Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
21i6432 PCr/US93/0950~
INSULIN-DEPENDENT YEAST OR FUNGI
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is related to the following
copending applications of Maureen A. McKenzie: Serial
No. 07/956,342 filed on October 5, 1992 entitled
"INSULIN-LIKE PEPTIDE" (Attorney Docket No. 1828-102P)
and Serial No. 07/956,290 filed on October 5, 1992
entitled "INSULIN RECEPTOR-LIKE PROTEIN" (Attorney
Docket No. 1828-103P). The entire contents of both of
these applications are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
Field of the invention
The present invention relates to mutant strains of
yeast or fungi.
Description of the Related Art
Fundamental discoveries throughout the past
half-century have elucidated the physiological effects
of insulin on glucose homeostasis and intermediary
metabolism in vertebrates. At the cellular level, these
effects include stimulation of hexose, ion and amino
acid uptake, [Wheeler et al, Annu. Rev. Physiol. 47:503
(1985)]; modulation by net dephosphorylation of the
activities of rate-limiting enzymes including glycogen
synthetase, hormone-sensitive lipase and pyruvate
W094/07994 ~2 14B ~3~ PCT/US93/0950
dehydrogenase, and modification by phosphorylation of
seryl residues in proteins such as acetyl coenzyme A
carboxylase, adenosine triphosphate citrate lyase and
ribosomal protein S6, [Haring, Diabetologia, 34:848
(1991); Kahn, Annu. Rev. Med., 36:429 (1985); Czech,
Annu. Rev. Physiol., 47:357 (1985); Denton, Adv. Cyclic
Nucleotide Protein Phosphorylation Res., 20 :293 (1986);
Cohen et al, In Molecular Basis of Insulin Action, M.P.
Czech, Ed, Plenum Press, New York, pp. 213-233 (1985) ];
alteration of a subset of phosphoproteins on tyrosyl
residues, [Becker and Roth, Annu. Rev. Med., 41: 99
(1990)]; regulation of gene expression for specific
regulatory enzymes such as phosphoenolpyruvate
carboxykinase, [Sasaki et al, J. Biol. Chem., 259:15242
15 (1984); Bridges and Goodman, Cell Biochem., lle
(suppl) :64 (1987)]; redistribution of membrane proteins
including the glucose transporter, insulin-like growth
factor II and transferrin receptors, [Karnieh et al, J.
Biol. Chem., 256 :4771 (1981); Kono et al, ibid.,
257:10942 (1982); Oka et al, Proc. Natl. Acad. Sci.
U.S.A., 81:4082 (1984); Davis et al, EMBO J., 5:653
(1986) ]; and stimulation of cell growth,
[Straus, Endocr. Rev., 5 :356 (1984) ] . The chronology
varies, with some of the processes occurring within
25 seconds (e.g. insulin receptor autophosphorylation on
tyrosyl residues and inhibition of transcription of the
phosphoenolpyruvate carboxykinase gene, [Rosen, Science,
237: 1452 (1987) ] . Many of the rapid effects on cellular
processes, such as stimulation of hexose transport, do
30 not require alterations of enzyme activities nor
synthesis of new protein or nucleic acid species in
response to insulin. Effects on cell processes such as
macromolecular synthesis and cell proliferation require
hours to days to become manifest. Most of the effects
35 of insulin are cell- and tissue-specific and i~nvolve
only a discrete subset of proteins in differentiated
systems. In mammals, insulin synthesis and secretion
=
~ 94/07994 ~ 4 64t3 2
coupled to nutrient sensing is compartmentalized to the
beta cells of the pancreas, whereas insulin action is
primarily exerted in peripheral target tissues.
Although the effects of insulin recognized at the
cellular level are numerous, the molecular mechanism of
insulin action is not known. In recent years,
simplifying assumptions have been made to invoke a
single mechanism in the initiation of the biological
effects of insulin, [Rosen, Science, 237:1452 (1987)].
The first essential and common step in insulin action
begins at its cognate receptor. Rapid
autophosphorylation of the insulin receptor on tyrosyl
residues in response to insulin binding activates a
cascade of "downstream" proteins through
receptor-mediated tyrosine phosphorylations. Observed
in many types of mammalian cells, the identities and
roles of these phosphotyrosine proteins remain obscure.
The downstream insulin signal transducing proteins
appear to be low abundance, transient species that are
exceptionally difficult to isolate from mammalian
sources.
Diabetes mellitus and sequelae (e.g.
cardiovascular, renal, ocular, neural and congenital
disorders) and proliferative diseases
related to insulin (e.g. insulinomas) have
multifactorial etiologies. Some of ~hese diseases
result from aberrant insulin secretion or defects in
insulin structure or processing. In other forms of
disease, the receptor is defective in number, hormone
binding properties or protein tyrosine kinase activity.
Some may be caused by dysfunctional interactions of the
receptor with downstream signaling proteins. Various
genetic systems are available for studying insulin
action in this complex group of diseases. The first is
represented by human diabetics (or animal models of
diabetes) whose disease possesses a heritable component.
In the recent past, transgenic mice have been developed
W094/07994 ~2~643~ PCT/US93/0950 ~
to address specific genetic lesions associated with the
diabetic phenotype. Another genetically amenable
organism for analyzing insulin action is the fruit fly
Drosophila melanogaster. Homologues of insulin
signaling elements have been identified in Drosophila,
[Kramer et al, Insect Biochem., 12:91 (1982); LeRoith et
al, Diabetes, 30:70 (1981); Tager et al, Biochem. J.,
156:525 (1976); Duve et al, Cell Tissue Res., 200:187
(1979) and Petruzzelli et al, J. Biol. Chem., 260:16072
(1985); Fernandez et al, Mol. Cell Biol., in press;
Petruzzelli et al, Cold Spring Conf. Cell Proliferation,
3:115 (1985); Petruzzelli et al, Proc. Natl. Acad. Sci.
USA, 83:4170 (1986)], although they have not been
completely characterized.
SUMMARY OF THE INVENTION
The present invention is therefore directed to a
yeast or fungal strain which possesses different growth,
morphology or viability properties in the presence of
insulin than the parent strain from which it was
derived. A preferred yeast or fungus is one which grows
better than Saccharomyces cerevisiae (S. cere~isiae)
strain S288c in the presence of insulin at a
concentration of 10~ M at a temperature between 17C to
37C. Preferably, the yeast or fungus grows at least 10
better, more preferably at least 50~ better and most
preferably at least 100~ better (2 fold better) than S.
cerevisiae strain S288c when cultivated in a rich medium
containing insulin (YPD+I medium) for 24 hours at 30C
when 1 x 105 cells are inoculated into 100 ml of media to
yield a final density 1 x 103 cells/ml.
The mutant yeast strain may also have at least one
of the following characteristics: a response to a
compound known to be an insulin secretagogue for mammals
(e.g. certain nutrients) is altered compared wit~ said
response observed in the strain from which it was
derived; transport of an insulin secretagogue is altered
~ 94/07994 PCT/US93/0950~
compared with transport of said secretagogue observed in
wild-type yeast; secretion of insulin-like peptide is
altered compared with secretion of insulin-like peptide
in the strain from which it was derived; it has a mutant
insulin-like peptide receptor protein; post-
translational modification of the insulin-like peptide
receptor is altered compared to the post-translational
modification of the insulin-like peptide receptor
observed in wild-type yeast; has a reduced number of
insulin-like peptide receptor molecules per cell
compared to the number of insulin-like peptide receptors
observed in wild-type yeast; cell wall synthesis is
altered so as to retain all of the insulin-like peptide
made by a cell of said mutant yeast strain within the
periplasmic space encompassed by said cell wall; cell
wall synthesis is altered so as to fail to retain the
insulin-like peptide made by a cell of said mutant yeast
strain within the periplasmic space encompassed by said
cell wall; alteration in the regulation of the RAS/cAMP
pathway; does not produce a f~ully active endogenous
insulin-like peptide; and does not produce one or more
functional effectors in the insulin sensing/signaling
system.
The present invention is also directed to a method
for isolating a mutant yeast strain which
comprises mutating yeast cells and selecting a mutant in
said yeast cells which possesses different growth,
morphology or viability properties in the presence of
insulin than the parent strain from which it was
derived.
The present invention is also directed to a method
for production of insulin or an insulin-like peptide
comprising culturing a yeast or fungus which
overexpresses said insulin or insulin-like peptide and
recovering insulin or insulin-like peptide from said
strain or said culture medium.
The present invention is also directed to a method
W094t07994 ~ PCT/US93/0950~ ~
2i4~432
for production of insulin, which comprises i) providing
a mutant yeast strain in which the response to insulin
observed for wild-type yeast has been abrogated; i i )
transforming said mutant yeast strain with DNA which
expresses an insulin gene; iii) culturing the
transformed mutant yeast strain from step (ii) under
conditions which provide for efficient expression of
said insulin gene; and iv) recovering the insulin
produced by the culture of step (iii).
The present invention is also directed to a method
for detecting an insulin secretagogue which comprises i)
culturing cells of a mutant yeast strain wherein a
response to said secretagogue is altered so as to be an
exaggerated response, in a medium lacking said
secretagogue; ii) contacting a sample of cells from step
i) with a sample to be assayed for the presence of said
secretagogue; and iii) measuring the response of said
cells after contacting them with said sample.
The present invention is also directed to a method
for identifying elements in the insulin
sensing/signaling pathway or to detect agents which
modulate the activity of the components or elements of
the pathway which comprises culturing the yeast; and
analyzing basal status of said componentsi and adding an
agent to said yeast; and measuring the status of said
components; and comparing said status with said basal
status.
The present invention is further directed to a
method for making a mutant yeast strain which comprises
mutating a yeast culture and selecting a mutant in said
yeast culture which grows better in the presence of
insulin than the yeast from which said mutant was
derived.
The present invention is further directed to method
for production of insulin comprising culturing a~nutant
yeast strain which is transformed with a gene for
mammalian insulin in a culture medium and recovering
~ 94/07994 2 4 G ~ 3 2 PCT/USg3/ogSO~
insulin from said mutant strain or said culture medium.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of a four phase model
for an insulin sensing/signaling pathway wherein the
following abbreviations apply:
Genes
GGPI = glycosylphosphatidylinositol protein
PLC = phospholipase C
PIK = phosphatidylinositol kinase
CYR1 = adenylate cyclase
RAS = GTP-binding protein
CDC25 = cell division cycle control protein and
glucose sensor
GTs = glucose transporters
ScILP = Saccharomyces cerevisiae insulin-like
peptide
PDEs = phosphodiesterases
CDM1 = calmodulin
Second Messengers and Substrates
DAG = diacylglycerol
DPI = diphosphoinositide
TPI = triphosphoinositide
InsPglycan = inositol-phosphate glycan
ATP = adenosine triphosphate
AMP = adenosine monophosphate
cAMP = cyclic-3'-5'-adenosine
G = glucose
Ca~ = intracellular/extracellular calcium;
FIG. 2 is a summary of a procedure for mutagenizing
yeast and isolating insulin-dependent mutants;
FIGS. 3A, 3B and 3C are graphs showing plating
efficiency curves at three different plating densities,
respectively;
FIGS. 4-9 illustrate representative plates having
(YPD+I)/~PD or (YNB+I)/YNB ratios as indicated in the
Figures.
