QP552. G\6  , I>99bHIir C.J G347474
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G-PROTEIN MEDIATED SIGNAL TRANSDUCTION IN SACCHAROMYCES CEREVISIAE
BARTHOLOMEW DZUDZOR
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF A MASTER OF PHILOSOPHY DEGREE IN BIOCHEMISTRY AT THE UNIVERSITY OF GHANA, LEGON
DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF GHANA LEGON, GHANA
SEPTEMBER, 1995
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DECLARATION
The work presented in this report was carried out by me at 
the Department of Biochemistry, University of Ghana, Legon and at 
the Department of Biological Chemistry, University of California, 
Los Angeles, USA under the supervision of Professors F.N. Gyang 
and John Colicelli .
Si gned:
B. DZUDZOR
(CANDIDATE)
PROF. F.N. GYANG
(SUPERVISOR)
SEPTEMBER, 1995
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i i
DEDICATION
To my father,
Mr. Avedezi Dzudzor
and
to the memory of my beloved son,
Frederick Kwasi Selom Dzudzor
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i i i
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to all who 
contributed in making this work a success.
I am especially grateful to my supervisors, Prof. F.N. Gyang 
and Prof. J. Colicelli for their patience, invaluable suggestions 
and relentless effort in seeing to the successful completion of 
this work. I am also greatly indebted to Prof. M.E. Addy, Head of 
Department and a member of the supervisory committee for her 
criticism, suggestions and always keeping me on my toes. I also 
wish to thank Drs. R.A. Acquah, N.A. Adamafio, S.K. Gbewornyoh, 
J.P. Adjimani and Prof. K.K. Oduro, all of the Department of 
Biochemistry Legon, for their criticisms and support.
A special note of thanks is due Meenakshi Ramakrisman, who did 
the original suppressor cloning and Brian Spain for the continuous 
work on YGC1 and MCM1. Both Meena and Brian were students under 
Prof. J. Colicelli at the Department of Biological Chemistry, UCLA, 
Los Angels. My sincere thanks also goes to Limin Han, Raji Pillai, 
both students at Colicell’s laboratory for their support. I also 
owe a debt of gratitude to Prof. E. Neufield, Head, Department of 
Biological Chemistry, UCLA and Mrs. Antoinette Green for their 
support and generosity to me when I was undertaking this research. 
I thank the entire staff of Biological Chemistry Department, UCLA 
for their co-operation.
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Last, but not the least, I will like to thank Miss Christine 
Kamasah, my wife for her moral support. I have also not forgotten 
my colleagues, Charles Brown, Lambert Faabuluom and Ernest Agbovi 
(a dedicated and devoted friend) for their support. Finally I wish 
to thank the entire technical staff at the Biochemistry Department 
for their assistance.
The manuscript was typed by Ms. H.P. Agbesi of ISSER, Legon. 
I deeply appreciate her help.
I am, however, wholly responsible for any shortcoming with 
regard to this work.
i v
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ABSTRACT
V
Yeast mating type locus gene alpha2 (MATa2), Yeast G protein 
complementing gene (YGC1) and minichromosome maintenance gene 
(M C M 1) have been identified by isolation of plasmids that are able 
to complement or suppress a gpa1::HIS3 mutation. MATa2 and YGC1 
rescue both MATa and MATa-gpa1::HIS3 haploid cell types whereas 
MCM1 complements only MATa gpal::HIS3 cell type. MATa2 is known to 
be a general repressor and a determinant of both haploid and 
diploid cell types. MCM1 is known to be a general transcriptional 
activator. YGC1 has not been characterised, hence its function or 
mode of action is not previously known.
G protein alpha subunit (GPA1) is a yeast G protein a subunit 
that negatively controls the budding yeast pheromone signal 
transduction pathway. Disruption of GPA1 results in constitutive 
arrest of the signal pathway that leads to cell cycle arrest at the 
early G1 phase of the cell cycle.
Both Southern analysis and sequencing showed that MATa2, YGC1, 
MCM 1 have no homology to GPA1. Disruption of MATa2 (that is 
mata2::URA3) leads to constitutive arrest of the cell cycle at the 
G 1 phase. MATa2 also has no sequence homology to GPA2, the other G 
protein a subunit in yeast, known to be involved in cAMP pathway in 
yeast. It has been shown here that MATa2 rescues gpal::HIS3 cells 
even in single copy, centromere plasmids. Mating efficiency is 
largely reduced in cells kept alive with MATa2. MATa2 does not have
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the pheromone response elements (PREs) common to the STE genes 
(whose disruption leads to insensitivity to mating factors).
The plasmid TGC was also constructed and used in creation of 
the yeast haploid strains LG1 and LG2. This was an attempt to 
screen a mammalian cDNA library for possible analogs of GPA1. These 
strains were used to isolate two mammalian analogs that complement 
the gpa1::HIS3 mutation.
The results indicate that MATa2, YGC1 and MCM1 are components 
or modulate component(s) of the signaling pathway. It also showed 
that MATa2 is even a more potent negative regulator of the 
signaling pathway than GPA1, since overexpression is not a
prerequisite for negatively regulating the pathway. MATa2 does not
\
belong to the G protein family since it has no GTP/GDP binding 
and/or exchange domains.
vi
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v i i
TABLE OF CONTENTS
DECLARATION ............................................................  i
D E D I C A T I O N ............................................................  ii
ACKNOWLEDGEMENTS ..................................................... iii
ABSTRACT ...............................................................  v
TABLE OF C O N T E N T S .....................................................vi i
LIST OF F I G U R E S ............................................................x
LIST OF T A B L E S ..................................................... xi i
ABBREVIATIONS ..................................................... xiii
CHAPTER I
INTRODUCTION AND LITERATURE REVIEW ..............................  1
1.1 General Introduction ........................................... 1
1.2 Literature Review
1.2.1 Overview of Pheromone Response ...........................  7
1.2.2 Responses and Assays for Signaling ......................... 7
1.2.3 The a-Factor R e c e p t o r ......................................  11
1.2.4 The a-Factor Receptor ................................ 13
1.2.5 Receptor Structure and Function .......................... 14
1.2.6 Receptors Control the Ability to Respond to
Specific Factors ................................................ 14
1.2.7 Yeast G P r o t e i n ................................................ 16
1.2.8 Ga Subunit of Yeast G p r o t e i n ..............................  17
1.2.9 Cyclic-AMP Pathway and G P A 2 ................................. 21
1.2.10 GBy Subunits of Yeast G protein .........................  22
1.2.11a Additional Components Involved in G Protein
and Receptor Function ................................ 25
1.2.11b Adaptive Response to the Mating Factors ............  29
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1.2.12 Downstream From the G Proteins ^ 31
1.2.13 STE12 and Transcriptional Activation .................  37
1.2.14 Interfacing with the Cell C y c l e ............................ 41
1.2.15 MATa2 and MCM1 ................................................ 42
CHAPTER TWO
MATERIALS AND METHODS ................................................ 46
2 . 1 Mater i al s .......................................................... 46
2.2 M e t h o d s ............................................................. 50
2.2.1 Construction of the Yeast C l o n e s ............................ 50
2.2.2 Selection for the Yeast G P A 1 Complementary (Y G C ) 
C l o n e s ............................................................... 50
2.2.3 Southern Analysis .............................................  53
2.2.4 Hybridization of Clone 9 to the Other Clones ..........  54
2.2.5 Mapping of Clones 5,9 and 1 2 ................................. 55
2.2.6 Clone D e l e t i o n s ...................................................56
2.2.7 Creation of Yeast Strains ...................................  56
2.2.8 Construction of TRP/GPA/CAN plasmid (pTGC) ............ 60
2.2.9 Construction of Y E p 9 ........................................... 62
2.2.10 Construction of plasmid RS416 "9"   62
2.2.11 Disruption of MATa2 .......................................... 64
2.2.12 Mating Assays .   67
2.2.13 Quantitative Mating Assay ..................................  68
2.2.14 Sequencing ......................................................  68
2.2.15 Transformation of Bacteria with Plasmids ..............  69
2.2.16 Transformation of Yeast with Plasmids .................  70
2.2.17 Mini Plasmid Preparation Procedure ........................  71
2.2.18 Preparation of Yeast Genomic DNA ..........................  71
vi i i
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2.2.19 Qiagen Plasmid Midi Preparations ........................... 72
2.2.20 Revertants ......................................................  73
2.2.21 Mock T r a n s f o r m a t i o n s ........................................... 73
CHAPTER 3
R E S U L T S ................................................................ 74
3.1 Isolation of the MATa2, YGC1 and MCM 1 G e n e s ................. 74
3.2 The Southern A n a l y s i s .......................................75
3.3 Phenotype of MATa2 Disruption .............................. 76
3.4 Overexpression of MATa2 is Not a Prerequisite
for GPA1 C o m p l e m e n t a t i o n .....................................76
3.5 MATa2 has no Sequence Homology to G - p r o t e i n s ........... 85
3.6 MATa2 Suppresses Mating in both MATa and
MATa gpal yeast c e l l s ....................................... 85
3.7 The Loss of MATa2 Function Results in late G1 Arrest . . 85
3.8 Plasmid Constructs and Creation of New Yeast Strains . . 95
CHAPTER FOUR
DISCUSSIONS & CONCLUSIONS .........................................  104
4.1 General Strategy for Selecting the High
Copy Suppressor Clones ........................................  104
4.2 Identification of MATa2, Y G C 1 and M C M 1 Genes
Involved in the Pheromone Response Pathway ...............  104
4.3 Implications for the Involvement of MATa2 and YGC1
in the Pheromone - Induced Signaling Pathway ........  106
4.4 Possible Models of M A T a 2 , Y G C 1 and M C M 1 Actions . . . .  109
4.5 New Yeast S t r a i n s ........................................... 114
A P P E N D I X .................................................................115
R E F E R E N C E S .............................................................. 118
i x
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XLIST OF FIGURES
Page
1. Differentiation and cell cycle arrest in response to
pheromone..........................................................9
2. Yeast cell t y p e s ................................................  10
3. Regulation of cell types by MAT locus gene and M C M 1 . . . 45
4. Complementation of g p a l ............................................ 51
5. Selection of high copy suppressors of g p a l ................... 52
6. Creation of G L 1(G L 2 ) - 5, 9, 12, UGC and UC strains. . . 58
7. Creation of LG1(LG2) - TG s t r a i n s .............................. 59
8. Construction of p T G C ...............................................61
9. Construction of Y E p 9 ...............................................63
10. Construction of CEN plasmid RS416"9"..........................65
11. Construction of UGC, U5C, UVC, U9C and U12C plasmids. . 66
12. High copy suppressors are not G P A 1 .............................77
13. Suppressor 9 hybridizes to most other suppressors . . .  78
14. Suppressors 5 and 12 are each unique.......................... 79
15. Restriction mapping of clone 9 ..................................80
16. 19, Restriction map of clone 9
Restriction map of MATa2 .................................. 81
17. Restriction map of clone 5 ..................................... 82
18. Restriction map of clone 1 2 .................................... 83
20. Clone 9 deletions and suppression of g p a l ...................84
21. Sequence of M A T a 2 ................................................. 86
22. Clone 9 (a2) rescues both MATa and MATa cells ........... 87
23. Confirmation of RS416"9" plasmid............................... 88
24. Map of plasmid R S 4 1 6 " 9 " ......................................... 91
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25. Rescue of cells in single copies by clone 9 ................92
26. ■ Disruption of clone 9 ........................................... 93
27. Disruption of MATa2 leads to constitutive cell-cycle 
arrest (death of the c e l l ) ...................................... 94
28. Map of plasmid T G C ................................................96
29. Map of plasmid K S E X ................................................97
30. Map of plasmid K S X S ................................................98
31. Map of plasmid K S E K ................................................99
Plate 1 Morphology of clone 9 revertants..........................101
Plate 2 Differentiation of YGC1 c e l l s ............................ 102
x i
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x i i
LIST OF TABLES
Page
1. Genes involved in pheromone signal transduction in
budding yeast ..................................................  28
2. Yeast strains used in this s t u d y ............................... 46
3. Plasmids used in this w o r k .......................................47
4. Drop-out m e d i a ...................................................... 48
5. Transformation results............................................ 89
6. Mating assay results.............................................. 90
7. Morphology of revertants of gpal ce l l s ........................ 100
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x i i i 
ABBREVIATIONS 
G protein Guanine nucleotide binding protein
FUS Genes whose activation leads to the Fusion of the
Cel 1
GP A 1 Yeast G protein alpha subunit
MATa2 Mating type locus alpha 2 gene
YGC Yeast G protein complementing clones
MC M 1 Minichromosome maintenance gene
STE Genes whose inactivation leads to sterility of the
cel 1
CDC Cell division cycle genes
(3ME [3-mecarptoethanol
SDS Sodium dodecyl sulfate
LB Luria-Bertani medium
MES 2 (-N-morpholino) ethanesulfonic acid
YPD Yeast peptone dextrose media
SCG1 Sa cc haromyces cerevisiae
G protein alpha subunit
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CHAPTER ONE 
INTRODUCTION AND LITERATURE REVIEW
1.1 GENERAL INTRODUCTION
The fundamental goal of Molecular Biology is to understand the 
metabolic processes that govern growth and development,
differentiation and diseases in plants and animals. To achieve this 
end, the enzymatic and structural functions of proteins must be 
recreated and characterised in vitro. A comprehensive understanding 
of a protein’s function requires that the gene encoding the protein 
be cloned for further manipulation and characterization. Cloning a 
gene allows one to:
(a) Sequence it and determine if the encoded protein (or RNA) 
contains particular motifs which will help us understand its 
function.
(b) Use mutagenesis to introduce nucleotides substitutions,
insertions or deletions into the gene. The effect of these 
mutations on the activity of the protein reveal important
insights into its mechanism of action.
(c) Express the protein at high levels so that it can be purified 
and characterised further to;
(i) Determine its structure by X-ray crystallography or Two­
Dimensional Nuclear Magnetic Resonance (2-D NMR).
(ii) Use as a therapeutic agent to cure or control a 
particular disease brought about by defective gene and 
hence gene product.
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iii) Perform extensive biochemical studies.
Molecular biologists employ plasmids as vectors for numerous 
purposes, hence construction of plasmid vectors is important tasks 
for the geneticist. Construction of plasmid vectors has involved 
the incorporation of ancillary sequences that are used for a 
variety of purposes, including visual identification of recombinant 
clones by histochemical tests, generation of single-stranded DNA 
templates for DNA sequencing, transcription of foreign DNA 
sequences in vitro, direct selection of recombinant clones, and 
expression of large amounts of foreign proteins. Bacteria contain 
certain mechanisms to control the copy number of the plasmid to a 
level that affords them protection from the antibiotic but not at 
the expense of cellular functions. The control of plasmid copy 
number resides in a region of the plasmid DNA that includes the 
origin of DNA replication. For example, (2|jm) and centromere (CEN) 
gene plasmids are multi and single copy plasmids respectively which 
were constructed and used in this work.
All biological systems have the ability to process and respond 
to enormous amount of information. Much of this information is 
provided to individual cells in the form of changes in 
concentration of hormones, growth factors, neuromodulators, or 
other molecules. These ligands interact with transmembrane 
receptors, and this binding event is transduced into an 
intracel1ular signal. Several families of cell surface receptors 
and ligands that are coupled to different mechanisms of signal 
transduction have been characterized.
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Response to pheromones during the process of yeast mating 
provides an opportunity to study signal transcription in a 
unicellular eukaryote. Haploid a and a cells of the budding yeast, 
Saccharomyces cerevisiae, are able to grow vegetative! y or can mate 
to form a diploid a/a cell. The process of mating is mediated by 
extracellular peptide mating pheromones and integral membrane 
protein receptors. This programme of signaling and response leads 
to cellular differentiation in preparation for mating, which is 
manifested by transcriptional induction of numerous genes, by 
morphological changes, and by arrest of the cell cycle in the G1 
phase. The study of the cell cycle or its mutants has increased the 
understanding of how individual cell cycle steps (such as DNA 
synthesis and mitosis) are coordinated so that the events occur in 
the right order. The analysis of cell cycle mutants has also 
revealed how cells maintain a constant average size over many cell 
divisions. This size regulation requires that the continuous events 
of the cell cycle collectively referred to as cell growth, are 
coordinated with the cycle of stepwise events that includes DNA 
synthesis, centrosome duplication, and mitosis. If there is no 
coordination between growth and the stepwise events, the average 
cell size can only be maintained if the doubling time for cell mass 
is exactly equal to the length of the cycle of stepwise events 
(Murray and Kirschner, 1989).
This signal transduction pathway leading eventually to the 
arrest of the cell cycle at the G1 phase is very crucial in our 
understanding of cellular division and growth control, because we
3
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sometimes think of tumor cells as uniform, completely 
undifferentiated and fast growing but this is not really true. 
There are in fact many kinds of tumors arising from many tissues 
and they may retain some of the characteristics from their tissue 
of origin. In addition, they need not grow at a rapid rate. They 
have simply exited from a no growth (cell cycle arrest) or 
controlled growth (i.e stem cell) state or they have escaped 
controlled cell death (apoptosis). In other words, tumor grows 
because fewer cells exit the cell cycle, whereas in normal tissue, 
fewer cells are cycling; more cells exit the cell cycle. In 
cancers, a great number of progeny cells continue to cycle, because 
they have lost the growth control mechanisms. In the yeast 
Saccharomyces cerevisiae, cells of a mating type produce a-factor 
and respond to a-factor, and cells of a-mating type produce a- 
factor and respond to a-factor. Because the ability to produce 
mating factors and respond to them is required for mating, it has 
been possible to identify many of the genes and proteins that play 
roles in this signaling process by isolation of mutants that are 
defective in mating. Attention has been given to the negative 
growth factors such as TGF-13 (Moses et al. , 1990) that trigger
differentiation and cel 1-cycle arrest. Thus the yeast signaling 
pathway provides an experimental model to study pathways in 
mammalian signaling systems with the techniques of manipulative 
molecular genetics. For example, some of the components of the 
yeast pathway are also found in mammalian signaling systems - G 
proteins with their distinctive receptors that have seven
4
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membrane-spanning regions as well as severa
serine/threonine protein kinases. For yeast, we now 
outline of the complete pathway beginning with ligand binding to 
the receptor at the cell surface and culminating in events within 
and affecting the nucleus, that is differentiation and cel 1-cycle 
arrest.
An important backdrop for the research studies described here 
is the extensive knowledge of the molecular basis for cell 
specialization in yeast, a and a cells produce different receptors 
and different mating factors, a/a cells lack these specialized 
products and others involved in response (and several more). These 
differences result from cell-type-specific regulation of gene 
expression by identified transcriptional regulatory proteins and 
have been the subject of several reviews (Herskowitz, 1989; Dolan 
& Fields, 1990). Other reviews focus on aspects of the signaling 
system (Kurjan , 1990;Dohlman et al. , 1991; Marsh, 1991) and the 
mating process itself (Cross et al., 1988).
The purpose of this literature review is to describe the yeast 
signal transduction pathway at our present state of knowledge 
indicating both what is known and what is less certain or unknown. 
Beginning with an overview of our current view of signaling in the 
pheromone response pathway of yeast, a brief mention is made of 
responses and assays for signaling. These are followed by detailed 
description of receptors, the G protein, other signal transduction 
components, MATa2 and MCM1 genes all of which are involved in the 
pathway. Mention is also made of transcriptional activation and
5
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genetic and/or biochemical evidence for the specific roles of the 
above mentioned genes or gene products when known. Description of 
how the pathway culminates in cell-cycle arrest concludes the 
literature review.
6
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71.2 LITERATURE REVIEW 
Overview of Pheromone Response
Pheromone response in yeast starts with extrace11ular peptide 
mating factors (a-factor and a-factor) binding to integral membrane 
protein receptors, and cells of a-mating type produce an a-factor 
receptor. Both receptors are coupled to the same heterotrimeric G 
protein, GaBy. The a subunit has GDP bound in one state and GTP 
bound in the other state. Stimulation of the receptor causes a 
switch to the GTP - bound state of Ga, which leads to release of 
the 3y subunit of the G protein. G(3y then activates downstream 
components of the signaling pathway (refer to figure 1). Proteins 
required for further signaling include several serine/threonine 
protein kinases and other products. Ultimately a transcriptional 
activator, STE12, is activated, which leads to differentiation, 
that is, increased transcription of several genes (figure 1) 
including those encoding cel 1-surface proteins involved in cell­
cell interaction and fusion (figure 1). G1 cyclins are inactivated, 
leading to cell cycle arrest. The signaling pathway is similar in 
a and a cells except for the receptors.
