGAREIGYLFGQYKRITKN YTGVLTPKGQE 237
I I I I I II . I I II I III: I II II I : II I I I I I I I :: I I I::I. . I .I I I I . I : . .
REVMRFCQAFMTELYRHIGPDIDVPAGDLGVGAREIGYMYGQYRKIVGGFYNGVLTGKARS
a,
B
a
1 o
* * *** **** *
YGGSEIRPEATGYGAVLFVENVLKDKGESLKGKRCLVSGAGNVAQYCAELLLEKGAIVLSL 298
: I I I : I I I I II I I . I . : I I . I . I . . . : . I II . : . I II II . I . I I II . : . I
FGGSLVRPEATGYGSVYYVEAVMKHENDTLVGKTVALAGFGNVAWGAAKKLAELGAKAVTL
a
1 1
B-KiiH=5> G3
SDSQGYVYEPNGFTREQLQAVQDMKKKNNSARISEYKSDTAVYVGDRRKPWELDCQVDIAF 359
: I
SGPDGYIYDPEGITTEEKINYMLEMRASGRNKVQDYADKFGVQFFPGEKPWGQ KVDIIM
t>—( (H=?>—Q7T&-KX3IÜÜ
** * *** *****
PCATQNEIDEHDAELLIKHGCQYWEGANMPSTNEAIH KYNKAGIIYCPGKAANAGGVAV 419
I I I I I I::I . : I . : : : . . . I : I . I I I I . I I I I : : .1:11.11111 I
PCATQNDVDLEQAKKIVANNVKYYIEVANMPTTNEALRFLMQQPNMVVAPSKAVNAGGVLV
ÃœZEUZIDK
a
1 6
B-C
a
1 7
“7
SGLEMTQNRMSLNWTREEVRDKLERIMKDIYDSAMGPSRRYNV DLAAGANIAGFTKVAD 478
I I : I I . I I . . -I . I I III . I I . . : I . I I . I : . : : . II.: : I . II I I I . I I I : II
SGFEMSQNSERLSWTAEEVDSKLHQVMTDIHDGSAAAAERYGLGYNLVAGANIVGFQKIAD
E3
AVKAQGAV
I : . I I I .
AMMAQGIAW
486

113
that is conserved among all GDHs, whereas domain two contains
the characteristic |3«(3 dinucleotide-binding fold (|3g«iof3h)
responsible for NAD(P)+ binding (Rice et al., 1987). This
fold contains the consensus GXGXXA/G (residues 276 to 281)
nucleotide binding fingerprint. The preference for alanine
(281) as the last residue observed in the C. sorokiniana
sequence (Fig. 18) is indicative of NADP+-specific
dehydrogenases, whereas a glycine residue is typical in NAD+-
specific enzymes (Baker et al. 1992). The two domains are
separated by a deep cleft that in the tertiary structure of
the subunit forms a catalytic pocket to bring the two
substrates in close proximity (Baker et al., 1992). The
lysine 234 and glutamate 423 (Fig. 18) are predicted to form
a salt bridge that is responsible for stabilizing the
tertiary structure of the NADP-GDH subunits (Britton et al.,
1992). Additionally, regions of domain one («i-«5, (3a-(3br«17
[Fig. 18]) were shown to play a critical role in the
dimerization and trimerization processes that lead to the
catalytically active hexameric GDH (Baker et al., 1992).
Comparison of the structural predictions of the «-
subunit and (3-subunit revealed the a-subunit possesses an
additional amino-terminal «-helical domain that is lacking in
the (3-subunit (Fig. 18). Comparison of NADP-GDH «- and (3-
subunit amino acid sequences to the greater than 35 GDH
sequences in the protein data bases revealed that the C.
sorokiniana NADP-GDH a- and (3-subunits possessed an
additional 41 and 30 unique amino-terminal residues,

114
respectively. Comparison of the unique regions to all other
proteins in the data bases yielded no evidence of a conserved
functional domain. However, visual inspection revealed that
the residues 33 to 40 (GTEGLVHG or GXXGXXXG [Fig. 18]) of the
unique region form a glycine-rich turn that closely resembles
the motif GXGXXG of the nucleotide-binding fingerprint.
Comparison of the Induction Patterns of the NADP-GDH
Antigens, Activities, and mRNAs in 29 mM Ammonium Medium
The cellular levels of the NADP-GDH antigens, enzyme
activities, and mRNAs were measured throughout a 240 min
induction in ammonium medium to determine if a correlation
exists between a specific NADP-GDH mRNA and a specific
activity or subunit type. Synchronous nitrate-cultured C.
sorokiniana cells were transferred to 29 mM ammonium medium
and cultured in continuous light for 240 min according to
Cock et al. (1991). Samples were collected at various time
intervals throughout the induction for the analysis of enzyme
activity, antigen, and mRNA.
During the 240 min induction period, culture turbidity
increased 1.87 fold (Fig. 19); however, the culture cell
number remained constant at 2.89 x 107 cells per mL. Total
soluble protein showed an initial decrease in the first 20
min followed by a rapid linear increase between 40 and 140
min that slowed at 240 min (Fig. 20).
The pattern of NADP-GDH antigen accumulation was
examined by use of a western blot immunodetection procedure.

Figure 19. Increase in culture turbidity of Chlorella cells
cultured for 240 min after the addition of 29 mM ammonium.
At zero time, ammonium was added to synchronous daughter
cells and the culture was illuminated. Cell number remained
constant throughout the induction time period.

116

Figure 20. Pattern of the total soluble protein in
synchronized daughter cells induced for 240 min in 29 mM
ammonium medium. At zero time, ammonium was added to
synchronized cells and the culture was illuminated. Culture
samples were collected at various time intervals, ruptured,
and the clarified supernatants were analyzed according to
Bradford (1976) for total protein content.

mg Protein/mL Culture
118

119
The NADP-GDH p-subunit (52.3 kD) was detected at To and
increased for the first 40 min followed by a gradual decrease
that continued for the remainder of the induction period
(Fig. 21, Fig. 22). The a-subunit (53.5 kD) was first
detected at 20 min and gradually increased for the first 80
min followed by a marked increase that continued for the
remainder on the induction period (Fig. 21, Fig. 22). The
transition from the p-subunit being the prominent species to
the a-subunit being prominent occurred between 60 and 80 min.
The NADPH-GDH (aminating) and the NADP+-GDH
(deaminating) activities were monitored throughout the 240
min induction period. Both the NADPH- and NADP+-GDH
activities showed an initial lag of 20 and 40 min,
respectively (Fig. 23). The NADPH-GDH activity increased
rapidly for the first 80 min and declined sharply at 100 min
before increasing at 240 min. The NADP+-GDH activity
accumulated at a linear rate throughout the induction after
an initial lag (Fig. 23). The NADPH-GDH:NADP+-GDH activity
ratio and the a:p subunit ratio were calculated to determine
if the a- or p-subunit ratio was correlated with a particular
GDH activity (Table 6).
Table 6.
Ratios Of NADP:NADP+
activities and a:p subunits
Time
(min)
Ratio NADPH:NADP+
GDH Activity
Ratio a:p Subunit
0
2.87
0.28
20
2.96
0.58
40
3.81
0.49
60
4.51
0.80
80
3.49
1.57
100
2.73
8.74
140
1.61
11.23
240
1.13
34.79

Figure 21. Patterns of accumulation of NADP-GDH antigens in illuminated cells cultured in
29 mM ammonium medium for 240 min. At zerp time, ammonium was added to synchronous
daughter cells and the culture was illuminated. The proteins in equal volumes of
homogenates, from cells harvested at the various time points, were resolved by SDS-PAGE and
transferred to nitrocellulose. The resulting blot was immunoreacted with polyclonal rabbit
anti-NADP-GDH antibodies labelled with 125l-Protein A. Lanes are labelled with
corresponding time points. The positions of the a-subunit (53.5 kD) and (5-subunit (52.3
kD) are noted.

O 20 40 60 80 100 140 240
121

Figure 22. Patterns of accumulation of NADP-GDH antigens in
illuminated cells cultured in 29 mM ammonium medium for 240
min. At zero time, ammonium was added to synchronous C.
sorokiniana daughter cells and the culture was illuminated.
Autoradiographs of Western blots (Fig. 21) were analyzed by
laser densitometry to determine the relative levels of the
NADP-GDH a-subunit (•) and (3-subunit (o) throughout the 240
min induction period.

Relative NADP-GDH
Antigen/Cell
co cn ~si co —*• co
123

Figure 23. Pattern of NADP-GDH activities in homogenates of
synchronous C. sorokiniana cells cultured for 240 min in 29
mM ammonium medium in continuous light. Aliquots of
clarified homogenates, from cell collected at various time
intervals, were analyzed spectrophotometrically for both
aminating (•) and deaminating (o) NADP-GDH activities.