SU~5TITUTE SHE~T (RULE 26)
W094/07994 , :-~ ' PCT/US93/0950 ~
2 i ~ ~ 4 7A
FIGS. 10A through 10G shows the elevation of
intracellular cAMP of cultured S. cerevisiae S288c in
response to the addition of various nutrients.
FIGS. llA through llD shows the proliferation
response of cultured S. cerevisiae S288c in response to
the addition of various nutrients.
FIGS. 12A through 12D shows the TCA precipitable
phosphate in actively growing versus phosphate arrested
yeast cultures.
FIGS. 13A through 13F show the results of DNA
synthesis of growing yeast cultures and cultures
arrested by phosphate depletion analysed by cell
sorting.
FIG. 14 shows the results of phosphoamino acid
analysis of actively growing yeast cultures compared to
cultures arrested by phosphate depletion.
FIG. 15 shows a Western blot with anti-
phosphotyrosine antibody of total proteins of yeast
throughout the life cycle (inoculation-exponential
phase-stationary phase-redilution) of a yeast culture.
DETAI~ED DESCRIPTION OF THE lNv~NlION
The invention comprises mutant yeast cells,
preferably Saccharomyces cerevisiae, which have an
altered response to insulin, insulin secretagogues and
insulinomimetic compounds compared to wild-type yeasts.
S~T~ S~ E ~6)
4/07994 2 1 ~ 6 ~ 3 2 PCT/US93/0950~
The response to insulin and insulinomimetic compounds
can be abrogated or enhanced. The mutations which
affect response to insulin and insulinomimetic compounds
may be distributed throughout the biochemical pathways
which act to bind the compound and then produce a
physiological change in metabolism in response to that
binding. Elements of this pathway include (but are not
limited to) secretagogue receptors, such as glucose
transporter and proteins which bind to sulfonylurea
compounds, protein kinase C, phospholipase C, proteins
involved in phosphatidylinositol turnover, other
proteins involved in second messenger signaling, such as
adenylate cyclase and phosphodiesterase, enzymes
involved in synthesis, intracellular transport and
secretion of the yeast insulin-like protein, the
membrane-localized insulin receptor-like protein ~IRP)
for insulin and insulin-like proteins, and proteins
which act downstream of the IRP that transduce the
signal of ligand binding to the IRP to reprogram cell
physiology, such as the product of the CDC25 gene,
phosphatidylinositol kinase, and other as yet
unidentified proteins. The "downstream" proteins can be
of two classes, those which interact directly with the
IRP and those which are even further "downstream~', such
as transcription factors and the proteins which carry
biochemical signals from the plasma membrane to the
nucleus. Other sites of mutation include enzymes
involved in cell wall biosynthesis, as permeability of
the cell wall to insulin has an influence on insulin
transport from the culture medium to the cell membrane.
In the mutant yeast strain, the secretion of
insulin-like peptide may be altered so as to decrease
secretion of insulin-like peptide in response to
secretagogue stimulation or the secretion of insulin-
like peptide may be altered so as to res~lt inconstitutive secretion of insulin-like peptide.
In the mutant yeast s~rain, administration of an
W094/07994 ~ PCT/US93/09S0~ ~
~1~6 432
insulin secretagogue may not result in said yeast
entering a cycle of DNA replication. This may be
because the yeast does not commit to the start portion
of said cycle of DNA replication.
In the mutant yeast strain, regulation of a second
messenger response to an insulin secretagogue may be
altered.
If the mutant yeast strain has a mutant insulin-
like receptor, the insulin-like receptor may not be able
to interact with downstream effector proteins.
"Secretagogues" generally are compounds which
elicit secretion of a hormone or other factor from a
cell when the cell is exposed to them. In the present
case, "secretagogue" is used to denote a compound which
15 elicits secretion of "insulin-like protein" from a yeast
cell. Examples of such secretagogues are nutrients such
as glucose, oleic acid, amino acids such as leucine,
lysine and arginine, nucleotides such as adenine, and
sulfonylurea compounds and derivatives of sulfonylurea
compounds: [Caro, Am. J. Med., 89:17S-25S (1990); Easom
et al, J. Biol. Chem., 265:4938-14946 (1990); Fleischer
et al, In Molecular and Cellular Biology of Diabetes
Mellitus: Insulin Secretion. Volume I, pp. 107 - 116
(1989); Floyd et al, J. Clin. Invest., 45 : 1487-1502
(1966); Grapengiesse et al, J. Biol. Chem., 266 : 12207 -
12210 (1991); Malaisse et al, J. Lab. Clin c. Med.,
72 :438-448 (1968); and Sener et al, E~perientia,
40: 1026-1035 (1984) ] .
In wild-type yeast, exposure to a secretagogue
30 results in many of the physiological responses
associated with the "early response" seen in mammalian
cells provided with growth factors, Fleischer et al,
Molecular and Cellul~r Biology of Diabetes Mellitus:
Insulin Secretion, Volume I, pp. 107-116 (1989) . In
35 particular, there is a prompt phosphorylation ~f many
cellular proteins, a transient increase in intracellular
3',5'-cyclic adenosine monophosphate (cAMP) levels, an
~ 94~0,994 2 1 ~ 6 4 3 2 PCr/US93/09504
? ~ ~'
increase in phosphatidylinositol (PI) turnover with
associated release of diacylglycerol and a transient
flux of calcium across cell membranes, i.e. mobilization
of calcium: [Auger et al, J. Biol. Chem., 264:20181-
20184 (1989); Broach, Trends in Genetics, 7:28-33
(1991); Cameron et al, Cell, 53:555-565 (1988); Davis et
al, Cell, 47:423-431 (1986); Frascotti et al, FEBS,
274:19-22 (1990); Huber et al, The Endocrine Society,
74th Annual Meeting, San Antonio, TX (1992); Iida et al,
J. Biol. Chem., 265:21216-21222 (1990); Kaibuchi et al,
Proc. Natl. Acad. Sci. USA, 83:8172-8176 (1986); and
Mbonyi et al, Molec. Cell Biol., 8:3051-3057 (1988)].
One link in the physiological response to a
secretagogue is the secretion by the yeast of an
"insulin-like protein" (ILP~ which binds in an
autocrine/paracrine fashion to its receptor localized at
the cell membrane. This receptor-ligand interaction
begins a physiological response which alters
carbohydrate and lipid metabolism in the yeast and also
stimulates entry into the cell cycle culminating in DNA
replication and budding. Over-exposure o~ the cell to
insulin or insulinomimetic compounds that can bind to
the receptor can result in insulinemia or insulin
resistance (i.e. a diabetes-like state) in a culture of
yeast, with concomitant disruption of the normal
cellular metabolism and death of the cells.
Accordingly, for the production of insulin by
recombinant DNA techniques wherein the insulin is
secreted by the yeast host cells, it might be
advantageous to employ a mutant yeast of the present
invention, which is insensitive to the toxic effects of
insulin, [Shuster et al, Gene, 83:47 (1989)], as the
host cell.
Yeast, unlike higher eukaryotic cells, can be grown
to high densities in inexpensive, defined nutrient
media. Thus, scale-up of the culture can be used to
overcome the limited quantities of starting materials
W094/07994 ~ .~ PCT/US93/0950 ~
2146432 11
from which to isolate the insulin signaling/sensing
proteins. Furthermore, the precise role of these
proteins can be determined in mutant strains of yeast
defective in normal insulin related functions. Finally,
molecular biology techniques can be employed to
introduce into yeast m~mm~l ian homologues of the insulin
signal transducing proteins for identification of
common, fundamental functions in evolutionarily diverse
organisms. Insight into the molecular mechanism of
insulin action should pave the way for discovery and
development of novel classes of therapeutic agents that
circumvent the receptor in mediating an insulin signal.
The availability of such agents would be useful for
treatment of
diabetes mellitus and other diseases of cell
proliferation and metabolism related to insulin.
A mutational analysis of gene products involved in
the mechanism of insulin action should be feasible in
the lower eukaryote S. cerevisiae. Furthermore, yeast
is known to possess many of the proteins proposed to
transduce the insulin signals in mammals. Therefore,
advanced molecular biological techniques available with
this organism could be used to establish the role of the
proposed transducing proteins, and would permit
identification of second-site mutations in as yet
unknown elements of the insulin signal transduction
pathway. Yeast has been exploited for study of
fundamental aspects of cell biology including control of
cell division, protein secretion and signal transduction
through a mating factor receptor. However,
demonstration of endogenous, molecular components
related to insulin production and action was required
before yeast could be used to study the mechanism of
insulin action.
Although the experimental work to date has been
performed with S. cerevisiae, it is anticipated that
mutants of other yeast and fungi can be made in
94/07994 21 ~ 6~32 PCTtUS93/0950
i2
accordance with the general techniques described in this
application. It is expected that all yeast and fungi of
the class Ascomycetes may be useful parent strains to be
mutated. Representative yeast include yeasts from the
genera Saccharomyces, Schizosaccharomyces, Aspergillus,
Penicillium, Neurospora, Candida, Torulaspora, and
Torulopsis. Other species of S. cerevisiae may include
carlsbergensis and ellipsoideus var. Various yeast and
fungal strains which may be useful are described in the
American Type Culture Collection CATALOGUE OF
FUNGI/YEASTS, Seventeenth Edition (1987).
Mutations were generated using ethyl
methanesulfonate as the mutagenic agent. However, other
mutagens can be employed to create the mutations. Such
mutagens include nitrous acid, N-nitrosoguanidine,
ultraviolet radiation and transposon insertion
mutagenesis.
The insulin dependent mutants of the present
invention include any mutant which grows better in the
presence of insulin than the parent strain from which it
was derived. Preferred mutants are those which have a
YPD+I/YPD and a YNB+I/YNB ratio of 2.0 or more as
measured by the plating assays described herein at a
temperature of 17C, 23C, 30C or 37C. Some yeast
mutants have the same YPD+I/YPD ratio and YNB+I/YNB
ratio at all four of the above temperatures. These
yeast show a "temperature independent insulin response~.
Other yeast show a progressive increasing or decreasing
ratio with increasing temperature. This may be a result
of a single site temperature sensitive mutation. These
yeast show a "temperature dependent insulin response".
Some mutants demonstrate fluctuating ratios as a funtion
of temperature suggesting mutations at multiple loci.
One possible use of the mutant yeast or fungi of
the present invention is as a parent (host) strain which
is transformed with a gene for vertebrate or mammalian
insulin (e.g. human insulin, bovine insulin, etc.) or
W094/07994 ~46 4~` PCT/US93/0950 ~
for elements of the insulin sensing/signaling system.