1.2.2 Responses and Assays for Signaling
The ability to mate exemplified by the formation of zygotes or 
prototrophic colonies (Sprague , 1991), serves as one assay for
ability to carry out signal transduction in response to pheromones,
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since production of mating pheromones and the ability to respond to 
them are essential for mating. There are also several single assays 
for different steps in the mating process. In particular, haploid 
cells respond to purified or synthetic pheromones of the opposite 
mating type. Both a cells and a cells arrest in the G1 phase of the 
cell cycle as unbudded cells, undergo morphological changes (from 
an ovoid cell to a pear-shaped shmoo (figure 2), and exhibit 
transcriptional induction of several genes (Cross et al. , 1988).
The FUS1 gene (or a Ft/S7-lacZ hybrid gene) provides a particularly 
convenient assay for this process since its expression is increased 
several hundredfold by mating factors (Trueheart et al., 1987). 
Several different assays for pheromone production or response 
examine growth arrest (Sprague , 1991). One of these is the zone- 
of-inhibition assay in which the ability of a purified mating 
factor to inhibit growth when spotted on a lawn of test cells is 
determined. Another assay involves formation of cells of aberrant 
morphology in response to a-factor (shmoo formation). Both zone-of- 
inhibition and aberrant norphology assays are used in this study.
Many of the genes involved in the pheromone response pathway 
were identified because mutations in these genes confer resistance 
to the growth - inhibitory effect of the mating factors.
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STE11. STE7, FUS3, K'SSl
STE12 STE12'
X  *
X
1
FUS3 F A R 7 
I I
1
CLN1
1  1  
CLN2 CLN3
CDC28
G1 -----— P- S f
Cell cycle arrest
Transcription 
induction
I I
Figure li Differentiation and call cycle arrest in response to pheromone.
Binding of pheromone (hatched circle) alters the conformation of the 
receptor. As a result of an attendant conformational change Ga.GTP 
replaces GDP, and Gfly is released. Activated GJ3y then initiates a signal 
that passes through STE5 to four protein kinases (STEll, STE7, FUS3 and 
KSS1) •• The transcriptional activator STE12 is rapidly phosphorylated and 
transcription of target genes is stimulated. Some target genes are 
directly involved in [differentiation (FUSl, KAR3), others in cell arrest 
(FUS3, FAR1). As a result of the action of FUS3, FAR1, and other 
hypothetical proteins(s) (X), CLN products do not accumulate and cell
cycle arrest in GI ensues. Arrow heads indicate stimulation and terminal 
bars inhibition of the signal. The parallel lines represent the plasma 
membrane, the serpentine line represents a pheromone receptor and the 
three boxes represent the a,Ih and y subunits of the O protein.
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figure 2i leant Coil Typos
Binding of mating factors to cognate receptors induces cellular and 
morphologiaal dlfferenti^ti^A. As a result the oella mate and form 
an a/a zygote. The arrow heads show the cycle of haploid and diploid 
cell types formation. Double circles indicate the budding of yeast 
and the ovoid shows the ohmoo formation in response to the mating 
signal.
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In particular, mutations in STE2, STE4, STE5, STE7, STE11, STE12, 
STE18, STE20, and FUS3 all confer resistance to a-factor by 
disrupting the signaling pathway (Mackay & Manney,1974; Hartwell, 
1980; Whiteway et a l ., 1989; Leberer et al., 1992; Elion et a l . ,
1990). Many of the genes involved in phenomone production were 
identified as mutants defective in mating (Wilson & Herskowitz ,
1987).
11
1.2.3 The a-Factor Receptor
a-Factor is an unmodified peptide of thirteen amino acid 
residues (Duntze et a l ., 1970) and is necessary for mating by a
cells (Kurjan, 1985). It activates a cells via the a-factor 
receptor encoded by the STE2 gene (Nakayama et al., 1985) and is 
degraded by a specific extracellular peptidase encoded by the BAR1 
gene (Sprague & Herskowitz, 1981). Both of these genes are 
expressed only in a cells (Kronstad et al., 1987). Mutations in 
STE2 affect pheromone response only of a cells. The main structural 
features of the receptor are seven hydrophobic domains that are 
thought to span the membrane, leaving the N-terminus outside the 
cell and the C-terminus inside the cell (Nakayama et al., 1985). A 
diverse family of integral membrane protein receptors coupled to G 
protein has seven hydrophobic, potential membrane-spanning domains 
(Ross , 1989; Dohlman et al. , 1991). Supporting the notion that 
these hydrophobic domains are important for a-factor receptor 
function,their size and position is conserved in the Saccharomyces 
kluyveri a-factor receptor, which is only 50% identical to the S.
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cerevisiae product (Marsh & Herskowitz, 1988). Despite structural 
conservation, the a-factor receptor has no obvious sequence 
identity with known mammalian receptors.
a-Factor binds specifically to a mating-type cells with an 
equilibrium dissociation constant of 6x10-3 (Jenness et al., 1986) 
to 2x10"8 (Raths et a l ., 1988). The receptor is localized to the
cell surface as shown by indirect immunofluorescence of a STE2-lacZ 
fusion protein that retains receptor activity (Marsh & Herskowitz, 
1988). Genetic evidence shows that a cells have only one receptor 
gene, and Scatchard analysis indicates that all detected receptors 
have a single affinity (Marsh et a l . , 1991). There are roughly 10* 
binding sites per cell, as determined by binding of 35S - or 3H - 
labelled a-factor, or by binding competition studies (Raths et al., 
1988). Temperature-sensitive mutations in the STE2 gene lead to 
temperature-sensitive binding of a-factor (Jenness et al., 1983). 
The a-factor has also been cross-linked to the STE2 gene product, 
albeit with low efficiency (Blumer et al., 1988).
a-factor is internalized and degraded by a cells in a process 
that is dependent on the presence of the a-factor receptor (Jenness 
& Spatrick, 1986). Surface a-factor binding sites are also down- 
regulated after exposure to a-factor in a process that does not 
require G proteins (Jenness & Spatrick, 1986). The receptor is the 
only protein in yeast currently known to be subject to specific 
endocytosis (Marsh et al., 1991).
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1.2.4 The a-Factor Receptor
a-Factor is a farnesylated and carboxy-methy1ated peptide of 
twelve amino acid residues, unrelated to a-factor (Xue et al. , 
1989; Schafer et al., 1990) and is necessary for mating by a cells 
(Michaelis & Herskowitz, 1988). Its synthesis and secretion follow 
a strikingly different route from a-factor (Kuchler et al., 1989). 
The a-factor receptor is encoded by STE3 and expressed only in a 
cells (Nakayama et al., 1985). Mutations in STE3 block mating and 
mating-factor response only in a cells. Binding studies have not 
been performed with a-factor since it is hydrophobic and exhibits 
a high level of nonspecific binding. Synthetic a-factor is active 
in inducing cellular responses at nanomolar concentrations, thus 
suggesting that affinity of the a-factor receptor for its ligand is 
in the same range as that of the a-factor receptor for a-factor 
(Xue et al., 1989).
The STE3 gene encodes a hydrophobic protein with a predicted 
molecular weight of 54kd (Nakayama et al., 1985). The a-factor 
receptor, like the a-factor receptor, has seven potential membrane- 
spanning domains and a long hydrophilic carboxyl-terminus. A 
variety of experimental data are consistent with a receptor 
topology similar to that of a-factor receptor (Clark et al. , 1988). 
Despite the potentially similar structure, the a-factor and a- 
factor receptors have very little amino acid sequence homology; 
some similarity is however observed between position 222-268 in 
STE2 and 117-163 in STE3 (Marsh et a l . , 1991).
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1.2.5 Receptor Structure and Function
A working model for the a-factor receptor and other G protein- 
coupled receptors is that they consist of a central core made up of 
a bundle of seven membrane-spanning helices that contacts ligand 
towards its outer face and G protein on its inner face (Dohlman et 
al., 1991). Ligand specificity determinants for distinguishing S. 
cerevisiae and S. kluyveri a-factors, as revealed by studies with 
receptor hybrids, lie in the central region that includes receptor 
hydrophobic domains (Marsh et al. , 1991). Studies of receptor
mutants show that residues that control receptor activation also 
lie in hydrophobic domains. These studies suggest that the a-factor 
peptide may activate the receptor in a manner analogous to the 
activation of the [3-adrenergic receptor by epinephrine, where it 
has been shown that ligand binding and receptor activation involve 
the membrane-spanning domains (Ross , 1989). Most of the large
hydrophilic, carboxy-terminal domain of the a-factor receptor is 
not required for ligand binding or G protein activation. Cells 
lacking this domain are hypersensitive to a-factor, which suggests 
a role in desensitization (Reneke et a l . , 1988; Konopka et a l . ,
1988). Some residues immediately following the seventh hydrophobic 
domain (beyond residue 295) may be required for signaling, since 
truncation at this point reduces mating efficiency (Marsh et al., 
1991 ).
1.2.6 Receptors Control the Ability to Respond to Specific 
Factors
In theory, the ability of each mating type cell to respond
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only to the pheromone of the opposite cell type could be determined 
in at least two ways: the receptor for a given pheromone could be 
expressed only by the appropriate cell type, or receptors for both 
types of pheromone could be expressed, in which case the necessary 
coupling proteins could be cell-type specific. Yeast uses the 
first scheme - each cell transcribes only one receptor gene. 
Further analysis demonstrates that both receptors are coupled to 
the same intracellular machinery and downstream responses. An a 
cell engineered to produce the a-factor receptor instead of the 
usual a-factor receptor responds to a-factor- instead of a-factor 
(Nakayama et a l . , 1987; Bender & Sprague , 1989). An a cell
engineered to produce the a-factor receptor undergoes autocrine 
arrest (Nakayama et al., 1987).
Other receptors expressed in cells of S. cerevisiae are also 
able to function. S. kluyveri is a yeast with a mating system 
resembling that of S. cerevisiae. Each of these yeasts responds 
better to its own a-factor than to the heterologous a-factor 
(McCullough & Herskowitz , 1979). When STE2 in S cerevisiae was 
replaced with the STE2 homologue from S. kluyveri, the resulting 
strain responded preferential1y to S. kluyveri a-factor(Marsh & 
Henskowitz , 1988), thereby demonstrating that the STE2 protein is 
the specificity determinant for a-factor. The ability of the S. 
kluyveri receptor to function in S. cerevisiae indicates that the
S. cerevisiae G protein is compatible with the receptor from S. 
kluyveri.
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Mammalian receptors can also function in yeast. A |3-adrenergic 
receptor expressed in yeast displayed the binding characteristics 
of the receptor found in mammalian cells (King et a l ., 1990).
Agonist-induced activation of the yeast pheromone response pathway 
(measured by induction of FUS1-lacZ expression) required co­
expression of mammalian GQS. The yeast system may prove useful for 
genetic analysis of mammalian receptors, since mutant receptors 
with altered signaling properties are easily identifiabde by plate 
assay.
1.2.7 Yeast G Protein
The a-factor and a-factor receptors appear to be coupled to a 
single heterotrimeric yeast G protein (guanine nucleotide-binding 
protein) present in both cell types (Kurjan , 1990). In the
mammalian systems that have been studied, activation of receptor 
leads to replacement of GDP with GTP on the a subunit of G protein 
and separation of G0 subunit from the G0^  subunit (Stryer & Bourne 
, 1986; Kaziro et al., 1991). A similar coupling process is thought 
to occur in yeast, although biochemical studies have not yet 
confirmed it in yeast. In budding yeast, unlike some other well- 
studied mammalian systems, it is the free GI3y subunit rather than 
the Ga subunit that is responsible for activating signaling targets 
(Dietzel & Kurjan , 1987; Whiteway et al. , 1987). Activation of 
phosphol ipase A2 by GBy has been reported to occur in bovine rod 
outer segments (Jelsema & Axelrol, 1987). Also GBy derived from Gs 
Gj and G0 stimulates protein kinase dependent phosphorylation of
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both muscarinic acetylcholine receptor and rhodopsin (Haga & Haga, 
1992).
Association of receptor and G protein increases the ligand 
affinity of many G protein-coupled receptors (Stryer & Bourne,
1986). The yeast a-factor receptor apparently behaves similarly: 
alteration in affinity of the a-factor receptor for its ligand 
therefore provides an assay for interactions between the G protein 
and receptor. Mutants defective in G0 or G^  show reduced a-factor 
binding (Jenness et al., 1987; Blumer & Thorner , 1990) which
suggest that G0^  is necessary for proper association of G„ with the 
receptor. Yeast membranes exposed to the non-hydrolyzable GTP 
analogue, GTP^S, which locks G protein into the GTP-bound state, 
have a ninefold lower affinity for a-factor (Blumer & Thorner , 
1990). This observation supports the model that in yeast, as in 
mammalian cells, the receptor is not associated with the GTP bound 
form of G protein.
1.2.8 Ga Subunit of Yeast G protein
Yeast Ga genes were identified by cross-hybridization, to a rat Ga i 
DNA probe (Nakafuku et al., 1987, 1988) and by selecting for genes 
whose overexpression confers resistance to mating factors (Dietzel 
& Kurjan , 1987). One of the G„ genes of yeast, G P A 1 (also known as 
SCG1) , is involved in mating factor response (Miyajima et al.,
1987). On the other hand, genetic and biochemical analyses have 
suggested that GPA2, the other gene coding for GQ may participate 
in regulation of the intracellular levels of cAMP. The first clue
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for solving the function of GPA1 in yeast cells was obtained by the 
analysis of its expression. Northern blot analysis indicated that 
GPA1 was expressed only in haploid cells (Miyajima et a l . , 1987), 
whereas GPA2 was expressed both in haploid and diploid cells 
(Nakafuku et a l ., 1988). Later, it was found that the level of GPA1 
transcript was increased several fold in response to mating factors 
as in the case of other hapl oi d-speci f i c genes (Jahng et a l .,
1988). The apparent differences of the expression pattern of the 
GPA1 and GPA2 genes strongly suggest differential function for 
these two genes. GPA1 (SCG1) gene product would be referred to as 
Ga in this study.
The yeast Ga subunit contains 472 amino acid residues and is 
45* identical to rat Gai (Miyajima et a l . , 1987). Like the
mammalian Ga, the yeast protein is membrane associated (Blumer & 
Thorner , 1990) and has also been shown to be myristoy1ated (Marsh 
et a/., 1991).
The regions of strongest identity between the GPA1 (SCG1) 
product and other Ga subunits include the guanine-nucleotide- 
binding consensus region and GTP-hydrolysis region. Similarity in 
other regions is.also generally high, although the yeast protein 
has 110 extra amino acid residues (126-235) not found in the 
mammalian GQ subunits (Marsh et a l . , 1991). The role, if any of
this extra domain is not known.
Deletion of the GPA1{SCG1) gene results in constitutive 
activation of all mating pheromone responses: induction of FUS1-
lacZ and cell-cycle arrest is observed in the absence of pheromone
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and receptor (Miyajima et al., 1987). Since absence of Ga causes 
the pathway to become activated, Ga is obviously not required to 
propagate signal for activation. Rather, Ga is formally a negative 
regulator of signaling necessary to maintain the pathway in a 
quiescent state, apparently by binding to G|3y.
Studies of mammalian Ga and receptors in yeast demonstrate 
specific interactions between components involved in signaling. 
Yeast cells lacking yeast Ga but expressing mammalian Ga are not 
constitutively activated (Dietzel & Kurjan , 1987). It thus appears 
that the mammalian Gas can interact with the yeast GI3y. These 
cells, however, are not inducible by a-factor, which indicates that 
the activated receptor cannot interact appropriately with the 
mammalian Gas subunit. This finding is complementary to the 
observation that function of the (3-adrenergic receptor expressed 
in yeast requires coexpression of Gas (King et al., 1990). Hybrid 
studies with yeast and mammalian Ga suggest that the C-terminus of 
Ga may be required for interaction with the receptor (Kang et al.,
1990). Some point mutations in the C-terminus of Ga also block 
signaling (Stone & Reed , 1990; Hirsch et al., 1991). Of special 
interest are two substitutions in this region that exhibit 
different phenotypes in different cell types and suggest that these 
residues may be specially involved in receptoi— Ga interactions 
(Hirsch et al., 1991). The Pro467 mutant exhibits a much more
severe defect in a cells than in a cells, which suggest that it 
interacts less well with the a-factor receptor than with the a- 
factor receptor. In contrast, the Pro466 mutant exhibits a somewhat
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more severe defect in a cells (Marsh et a l . , 1991). Such mutations 
may lead to a further understanding of how a single yeast Ga can 
interact with two receptors that lack obvious sequence homology. It 
may also be possible to identify determinants on the receptor that 
interact with Ga by exchanging regions of STE2 and STE3 and 
determining their ability to function with these Ga mutants. Also, 
again using genetic approaches, several kinds of "activated" 
mutations of the GPA1 gene have been characteri zed. A GPA 7va'"50 
mutation, which has a substitution of Gly-50 with valine, was 
introduced by site-directed mutagenesis based on the analogy with 
the val-12 mutation of Ras (Miyajima et al., 1989). This mutation 
of Ras decreases GTPase activity and increases transformation 
activity (Seeburg et al., 1984). The other mutations, GPA 7^ s'355 and 
were selected from a pool of mutants based on phenotypic 
changes (Stone and Reed , 1990). The alignment of Ga primary
structures shows that the mutations correspond to Val-49 mutation 
of Gas proteins. According to the model described above, 
constitutive activation of GP1a protein would cause phenotypes 
supersensitive to the mating factors. This has turned out to be the 
case in short-term responses: i.e growth arrest and gene inductions 
of cells carrying these GPA1 mutations were elicited by a 100-fold 
lower concentration of mating factors than required for wild-type 
cells. More interestingly, however, these mutations also enhanced 
recovery from factor-induced growth arrest, and after long-term 
incubation with factors, mutant cells finally showed phenotypes of 
factor-resistant growth (Stone & Reed , 1990). One possible
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explanation is that independent of growth arrest and gene-induction 
pathways driven by the By-subunit, GP1a can turn on another 
signaling pathway which leads to a recovery from mating factor 
responses. All of the evidence described above has relied on 
genetic studies. Biochemical studies are necessary to elucidate 
the precise molecular mechanisms of G protein function in mating 
factor signal transduction.
1.2.9 Cyclic-AMP Pathway and GPA2
In addition to the mating factor signaling system, S. 
cerevisiae has another signal transduction pathway, which operates 
in the early G1 phase of the cell cycle, this is mediated by 
nutrients such as glucose, which serves as an extracel1ular signal 
for the activation of adenylate cyclase, and cAMP plays a crucial 
role in cell cycle progression at this stage (Matsumoto et al.,
1985). It is well known that GTP-binding proteins encoded by RAS1 
and RAS2, which are yeast counterparts of mammalian Ras, 
participate in the control of the activity of adenylate cyclase. In 
contrast to yeast adenylate cyclase, mammalian adenylate cyclase 
activity is regulated by two G proteins, Gs and G i . A recent study 
reports that a yeast G protein GPA2, in addition to Ras protein, is 
involved in the regulation of cAMP levels in the cell.
Yeast cells cultured under starvation conditions transiently 
accumulate cAMP in response to glucose (Eraso et al., 1985).
Introduction of YEpGPA2 (a multicopy plasmid carrying the GPA2 
gene) in wild type cells was found to enhance glucose-induced cAMP
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accumulation remarkably (Nakafuku et al. , 1988). In addition,
YEpGPA2 suppressed the growth defect by a temperature-sensitive 
(ts) mutation of the RAS2 gene[ras2-101(t s )]. In ras2-101(ts) 
cells, mutant RAS2 proteins would not support the activation of 
adenylate cyclase at nonpermissive temperature and therefore 
glucose could not induce cAMP formation. Introduction of YEpGPA2 
restored the cAMP response in the mutant cells at high temperature. 
These results suggest that GPA2, in addition to Ras proteins, is 
involved in the regulation of cAMP levels in S. cerevisiae.
Since GPA2 could not restore gpal phenotypes (Kaziro et a l .,
1991), STE4- and STE18-e ncoded 13- and y- subunits respectively 
cannot interact with GPA2. This implies that an additional set of 
genes that code for (3-and y-subunits interacting with GPA2 must be 
present in yeast cells.
