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A model for the mechanism of agonism and antagonism in steroid receptors

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A model for the mechanism of agonism and antagonism in steroid receptors

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A MODEL FOR THE MECHANISM OF AGONISM AND ANTAGONISM IN
STEROID RECEPTORS
by
Sujatha Tanjore Subramanian
A Dissertation presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
in partial fulfillment o f the
requirements for the
MASTER’S DEGREE
(Biochemistry and Molecular Biology)
AUGUST 1998
Copyright 1998 Sujatha Tanjore Subramanian
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UMI Number: 1393189
UMI Microform 1393189
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UNIVS^SrTY O F SOUTHSHN c a u k c r n i a
r v » £ c ^ A C U A r s s c h o o l .
U N i v c H s r r r * a a k
- c s A N c r t rs. C a l i f o r n i a j o c o t
thesis, virzsten by
Sujatha Tanjore Subramanian
under the d irection o f h .,^ Thesis C om m ittee,
and a p p ro ve d by a ll its members, has been p re
sented to an d a ccep ted by the D ean o f T h e
G raduate S ch ool, in p a rtia l fu lfillm ent o f th e
requirem ents fo r the degree of
Master o f Science
06/30/1998
THESIS COMMITTEE
C f c iiM 'im n
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ACKNOWLEDGEMENT
I express my gratitude to the following luminaries and intellectually eminent
personalities without whose guidance, assistance and unstinted selfless cooperation,
this project study would have been impossible.
A word about my thesis advisor Dr. Michael R.Stallcup. He has been my friend,
guide and philosopher from the beginning o f my work in his laboratory. My special
thanks goes to the members o f the graduate committee Dr. Zoltan A.Tokes and
Dr. Robert H.Stellwagen. Their spontaneous understanding o f my requirements and
subsequent masterly guidance led to the frnalisation o f this project.
I would like to thank all of my colleagues for their friendship, advise, assistance and
moral support during my years in the laboratory.
I owe my sincere gratitude to my husband for all the support. I am forever indebted to
my parents for their love and guidance.
ii
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. , y v .
TABLE OF CONTENTS
TABLE OF ABBREVIATIONS.......................................................... vii
ABSTRACT viii
Chapter 1
INTRODUCTION.----------------------------------------------------------- 1
1.1 Honnone-receptor complexes........................................................... 3
1.2 Receptor transformation................................................................... 3
1.3 Anatomy o f the transcription apparatus............................................ 5
Chapter 2
MATERIALS AND METHODS________________________ 10
2.1 Yeast transformation............................................................................ 10
2.1.1 Lithium acetate transformation o f yeast (trp and leu marked
plasmids............................................................................................10
2.12 LiAc transformation of yeast (ura marker)......................................12
2.2 Liquid culture (3-galactosidase assays.................................................12
2.3 Yeast two-hybrid assays..................................................................... 14
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Chapter 3
3.1 Coactivator assays and Dose response curves.................................... 15
3.2 Yeast two-hybrid assays to assess SR-coactivator
interactions in the presence o f SR agonists and antagonists 32
3.2.1 The effect o f agonists....................................................................... 32
3.22 The effect o f antagonists.................................................................. 38
3.2.3 Competition between agonists and antagonists............................... 42
CHAPTER 4
DISCUSSION........................................................................................ 44
4.1 Does SR with antagonist dissociate from hsp 90 and bind DNA?. 46
4.2 Structural differences in SR’s w ith agonist and antagonist: 3D
data......................................................................................................... 46
4.3 Does data on each antagonist correspond to in vivo activity? 47
REFERENCES__________________________________________ 49
List of figures................................................................................................ 2
Figure 1. The human nuclear receptor family.......................................... 2
Figure 2. A simplified model o f steroid receptor action.......................... 4
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Figure 3. Model o f 3teroid receptor action............................................... 6
Figure 4. Model showing how the coactivator works............................. 8
Figure 5. Explains the strategy for yeast two-hybrid assay..................... 16
Figure 6. Agonists cause SRs to recruit coactivators to the gene promoter,
antagonists do not....................................................................... 45
List o f Graphs............................................................................................. 18
Legend to Graph 1..................................................................................... 18
Graph 1A.................................................................................................... 19
Legend to Graph 2...................................................................................... 20
Graph 2A.................................................................................................... 21
Graph IB .................................................................................................... 23
Graph 2B.................................................................................................... 24
Legend to Graph 3.......................................................................................25
Graph 3..................................................................................................... 26
Legend to Graph 4.......................................................................................27
Graph 4..................................................................................................... 28
Legend to Graph 5..................................................................................... 30
Graph 5..................................................................................................... 31
Legend to Graph 6..................................................................................... 33
Graph 6A.................................................................................................. 34
Graph 6B.................................................................................................. 35
Legend to Graph 7.................................................................................... 36
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Graph 7.................................................................................................... 37
Legend to Graph 8................................................................................... 39
Graph 8A................................................................................................. 40
Graph 8B................................................................................................. 41
V I
■ A -
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TABLE OF ABBREVIATIONS
hsp 90 Heat shock protein 90.
SRE Steroid hormone response element.
GRE Glucocorticoid response element.
ERE Estrogen response elem ent
NR (s) Nuclear receptor.
SR (s) Steroid receptor.
MR Mineralocorticoid receptor.
GR Glucocorticoid receptor.
AR Androgen receptor.
ER Estrogen receptor.
PR Progesterone receptor.
DBD DNA binding domain.
HBD Hormone binding domain.
DNA Deoxyribonucleic acid.
RNA Ribonucleic acid.
mRNA Messenger RNA.
GRIP 1 Glucocorticoid receptor interacting protein-1.