The yeast can be mutated to increase its ability to grow
in the presence of insulin either before or after
transformation with the m~mmAlian insulin gene. Such
5 transformed mutants are considered to be within the
scope of this patent. Techniques for transforming yeast
with foreign (heterologous) DNA are described in U.S.
Patent 5,108,925 to Enari et al which issued on April
28, 1992. Preparation of transformant yeast which
express the human insulin gene is described in U.S.
Patent 4,916,212 to Markussen et al which issued on
April 10, 1990. The entire contents of both of these
patents are hereby incorporated by reference.
The invention is described in detail by the various
15 examples below. The examples set forth are meant to be
illustrative, rather than limiting, of the scope of the
invention set forth therein.
EXAMPLE 1: Isolation and characterization of Yeast
strains havinq an altered res~onse to insulin
Newly created and isolated mutant strains of the
present invention were derived from S. cerevisiae
wild-type strain S288c (MATa mal mel gal2 CUP1 SUC2)
available from the Yeast Genetics Stock Center
(University of California, Berkeley) (ATCC No. 26108) .
Mutagenesis was conducted with ethyl methanesulfonate
essentially as described in Methods in Yeast Genetics
(Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York, pp. 9-17 (1989)).
Ethyl methanesulfonate is an alkylating agent that
can generate as many as 102-104 mutations per gene
without significant inactivation. Thus, isolates may be
defective at multiple genetic loci. The primary targets
of ethyl methanesulfonate are keto oxygens at position
six of guanine to yield O- 6 ethyl guanine and at
35 position four of thymine to yield 0-4 ethyl thymine.
Modification at these positions leads to mispairing of
~ 94/07994 r 2 1 9 ~6 1 pcr/us93/o95o4
14
guanine with thymine resulting in GC ~ AT transitions.
The mutagenesis procedure is initiated with parent cells
grown to a mid-logarithmic phase density of 2 x 108/ml in
YPD broth consisting of 1~ (w/v) yeast extract, 2~ (w/v)
Bacto-peptone and 2~ (w/v) glucose. Cells harvested by
- centrifugation at 3,000 x g at room temperature were
washed and resuspended in sterile, phosphate buffered
saline (PBS) to a concentration of 2 x 108/ml.
Cells were mutagenized in the presence of 30 ~l of
ethyl methanesulfonate per ml for 1 hour at 30C to
achieve approximately 70~ killing. Control cells were
incubated similarly in the presence of PBS. Following
incubation, samples were transferred to fresh tubes,
collected by centrifugation as above and washed once
with sterile distilled, deionized water. The
mutagenized cells were treated with 5~ (w/v) sodium
thiosulfate, washed and resuspended in the same solution
to neutralize ethyl methane-sulfonate. Unmutagenized
control samples were washed twice in PBS.
To select for insulin dependent mutants, YPD agar
plates (15 ml) are supplemented with 0.15 ml of bovine
insulin (10~ M) which was dissolved in 10 mM HCl,
neutralized in PBS and emulsified with 1 ~ Emulphor~
(Hoffmann-LaRoche). The thus prepared insulin stock
solution is sterilized through 0.2 ~m cellulose acetate
filter and spread with a sterile glass rod over the
surface of the plate. Under no circumstances is insulin
added to the medium prior to autoclaving the medium.
Control plates were spread with a similar solution
lacking insulin. Mutagenized and control cell
suspensions were diluted by a factor of 105 and aliquots
of 0.1, 0.2 and 0.4 ml were plated in 10 replicates on
both types of media. The plates were inverted and
incubated at room temperature (approximately 23C).
After 24, 48, 72 and 96 hours of incubation, plates were
examined for growth. Representative recoveries (plating
efficiencies) of mutagenized cells are presented in
SUE3ST~ 1~ U~ L t~ LE 2~/
W094/079g4 PCT/US93/0950 ~
21~ 14A
,~ .. .... .
Figures 3A, 3B and 3C wherein 1200, 2400 and 4800 yeast
cells, respectively, were plated. Following 72 hours of
5~TITUTE SHEET (RULE 26)
~ 94/07994 PCT/US93/09501
21~6~32 ~
incubation, isolates appearing as colonies on YPD plus
insulin (YPD+I) plates were streaked on YPD or YPD+I
plates to demonstrate the degree of insulin dependence.
To assess dependence, single colonies of designated
mutants were picked with a standardized (0.01 mL)
- sterile inoculating loop and streaked across a petri
plate in 4 quadrants (12 passes per quadrant). As a
control, mutagenized cells recovered on YPD were tested
on YPD+I plates to determine if they were insulin
sensitive.
The mutants demonstrated varying degrees of
dependence or insensitivity to supraphysiological
concentrations (e.g. 10- M) of insulin, and some of the
phenotypes observed between the permissive
temperatures of 23C and 30C could be exacerbated at
extreme temperatures of 17C and 37C. Because YPD may
contain insulin or insulinomimetic substances, the
mutant strains of S. cerevisiae were also tested for
insulin dependence by cultivation on complex, defined
medium, YNB. The chemical composition of the medium
designated YNB is presented in Table I.
W094/07994 . '~ ' PCT/US93/0950 -
2~4~43~
16
TABLE I
Constituent Final mg/l
adenine sulfate 20
uracil 20
L-tryptophan 20
L-histidine-HCl 20
L-arginine-HCl 20
L-methionine 20
L-tyrosine 30
L-leucine 30
L-isoleucine 30
L-lysine-HCl 30
L-phenylalanine 50
L-glutamic acid 100
L-aspartic acid 100
L-valine 150
L-threonine 200
L-serine 400
L-cysteine 150
L-alanine 100
L-asparagine 50
L-proline 100
L-glutamine 100
L-glycine 100
The above components are added to yeast nitrogen base
without amino acids (Difco, Catalog No. 0919)
supplemented with 5 g/L ammonium sulfate and 20 g/L
glucose.
~ 94/07994 2 1 ~ 6 4 3 2 PCT/US93/0950~
~1' ~' k ~
,.l 17
The results of incubation at various temperatures on
rich, complex YPD or defined YNB media are presented in
Table II.
WO 94/07994 PCr/US93/0950~
2~ 32 18
TABLE
1 7C
MU~ANT YPDYPt)+l YPD~IIYPDt~U~ANT YNB YNB~I YN8+1/YNB
M1.2D3 1.0 4 0 4 0 Ml.2D1 1 0 4 0 4 0
Ml.4A3 1.0 4 0 4 0 M2.1D2 1 0 3 0 3 0
M1.2Bl 1 0 3 0 3 0M2 2C5 1 0 3 0 3 0
M1.2D4 1 0 3 0 3 0M1.4A4 o 5 1.0 2 0
M1.4B1 1 0 3 0 3 0M2.4B2 0 5 1 0 2 0
M1 4B2 1 0 2 0 2 0M1 2B1 2 0 3 0 1 5
M2.1B2 1 0 2 0 2 0M1.4E1 2 0 3 0 1 5
M2.2C5 1 0 2 0 2 0M2.1D1 2 0 3 0 1 5
M2 4A3 1.0 2 0 2 0 M2.4A2 1 0 1 5 1.5
M1 4A1 1 0 1 5 1 5 M2.4C1 2 0 3 0 1 5
M1.4A4 1.0 1 5 1 5 M2 4E1 1.0 1 5 1 5
M1.4C5 1.0 1 5 1 5 Ml.2D2 3 0 4.0 1.3
M1.4E1 2.0 3 0 1 5 M1.4B1 3 0 4 0 1 3
M2.181 1 0 1 5 1.5 M2.2E3 3 0 4.0 1.3
M2.1D1 2 0 3 0 1.5 M1.2B3 1.0 1.0 1.0
M2.4D4 2 0 2 5 1 3M1.2D3 3 0 3 0 1.0