1.2.10 GI3y Subunits of Yeast G protein
The 6 and y subunits of yeast G proteins can be considered as 
a unit since they function together: null mutations in the genes 
encoding these proteins lead to similar phenotypes (Whiteway et 
al., 1989, 1990). Mammalian G protein 13 and y subunits copurify as 
a tight complex and likewise function as a unit (Stryer & Bourne ,
1986). The STE4 gene encodes a product with similarity to mammalian 
G|3 (Whiteway et al., 1989). The yeast analogue to the Gy subunit is 
encoded by the STE18 gene, which has only weak sequence similarity 
to the mammalian Gy, but is of similar size (Whiteway et al. ,
1989).
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The STE4 gene product is predicted to be a protein of 423 
amino acid residues. STE18 encodes a predicted product of 110 
amino acid residues and shares an important feature with mammalian 
Gy subunits (Whiteway et al. , 1989): both end with a consensus
amino acid sequence (Cys-aliphatic-aliphatic-X amino acids) for 
isopreny1ation, a lipid modification that may localise the subunit 
to the membrane (Whiteway et al., 1989). Isoprenylation may be 
required for GI3y to function in signaling (Schafer et al., 1989). 
Mammalian Gy subunits have a related lipid modification (Mumby et 
al. , 1990). Both STE4 and STE18 are required for response to 
pheromones (Whiteway et al., 1989): mutants defective in these
genes are unresponsive to mating factors. As noted above, 
inactivation of the GPA 1 (SCG1) gene causes constitutive response 
of the pathway, presumably because an activator of the response 
pathway is liberated. The behaviour of mutants defective in both 
GPA1 and in the STE4 or STE18 gene indicates that it is the STE4 
and STE18 products that are responsible for activating the pathway: 
gpal (scgl) ste4, and gpal (scgl) ste18 mutants exhibit the 
phenotype of ste4 and ste18 mutants - the response pathway is not 
activated (Nakayama et al., 1988; Whiteway et al. , 1989). The role 
of Ga is apparently to prevent signaling by G|3y in the absence of 
receptor/1igand interaction (Whiteway et a 7., 1989).
Blinder et al. (1989) identified mutations of STE4 that lead 
to constitutive expression of the signaling pathway. These 
mutations, termed haploid-specif ic lethals ( S T E 4 ), cause 
lethality only in a and a haploid cells but not in a/a diploids in
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which several essential components of the signaling pathway (such 
as STE5, STE12, and FUS3) are turned off.
The mutations may cause synthesis of a G(3 subunit that
is insensitive to inhibition by the Ga subunit, but which preserves 
its ability to interact with downstream components of the pathway.
The importance of the balance between Ga and GI3y has also been 
demonstrated by studies in which different subunits are 
overproduced. Overexpression of GI3(STE4) alone, or with Gy (STE18), 
leads to constitutive mating-factor responses (Whiteway et a l ., 
1990; Cole et a l ., 1990). This induction is overcome by
overproduction of Ga, (Whiteway et a l ., 1990: Cole et a l ., 1990), 
presumably by converting free GBy subunits back to GaBy 
heterotrimers and restoring the normal ratio of the subunits. Over 
expression of Ga(SCG7) has also been observed to counter the growth 
inhibition of certain a strains (mutants defective in the SST2 
gene) exposed to a-factor (Kang et a l . , 1990; Cole et al., 1990).
At the C-terminus of Ste18 proteins, a cys-A-A-X sequence (A 
represents aliphatic amino acid and X is the last amino acid) 
common to all the Gy-subunits is found. In Rasp21, this motif 
signals postranslational modification of the C-terminus, which is 
required for membrane association and biological activity 
(Willumsen et al., 1984). Studies have demonstrated that the
conserved cysteine in the motif is the site of polyisoprenylation 
of both Ras (Casey et al., 1989) and Gy proteins (Mumby et al.,
1990). It was also shown that a mutational change of the cysteine 
(Cys-107) to serine resulted in the loss of function of Ste18
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(Finegold et al., 1990).
Furthermore, a yeast dpr1/ram1 mutation, which was originally 
isolated as a defective mutation in postranslational processing of 
yeast Ras proteins, was found to affect the membrane association 
and biological activity of Ste18 protein (Kaziro et a l ., 1991).
These results indicate that G protein Y-subunits and ras proteins 
may share a set of the same modification process.
1.2.11a Additional Components Involved in G Protein 
and Receptor Function
Although in vitro studies with mammalian receptors and G 
proteins suggest that pheromone, receptor, and G^Sy are sufficient 
to permit GDP/GTP exchange and coupling to downstream responses, it 
is possible that other components are involved in vivo in 
modulating the signaling response. In yeast, genetic screens have 
identified a number of genes whose products may modulate activity 
of the G protein. Inactivation of these genes leads to activation 
of the signaling pathway; hence, these products can be formally 
considered as negative components of the pathway. The cell division 
cycle (CDC) genes with this behaviour are CDC36 and CDC39 (Neiman 
et al., 1990; de Barros Lopes et al., 1990), CDC72 and CDC73 (Reed 
et al., 1988), and SRM1 (Clark & Sprague , 1989) (See table 1).
Mutants with temperature-sensitive defects in any of these genes 
exhibit cell-cycle arrest in G1 and induction of FUSI-lacl 
expression at non-permissive temperature. Activation of the 
response pathway in these mutants is blocked by inactivation of the
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STE4 gene, as observed for mutants defective in Ga itself. Thus 
these proteins might be regulators of G protein activity, receptor 
- G protein adapters, new G protein subunits or modifiers of the G 
protei n s .
CDC72 has been shown to be identical to the NMT1 gene, which 
codes for N-myristoyl transferase (Duronio et al., 1989). The yeast 
Gj polypeptide is myristoylated in wild-type strains, but not in 
the cdc72 mutants (Marsh et a l ., 1991). CDC36 may also control
synthesis of a functional Ga subunit (Neiman et al., 1990). 
Transcription of GPA1{SCG1) is normal in these strains (de Baros 
Lopes et al. , 1990); hence CDC36 might be involved in post-
translational modification of GQ. Physiological analysis of a cdc 
39-ts mutant indicates that CDC39 does not control synthesis of a 
functional Ga subunit, but rather raises the intriguing possibility 
that it might play a role in communication between the activated 
receptor and Ga, or be involved in stabilizing the GDP-bound form 
of Ga (Neiman et al., 1990).
The SRM1 gene (Clark & Sprague , 1989) shares extensive
similarity with the mammalian gene, RCC1 (Uchida et a 1., 1990), and 
is identical to the PRP20 gene, which is involved in messenger RNA 
metabolism (Aebi et al., 1990). The relationship between the SRM1 
product and the response pathway is obscure and might be very 
indirect (Marsh et al., 1991).
It should be noted that the genes CDC36, CDC39, CDC72 and SRM1 
are known to play roles above and beyond their roles in the signal 
transduction pathway. This is in contrast to the GPA1(SCG1) gene,
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which is essential only for the signal transduction pathway. This 
difference can be readily discerned by the observation that a/a 
strains defective in GPA1(SCG1) are viable whereas a/a strains 
defective in CDC36 etc are inviable (Marsh et al., 1991).
Several other genes have been identified that may represent 
other components involved in early steps in the signal transduction 
pathway. The DAF2 product may be involved in STE4 function (Cross 
, 1990). Certain mutations in the RAM1 gene (also known as DPR1, 
SGP2, or STE16), which is necessary for farnesylation of RAS and 
the a-factor precursor, also cause defects in the response pathway 
(Nakayama et al., 1988); Matsumoto et al., 1988), perhaps because 
of a failure to modify STE18 or some other product.
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T o b i o  1:
G e n e s  i n v o l v e d in I M i e r o m o n e  S i g n a l T r a n s d u c t i o n  inB u d d i n g  Y e a s t
G c n  e F u n c t i o n N u l l  in u 1 a t i o n p h e n o t y p e * C e 11 - t y  p (2 e x p r e s s  ionSTE2 a - f a c o r  r e c e p t o r U n r e s p o n s i  v e aSTE3 a - f a c t o r  r e c e p t o r U n r e s p o n s i v e aGPA1 (SCGI) G a  s u bu n i t C o n s t i t u t i v e  ( l e t h a l ) a ,  aSTE4 G[3 s u bun i t U n r e s p o n s i v e a , aS'l'E IS G y s u bun i t U n r o s p o n s i  v e a ,(xSTE20 Pr o t e i n  K i n a s e U n r e s p o n s i  v e Al lSTE5 U n k n o w  n U n r e s p o n s i v e a,<x
FUS3 Pr o t e i n  K i n a s e U n r e s p o n s i v e a , aSTE7 P r o t e i n  K i n a s e U n r e s p o n s i v e Al lSTE11 P r o t e i n  K i n a s e U n r e s p o n s i v e Al lKSSI P r o t e i n  K i n a s e S e e  t ex t No t  rcpoi tet fSTE12 T r a n s c r i p t i o n a l U n r e s p o n s i  v e a , aa c t i v a t o r No t  r e p o r t ^SGVI Pr o t e i n  K i n a s e H y p e r s e n s i t i v eSST2 U n k n o v n M y p e r s e n s i t v e a , aCDC36 U n k n o w n C o n s t i t u t i  v e ( l e t l i  a l ) Al lCDC39 U n k n o w n C o n s l i t u t i v e ( l e t l i a l ) Al lCDC72(NMTI) My r i s t o y l  t r a n s f e r a s e C o n s t i t u t i  v e ( l e l l i a l ) Al lCDC73 U n k n o w n C o n s t i t u t i  v e ( I e t h  a l ) Al lSRMI U n k n o w n C o n s t i t u t i  v e ( l e t h  a l ) Al l
P h e n o t y p e  is w i t h  r e g a r d  to e x p r e s s i o n  o f  p h e r om o n e - i n d u c e d  r e s pon s e s .  C o n s t i t u t i v e  a c t i v a t i o n  o f  t h e s e  r e s p o n s e s  l e ads  to cel  I - c y c l e  a r r e s t  a nd  d e a t h .  S e e  t ex t  f o r  r e f e r e n c e s .
University of Ghana          http://ugspace.ug.edu.gh
1.2.11b. Adaptive Response to the Mating Factors
Yeast cells exposed to the mating-factors recover from G1 
arrest after a period of time and resume growth. Thus arrest is 
transient. There are several different mechanisms for adapting to 
the mating factors. As noted above, the a-factor receptor is 
subject to internalization (Jenness & Spatrick , 1986). In
addition, a cells produce an-extracellar protease, coded by the 
BAR1 gene, that inactivates a-factor by degradation, thus allowing 
enhanced recovery to a-factor (Sprague & Herskowitz , 1981; Mackay 
et al., 1988). It was discovered that a-cells inactivate a-factor 
(Marcus et al., 1991).
Yeast cells are able to adapt even in the absence of a-factor 
inactivatin (Moore , 1984). This adaptive response appears to
function by several independent pathways and to involve the 
receptor, GQ, Gg and the SST2 gene. Receptor mutants lacking the C- 
terminal segment are supersensitive to mating pheromones (Konopka 
et al., 1988; Reneke et al., 1988). The C-terminal segment is rich 
in serine and threonine residues and is hyperphos-phorylated in 
response to a-factor (Marsh et a l ., 1991).
Mutants of GQ with a Gly to val substitution at position 50 
(analogous to the rasva^  mutation that reduces the intrisic GTPase 
activity of ras) exhibit a complex phenotype that appears to 
indicate that Ga plays a role in adaptation (Cole et a l ., 1990). 
Another interpretation of such mutants is given by Kurjan et a l ., 
(1991). These strains are partially constitutive for the pathway 
and supersensitive to growth arrest by a-factor. However, they also
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appear to exhibit a stronger ability to recover from arrest than 
do wild-type cells. Miyajima et al., (1989) have proposed that the 
activated Ga subunit (Ga-GTP) provokes a recovery process. Irie et 
al., (1991) identified a gene SGV1, which encodes for a protein 
kinase related to CDC28 (42% identical), that may play a role in 
this Ga-stimulated adaptive response.
Cole & Reed (, 1991) reported that the STE4 protein (GI3) is
rapidly phosphory1ated after a-factor treatment of a cells. This 
phosphorylation appears to play a role in adaptation, based on the 
observation that deletion of a segment of STE4 eliminates
pheromone-induced phosphorylation and causes cells to become
hypersensitive to mating factors (Marsh et a l ., 1991). It has been 
further shown that an intact Ga subunit is required for this 
phosphorylation. These observations lead to the hypothesis that the 
Gq- and SGV1- dependent recovery process proposed by Miyajima et 
al., ( 1989) may function by stimulating phosphorylation of GI3 (Irie 
et al., 1991 ).
The product encoded by the SST2 gene is involved in
desensitization or recovery from signaling. Mutations in SST2 cause
strains of either mating type to be hypersensitive to mating factor 
and to have prolonged responses (Chan & Otte , 1982). Transcription 
of SST2 is highly pheromone-inducible and thus serves to turn off 
response to pheromone and promote recovery (Dietzel & Kurjan ,
1987). The target of SST2 does not appear to be the receptor C- 
terminus since hypersensitivity resulting from receptor truncation 
and sst2 mutations is additive (Konopka et al. , 1988; Roneke et
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al. , 1988). SST2 may act on Ga or some other G protein subunit
since certain Ga mutant alleles are epistatic to sst2 mutations 
(Kurjan et al. , 1991). Also, overexpression of Ga partially
overcomes an sst2 mutation (Dietzel & Kurjan , 1987). The SST2
product may stimulate the intrinsic GTPase activity of Ga and thus 
be analogous to GAP acting on RAS in mammalian cells (McCormick et 
al., 1988), or to IRA1 and IRA2 acting in yeast (Tanaka et al.,
1990).
The recovery processes mediated by SST2, GI3, and the C- 
terminus of the receptor appear to be independent of each other. 
This conclusion is drawn from the observation that mutants doubly 
defective in these components exhibit more severe defects than do 
mutants with mutations in individual components (Marsh et al.,
1991). For example, the extremely hypersensitive phenotype of 
strains with a C-terminal deletion of STE2 and a mutation in SST2 
makes it unlikely that SST2 function by phosphory1ating or other 
wise affecting the C-terminus of the receptor (Konopka et al. , 
1988; Reneke et al., 1988).
1.2.12 Downstream From the G Proteins
Six genes (STE5, STE7, STE11, STE12, STE20 and FUS3) that have 
been identified as necessary for signal transduction appear to 
function after or at the level of the G protein. This placement is 
based on the behaviour of double mutant strains with alterations in 
Gq and Gf3 and STE5 or other STE genes. As noted earlier, null 
mutations in GPA1(.SCG1) cause constitutive behaviour of the
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pathway, whereas null multations in the STE genes are nonresponsive 
to a-factor. The important result (Nakayama et al., 1988) is that 
strains defective in both GPA1 and any of the STE genes or FUS3 
(Elion et al., 1990) are nonresponsive for example, a gpal stell 
strain exhibits the properties of the ste11 mutant. These 
observations are interpreted to indicate that the STE11 protein 
acts after Ga in a simple linear pathway. Another interpretation is 
that GPA1 and STE11 function in separate pathways that intersect 
downstream from GPA1 (Elion et a l ., 1990). One cannot distinguish 
between these two possibilities at present, but for simplicity, we 
consider these STE genes products as functioning downstream of the 
G protein. Analogous epistasis results have been obtained using the 
special mutation of the GG subunit, encoded by the S T E ^  mutation 
described earlier, which leads to constitutive expression of the 
pathway (Blinder et al ., 1989). These findings place the STE
products and FUS3 downstream of G(3 (or functioning at the same step 
in the pathway as G(3). Additional epistasis tests with mutants 
defective in CDC36 and CDC39 genes are consistent with these 
findings (Neiman et a l ., 1990; de Barros Lopes et a l ., 1990).
After activation of the G protein, the liberated G(3y subunit 
is presumed to interact with one or more proteins to propagate the 
signal. The gene product that lie immediately downstream of GGy - 
STE20, STE5, STE7, STE11 and FUS3 are candidates for this target. 
Leberer et a7.,(1992) demonstrated through epistasis relationship 
that STE20 could lie closest to the Gf3y subunits. This supposed 
interaction between the G(3y and STE20 product is however not clear
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and must be substantiated by further genetic and biochemical 
studies. Thus, identification of the target for G(3y is one of the 
most important challenges in understanding the yeast signaling 
pathway.
In several mammalian systems, synthesis or degradation of a 
second messenger molecule such as cAMP, cGMP or IP3 is controlled 
by activation of the G protein (Iyengar & Birnbauner , 1990). No 
such second messenger molecule has been identified in yeast. An 
early contention that cAMP was the second messenger for this 
pathway (Thorner , 1982) has not been substantiated (Casperson et 
al. , 1983). None of the genes in the signaling pathway has features 
of known enzymes involved in second messenger production or 
regulation. Studies of the genes that lie downstream of the G 
protein have, however, provided some information as to their 
function: STE7, STE11, STE20 and FUS3 appear to code for protein 
kinases and STE12 code for a transcription factor that is subject 
to phosphorylation (see table 1).
STE7, STE11, FUS3 and STE20 all contain significant si mi 1arity 
to the catalytic domains of serine/threonine protein kinases 
(Teague et al., 1986); Rhodes et a l ., 1990; Elion et al., 1990;
Leberer et al., 1992). They contain all of the 15 amino acid 
residues conserved in most protein kinases (Rhodes et al.,
1990). STE7, STE11 and STE20 share 25-30% identity with each other 
(Leberer et al., 1992) and 22-27% identity with FUS3 (Rhodes et
a 7. , 1990). FUS3 has 34-36% identify to the CDC28/cdc2 kinase
family, which plays important roles in cell-cycle regulation. FUS3
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bears even more similarity (54% identity) to another putative yeast 
protein kinase, which is encoded by the KSS1 gene (Courchesne et 
al. , 1989). A mammalian protein kinase with particular similarity 
to FUS3 and KS S 1 has been identified (Boulton et al., 1990). This 
protein, the insulin-stimulated protein kinase (E R K 1, extracellular 
signal-regulated protein kinase), is 50-52% identical to FUS3 and 
KSS1 and shares a C-terminal extension that is not present in the 
CDC28/cdc2 family. It thus appears that these protein kinases 
identify a new structurally related group of protein kinases, which 
appear to play roles in response of cells to extracellular signals.
The FUS3 and KSS1 genes are functionally redundant in some 
respects and not in others (Elion et al., 1990, , 1991). Strains
deleted for either KSS1 or FUS3 respond to a-factor (exhibiting 
induction of FUS1-lacZ), whereas strains defective in both genes do 
not (Elion et al., 1991). Thus it appears that doubly defective
strains are unable to propagate the signal needed to activate 
transcription (which appears to be activation of STE12). In 
contrast, FUS3 and KSS1 are both able to propagate the signal 
leading to transcriptional activation. FUS3 and KSS1, however, are 
not functionally interchangeable in all respects. This difference 
can be seen from the observation that FUS3+kss1 strains arrest in 
response to a-factor, whereas fus3'KSS1+ strains do not (Elion et 
al., 1990, , 1991). An important clue as to how this occurs comes 
from the observation (Elion et al., 1991) that the ability of fus3 
strains to undergo cel 1-cycle arrest is restored if they are also 
defective in the CLN3 gene, which codes for a G1 cyclin. It has
34
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been proposed that the normal role of FUS3 is to inactivate the 
CLN3 protein perhaps by phosphory1ating it (Elion et a l ., 1990). It 
is not possible to know from these observations if CLN3 is a direct 
substrate of FUS3. These studies of FUS3 and KSS1 reveal some of 
the complexities likely to be encountered in studying protein 
kinases with multiple substrates and overlapping specificities; it 
appears that FUS3 has at least two substrates, only one of which is 
a substrate for KSS1 (Marsh et al. , 1991).
KSS1 was identified because its expression, when carried on a 
high copy number plasmid, allows cells defective in the SST2 gene 
to become partially resistant to a-factor (Courchesne et al. , 
1989). Given that KSS1 now appears to play a role in propagating 
the signal through the pathway, it is not clear why a high copy 
number plasmid carrying KSS1 should inhibit the functioning of the 
pathway.