SRC-1 Steroid receptor coactivator-1.
AD(s) Activation domain.
LBD Ligand binding domain.
vii
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ABSTRACT
When target tissues are exposed to steroid hormones, in vivo or under
physiological conditions in vitro, steroid-receptor complexes are formed. The steroid
hormones or the ligand can be an agonist or an antagonist. An agonist is responsible
for stimulating the activity o f a receptor. An antagonist is pure when it completely
suppresses activity and is a partial agonist when it is able to suppress activity only
partially. In other words, there is some residual activity. M ost receptors are active in
the presence o f an agonist but are partially active or completely inactive in the
presence o f an antagonist Both agonists and antagonists can bind with high affinity to
steroid receptors, cause their dissociation from hsp 90 and promote binding o f steroid
receptors as homodimers to the hormone response element (HRE) associated with
target genes. However, only agonists can induce the ability o f steroid receptors to
activate transcription o f target genes, while antagonists cannot
The mechanism by which nuclear receptors (NRs) regulate the efficiency o f
transcriptional initiation could be through direct interactions between NRs and basal
transcription factors. A yeast-based genetic system demonstrated that NR effects on
the transcriptional initiation complex may be transmitted through indirect interactions
mediated by intermediary proteins called transcriptional coactivators. The ligand-
activated NR recruits a coactivator, and the coactivator communicates with the core
transcription machinery. This results in transcriptional activation.
V U l
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Each class o f ligand induces a distinct conformation in the nuclear receptor.
This results in the release o f hsp 90. The conformation induced by an agonist is able
to recruit a coactivator and results in increased transcriptional activation. An
antagonist is able to bind the nuclear receptor and cause release of hsp 90 resulting in
a conformational change. But this conformation is not favorable to recruit a
coactivator. Hence there is no transcriptional activation. There are some exceptions
where an antagonist is able to act as a partial agonist, hi that case the conformation is
closer to the active state. This provides structural evidence o f the mechanism o f
antagonism.
ix
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CHAPTER 1
INTRODUCTION
Steroid hormones are produced by endocrine glands. The hormone reaches die
target cell by traveling through the blood stream. The hormone then enters the cell and
binds a specific receptor (Tsai and O'Malley, 1994). Steroid hormone receptors
(Androgen receptor, Estrogen receptor, Progesterone receptor, Glucocorticoid receptor
and Mineralocorticoid receptor) belong to a structurally and functionally related group
o f intracellular proteins known as the nuclear receptor or the steroid/thyroid hormone
receptor superfamily. The nuclear receptors are present within the cell rather than on the
cell surface. After associating with their respective ligands, they eventually act as
transcription factors in the cell nucleus to enhance the expression o f specific genes
(Beato et al., 1995).
Steroid/thyroid hormone receptors are ligand-dependent transcription factors that
regulate diverse aspects o f growth, development and homeostasis. They bind as
homodimers or heterodimers to their cognate DNA response elements to modulate
transcription o f target genes.
The steroid receptors can be divided into several functional domains (Figure 1).
1. An N-terminal A/B domain. This region contains a transactivation function called
AF-1. When the hormone binding domain is deleted, this can function in a
hormone-independent manner.
2. A central C-region called DNA-binding domain. The C region contains two zinc
fingers which are responsible for DNA recognition and dimerization (Evans, 1988).
1
4
. .
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Figure L . The human nuclear receptor family.
A/B
i_____ !
i !
185
5 15
595
E R
3 0 /
687
! 933
P S .
4 2 1
532
603 738
///
669 558
984
918
G R
M R
AR
A/B, F : Modulating regions.
C : DNA-binding region.
D : ‘hinge’ region.
E : ligand-binding region.
Boxes indicate highly conserved domains.
Thin black lines are regions o f low homology.
The position of each domain boundary is given as the
number of aminoacids from the amino terminus.
Modified from Jensen, 1991.
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3. D region also known as die hinge region. It allows the protein to bend or alter
conformation. It contains a nuclear localization domain and or a transactivation domain.
4. A C-terminal hormone binding domain (HBD) or E region. It contains a
transactivation function called AF-2. This is hormone dependent
5. The last region is the variable F region. No specific function has been identified.
1.1 Hormone-receptor complexes:
A simplified model o f steroid hormone action: hi the 1970s it was thought that steroid
hormone (S) enters target cells and binds to the steroid receptor to form a hormone
receptor complex (SR). Binding o f steroid hormone activates the receptor (SR*), which
can then bind to DNA target element to stimulate mRNA synthesis. The new mRNAs
code for new proteins that result in the change of cellular function. This model, although
lacking detail, is still largely correct (Shibata et al., 1997 and Figure 2).
1.2 Receptor transformation.
Before the hormone binds the receptor, SRs remain inactive in a complex with hsp 90
and other stress family proteins. It was demonstrated that the hsp 90 obscures the
molecular domain that is responsible for DNA binding. Once the hormone binds the
receptor, the receptor undergoes a conformational change. This causes the receptor to
dissociate from hsp 90 (Pratt et al., 1989). The transformed receptor then binds as a
homodimer or a heterodimer to specific DNA enhancer elements associated with target
genes to modulate transcription.
3
. f lW V . • • •
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Figure. 2 A simplified model of steroid receptor action.
S > SR > SR* > SR*-DNA--------- > TmRNA
TProtein--------- > “Function”
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Thus the steroid hormones play die crucial role o f converting the native receptor to a
biochemically functional state. This can then bind in the nucleus and enhance gene
transcription (Jensen and Desombre, 1973). This receptor transformation step is a
general phenomenon that is seen in all classes o f steroid hormones.