M1.283 1 5 1 5 1 0M1.2D4 1.0 1.0 1 0
M1.2D1 1.0 1 0 1 0M1.4A3 2.0 2 0 1 0
M1.2D2 2 0 2 0 1 0M1.4E32 1 0 1 0 1.0
Ml.4B2 1 0 1 0 1 0Ml.4B2 1 0 1 0 1 0
Ml.4C1 1.5 1.5 1 0M1.4C1 1.0 1 0 1.0
M1.4C2 1.0 1.0 1.0 Ml.4C2 1 0 1.0 1 0
M1.4C3 2 0 2 0 1 0 M1.4C3 3.0 3 0 1.0
M1.4C4 4.0 4 0 1.0 M1.4C4 4.0 4 0 1.0
M1.4D1 1.0 1 0 1.0 M1.4D3 1 0 1.0 1 0
M1.4D2 1.0 1 0 1.0 M2.1A1 1 0 1 0 1.0
M2.1A1 3.0 3.0 1.0 M2.1B1 1.5 1.5 1.0
M2.1C1 4 0 4 0 1 0M2.182 2.0 2.0 1.0
M2.1D2 1.0 1.0 1 0M2.1C1 4.0 4.0 1.0
M2.1D3 1.0 1 0 1 0M2.2A1 1.0 1.0 1.0
M2.2A2 1 5 1 5 1 0M2.281 1.0 1.0 1.0
M2.2B2 1 0 1 0 1 0M2.2B2 1 0 1.0 1.0
M2.2B3 1 0 1 0 1.0M2.2C1 1.0 1.0 1.0
M2.2B4 1.5 1 5 1 0M2.2C3 1.0 1.0 1 0
M2.2C2 1 01.0 1 0 M2.2D2 1.0 1.0 1.0
M2.2C3 1.01 0 1.0 M2.2D3 0.5 0.5 1.0
M2.2C4 1 01.0 1 0 M2.4A1 1.0 1.0 1.0
M2.2D1 1.01 0 1 0 M2.4A3 1 0 1.0 1 0
M2.2D3 0.50.5 1 0 M2.482 1.0 1.0 1.0
M2.2E2 1.01 0 1.0 M2.4C2 1.0 1.0 1.0
M2.4A1 1.01 0 1 0 M2.4C4 1.0 1.0 1.0
M2.4A2 1.01.0 1.0 M2.403 4.0 4.0 1.0
M2.4B2 1.01 0 1 0 M2.4D4 3.0 3.0 1 0
M2.4C1 4.04.0 1.0 M2.4E2 0.5 0.5 1.0
M2.4C2 1.01.0 1 0 M2.4E3 2 0 2 0 1.0
M2.4C3 2.02.0 1 0 M1.282 2.5 2.0 0.8
M2.4C4 1.01.0 1 0 M1.4A1 1.5 1.0 0.7
M2.4D1 2.02.0 1.0 M1.4C5 1.5 1.0 0.7
M2.4D3 4.04 0 1 0 Ml.4D2 1.5 1.0 0.7
M2.4E2 1.01 0 1 0 M2.2A2 1.5 1.0 0.7
M2.4E3 2.02 0 1.0 M2.4C3 1.5 1.0 0.7
M2.2A1 1.51.0 0.7 M2.2B3 2.0 1.0 0.5
M2.4E1 1.51 0 0.7 M2.284 2.0 1.0 0.5
Ml.2B2 2.51 5 0 6 M2.2C2 2.0 1 0 0.5
M2.2E3 3.52.0 0.6 M2.4D1 4.0 2.0 0.5
M2.4B1 0.01.0 -~ Ml.4D1 4.0 1.0 0.3
Ml.4D3 1.00.0 1~1 M2.2D1 4.0 1.0 0.3
M2.2B1 1.00 0 ~I M2.2C4 0.0 1.0 1
M2.2C1 1.00.0 ~I M2.2E1 0.0 1.0 1~
M2.2D2 1.00.0 1~1 M2.2E2 1.0 0.0 1~1-
M2.4B2 1.00.0 1~1 M2.1D3 0.0 0.0 N~
M2.2E1 0.00 0 1~3 M2.4B1 0.0 0.0 NSB
1S288C 3.82.5 0.7 1S288C 4.0 3.0 0.8
2S288C 4.03 5 0 9 2S288C 3.5 2.5 0.7
94/07994 - ` - PCI /US93/0950 1
21~-5~ 3~
TABliE Il ( COhT ' D . )
23C
MUTANT YPt~ YPD+I YPD+I/YPD MUTAN~ YNB YNBI~ YN8+1/YN8M1.21~2 1 0 4 0 4 0 M2 lB1 1 0 4 0 4.0
M2.2C1 1 0 4 0 4 0 M2.4E1 1 0 3 0 3 0
M1.2D3 1 0 3 0 3 0 M1.4A4 1 0 2 0 2 0
M1.4C1 1 0 3 0 3 0 M1.4C5 2 0 4 0 2 0
M2 2B4 1 0 2 5 2 5 M1.4D1 2 0 4.0 2 0
M2.2D3 1 0 2 5 ^ 5 M2.1D2 2 0 4 0 2 0
M1.4D2 1 0 2 0 2 0 M2.2D3 0 5 1 0 2 0
M2.283 1.0 2 0 2 0 M2.2E2 1 0 2.0 2 0
M2.4A1 1 0 2 0 2 0 M2.4C1 2.0 4 0 2 0
M2.4Ct 3 0 5 0 1.7 M2.4c3 1.0 2 0 2 0
M2.2A1 2 0 3 0 1 5 M1.2D2 2.0 3 0 1 5
M2.2B2 2 0 3.0 1 5 M2.4A2 2.0 3 0 1 5
M2.2C3 2 0 3 0 1 5 M2.4A3 2.0 3.0 1.5
M2.4A3 2.0 3 0 1.5 M1.4C2 3.0 4.0 1 3
Ml.4A4 1.~; 2.0 1.3 M2.4C4 3 0 4.0 1 3
M2.4E3 3 0 4 0 1 3 M1.2B3 2.0 2.5 1 3
M2.2C5 2 0 2 5 1 3 M2.2C5 2.0 2.5 1.3
M2.4C3 3 0 3 5 1 2 M1.2B1 4 0 4.0 1.0
Mæ2E3 4 0 4 5 1.1 M1.2D1 4.0 4.0 1.0
M1.2B3 2 0 2 0 1.0 Ml.2D3 4.0 4.0 1 0
Ml.2D4 1.0 1 0 1 0 M1.2D4 4.0 4.0 1 0
M1.4A1 2.0 2.0 1 0 Ml.4B1 4.0 4 0 l.o
M1.4A3 4.0 4.0 1 0 Ml.482 4 0 4.0 1.0
Ml.4B2 4.0 4.0 1 0 Ml.482 2 0 2.0 1 0
M1.4B2 2.0 2.0 1.0 M1.4C3 3.0 3.0 1 0
M1.4C2 3 0 3 0 1.0 M1.4C4 4.0 4.0 1 0
M1.4C3 4 0 4.0 1.0 M1.4D2 1.0 1.0 1.0
M1.4C4 4.0 4.0 7.0 M2.1C1 4.0 4.0 1.0
M1.4C5 2.0 2.0 1 0 M2.tD1 2.0 2.0 1.0
M1.4D1 4.0 4.0 1.0 M2.1D3 4.0 4.0 1 0
M1.4E1 3.0 3.0 1 0 M2.2A1 2.0 2.0 1.0
M2.1B1 2.0 2.0 1.0 M2.2B1 1.0 1.0 1.0
M2.1B2 2 0 2.0 1.0 M2.2B2 2.0 2 0 1.0
M2.1C1 4 0 4.0 1 0 M2.2B3 2.0 2.0 1 0
M2.1D1 4 0 4.0 1 0 Mæ2C1 1.0 1.0 1.0
M2.1D2 4 0 4.0 1 0 M2.2C3 3 0 3.0 1 0
M2.1D3 4.0 4.0 1.0 Mæ2E3 3.0 3.5) 1.0
M2.2A2 2.0 2 0 1.0 M2.481 1.0 1.0 1 0
M2.2B1 1.0 1 0 1.0 M2.4B2 3.0 3.0 1 0
M2.2C2 4.0 4.0 1.0 M2.4D1 4.0 4.0 1.0
M2.2D1 1.0 1.0 1 0 M2.4D3 4.0 4.0 1.0
Mæ2E1 1.0 1.0 1.0 M2.4D4 4.0 4.0 1.0
Mæ2E2 1 0 1.0 1.0 M2.4E2 1.5 1.5 1.0
M2.4A2 2.0 2.0 1.0 M~ 4E3 3.0 3.0 1.0
M2.4B2 2.0 2 0 1 0 M1.4D3 5.0 4.0 0.8
M2.4B2 2.0 2 0 1.0 Ml.4A3 4.0 3.0 0.8
M2.4C4 3.0 3.0 1.0 Ml.282 3.0 2.0 0.7
M2.4D1 4.0 4.0 1.0 M1.4C1 3.0 2.0 0.7
M2.4D3 4.0 4.0 1.0 M1.4E1 3.0 2.0 0.7
M2.4D4 3.0 3.0 1 0 M2.1A1 3.0 2.0 0.7
M2.4E2 1.0 1.0 1 0 Mæl82 3.0 1.5 0.5
M1.2B2 3 0 2.5 0.8 M2.2C2 4.0 2.0 0.5
M1.2D1 5.0 4.0 0.8 M2.2C4 4.0 2.0 0.5
M1.4B1 4.0 3.0 0 8 M2.4B2 2.0 1.0 0.5
M2.1A1 4.0 3.0 0.8 Ml.4A1 2.5 1.0 0.4
Ml.2B1 3.0 2.0 0.7 M2.2B4 2.5 1.0 0.4
M2.4E1 3.0 2.0 0.7 M2.2A2 3.0 1.0 0.3
M1.4D3 2.0 1.0 0.5 M2.4C2 4.0 1.0 0.3
M2.2D2 4.0 2.0 0.5 M2.2Dl 0.0 1.0 N~
M2.4C2 4.0 1.0 0.3 M2.4A1 0.0 1.5
M2.2C4 0.0 4.0 ~; M2.2D2 3.0 0.0 t~
M2.4B1 0.0 0.0 ~B M2.2E1 1.0 0.0 ~;1
lS288C 4.3 4.3 1.0 lS288C 4.0 4.3 1.1
2S288C 3.5 4.3 1.2 2S288C 4.0 4.0 1.0
WO 94/07994 2~ ~ PCI /US93/0950~O
TA~ II (COl~T' D. )
30cc
MUTANT YPD YPD~I YPD+IIYPD MUTANr YN8 YN8~1 YN8+1/YNR
M1.2821 0 3 0 3 0 M2 2B1 1 0 4 0 4 0
M2.2C51 7 3 5 2 1 M1.4C3 1 1 3 5 3 2
Ml.2B31 ~ 3 0 2 0 M1.2D2 1.0 3 0 3 0
M2.2D22 0 4 0 2 0 M1.282 1 0 2 5 2 5
M2.1811 5 2 8 1 9M1.281 2 0 4 0 2 0
M2.4E21 0 1 7 1 7M2.2C3 2 0 4 0 2 0
M2.4A31.3 2 0 1 5Ml.4C5 1.0 1.8 1 8
M1.2D42 0 3 0 1 5M2.4C4 2.0 3 0 1 5
Ml.4C51 0 1 5 l SM2.4E2 1.0 1.5 1 5
Ml.4E12.0 3 0 1 5Ml.2D1 3.0 4.0 1 3
M2.2812 0 3 0 1 5M2.2A2 2 5 3 3 1 3
M2.4D42 0 3 0 1 ~Ml.283 2 0 2 5 1.3
M2.2A1 1.5 2 2 1 5Ml.481 4 0 5.0 1 3
M2.4E12 0 2.8 1 4Ml.4D1 4 0 5 0 1.3
M2.4C32.0 2 7 1 4M2.1D1 4.0 5 0 1 3
Ml.481 3 0 4 0 1 3Ml.4C1 3.0 3 5 1.2
Ml.4C23 0 4 0 1 3M2.283 2.0 2 3 1.2
Ml.4D2 1 5 2 0 1 3M2.2E2 1.5 1.7 1 1
M2.2C4 3 0 4 0 1 3M2.2A1 1.8 2 0 1 1
M2.4A2 1.5 2 0 1 3M2.4E1 2 0 2 2 1 1
M2.4E3 3 0 4 0 1 3Ml.2D4 3.0 3 0 1.0
Ml.4C1 2 5 3 2 1 3Ml.4A1 2.0 2.0 1.0
M2.1C1 4.0 5 0 1 3Ml.4B2 4.0 4 0 1 0
M2.2E2 1.5 1 8 1 2Ml.482 1.8 1.8 1.0
M2.4A1 2 5 3 0 1 2Ml.4C4 4.0 4.0 1.0
M2.4B2 2 5 3 0 1 2Ml.4D2 2.0 2.0 1.0
M1.4C3 3.0 3 5 1 2M2.1A1 4.0 4.0 1.0
M2.182 3.0 3.5 1.2M2.181 1.0 1.0 1.0
M2.2E3 3.0 3 5 1 2M2.1C1 5.0 5.0 1.0
Ml.2B1 2.0 2.0 1 0M2.1D2 4.0 4.0 1.0
M1.2D1 4.0 4 0 1.0M2.2B2 1.0 1.0 1.0
Ml.4A3 5.0 5 0 1 0M2.2Cl 1.0 1.0 1.0
Ml.4A4 2 0 2.0 1~0M2.2D1 4.0 4.0 1.0
Ml.4B2 4.0 4 0 1 0M2.2E3 3.0 3 0 1.0
Ml.4D1 4.0 4.0 1 0M2.4A1 1.5 1.5 1.0
M2.1A1 4 0 4.0 1 0M2.4B1 1.0 1.0 1.0
M2.1D1 4 0 4 0 1.0M2.4B2 4.0 4.0 1.0
M2.2A2 1.5 1 5 1 0M2.4C1 4.0 4.0 1.0
M2.2C1 2.0 2 0 1.0M2.4C3 2.0 2.0 1.0
M2.2C3 4.0 4.0 1.0M2.4D1 4.0 4.0 1 0
M2.2D1 4.0 4.0 1 0M2.4D3 4.0 4.0 1.0
M2.2E1 1.0 1.0 1.0M2.4D4 3.0 3.0 1.0
M2.4B1 1.0 1.0 1 0M2.4E3 4.0 4.0 1.0
M2.4B2 4.0 4 0 1.0Ml.4E1 2.3 2.0 0.9
M2.4C2 4.0 4.0 1.0M2.482 3.0 2.5 0.8
M2.4D1 4.0 4.0 1 0M1 2D3 4.0 3.0 0.8
M2.2B4 2.3 2 0 0.9M2.2C4 4.0 3.0 0.8
M1.4B2 3.0 2.5 0.8M2.2D2 4.0 3.0 0.8
M2.2D3 3.0 2.5 0.8M2.2D3 2.0 1.5 0.8
Ml.2D3 5.0 4.0 0 8M2.4A3 2.0 1.5 0.8
M2.4C1 5.0 4.0 0.8M2.4C2 4.0 3.0 0.8
M1.4A1 1.7 1.3 0.8M2.4A2 2.8 2.0 0.7
M1.4C4 4.0 3.0 - 0.8M1.4A4 2.2 1.5 0.7
M2.1D2 4.0 3 0 0.8M2.1B2 3.0 2.0 0.7
M2.4D3 4.0 3.0 0.8M1.4A3. 5.0 3.0 0.6
M2.1D3 3.0 2.0 0.7M2.2B4 2.5 1.3 0.5
M2.4C4 3.0 2.0 0.7M1.4C2 4.0 2.0 0.5