The STE11 product is the only one of this group of putative 
protein kinases of yeast that has been shown to have kinase 
activity. This demonstration has come from an immune-complex 
phosphorylation assay using an epitome-tagged STE11 protein (Rhodes 
et al., 1990). These in vitro studies identify a substrate (p78) of 
78kd, which does not correspond to any known gene product, such as 
STE4 (47 kd), STE7 (55kd), STE12 (112 kd) or FUS3 (40kd) Rhodes et 
al. , 1990), and neither does it correspond to STE20 (102kd) Leberer 
et al. , 1992). Whether p78 has a role or not in signal transduction 
is unknown. There are numerous possible substrates for STE11 and 
the putative protein kinases in the response pathway. The following
35
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gene products have been shown to be phosphory 1 ated. STE2 (Reneke et 
al. , 1988), STE4 (Cole and Reed , 1991), STE5 (Marsh et al ., 1991), 
STE7 (Marsh et a l . , 1991), STE11 (Rhodes et al., 1990), STE12 (Song 
et a l . , 1991) and FAR1 (Marsh et a l . , 1991).
The steps mediated by STE4, STE7, STE11, STE20 and FUS3 have 
not been ordered biochemically with respect to each other. However, 
the behaviour of double mutants carrying these activated alleles 
and null mutations in other genes suggests that the signaling 
pathway may not be a simple linear kinase cascade. These protein 
kinases differ from each other with respect to their regulation. 
Transcription of the FUS3 gene is activated several-fold after 
treatment with a-factor (Elion et a l . , 1990). Although the STE7
gene contains three PRE (pheronome response elements) in its 
upstream regulatory region, synthesis of STE7 protein is not 
increased by exposure to a-factor (Marsh et a l . , 1991).
Hyperphosphorylation of STE7 however is rapidly induced by this 
treatment (Marsh et al., 1991). Neither transcription of STE11 nor 
activity of its product are increased by treatment of cells with a- 
factor (Rhodes et al . , 1990). The STE7, STE11 and STE20 genes are 
expressed in all cell types, even in a/a cells which do not respond 
to mating factors. Their role in a/a cells is not known; mutants 
defective in STE7, STE11 and STE20 genes do not exhibit any 
additional phenotypes. In contrast, the FUS3 gene, like many genes 
in the si gnal-transducti on pathway is expressed only in a and a 
cells and not in a/a cells. Nothing is known about the STE5 except 
that it appears to function downstream from G protein and upstream
36
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of STE12 (Marsh et a l . , 1991).
1.2.13 STE12 and Transcriptional Activation
The target for transcriptional activation of genes by the 
mating factors is the STE12 product (Dolan et al., 1989; Errede & 
Ammerer, 1989). Two pieces of information indicate that STE12 is at 
the end of the pathway for transcriptional activation. First, Ste12 
protein binds to the DNA sequence that is responsible for mating- 
factor inducibility (Dolan et al., 1989; Errede & Ammerer, 1989). 
Secondly, plasmids that express STE12 at high levels from a pGAL 
regulatory region bypass the need for various STE genes in the 
response pathway: substantial expression of FUS1 is observed in
strains overexpressing STE12 in ste7 and ste11 mutants (Dolan & 
Field , 1990), as well as in mutants defective in STE4, STE5, and 
FUS3 (Marsh et al., 1991). These observations indicate that STE12
is at the end of the si gnal-transduction pathway for
transcriptional induction and lead to the hypothesis that its 
activity is controlled by phosphorylation, perhaps by STE7, STE11 
or FUS3 (Dolan et al., 1989; Errede & Ammerer, 1989).
Transcription of many genes is induced by the mating 
pheromones (Appletauer & Achstetter , 1989). These include genes 
involved in cell fussion and other aspects of cell-cell
interactions: FUS1, which is induced more than 100-fold (Trueheart
et al., 1987), CHS 1 (coding for chitin synthase 1), which is
induced 10-fold (Appeltauer & Achstetter , 1989), and A G a 1 (coding 
for a-agglutinin), which in induced more than 20-fold by a-factor
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(Lipke et al. , 1989), AGA1 (coding for an a-agglutinin subunit); 
(Roy et al. , 1991) and KAR3 (coding for a kinesis-like protein
necessary for nuclear fusion) (Meluh & Rose , 1990). Genes involved 
in pheromone biosynthesis, such as structural genes for a-factor 
(MFa1 ) and STE13, are induced two to fivefold (Achstetter , 1989). 
Many of the components of the response pathway are inducible to 
similar extent; these include STE2, STE3, STE4, STE5, GPA1 and 
FUS3. The sequence TGAAACA (termed the PRE, pheromone response 
elements) is present in two to nine copies in the upstream 
regulatory regions of genes whose transcription is induced by 
mating pheromones (Trueheart et a l ., 1987; Konstad et al., 1987)
and was shown to be necessary for induction (Kronstad et a l . , 
1987). Studies have also shown that multiple, tandem PRE sequences 
are sufficient to confer pheromone inducibility to test plasmids 
(Hagen et a/., 1991). Presence of PRE sequences, however, is not 
always sufficient to confer inducibility to mating factors. Even 
though they contain multiple PRE sequences in their upstream 
regions, STE7 and STE12 do not appear to be inducible by a-factor 
(Marsh et al., 1991 ).
Although STE12 can bind weakly to individual PRE sequences 
(Dolan et al., 1989), its binding is greatly enhanced by
association with other proteins, such as the general transcription 
factor, MCM1, and by other as-yet uncharacterized factors (Errede 
& Ammerer , 1989). PRE sequences are often found in upstream
regulatory regions adjacent to MCM1 - binding sites (Krostad et 
al., 1987; Errede & Ammerer , 1989).
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STE12 is essential both for induction of transcription by 
pheromones and for setting the basal level of transcription of 
genes in the signal transduction pathway. This can be seen from the 
observation that transcription of STE2 and STE3 is reduced fivefold 
in a ste12 mutant (Fields et al. , 1988; Hagen et al., 1991). Five 
to twenty-fold decreases in transcription of all a-specific and a- 
specific genes analysed (M F a 1, MFa2, MFA1, MFA2 and STE6) are also 
observed in ste7 and ste11 mutants (Fields et al., 1988). It 
appears that the basal level of expression of these genes is due to 
some spontaneous activity of the pathway (for example, partially 
active STE12 protein or free GBy) rather than to the presence of a 
low level of mating factor in cultures (Hagen et al., 1991).
Studies on STE12 show that it is phosphory 1 ated after 
pheromone treatment and provide information on its functional 
domains. For technical reasons (to increase the amount of STE12), 
the phosphorylation studies were carried out with hybrid proteins 
that contain the DNA-binding domain of GAL4 (amino acid residues 1­
147) attached to all or parts of STE12. The GAL4-S7"£7i? (1-688) 
hybrid (containing all of STE12) is rapidly phosphory1ated after 
addition of a-factor, with kinetics similar to that for induction 
of pheromone-responsive genes (Song et al., 1991). Both
phosphorylation of STE12 (Song et al. , 1991) and transcriptional
induction (Achstetter , 1989) can occur in the absence of protein 
synthesis. The five protein kinases upstream of STE12 in the 
response pathway are obvious candidates for being responsible for 
phosphorylation of STE12. The observation that over-production of
39
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STE12 allows induction of FUS1 in ste4, ste7, ste11 and fus3 
mutants (Dolan & Fields , 1990) indicates that STE12 must have a 
basal activity independent of the response pathway.
The GAL4-STE12 hybrids have revealed information on functional 
domains of STE12. Hybrids containing the entire STE12 polypeptide 
exhibit some transcriptional activation ability, tenfold above 
background; this activity is observed only when cells are treated 
with a-factor. Hybrids containing residues 214-688 (which lack the 
STE12 DNA-binding domain) or 1-473 (which lack the putative MCM1 
interaction domain) exhibit similar induction (Marsh et al. , 1991). 
In contrast, the GAL4-STE12 hybrid containing residues 214-473 
behaves quite differently. It exhibits potent, constitutive 
activation activity, which is 250-fold above background (Marsh et 
al., 1991). The potent activity of the GAL4-STE12 (214-473) hybrid 
in comparison with the other hybrids suggests that the 1-214 and 
474-788 segments of STE12 contain inhibitory domain. An attractive 
possibility is that phosphorylation of such an inhibitory domain 
leads to activation of STE12 (Marsh et al., 1991).
The observation that transcriptional activation by several 
hybrids is stimulated by a-factor has different possible 
explanations. If one assumes that the GAL4 domain is sufficient for 
DNA-binding and localization to the nucleus, these observations 
indicate that the STE12 activation domain is regulated by the 
mating response pathway (Song et a l ., 1991), in particular, by
phosphorylation. Other explanations can be envisaged if the GAL4 
domain is not sufficient for DNA binding and nuclear localization.
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For example, phosphorylation of STE12 could regulate its entry into 
the nucleus (Baeuerle & Baltimore, 1988).
1.2.14 Interfacing with the Cell Cycle
One of the responses to mating pheromone is arrest of the cell 
in the G1 phase of the cell cycle. This is a transcient arrest that 
ensures that nuclear fusion will occur between nuclei containing a 
1N complement of chromosomes. The targets for this control appear 
to be the three G1 cyclins of yeast CLN1, CLN2, and CLN3, which are 
required for progression from G1 to S and are thought to be 
required for activity of CDC28, the budding yeast homologue of 
p34cdc2 (Wittenberg et a l . , 1990). Arrest in the cell cycle requires 
that all three G1 cyclins be inactivated (Richardson et al ., 1989), 
which may occur through the action of three separate inhibitors 
(Chang & Herskowitz , 1990). Two of the inhibitors (FUS3 and FAR1) 
have been identified because mutants defective in these genes have 
an intact, functioning response pathway but do not undergo growth 
arrest in response to mating factors (Elion et al., 1990; Chang & 
Herskowitz , 1990). FUS3 appears to be responsible for inactivating 
CLN3 (Elion et a l ., 1990, , 1991 ), and FAR1 for inactivating CLN2 
(Chang & Herskowitz , 1990). The compound responsible for
inhibiting CLN1 is unknown. The response pathway apparently 
triggers cell-cyle arrest by enhancing activity of these inhibitors 
of the G1 cyclins. Transcription of both FUS3 and FAR1 is induced 
several fold by mating factors Elion et al., 1990; Chang & 
Herskowitz , 1990). In addition, FAR1 is rapidly phosphory1ated
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after treatment of a cells with a-factor (Marsh et al., 1991). The 
existence of mutants such as strains that lack FAR1 (Chang & 
Herskowitz , 1990) or that carry the CLN3-1 mutation (Cross , 1988) 
that have an intact signal response system, but that do not arrest 
in response to mating factors, demonstrates that cel 1-cycle arrest 
is not a requirement for differentiation in yeast.
1.2.15 MATa2 and MCM1
The mating type of yeast cell, a or a, is determined by a 
single locus on chromosome III, called the mating-type locus (MAT), 
which may contain either of two types of sequences. MATa differs 
from MATa in the substitution of a 650-bp a-specific sequence for 
a non-homologous 750-bp a-specific sequence (Nasmyth and Tatchell 
, 1980). The two genes at MATa, a1 and a 2 , are transcribed
divergently from a central promoter and regulatory region (Johnson 
& Herskowitz , 1985). The MATa 2 gene is required for the
determination of both haploid and diploid cell types. In haploid 
cells, it is necessary for inhibiting the expression of a mating 
functions, which are otherwise antagonistic to a mating functions 
(Strathern et al., 1980). In diploid cells, the same inhibition of 
a mating functions is exerted, but in addition, it acts in 
conjunction with the a1 gene to regulate MAT transcription, 
principally the repression of a1 transcription and to allow 
sporulation (Nasmyth et al., 1981). The MATa2 gene product is 
therefore necessary for preventing the expression of both a and a 
mating types in a/a diploids (figure 3), (also Nasmyth et al . ,
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1981). In vivo, aZ represses transcription of two sets of cell 
type-specific genes by binding to operator sites together with 
either the a1 or Mcm1 proteins (figure 3). Each protein targets a2 
to a different set of operators (Herskowitz , 1989). The yeast a2 
repressor is also a particularly wel1-characterised member of the 
homeodomain superfamily of DNA-binding proteins (Wolberger et al. , 
1991). First identified in a series of Drosophila genes that 
regulate development, the homeodomain is a conserved sequence of 60 
amino acids. Homeodomain-containing proteins have now been found in 
virtually all eukaryotes examined (Scott et al., 1989).
MCM1 is a yeast transcription factor with homologs throughout 
the metazoa. MCM1 was first identified as a gene involved in 
maintenance of artificial minichromosomes in yeast (Elbe and Tye ,
1991). It has also been shown to serve as a transcri pti onal 
regulator of mating-type-specific genes (Elbe and Tye, , 1991).
Biochemical data suggests that MCM1 coactivates a-specific genes 
and corepresses a-specific genes by binding to a 10-base pair dyad 
symmetry element in their upstream regions (Elbe and Tye, , 1991). 
Intrigued by the cells capacity to tightly control its growth, and 
by the consequences of its inability to do so, the objective of 
this study is to isolate and characterize additional genes of the 
signal transduction pathway that are involved in this cellular 
growth control. This goal can only be accomplished by analyzing 
these genes structural 1y , genetically and biochemically in other 
to understand their mode of action. The importance of these growth 
control points in the cell cannot be overemphasized since they
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presumably exist to prevent both replication of a damaged DNA 
template and segregation of damaged chromosomes. It is thought 
that the transient delays at these growth control points permits 
repair of damaged DNA prior to these critical cellular functions 
and should thus enhance cell survival and limit propagation of 
heritable genetic errors (Weinert & Hartwell , 1988). Since the
yeast provides an experimental opportunity with all of the 
techniques of manipulative molecular genetics, the yeast pheromone 
response pathway offers opportunity to explore signal transduction 
in a genetically tractable organism. This is so because many of the 
features that have been found to date are remarkably similar to 
those found in multicel 1ular eukaryotes.
In this study, the focus on the search for possible GPA1(SC G 1) 
homologs was inferred through hybridization studies that there 
could be at least two additional genes homologous to GPA1(SCG1) in 
the yeast genome (Dietzel & Kurjan , 1987). This was coupled with 
the possibility that other gene products are involved in vivo in 
modulating the signaling response.
44
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4 5
M A T  locus t
MCM1
(a) a Cel
cx I +  CO
+  o o
asg
a sg
0.2 hsg
O O
(b) a Cell asg
asg
GO
CO
hsg
(c) a/a Cell
asg
asg
hsg
Figure 3 i Regulation of cell types by MAT locus genes and MCMl
Regulation of a-specific genes (asg), a-specific genes 
(asg), and haploid specific genes (hsg) in a, a and a/a 
yeast cells by regulatory proteins encoded at the MAT 
locus together with M C M 1 , a constitutive DNA-binding 
protein. 3a shows transcription of asg by al-McMl pro­
teins and repression of aug by a2-Mcml protein complex. 
3b shows repression of asg by a2-Mcml proteins. 3a shows 
repression of asg by a 2 -Mcml proteins, hsg by al-a2 
proteins and al transcription repression by al-a2 
proteins complex.. Solid bars indicate repression of gene 
transcription wh’ereas arrowheads or wavy arrows denote 
transcription activation. As a result of this regulation, 
each diploid or haploid cell type exhibits a distinctive 
pattern of gene expression.
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CHAPTER TWO 
MATERIALS AND METHODS
2.1 Materials
46
Table 2: Yeast Strains Used in this Work
Strain fielevant Genotype Source
SP1 MATQ l e u 2 ,  u r a 3 t h i s  3 a d e 8  t r p l t TYR 1 ,  g a l 2 ,  ca .n l Colieelli
FY250 MATa u r a 3 , h i s 3 ,  l e u 2 , t r p l Colicelli
DC14 MATa h i s l Colieelli
DC17 MATa h i s l Colicelli
DC124 MATa l e u 2 , u r a 3 ,  h i s 4 , t r p l  a d e 8 Colicelli
SP1/DC124 MATa/MATZ, I e u 2 / l e u 2 1 h i s 3 /H I S 3 ,H I S 4 / h i s 4 , u r a 3 / u r a 3 ,  t r p l / t r p l ,  a d e 8 / a d e 8 Colicelli
GUI MATa g p a l : H I S 3  l e u 2 , u r a 3 , c a n l  (pTLCG ) Colicelli
GU2 MATa g p a l : :H IS 3 ,  l e u 2 t u r a 3 , c a n l  (pTLCG ) Colicelli
LG1-TG GUI with plasmid replacement for TRP1/GPA1/CAN1 This work
LG2-TG GU2 with plasmid replacement for TRP1/GPA1/CAN1 This work
LG1-UG GUI with plasmid replacement for URA3/GPA1 This work
LG2-UG GU2 with plasmid replacement for URA3/GPA1 This work
GL1-5 GUI with plasmid replacement for URA3/MCM1/CAN1 This work
Gll-9 GUI with plasmid replacement for URA3/MATa3/CAN1 This work
GL1-12 GUI with plasmid replacement for URA3/YGC1/CAN1 This work
GI1-UGC GUI with plasmid replacement for URA3/GPA1/CAN1 This work
GIl-UC GUI with plasmid replacement 
for URA3/CAN1 This work
GL2-5,9 
12, UGC and UC
All created by GU2 replacements for 
the respective plasmids as in 
GLl-strains
This work
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47
Table 3 
Plasmids Used in this Study
Plasmid Relevant Genotype Source
pTLGC LEU2/GPA1/CAN1 Colicelli
pUV2 pUC118 JURA3 Colicelli
YEP13M4 LEU2 vector Colicelli
pRS416 URA3/CEN plasmid Colicelli
pYeCAN CAN1 Source Colicelli
pKS pBluescript vector Colicelli
pTGC TRP1/ GPA1 / CAN1 This work
pUGC URA3/G PA1 / CAN This work
pU5C URA3/MCM1/ CAN 1 This work
pU9C URA3/M ATa2 / CAN This work
pU12C URA3/YGC1/CAN This work
pUC URA3/CAN1 This work
YEp9 LEU2/MATa2 This work
pRS416M9 M CEN/URA 3 /MA To. 2 This work
pUVEK pUV2/.E!a£i - k p n l  
fragment of MATa2
This work
PUVEK-URA3 pUV2/mata2::URA3 This work
pKSEK pK S /M ATa2 This work
pKSSX pKS J S m a l -X b a l  
fragment of MATa2
This work
The E coli strain DH5a purchased from New England Biolabs, Beverly, MA was 
used throughout this work for the propagation of the plasmid DNAs.
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48
Table 4
Drop-Out Medium (Synthetic Complete (SC) Medium)
To lOOg of Yeast Nitrogen Base without Amino Acids (Difco), were added:
Adenine 0.75g
Arginine 1 • 5g
Asparagine 1. 5g
Aspartic Acid 1. 5g
Histidine 1. 5g
Isoleucine 1.5g
Leucine 3. Og
Lysine 1. 5g
Methionine 1. 5g
Phenylalanine 1 • 5g
Proline 1.5g
Serine 1. 5g
Threonine 1. 5g
Tyrosine 1 • 5g
Tryptophan 1.5g
Uracil 1.5*
Valine 1. 5g
For the appropriate drop-out recipe, the corresponding constituent was 
omitted. The constituents were then mixed well. For plates preparations;
9.0g drop-out mix was added to 350ml 
of water in 1 litre flask (A)
20g glucose was added to 50 ml of water in 250ml 
flask (B)
and 20g of agar was added to 600ml of water in 2 litre 
flask with stirbar (C).
All flasks were autoclaved for 30 min at 121°C. Contents of flask A and B 
were mixed, then added to flask C with slow stirring, cooled and poured. YPD
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medium contained 1% Bacto-yeast extract 2% Bacto-peptone, and 2% dextrose 
(Difco). LB medium contained 1% Bacto-tryptone, 0.5% Bacto-yeast extract and 1% 
Nacl (pH 7.0)
49
ENZYMES AND REAGENTS
Restriction endonucleases, DNA modifying enzymes and DNA markers were 
purchased from New England Biolabs Inc., Stratagene and Boehringer Manhein.
Sequencing kits (reagents) were purchased from United States Biochemicals, USA.
3 2  «(a- P)dATP was obtained from Amersham Corp. Restriction endonuclease linker 
oligonucleotides were ordered from Stratagene. Bacteriophage T7 and T3 RNA 
polymerase primers were purchased from New England Biolabs Inc. Other Chemicals 
and Reagents used were purchased either from Sigma Chemicals Company or from 
British Drug House Chemicals Ltd (BDH), and were of the highest purity grade 
commercially available.