125
Time (min)

126
The peak NADPH:NADP+-GDH ratio occurred at 60 min when the
subunit ratio showed the (3-subunit to be the prominent NADP-
GDH antigen, whereas the a-subunit was the prominent form
when the NADPH:NADP+-GDH ratio was the lowest.
RPA of total RNA from each time point was used to
elucidate the relative induction patterns of the +42 nt and
-42 nt NADP-GDH mRNAs. Total RNA from each time point,
containing equal amounts of poly(A)+ mRNA, was hybridized
with an antisense protecting probe corresponding to the 5'-VR
of the +42 nt mRNA, 5'-VR of the -42 nt mRNA, HCR, or 3'-UTR.
The hybridized products were treated with a ribonuclease
mixture, and the resulting products were analyzed on a 5%
sequencing gel.
Two unique protected regions (Fig. 24A, B) corresponding
to the 5'-VR were detected throughout the induction period
with the +42 nt 5'-VR probe (ca. 367 and 170 nt) and -42 nt
5'-VR probe (ca. 309 and 200 nt). A single protected region
(Fig. 24C, D) was detected throughout the induction with the
probes corresponding to the HCR (ca. 154 nt) and 3'-UTR (ca.
388 nt). Each protected region occurred as a doublet,
separated by 4 to 11 nt, that resulted from the cleavage of
the protecting fragment by RNase A (cleaves after C, U) or
RNase Ti (cleaves after G) in the RNase mixture. To verify
the doublet represented a single protected region, RNase I, a
ribonuclease that cleaves in a nucleotide independent manner,
was utilized in a single set of RPAs with the -42 nt 5'-VR
protecting probe. Two protected regions, occurring as single

Figure 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. Total RNA, containing equal amounts of poly(A)+ RNA,
isolated from cells collected at various time intervals, was
hybridized with antisense RNA probes corresponding to the 5'-
VR of the +42 bp mRNA (A), 5’-VR of the -42 bp mRNA (B), HCR
(C), and 3'-UTR (D). The resulting hybrids were treated with
a RNase mixture and the RNase resistant fragments were
resolved on a 5% sequencing gel and analyzed by
phosphorimaging. A, +42 bp protected region: 373 nt, 362 nt
(ca. 367 nt); -42 bp protected region: 172 nt, 167 nt (ca.
170 nt). B, -42 bp protected region: 311 bp, 306 nt (ca. 309
nt); +42 bp protected region: 202 nt, 198 nt (ca. 200 nt).
C, HCR protected region: 155 nt, 152 nt (ca. 154 nt). D, 3'-
UTR protected region: 392 nt, 381 nt (ca. 388 nt). Lanes U,
unprocessed antisense probe; T, tRNA control hybridized with
probe and Rnase treated; 0-240 min sample times; P, poly(A)
RNA used in RACE-PCR.

128
U T 0 20 40 60 SO 100 140 240 P
♦
•mu >
if»?:: >
tfftti->
B

U T O 20 40 60 80 100140 240 P
• ft it*
Figure 24 continued

130
fragments, were detected. The RNase I resistant
fragments corresponded to identical regions detected as
doublets with the RNase A-Ti mixture (data not shown).
Because of the quantity of units required and the expense of
the RNase I, it was not feasible to perform all of the RPAs
with this enzyme. The +42 nt 5'-VR probe yielded a protected
region (170 nt) corresponding to the -42 nt mRNA (Fig. 24A)
that was approximately 30 nt shorter than the protected
fragment predicted by the cDNA sequence (200 nt). However,
the reciprocal probe, -42 nt 5'-VR, yielded two protected
regions of predicted size (309 and 200 nt). This paradox may
reflect a probe-specific artifact due to the mis-
incorporation of a nucleotide in the PCR generated +42 nt 5'-
VR probe template, or a unique hybridization artifact.
The relative induction pattern of each protected region
was determined by analysis on a phosphoimager. No NADP-GDH
mRNA was detected with any of the probes in the To sample.
All four of the the protected regions showed a similar
induction pattern for the first 140 min (Fig. 25). The
protected region corresponding to the -42 nt NADP-GDH mRNA
showed a decrease at 240 min that contrasted with an increase
of the +42 nt mRNA (Fig. 25). The 3'-UTR probe, that
hybridizes to both mRNAs, showed a peak mRNA abundance at 80
min in contrast to the 60 min peak observed for the other
regions. RPA only detects RNA degradation in the region
corresponding to the antisense probe and is not affected by
degradation outside of this region. Therefore, the observed

Figure 25. Relative abundance patterns of NADP-GDH mRNA in
cells induced in 29 mM ammonium medium. The abundance
pattern of the +42 bp mRNA (•) and -42 bp mRNA (o) were
determined by quantifying 32P-labeled RNase resistant
fragments resulting from differential digestion of the -42 bp
5'-VR RPA probe. The abundance patterns for total NADP-GDH
mRNA was determined by quantifying 32P-labeled RNase resistant
fragments corresponding to the HCR (a) and 3 '-UTR (â– ). The
relative cpm of each protected fragment was quantified on a
phosphorimager.

132

133
shift may reflect the 5' to 3’ degradation of eukaryotic mRNA
reported by Brawerman (1993). The induction patterns
observed with the HCR probe were equivalent to the pattern
detected by Cock et al. (1991) by northern blot analysis.
Quantitative comparison of the relative abundances of
the +42 nt mRNA and the -42 nt mRNA with respect to each
other could not be easily accomplished with this procedure.
Since the antisense protecting probes were radiolabeled with
a single nucleotide (dCTP), the specific activity was
affected by the number of cytosine residues remaining in the
processed product. The processing of the -42 nt protecting
fragment hybrized to the +42 nt mRNA preferentally removed
regions rich in cytosine that artificially lowered the cpm
emmitted by this protected region relative to the -42 nt mRNA
protected fragment.
RT-PCR Analysis of the NADP-GDH mRNAs
To determine if the +42 nt and -42 nt mRNAs differ in
any region other than the 5'-VR and to quantify the relative
abundance of each mRNA, mRNA from various time points of the
240 min 29 mM ammonium induction was analyzed by RT-PCR
(Kawasaki, 1990). Total RNA, containing equal amounts of
poly(A)+ RNA, from each time point was amplified using a
series of primers spanning the entire length of the NADP-GDH
mRNAs. The final PCR products were analyzed on ethidium-
stained agarose gels and compared to control reactions that
utilized cDNA clones bearing the region(s) of interest as PCR

134
templates. Photographic negatives of the ethidium-stained
gels were quantified using a laser densitometer.
RT-PCR analysis revealed that the +42 nt and -42 nt
NADP-GDH mRNAs differed only in their 5'-VRs (Fig. 26). A
single RT-PCR product was detected for all other regions of
the mRNAs (Fig. 26C-F). The PCR products were identical to
the sizes predicted from the cDNA sequences and to the
plasmid DNA control products. No PCR products were detected
in control mRNA samples in which no reverse transcriptase was
added, verifying the observed products did not result from
amplification of contaminating DNA. NADP-GDH mRNA was not
detected in the To sample and the relative induction pattern
of each region was equivalent to those determined by RPA
(Fig. 25).
Quantitative comparison of the two NADP-GDH mRNAs
revealed the relative abundance patterns of the two mRNAs
(Fig. 26A,B) differed significantly (Fig. 27). The +42 nt
mRNA was approximately three times more abundant than the -42
nt mRNA throughout the induction time course. Both mRNAs
showed a similar induction pattern from 20 to 140 min;
however, the +42 nt mRNA showed an increase at 240 min that
contrasted with a decrease of the -42 nt mRNA (Fig. 27).
This observation is in agreement with the contrasting shift
observed for the two mRNAs at 240 min using the RPA (Fig.
25).

Figure 26. RT-PCR analysis of the NADP-GDH mRNAs synthesized
in synchronous C. sorokiniana cells throughout a 240 min
induction period in 29 mM ammonium medium. Total RNA,
containing equal amounts of poly(A)+ RNA, isolated from cells
collected at various time intervals, was amplified using a
series of primer pairs spanning the entire length of the
NADP-GDH mRNAs. The resulting PCR fragments were resolved on
agarose gels and the molecular weight of the products were
determined by comparison to commercial standards (M). A, 147
bp and 105 bp. B, 311 bp and 269 bp. C, 464 bp. D, 155 bp.
E, 760 bp. F, 745 bp. The RT-PCR products from each time
point (0 to 240 min) were compared to products amplified from
cDNA plasmid templates bearing the regions of interest
(control).

ti m o o
03 >
II
II
II
II
I
»
Â¥
0
20
40
60
80
1 00
1 40
240
control
136

Figure 27. Relative abundances of the NADP-GDH mRNAs
synthesized in synchronous C. sorokiniana cells throughout a
240 min induction period in 29 mM ammonium medium. RT-PCR
products corresponding to the +42 bp (#) and -42 bp (o)
mRNAs, derived from primer pairs spanning the 5'-VR (Fig. 26
A,B), were quantified by laser desitometry to determine the
relative abundances of each specific NADP-GDH mRNA during the
induction period.