Once the receptor has been transformed to its biochemically functional form, it
recognizes DNA sequences in the vicinity o f the regulated promoter. These nucleotide
sequences called hormone response elements or HREs must be present for the adjacent
gene to respond to hormone. HREs are binding sites for hormone activated steroid
receptors found in the 5-flankmg region o f hormone-responsive genes (Tsai et al.,
1991). Deletion or mutation o f SREs causes a dramatic decrease in the inducibility o f
target genes by hormones (Chalepakis et al., 1988).
1.3 Anatomy of the transcription apparatus :
The mechanism by which DNA-bound steroid receptors can activate transcription
initiation from associated promoters is by facilitating the assembly of basal transcription
factors into a stable pre-initiation complex. In addition to RNA polymerase II, foe pre-
initiation complex consists o f seven basal transcription factors, namely TFIIA, i n IK,
TATA-box binding protein (a subunit o f TFUD), TFHE, T F E D F , TFHH and TFEJ. This
complex alone can initiate transcription at a basal rate. Increase or decrease in
transcription is brought about by regulatory molecules known as activators or
repressors. Activators bind at sites on foe gene known as enhancers and they increase
foe efficiency o f transcription initiation. Repressors bind at sites known as silencers and
they decrease transcription initiation (Figure 3).
5
• • ~ ~ . /* • ;
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Figure 3. Model of steroid receptor action.
P23j P53 ;
R c
H 90
H 70
Salt
Rc
Rc
Rc
Rc
SRE
p o l h
R c
R c
E/F
SRE
T A T A
NTPs
PO LH
R c R c
E /F
I D
SRE
d >
Rc : Receptor
SRE : Steroid response element
B, D, E/7 and Pol II denote the transcription factors
TFIIB. I F LIU, TFHE7 and RNA polymerase II respectively.
P23 and P53 are 23 and 53 kD proteins respectively.
Modified from Tsai et al.. 1991.
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Activators and repressors communicate with basal factors through coactivators.
Various coactivator proteins may be linked in a tight complex with the TATA-box
binding protein (TBP) or recruited directly by the activators. The coactivators are
"adapter" molecules that integrate signals from activators and relay the results to the
basal factors (Verrijzer and Tjian, 1996 ; Glass et al., 1997 ).
Coactivators associate with SRs and enhance their ability to transactivate target
genes. They mediate transcriptional activation by DNA-bound activator proteins by
forming a bridge (Figure 4). One such coactivator is the Glucocorticoid receptor
interacting protein (GRIP1) that interacts with steroid receptor HBDs in a hormone-
dependent manner. GRIP1 can interact with the transcription machinery in vivo and
serve as a coactivator for the transcriptional activation function of steroid receptor
HBDs (Hong et al., 1996). GRIP1 belongs to a family o f three related coactivator
proteins that includes SRC-1 and pCIP (Onate et al., 1995 and Torchia et al., 1997).
The role o f SRC-1 was evaluated using transient transfection assays.
Transcriptional activation by the progesterone receptor (PR) was inhibited by a
dominant-negative PR-HBD interacting region o f SRC-1. This indicated that SRC-1
was required for actual transcriptional processes (Onate et al., 1995).
* I * r * • ■
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Figure . Mode: snowuig aow coac::va:or works.
KSD-
Gal4. site
S
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Previous studies (Onate et al., 1995 and Hong et al., 1997) showed that agonists
cause SRs to recruit coactivators to the gene promoter but antagonists cant recruit
coactivators, hi an attempt to determine whether this conclusion applies to all
antagonists and SRs and to understand the mechanism o f antagonism, the following
strategy was adopted: Dose response curves were conducted to find out the saturating
concentrations o f ligand. This will also answer the question about the role o f GRIP1 and
SRC-1 as a coactivator. Is it able to act in a ligand-dependent manner and activate
transcription in the presence o f an agonist? Is there a difference in die ability o f a pure
antagonist, a partial agonist, or an agonist to transform the SRs into a shape that can
recruit coactivators?
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CHAPTER 2
MATERIALS AND METHODS
2.1 Yeast transformation:
A series o f yeast transformations were performed (lithium acetate protocol, Gietz et al.,
1995) and the transformants were used to conduct dose response experiments. This will
give an indication of the saturating concentration (the concentration of the ligand in
which p-galactosidase activity is the maximum).
The yeast strain into which the plasmids were transformed for the whole study
was YNK100. This was previously known as Ieml-1 (Kralli et al., 1995).
The following paragraphs describe the various transformations :
2.1.1 Lithium acetate transformation of yeast (trp and leu marked plasmids):
The receptor PR full length was encoded on YEp (yeast episomal plasmid) expression
vector and ERhbd was on pGBT9. pGBT9 is a vector for expressing GAL4-DBD fusion
proteins. These plasmids contain a trp marker. For dose response curves, pGRIPl
(expressing GRIP1 full length) was on a modified pGAD424 vector and for yeast two-
hybrid assays, GRIP1 was on pGAD424 which is a vector for expressing GAL4-AD
fusion proteins. The vectors for pGAD424 and GR1P1 contained leu marker (Hong et
al., 1996).
10
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Steps involved in transformation are as follows:
Cells were inoculated from a single colony (2-3 mm in diameter) into 10 ml YPD. It
was incubated at 30°C with shaking for 16-18 hours. The yeast strain used was YNK
100. Since this yeast strain grows slowly it may take more time. OD600 should be >1.5
and in this case OD600 was 1.9. 8 ml o f this overnight culture was transferred to 100 ml
YPD in a 250-ml flask to get an OD600 o f 0.2-0.3. It was incubated at 30°C with
shaking for 3 hours. The OD600 was 0.5.