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50
2.2 Methods
2.2.1 Construction of the Yeast Clones
Genomic DNA libraries of Saccharomyces cerevisiae were constructed by 
ligating partial Sau3A digests of genomic DNA into the BamHl sites of a URA3 
marker vector (pUV2). The library was composed of 4 kilobase or larger Sau3A 
restriction fragments, selected by running the restriction fragments on 0.7% 
agarose gel alongside a Lambda DNA - Hind III digested marker. Fragments selected 
were then cut-off from the gel, electroeluted, using dialysis tube and TBE (See 
appendix) buffer, purified with phenol and chloroform, precipitated with 0.5ml 
of 100% cold ethanol plus 0.1ml of 3M NaOAc (pH 5.2), washed with 1ml 70% cold 
ethanol and then randomly ligated to pUV2 cut with BamHl using Bacteriophage T4 
DNA ligase modifying enzyme.
2.2.2 Selection for the Yeast GPA1 Complementary (YGC) Clones
Screening of the clones or library was based on the complementation of 
gpal, First, the GU cells were transformed with the URA3 marker based YGC 
library. One percent of the transformed cells were then plated on SC-URA 
(synthetic complete medium without uracil) to test the efficiency of the 
transformation. The rest, 99% of the transformed GU cells were grown in 1.5 ml 
SC-URA culture media for 2 days at 30°C with shaking at 250rpm on a rotary 
platform. This was to enable the GU cells lose the GPA1/LEU2/CAN1 maintenance 
plasmid.
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igure 4: Complementation of GPA1
The diagram shows the selection procedure based on the 
complementation of gpal strains of GUI and GU2. The 
strains were maintained by the plasmid GPA1/LEU2/CAN1. 
Other plasmids (Clones) labelled X/URA3 were introduced 
into the strain and the GPA1/LEU2/CAN1 plasmid destroyed 
by the uptake of the drug canavanine. Larger circles 
indicate the GUI and GU2 strains whereas the smaller 
inner circles denote the maintenance plasmids. The 
genotype of the GU cell is underlined.
The signaling pathway leading to differentiation and cell 
arrest is indicated by the arrow. The yeast G protein 
(afly) is also shown. Where the fly subunit is involved in 
the signal transduction is denoted by a circle and the 
signal inhibitory a subunit is indicated with an ovoid. 
The wavy lines on the largest circle denote the seven 
transinembrane receptors of the a-or a-factor.
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52
T ran s fo rm a t io n  of  GU ce l ls  
(gpa l  Ieu2 ura3 can l  pGPA1 /LEU2 /CAN1)  
w i th  URA3 based l ib ra ry
Figure 5: Flow Chart describing the selection of high copy 
suppressors of gpal
Plasmid dependence was checked by streaking single 
colonies from the SC-URA-ARG+CAN plate onto SC-LEU and 
SC-URA plates to look for no growth and growth on the 
respective plates. Plasmids containing the CAN1 gene 
cannot survive or grow on canavanine plate or media 
because the CAN1 gene selects against itself. Thus, 
colonies that survive on the canavanine plate contain the 
yeast GPA1 complementary clones. No growth on the SC-LEU 
plate indicates the lost of GPA1/LEU2/CAN1 plasmid since 
LEU2 gene is its marker.
plate 1% on SC-Ura 
( e f f i c i e n c y  t e s t )
g row  99% in SC-Ura 
( p la sm id  loss )
p la te  on SC-U ra -A rg  + Canavan ine  
( s u r v i v o r s  have los t  
GPA1 /LEU2 /CAN1  p la sm id )
check  fo r  p lasm id  dependence  
on SC-LEU and SC-URA plates
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After 2 days of culturing the cells in SC-URA media, 5pl of the cells were plated 
on SC-URA-ARG+CAN (SC media without uracil and arginine but has a drug canavanine 
added). Survivors on this plate lost the GPA1/LEU2/CAN1 maintenance plasmid since 
CAN1 gene allows the drug canavanine (an arginine analogue) to counter select 
against itself. Plasmid dependence of the GU cells were determined by streaking 
single survival colonies from the SC-URA-ARG+CAN plate onto SC-URA and SC-LEU 
plates. (Jrowth' and no growth on these plates respectively confirms the loss of 
the GPA1/LEU2/CAN1 plasmid as opposed to possible survival due to reversion 
(Fig.5). This procedure was used to successfully select 8 high copy suppressors 
of gpal from the yeast library. These clones were pYGC5, 6,7,9,11,12,14 and 20 
(Fig.12). This method was routinely used to retest and confirm the clones before 
further analyses were carried out. Figures 4 and 5 show the diagramatic 
presentation and flow chart of the selection procedure respectively.
2.2.3 Southern Analysis
In order to determine the genes responsible for complementing the gpal 
strain of GU cells, Southern blottings were done as described by Maniatis et. 
al., (, 1982). Ten micrograms (lOyg) of the high copy suppressor plasmids were 
digested with Ecc&l and Hindlll restriction endonucleases. The resulting 
restriction fragments were separated according to size by electrophoresis on 0.8% 
agarose gel. The gel was photographed to make sure that the restriction digests 
were complete and also to note the size of the various DNA fragments. DNA 
fragments were then capillary transferred from the gel onto a nitrocellulose 
filter (Maniatis et. al., 1982), using lOx SSC buffer, Whatman 3MM paper, paper 
towels, plexiglas, Saran wrap, glass plate and a 500g weight. The DNA fragments 
were denatured by soaking the gel for 45 minutes in 200 mis mixture of 1.5M Nacl
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and 0.5N NaOH with constant and gentle agitation on a rotary platform. The gel 
was then rinsed in 250mls of deionized water, and then neutralized by soaking for 
35 min in 200 mis of 1M Tris (pH 7.4) containing 1.5M Nacl at room temperature
with constant, gentle agitation. Meanwhile, 1.9Kb EccMl fragment of GPA1 was
3?labelled with P using the modifying enzyme T4 polynucleotide kinase reaction.
The (?R4i-labelled probe was then denatured by heating at 100°c for 8 min and
rapidly chilled in ice water. The nitrocellulose filter containing the
immobilized single-stranded fragment was then wetted in 6X SSC, slipped into a
heat-sealable bag (Sears Seal-A Meal bag) and 2.5 ml of prehybridization solution
(see appendix) added after which the bag was sealed. The sealed bag was then
submerged in 68°C water bath for 2 hours with gentle agitation. The bag
containing the filter was then removed from the water bath, opened with scissors 
Itand the P-labelled denatured probe added to the prehybridization solution and 
the bag resealed with a heat sealer. The bag was then incubated, for 2 hrs at 
68°C. At the end of the incubation period, the filter was washed eight times in 
500ml of 2xSSC containing 0.5% SDS, and in 500 ml of 0.1XSCC containing 0.5% SDS, 
at room temperature. The filter was incubated for 45 min at 37°C with gentle 
agitation and then transferred into a 68°C water bath for another 45 min. The 
filter was washed again briefly with 250ml of 0.1XSSC at room temperature. The 
excess liquid was removed by placing the filter on a pad of paper towels. The 
filter was then covered with Saran wrap and exposed to Kodak (XAR-2)X-ray film
for 24 hrs (Fig 12).
2.2.4 Hybridization of Clone 9 to the Other Clones
This Southern blot was done to classify the various clones. The same 
nitrocellulose blot described above (Fig. 12) was stripped in a boiling water
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bath. This was done by heating 400ml of 0.05X SSC containing 0.01M EDTA (pH 7.9) 
(elution buffer) to boiling. The fluid was then removed from the heat and SDS 
added to a final concentration of 0.1%. This was followed by immersion of the 
filter in the hot elution buffer for 17 min. The filter was briefly rinsed in 
0.01X SSC at room temperature after the immersion step was repeated with a fresh
batch of boiling elution buffer. Excess liquid was removed from the filter with
32paper towels, dried and rehybridized to a P-labeled probe made from Clone 9 
(Figure 13).
To determine the uniqueness of clones 5 and 12, the high copy suppressor
plasmids 5 and 12 were digested with E'coRl and Hindlll, run on 0.8% agarose gel
35and transferred to a nitrocellulose filter. The filter was then probed with P- 
labeled clone 12 in a similar way as described above (Figure 14).
2.2.5 Mapping of Clones 5.9 and 12
The high copy suppressor plasmids were restriction mapped using several 
restriction endonucleases. This involved the setting up of digests with 
restriction enzymes that cut only once or not at all in the pUV2 vector. Double 
enzyme digests were then carried out to locate the restriction sites on the 
inserts and their respective distances apart by running the restriction fragments 
alongside vector digests, lambda DNA #indIII digest and $X174 DNA-Haelll digest 
markers on agarose and polyacrylamide gels respectively. The restriction digests 
were usually set up in 30pl total volumes comprising the plasmid DNA, enzyme 
buffer, RNAase (when miniprep DNA was used) and water in eppendorff tubes 
incubated at 37°C for 1 to 24 hours depending on the activity of the enzyme(s) 
used. In some cases, the inserts were subcloned into other plasmids such as pKS 
for further mappings, especially if some restriction sites exist on both the
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56
vector as well as on the insert, more than once (Figures 15 and 16).
2.2.6 Clone Deletions
The clones 5, 9 and 12 were deleted to locate the smallest possible 
fragment capable of complementing the gpal. Deletions were done using 
restriction enzymes based on the restriction sites mapped on the three clones. 
After these restriction endonuclease site-specific deletions, the fragment ends 
were either religated in the same plasmid clone using T4 DNA ligase or subcloned 
into pUV2 and transformed with GUI and GU2 (MAta and MATa) haploid yeast strains 
(Figure 20).
2.2.7 Creation of Yeast Strains
First, the yeast strains whose plasmid replacement were to be carried out 
were transformed with the plasmid which was to replace the original strain 
maintenance plasmid. The transformed cells were then grown in the media where the 
replacing plasmid could propagate at the expense of the original strain 
maintenance plasmid at 30°C for 2| to 3 days. Then 5pl of the culture media was 
plated on the SC-plate where the replacing plasmid could grow better for to 
3 days at 30°C. The cells on the "master" plate were then replica-plated onto a 
velvet and then from the velvet onto an SC-plate containing the introduced 
plasmid marker gene and another plate containing the marker gene of the original 
strain maintenance plasmid. The replica plates were then incubated at 30°C for 
12-15 hours. Plasmid loss on the original plasmid marker plate (observed by the 
presence of colonies footprint) was looked for by comparison with either the 
'master* plate or the introduced plasmid marker replica plate, on which there 
would be no loss of colonies. Single colonies were then picked from the plate on
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which no loss had occured by it being matched with the lost colonies on it’s 
replica plate. The single colonies were then appropriately purified by growing 
them in the Sc-raedia of their marker genes. Plasmid dependence was then 
confirmed by no growth when about 4yl of the cells were diluted with lOOpl of the 
culture media and plated on the original plasmid marker Sc-media and grown on the 
replacing plasmid marker Sc-media. The newly created strains were then used for 
a mock library transformation with the appropriate plasmid to test for the 
usefulness or otherwise of the strain in library screening. (See the flow charts 
on creation of yeast strains for the creation of the specific yeast strains in 
Figures 6 and 7).
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Transformation of GUI and GU2 Strains with plasmids,U5C,U9C,U12C,UGC,and UC.
Plate on Sc-URA3 and grow for 2.5days
Pick single colonies and grow in Sc-URA3 for 2.5days
Plate 200ul o f  l/lO 1^ . dilution of the culture onto Sc-URA3 for 2.5days
___________________Replica plate onto Sc-URA3 and Sc-LEU2
i , i . .Sc-LEU2 (look for plasmid loss from here) , . , .^c ' f , _ ^ (pick leu2 colonies from here
Recheck for growth and no growth on Sc-URA3 and Sc-LEU? respectively(Plasmid^dependence).
Mock library transformation with YEpl3 and YEp^
Figure 61 Flow Chart showing the creation of haploid strains 
GL1 (GI<2) of clones 5,9 12, UGC and UC.
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Creation o f  LG 1 fLG2)-TG Strains
Transformation o f  GL l-9 ,GL l-12 ,GL2-9  and GL2-I2  Strains with pTGC
flate on Sc-TRP and grow at 30°c for 2.5dnys
Replica plate onto Sc-TRP and Sc-URA3  
Sc-T*RPSc-URA3  look for ura3 lost colonies here) (Pick ura3 and TRP colonies here)
Confirm plasmid dependence
Mock transformation
figure 7: Creation of LOl(LG2)-TO Strains
The strains GLl(GL2)-9 and 12 were transformed with 
plasmid TGC and plated on SC-TRP. Selection of the 
strains were based on growth on SC-TRP and no growth on 
SC-UUA media since the new strains have TRP1 as theirs 
marker gene.
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Plasmid Constructs
The plasmids were constructed as illustrated in the figures 
(diagrams)(8,9,10,11). Refer to legends in each case for description of the 
constructs.
2.2.8 Construction of TRP/GPA/CAN plasmid (dTGC)
Restriction endonuclease XhOl oligonucleotide linker ligation was used to 
change a Smal to a Xhol site in pTGS to give pTGX. This was performed by cutting 
pTGS with Smal enzyme. This was followed by phosphatasing the cut ends with calf 
intestine phosphatase (CIP). The cleaved and phosphatased pTGS was then purified 
and pelleted with chloroform/phenol and cold 100% ethanol respectively. 
Meanwhile, the Xhol oligonucleotide linker was kinased using T4 DNA kinase. The 
phosphatasing was necessary to prevent the religation of the compatible Smal 
ends. The phosphatased pTGS and the kinased Xhol oligonucleotide linker were then 
ligated using T4 DNA ligase.
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Xhol/Pstl 
fragment (ARS>
Xhol and Patl 
cut and gel 
Purified
  4-ion
Figure .8: Construction of pIOC
This illustrates the various steps involved in the 
construction of plasmid TGC. Restriction enzyme digests and 
ligation reactions were mainly used in the construction. The 
Smal site was changed to a Xhol site using a Xhol 
oligonucleotide linker to form plasmid TGX. The autonomous 
replicating sequence (ARS) was then cut from plasmid KSars. 
pKSars was formed by ligating the Patl/Xhol fragment of ARS to 
Bluescript KS+. The* ARS fragment was then ligated to TGX which 
had been cut with P&tl and Xhol to form plasmid TGSXars. 
pTGSXars was cut with Xhol phosphatased and then ligated to 
the Sall/Xhol fragment of CAN1 gene to form plasmid TGC. pTGC 
has TRP1 marker, ARS, CAN1 and CPA1 genes as its relevant 
genotype.
pUCU 8
CANI
Cut from plasmid pYeCAN
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The Pstl to Xhol restriction fragment of autonomous replicating sequence (AES) 
was cut from plasmid ksARs and ligated to pTGX which had been cut with Xhol and 
Pirtl and gel purified to give pTGSXars. The plasmid TGSXars was then cut with 
X/joI, phosphatased and ligated to Sall/Xhol fragment of canavanine gene (CAN1) 
which was cut from the plasmid pYeCAN to give pTGSXarsCAN which was shortened to 
pTGC (Figure 8). Sail and Xhol cut to leave compatible sticky ends, but when 
ligated, neither site is retained.
All the constructs on the pathway leading to the final plasmid were 
confirmed using appropriate restriction digests and run on agarose gels alongside 
DNA markers and vector digests. The pTGC was used to create the LG1(LG2)-TG 
strains (Figure 7).
2.2.9 Construction of YEd 9
YEp9 was constructed using YEpl3M4 which has LEU2 as its marker gene, as 
the vector. YEpl3M4 was cut with Sacl and Smal restriction enzymes. The digested 
YEpl3M4 was then agarose gel-purified and ligated to a purified Sacl/Sisal 
fragment of Clone9 (Figure 9).
2.2.10 Construction of plasmid RS416 "9"
Plasmid RS416, a centromere (CEN) plasmid and a URA3 based vector was used. 
The vector RS416 was Kpnl and Cial double digested and gel purified. Similarly, 
clone9 was Kpnl and Cla.1 double digested using their compatible buffer, and gel 
purified.
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Figure
63
Siicl Xbal Smal
r^vcJ/Smal KrapmcM of CloncV
Cut w ill)  SacI
Ligation
Xbal
9: Construction of YEp9
This shows steps involved in construction of YEp9. First, 
the vector YEpl3M4, which has LEU2 as its marker was cut 
with Saal and Smai double digest. This was then purified 
and ligated to the S a d / S m a l  fragment of clone9 to form 
YEp9. YEp9 has M A T a 2 , LEU2 marker and a 2pm origin of 
replication genes.
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The gel purified RS416 was then ligated to the Kpnl/Cla.1 fragment of MATa2 using 
T4 DNA ligase to give pRS416"9", a CEN based plasmid (Figure 10).
All other plasmids were similarly constructed by ligating cohesive 
restriction ends of purified fragments and vectors. Restriction enzyme digests 
were mainly used to confirm the plasmids. However, the yeast plasmids were
further confirmed by marker gene prototrophy.
2.2.11 Disruption of MATa2
In order to test the importance of MATa2 in recovering the gpal strains,
the MATa2 transcript was disrupted using URA3 marker gene.
A Hind III restriction endonuclease site was inserted into the Xbal 
recognition site on the gene subcloned into pBluescript (pks) vector forming the 
plasmid KSEK using Hindlll oligonucleotide linker (Figure 31). The plasmid KSEK 
was cut with Xbal, phosphatased using calf intestine phosphatase and the DNA 
pelleted with ethanol. l.Oyg of the Hindlll linker was phosphorylated using T4 
polynucleotide kinase reaction and ligated to the pKESK phosphatased plasmid. 
This was followed by insertion of the 1.17Kb Hind III fragment of the URA3 marker 
gene into the newly created Hind III site based on Hindlll cohesive end 
compatibility and T4 DNA ligase reaction (Fig 26).
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65
H in d IU  E c o R J  S n u l  S m I H a l l  SSCH  f w c l
Koni
+  Kpnl
Clal Eagl Xbal Smal\I I  I w  a
ligation
Figure 10: Construction of Centromere plasmid.
This illustrates the Steps involved in construction of 
plasmid RS416"9"- The vector pRS416 was cut with Clal and 
Kpnl and ligated to the Clal/Kpnl fragment of clone 9 to 
form pRS416"9" .
Plasmid RS416"9" has CENS origin of replication, URA3 
marker and MATa2 genes.
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66
Sinai
Knn-lXlXho1
Sail
CAN1
Ligation of Kpnl/Saol 
fragment of CAN1 to 
pUV2 cut with Kpnl 
and Sacl
Ligation of Kpnl/Baml 
fragment of CAN1 to 
pYGC5 cut with Kpnl
Baml_______________ Sacl1 \  iKpnl
Ligation of Smal 
fraqment of CAN1 
to pUG cut 
with Smal 
*
Ligation of Xhol/Sacl 
rragment of CAN1 to 
PYGC9 °
/
/Jnml
Snial
Ligation of Kpnl/Clal 
fragment of CAN1 t o  Smal pYGC12 cut with 
Kpnl and Clal
Figure lit Construction of UGC, U5C, UVC, U9C and 
U12C plasmids
The various restriction fragments of CAN1 were transferred 
into pUV2, pUG, pYGC5, pYGC9, pYGC12 from pKCC to form the 
CAN1 versions of the respective plasmids. First, Xhol/Sall 
fragment of CAN1 was subcloned into the multiple cloning site 
of Bluescript Ks(+/-) to form pkCC. This increased and varied 
the restriction sites flanking the CAN1 gene. The plasmids 
U V 2 , UG/YGC5, YGC9 and YGC12 were then cut with Kpnl/Sacl, 
Smal/ Kpnl/Baml, Xhoi/Sacl and Kpnl/Clal respectively and 
.jLlgatgd ±q—fchfl—yarious fragments of CAN1 which were cut with 
the corresponding* enzymes to enhance sticky ends compatibility 
1 1 rrnt 1 onn .
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The Sacl/Kpnl restriction fragment of the URA3 disrupted gene (mata2::URA3) was 
then subcloned into pUV2 vector to give the plasmid UVEK-URA. The pVEK-URA was 
then transformed with GUI and GU2 haploid yeast strains and plated on SC-URA 
(fig.27). Single colonies were picked after 2 days and inoculated into SC-URA 
media for another 2 days and plated on SC-URA-ARG+CAN. The disruption of the 
MATa2 gene was confirmed with restriction endonuclease digests of both the 
vectors and the vectors plus the inserts.