Relative RNA Abundance
L L 1 L k [\3
oro^cDCDoro-P^CDOoo
138

DISCUSSION
The results presented in this study reveal the C.
sorokiniana nuclear genome possesses a single NADP-GDH gene
that is transcribed to yield, via alternative pre-mRNA
splicing, two NADP-GDH mRNAs. Cock et al. (1991) provided
Southern blot and genomic cloning evidence that was
consistent with the existence of a single NADP-GDH gene in
the C. sorokiniana genome. However, the significance of
these findings was limited by the fact that none of the NADP-
GDH cDNA clones isolated possessed complete 5'-terminal
sequences and many lacked a 3' terminus. The lack of
terminal sequences in the cDNAs prevented the researchers
from determining whether the NADP-GDH gene yields a single or
multiple mRNAs that encode the NADP-GDH «- and p-subunits.
In this study, experimental evidence was obtained for
two NADP-GDH mRNAs that are identical except for the presence
of a 42 nt insert located in the 5’-coding region of the
longest mRNA. The full-length NADP-GDH mRNA sequences were
assembled by combining overlapping regions of identical
sequence of various cDNA clones (Figs. 1,3). Several lines
of evidence support the inference that the two NADP-GDH mRNA
sequences actually exist in vivo.
RPA of total cellular RNA isolated from cells
synthesizing both the a- and (3- subunits detected a single
139

140
RNase resistant region with the HCR and 3'-UTR probes,
whereas two resistant regions were detected with both the +42
bp and -42 bp 5'-VR probes (Fig. 24). These results provide
strong evidence that the two unique 5'-VRs do exist in vivo
and are not an artifact of the 5' RACE-PCR cloning. RT-PCR
analysis of mRNA isolated from cells synthesizing both
subunits, using overlapping primer pairs that span the entire
lengths of the NADP-GDH mRNAs, yielded a single PCR product
for each region of amplification downstream of the 5'-VR
(Fig. 26 C-F). Primer-pairs, designed to anneal to regions
proximal and distal to the 5'-VR, yielded two PCR products
differing by 42 bp (Fig. 26 A,B). The molecular weights of
the mature a- and p-subunits deduced from the combined cDNA
clones were nearly identical to the molecular weights of the
in vivo subunits determined by SDS-PAGE. These results
support the authenticity of the +42 bp and -42 bp NADP-GDH
mRNA sequences.
Although both mRNA sequences could be accounted for in
the previously determined nuclear NADP-GDH gene sequence, the
possiblity remained that two closely related NADP-GDH genes
might exist that differ only at their 5'-termini
corresponding to the 5'-VR of the mRNAs. Southern blot
analysis using the 5'-VR, HCR, and 3'-VR probes yielded
hybridization patterns consistent with patterns predicted by
the single NADP-GDH gene sequence (Fig. 12). In addition,
allele-specific PCR analysis of C. sorokiniana genomic DNA
yielded single PCR products corresponding to the 5’-VR, HCR,

141
and 3'-UTR identical in size to corresponding products
amplified from the NADP-GDH genomic clones (Fig. 13). These
results are consistent with the existence of only a single
NADP-GDH gene in the C. sorokiniana nuclear genome.
Comparison of the NADP-GDH +42 and -42 bp cDNA sequences
to the NADP-GDH gene sequence revealed the mRNA sequences
span a 7178 bp region of the C. sorokiniana nuclear genome
(Fig. 19). The +42 nt mRNA is divided over 22 exons ranging
in size from 18 to 550 bp, whereas the -42 nt mRNA is divided
over 23 exons ranging in size from nine to 550 bp. The
intron/exon splice junctions are highly conserved and closely
match the consensus sequences identified for constitutively
spliced exons in higher plants and animals (Brown, 1986;
Hawkins, 1988).
The 42 bp intron (auxon), which is alternatively spliced
to yield the two NADP-GDH mRNAs, lacks the consensus splice-
site elements. Since the splice-site junction sequences of
the 42 nt auxon lack conserved sequence elements, there might
be a different recognition involved in the splicing
mechanism. The splice elements involved in alternative RNA
splicing often do not appear to differ significantly from
constitutively-spliced elements (Breitbart et al., 1987a),
though in some reported cases extreme variants of the
sequence and arrangement of the elements influence alternate
splice-site utilization. In addition to cis-elements,
regulation of alternate splice-site utilization also involves
differences in cellular trans-factors as evidenced by

142
developmentally and cell-specific regulated splicing
(Breitbart et al., 1987b). The retention or removal of the
42 bp auxon might be determined by specific trans-factors
that are only present under certain physiological conditions.
The additional trans-factors could simply be variants of the
snRNPs associated with the constitutive sliceosome complex
(Smith et al., 1989). In mouse and Xenopus, multiple U1
snRNAs associated with U1 snRNP have been isolated and
characterized and have been shown to be developmentally
regulated (Lund et al., 1985; Lund and Dahlberg, 1987).
Since the Ul snRNP is responsible for 5' splice-junction
recognition, it has been proposed that sequence differences
in the multiple Ul snRNA allow for recognition of alternative
5' splice-sites in developmentally regulated alternative RNA
splicing. Heterogeneity in snRNAs associated with other
snRNPs of the spliceosome complex have also been implicated
in alternate splice-site utilization (Smith et al., 1989).
The possiblity cannot be overlooked that the 42 bp auxon
could be alternately removed by a unique processing mechanism
that does not involve the spliceosome complex.
The small size of the 42 nt auxon and 9 nt exon is
smaller than any intron or exon reported for a plant gene.
In addition to specific cis-elements and trans-factor
requirements for RNA splicing, there also appears to be
minimal distance requirements that reflect steric constraints
for the binding of splicing factors to the pre-mRNA (Chabot
and Steitz, 1987). In mammalian genes, a minimal intron

143
length of 66 nt has been determined; however, introns as
small as 31 nt (Craig et al., 1989) and 43 nt (DeWet et al.,
1987) have been shown to be effectively spliced in nematodes
and Photinus, respectively. Therefore, there is no
biochemical precedent to preclude the processing of the 42 nt
intron. There appear to be no restrictions on exon length,
as exons of 3 nt (Santoni et al., 1989) and 6 nt (Cooper and
Ordahl, 1985) have been identified in mammals.
To identify the putative NADP-GDH promoter region, the
5'-terminal sequences of the NADP-GDH mRNAs were compared to
the GDH gene sequence. Comparison of the sequences
identified the transcriptional initiation site. An
additional 1285 bp of genomic DNA sequence was identified
upstream of the initiation site. Since the induction rate of
the NADP-GDH isoenzymes is influenced by various carbon and
nitrogen compounds, it is anticipated the NADP-GDH promoter
might have cis-elements sensitive to these various molecular
signals. The putative NADP-GDH promoter region (Fig. 11)
does not possess a consensus TATA box nor CCAAT box normally
located at position -25 to -35 bp and -50 to -100 bp,
respectively. No positive or negative cis-regulatory
elements common to other previously identified plant promoter
elements were detected (Kuhlemeier, 1992; Katagui and Chua,
1992). The promoter region of the Chlorella kessleri
H+/hexose cotransporter gene (Wolfe et al., 1991), the only
other Chlorella gene published to date, also lacks these cis-
regulatory elements. Comparison of the C. kessleri promoter

144
region to the putative NADP-GDH promoter region revealed no
common upstream elements. Although no common promoter
elements were identified in the 1285 bp upstream sequence,
the evidence presented herein supports the identification of
the transcriptional start site. The 5'-terminal sequences of
the NADP-GDH mRNAs possessed a 32 bp pyrimidine rich (89%)
5'-UTR and a translation initiation sequence indicative of
eukaryotic mRNAs (Kozak, 1984). In addition, the presence of
unique 5' guanine residues in the 5' RACE-PCR clones provides
a definitive means for the identification of the 5'-capping
points of the two NADP-GDH mRNAs (Bahring et al., 1994). The
identification of the transcriptional start-site and putative
promoter region should allow analysis of sequence elements
involved in the metabolic regulation of transcription of the
NADP-GDH gene by in vivo footprinting.
The physiological significance of the 42 nt insert in
the 5'-coding region of the +42 bp NADP-GDH mRNA is unclear
(Fig. 6). The 42 nt insert or the lack thereof could
potentially influence translational efficiency, mRNA
turnover, precursor-protein import, or precursor processing
by the SPP. Analysis of the secondary structural predictions
of the two NADP-GDH precursor proteins indicates that the 42
nt insert introduces 14 amino acids residues that alter the
structural characteristics of the transit peptide domain
(Figs. 7,8). Transit peptide sequences differ considerably
from one another even within a single organism and no strong
secondary structural requirements seem to be necessary for

145
proper import or processing. Although the structural and
sequence determinants for precursor-protein processing have
not been well established, alterations in the carboxy-
terminal regions of the transit peptide have been shown to
influence processing (Smeekens et al., 1990). Therefore, the
additional amino acids or secondary structure may influence
processing of the precursor protein by the SPP.
One possiblity is the insertion of the additional amino
acid residues influences the location of the SPP binding site
in relation to the two SPP cleavage sites (Fig. 28). If the
polypeptide lacks the 14 amino acid insert, the SPP cleavage
domain might be placed in close proximity to the p-subunit
cleavage site and yields the mature p-subunit (Fig. 28A).
The presence of the 14 amino acid residues in the larger
polypeptide might shift the downstream cleavage sites in
relation to the SPP binding site and position the SPP
cleavage domain in contact with the a-cleavage site, thus
yielding the mature a-subunit (Fig. 28B). The possibility
also exists that if the insert does influence processing it
may do so by creating or eliminating a cleavage recognition
domain. The possibility remains that the a- and p-subunits
arise via differential processing of the precursor proteins
by two different SPP; however, only a single general SPP has
been identified in chloroplasts of all plants analyzed to
date (Musgrove et al., 1989). The single SPP has been shown,
by in vitro processing analysis, to process all stromal
proteins to authentic mature proteins.