Cells were centrifuged at 2500 rpm for 15 min. at 4°C and the pellet was
resuspended in 20 ml water. It was centrifuged as before and the pellet was resuspended
in 0.5 ml fresh sterile 1 X TE/LiAc. For each transformation, the following materials
were added in a microtube: (i) Each type o f plasmid DNA, 0.1 pg. (ii) Herring Salmon
sperm DNA (10 pg/pl), lOpl. The carrier DNA was heated at 100°C for 3 min and then
placed on ice. Then it was added to the microtube, (iii) Yeast competent cells, 100 pi.
The materials were mixed well and then 600 pi sterile PEG/LiAc solution was
added. It was vortexed to mix and then incubated at 30°C for 30 min. 70 pi DMSO was
added and mixed gently by inversion. It was heat shocked at 42°C for 15 min. Cells
were chilled on ice and centrifuged for 5 sec in a microcentrifuge. Cell pellets were
resuspended by pipetting with 0.5 ml o f 1 X TE.
1 1
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When two plasmids are transformed at the same time, it is a double plasmid
transformation. 250 pi (double plasmid) o f the transformation mixture was spread onto
each 100 mm plate with proper selection medium (-trp-leu). Since the plasmids
contained trp and leu markers, the selection medium used was -trp-leu so that the
transformants will grow on -trp-leu plates. It was incubated at 30°C for 2 days.
2.1.2 LiAc transform ation o f yeast (ura m arker): The steps involved are:
After transforming plasmids with trp and leu markers, foe next step is transformation of
a third plasmid with a ura marker and a reporter gene. In a triple plasmid transformation,
foe transformation is done in two steps because this way the efficiency is better than
when all foe plasmids are transformed at foe same time. The transformation protocol
was foe same as foe steps involved in transformation o f trp and leu markers. 250 pi of
foe transformation mixture was spread onto each 1 0 0 -mm plate with proper selective
medium (-trp-ura-leu). Since this is a triple plasmid transformation, foe selection
medium is without trp, ura and leu. The plates were incubated at 30°C for 2 days. Once
foe transformants were ready, foe next step was to perform liquid culture (3 -
galactosidase assay.
2.2. Liquid culture (3-galactosidase assay:
The assay (Bohen and Yamamoto, 1993) was performed as follow s:
5 ml liquid SD medium (- trp-ura-leu) was inoculated with yeast colony. It was grown
overnight at 30°C with shaking sc that cultures could get saturated. 20 pi was
transferred to 180 pi ofSD medium plus hormone, ft was incubated at 30°C for 12 to 16
hours. To prevent evaporation, plates were placed on a wet paper towel and foe two
12
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were put in a folded plastic bag. Yeast would have grown to mid-log phase. Cells in
each well were resuspended with pipette action. 80 pi was transferred into new
identically labeled plate. 80 pi o f 2 X Z buffer was added to each well with
resuspension. The plate was read at OD595 at this point The new plate was set at -70°C
for 30 min. to lyse the cells. Frozen cells were removed and thawed at 37°C for 15 min.
Using the multipipetter, 20 pi Z buffer/ (3-mercaptoethanol was added to the entire plate.
20 pi ONPG was added quickly to the entire plate. Hie time was started immediately.
The initial time point OD 410-595 reading (dual wavelength) was taken.
Test filter = 410 nm and Reference filter = 595 nm. Plates were incubated at 37°C. After
the development of yellow color, OD 410-595 was read again (second time point
reading). P-galactosidase units were calculated as follow s:
Activity units are defined as: A = 1000 x (AOD410-595 x cv) / (OD595 x sv x min)
where AOD410.595 = change in OD410.595 (ODuwr - O Dtim eA)
OD595 = absorbance o f the cultures at 595 nm.
OD410 = absorbance o f P-galactosidase assay at 410 nm.
cv = volume of culture used to determine OD595 » 160 pi.
sv = volume of culture used for the assay » 2 0 0 pi.
Serial dilutions o f steroid agonists and antagonists were used. The concentration
range was from 10' 5 M to 10*10 M.
13
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2 3 Yeast two-hybrid assays: This assay is an in vivo system for detecting protein-
protein interactions and was performed as described previously (Hong et al., 1996). The
nuclear receptor hormone-binding domain was fused with a GAL4 DNA binding
domain in pGBT9 expression vector. The coactivator and its fragments were fused with
the GAL4 activation domain in pGAD424 vector. The pLGSD5 reporter has a GALA
binding sequence. The assay throws light on whether GRIP1 is able to interact with the
nuclear receptor in a ligand-dependent manner.
The dose response curves were performed for each steroid receptor with empty
vector (pGAD424) and with GRIP1. The tests were performed in the presence and
absence o f ligands (either an agonist or an antagonist).
14
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CHAPTER 3
RESULTS
The idea behind this study is to explain the mechanism o f antagonism and to test
whether all antagonists act by the same mechanism. The coactivator is able to increase
transactivation in the presence o f an agonist But in the presence o f an antagonist the
coactivator is not able to increase transcriptional activity. So there could be a difference
in the ability of a partial agonist a pure antagonist or an agonist to transform the SRs
into a shape that can recruit a coactivator. To evaluate this hypothesis the following
strategy was used: Plot dose response curves to find out the saturating concentration o f
ligand. This can be done by performing a coactivator assay in yeast This will also
confirm the role of GRJP1 and SRC-1 as a coactivator. Then yeast-two hybrid assays
will test for protein-protein interactions between the coactivator and the nuclear
hormone receptor in the presence of various ligands. The results will enable us to come
up with a model for the mechanism o f antagonism.
3.1 Coactivator assays and Dose response curves:
The different components that will be put into yeast for the test are : Either full length
receptor or the SR HBD fused to a GAL4 DNA binding domain is used for the assay.