M2.2B2 2.0 1.0 0.5M1.4D3 4.0 2.0 0.5
M2.2B3 3.0 1.5 0.5M2.2C5 3.0 1.5 0.5
M1.2D2 3.0 1.0 0.3M2.2C2 4.0 1.0 0
M1.4D3 4.0 1.0 0.3M" 2El 0.0 1.0 ~
M2.2C2 4.0 0.0 1\~1 M2.1D3 2.0 0.0 ~1
1S288C 3.0 3 8 1.31S288C 3.5 4.0 1.1
2S288C 3.8 3.5 0.92S288C 3.3 4.0 1.2
~94/07994 211~6~3`2 PCI/US93/0950~
q~AB~E lI ( c()~ D. )
37'C
MUTANT YPo YPD+I YPD~I/YPD MUTANT YNB YNf3+l YN8+1/YN~3
M2 1D21 0 4 0 ~ 0 M1 4E1 1 0 2 0 2 0
M1.4A41.0 3 0 3 0 M2.2C3 2 0 4 0 2 0
M2.2C31 0 3 0 3 0 M2.2D3 1 0 2 0 2 0
M2.2C51 0 3 0 3 0 M2.4A2 1.0 2 0 2 0
M1.2D42 Q 4 0 2 0 M2.4C3 1 0 2 0 2 0
Ml.4B22 0 4 0 2 0 M1.282 2 0 3 0 1 5
M1.4El1.0 2 0 2.0 Ml.4A1 2 0 3.0 1 5
M2.2B42 0 4 0 2 0 M2.482 3.0 4.0 1 3
M2.2D31 0 2 0 2 0 Ml.4D1 4 0 5 0 1 3
M2.4A12.0 4.0 2 0 M2.1A1 4 0 5.0 1.3
M2.4A21 0 2 0 2 0 M1.2B1 1 0 1.0 1.0
Ml.4C12 0 3 0 1 5 Ml.283 2.0 2.0 1 0
Ml.4C32 0 3 0 1 5 Ml.2D1 4 0 4 0 1 0
M2.4C32.0 3 0 1 5 Ml.2D3 5.0 5.0 1.0
Ml.4D13 0 4 0 1 3M1.4A3 5 0 5 0 1.0
M2.4D43.0 4.0 1 3Ml.4A4 1.0 1.0 1.0
M2.1A14 0 5 0 1 3M1.482 4 0 4.0 1 0
M1.2B11.0 1 0 1 0M1.4B2 2.0 2 0 1.0
Ml.2B22 0 2 0 1 0M1.4C1 2.0 2 0 1.0
Ml.2B32 0 2 0 1 0M1.4C2 1 0 1 0 1.0
M1.2D14.0 4 0 1 0M1.4D2 2 0 2.0 1 0
Ml.2D35 0 5 0 1 0M2.1B1 1 0 1 0 1.0
M1.4A13.0 3 0 1 0M2.1B2 1.0 1.0 1.0
M1.4A35.0 5 0 1 0M2.1C1 5.0 5 0 1.0
M1.4C21 0 1 0 1 0M2.1D2 1 0 1.0 1.0
M1.4C41.0 1 0 1 0M2.2A1 3.0 3.0 1.0
M1.4C52 0 2 0 1 0M2.2A2 1.0 1.0 1.0
M1.4D22.0 2 0 1.0 M2.2B4 2.0 2.0 1.0
M1.4D31 0 1 0 1.0 M2.2C2 1.0 1.0 1.0
M2.1 Bl 1.0 1 0 1.0M2.2C4 1.0 1.0 1.0
M2.1B21 0 1 0 1 0 M2.2E3 3.0 3.0 1.0
M2.1C15 0 5 0 1.0 M2.4Al 1.0 1.0 1.0
M2.1D14 0 4.0 1.0 M2.4A3 1.0 1.0 1 0
M2.2A13.0 3 0 1.0 M2.4B1 1.0 1.0 1 0
M2.2A21.0 1 0 1 0 M2.4C1 4.0 4.0 1.0
M2.2B12.0 2.0 1 0 M2.4C2 4.0 4.0 1.0
M2.2B2M0 1 0 1.0 M2.4C4 3.0 3.0 1.0
M2.2831.0 1 0 1 0 M2.4D3 5.0 5.0 1.0
M2.2C24.0 4 0 1.0 M2.4D4 3 0 3 0 1.0
M2.2D14.0 4.0 1.0 M2.4E1 1.0 1.0 1.0
M2.2E11.0 1 0 1.0 M2.4E2 1.0 1.0 1.0
M2.2E34 0 4.0 1.0 M2.4E3 3.0 3.0 1.0
M2.4A31.0 1.0 1.0 M1.4B1 5.0 4.0 0.8
M2.4C14.0 4.0 1.0 M2.1D1 4.0 3.0 0.8
M2.4C24.0 4.0 1.0 M2.4D1 4.0 3.0 0.8
M2.4C43.0 3 0 1.0 M1.4C3 3.0 2.0 0 7
M2.4D35.0 5.0 1.0 M1.2D4 4.0 2.0 0.5
M2.4E12.0 2.0 1 0 M1.4C5 2.0 1.0 0.5
M2.4E21.0 1.0 1.0 M2.2B1 4.0 2.0 0.5
Mæ4E3 2.0 2.0 1.0 M2.2B3 2.0 1.0 0.5
M1.4B14.0 3.0 0.8 M2.4B2 2.0 1.0 0.5
M1.4B24.0 3.0 0.8 Ml.4D3 3.0 1.0 0.3
M2.4B24.0 3.0 0.8 M2.2C5 3.0 1.0 0.3
M2.2C44.0 2.0 0.5 M2.1D3 4.0 1.0 0.3
M2.2D24.0 2.0 0.5 Ml.4C4 0.0 1.0 N~;
M2.4B22.0 1.0 0.5 M2.2B2 0.0 2.0 1
M2.4D13.0 1.0 0.3 M2.2E2 0.0 2.0 1\~
M2.1D30.0 4.0 ~ M2.2D1 4.0 0.0 ~;7
M2.2C10.0 1.0 N~ M2.2D2 4.0 0.0 ~1
Mæ2E2 0.0 1.0 t~ Ml.2D2 0.0 0.0 NGB
M2.4B11.0 0.0 ~I M2.2C1 0.0 0.0 t~B
Ml.2D20.0 0.0 1~8 M2.2E1 0.0 0.0 ~B
lS288C 4 3 4.0 0.9 lS288C 4.0 3.0 0.8
2S288C 3.5 4 0 1.1 2S288C 4.0 3.0 0.8
W094/07994 ~ 2 ~ PCT/USg3/og~O~
22~-
The wild-type parental strain S288c is
characterized by vigorous growth with a doubling time of
2 to 2.5 hours in YPD and YNB media, respectively. The
colonies are pearly white to buff colored, relatively
large (1-5 mm) in diameter, luxurious and thick. Each
of the mutants possess a distinct colony or individual
cellular morphology (See Table III). Whereas single
cells of S288c are ellipsoid or round and approximately
5-10 ~m in diameter, the mutants do not conform to the
wild-type characteristics. The growth rates are
generally slower than for S288c, although for some
mutants, Io-6 M insulin restores growth characteristics
of the wild-type. The morphology of selected insulin-
dependent mutants which have a YNB+I/YNB growth rate
ratio of 2 or more is described in the following Table.
-- ~14~6~g,32 Pcr/US93/09504
23
TABLE III
MORPHOLOGY OF S~LECTED INSULIN-DEPENDENT M~JTANTS
MUTANT CELL MORPHOLOGY COLONY MORPhriOLOGY
M1.2B1 medium, budded small, fused
M1 2D1clumped, heterogeneous small, fused
M1.2D2clumped, irregularly shaped small, fused
M1 2D4clumped, budded, elongated small, fused, individual
M1.4A1 medium, clumped small, fused, individual
M1.4A4 large small, fused, dense
M1.4C2 medium, budded small, fused, individual
M1.4C~medium, heterogeneous small, fused, individual, sparse
M1 4D1medium, clumped, heterogeneous small, ~sed
M1.4D3large, heterogeneous small, fused, ndividual
M1.4E1medium, budded, round small, fused, ndr~idual
M2.181 large, clumped small, fused, individual
M2.1B2clumped, heterogeneous, elongated small, fused
M2.1 D2large, clumped small, fused, individual
M2.1D3medium, elongated small, fused, individual
M2.2A2clumped, budded, irregularly shapedsmall, fused, individual
M2.2B1 medium, round small, fused, individual
M2.2B3medium, clumped, budded small, fused
M2.2B4clumped, heterogeneous, budded individual, small, sparse
M2.2C2 medium, clumped smail, fused, individual
M2.2C3 medium, budded small, fused, individual, sparse
M2.2C4 medium, clumped ~mail, fused, individual
M2.2C~clumped, irregulariy shaped sma, fused, individual, sparse
M2.2D1 clumped, heterogeneous, budded, irregulariy shaped smal, fused, individual, dense
M2.2D2small, clumped, budded small, fused, dense, individuai
M2.2D3medium, round small, fused, individual
F~ small, clumped small, fused, dense
M2.4A2clumped, heterogeneous, buddedsmall, fused, individual
M2.4B2clumped, heterogeneous small; fused
M2.4C1medium, budded, round, healthy small, fused, dense
M2.4C2clumped, heterogeneous small, fused
M2.4C3 medium small, fused,dense
M2.4D1medium, clumped, budded small, fused
M2.4E1medium, heterogeneous small, fused, individual
M2.4E2large, clumped small, fused
~lutants defined as "Class I", i.e. having a growth rate score of 2 or higher
for YNB+I/YNB
~ i:
W094/07994 2146 ~32 PCT/US93/0950 ~
24
Mutant strains are maintained by freezing using the
following protocol. 200 ~l of a previously frozen stock
or stock YPD+I plate stored at 4C for less than one
month, is inoculated into 200 ml of YPD media with
insulin. The inoculated media is put on a shaker at 30C
until cells reach the mid-logarithmic phase of growth
(optical density at 600 nm between 0.5-1.0). The
culture is transferred to four 50 ml sterile tubes and
centrifuged at 3,000 x g for 15 min. The supernatant is
removed by pipetting until between 10-15 ml i8 left in
each tube. The pellet is resuspended in the same YPD+I
media (the 10-15 ml). The suspensions are transferred
to one 50 ml sterile tube and centrifuged as above for
15 min (to obtain 1 pellet). The supernatant is removed
and resuspended in 5.5 ml of YPD media supplemented with
insulin and 20~ glycerol. 500 ~l of suspension is
dispensed into each vial. The cultures are checked for
contamination by light microscopy under oil immersion.