Figure 28. Model for the regulation of the processing of the
two NADP-GDH precursor proteins. A, The NADP-GDH precursor
protein lacking the 14 amino acid insert is cleaved at the p-
subunit cleavage site downstream of the SPP binding domain
(crosshatched box). B, The larger NADP-GDH precursor protein
possessing an additional 14 amino acid insert (stippled
region) is cleaved at the a-subunit cleavage site, due to a
shift in the position of the cleavage sites relative to the
SPP binding site. Asterisks denote the a- and p-cleavage
site as noted.

147
A
a p
B
a f,

148
There are several other possiblities of how the 42 nt
insert might affect the regulation of the NADP-GDH gene
expression; however, there is some precedent to support the
model proposed in Figure 28. As will be discussed later,
there is a correlation between the relative abundance of the
+42 nt mRNA and the level the NADP-GDH cx-subunit. It is also
worth noting that the two precursor proteins differ by
approximately the same number of amino acid residues and
molecular weights as the mature a- and (3-subunits, 14 amino
acids (1.5 kD) and 11 amino acids (1.2 kD), respectively.
The possibility also exists that the alternative processing
of the 42 nt insert is a nuclear splicing event that has no
influence on the subunit type encoded by the mRNA. Since the
variable splicing does not greatly alter the primary
sequences of the precursor polypeptides, the splicing may
have no deleterious effect and therefore has not been subject
to any strong selective pressures to eliminate the splicing
event. By use of in vitro transcribed/translated precursor
proteins, one should be able to determine unequivocally
whether a given precursor is processed to the a- or 13-
subunit .
Results presented previously (Yeung et al., 1981;
Bascomb and Schmidt, 1987) and in this study indicate that
the NADP-GDH a- and (3-subunits have a high amount of primary
sequence identity. Both MAbs (Fig. 16) and polyclonal
antibodies (Fig. 21) produced against the a-subunit are able
to detect the (3-subunit on immunoblots. Yeung et al. (1981)

149
showed that rabbit anti-NADP-GDH p-subunit antibodies
immunoprecipitated the a-subunit. The molecular weights of
the mature a- and p-subunits predicted from the nearly
identical amino acid sequences deduced from the NADP-GDH cDNA
clones (53.5 and 52.34 kD, respectively) is in close
agreement with the in vivo molecular weights determined by
SDS-PAGE (53.48 and 52,27 kD). Furthermore, the amino-
terminal residues identified for the a-subunit overlapped
with nine identical residues identified in the p-subunit
amino-terminus. These results indicate that the a-and p-
subunits are identical except for the additional 11 amino
acid residue extension identified in the a-subunit amino
terminus (Fig. 18).
Comparison of the NADP-GDH a- and p-subunit sequences to
the GDH sequences reported in the data bases revealed the two
subunits possess an additional 41 and 30 amino-terminal
residues that are unique to the C. sorokiniana NADP-GDH a-
and p-subunits, respectively. Visual inspection revealed
that residues 33 to 40 of the unique region (Fig. 18) form a
glycine rich turn, GXXGXXXG, that closely resembles the
motif, GXGXXG, of the dinucleotide-binding fingerprint.
Although not an exact consensus, it is possible this region
still interacts with pyridine dinucleotides (i.e. NADPH). A
similar dinucleotide-binding site, GXXGXXA, has been
identified in porcine malate dehydrogenase and the tetrameric
NADP-GDHs (Britton et al., 1992). This motif might not be
sufficient for strong NADP+/NADPH binding; however, it might

150
provide for a weak interaction such as would be required for
the NADPH allosteric regulation observed for the C.
sorokiniana NADP-GDH a-homohexamer (Bascomb and Schmidt,
1987) .
In an attempt to explain the kinetic and biochemical
differences reported previously between the a- and p~
homohexameric forms of the NADP-GDH isoenzymes (Bascomb and
Schmidt, 1987), a comparison was made between the structural
elements of the a- and p-subunits (Fig. 18). Both the a- and
P-subunits possess an additional a-helical domain (a'2)
upstream of the putative dinucleotide-binding fingerprint
(Fig. 18). The additional 11 amino-terminal residues of the
a-subunit form a second a-helical domain (a'i) not found in
the p-subunit. Helical wheel projections of the two helical
domains reveal that both domains possess a hydrophobic and
hydrophilic face (Fig. 29). The hydrophobic faces of the «-
helical domains may implicate these helicies as being
important in subunit-subunit contacts, since the downstream
ai helix common to all hexameric GDHs has been shown to be
involved in subunit dimerization and trimerization (Baker et
al., 1992). Comparison to the three-dimensional structure of
the C. symbiosum GDH revealed the additional helical domains
would be predicted to lie in the large central core formed in
the assembled hexamers (Fig. 30), thus exposing the
hydrophilic faces to the central cavity. In the non-
allosterically regulated C. symbiosum GDH, all contacts
between subunits involve domain one structures. However, the

Figure 29. Helical wheel projections of the unique
C.sorokiniana NADP-GDH amino-terminal helical domains. A,
Helical wheel projection of the a'i helix of the a-subunit.
B, Helical wheel projection of the a'2 helix identified in
both the a- and (3-subunits. Boxed residues identify
hydrophobic amino acids.

152

Figure 30. Diagramatic representation of the assembled
hexameric NADP-GDH. Arrows denote the large central core
where the putative dinucleotide binding-domain and the a'i and
a'2 helical domains are proposed to interact with the NADPH
ligand. Dimerization and trimerization contacts involve
domain one contacts. Diagram after Baker et al. (1992).

154
NADPH
NADPH

155
vertebrate GDHs possess approximately 50 carboxy-terminal
resides proposed to be involved in domain two contacts (Baker
et al., 1992). Interestingly, the domain two (catalytic
dinucleotide-binding domain) contacts have been implicated in
subunit-subunit communication important in the complex
dinucleotide allosteric regulation of the enzyme.
Bascomb and Schmidt (1987) showed that the affinity of
the a-homohexamer for ammonium was allosterically regulated
by NADPH. The mechanism of the NADPH allosteric regulation
may involve the unique a-helical domain of the a-subunit.
The additional amino-terminal extension of the a-subunit may
allow for contacts with the dinucleotide-binding domain two
or neighboring subunits. Upon binding of NADPH at the
putative amino-terminal dinucleotide-binding site, a
conformational change may occur that influences the binding
of ammonium to the subunit (Fig. 30). Substrate mediated
conformational rearrangement of the C. symbiosum hexameric
NAD-GDH has been shown to be an important step in the
catalytic cycle of the enzyme (Stillman et al., 1992).
In addition to differences in allosteric properties, the
a-subunit been shown by pulse-chase studies to be degraded
three times faster than the p-subunit in vivo (Bascomb et
al., 1987). Since the only observed differences in the two
subunits is the additional 11 amino acid residues in the a-
subunit, it is likely this region plays a critical role in
subunit turnover. Analysis of the amino-terminal residues
and structures yielded no consensus motifs that implicated

156
any specific degradation pathway. Previous researchers (Cock
and Schmidt, unpublished data) have shown that C. sorokiniana
NADP-GDH cDNAs subunits expressed from cDNAs in a GDH- strain
of E. coli are assembled into functional NADP-GDH hexamers.
Therefore, it is possible to synthesize preferentially the a-
or p-homohexamers in GDH- E. coli for crystallagraphic
analysis and mutational analyses to determine the
significance of the additional protein domains of the two
subunits.
The cellular abundances of the NADP-GDH mRNAs were
measured throughout a 240 min induction in 29 mM ammonium
medium. The induction patterns of the two NADP-GDH mRNAs
were identical with the exception of a decrease observed at
240 min in the -42 nt mRNA that contrasted with an increase
in the +42 nt mRNA (Fig. 27). It was evident that between 60
and 100 min a net loss of both mRNAs occurred indicating both
mRNA synthesis and degradation regulate the cellular levels
of both NADP-GDH mRNAs. Although both mRNAs decreased during
this period, the NADP+-GDH activity continued to accumulate
in a linear fashion (Fig. 23). A similiar phenomena has been
reported for the inducible NR mRNAs of barley (Melzer et al.,
1989) and Chlorella vulgaris (Cannon et al., 1992). In both
organisms, the NR activity continued to accumulate during
induction with nitrate, whereas the NR mRNA showed an initial
increase followed by a rapid decrease during the early stages
of induction. Cannon et al. (1992) showed that the NR mRNA
isolated from cells in early induction time periods was