The test is performed both in the presence and absence o f full length GRIP1 or GRIP1
fused to the GAL4 activation domain. To confirm the interaction, a (3-galactosidase
reporter gene with either one or three SREs (GRE 1 or GRE 3) or a pLGSD5 reporter
gene with a GAL4 binding sequence will be used (Figure 5). The assay is performed in
the presence of varying concentrations o f ligand.
15
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Figure 5. Explains the strategy tor yeast two-hybrid assay.
16
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The above experimental setting will indicate the saturating concentration o f die
ligand that will be needed to carry out the assay. It will indicate whether die coactivator
is able to increase transactivation in a ligand-dependent manner. It will also explain why
there is a difference in activity in the presence o f GRIP1 when compared to activity in
the absence o f GRIP1.
For full length PR, the assay was performed using different concentrations of
ligand (PR agonist Progesterone) and in the presence and absence o f full length GRIP1.
The hormone response element was present in a single copy (GRE 1) on the reporter
gene. The (3-galactosidase activity saturated at 10' 5 M Progesterone (Graph 1A). In the
presence of agonist Progesterone, full length GRIP1 was able to potentiate activity when
compared to activity without full length GRIP1 by about 2-fold. When the assay was
performed using different concentrations o f PR antagonist RU 486 in the presence and
absence o f full length GRIP1, the activity did not saturate even at 1 0 * s M RU 486
(Graph 2A). There is potentiation o f activity by full length GRIP1 in the presence of
antagonist when compared to activity in the absence o f full length GRJPl. So RU 486
may be acting as a partial agonist although the P-galactosidase units were small
compared with activity in the presence o f agonist (Graph 1 A).
17
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Graph 1: GRIP1 Coactivator assay using full length PR and an agonist
A. Full length PR was expressed in yeast both in the presence and absence o f GRIP1.
The P-galactosidase reporter gene contained the hormone response element in a
single copy. Serial dilutions o f the agonist Progesterone were used and as control,
the test was done in the absence o f hormone.
B. The above experiment was repeated with the hormone response element being
present in three copies.
p-galactosidase activities of extracts from the liquid yeast cultures are expressed as the
mean and standard deviation o f ( 3-galactosidase units from assays done in
quadruplicate.
18
- - ■ . ■ .
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G ra p h IA
+ V
t r c :
a c .
© © G
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m
CM
ir> t /:
rjrun |b 5 - C
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tO-1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -8 10-5
(Progesterone] M
Graph 2 : GRIP1 Coactivator assay using full length PR and an antagonist.
A. Full length PR was expressed in yeast in the presence and absence o f GRIP1.
(3-galactosidase reporter gene contained the hormone response element in a single
copy. The antagonist RU 486 was used in serial dilutions and as a control the assay
was conducted in the absence of hormone.
B. The above experiment was repeated with the hormone response element being
present in three copies.
(3-galactosidase activities o f extracts from the liquid yeast cultures are expressed as the
mean and standard deviation o f |3-galactosidase units from assays done in quadruplicate.
20
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[R U < 1 8 6 ] M
The above assay was repeated with PR when the hormone response element was present
in three copies (GRE 3). The activity saturated at 1 0 " * M Progesterone. Full length
GR1P1 potentiated activity by as much as 13 fold when compared to activity in the
absence o f GRIP1 (Graph IB). With antagonist RU 486, the activity almost saturated at
10' 5 M RU 486. Not too much difference was observed in activity with and without foil
length GRIP1. The p-galactosidase units (activity) was higher than that which was seen
when the hormone response element was present in a single copy (Graph 2B).
The ER dose response curve was performed with ER HBD fused to GAL4 DBD
in a yeast two-hybrid assay form at The assay was performed using different
concentrations o f ligand and in the presence and absence o f foil length GRTP1. In the
presence o f foil length GRIP1 the activity saturated at 3 x 10*10 M Estradiol. There was
no activity in the absence o f GRIP1 (Graph 3).
ER antagonist 4-hydroxy Tamoxifen was tested with foil length ER in the
presence and absence o f foil length GRIP1 in a coactivator assay form at Activity nearly
saturated at I O ' 5 M 4-hydroxy Tamoxifen. Full length G R J0P 1 was able to potentiate
activity when compared to activity in the absence of foil length GRIP1. So 4-hydroxy
Tamoxifen was able to act as a partial agonist (Graph 4).
22
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G ra p h 1 1 3
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M l [981' n ti)
Graph 3. Yeast two-hybrid assay for ER HBD binding to GRIP1 in the presence of
an agonist:
ER and GRJP1 proteins were expressed in yeast as follows :
The ER HBD was fused to the GAL4 DBD. The coactivator GRIP1 was fused to the
GAL4 activation domain. The reporter gene pLGSD5 has a GAL4 binding sequence.
The test was done in the presence of GRJP1-GAL4 AD fusion protein (+ G R D P 1 full
length) or in the presence of GAL4 AD alone (+ pGAD424). Different concentrations of
ER agonist Estradiol were used for the assay.
P-galactosidase activities o f extracts from the liquid yeast cultures are expressed as the
mean and standard deviation of P-galactosidase units from assays done in
quadruplicate.
25
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U J U J
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G raph 4. GRIP1 Coactivator assay using full length ER and an antagonist:
ER antagonist 4-hydroxy Tamoxifen was tested in yeast with full length ER in the
presence and absence of GR1P1. The reporter gene contained the estrogen response
element in a single copy. Serial dilutions o f the antagonist were used to do the assay.
(3-galactosidase activities of extracts from the liquid yeast cultures are expressed as the
mean and standard deviation o f p-galactosidase units from assays done in
quadruplicate.