2.2.12 Mating Assays
Patch mating tests were performed by replica-plating patches of cells to 
a lawn of the tester strains on permissive plates. Patches of the haploid strains 
carrying these respective plasmids; SPI(pUV2), SPl(pYGC9) GUl(pUV2), GUl(pYGC9), 
FY250(pUV2), FY250(pYGC9) GU2(pUV2) and GU2(pYGC9) were streaked on YPD plate to 
give the "master" plate. The master plate was then incubated at 30°C for 60hours 
for the cells to grow. The plate was then replicated onto a velvet, then from the 
velvet onto SC-URA plate and SC-HIS-TRP plate which had been spread with DC14 
cells. The master plate was re-replica plated onto a new velvet followed by 
replication from the velvet onto SC-HIS-TRP spread with DC17 cells. Diploids were 
selected on the SC-HIS-TRP plates whilst the SC-URA plate served as the control. 
The plates were incubated at 30°C overnight.
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2.2.13 Quantitative Mating Assay
Single colonies of the strains with their plasmids were picked from the YPD 
plate and grown in YPD media for 60 hrs. 10S cells of each strain were counted 
and mixed with 10 cells of the tester strains DC14 and DC17 in TE buffer and 
incubated at 30°C for 3 hours to allow the cells to mate. 1.5 mis of SC-HIS-TRP 
was added to each tube to select for the diploids. The diploid cells were counted 
from drops of the cell culture fixed on Hematocrit under a Nikon phase-contrast 
microscope.
2.2.14 Sequencing
Both DNA strands of MATa2 were sequenced by the dideoxy chain termination 
method (Sanger et. al., 1977). The sequenase version 2.0, a genetic variant 
of bacteriophage T7 DNA polymerase and [a-3ZP]dATP were used. The MATa2 gene was 
subcloned into pBluescript 11KS(+/-) using cohesive restriction ends. MATa2 was 
cleaved into almost two equal parts and subcloned into multicloning sites of pks 
forming the plasmids KSEX and KSSX (Figure 29 and 30). Unlike Sanger’s method 
which stressed on single stranded template sequencing vectors such as bacterio­
phages, the use of plasmids called for denaturation of the plasmids prior to the 
annealing reaction to create single stranded templates.
The purified RNA-free plasmid DNAs were prepared using the Qiagen method. 
7yg of plasmid DNA in 200yl total volumes were alkaline-denatured by adding, 20jil 
of 0.2M NaOH containing 0.2mM EDTA and incubated at 37°C for 30 minutes. The 
denatured DNAs were neutralized by adding 20iil of 3M sodium acetate (pH5.2) and 
the DNA precipitated with 600yl of ethanol (-70°C, 15 min). After washing the 
pelleted DNA with 70% ethanol, it was redissolved in 6pl of distilled water. 2pl 
of sequenase reaction buffer and 2yl of T7 and T3 DNA polymerase primers were
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then added. Annealing of primers to the template was done by warming the capped 
tubes containing the DNA templates and primers to 65°C for 2 min and leaving to 
slowly cool from 65°C to 30°C in a temperature block. The rest of the reactions
were as described by Sanger et. al., (1977).
2.2.15 Transformation of Bacteria with Plasmids
Plasmid DNAs and their DNA inserts were routinely propagated by 
transforming E. Coli strain DH5a with the plasmids. The DH5a cells were cultured 
in 250ml flasks (OD^ = 250), usually in 100ml LB+ ampicillin media. The cells 
were then spun down at 3,500 rpm in a Sorvall GS3 rotor set at 4°C. The 
supernatants were then decanted and the pellets combined in 20ml transformation 
buffer (TFB) (see appendix) kept on ice for 10 min and then centrifuged at 4°C 
for 10 min at 3,500 rpm in a Sorvall GS3 rotor. The supernatant was well decanted 
and 2ml TFB added to the pellets. 70pl of DnD (See appendix) and water to 10ml 
total volume was added gently, swirled and kept on ice for 15 min. Another 70jil 
DnD aliquot was added, swirled and again kept on ice for 15 min. 200pl of cells 
were then aliquoted into eppendorf tubes and either 4pl of ligation reaction 
added or a lesser concentration of purified plasmid DNA added and kept on ice for 
30 min. These were followed by 90 seconds heat shocking at 42°C after which the 
cells were kept on ice for 1 min and quickly spun at 6,000xg for 3 seconds in a 
microfuge. The supernatant was then removed and the cells resuspended in 200pl 
of SOC (see appendix) and incubated for 50 min at 37°C thus allowing the cells
to recover. After the incubation period, the cells were spread on LB+ Amp plates
and incubated at 37°C overnight. However, at certain times part of these 
competent cells were frozen at -80°C and readily used when needed except that the 
efficiency of transformation slightly fell below that of the freshly prepared 
competent cells.
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2.2.16 Transformation of Yeast with Plasmids
Most often, yeast transformations were used to determine the function of 
genes cloned into plasmids and also for marker genes prototrophy. In this work, 
most of the yeast strains used for the transformations were haploid strains.
Cells were cultured to 0.5x10^ to 3.0x10^ cells/ml usually in 100ml YPD 
culture media in 250ml flasks. The cells were spun for 5 min at 2,000rpm using 
Beckman bench-top centrifuge at room temperature. After decanting the 
supernatant, the cells were resuspended in 20ml 0.1M LiOAC, containing 1M 
sorbitol per 50 ml culture and kept at room temperature for 10 min. The cells 
were then spun at 2,000rpm for 5 min at room temperature in a Beckman bench-top 
centrifuge and resuspended as before in 0.1M LiOAC, 1M sorbitol. The cells were 
then pelleted again by spinning at 2,000rpm for 5 min at room temperature and 
6.5x10" cells were resuspended per ml in 0.1M LiOAC+lM sorbitol followed by 
addition of 20yl denatured carrier DNA per ml (Salmon sperm DNA 5mg/ml stock). 
0.3ml cells were aliquoted into 1.5 ml eppendorf tubes and about lOpg (2Ojil) DNA 
added, mixed and kept at 30°C for 15 min without shaking. 0.7ml of 50% PEG in 
TE (Polyethylene glycol in Tris EDTA) was added to each tube, mixed by inversions 
and incubated at 30°C for 30 min with inversions every 10 min. This was followed 
by the addition of 0.1M DMSO (Dimethyl sulfoxide) with immediate mixing and heat 
shocking at 42°C for 5 min. The cells were then spun down at 7,500xg for 5 sec 
in a microfuge and resuspended in 200pl TE plus penicillin/streptomycin after the 
supernatants were removed. The cells were then plated on the appropriate plate 
and grown at 30°C for 60 hours.
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2.2.17 Mini Plasmid Preparation Procedure
Two-and-half (2|) mis of LB+AMP bacteria culture was grown at 37°C 
overnight with shaking. 1.5mls of the culture was then spun in microcentrifuge 
tubes for 10 seconds and the supernatant decanted such that about 6O11I of it was 
left in the tube into which the cells were resuspended completely (using 200iil 
pipet). 300pl of TENS solution (lOmM Tris-Hcl, pH 7.5, ImM EDTA, O.IN NaoH, 0.5% 
SDS) was added and vortexed for 4 sec until the cells lysed and the mixture 
became viscous (up and down pipetting also lyses the cells). 150pl of 3.0M 
sodium acetate, (pH5.2) was then added and vortexed for 5 seconds to mix 
completely. Cell debris and chromosomal DNA were pelleted by spinning for 2 min 
at 12,000xg in a microfuge, the supernatant transferred to a fresh tube and 900pl 
of cold 100% ethanol added and mixed well by inverting the eppendorf tubes. 
Plasmid DNAs and RNAs were pelleted by spinning for 5 min at 12,000xg in a 
microfuge. The supernatants were then discarded and the pellet, having a white 
appearance rinsed twice with 1ml 70% cold ethanol. Residual ethanol was removed 
after another quick spin and the DNA pellets were then resuspended in 30-40yl of 
TE buffer or sterile deionised water.
2.2.18 Preparation of Yeast Genomic DNA
The cells were cultured in a rich medium such as YPD overnight at 30°C in 
an incubator with shaking at 250rpm. 10ml portions of the cultured cells were 
spun down at 2,000rpm in Beckman bench-top centrifuge for 5 min and resuspended 
in 1 ml TE, and transferred into eppendorf tubes. The cells were then spun down 
for 30 min in a microfuge at 7,500xg. Cells were then resuspended in 1 ml buffer 
containing 1M sorbitol, 0.1M Tris (pH 7.5), 50mM EDTA, 50mM 13ME (3.5ml/ml) plus 
0.5mg zymolyase/ml, and incubated for 40 min at 37°C, pelleted for 30 sec in
71
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microfuge at 6,000xg and the sphereoplasts resuspended in 0.5ml buffer containing 
0.1M Tris(pH7.5) and 50mM EDTA. 25jil of 10% SDS was added, mixed and heated at 
50°C for 10 min, followed by the addition of 200pl of 5M KOAC and kept on ice for 
30 min. This was followed by a 10 min spinning in the cold at 12,000xg in a 
microfuge and the supernatant transferred into a new tube, 1ml ethanol added, 
mixed and kept at room temperature for 5 min. It was then spun at 12,000xg in a 
microfuge for 5 min, the pellet washed with 70% ethanol and resuspended in 300pl 
TE. 3ml of 10% SDS was added, mixed and also 10yl of 5mg/ml Proteinase K was 
added, mixed and incubated at 37°C for 1 hr. The mixture was then phenol and 
chloroform extracted and 30pl of 3M NaOAC or 75pl of 6M NH^OAC added. 600yl of 
ethanol was then added and the mixture kept at -20°C for 5 min. The genomic DNA 
was then pelleted by spinning at 12,000xg in a microfuge for 5 min. The pelleted 
DNA was then washed with 70% ethanol, dried in a speed vacuum and dissolved in 
50pl TE.
2.2.19 Qiagen Plasmid Midi Preparations
The protocol described below is as given in the QIAGEN plasmid Midi and 
Maxi preparations. This protocol involves elution of DNA through columns making 
the plasmid DNA free from chromosomal DNA and RNA. The cultured DH5a cells 
usually in 200ml LB plus ampicillin medium were spun at 3,500 rpm in a Sorvall 
GS3 rotor for 10 mins and the pellets suspended in 4ml PI buffer. Four mis of 
buffer P2 was added, mixed gently and incubated at room temperature for 5min. 
Then 4ml of buffer P3 was added, mixed immediately but gently and centrifuged at 
4°C for 30 min at 12,000xg. The supernatant was promptly removed and applied to 
QIAGEN-tip 100 which has been already equilibrated with 5ml of buffer QBT and 
allowed to enter the resin by gravity flow. The QIAGEN-tip 100 was then washed
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with 10 ml of buffer QC and the DNA eluted with 5ml of buffer QF into a new tube. 
The DNA was then pelleted with 3.5ml of isopropanol which had been previously 
equilibrated to room temperature by centrifuging at 4°C using the Sorvall 
ultracentrifuge set at 12,000xg. The DNA was washed with 70% ethanol, vacuum 
dried for lOmin and redissolved in lOOpl TE. This was mainly the protocol used 
for purifying large quantities of plasmid DNA.
2.2.20 Revertants
Clone 9 revertants were selected to compare their morphology with those of 
normal cells. GL1 (GL2)-9 or GU1(GU2)-U9C strains were grown in 3ml YPD or SC - 
complete media at 30°C for 3 days. About lOyl of the culture was then plated on 
SC-URA-ARG+CAN and incubated at 30°C for 2 days. The morphology of the revertants 
was then observed under a Nikon phase-contrast microscope after fixing the cells 
on microslides. The fixing was done by aliquoting l.Opl of the cells onto a 
microslide and 1.5 ml of a buffer containing 1M sorbitol and O.IMTris EDTA 
(pH7.5). These were then covered with a microslide cover and viewed under the 
microscope.
2.2.21 Mock Transformations
Mock transformations were done as described by the flow charts to check the 
background of strains which were to be used for screening libraries. The strains 
were transformed with the appropriate vector plasmid. 5% of the transformation 
was then plated on the appropriate media and the rest grown in 2.5mls of the same 
media. 5pl of the cells in the media were then plated on the selective plate that 
destroyed the strain maintenance plasmids. The 5% of the transformed cells on the 
plate were just to determine the efficiency of the transformation. A clean 
background as indicated by no colony or 1-3 colonies confirmed the usefulness of 
the strains; colonies of more than 10 had a poor background and were discarded.
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74
CHAPTER THREE 
RESULTS
3.1 Isolation of the MATa2. YGC1 and MCM1 Genes
High copy number plasmids that were able to complement the gpal defect in 
gpal: :HIS3 cells were isolated from a yeast library in the plasmid UV (a URA3 
marker based vector). Several plasmids containing nonhomologous inserts (Fig.4 
and 12) were able to complement the gpal mutation (in some cases only partially). 
Two nonhomologous plasmids that allowed complemen-tation in both MATa and MATa 
gpal strains were analyzed in detail. The first plasmid, pYGC9 contains the MATa2 
gene which has previously been characterized but its function of complementing 
gpal yeast strains has not been unraveled. The second plasmid, pYGC12 contains 
what is known as the yeast G - protein complementation genel (YGC1) which has 
hitherto not been characterized. Another plasmid pYGC5 contains the MCM1 gene 
which was known to be a general transcription activator. However, because the 
MCM1 product complements only MATa gpal cells but not MATa gpal strains unlike 
MATa2 and YGC1, there was little characterisation of it here. The ability of the 
YGC9 plasmid to suppress the gpal mutation suggested that it might encode a 
component of the pheromone response and/or recovery pathway or have a function 
similar to a component of this pathway; therefore, this gene was characterized 
further. From the Southern hybridization results (figures 12, 13 and 14) it was 
clear that none of the inserts in plasmids YGC5, YGC9 and YGC12 had a sequence 
homology to G-protein alpha subunit (GPA1) gene of Saccharomyces cerevisiae. 
Another reason why much attention was paid to the pYGC9 insert was that its gpal- 
complemented cells looked more viable. Additionally, the size of the insert was 
comparatively smaller. The restriction map of the pYGC9 insert is shown in
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figures 16, 19 and 20, whilst those of pYGC5 and pYGC12 are shown in figures 17 
and 18, respectively. Comparing the three restriction maps of the inserts to that 
of GPA2 (another G-protein alpha subunit in yeast) also showed no sequence or 
restriction mapping similarity. It was important to compare the sequences and/or 
restriction maps of the inserts to that of GPA1 since GPA1 is known to be the 
inhibitor of the pheromone signal transduction pathway in Saccharomyces 
cerevisiae.
Plasmids derived by deletions or subcloning of fragments of the pYGC9 
inserts were constructed and tested for their ability to suppress the gpal 
mutation, as described in figure 20. Multicopy plasmids containing a 4.8 kb Hind 
III/EcoRl fragment of pYGC9 were able to complement the gpal mutation (figure 
22). When the size was narrowed down to 1.8kb Eagl/Kpnl (Figure , 19) or
Clal/Kpnl (Figure 20) fragments of pYGC9, it was still able to complement the 
gpal lethality both in MATa and MATa cell types. Thus the active part of pYGC9 
involved in the complementation is located between Eagl and Kpnl.
3.2 The Southern Analysis
The Southern blot showed that GPA1 does not hybridize to the high copy 
suppressor plasmids (Figure 12). In Figure 12, the faint signals seen in lanes 
2-10 correspond to plasmid sequences because the probe fragment contained a small 
amount of contaminating vector fragment that was also radioactively labeled. 
Figure 13 indicated that clone9 hybridized to all the other clones except clones 
5 and 12. the band in lane 10 (Figure 13) was due to a residual signal from the 
hybridization on (Figure 12) which was not completely removed by the stripping 
procedure. Figure 14 showed that clones 5 and 12 are each unique since they do 
not hybridize to each other.
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3.3 Phenotype of MATa2 Disruption
To test for the importance of MATa2 to the pheromone signal transduction 
pathway, the gene was disrupted with URA3 marker gene (Figure 26). Insertion of 
a Hindlll fragment of the URA3 marker gene into the Xbal site changed to Hindlll 
site within the 1.8kb Eagl/Kpnl fragment eliminated the ability of the plasmid 
to complement gpal (Figures 26 and 27). This result indicated that the- gene 
required for complementation is contained within the Eagl-Kpnl fragment, since 
its disruption led to the cells lethality (Figure 27).
3.4 Overexpression of MATa2 is Not a Prerequisite
for GPA1 Complementation
In order to test whether overexpression of the MATa2 product is necessary 
for the suppression of gpal strains, the 1.8kb Eagl/Kpnl fragment of pYGC9 was 
cloned into a centromere plasmid vector RS416 to obtain pRS416"9" (Figures 10 and 
24). Centromere plasmids maintain the plasmid copy number at one per cell. A 
selection was imposed for uracil prototrophy and growth at 30°C (Figure 25). The 
ability of the centromere plasmid to suppress the gpal mutation suggests that 
overexpression was not required for complementation by MATa2 (Figure 25).
No GR4.Z-containing plasmid was isolated in the original screen for 
suppression of gpal mutation (Figure 12). To be certain that suppressing the gpal 
did not require a mutation that had arisen during cloning, overlapping clones 
were isolated by hybridization to the pYGC9 insert (Figure 13) and shown to be 
capable of suppression of the gpal mutation. Figure 15 shows one of the pictures 
taken during restriction mapping of clone 9. In the picture, double digests of 
clone9 using some restriction endonucleases are shown.
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77
1 2 3 4 5 6 7 8 9  10
Figure 12: High Copy Suppressors are not QPAl
High aopy suppressors plasmids were digested with EcoRl and 
Hindlll, run on a 0.8% agarose gel, blotted onto a 
nitrocellulose filter and probed with the 32P labeled 1.9kb 
EcoRl fragment of G P A 1. Lane 1: Lambda H i ndlll digest marker: 
Lanes 2-9: suppressors 5,6,7,9,11,12,14 and 20, respectively. 
Lane 10: GPA1 E c oRl fragment.
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7 8
1 2 3 4 5 6 7 8 9  10
Figure 13i Suppressor 9 Hybridizes to Most Other Suppressors
The same nitrocellulose blot (used in fig.12) was stripped in 
a boiling water bath and rehybridized to a ” P-labeled probe 
made from suppressor 9. The vector fragment hybridizes in each 
lane except lanes 1 . . and 10. The insert fragments of
suppressors 6,7,9,11,14 and 20 also hybridize but not 
suppressors 5 and 12. The band in lane 10 is a residual signal 
from the previous hybridization that was not completely 
removed by the stripping procedure.
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'79
1 2 3
P'.i.yure 14: Suppressors 5 and 12 are each unique
High copy suppressor jplasmids 5 and 12 were digested with 
EcolU and IlindXXX, run on an agarose gel and transferred to a 
nitrocellulose filter. The filter was then probed with ” P 
labeled clone 12. Lane 1, Lambda DNA-Hindlll digest marker;
. lane 2, clone 5; lane 3, clone 12. Thg.se results show that 
clones 5 and 12 have 2 different inserts.
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80
i o u  t; f i m o i n i i n n i  £1^1^1718 19
Figure 15: Restriction Mapping of Clone 9
This shows an example of several double restriction digests 
during the mapping of clone 9. Lane 1 Lambda DNA-HindlII 
digest marker [sizes of fragments from origin are?f’y.42, 6.56, 
4.36, 2.32*n-2.03, respectively. _ •; A
Lane 2, Clal EcoRl; Lane 3, Clal+Xhol; Lane 4, Clal+Pstl; Lane 
5, Clal+Sacl; Lane 6, Clal+Apal Lane 7, Pst+ Hind III; Lane 8, 
Xhol+Hind III; Lane 9, Xhol+Sacl; Lane 10, Xhol+EcoRl; Lane 
11, Xhol+Apal; Lane 12, Apal; Lane 13, Apal+Kpnl; Lane 14, 
Apal+EcoRl; Lane 15, Apal+Sacl; Lane 16, Sacl; Lane 17, 
Sacl+Hind III; Lane 18, EcoRl+Hind III; Lane 19, EcoR+Sacl. 
Digests were run on 0.8%agarose gel.
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H ind lll
\  Sail
nJ
Xhol
Pstl Sacl
EcoRI EcoRI
/c ia l Eagl
Kpnl
Xbal Smal\Sacl
I _ \ \ l EooRI
Figure 16s Restriction Map of Clone 9
Clone 9 was mapped using restriction endonuclease 
digests run on agarose and polyacrylamide gels 
alongside marker DNAs. Restriction sites 
highlighted are unique on the insert.
The Zi'coRl site highlighted ia almost at the centre 
o tlle insert. The size of the insert is 4.0kb.
Eagl
Spel Xbal Kpnl
Figure 19: Restriction Map of MATa2
This Eag/I<pnl fragment of clone9 Complements the 
gpal lethality both in MATa and MATa Yeast strains.
I
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0 2
If
Kpnl
m a l Clal  |R I
Kpnl
Sph I
S a c I
jXhoI RI .Xbal S a d .ClalJ____
Xba l
P v u l l \ III
ORF o f  CIone5
Sms I SphI
jXlioISmal
figure 17i Restriction]Map of Clone 5
The Smal/Spill fragment and Die Sjnal/X/iol segments 
are both able to complement the g p a l  in MATa cells. 
The restriction sites are: E v o l i l (IU) , Kpnl, SjiiuI, 
C l a 1, SpliX r S a d , X h o l  t Xbcxl r C l a l /  FvuII and 
H i n d l l l .
ORl? stands for the open reading frame, and its 
direction is shown by the arrow.
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HI ac4 <pnlmul jClal |RI Kpnl .Sphl
SacI
.Xhol ,RI ,XbaI 
I I
SacI ^bal 
jCkl1 tPvuir\|111
ORF o f  Clone5
Smal  Sphl
XholSmal
figure 17: Restriction!Map of Clone 5
The Smal/Spill fragment and the Siual/JfTaol segments 
are both able to complement the gpal in MATa cells. 
The restriction sites are: EcoKl (1U), Kpnl, Sjiial, 
Clal, Sphl, S a d ,  Xhol, Xbal, Cli il, fvull and 
H i n d l l l .
ORF stands for the open reading frame, and its 
direction is shown by the arrow.
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S a c l  r v  N l ie l  .Psl1 I IIII
^ mmmmm^ ^ M M M B — I— —A --------------------------------------------------ORF  o f  KGC/
' 5 phiR 1 Sm a l  Ps t I  q ,,.,\  S i c /  W I  Xbi'T I<PnI ^  / H I )
b  M /  ^acl *}v  1 / 'i111?  fig111 B>iml FgH1 ft1 I I'1111 t y i X /
Figure :L0: Restriction Map of Clone 12
Figure A shows the ORF of YGCl from the N-terminal 
end. This fragment of YGC1 is fully capable of 
complementing the gpal lethality: Fig. B shows
restriction map of the original clone 12 (Y G C 1 ).
Restriction sites are indicated by the names of the 
enzymes.
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Ii
,1-1 III_______________________________________ C la l
Sa i l  .Xho l
Con iD lem cn liilion
,1-1 i lu l l I I___________|SacI______________ RI  / ClaI__________Xba l _____________ Kpn 1
RI X b a l  R j
I
a c l  X b a l  S a c l
,ClaI ^vbal K p n l
"'‘Figure'5 20: ' '’'Clone" 91l‘tielot'ioi!is,,ancl*'BuppreBrfic>nvof,<gjJbl'“
Figure indicates that the active fragment of cl‘one9 which is 
complementing the gpal is between Clal and K p n l . 
Complementation is basied on the rescuing of gpal cells. 
Regions tested for their ability to complement the gpal 
mutation in pUV plasmids are indicated in rectangular bars. 
Restriction sites IU (ii’coRl) Hindlll, Sac 1, Clal, Xbal and 
Kpnl. Ability to complement the gpal is indicated with 
positive signs (-I-) and inability to do so with negative; signs
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3. 5 MATa2 has no Sequence Homology to G-proteins
The pYGC9 insert could not be identified from its 
restriction map. However, when about 500 bp of the Eagl/Kpnl fragment was 
sequenced using the Sanger dideoxy method and the sequence fed into the BLAST 
Database search for homology, the MATa2 gene was identified (Figure 21). The 
MATa2 sequence had already been reported by Nasmyth and Tatchell (, 1980). The 
MATa2 gene had no homology to any known G-protein and/or its subunit(s). It had 
no classified region implicated in guanine-nucleotide binding and GTPase 
activity. The MCM1 product is also not known to have either GDP/GTP-binding site 
or GTPase activity. However, in the case of the YGC1, the sequence did not have 
any GDP or GTP binding domain, but further characterization is going on currently 
to classify this gene.
3.6 MATa2 Suppresses Mating in both MATa and 
MATa gpal yeast cells
Table 6 shows the average of two qualitative mating assay results of pYGC9 
in both MATa and MATa cell types. Judging from the number of diploid cells 
selected on the SC-His-Trp prototroph, there seemed to be suppression of mating 
by pYGC9 in both GUI and GU2 cells as compared to SP1 and FY250 haploid cells. 
GUI and GU2 are gpal strains whereas SP1 and FY250 are GPA1 haploid strains. 
Suppression of the mating seemed to be more pronounced in MATa gpal cells than 
MATa gpal cell types.
3.7 The Loss of MATa2 Function Results in late GI Arrest.
Microscopic examination of the spores containing a disrupted MATa2 showed 
that they germinated and went through several cell divisions.
85
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10  2 0  30  40  50  60
1 CAGTTACAAA CATCTTAGTA GTGTCTGAGG AGAGGGTTGA TTGTTTATGT ATTTTTGCGA
61 AATA l'A TATA  TATATATTCT  ACACAGATAT ATACA TA TTT  GTTTTTCGGG CTCATTCTTT
121 CTTCTTTGCC AGAGGCTCAC CGCTCAAGAG GTCCGCTAAT TCTGGAGCGA TTGTTATTGT
181 TTTTTCTTTT  CTTCTTCTAT  TCGAAACCCA GTTTTTGATT TGAATGCGAG ATAAACTGGT
241  ATTCTTCATT  AGATTC TC TA .GGCCCTTGGT ATCTAGATAT GGGTTCTCGA TGTTCTTCTT
310 320 330 340 350 360
301 TGCAAACCAA CTTTCTAGTA TTCGGACATT T TC T T T T G TA AACCGGTGTC CTCTGTAAGG
361 TTTAGTACTT TTGTTTATCA TATCTTGAGT TACCACATTA a a t a c c Aa c c CATCCGCCGA
421 TTTATTTTTC TGTGTAAGTT GATAATTACT TCTATCGTTT TCTATGCTGC GCATTTCTTT
481 GAGTAATACA GTAATGGTAG TAGTGAGTTG AGATGTTGTT TGCAACAACT TCTTCTCCTC
541 ATCACTAATC TTACGGTTTT TGTTGGCCCT a g a t a A g a a t AGTAATATAT CCCTTAATTC
610 620 630 640 650 660
601 AACT'I’CTTCT TCTGTTGTTA CACTCTCTGG TAACTTAGGT AAATTACAGC AAATAGAAAA
661 GAGCTTTTTA TTTATGTCTA GTATGCTGGA TTTAAACTCA TCTGTGATTT GTGGATTTAA
721 AAGGTCTTTA ATGGGTATTT TATTCATTTT TTCTTGCTTA TCTTCCTTTT TTTCTTGCCC
781 ACTTCTAAGC TGATTTCAAT CTCTCCTTTA TATATATTTT TAAGTTCCAA CATTTTATGT
041 TTCAAAACAT TAATGATGTC TGGGTTTTGT TTCGGATGCA ATTTATTGCT TCCCAATGTA
910 920 930 940 950 960
901 GAAAAGTACA TCATATGAAA CAACTTAAAC TCTTAACTAC TTCTTTTAAC CTTCACTTTT
961 TATGAAATGT ATCAACCATA TATAATAACT TAATAGACGA CATTCACAAT ATGTTTACTT
1021 CGAAGCCTGC TTTCAAAATT AAGAACAAAG CATCCAAATC ATACAGAAAC ACAGCGGTTT
1081 CAAAAAAGCT GAAAGAAAAA CGTCTAfiCT f; AGCATGTGAG GCCAAGCTGC TTPAATATTA
1141 TTCGACCACT CAAGAAAGAT ATCCAGA1TC CTGTTCCTTC CTCTCGATTT TTAAATAAAA
1210 1220 1230 1240 1250 1260
1201 TCCAAATTCA CAGGATAGCG TCTGGAAGTC AAMTftCTCA GTTTC'fiACAR TTCAATAAGA
Figure 21: Sequence_of MATa2.
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AB
Figure 22: Clone 9(a2) rescues both MATa and MATa cells
Plates shoW growth on Sc-URA-ARG+CAN1. Survivors 
lost the pLEU2/GPA1/CAN1 plasmid as indicated by no 
growth on the pUV2 plates. (A) GUI transformed with 
clone9 and pUV2 at left and right respectively. (B) 
GU2 transformed with clone9 and pUV2 at left and 
right respectively. ,
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iguro 23 : Confirmation of Plasmid RS416'9'.
This shows restriction enzyme digests of the
plasmid RS416"9", a CEN plasmid. The various
plasmid constructs were similarly confirmed. Here, 
pRS416'9' was cut with Clal and Kpnl. Lanel is the 
Lambda DNA-tfindIII digest marker (sizes from origin 
ar eg $J.42, 6.56, 4.36, 2 . 32^rt7 .03). . The
smaller (lower) band corresponds'to the MATa2 gene, 
whilst the larger (upper) band is the CBN plasmid
vector RS416' ' (lanes 2 to 1£) .
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Table 5:
T rans fo rm a t io n  Resu lts
Y e a s t  S t r a i n  Ma t i ng  T vp£  
GU1 MATa
GU2 MATa
P l a s m i d
c l o n e  9 ( a2)  
c l o n e  5{MCM1)  
c l o n e 1 2  ( YGC1 )
c l o n e 9 ( a 2 )  
c l o n e 5  (MCM1)  
c l o n e 1 2  ( YGC1 )
O n m n l e m e n t a t i o n
qU lomI
+
+
+
The transformation results indicate: that clones 9 and 12
complement gpal in both MATa and MATa cell types whereas 
clone5 only complement gpal in MATa cells. The positive signs 
indicate the ability of the plasmids to keep the gpal cells 
alive whereas the negative sign shows the inability to do so.
+
+
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Table 6
MATING ASSAY RESULTS
Mating
plasmid
Mating type 
of yeast 
strain
DC17(a-cells) 
Number of 
colonies
GU2 with pUV2 MATa 0
GU2 with pYGC9 MATa 0
FY250 with MATa 2
pUV2
FY250 with MATa 1
pYGC9
GUI with pUV2 MATa 612
GUI with pYGC9 MATa 32
SPI with pUV2 MATa 1210
SPI with pYGC9 MATa 1500
DC14(a-ceHs/ 
Number of 
diploid 
colonies
75
5
2,540
996
0
0
0
1
The results show' that ,clone9 complemented strains mated with 
both MATa and MATa mating type cells. The assay results also 
indicate, however, that MATa cells containing pYGC9 mate 
better than MATa cells. Clone9 suppresses mating in wild type 
MATa cells (FY250) but no(t in wild type MATa cells (SP1) as 
uhown. The tester or mating type strains are DC17(a-cells) and 
DC14(a-cells)
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CENS
Figure 2jt/t Map of plasmid RS416"9"
This shows a centromere plasmid RS416"9" map which 
was constructed and transformed with the GUI and 
GU2 yeast strains to find out whether complemen­
tation of gpal by MATa?. depended on the copy number 
or not. The URA3 marker, the CEN centre and ths 
alpha2 genes are shown with the arrows indicating 
their direction of transcription.
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9 2
'’igure 25 s Rescue of Cells In Single Copies by Clone 9
pRS416"9" is a CEN plasmid containing crb"ne9 -.which 
is capable of complementing the gpal both in MATa 
and MATa cells. Thus, complementation is 
independent of the copy number. A; GUI transformed 
with the plasmids RS416"9" and O S m t  at left and 
right respectively. B; GU2 transformed with the 
plasmids RS4169* and t at left and right
respectively. Cells were plated on Sc-URA-ARG+CAN.
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9 a
■Engl Xbalj____
Kpn l
ORF o f  Alpha2
I
'Engl jKpnl
mi
Figure 26 t Disruption of Clone9
Figure 2 6 shows the disruption of alpha2 with the URA3 Marker. 
The H i n d III site was inserted at the Xbal site and the Hindlll 
fragment of U R A3 was used to frame-shift the alpha2 gene. The 
disrupted gene was confirmed by restriction enzyme digests. 
The Xbal' site was cut with Xbal endonuclease, phosphatased 
using calf in testine phospha tase (CI.1?) and the Hindi II 
oligonucleotide linker which had been phonphorylated uning T'l 
polynucleotide kinase, inserted at the original Xba 1 siti-j 
using the T4 DNA ligase reaction. The introduced Hindlll sit 
was then cut with H i n d III endonuclease and the H i n d II 
fragment of U11A3 inserted there to frame-shift the open 
reading frame (ORF) of the alpha2 gene. Figure 27 3hows that 
this disruption is lethal to the cells.
Q) H
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Figure 27 1 Disruption of MATa.2 Leads to Constitutive Cell-Cyale 
Arrest.
(A) ; GUI transformed with clone9 and pUVEK-URA showing at 
the left and right respectively. (B) ; GU2 transformed 
with clone9 and pUVEK-URA at left and right respectively. 
Transformed colonies were shown on SC-URA-ARG+CAN plates. 
This figure indicates that the disruption is lethal to 
both MATa and MATa cell types. Plasmid UVEK-URA contains 
MATa2 which has been disrupted with URA3 marker. Growth 
of transformed GUI and GU2 cells with M A Ta 2 and no growth 
with UVEK-URA show that disruption of MATa.2 is lethal to 
the cells indicating that the signaling pathway is 
constitutively arrested.
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Micromanipulation of these cells revealed that most of the cells were unbudded 
and some of them showed an aberrant cell morphology, similar to the "shmoo" of 
cells arrested by mating factors (table 7 and plate 1). This phenotype is 
characteristic of cells arrested in late GI phase. Also, figure 27 shows that 
disruption of the MATa2 gene product is lethal to both MATa and MATa cell types.
3.8 Plasmid Constructs and Creation of New Yeast Strains
In an attempt to screen mammalian cDNA library for possible pYGC9, pYGC5 
and pYGC12 analogs, plasmids U5C, U9C, U12C, UGC and UC were constructed (figure 
11). These were used to create new hosts for GUI and GU2, forming the GL1(GL2)- 
5,9,12, UGC and UV strains all of which had the URA3 gene as their selective 
marker and canavanine 1 gene for the inhibition of these plasmids in the strains 
(Figure 6, 11). It, however, turned out that the background for these strains 
obtained from the mock transformations were too high to be used for screening 
purposes.
Another plasmid TGC (TRP1/GPA1/CPM1) was constructed which had the 
autonomous replicating sequence (ARS) giving it an advantage to be lost easily 
(Figure 7, 8). The TRP1 is the marker gene since the cDNA library had the LEU2 
gene as its selective marker. Construction of pTGC initially involved a lot of 
techniques including PCRings out the CAN1 gene from the wild type yeast genomic 
DNA.
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gflll/X ho l
Figure 28: Map of plasmid TGC
Plasmid TGC was constructed and its strain created. The 
idea was to use this strain for screening the mammalian 
library. EaoRl fragment of GPA1 is shown as shaded part. 
TRP1 marker, autonomous replicating sequence (A R S 1 ), and 
the CAN1 marker genes are also shown.
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Figure 29: Plasmid KSEX.
The arrows indicate the direction of sequencing of the 
Eagl/Xbal fragment of MATa2. The Eagl/Xbal fragment of 
M A Ta 2 was subcloned into Bluescript (pKS+1/-) for 
sequencing purposes. The T7 and T3 promoters of pKS were 
useful since T7 and T3 sequencing primers are readily 
available in the market. ,
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Figure 30: Plasmid KSXS
Direction of the arrows indicates direction of sequencing 
by the sequence version 2.0 enzyme. In plasmid KSXS, the 
Xbal to Smal fragments of MATa2 were subcloned into 
vector pks. The T7 and T3 promoters were helpful since T7 
and T3 sequencing primers are commercially available.
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pKSEK 
(~4.7kb)
Outline of plasmid KSEK.
This plasmid was constructed and used for the 
disruption of M A T a 2 with URA3 Marker gene. The 
marker gene was inserted at the Xbal site after the 
tfindlll restriction site was introduced. First, 
Xbal endonuclease was used to digest KSEK. Since 
Xbal is unique on KSEK, a HindiII restriction 
endonuclease oligonucleotide that fitted into the 
sticky ends of the Xbal restriction site was 
introduced and ligated to it using T4 DNA ligase.
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100
Table 7
MORPHOLOGY OF REVERTANTS OF gpa l CELLS
S t r n i n CeMs C o l o n i e s
9Rov1 some shmoos, small large
9 R o v 2  • high percent, large shmoos small9 R e v 3 some shmoos, small medium9 R e v 4 high percent, large, long shmoos small
9 R e v 5 some shmoos, small big
9 R g v 6 very high percent small9 R e v 7 high percent, large shmoos small-med.
9 R e v 8 some shmoos, small big
9 R e v 9 high percent, small, some long shmoos big
9 R e v 1  0 high percent big9 R e v 1 1 high percent, large shmoos small-med.
9 R g v 1 2 moderate to high percent, small shmoos big
9 R e v 1  3 some shmoos, small medium
9 R e v 1  4 moderate to high percent medium9 R e v 1  5 high percent, large shmoos small-med.9 R e v 1  6 high percent, small shmoos big
9 R e v 1  7 high percent,, small semi-shmoos big
9 R e v 1  8 high percent, small shmoos big9 R e v 1  9 high percent, large, round shmoos small
9Rev20 high percent, large semi-shmoos small
9 R e v 2 1 high percent, very large, round shmoos small-med.9 R e v 2 2 high percent, round shmoos, 2 5 %  cell death small9 R e v 2 3 high percent, shmoos, some cell chains big
Reversion of the strains may occur as a result of a switch of the uraj of 
the chromosome with the URA3 of the U9C plasmid.
Reversion also occurs as a result of mutations In the genes involved in 
the pathway. The cells were observed under the Nikon phase-contrast 
microa-oope. The revertants Were created from olone9 by growing a number 
of colonies picked from SC-URA-ARG+CAN plate in SC-URA media. The strain 
example 9revl means revertant number one from clone 9 etc. Cells are 
described by their sizes, percentage of the cells slimooed per colony and 
the shape of the shmoos as viewed under the microscope. The colonies grown 
are also described by their sizes, whether big, small or medium.
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Plate 
1 
Shows 
the 
morphology 
of 
Wild 
type 
and 
clone 
9 
revertant 
cells 
the 
" 
£hmoo" 
shaped 
cells 
can 
be 
observed 
on 
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10]
j Type
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.the 
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However, the PCR CAN was truncated and not active because the 
oligonucleotide primers used did not flank the CAN1 gene completely. Plasmid 
YepCAN was however obtained and the entire CAN gene cut out for the construction 
of pTGC (figure 8). The LG1 (LG2)-TG strains created from the TGC gave a very 
good mock transformation background.
These LG1 (LG2)-TG strains were used to isolate two genes from a mammalian 
cDNA library (Colicelli, personal communication) by virtue of their ability to 
complement gpal yeast cells. Both genes were not previously characterized. These 
mammalian genes have no sequence homology to GPA1.
Reversion of strain occured as a result of switch of the ura3 of the 
chromosome with the URA3 of the U9C plasmid as shown in table 7. Reversion may 
also have occured as a result of mutations in the genes involved in the pathway 
(table 7 and plate 1).
Plate 1 shows the shmoo morphology of revertants derived from clone 9. 
Plate 2 shows differentiation of YGC1 cells compared with wild type cells. Plate 
2 also showed that cell cycle arrest can be distinguished from cell 
differentiation.
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104 
CHAPTER FOUR 
DISCUSSION AND CONCLUSIONS
4.1 General Strategy for Selecting the High 
Copy Suppressor Clones
The yeast strain GUI having the genotype MATa/gpal::HI33/ Ieu2/ura3/canl 
was kept alive with the plasmid TLCG whose relevant genotype is GPA1/LEU2/CAN1, 
since the gpal strain would have remained in the constitutively cell arrested 
state (death). Transforming the GUI strains with the clones (X/URA3) caused the 
GUI to harbor two plasmids at the same time, that is plasmids TLCG and the clones 
(figure 4). When the transformed cells were plated on Sc-URA-ARG+CAN media, the 
plasmid TLCG was destroyed because CAN1 is a toxic arginine (ARG) analog which 
allows the arginine permease to accumulate canavanine drug (Figure 5). canl is 
a recessive mutation which eliminates the arginine permease, thus preventing 
canavanine drug from entering the cell. The GUI cells and the clones are 
therefore resistant to canavanine. Because the CAN1 gene is tagged to the GPA1 
maintenance plasmid, survival of cells after the destruction of the GPA1 means 
that the high copy suppressor plasmids were responsible for keeping the GUI cells 
alive or complementing the gpal::HIS3 lethality (figures 4, 5, 22 and table 5).
4.2 Identification of MATa2. YGC1 and MCM1 Genes 
Involved in the Pheromone Response Pathway
Saccharomyces cerevisiae genes MATa2, YGC1 and MCM1 have been identified 
by isolation of plasmids that were able to complement or suppress a gpal::HIS3 
mutation.
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MATa2 gene is known to be required for the determination of both haploid 
and diploid cell types. In haploid cells, it is necessary for inhibiting the 
expression of a mating functions which are otherwise antagonistic to a mating 
functions (Strathern et al., 1980). In diploid cells, the same inhibition of a 
mating functions is exerted by MATa2, but in addition, it acts in conjunction 
with the al gene to regulate MAT transcription, principally the repression of al 
transcription and to allow sporulation (Figure 3). In vivo, a2 protein represses 
transcription of two sets of cell type-specific genes by binding to operator 
sites together with either the al or MCM1 proteins (Figure 3). The a2 protein is 
also a member of the homeodomain superfamily of DNA-binding proteins that 
regulate development in eukaryotic cells (Wolberger et al., 1991).
MCM1 was first identified as a gene involved in maintenance of artificial 
minichromosomes in yeast. It has also been shown to serve as a general 
transcriptional regulator of several genes including mating-type specific genes. 
Biochemical data suggest that the Mcml protein coactivates a-specific genes and 
corepresses a-specific genes by binding to a 10 base pair dyad symmetry element 
in their upstream regions (Elble and Tye , 1991).
Yeast G-protein complementing gene (YGC1) has not been characterised, hence 
its function or mode of action is not known. However, it has been established 
here that this novel gene complements or suppresses a gpal: :HIS3 mutation in high 
copy plasmids (table 5).
In this study, another important function of MATa2 gene has been 
established, that is complementation of gpal yeast strains (figure 20,22 and 25; 
table 5). The a2 protein can suppress gpal mutations, including null mutations 
not only when overexpressed in multicopy plasmids but also in single copy 
centromere plasmids (figures 22 and 25). This result is however contrary to the
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earlier findings that SCG1 (also known as GPA1) can only complement 
supersensitive strains (sst2-l) mutations when overexpressed (Dietzel and Kurjan, 
1987). The ability of MATa2 to suppress the pathway even in single copy (figure 
25) not only show its negative regulatory influence on the pathway but also that 
it is an even more potent inhibitor of the signal pathway than GPA1. The gpal 
mutation results in hypersen-sitivity to pheromone and a defect in recovery from 
the cell-cycle arrest caused by exposure to pheromone. The ability of MATa2 and 
YGC1 to suppress gpal: :HIS3 mutation in both MATa and MATa cells (table 5, figure 
22) and MCM1 in MATa cells (table 5) suggests that MATa2, YGC1 and MCM1 might be 
components of the pathway involved in pheromone response and/or recovery or that 
they might have a biochemical function similar to GPA1 or some other components 
of this pheromone signal transduction pathway.
4.3 Implications for the Involvement of MATa2 and YGC1
in the Pheromone - Induced Signaling Pathway
It is very surprising that MATa2 (figure 21) and YGC1 which does not show 
any level of sequence homology to GPA1 or to the a subunits of G-proteins which 
are involved in a number of different signal transduction systems should suppress 
GPA1 mutants in haploid cells (Figure 22). The fact that some of the suppressor 
mutations may occur in the effector and/or other molecules in the pathway has not 
been ignored. In this case, the mutant effector and/or molecule(s) may be altered 
not to generate the arrest signal. Therefore this type of mutation may be able 
to suppress the lethality of gpal::HIS3 mutations. This possibility has however 
been ruled out based on the control transformation results and the consistency 
of the complementation results obtained (Figure 22).
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To investigate further the possibility of a2 protein involvement in the 
pathway, mata2 disruption mutant was created (figure 26). This shows a 
constitutive arrest of the cell-cycle at the late GI phase (figure 27). The q2 
revertants also showed the peculiar "shmoo" shaped cells which are very familiar 
with the arrested cells (plate 1). The cellular morphology of mata2:: URA3
suggests that the MATa2 is involved in the pheromone response pathway. The
ability of q2 to suppress gpal mutations further supports the hypothesis of being 
involved in the signaling pathway. Although the possibility of being involved in 
the pathway is favoured, one has not eliminated the probability that MATa2 is 
required for growth per se, and that suppression of the gpal phenotype is
indirect. The results and observations are consistent with a simple model (Figure
1) for the role of the yeast Ga (Gpal), GI3 (Ste4) and Gy(Stel8) subunits in the 
activation of the pheromone response pathway. Genetic results indicate that GRy 
functions downstream of Ga to activate the pathway, presumably by activating a 
downstream effector which is currently unidentified. In the absence of pheromone, 
Ga is presumed to bind GDP tightly and interacts with G/3y to inhibit the pathway. 
In the presence of pheromone, the pheromone - receptor interaction relieves this 
negative control by promoting GDP and GTP exchange on Ga, resulting in 
dissociation of Ga from Gf3y and the free GBy then activates the pathway. In a 
gpal null mutant, free GBy is present and constitutively activates the pathway 
leading to GI arrest and morphological alterations. In analogous manner, the 
MATa2 and YGC1 gene products seem to be playing the role of GPA1 in gpal mutant 
cells either directly or indirectly on the pathway. It is however amazing and 
unexpected that MATa2 and YGC1 should play the role of GPA1 in the pathway 
because apart from not having sequence homologies, MATa2 is not known to bind 
GDP/GTP and neither can it respond to conforma-tional changes after guanine
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nucleotide treatment since it has no GTP binding, exchange and hydrolysis 
domains. It is however pertinent to note that since the two receptor-pheromone 
interactions are interchangeable (Nakayanua et al., 1987) and the phenotypes 
associated with a2 are the same in both a and a cells, the mechanism of MATa2
action is likely to be the same in both mating types.
The partial sterility of gpal cells expressing q2 proteins (table 6)
suggested that the protein was able to interact with a downstream component of
the pheromone response pathway to keep the pathway inactivated but was unable to 
interact effectively with pheromone receptors to elicit activation of the pathway 
in response to pheromone. Another possible explanation is that the protein is 
able to interact with yeast Gfty as a conformational analog of Gpal (GDP), thus 
preventing activation of the pathway. Since the resulting cells are sterile and 
unable to respond to pheromone, this suggests that the a2 protein cannot interact 
functionally with the pheromone receptors. From table 6, it is obvious that there 
is a very strong reduction in mating efficiency in both GUI and GU2 cells kept 
alive with MATa2. GUI cells that are kept alive with MCM1 and YGC1 also showed 
similar pattern of suppression of mating efficiency. It could also be inferred 
from table 6 that wild type MATa cells (FY250) show a large reduction in mating. 
This observation could further be explained on the basis of non-specific and 
noneffective, interaction with the pheromone receptors to elicit activation of 
the pathway in response to pheromone.
Lethality of the cell with disrupted MATa2 (figure 27) can be explained by 
the uncoupling of the effector molecule from q2 protein. The effector, which may 
be unlocked from the mating factor receptor complex, may elicit a constitutive 
signal from cell-cycle arrest regardless of the presence of mating factors 
(figures 26 and 27). It was also observed, through microscopic examination of the
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MATq2 disrupted spores and micromanipulation of the mata2::URA3 tetrapods, that 
most of the cells were unbudded and some of them showed an aberrant cell 
morphology similar to the "shmoo" of cells arrested by mating factors (table 7 
and plate 1). This observation supports the idea that MATa2 disruption results 
in continuous production of a cell-cycle arrest signal (figure 27) and promotion 
of conjugation in the absence of a mating factor signal.
The clone9 revertants (table 7 and plate 1) observed are of interest 
because some are clearly in known pathway genes. These were the ones that yielded 
sterile, morphologically normal cells which were not examined further, but are 
probably genes such as STE4, STE18 and the Effector molecule. The revertants may 
also contain new genes (which give cells that are still differentiated but are 
going through mitosis (table 7 and plate 2). The differences in these growing 
shmoo revertants may reflect different phenotypes from various mutations in the 
same gene.
4.4 Possible Models of MATa2. YGC1 and MCM1 Actions
The results of this study that support the role of a2 and Ygcl proteins in 
the mating factor signal transduction pathway include; (a) the arrest phenotype 
of haploids when a2 and Ygcl expressions were turned off, is characteristic of 
cells arrested in late GI phase and some of these arrested cells exhibit shmoo 
morphology (figure 2, table 7 and plate 1) and (b) disruption of a2 is lethal in 
haploid cells (figure 27) which indicates a2 is a haploid essential gene for 
cellular growth.
To explain the mechanism of action of a2, Ygcl and Mcml, one could say that 
they are either involved directly in the signaling pathway and/or that these 
proteins act to modulate a component of the pathway, most probably G(3y or the
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effector molecule. Infact some other products are known to be involved in vivo 
in modulating the signaling response. Inactivation of these genes leads to 
activation of the signaling pathway; hence, these products, just like a2, can be 
considered as negative components of the pathway. The genes with this behaviour 
are CDC36 and CDC39 (Neiman et al., 1990; de Barros Lopes et al., 1990), CDC72
and CDC73 (Dietzel & Kurjan , 1987). The abbreviation CDC stands for cell
division cycle genes. The a2 protein might play a role in communication between 
the activated receptor and GBy or be involved in stabilizing the putative 
effector and/or G!3y proteins. In this way, inactivating a2 and Ygcl would 
definitely have an effect on the pathway (figure 27).
There are a group of genes whose transcripts are expressed in haploids but 
repressed in a/a diploids. Repression requires both the al protein, encoded at 
MATa, and the a2 protein encoded at MATa, which make up al-a2 activity (figure 
3, Jensen et al., 1983). It is proposed that the transcription of GPA1 would be 
under negative control by al-a2 activity (figure 3 and Miyajima et al., 1987). 
GPA1 would be expressed in haploid a cells because of the absence of a2 products 
and in a cells because of the absence of al product. However, a 20 base pair 
consensus sequence common to the 5’ ends of haploid specific genes that are 
negatively regulated by al-a2 has not yet been found upstream of GPA1 except for 
some sequence homology 50 base pairs upstream of the translation start (Miller 
et al., 1984).
If al-a2 can regulate GPA1 negatively, and if the regulation involves a 
direct contact between al-a2 and GPA1, then one could argue that probably 
the region of contact between the al-a2 complex and GPA1 might be structurally
similar to that of GBy subunits of yeast G-protein. If this is possible, then in
the absence of GPA1, that is in gpal: :HIS3 yeast strains, al-a2 or probably Mcml-
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a2 could bind the GBy nonspecifically because of the assumed similarity in the 
region of contact between GPA1 and GBy. Thus in MATa cells, al and/or Mcml might 
repress the Gfiy activity whereas in MATa cells, the a2 component of the complex 
is expected to functionally repress the GBy activity. One could however be 
tempted to say that this model could be inadequate because only the a2 is able 
to suppress the pathway both in MATa and MATa cell types. The argument could have 
been more valid if gpal::HIS3 yeast strains are complemented by a2 only in MATa 
cells (table 5). However, one did not overlook the fact that probably, high copy 
expression of the a2 proteins activated the transcription of its putative 
complexing counterpart in MATa cells to suppress the signaling pathway. Here, it 
is pertinent to note that a2 protein does not repress the pathway only when 
expressed in a high copy plasmid but also in a single copy centromere plasmid 
(figure 25). MCM1 complements gpal only in MATa cells probably because it forms 
a complex with a2 (figure 3) to modulate the pathway by repressing a-specific 
genes involved in the transduction of the pathway whilst depressing a-specific 
genes.
Another plausible mechanism through which MATa2 can complement gpal 
lethality would be to bind to the putative biological effector of GBy which might 
act at the same level as GBy thereby preventing the transduction of the signal 
from GBy to the effector molecule. In this way, the signaling pathway would be 
suppressed due to the competition between a2 and GBy for binding to the effector. 
Logically if the a2 is transcribed in higher copies, it would advantageously out 
compete the GBy thereby suppressing the pathway. This model can only be validated 
after the effector molecule is isolated, cloned and shown both genetically and 
biochemically to bind to a2 protein.
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One could also envisage that the possibility of a.2 and Ygcl suppressing the 
pathway could be that the pathway is not simply linear, at least upstream, that 
is at the level of the G proteins. The possibility of the pathway being branched 
at the G proteins level is one of the main objectives of this study. 
Hybridization analysis by Dietzel and Kurjan (, 1987) indicates that there are 
at least two additional GPA1 homologs in S. cerevisiae. Isolation of these 
homologs could have thrown more light in understanding the pathway if they could 
be shown to be involved. Ascertaining the possible involvement of these homologs 
may confirm the branching of the pathway upstream and also show whether there is 
a common effector in the pathway, similar to the cAMP pathway as described by 
Stryer and Gilman (, 1986). If it is confirmed that the pathway is branched, then 
a2 and Ygcl proteins could be components or modulators of the branched pathway. 
This stem from the fact that disruption of these proteins leads to similar 
phenotypes as in GPA1 disruption (figure 27 and table 7). It is rather 
unfortunate that the screening of the GPA1 homologs does not yield any of the 
supposed Ga subunits present in S. cerevisiae. Surprisingly, the screening does 
not even produce GPA1 (figure 12).
The emerging view of gene regulation is one of combinatorial control. The 
specific level of expression of a given gene may result from the interplay of a 
multiplicity of factors, each contributing differently to the final level of 
transcription. Thus, from a limited pool of DNA-binding factors, a virtually 
unlimited range of binding specificities and levels of expression may be 
generated. The same factor may act positively or negatively, depending on the 
context of its binding sites and/or on the other factors with which it interacts 
(Berk and Schmidt , 1990). For example, Serum response factor (SRF), the 
vertebrate homolog of the yeast MCM1 gene binds together with the ternary complex
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factor to activate transcription of c-fos (Norman et al., 1988). Repression of 
c-fos is mediated by SRF acting at the same site (Rivera et al., 1990) -
presumably in conjunction with other transcription factors that interact with 
other transcription factors, MCM1 may interact with some other factor in MATa 
cells to suppress the pathway and may bind in the same site or interact with 
other factors in MATa cells to enhance the transduction of the pheromone signal. 
Also an increase in the abundance of these transcription factors may shift 
equilibrium in the cell and reduce the expression of proteins required for cell 
arrest and differentiation. This could result in the ability of a2 and Mcml to 
suppress gpal lethality. This may be unraveled by doing Northern blots. The fact 
that the q2 protein has no particular motif to bind GTP/GDP unlike Gpal and has 
no sequence homology to any of the known Ste proteins indicates that the pathway 
might be branched whereby a2 would be involved in the branched pathway or if the 
pathway is simply linear as envisaged, then a2 might modulate some component(s) 
on this linear or the branched pathway either directly or indirectly. The fact 
however remains that a2 protein is strongly involved in the signaling pathway 
that leads to cell-cycle arrest at the GI phase since disruption of the MATa2 
gene results in constitutive arrest of the pathway (figure 26 and 27). Thus, this 
study shows that the signaling pathway seems to be more complex and one of 
combinatorial control by structural and functional genes than simply by G- 
proteins and the Ste proteins most of which are known to be protein kinases with 
homology to protein kinase C. Hence the involvement of a2, Ygcl and Mcml has 
given more impetus to understanding GPA1 signal transduction in S. cerevisiae. 
Generation of several point mutants of MATa2 and the study of these mutants in 
terms of arresting the signaling pathway would throw more light into the 
mechanism of suppression of a.2 protein.
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4•5 New Yeast Strains
The plasmid TGC was constructed (figure 8) and subsequently used to create 
a new host for GUI and GU2 haploid cells forming LGl-and LG2-TG respectively 
(Figure 7). These yeast strains were very useful in screening a mammalian cDNA 
library leading to the isolation of two mammalian analogs of GPA1 which were able 
to complement gpal haploid cells (Colicelli, personal communication). The yeast 
strains have ARS origin of replication enabling the plasmid to be easily lost 
when desired. It also has canavanine gene (an arginine analog) for selection 
purposes and TRP1 as its selective marker since the cDNA library available was 
LEU2 based.
Analysis of the signal transduction pathway whereby MATa2 and YGC1 genes 
elicit physiological changes in the responding cells is likely to provide 
important insights into the mode of action of other hormonal factors in higher 
eukaryotes. It is also not unexpected that the identification and involvement of 
MATa2, YGC1 and MCM1 would throw more light on the intricacies of the mechanism 
of signal transduction in yeast and subsequently in multicellular eukaryotes such 
as mammals. Understanding the mechanism of the signal pathway would further 
reveal how growth is stringently controlled and how cancer cells occur due to the 
defect in this cellular growth control mechanism.
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APPENDIX
Tris-borate (TBE) Working Solution
0.5x:0.045M Tris-borate 
0.001M EDTA
10% Sodium dodecvl sulfate (SDS)
Dissolve lOOg of electrophoresis-grade SDS in 900ml of H^o Heat to 68°C to assist 
dissolution. Adjust the pH to 7.2 by adding a few drops of Conc.HCL. Adjust the 
volume to 1 liter with H^o.
i
20x sodium citrate fSSCT
Dissolve 175.3g of Nacl and 88.2g of sodium citrate in 800ml of H^o. Adjust the 
pH +7.0 with a few drops of a 10N NaoH, Adjust the volume to 1 liter with HjO.
20 x SSPE
Dissolve 175.3 of Nacl, 27.6g of NaH^PO^.HjO and 7.4 g of EDTA in 800ml of H^o. 
Adjust the pH to 7.4 with NaoH (— 6.5ml of a ION solution). Adjust the volume to 
1 liter with H^o.
DnD Solution 
To prepare 10ml of DnD 
dithiothreitol 1.53
DMSO 9ml
1M potassium Accetate (pH7.4) 100ml
Hjo to 10ml
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116
Sterilise the DnD solution 
by filtration through millex 
SR membrane unit (Millipore) 
and store at -20°C 
in sterile 0.5ml microfuge 
tubes.
Preparation of 1 liter TFB
Reagemt Amount required/liter Final Concentration
1M MES (pH63) 10ml
MnClj.4HjO 8 .91g
CaClr 2H2o 1.47g
KCL 7.46
Hexamminecobalt 
chloride 0.80g
lOmM
45mM
lOmM
lOOmM
3mM
SOC Medium 
Per liter:
To 950ml of deionized HjO, add: 
bacto-tryptone 20g
bacto-yeast extract 5g
Nacl 0.5g
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Shake until the solutes dissolve. Add 10ml of a 25mM solution of KC1. Adjust the 
pH to 7.0 with 5N NaoH (.2ral). Adjust the volume of the solution to 1 liter with 
deionized HjO. Sterilize by autoclaving for 20 minutes at 121°C on liquid cycle. 
Allow it to cool to 60°C or less and then add 20ml of a sterile 1M solution of 
glucose. Just before use, add 5ml of sterile solution of 2M MgClj.
Prehybridization solution 
50% formamide 
6xSSC (or SSPE)
0.05xBLOTTO (Bovine Lacto Transfer Technique Optimizer 5% nonfat dried
milk dissolved in water containing 0.02% sodium azide).
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118
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PRF90, altered in mRNA metabolism and maintenance of the nuclear 
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Appeltauer, U., Achstetter, T. (1989). Hormone-induced
expression of the CHS1 gene from Saccharomyces cerevisiae. Eur. J. 
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Baeuerle, P.A. Baltimore, D. (1988). Activation of DNA-binding activity in an 
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Bender, A.; Sprague G.F. Jr. (1989). Pheromones and pheromone receptors are the 
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Berk, A.J, Schmidt, M.C.; (1990) Genes Dev. 4:151-155.
Blinder, D. Bouvier, S., Jenness, D.D. (1989). Constitutive mutants in the yeast 
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