157
resistant to cellular RNases in vitro, whereas NR mRNA from
fully induced cells was susceptible. The results indicate
that the change in NR mRNA turnover was in part due a change
in mRNA stability. The degree of NR mRNA stability was
inherent to the NR mRNA or factors associated with the early
induced NR mRNAs. A similar mechanism might be involved in
the NADP-GDH mRNA regulation and the stabilizing factor(s)
may be inherent to inducible mRNAs or mRNAs of nitrogen
metabolism genes.
NADP-GDH mRNA was not detected in the To total RNA
sample; however, NADP-GDH antigen and activity were detected
indicating that protein turnover plays a significant role in
the regulation of the NADP-GDH activity. Bascomb et al.
(1987) showed by pulse-chase analysis that both the a- and (3-
subunits were turned over rapidily in Chlorella cells induced
in 29 mM ammonium medium. The fact that no mRNA was detected
in the nitrate cultured To sample, but detectable levels of
NADP-GDH activity and antigen were present, might reflect a
difference in subunit turnover half-life that is influenced
by the metabolic state of the cell.
Comparison of the induction patterns of the NADP-GDH
mRNAs to the patterns of the NADP-GDH antigens did not reveal
a strong correlation between a specific mRNA and antigen
(Figs. 22, 27). However, the -42 nt mRNA showed a continued
decrease at 240 min that correlated with a continued loss of
the p-subunit antigen, whereas the +42 nt mRNA showed a
marked increase that correlated with the continuous

158
accumulation of the a-subunit (Figs. 22, 27). By pulse-chase
studies, previous researchers determined the a-subunit was
synthesized five times faster than the p-subunit under
comparable induction conditions (Bascomb et al. 1987). Based
on the faster rate of synthesis of the a-subunit, its mRNA
would be predicted to be more abundant than the p-subunit
mRNA. RT-PCR analysis of the NADP-GDH mRNAs revealed the +42
nt mRNA was on average three times more abundant than the -42
nt mRNA. Although not conclusive, the aforementioned results
implicate the +42 nt and -42 nt mRNAs as encoding the a- and
P-subunit precursor proteins, respectively.
Comparison of the NADPH:NADP+-GDH activity ratios to the
subunit ratios throughout the 240 min induction in 29 mM
ammonium medium revealed a correlation between a particular
subunit type and activity ratio (Table 6). The peak
aminating ratio occurred at 60 min when the p-subunit was the
prominent antigen (but not exclusive), whereas the a-subunit
was prominent when the aminating ratio was lowest.
Interestingly, the aminating activity was highest when both
subunits were present suggesting that the heterohexamers,
formed by various combinations of the a- and p-subunits,
might have a stronger influence on the catalytic activity
than either subunit alone. However, the in vivo function of
the NADP-GDH isoenzymes may not be reflected in the
aforementioned in vitro assay, since both NADPH-GDH and
NADP+-GDH activities were determined in the presence of
saturating amounts of substrates. This substrate saturation

159
might particularly affect the cx-homohexamer that shows a
decreased affinity for ammonium as the NADPH concentration is
increased (Bascomb and Schmidt, 1987).
The induction patterns of the NADP-GDH a- and p-
subunits observed in this research (Fig. 21) differed from
the patterns reported by previous researchers (Prunkard et
al., 1986; Bascomb and Schmidt, 1987). Previous studies
showed that C. sorokiniana cells induced in 29 mM ammonium
medium accumulated both the a- and p-subunits for the first
120 min with only the p-subunit accumulating thereafter
(Bascomb and Schmidt, 1987). In this study, both subunits
accumulated for the first 40 min after which the p-subunit
continued to decrease, whereas the a-subunit increased
rapidly for the remainder of the induction period (Fig. 22).
Furthermore, cells cultured continuously in 29 mM ammonium
medium synthesized primarily the a-subunit (Fig. 14), whereas
previous researchers reported the p-subunit primarily
accumulated at this ammonium concentration (Yeung et al.,
1981; Bascomb and Schmidt, 1987). Chlorella cells cultured
in 1 mM ammonium medium accumulated only the a-subunit (data
not shown) which is consistent with the results of Bascomb
and Schmidt (1987).
These apparent inconsistencies imply the regulation of
the NADP-GDH isoenzyme pattern is likely influenced by
multiple metabolic signals rather than strictly the nitrogen
status of the cells. Prunkard et al. (1986) showed that the
NADP-GDH a:p-subunit ratio was influenced by both the carbon

160
source and light conditions under which the C. sorokiniana
cells are cultured. GDH activity and isoenzyme pattern in
Nicotiana (Maestri et al., 1991) and carrot (Athwal, 1994)
cell suspension cultures are influenced by nitrogen and
carbon source as well as energy status of the cell. It is
hypothesized that the suspension cultures may exhibit a
regulatory mechanism similiar to the catabolic repression
observed in prokaryotes and eukaryotes (Magasanik, 1961;
Polakis and Bartley, 1965) and only recently detected in
higher plants. In addition, the GDH levels and isoenzyme
patterns have been shown to be influenced by growth
regulators and environmental stress conditions in higher
plants (Srivastava and Singh, 1987). Since the C.
sorokiniana NADP-GDH isoenzymes are regulated by both
carbohydrate and nitrogen metabolism, it is possible that the
regulatory signal involves the common metabolic link between
the two pathways, glutamate. With the molecular probes
provided in this research, it should be possible to further
elucidate the metabolic regulation of the NADP-GDH
isoenzymes.
The model presented in Figure 31 was proposed to
summarize the data presented herein. The C. sorokiniana
nuclear genome possesses a single NADP-GDH gene in which the
transription rate is increased upon the addition of ammonium
to the culture medium. The intracellular inducer is not
likely ammonium, but rather an organic metabolite (eg.
glutamate) derived from a nitrogenous or non-nitrogenous

161
compound whose intracellular pool is influenced by both
carbon and nitrogen metabolism. The resulting pre-mRNA
undergoes alternative processing that is either directly or
indirectly regulated by the organic metabolite. Under
conditions that favor the a-homohexamer, the pre-mRNA is
processed to 22 exons that yields the +42 nt NADP-GDH mRNA
(2116 nt) that encodes a 57850 D precursor protein. Under
conditions favoring the (3-homohexamer, the pre-mRNA is
processed to 23 exons that yields the -42 nt NADP-GDH mRNA
(2074 nt) that encodes a 56350 D precusor protein. Upon
import into the chloroplast, the 57850 and 56350 D precursor
proteins are processed by a general SPP to the mature cx-
subunit (53501 D) or [3-subunit (52342 D), respectively. The
mature subunits are assembled in the chloroplast stroma to
homo- and heterohexamers with different substrate affinities
and allosteric properties. Upon removal of the inducer from
the culture medium, the subunits comprising the isoenzymes
are rapidly inactivated by covalent modification to form
subunit dimers that target them for degradation by
chloroplastic proteases (Bascomb et al., 1987). Although the
proposed molecular regulation has not been demonstrated
unequivocably, the proposed model provides a scaffold to
direct future research into the regulation of the C.
sorokiniana NADP-GDH isoenzymes.

Figure 31. Model for the regulation of the C. sorokiniana
chloroplastic NADP-specific GDH isoenzymes. In the diagram
of the model, black boxes represent exons, the white area,
denoted by the arrow over exon two, indicates the
alternatively processed 42 nt auxon. The mRNA sizes do not
include the poly(A) tail. The white box in the 57850 D
precursor protein represents the additional 11 amino-terminal
residues of the a-subunit. The large open and filled circles
depict the mature a- and p- subunits, respectively.

163
P
( 52342 D ) p - Subunit
V.
O “ ■ Subunit ( 53501 D )
- Homohexamer
Low NH3- Affinity
(K - 75 mM )
Seven Isozymes
a - Homohexamer
High NH3- Affinity
( Km- 0.02 - 3.5 mM )
Covalent Modification /
Inactivation
Q-Q
NH3 Removal
Subunit Dimers
Degradation
Non - Antigenic Products

LIST OF REFERENCES
Alt FW, Bothwell ALM, Knapp M, Siden E, Mather E (1980)
Synthesis of secreted and membrane bound immunoglobulin
H heavy chains is directed by mRNAs that differ at their
3’ ends. Cell 20:293-302
Amersham (1985) Membrane Transfer and Detection Methods.
Amersham International pic, Arlington Heights, IL
Athwal GS, Laurie S, Pearson J, Phillips R (1994) The use of
carrot cell suspension culture to investigate the
regulation of glutamate dehydrogenase (abstract No.
168). Plant Physiol 105:S-40
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith
JA, Struhl E (1989) Current Protocols in Molecular
Biology. Wiley, New York
Bahring S, Sandig V, Lieber A, Strauss M (1994) Mapping of
transcriptional start and capping points by a modified
5' RACE technique. Biotechniques 16:807-808
Baker AL, Schmidt RR (1963) Intracellular distribution of
phosphorus during synchronous growth of Chlorella
pyrenidosa. Biochim Biophys Acta 74:75-83
Baker BS, Wolfner MF (1988) A molecular analysis of double¬
sex, a bifunctional gene that controls both male and
female sexual differentiation in Drosophilia
melanogaster. Genes Dev 2:477-489
Baker PJ, Britton KL, Engel PC, Farrants GW, Lilley KS, Rice
DW, Stillman TJ (1992) Subunit assembly and active site
localization in the structure of glutamate
dehydrogenase. Proteins: Struct Func Genet 12:75-86
Bark IC (1993) Structure of the chicken gene for SNAP-25
reveals duplicated exons encoding distinct isoforms of
the protein. J Mol Biol 233:67-76
Bascomb NF, Prunkard DE, Schmidt RR (1987) Different rates of
synthesis and degradation of two chloroplastic ammonium-
inducible NADP-specific glutamate dehydrogenase
isoenzymes during induction in Chlorella sorokiniana
cells. Plant Physiol 83:85-91
164