27
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The androgen dose response curve was conducted with the AR HBD fused to
GAL4 DBD in a yeast two-hybrid form at The assay was performed using different
concentrations of AR agonist Dihydrotestosterone in the presence and absence o f foil
length GRIP1 fused to GAL4 AD. Activity saturated at JO'7 M Dihydrotestosterone. The
GRIP1 fusion protein was able to potentiate activity whereas there was no activity in the
absence o f GRIP1 (Graph 5). GRJPl binds to the Androgen receptor in a ligand
dependent manner.
The saturating concentration for the GR agonist deoxycorticosterone was
determined to be 10-6 M by previous experiments (Nam K. Bui, personal
communications).
GRIP1 is able to increase transcriptional activation in a ligand-dependent manner
with full length steroid receptors. The activity in the absence o f GRJPl is because yeast
has an endogenous mechanism to support transactivation of AF-1 domain but not the
AF-2 of SRs (Hong et al., 1997). The enhancement of activity is because GRJPl is able
to act as a coactivator o f the AF-2 domain.
Potentiation of transcriptional activation was generally more dramatic in the
presence o f agonist than in the presence o f antagonist This means that the mechanism
o f agonism is different from the mechanism o f antagonism. The ability o f GRJPl to act
as a coactivator of the AF-2 domain with different ligands may depend on the ability of
the ligand to promote receptor binding to GRJPl. This hypothesis will be tested by two-
hybrid assays which is an in vivo system to study protein-protein interactions.
29
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Graph 5. Yeast two-hybrid assay for AR HBD binding to GRIP1 in the presence of
an agonist:
The Androgen dose response curve was conducted in yeast with the AR HBD fused to
the GAL4 DBD. GRIP 1 was fused to a GAL4 activation domain. The reporter gene
pLGSD5 has a GAL4 binding sequence. The assay was performed using different
concentrations o f AR agonist Dihydrotestosterone in the presence o f GRIP1-GAL4 AD
fusion protein (+ GRIP1 full length) or in the presence of GAL4 AD alone
(+ pGAD424).
(3-galactosidase activities o f extracts from the liquid yeast cultures are expressed as the
mean and standard deviation of |3-galactosidase units from assays done in
quadruplicate.
30
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c © ^
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10-10 1 0 -9 1 0 -8 10-7 10-6
[Dlliyrirolcstosterone] M
3.2 Yeast two-hybrid assays to assess SR-coactivator interactions in the presence of
SR agonists and antagonists :
3.2.1 The effect of agonists :
Yeast two-hybrid system is an in vivo assay to test protein-protein interactions. The
strategy is to fuse the SR HBD with the GAL4 DNA-binding domain. The full length
GRIP1 or SRC-1 is fused to the GAL4 activation domain. The reporter gene pLGSD5
has a GAL4 binding sequence. Interaction o f the two fusion proteins results in the
reconstitution o f a functional GAL4 protein which can subsequently activate a (3-
galactosidase reporter gene containing an enhancer element recognized by the GAL4
DBD. The f3-galactosidase assay gives an indication of the amount of protein-protein
interaction.
Agonists o f AR, GR and PR were tested in the yeast two-hybrid system to check
the ability to promote interaction between the GAL4dbd-NRhbd and GRJPl or SRC-1
fused to GAL4 AD. The AR agonist Dihydrotestosterone (Graph 6A) promoted a strong
interaction between ARhbd and GRIP1. In the same way, the GR agonist DOC
promoted a strong interaction between GRhbd and GRIP1 (Graph 6B). The PR agonist
Progesterone also promoted a strong interaction between the PR HBD and SRC-1 full
length (Graph 7).
32
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G raph 6 . Yeast two-hybrid assay using AR o r GR HBD with an agonist,
antagonist and com petition:
Li these yeast two-hybrid assays, the AR or GR HBD was fused to the GAL4 DBD.
GRIP1 was fused to a GAL4 activation domain (GRIP1- full length). pGAD424
expresses the GAL4 AD alone. The pLGSD5 reporter has a GAL4 binding sequence.
Sub saturating concentration o f agonist was used and a saturating concentration of
antagonist was used to perform the assay.
A. This shows AR HBD and ligands. The agonist used was Dihydrotestosterone (DHT)
and the antagonist used was Hydroxyflutamide (HF).
B. This shows GR HBD and ligands. The agonist used was Deoxycorticosterone
(DOC) and the antagonist used was RU 486.
( 3-galactosidase activities o f extracts from the liquid yeast cultures are
expressed as the mean and standard deviation o f (3-galactosidase units from assays done
in quadruplicate.
33
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G ra p h 6A
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Graph 7. Yeast two-hybrid assay using PR HBD with an agonist, antagonist and
competition:
In this yeast two-hybrid assay, the PR HBD was fused to a GAL4 DBD. The assay was
performed with SRC-I full length fused to the GAM activation domain (SRC-1 full
length). pGAD424 expresses GAM AD alone. The pLGSD5 reporter gene has a GAM
binding sequence.
The agonist used was Progesterone (Prog) and the antagonist used was RU 486.
|3-galactosidase activities o f extracts from the liquid yeast cultures are
expressed as the mean and standard deviation o f P-galactosidase units from assays done
in quadruplicate.
36
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Agonists o f MR and ER were also tested in the yeast two-hybrid system to test the
ability to promote interaction between the GAL4 DBD-NR HBD and full length GRIP1
or SRC-1. The ER agonist Estradiol was able to potentiate interaction strongly between
the HBD and the coactivators (Graph 8A). The MR agonist Aldosterone also
potentiates interaction between the HBD and the coactivators (Graph 8B).
This confirms that the coactivator (Full length GRIP1 or full length SRC-1) is able
to bind to the NR HBD in the presence o f an agonist This is consistent with its role as a
coactivator.