The cultures are then frozen at
-80C.
Yeast strains 2.lD2, 2.2C5 and 2.4B1 described
above were deposited at the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland
20852, USA, on October 5, 1992 under the conditions of
the Budapest Treaty. The strains were assigned the
following designations ATCC 74189 (2.1D2), Arcc 74190
(2.2C5) and ATCC 74191 (2.4B1), respectively. Strain
2.lD2 was selected because it was insulin sensitive.
Strain 2.2C5 was selected because it demonstrated a
strong insulin- and temperature-dependent phenotype on
YNB. Strain 2.4B1 was selected because it was
unaffected or improved with respect to viability by
extended term culture in the presence of
supraphysiological concentrations of insulin.
V ~ ~.i ~ t 'F~ 3 ~ PCT/US93/0950~
EXAMPLE 2: Use of yeasts as a secretaqoque sensinq
system
In each of the systems described in Examples 2 and
3, some of the mutant yeasts as isolated and described
in Example 1 may be employed. In particular, those
mutants which show an enhanced or stabilized response
would be desirable to be used. For example, a mutant
which demonstrated a rapid rise in 3',5'-cyclic
adenosine monophosphate (cAMP) level which did not
subsequently decay, and thus maintained a high, stable
level of intracellular cAMP, would be particularly
useful. Similarly, a mutant which showed an increased
amount of tyrosine phosphorylation of total cellular
proteins or of the IRP would be most useful as a
nutrient sensor cell.
A. General technique; sensing glucose by monitoring of
cAMP response.
A newly discovered nutrient sensing system coupled
to insulin secretagogues has been documented in S.
20 cerevisiae strain S288c. The effects of various
insulin-secretagogues, including metabolizable and
nonmetabolizable analogs of carbon and nitrogen sources
on intracellular levels of cAMP, the primary second
messenger associated with insulin secretion in mammals,
was analyzed as follows.
1. Media
For minimal YNB without glucose medium (YNBM), 1.7
grams of Difco Yeast Nitrogen Base and 5.0 grams of
ammonium sulfate were dissolved in 1 liter distilled,
deionized water. When all solids have dissolved the
solution is made up to 1 liter with distilled water and
sterilized through a 0.2 micropore Fisher filter and
stored in an autoclaved bottle in a refrigerator. For
YNBM plus glucose, 20 grams of dextrose (Sigma) was
added per liter.
W094/07994 2 1`~$~4~ PCT/US93/0950 ~
.~ . .
26
2. Assay of cAMP level following glucose exposure
Cells of strain S288c (stored for less than one
month at 4C) were transferred from YPD plates to a 15 ml
sterile, conical tube containing minimal YNBM plus 2~
(w/v) glucose to make a starter culture with a density
of 3 x 107 cells/ml. Cells were propagated at 30C with
shaking at 250 rpm on a New Brunswick Scientific G76
water bath to a density of 3 x 106/ml in 100 ml of YNBM
in 250 ml Erlenmeyer flasks with screw caps. At this
point, aliquots of the starter culture were transferred
into two 25Q ml flasks; one contained YNBM with 2~ (w/v)
glucose while the other contained YNBM without glucose.
Yeast incubated in the presence of glucose were diluted
frequently in YNBM before assay to maintain a maximum
cell density of 3 x 106/ml. Cells prepared in the
absence of glucose were diluted in YNBM minus glucose to
allow six to seven generations of growth on
intracellular glycogen before arrest at 3 x 106/ml.
Following arrest, in YNBM medium lacking glucose, cells
were maintained at 30C with shaking at 250 rpm for a
period of sixteen hours to ensure a starved depleted
state.
At this time, D-glucose solution was added to each
flask containing 100 ml of YN3M to give a final
concentration of 110 mM glucose. In separate flasks,
a n a l o g s o f g l u c o s e , i n c l u d i n g
3-O-methyl-D-glucopyranoside (110 mM) and
2-deoxy-D-glucose (110 mM) were added to glucose starved
and continuously fed cultures. Each of the cultures
were incubated for 0.25, 0.5, 1, 5, 10, 30, 60, 120, 180
and 240 minutes after which 5 ml aliquots were withdrawn
and filtered through a microanalysis filter unit fitted
with a 0.6 ~m polycarbonate copolymeric (PCTE) membrane.
The filter was transferred to a 5 cm sterile plastic
petri dish (Falcon) and submerged in 1 ml of 1 M ~ormic
acid saturated with n-butanol for 15 minutes to extract
cAMP. The extract and two 250 ~l washes of 1 M formic
g~/07994 ~tlt~6~3,~ Pcr/US93/09504
27
acid saturated with n-butanol were pooled into sterile
microfuge tubes. The tubes were centrifuged for 5
minutes at 13,000 x g at room temperature. The
supernatants were then transferred to another Eppendorf
and immediately frozen at -20C.
The cAMP content of the supernatants was
quantitated by a scintillation proximity technique using
a kit, (Amersham, Catalog No. RPA.538, Arlington
Heights, IL) as described by the manufacturer. Prior to
assay, supernatants were vacuum evaporated to dryness,
resuspended in assay buffer and acetylated with acetic
anhydride and trimethylamine to stabilize the cyclic
nucleotide.
To assess the effect of secretagogues on budding
kinetics, samples (1 ml) were also withdrawn from flasks
at each time point and fixed with 0.05~ glutaraldehyde
in YNB. The contents fixed for 3 minutes and were then
centrifuged for 2 mins at 13,000 x g. The pellets were
resuspended in 4~ formaldehyde in PBS, and stored frozen
at -20C. Budding was quantitated by counting, under a
light microscope, the number of buds per one hundred
cells sampled.
Sensing of amino acids and amino acid analogs and other
nutrients:
To measure formation of cAMP transients in response
to amino acids, a similar experiment was performed as
above. The following amino acids and analogs were
tested in cultures grown to a density of 3 x 10-6 cell/ml
in YNBM supplemented with 2~ glucose: ~-lysine 0.164
mM, L-isoleucine 0.229 mM, L-leucine 0.229 mM,
L-cysteine 1.240 mM, I,-glutamic Acid 0.591 mM,
L-glutamine 0.684 mM, L-ornithine 0.119 mM, L-arginine
0.095 mM, ~-histidine 0.129 mM, L-methionine 0.134 mM,
L-tryptophan 0.098 mM, ,~-aminoisobutyric acid, 0.t~10 mM.
All steps of the cAMP extraction were carried out as
described above.
W094/07994 2 1 ~ PCT/US93/0950
28
Sulfonylureas were also analyzed for a cAMP
transient except in step four sulfonylureas supplemented
glucose in the following concentrations: into first set
of cultures (YNB with glucose), tolbutamide 0.025 mM
(with 110 mM glucose), chlorpropamide 0.025 mM (with 110
mM glucose), and glyburide 2.5 ~M (with 110 mM) were
added; into the second set o~ cultures (starved) the
same concentrations of sulfonylureas were added but with
5 mM of glucose instead of 110 mM. Again all steps of
the cAMP extraction were
carried out as described above. Also, an experiment was
performed wherein insulin at 1 x 10-7 M was added to a
culture in combination with 110 mM glucose.
Other nutrients similarly tested were oleic acid
0.070 mM, guanine 0.100 mM, adenine 0.108 mM, uracil
0.180 mM, galactose 100 mM, potassium phosphate 7 mM,
ammonium sulfate 38 mM.
C Sensing of sulfonylureas:
Yeast grown in the presence of glucose are
insensitive to further stimulation by additional
glucose. A period of starvation for glucose, followed
by refeeding results in a transient 5-to 10-fold
increase in intracellular cAMP, over the basal cAMP
level. Re~eeding with 2-deoxyglucose generates a 2- to
3-fold rise in cAMP in the starved cells. 3-O-
methylglucopyranoside and galactose do not alter cAMP
levels.
Amino acids recognized as secretagogues in
m~mm~l ian cells, including L-arginine, L-leucine and L-
lysine, induce cAMP transients of 2-, 10- and 2-fold,
respectively. Similar to transients induced by glucose,
the timing of the cAMP pulse is centered around 30
seconds. L-arginine also stimulates growth, but this
effect is observed clearly only after 6 ho~rs of
culture. k-glutamine and L-glutamate do not promote
cAMP pulses, but these amino acids effectively stimulate
~ . ' ~ PCI /US93/0950~
21,~
29
growth. Conversely, cysteine reduced cAMP levels as
compared to basal levels and interfered with traversal
through the cell cycle.
Of the other nutrients tested, adenine, oleic acid
and potassium phosphate induce a cAMP transient. The
magnitude of stimulation by adenine was 4-fold at 15
seconds. Oleic acid caused a protracted rise of 7- to
10-fold by 1 hr. of incubation, which was accompanied by
a stimulation of cell proliferation. Potassium
phosphate, which is required for insulin secretion in
mammals, supported a 15- to 30-fold elevation in cAMP
level within 30 seconds in cells starved for phosphate
in the presence of glucose.
Sulfonylurea treatment of yeast fed continuously
with glucose results in cAMP pulses comparable in
magnitude and duration as those observed in cells
starved and replenished with glucose. Glyburide
generates the highest response, stimulating a 5-fold
increase over basal levels. Tolbutamide also elevates
cAMP levels, though only 2- to 3-fold. Chlorpropamide
is able to stimulate cAMP transients in both starved
cells and cells continuously fed glucose. Stimulation
of cAMP elevation by chlorpropamide is preferentially
observed in continuously fed cells and apparently is
elicited by a mechanism different from that through
which glucose operates.