165
Bascomb NF, Schmidt RR (1987) Purification and partial
kinetic and physical characterization of two
chloroplast-localized NADP-specific glutamate
dehydrogenose isoenzymes and their preferential
accumulation in Chlorella sorokiniana cells cultured at
low or high ammonium levels. Plant Physiol 83:75-84
Bascomb NF, Turner KJ, Schmidt RR (1986) Specific polysome
immunoadsorption to purify an ammonium-inducible
glutamate dehydrogenase mRNA from Chlorella sorokiniana
and synthesis of full-length double stranded cDNA from
the purified mRNA. Plant Physiol 81:527-532
Batteiger B, Newhall WJ, Jones RB (1982) The use of Tween 20
as a blocking agent in immunological detection of
proteins transferred to nitrocellulose membranes. J
Immunol Methods 55:297-307
Bell LR, Maine EM, Schedl P, Cline TW (1988) Sex-lethal, a
drosophila sex determination switch gene, inhibits sex-
specific RNA splicing and sequence similarity to RNA
binding proteins. Cell 55:1037-1046
Beltzer JP, Morris SR, Kohlhau CTB (1988) Yeast LEU 4 encodes
mitochondrial and nonmitochondrial forms of a-
isopropylmalate synthase. J Biol Chem 263:368-374
Bingham PM, Chou TB, Mims I, Zachar Z (1988) On/off
regulation of gene expression at the level of splicing.
Trends Genet 4:134-138
Boggs RT, Gregar P, Idriss S, Bedote JM, McKeown M (1987)
Regulation of sexual differentiation in D. melanogaster
via alternative splicing of RNA from the transformed
gene. Cell 50:739-747
Blenis J, Marilyn R (1993) Subcellular localization specified
by protein acylation and phosphorylation. Curr Opin Cell
Biol 5:984-989
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein
utilizing the principle of protein dye binding. Anal
Biochem 72:248-254
Brawerman G (1993) mRNA degradation in eukaryotic cells. In J
Belasco, G Brawerman eds, Control of mRNA stability.
Academic Press, San Diego CA, pp 153-159
Breathnach R, Chambón P (1981) Organization and expression of
eukaryotic split genes coding for proteins. Annu rev
Biochem 50:349-383

166
Breitbart RE, Andreadis A, Nadal-Ginard B (1987a) Alternative
splicing; a ubiquitous mechanism for the generation of
multiple protein isoforms from single genes. Annu Rev
Biochem 56:467-495
Breitbart RE, Nadal-Ginard B (1987b) Developmentally induced,
muscle-specific trans factors control the differential
splicing of alternative and constitutive troponin-T
exons. Cell 49:793-803
Britton KL, Baker PJ, Rice DW, Stillman TJ (1992) Structural
relationship between the hexameric and tetrameric family
of glutamate dehydrogenases. Eur J Biochem 209:851-859
Brown JWS (1986) A catalogue of splice junction and putative
branch-point sequences from plant introns. Nucl Acid Res
14:9549-9559
Cammaerts D, Jacobs M (1985) A study of the role of glutamate
dehydrogenase in the nitrogen metabolism of Arabidopsis
thaliana. Planta 161:517-526
Cannon AC, Rendleton LC, Solomonson LP (1992) Regulation of
nitrate reductase mRNA in Chlorella. (abstract No.
35)Plant Physiol 99:S-6
Carlson M, Botstein B (1982) Two differentially regulated
mRNAs with different 5' ends encode secreted and
intracellular forms of yeast invertase. Cell 28:145-154
Carraway RE, Mitra SP (1990) Differential processing of
neurotensin/neuromedian N precursors in canine brain and
intestine. J Biol Chem 265:8627-8631
Carraway RE, Mitra SP, Duke GE (1993) A common precursor to
neurotensin and LANT6 and its differential processing in
chicken tissues. Peptides 14:1245-1251
Cavaner DR (1987) Comparison of the concensus sequence
flanking translational start-sites in Drosophilia and
vertabrates. Nucl Acid Res 15:1353-1361
Chabot B, Steitz JA (1987) Recognition of mutant and cryptic
5' splice-sites by the Ul small nuclear ribonuclear
proteins in vitro. Mol Cell Biol 7:698-707
Chatten B, Walter P, Ebel JP, Lacroute F, Fasiolo F (1988)
The yeast VAS 1 gene encodes both mitochondrial and
cytoplasmic valyl-tRNA synthetases. J Biol Chem 263:52-
57
Clark S (1993) Protein methylation. Curr Opin Cell Biol
5:977-983

167
Cock JM, Kim KD, Miller PW, Hutson RG, Schmidt RR (1991) A
nuclear gene with many introns encoding ammonium-
inducible chloroplastic NADP-specific glutamate
dehydrogeneases in Chlorella sorokiniana. Plant Mol Biol
17:17-27
Cock JM, Roof LL, Bascomb NF, Gehrke CW, Kuo KC, Schmidt RR
(1990) Restriction enzyme analysis and cloning of high
molecular weight genomic DNA isolated from Chlorella
sorokiniana (Chlorophyta). L Phycol 26:361-367
Cooper TA, Ordahl CP,(1985) A single cardiac troponin-T gene
generates embryonic and adult isoforms via
developmentally regulated alternative splicing. J Biol
Chem 260:11140-11148
Collomer A, Keen NT (1986) The role of pectic enzymes in
plant pathogenesis. Annu Rev Phytopathol 24:383-409
Davidson JN, Rao GN, Niswander L, Andreano C, Tamer C, Chen
K-C (1990) Organization and nucleotide sequence of the
3' end of the human CAD gene. DNA Cell Biol 9:667-676
Davis FC, Davis RW (1978) Polyadenylation of RNA in immature
oocytes and early cleavage of Urechis caupo. Develop
Biol 66:86-96
Davis RH (1986) Compartmental and regulatory mechanisms in
the pathways of Neurospora crassa and Saccharomyces
cerevisiae. Microbiol Rev 50:280-313
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set
of sequence analysis programs for the vax. Nucl Acid Res
12:385-387
Dobner PR, Barber D, Villa-Komaroff L, McKiernan C (1987)
Cloning and sequencing of cDNA for the canine
neurotensin/neuromedian N precursor. Proc Natl Acad Sci
USA 84:3516-3520
Enyedi A, Verma AK, Heims R, Adamo HP, Filoteo AG, Strehler
EE, Penniston JT (1994) The Ca2+ affinity of the plasma
membrane Ca2+ pump is controlled by alternative
splicing. J Biol Chem 264:41-43
Feinberg AP, Vogelstein B (1984) Addendum, A technique for
radiolabeling DNA restriction endonuclease fragments to
high specific activity. Anal Biochem 137:266-267
Fischer RL, Bennett AB (1991) Role of cell wall hydrolysis in
fruit ripening. Annu Rev Plant Physiol 42:675-703

168
Fischer R, Roller M, Flura M, Matthews S, Strehler-Page M,
Kribs J, Penniston JT, Carfoli E, Strehler E (1988)
Multiple divergent mRNAs code for a single human
calmodulin. J Biol Chem 263:17055-17062
Frohman MA (1990) Rapid amplification of cDNA ends. In DH
Gelford, JJ Snincky, TJ White, eds, PCR Protocols.
Academic Press, San Diego, CA, pp 28-38
Gaffe J, Tieman DM, Hamda AK (1994) Pectin methylesterase
isoforms in tomato (Lycopersicon esculentum) tissues.
Plant Physiol 105:199-203
Gamier J, Osguthorpe DJ, Robson B (1978) Analysis of the
accuracy and implications of simple methods for
predicting the secondary structure of globular proteins.
J Mol Biol 120:97-120
Gawienowski MC, Szymanski D, Perera IY, Zielinski RE (1993)
Calmodulin isoforms in Arabidopsis encoded by multiple
divergent mRNAs. Plant Mol Biol 22:215-225
Gray MW, Doolittle WF (1982) Has the endosymbiant hypothesis
been proven? Microbiological Review 46:1-42
Grotewold E, Athma P, Peterson T (1991) Alternatively spliced
products of the maize P gene encode proteins with
homology to the DNA-binding domain of myb-like
transcriptional factors. Proc Natl Acad Sci USA 88:4587-
4591
Hardwood JE, Kuhn AL (1970) A colormetric method for ammonia
in natural waters. Water Res 4:805-811
Harlow E, Lane D (1988) Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New
York
Harriman RW, Tieman DM, Handa AK (1991) Molecular cloning of
tomato pectin methylesterase gene and its expression in
Rutgers, ripening inhibitor, nonripening and never ripe
tomato fruits. Plant Physiol 97:80-87
Hawkins JD (1988) A survey of intron and exon lengths. Nucl
Acid Res 16:9893-9908
Henikoff S (1984) Unidirectional digestion with exonuclease
III creates targeted breakpoints for DNA sequencing.
Gene 28:351-359

169
Hirose T, Mamoru S, Sugiura M (1993) cDNA structure,
expression and nucleic acid-bind properties of three
RNA-binding proteins in tobacco: occurence of tissue-
specific alternative splicing. Nucl Acid Res 21:3981-
3987
Imamura K, Tanaka T (1982) Pyruvate kinase isozymes from rat.
Methods Enzymol 90:150-165
Israel DW, Granostajski RM, Yeung AT, Schmidt RR (1977)
Regulation of accumulation and turnover of inducible
glutamate dehydrogenase in synchronous cultures of
Chlorella sorokiniana. J Bact 130:793-804
Jain R, Gorner RH, Murtagh JJ (1992) Increasing specificity
from RACE-PCR technique. Biotechniques 12:58-59
Johnson DA, Gautsch JM, Sportsman JR, Elder JH (1984)
Improved technique utilizing non-fat dry milk for
analysis of proteins and nucleic acids transferred to
nitrocellulose. Genet Anal Tech 1:3-8
Jones ME (1980) Pyrimidine nucleotide biosynthesis in
animals: genes, enzymes and regulation of UMP
biosynthesis. Annu Rev Biochem 49:253-279
Julliard JH, Smith EL (1979) Partial amino acid sequence of
the glutamate dehydrogenase of human liver and a
reversion of the sequence of the bovine enzyme. J Biol
Chem 254:3427-3438
Katagui F, Chua N-H (1992) Plant transcription factors:
present knowledge and future challenges. Trends Genet
8:22-27
Kawasaki ES (1990) Amplification of RNA. In DH Gelford, JJ
Snincky, TJ White, eds, PCR Protocols. Academic Press,
San Diego, CA, pp 21-27
Keegstra K, Olsen W, Theg SM (1989) Chloroplastic precursors
and their trasport across the envelope membranes. Annu
Rev Plant Physiol 40:471-501
Kinnaird JH, Fincham JRS (1983) The complete nucleotide
sequence of the Neurospora crassa am (NADP-specific
glutamate dehydrogenase) gene. Gene 26:253-260
Kozak M (1984) Compilation and analysis of sequences upstream
from the translational start-site in eukaryotic mRNAs.
Nucl Acid Res 12:857-871
Kraft R, Tardiff J, Krauter KS, Leinwand LA (1988) Using
miniprep plasmid DNA for sequencing double stranded
templates with sequenase. Biotechniques 6:544-547

170
Kuhlemeier C (1992) Transcriptional and post-transcriptional
regulation of gene expression in higher plants. Plant
Mol Biol 19:1-14
Kwiatkowski D, Stossel TP, Orkin SH, Mole JE, Calten HR, Yin
HL (1986) Plasma and cytoplasmic gelsolins are encoded
by a single gene and contain a duplicated action binding
domain. Nature 323:455-458
Lai DML, Hoj PB, Fincher GB (1993) Purification and
characterization of (1-3, 1-4)-p-glucan endohydrolases
from germinated wheat (Triticum aesturim). Plant Mol
Biol 22:847-859
LeJohn HB, Cameron LE, Yong B, Rennie SL (1994) Molecular
characterization of an NAD-specific glutamate
dehydrogenese gene inducible by L-glutamine. J Biol Chem
269:4523-4531
Ling V, Perera I, Zielinski RE (1991) Primary structures of
Arabidopsis calmodulin isoforms deduced from the
sequences of cDNA clones. Plant Physiol 96:1196-1202
Loulakakis KA, Roubelakis-Angelakis KA (1991) Plant NAD(H) -
glutamate dehydrogenase consists of two subunit
polypeptides and their participation in the seven
isoenzymes occurs in an ordered ratio. Plant Physiol
97:104-111
Lovatt CJ, Cheny AH (1984) Aspartate transcarbamylase site of
end-product inhibition of the orotate pathway in intact
cells of Cucúrbita pepo. Plant Physiol 75:511-515
Lund E, Dahlberg JE (1987) Differential accumulation of Ul
and U4 small nuclear RNAs during Xenopus development.
Genes Dev 1:39-46
Lund E, Kahan B, Dahlberg JE (1985) Differential control of
Ul small nuclear RNA expression during mouse
development. Science 221:1271-1274
Maestri E, Restivo FM, Gulli M, Tassi F (1991) Glutamate
dehydrogenase regulation in callus cultures of Nicotiana
plumbagin folia: effect of glucose feeding and carbon
source starvation on the isoenzymatic pattern. Plant
Cell Environ 14:613-618
Magasanik B (1961) Catabolite repression. Cold Spring Harbor
Symposia on Quantitative Biology 26:249-257
Maley JA, Hyman BC, Lovatt CJ (1992) Evidence for two
carbomylphosphate syntheses in plants (abstract No.
535). Plant Physiol 99:S-90

171
Markoff AJ, Radford A (1978) Genetics and biochemistry of
carbomylphosphate biosynthesis and its utilization in
the pyrimidine biosynthetic pathway. Microbiol Rev
42:307-328
Markovic 0, Kohn R (1984) Mode of pectin deesterification by
Trichoderma reesei pectinesterase. Experientia 40:842-
843
Mattaj IW, McPherson MJ, Wooton JC (1982) Localization of a
strongly conserved section of coding sequence in
glutamate dehydrogenase genes. FEBS Lett 147:21-25
McBratney S, Chen CY, Sarnow P (1993) Internal initiation of
translation. Curr Opin Cell Biol 5:961-965
McPherson MJ, Wooten JC (1983) Complete nucleotide sequence
of teh Escherichia coli gdhA gene. Nucl Acid Res
11:5257-5266
Melzer JM, Kleinhofs A, Warner RL (1989) Nitrate reductase
regulation: effects of nitrate and light in nitrate
reductase mRNA accumulation. Mol Gen Genet 217:341-346
Meredith MJ, Gronostajski RM, Schmidt RR (1978) Physical and
kinetic properties of the nictonomide adenine
dinucletide-specific glutamate dehydrogenase purified
from Chlorella sorokiniana. Plant Physiol 61:967-974
Meredith MJ, Schmidt RR (1991) NAD-specific glutamate
dehydrogenase isoenzyme localized in mitochondria of
nitrate-cultured Chlorella sorokiniana cells. Plant
Physiol (Life Sci Adv) 10:67-71
Miflin BJ, Lea PJ (1976) The pathway of nitrogen metabolism
in plants. Phytochem 15:873-885
Miller ES, Brenchley JE (1984) Cloning and characterization
of gdhA, the structural gene for glutamate dhydrogenase
of Salmonella typhimurium. J Bact 157:171-178
Miller PW, Dunn WD, Schmidt RR (1994a) Preparative
nondenaturing gel electropheresis to purify NADP-
specific glutamate dehydrogenase from Chlorella.
BioRadiations (in press)
Miller PW, Russell BL, Schmidt RR (1994b) Transcriptional
initiation site of a NADP-specific glutamate
dehydrogenase gene and potential use of its promoter
region to express foreign genes in ammonium-cultured
Chlorella sorokiniana cells. J Appl Phycol 6:211-223

172
Miranda-Ham ML, Loyola-Vargus T (1988) Ammonia assimilation
in Canavalia ensiformis plants under water stress and
salt stress. Plant Cell Physiol 29:747-753
Moncrief ND, Kretsinger RH, Goodman M (1990) Evolution of EF-
hand calcium-modulated proteins. I. Relationships based
on amino acid sequences. J Mol Evol 30:522-562
Moustacos Am, Nari J, Bucel M, Noat G, Ricard J (1991) Pectin
methylesterase, metal ions and plant cell-wall
extensions: the role of metal ions in plant cell-wall
extension. Biochem J 279:351-354
Munoz-Bianco J, Cardenas J (1989) Changes in glutamate
dehydrogenase activity of Chlamydomonas reinhardtii
under different trophic and stress conditions. Plant
Cell Environ 12:173-182
Musgrove JE, Elderfield PD, Robinson C (1989) Endopeptidases
in the stroma and thylakoids of pea chlorplasts. Plant
Physiol 90:1616-1620
Neff NF (1993) Protein splicing: selfish genes invade
cellular proteins. Curr Opin Cell Biol 5:971-976
Nagoshi RN, McKeown M, Burtis KC, Belote JM, Baker BS (1988)
The control of alternative splicing at genes regulating
sexual differentiation in Drosophilia melanogaster. Cell
53:229-236
Nagy M, Le Gouar M, Potier S, Souciet JL, Herve G (1989) The
primary structure of the aspartate transcarbamylase
region of the URA2 gene product in Saccharomyces
cerevisiae: features involved in activity and nuclear
localization. J Biol Chem 264:8366-8374
Noguchi T, Inoue H, Tunaka T (1986) The Ml and M2 type
isozymes of rat pyruvate kinase are produced from the
same gene by alternative splicing. J Biol Chem
261:13807-13812
Noguchi T, Takada Y (1978) Purification and properties of
peroxisomal pyruvate (glyoxylate) aminotransferase from
rat liver. Biochem J 175:765-768
Nojima H (1989) Structural organization of multiple rat
calmodulin genes. J Mol Biol 208:269-282
Oda T, Funai T, Ichiyama A (1993) Generation from a single
gene of two mRNAs that encode the mitochondrial and
peroxisomal serine: pyruvate aminotransferase of rat
liver. J Biol Chem 265:7513-7519

173
Oda T, Yanagisawa M, Ichiyama A (1982) Induction of serine:
pyruvate aminotransferase in rat liver organelles by
glycogen and a high-protein diet. J Biochem 91:219-232
O'Neil KT, DeGrado WF (1990) How calmodulin binds its
targets: sequence independent recognition of amphiphilic
a-helices. Trends Biochem Sci 15:59-64
Perlman D, Halvorson HO (1981) Distinct repressible mRNAs for
cytoplasmic and secreted yeast invertase are encoded by
a single gene. Cell 25:525-536
Perlman D, Halvorson HO, Cannon LE (1982) Presecretory and
cytoplasmic invertase polypeptides encoded by distinct
mRNAs derived from the same structural gene differ by a
signal sequence. Proc Natl Accd Sci USA 79:781-785
Pickersky E, Gottlieb LD, Higgins RC (1984) Hybridization
between subunits of trióse phosphate isomerase isozymes
from different subcellular compartments of higher
plants. Mol Gen Genet 193:158-161
Plough M, Jensen AL, Barkholt V (1989) Determination of amino
acid composition and NH2-terminal sequences of peptides
electroblotted onto PVDF membranes from tricine-sodium
dodecyl sulfate-polyacrylamide gel electrophoresis:
application to peptide mapping of human complement
component C3. Anal Biochem 181:33-39
Polakis E, Bartley W (1965) Changes in the enzymatic
activities of Saccharomyces cerevisae during aerobic
growth on different carbon sources. Biochem J 97:284-297
Preiss T, Hall AG, Lightowlers RN (1993) Identification of
bovine glutamate dehydrogenase as an RNA-binding
protein. J Biol Chem 268:24523-24526
Prunkard DE, Bascomb NF, Molin WT, Schmidt RR (1986) Effect
of different carbon sources on the ammonium induction of
different forms of NADP-specific glutamate dehydrogenese
in Chlorella sorokiniana cells cultured in the light and
dark. Plant Physiol 81:413-422
Prunkard DE, Bascomb NF, Robinson RW, Schmidt RR (1986)
Evidence for chloroplastic localization of ammonium-
inducible glutamate dehydrogenase and synthesis of its
subunits from a cytosolic precursor-protein in Chlorella
sorokiniana. Plant Physiol 81:349-355
Recourt K, Ebellelaar MEM, Barbisan P, Laats MM, Wichers HW
(1992) Accession X68028, X68029, X68030. EMBL Databank

174
Rice DW, Baker PJ, Farrants GW, Hornby DP (1987) The crystal
structure of glutamate dehydrogenase from Clostridium
symbiosum at 0.6 nm resolution. Biochem J 242:789-795
Roberts DM, Lukas TJ, Watterson DM (1986) Structure,
function, and mechanism of action of calmodulin. CRC
Crit Rev Plant Sci 4:311-339
Rogers J, Early P, Carter C, Caíame K, Bond M (1980) Two
mRNAs with different 3' ends encode membrane bound and
secreted forms of immunoglobulin n chain. Cell 20:303-
312
Ross CW (1981) Biosynthesis of nucleotides In PK Stumpf, EE
Conn, eds, The Biochemistry of Plants. Academic Press,
New York, pp 169-205
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA (1986)
Analysis of enzymatically amplified p-globin and HLA-DQa
DNA with allele-specific oligonucleotide probes. Nature
324:163-166
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning:
A Laboratory Manual, eds, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA
74:5463-5467
Sarokin L, Carlson M (1984) Upstream region required for
regulated expression of the glucose-repressible SUC2
gene of Saccharomyces cerevisiae. Mol Cell Biol 4:2750-
2757
Sarokin L, Carlson M (1985) Upstream region of the SUC2 gene
confers regulated expression to a heterologous gene in
Saccharomyces cerevisiae. Mol Cell Biol 5:2521-2526
Santoni MJ, Barthels D, Vooper G, Boned A, Garidis C, Willie
W (1989) Differential exon usage involving an unusual
splicing mechanism generates at least eight types of
NCAM cDNA in mouse brain. EMBO J 8:385-392
Schagger H, von Jagow G (1987) Tricine-sodium dodecyl
sulfate-polyacrylamide gel electropheresis for the
separation of proteins in the range from 1 to 100 kDa.
Anal Biochem 166:368-379
Shibata H, Ochiai H, Sawa Y, Miyoshi S (1986) Localization of
carbomylphosphate sythase and aspartate
carbomyltransferase in chloroplasts. Plant Physiol
80:126-129

175
Smeekens S, Weisbeek P, Robinson C (1990) Protein transport
into and within chloroplasts. TIBS 15:73-76
Smith CWJ, Patton JG, Nadal-Ginard B (1989) Alternative
splicing in the control of gene expression. Annu Rev
Genet 23:527-577
Southern EM (1975) Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J Mol Biol
98:503-517
Srivastava HS, Singh RP (1987) Role and regulation of L-
glutamate dehydrogenase activity in higher plants.
Phytochem 26:597-610
Stillman TJ, Baker PJ, Britton KL, Rice DW, Rodgers HF (1992)
Effect of additives on crystallization of glutamate
dehydrogenase from Clostridium symbiosum: evidence for a
ligand-induced conformational change. J Mol Biol
224:1181-1184
Sun CW, Callis J (1993) Recent stable insertion of
mitochondrial DNA into an Arabidopsis polyubiquitin gene
by nonhomologous recombination. Plant Cell 5:97-107
Tabor S, Richardson CC (1987) DNA sequence analysis with a
modified bacteriophage T7 DNA polymerase. Proc Natl Acad
Sci USA 84:4767-4771
Tamplin ML, Martin AL, Ruple AD, Cook DW, Raspar CW (1991)
Enzyme immunoassay for identification of Vibrio
vulnificus in seawater sediment and oysters. Appl
Environ Micro 57:1235-1240
Tassig R, Carlson M (1983) Nucleotide sequence of the yeast
SUC2 gene for invertase. Nucl Acid Res 11:1943-1954
Tiam M, Maniatis T (1993) A splicing enhancer complex
controls alternative splicing of doublesex pre-mRNA.
Cell 74:105-114
Tischer E, DasSarma S, Goodman HM (1986) Nucleotide sequence
of an alfalfa glutamine synthetase gene. Mol Gen Genet
203:221-229
Towbin H, Gordon J (1984) Immunoblotting and dot
immunobinding: Current status and outlook. J Immunol
Methods 732:313-340
Towbin H, Stahlin T, Gordon J (1979) Electrophoretic transfer
of proteins from polacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc Natl Acad
Sci USA 76:4350-4354

176
Van Deusen RA, Whetstone CA (1981) Practical aspects of
producing and using anti-viral monoclonal antibodies as
diagnostic reagents. Ann Proc Am Assoc Vet Lab Diagn
24:211-228
Wallgrove JC, Hall NP, Kendall AC (1987) Barley mutants
lacking chloroplast glutamine synthetase: biochemical
and genetic analysis. Plant Physiol 83:155-158
Weiss RB (1991) Ribosomal frameshifting, jumping, and
readthrough. Curr Opin Cell Biol 3:1051-1055
Werenke JM, Chatfield JM, Ogren WL (1989) Alternative mRNA
splicing generates the two ribulosebisphosphate
carboxylase/oxygenase activase polypeptides in spinach
and Arabidopsis. Plant Cell 1:815-825
Werenke JM, Zielenski RE, Ogren WL (1988) Structure and
expression of a spinach leaf cDNA encoding rubisco
activase. Proc Natl Acad Sci USA 85:787-791
Williamson CL, Slocum RD (1994) Molecular cloning and
characterization of the pyrBl and pyrB2 genes encoding
aspartate transcarbamylase in Pea (Pisum satiuum L.).
Plant Physiol 105:377-384
Wolf K, Tanner W, Sauer N (1991) The Chlorella H+/hexose
cotransporter gene. Curr Genet 19:215-219
Yamaya T, Oaks A (1987) Synthesis of glutamate by
mitochondria. An anaplerotic function for glutamate
dehydrogenases. Physiol Plantarum 70:749-756
Yeung AT, Turner KJ, Bascomb NF, Schmidt RR (1981)
Purification of an ammonium-inducible glutamate
dehydrogenase and the use of its antigen affinity
column-purified antibody in specific immunoprecipitation
and immunoadsorption procedures. Anal Biochem 10:216-228

BIOGRAPHICAL SKETCH
Philip W. Miller was born in Cincinnati, OH, on August
9, 1962. He graduated from Lawrenceville Township High
School in Lawrenceville, IL, in May 1980, and entered Bob
Jones University in August of that year. He received a
Bachelor of Science degree in biology, with a minor in
English, in May 1984. After graduation, he worked as a
genetic research assistant for Agrigold Seed Company and as a
regional sales manager for PepsiCo Corporation before
entering the Master of Science program at Appalachian State
University in August of 1987. In January of 1990, he
transferred from Appalachian State University and entered the
Department of Microbiology and Cell Science at the University
of Florida where he began studies towards the Doctor of
Philosophy degree.
177

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
£
A
I¿1
Robert R. Schmidt, Chair
Graduate Research Professor
of Microbiology and Cell
Science
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
(l
in,
Phillipi/M. Achey
Professor of Microbiology
and Cell Science
r-
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Ddptpr of Philos
Richard P. Boyce
Professor of Biochemistry
and Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Francis C. Davis
Associate Professor of
Microbiology and Cell
Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
William B. Gurley7
Associate Professor of
Microbiology and Cell
Science
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philiosophy. Q zy
December, 1994 C7
Dean, College of Agriculture
Dean, Graduate School

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