3.2.2 The effect of antagonists :
Although RU 486 acts as a partial agonist with full length PR (Graph 2A and 2B) it
failed to promote interaction o f PR HBD or GR HBD with GRIP1 in yeast two-hybrid
assays (Graph 6B and 7). AR antagonist Hydroxyflutamide also failed to promote
interaction of AR HBD with GRIP1 (Graph 6A).
However, some of the antagonists did promote SR HBD-coactivator binding in
two-hybrid assays. 4-hydroxy Tamoxifen with full length ER acted as a partial agonist
in dose response curves (Graph 4) but did not promote ER HBD-coactivator binding in
two-hybrid assay (Graph 8A). However, MR antagonist Spironolactone (Graph 8B) and
ER antagonist ICI 182 780 (Graph 8A) promoted binding o f SR HBD to coactivators
although the binding was weaker than that observed with agonists.
38
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Graph 8. Yeast two-hybrid assay using ER or MR HBD with an agonist,
antagonist and competition:
In these yeast two-hybrid assays, the receptor HBD o f ER or MR was fused to the
GAL4 DBD. The test was performed using either GRIP1 or SRC-I fused to GAL4 AD
to check if the receptor could recruit the coactivators.
A. This shows ER HBD using an agonist Estradiol and the antagonists ICI and 4-
hydroxy Tamoxifen (4-OH Tam).
B. This shows MR HBD using an agonist Aldosterone (Aldo) and an antagonist
Spironolactone (Spiro).
p-galactosidase activities o f extracts from the liquid yeast cultures are
expressed as the mean and standard deviation of p-galactosidase units from assays done
in quadruplicate.
39
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3.23 Competition between agonists and antagonists :
The agonist was used in sub saturating level and the antagonist was used in saturating
level. The two were mixed in order to do this assay. In some cases the antagonist alone
did not promote any activity. This result could be interpreted in two ways.
1. The antagonist binds to the receptor but does not promote receptor-coactivator
interaction or
2. The antagonist is not able to bind to the receptor at the concentration used here.
hi the competition assays, if agonist plus antagonist has lower activity than agonist
alone, then this ensures that the antagonist is at a concentration that will enable it to bind
to the receptor. For MR (Graph 8B) when agonist and antagonist were mixed together
(aldosterone and spironolactone), in the presence o f GREP1 or SRC-1 the activity was
reduced when compared to activity with agonist alone. Spironolactone gave the same
amount o f activity in the presence o f SRC-1 or GRIP1 when it was used by itself and
when the agonist and antagonist were mixed together. For ER, in the presence o f GRIP1
or SRC-1, when the agonist Estradiol and antagonist 4-hydroxy Tamoxifen were
combined the activity was lower than when the agonist was used alone (Graph 8A). But
4-hydroxy Tamoxifen by itself promoted no interaction of ER HBD with GRIP1 and
SRC-1.
42
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For PR, when the agonist and antagonist were combined (Progesterone and RU
486), the activity was almost the same as when the agonist was alone. RU 486 by itself
promoted no interaction with SRC-1 full length (Graph 7). The reduction in activity that
was expected when the agonist and antagonist were combined was not seen and hence
this result did not indicate whether the RU 486 concentration was sufficient to bind PR.
For GR, there was reduced activity when the agonist and antagonist were combined
compared with activity when the agonist was used alone. The antagonist RU 486 did not
show any activity by itself (Graph 6B). For AR, die activity was reduced when the
agonist and antagonist were combined compared with activity when the agonist was
used alone; and the antagonist alone promoted no activity (Graph 6 A).
The result with agonist indicates that the coactivator is able to bring about
transcriptional activation in a ligand dependent manner. The agonist is able to induce a
conformational change in the nuclear receptor so that it is able to recruit a coactivator.
In the case o f AR, GR and PR, the antagonist didn’t bring about transcriptional
activation. The antagonist induces a conformational change in the nuclear receptor but
the conformation is not suitable to recruit a coactivator. In the case o f ER and MR, some
antagonists did promote binding to coactivators. In this case, the conformation o f the
nuclear receptor is closer to the active state and hence the receptor partially succeeds in
recruiting a coactivator. This could explain partial agonism in mammalian cells. The
fact that there is reduction in activity when die agonist and antagonist are combined
indicates that the antagonist is able to bind to the receptor. This enables us to come up
with a model to explain the phenomenon o f antagonism.
43
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CHAPTER 4
DISCUSSION
Yeast cells have an endogenous mechanism to support the transactivation activity
o f the AF-1 domain but not the AF-2 domain of SRs. AF-2 function in yeast requires
GRIP1 as a coactivator whereas AF-1 is GRIP1 independent (Hong et al., 1997). The
results presented here prove that both GRIP1 and SRC-1 bind to the AF-2
transactivation function.
NRs have different sensitivities to agonist and antagonist. There is enhanced
activity with agonist alone and no activity or partial activity with antagonist alone. The
competition assays showed that the antagonist is able to compete with agonist to bring
about reduced transcriptional activation. The agonist induces a suitable conformation in
the SR such that it is able to recruit the coactivator. Hence there is high SR activity with
agonist But when the antagonist binds, the SR assumes an unfavorable conformation
and this is not able to recruit a coactivator. In this case the antagonist is a pure
antagonist But sometimes the antagonist is able to assume a conformation that is closer
to the normal conformation. This enables some antagonists to act as partial agonists
because they are partially successful in recruiting a coactivator (Figure 6). This model
gives valuable insight into the binding of ligands to these receptors and provides the
basis for model-based designs o f unproved agonists and antagonists for the treatment of
diseases.