Insulin treatment of cells starved for glucose and
refed glucose suppressed the transient rise in cAMP
noted in the absence of the hormone. However, cells
exposed to insulin achieved a growth rate approximately
50~ more rapid than the starved and refed control
cultures. This observation correlates with the time of
appearance of their endogenous insulin-like peptide, ILP
in the growth medium, which occurs at approximately 1-2
hours. Furthermore, the highest specific activ;ity of
the ILP is greatest just at commencement of exponential
growth of the cells.
W094/07994 ~ ,; ' PCT/US93/o950 4
214~4~2
Glycogen biosynthesis in response to glucose
feeding was inhibited for approximately 10-30 minutes,
the time when cAMP transients were at their peak levels.
Addition of ILP and insulin at physiological
concentrations exacerbated the initial inhibition of
glycogen synthesis. However, once relieved, cells
synthesized glycogen at a rate 10- to 30-fold higher
than basal. Results of the cAMP response and
proliferation response to nutrients are presented in
Figures lOA-G and llA-D, respectively, and summarized in
Table IV-Summary of nutrients.
~94~07994 2 1 4 6~ 3-2 PCI/US9310950~
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W094/07994 ~4~3~ PCT/US93/0950
34
EXAMPLE 3: Monitorinq of phosphotvrosine content of
total Yeast ~roteins
Protein tyrosine phosphorylation is a central motif
in eukaryotic cell regulation, implicated in cell cycle
control, transformation, differentiation, neurotroph
signaling, and immune cell activation. Growth factor
receptor activation by autophosphorylation on tyrosine
residues is one well-documented physiological role. The
strongest link between protein tyrosine phosphorylation
and function is the activation of insulin, insulin-like
growth factor I (IGF-I), epidermal growth factor (EGF)
and platelet-derived growth factor (PDGF) hormone
receptors that possess intrinsic tyrosine protein kinase
activities in normal mammalian cells. An array of
possible tyrosine phosphorylated substrates has emerged
b u t f e w h a v e d e f i n e d r o l e s i n
phosphorylation/dephosphorylation cascades of the
receptor tyrosine kinases that control cellular
processes [(Hanks et al, Science, 241:42 (1988)]. The
relative abundance of protein tyrosine phosphorylation
in cellular proliferation and transformation suggests
that phosphotyrosine modification is requisite for
normal growth.
The occurrence of tyrosine phosphorylation in
growth factor receptor activation in phylogenetically
divergent organisms supports a conserved function in
mitogenesis and development. From an evolutionary
perspective, more ancient and simpler organisms
apparently possess less abundant, or more transient,
protein tyrosine modification. Protein phosphorylation
is documented in bacteria, but tyrosine-specific
phosphorylation remains in question. Nucleotidylation
has been implicated as a source of phosphotyrosine
detected in bacteria although authentic tyrosine
modification cannot be discounted before in vivo
processes receive more careful analysis.
Phosphorylation of serine and threonine residues has
94/07994 2~ ~ PCT/US93/0950
gained wide recognition in lower eukaryotes, yet
evidence for tyrosine phosphorylation is recent and its
physiologic role in these organisms is obscure. Novel
tyrosine kinases have been identified in the
multicellular slime mold Dictyostelium discoideum, and
the filamentous fungus, Neurospora crassa, has been
shown to possess tyrosine kinase activity and
phosphotyrosine- containing proteins.
The most conclusive assignment of a specific role
to a single tyrosine phosphorylated protein comes from
the fission yeast, Schizosaccharomyces pombe (S. pombe).
Tyrosine phosphorylation of the cdc2 protein
serine/threonine kinase in S. pombe, analogous to CDC28
of the budding yeast, S. cerevisiae, and pp34 of
vertebrates, was shown to ne~atively regulate
progression through the cell cycle.
A. Incorporation of P04 into TCA precipitable
macromolecules from early exponential phase S.
cerevisiae.
Strain S288c was grown in YNB medium (Difco) at 30C
with shaking at 250 rpm to a cell density of 3 x 106/ml.
For depletion of vacuolar phosphate, a starter culture
was inoculated at 4 x 103/ml into YNB, or comparable
medium formulated from separate components (Difco
Manual), and buffered with 50 mM
2-(N-morpholino)-ethanesulfonic acid (MES), pH 6.0, to
exclude KH2PO4. Cells cultured in phosphate-deficient
medium were maintained at 3 x 106/ml and were incubated
for an additional 16 hours following arrest. Cells
harvested by centrifugation were resuspended to 5 x
106/ml in fresh YNB or MES-buffered medium with indicated
concentrations of KH2PO4 and 0.125 mCi/MI 32po4. At
indicated times, aliquots of cells were removed and
phosphate uptake was terminated with ice-cold TCA (10
w/v). Extracts were washed 3x in ice-cold phosphate
buffered saline, neutralized with 0. lN NAOH and
W094/07994 ~ ~ 6 ~ . 36 PCT/US93/0950 ~
scintillation counted. Figure 12 shows TCA precipitable
counts measured from actively growing or phosphate-
arrested cultures supplemented with (A) 100~ KH2P04, (B)
2P04, (C) 10~ KH2P04 and (D) KH2PO4-deficient
MES-buffered medium.
B. Flow cytometric analysis of exponential phase yeast.
Actively growing and phosphate starved exponential
phase cells were grown to 3 x 106/ml as described in A.
At indicated times, 100 ~l aliquots of cells were
harvested and resuspended in citrate buffer-based
fixative. Cells treated with 100 ~g/ml RNaseA and 5
~g/ml propidium iodide were analyzed for red
fluorescence (DNA content) at 625 nm on a Coulter Epics
fluorescence activated cell sorter. Figure 13 depicts
cell cycle progression of actively growing and phosphate
arrested cells resuspended in MES-buffered medium with
100~ KH2P04 (FIGS. 13A-C at 0 h, 1 h and 2 h,
respectively) and 10~ KH2P04 (FIGS. 13D-F at 0 h, 1 h and
2 h, respectively).
C. Phosphoamino acid analysis of actively growing and
phosphate-arrested early exponential phase yeast.
Cells propagated as described in A were
resuspended to a density of 2 x 108/ml in MES-buffered
medium with 10~ KH2P04 containing 0.4 mCi/MI 32po4 in a
final volume of 2.5 ml. Cells were incubated for 90
minutes at 30C, collected by centrifugation for 10
minutes at 1500 x g, the supernatant was aspirated and
metabolism was terminated by addition of hot 2X sample
buffer, Laemmli, Nature, 227:680 (1970). Samples were
boiled immediately for 5 min, freeze-thawed, bead-beaten
in four 1 min. bursts with 0.5 mm glass beads (Biospec),
boiled again for 5 min. and clarified by
microcentrifugation for 5 min. Proteins were
concentrated by electrophoresis on a 7.5~ polyacrylamide
gel at 150V until the dye front migrated 1.5 cm into the
separating gel. Pieces (1 cm2)of the gel cut behind the
~? ~ Ç~L~ U~E 26~
2I~6~2 crus o
37
dye front were analyzed according to the method of
[Hunter et al, Proc. Natl. Acad. Sci. USA, 77:1311
(1980)]. Figure 14 shows results of the phosphamino
acid analysis.
D. Profiles of protein tyrosine phosphorylation in S.
cerevisiae .
Cells propagated in YNB as described in A were
collected by centrifugation throughout the life cycle
when density (cells/ml) reached (A)
3 x 106, (B) 7.5 x 106, (C) 1 . 5 x 107, (D) 3 x 107, (E)
6 x 107, (F) 1.2 x 108, (G) 2.4 x 108, (H) 3 x 108, and
(I) 3 x 106 from late stationary phase culture (H),
reinoculated into fresh ~3 medium at 1. 5 X 106 and
harvested after a 7 h lag. Yeast were resuspended to 5
x 107/ml, diluted in hot 2X sample buffer (Laemmli,
Nature, 227:680 (1970)) and processed for
electrophoresis as described in C. Phosphate-induced
with 10~ of standard medium phosphate (J) and starved
cells (K) were treated as above. Epidermal growth
factor activated A431 cell plasma membranes served as a
control (L). Protein extracts electrophoresed on a 7. 5
~ polyacrylamide gel at 30V for 28 h were transferred at
1.0 amp for 90 min. at 4C onto nitrocellulose (0.22 ~m;
Schleicher and Schuell, Keene, NH), probed with
antiphosphotyrosine monoclonal antibody 4G10 (Upstate
Biotechnology, Inc., Lake Placid, NY), and visualized by
ECL (Amersham) (See Figure 15).
Conditions were identified to allow measurement of
phosphate incorporation into macromolecules and to
supply sufficient phosphate to execute "START" of the
cell cycle (Johnston
et al, Exp. Cell Res., 105:79 (1977)). Exponential phase
yeast were transferred at low density to
phosphate-deficient medium. The yeast proli~erated
twelve to thirteen generations in phosphate-deficient
conditions and displayed a slightly greater specific
W094/07994 21~6 43~2 38 PCT/US93/095 ~
growth rate of 0.37, up to the point of arrest, than
cells grown on standard phosphate-containing medium
which demonstrated a specific growth rate of 0.35.
Vacuolar phosphate stores, constituting 35-40~ of yeast
cell dry weight (120 mm/kg wet weight; Griffin, Fungal
Physiology, John Wiley & Sons, NY (1981)), were
presumably mobilized during this period and seemed more
readily utilized than phosphate from external sources.
Yeast have been shown to liberate vacuolar polyphosphate
reserves when transferred into medium containing
disproportionately high nitrogen to phosphate levels.
Thus, arrest must occur when both external and internal
supplies are exhausted and intracellular phosphate pools
are equilibrated. Phosphate-arrested cells, positioned
at or before START of the cell cycle, were found to be
unbudded and thermotolerant.
To observe phosphate incorporation into tyrosine
residues, competition between cold and labelled
phosphate must be minimized to favor radiolabelled
product. However, sufficient phosphate was required to
promote growth in order to correlate production of
phosphotyrosine with growth. Therefore, conditions were
investigated to balance efficient labelling with
provision of an adequate phosphate source. Preliminary
studies confirmed that yeast cells arrested in
exponential phase by phosphate limitation transported
less phosphate than yeast cells grown throush
exponential phase without phosphate restriction (1.6
fold less). Expression of low affinity, high Km
phosphate transporters in phosphate unlimited cells, as
opposed to expression of high affinity, low Km phosphate
transporters in the phosphate starved cells could
account for this result. Consistent with this finding,
more total phosphate was incorporated into TCA
precipitable material in phosphate unl~mited,
exponential phase cells at the higher concentrations of
exogenous phosphate (FIGS. 12A and 12B) than in cells
~ 2 1 ~ 6 ~ 3 2~
grown at lower concentrations of exogenous phosphate
(FIGS. 12C and 12D). Furthermore, little difference was
observed in phosphate incorporation between phosphate
unrestricted and phosphate starved cells at high
concentrations of phosphate. However, at lower
exogenous phosphate concentrations, a significantly
greater percentage of the total radiolabelled phosphate
added was incorporated into TCA precipitable material.
Therefore, labelling conditions were favored that
exhibited differences between phosphate limited cells
and phosphate unrestricted cells in the transfer of
phosphate into macromolecules.
Flow cytometric analysis of phosphate- restricted
cells revealed that DNA synthesis was reinitiated
between 1-2 hours after fully replenishing medium
phosphate content (FIG. 13A). Addition of lx standard
medium phosphate was adequate to re-initiate
proliferation in 31~ of phosphate-starved cells (FIG.
13A) while use of O.lx standard medium phosphate induced
proliferation in 9~ of phosphate-limited cells (FIG.
13s). Tracer added alone to these cells was
insufficient for growth induction. Although labelling
macromolecules with radiolabelled phosphate is most
efficient when exogenous and internal phosphate stores
are negligible, these conditions are insufficient to
support growth. Therefore, phosphate-arrested cells,
positioned at the G1 phase, were stimulated with 0.1
standard medium phosphate. This condition achieved a
balance between efficient labelling and re-entry into
the cell cycle. Furthermore, arrest of logarithmic
phase cells by phosphate limitation establishes a fixed
metabolic position from which to initiate growth.
Under typical labelling conditions, phosphoamino
acid analysis of mid-exponential phase yeast cells has
demonstrated exceedingly weak signals ~ from
phosphotyrosine compared to those from phosphoserine and
phosphothreonine (Castellanos et al, J. Biol. Chem.,
WO 94/07994 . ~ PCI /US93/o9504
21~643~
260:8240 (1985); Dailey et al, Mol. Cell Biol., 10:6244
(1990); Schieven et al, Science, 231:390 (1986)).
Exponential phase yeast cells, depleted of vacuolar
phosphate reserves, when refed O.lx standard medium
phosphate, displayed phosphotyrosine more prominently
than the other phosphoamino acids. Under the conditions
reported, threonine phosphorylation appears to be less
actively involved in proliferative events, as evidenced
by its virtual absence in phosphoamino acid analysis
(FIG. 13) of growth-induced, phosphate restricted cells.
This finding has been corroborated by others [(Draetta
et al, Cell, 50:319 (1987); Gould et al, Nature, 342:39
(1989)], and may reflect the proliferation-related role
proposed for bifunctional tyrosine/serine kinases
detected in yeast and other organisms [(Levin et al,
Proc. Natl. Acad. Sci. USA, 84:6035 (1987); Tan et al,
Mol. Cell Biol., 10:3578 (1990); Ben-David et al, EMBO
J., 10:317 (1991)]. Exponential phase yeast
unrestricted for phosphate, and transferred to O.lx
standard medium phosphate, yielded a strong
phosphotyrosine signal (FIG. 3). Some investigators
report that 2'- and 3'-uridine monophosphate (UMP) or
cytidine monophosphate (CMP) comigrate with
phosphotyrosine (Cooper
et al, Methods Enzymol., 99:387 (1983)). Others report
that phosphotyrosine does not co-migrate under solvent
conditions not significantly different (Munoz et al,
Anal. Biochem., 190:233 (1990)) from the one reported
here. To assess the production of phosphotyrosine
relative to the possible cont~min~nts, each phosphoamino
acid spot from the sample lane was eluted from the
thin-layer chromatography plate and measured
spectrophotometrically at absorption maxima, 260 nm and
274 nm for 3'-UMP and 0-phospho-L- tyrosine,
respectively [(Cantor et al, Biophysical Chemistry (W.H.
Freeman, San Francisco, CA), Part II, p. 443 (1980)].
Exponential phase cells (5 x 108) unrestricted for
~ 214~? ~ 9504
41
phosphate yielded 2.1 x 10-7 moles of 3'-UMP and 1.2 x 107
moles of phosphotyrosine. In exponential cells (5 x
108), restricted for phosphate, 1 x 10-7 moles of 3'-UMP
and 1.1 x 1o-7 moles of phosphotyrosine were detected.
Cooper et al have noted the importance of
- equilibrating fully intracellular pools of exchangeable
phosphate in ATP, metabolic intermediates, and
macromolecules (1983). At best, this is difficult,
particularly in yeast, which mobilizes vacuoler
phosphate stores upon phosphate limitation. Labelling
the "steady- state" (Hunter et al, Proc. Natl. Acad.
Sci. USA, 77:1311 (1980)) of a growing population is not
usually balanced, and by normalizing the point of growth
initiation, mechanisms controlling differential
synthesis may be assessed. Our data revealed that
re-entry into the cell cycle occurred after 90 minutes
of phosphate replenishment. During this labelling
period, phosphate is preferentially directed to tyrosine
compared to serine or threonine in S. cerevisiae.
Therefore, under conditions of growth induction, the
ratio of phosphotyrosine to cont~mi n~nts in cells grown
to exponential phase and limited for phosphate prior to
labelling, was greater than in the unrestricted cells.
To further verify the authenticity of the
phosphotyrosine modification, yeast proteins were probed
with the monoclonal anti-phosphotyrosine antibody, 4G10
(Roberts, 1990). The specificity of the antibody was
confirmed by direct competition of immobilized yeast
proteins with 0-phospho-L-tyrosine. Displacement of
antibody by competing phosphoamino acids was ranked,
0-phospho-L-tyrosine > ~ 0-phospho-L-serine
0-phospho-L-threonine ~ ~ histidinol phosphate.
Furthermore, in the absence of primary antibody,
secondary antibody did not generate signal.
Protein targets with tyrosine-specific
phosphorylations, from successive phases of vegetative
growth, were analyzed by Western blotting with
W094/07994 ~ ' PCT/US93/0950 ~
`214`~32
42
monoclonal antibody 4G10 (FIG. 15). Yeast demonstrated
numerous tyrosine phosphorylated proteins in every phase
of growth. However, signals from bands at 95, 81 and 47
kD lost intensity as cells approached nutrientdependent
saturation density. The proteins regained exponential
phase intensity after introduction to fresh medium.
Although generally less intense, similar proteins from
starved cells were tyrosine phosphorylated upon
replenishment with O.lx standard medium phosphate and
resumption of growth. A protein of molecular weight 180
kDa was increasingly phosphorylated as cells entered
stationary phase, and another high molecular weight
protein at >220-240 kDa was phosphorylated to various
-extents in different phases of growth. Under varied
conditions of growth, the profile of tyrosine
phosphorylations in different phases were not
consistent. Cells grown to late stationary phase, then
transferred to fresh medium, exhibited intense
phosphotyrosine signals upon growth resumption. This
finding corroborates the result from phosphoamino acid
analysis in which increased tyrosine phosphorylation is
correlated with re-entry into the cell cycle.
Another possible mode o~ phosphoprotein turnover
implicated in cell growth was tested. Both
intracellular alkaline and acid phosphatase activities
were measured in early exponential and stationary phase
cells. The levels of alkaline and acid phosphatase
expressed in stationary phase cells were lower than in
early exponential phase cells. Amongst samples derived
from logarithmically growing cells, those incubated in
the presence of glucose demonstrated approximately
2-fold lower alkaline phosphatase activity in the
extracts derived from cells incubated in the absence of
glucose. Furthermore, addition of the general
phosphatase inhibitor, sodium vanadate, had little
effect on phosphatase activity. The phosphatase assay
measures relative activity for production of
~ ,3~2 PCT/US93/0950~
p-nitrophenyl from p-nitrophenyl phosphate. Phosphatase
activity may be the consequence of vigorous growth
regulation in early logarithmic phase cells.
Proteolysis was suspected to contribute to the
observed differences in the various phases of growth.
~ Thus yeast cells in early logarithmic and stationary
phases of growth were spiked with purified bovine serum
albumin, bead beaten in denaturing sample ~uffer and the
proteins analyzed by electrophoresis [(~aemmli, Nature,
227:680 (1970)]. This experiment revealed extensive
proteolytic digestion in stationary phase cells absent
in logarithmic phase cells. Siqnificant, though
slightly reduced proteolysis, was observed in
vanadate-treated, stationary phase extracts. Boiling in
sample buffer immediately after collection of yeast
cells dramatically increased recovery of phosphotyrosine
proteins and prevented proteolytic digestion and
phosphatase attack in later phases of growth.
Early exponential phase yeast were chosen for
studying phosphate limitation and growth resumption
because later phases o~ growth are characterized by
mixed nutrient diauxie and progressively altered
phosphotyrosine profiles (FIG. 15). Post-translational
protein modifications are related to culture conditions
such as nutrient availability, pH, age, cell density,
and in individual cells, at specific points in the
division cycle. In yeast, the modifications are
transient and their appearance is variable due to
extensive protease and phosphatase activities.
Culture conditions under which phosphotyrosine is
most efficiently isolated are not the conditions under
which the modification is maximally expressed in
response to environmental stimuli.
Undefined media contain a large proportion of
comDlex nitroqen substrates (e.q. PePtone) and certain
W094t~7~ PCT/US93/0950 0
21~6~3~
44
phosphate content is characteristically high when
compared to that of defined medium. Certain nitrogen
compositions promote protease activity, and a high
nitrogen to phosphate ratio forces phosphate
mobilization from the vacuole and enhanced scavenging
phosphatase activity. These findings may explain the
extremely low level or the failure to identify
phosphotyrosine by phosphoamino acid analysis or
antiphosphotyrosine antibodies.
The experiments reported here address a compromise
between effectively directing label to phosphotyrosine
and maintaining conditions supportive of growth. The
balance between depletion of vacuolar polyphosphate
reserves and restoration of sufficient phosphate for
growth induction generated a distinct subset of tyrosine
phosphorylated proteins. Thus, the level of
phosphotyrosine detected by phosphoamino acid analysis
and monoclonal anti-phosphotyrosine antibodies is a
physiological consequence of phosphate restriction
required to enhance radiolabel uptake. Furthermore,
phosphate restriction has been shown to influence
protein serine/threonine modifications in yeast, and
data herein indicate that protein tyrosine
phosphorylation is sensitive to phosphate levels.
Phosphate-starved yeast "sense" replenishment of
phosphate through the RAS/CAMP pathway, and this signal
transduction pathway, connected to nutrient
availability, may be integrated with protein tyrosine
phosphorylation-mediated signaling.
Example 4: Use of the mutant yeasts to screen for ant-
diabetic aqents
Any or all of the assay in the above Examples are
performed in the presence of an agent with anti-
diabetic, anti-proliferative and/or biological response
modifying activities.
All of the references cited in this application are
94/07994 214 6~ ~2 PCT/US93/09504
hereby incorporated by reference in their entirety.
The invention being thus described, various
modifications of the materials and methods shown by the
examples will be apparent to one skilled in the art.
Such modifications are to be considered as within the
scope of the invention as set forth in the claims below.