44
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Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission.
figure () Agonists cause Sits lo recruit coactivalors to the gene promoter, antagonists do
not
Basal transcription
factors
Transcription
Basal transcription
Transcription
S R b in d in g s ite C o re p ro m o te r SR binding site C ore prom oter
4.1 Does SR with antagonist dissociate from hsp 90 and bind DNA?
From the results, we can conclude that agonists o f ER, MR, AR, GR and PR promote a
strong interaction between the NR HBD and GRIP1 but antagonists generally do not
promote this interaction. The failure o f antagonists to stimulate N R binding to GRIP1
could be due to their inability to cause NR dissociation from hsp 90 or b) failure to
cause NR binding to DNA or c) the antagonists could cause dissociation o f NR from
hsp 90 and it could cause the NR to bind to DNA but the antagonist fails to induce a
conformation in the NR that can interact with GRIP1. Previous studies have shown that
antagonists do cause dissociation o f NR from hsp 90. Hong et aL , (1997) performed
coimmunoprecipitation assay to prove this.
Other studies have proved that the antagonist can cause the NR to dissociate from
hsp 90 and the NR is also able to bind DNA (El-Ashry et aL, 1989 for PR and Sabbah et
al., 1991 for ER). Since antagonists can cause NR to dissociate from hsp 90 and bind
specifically to DNA, it can be concluded from our studies that the antagonist is unable
to promote a strong interaction between the NR HBD and GRIP1 because the NR
conformation is not suitable to recruit a coactivator (Figure 6 ).
4.2 Structural differences in SR’s with agonist and antagonist: 3D data.
Our results indicate that the antagonist is unable to promote a strong interaction between
the NR HBD and GRIP1 because the NR conformation is not suitable to recruit a
coactivator. Structural evidence for this has been provided by Brzozowski et al, (1997).
The work was done on the estrogen receptor using an agonist (17 P-Estradiol) and an
antagonist Raloxifen.
46
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The LBD o f ER is folded into a three-layered antiparallel a-helical sandwich. This
arrangement o f helices creates a ligand-binding cavity. The LSD's transcriptional
activation function (AF-2) can interact with GRIP1 in a ligand-dependent m anner
(Hong et al., 1997). Agonist and antagonist bind at the same site w ithin the core o f die
LBD (ligand-binding cavity) but demonstrate different binding modes. Each class o f
ligand induces a distinct conformation in the transactivation domain o f the LBD.
When the agonist binds the receptor to form a hormone receptor complex, the C-
terminal a-helix (H12) is positioned in a way that it results in transcriptional activation
by sealing the ligand-binding cavity and generating a competent AF-2 shape that is able
to interact with a coactivator. But when an antagonist binds, this favorable alignment is
prevented because helix H12 is positioned differently relative to other helices o f die
LBD. Hence coacdvator is not able to bind and a transcriptionally competent AF-2 is
not formed (Brzozowski et al., 1997). This provides structural evidence for the
mechanism o f antagonism that this study has analyzed.
4 3 Does data on each antagonist correspond to in vivo activity?
4-hydroxy Tamoxifen with full length ER acts as a partial agonist in dose response
curves (Graph 4) but does not promote ER HBD-coactivator interaction in yeast two-
hybrid assay (Graph 8A). The reason 4-hydroxy Tamoxifen acts as a partial agonist with
full length ER is that full length ER has both AF-1 and AF-2. Hence although 4-
hydroxy Tamoxifen does not make AF-2 transcriptionally competent, AF-1 is still
competent enough to cause transcriptional activation. This is also true in m am m alian
cells (McDonnell et al., 1995).
47
: ,':S V
_
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
RU 486 acts as a partial agonist in dose response curves in yeast (Graph 2A and
2B) but it is not able to promote interaction in yeast two-hybrid assay between GRIP1
and GR or PR (Graph 6 B and Graph 7). The AR antagonist Hydroxyflutamide also fails
to promote interaction with coactivator (Graph 6 A). The reason that these antagonists
are unable to promote a strong interaction between the NR HBD and GRIP1 is because
the antagonist is able to freeze the AF-2 and hence it is unable to interact with a
coactivator. There is no AF-1 in this assay. Like 4-hydroxy Tamoxifen, RU 486 can also
act as a partial agonist in mammalian cells by silencing AF-2 but activating AF-1
(Zhang and Danielsen, 1995).
ICI 182,780 acts as a pure antagonist o f ER in mammalian cells (McDonnell et
al., 1995) but in our yeast two-hybrid assay (Graph 8A), it promotes a weak interaction
between the NR and coactivator. The same result is seen with the MR antagonist
Spironolactone (Graph 8B). The reason that these two compounds act as antagonists is
unknown, but may be because the interaction they promote between the NR HBD and
coactivator is not as strong as the interaction that is seen with agonist alone.
Thus, various ligands can induce a variety o f NR conformations, and these
different conformations determine whether the NR can recruit a coactivator to AF-2.
Full agonists activate both AF-1 and AF-2. Some partial agonists fail to activate AF-2,
but still activate AF-1. Pure antagonists fail to activate either AF-1 or AF-2.
48
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REFERENCES
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18. Shibata, H., Spencer, T.E., Onate, SA., Jenster, G., Tsai, S.Y., Tsai, and
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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Subramanian, Sujatha Tanjore (author)

Core Title A model for the mechanism of agonism and antagonism in steroid receptors

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Stallcup, Michael R. (committee chair), Stellwagen, Robert H. (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-26383

Unique identifier UC11341508

Identifier 1393189.pdf (filename),usctheses-c16-26383 (legacy record id)

Legacy Identifier 1393189.pdf

Dmrecord 26383

Document Type Thesis

Rights Subramanian, Sujatha Tanjore

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA