EP0666903A4 - Insulin-dependent yeast or fungi. - Google Patents

Abstract

The present invention relates to newly created and isolated insulin-dependent mutant strains of yeast or fungi such as (S. cerevisiae). The mutants grow on complex media of defined and undefined compositions. Optimal growth, morphology and viability of certain isolates demonstrate varying dependence upon supraphysiological concentrations of insulin. Other isolates are relatively insensitive to the same concentrations of insulin and can be maintained in the presence of the hormone for extended periods of time without toxic effects. The invention also pertains to a method for detecting insulin secretagogues which employs the mutant yeast cells. Furthermore, the invention encompasses methods for the production of recombinant insulin employing the mutant yeasts as host cells.

Description

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 . 4_.7:503 (1985)] ; modulation by net dephosphorylation θf the activities of rate-limiting enzymes including glycogen synthetase, hormone-sensitive lipase and pyruvate 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. , 16_.-429 (1985) ; Czech, Annu. Rev. Physiol . , 7:357 (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 (1984) ; Bridges and Goodman, Cell Biochem. , lie

(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 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 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 of insulin are cell- and tissue-specific and involve only a discrete subset of proteins in differentiated systems. In mammals, insulin synthesis and secretion 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 these 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 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, 2:115 (1985) ; Petruzzelli et al, Proc. Natl . Acad. Sci. USA, 2 :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 . cerevisiae) strain S288c in the presence of insulin at a concentration of 10^"° M at a temperature between 17°C to 37°C. 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 30°C 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 with said response observed in the strain from which it was derived; transport of an insulin secretagogue is altered 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 fully 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 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 components; 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 annuitant yeast strain which is transformed with a gene for mammalian insulin in a culture medium and recovering 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; FIG. 2 is a summary of a procedure for mutagenizing yeast and isolating insulin-dependent mutants;

FIG. 3 is a graph showing plating efficiency curves at three different plating densities;

FIGS. 4-9 illustrate representative plates having (YPD+D/YPD or (YNB+D/YNB ratios as indicated in the Figures .

FIGS. 10A through 10G shows the elevation of intracellular cAMP of cultured S. cerevisiae S288c in response to the addition of various nutrients . FIGS. 11A through 11D 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.

FIG. 13 shows 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.

DETAILED DESCRIPTION OF THE INVENTION

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. 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 result in constitutive secretion of insulin-like peptide.

In the mutant yeast strain, administration of an 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 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. Clinic. Med., 12.:438-448 (1968) ; and Sener et al, Experientia, 42:1026-1035 (1984)] .

In wild-type yeast, exposure to a secretagogue 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 Cellular Biology of Diabetes Mellitus: Insulin Secretion, Volume I, pp. 107-116 (1989) . In particular, there is a prompt phosphorylation of many cellular proteins, a transient increase in intracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels, an 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, 2:28-33 (1991) ; Cameron et al, Cell, 52:555-565 (1988) ; Davis et al, Cell, 4J7:423-431 (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, 22:8172-8176 (1986) ; and Mbonyi et al, Molec. Cell Biol., 2:3051-3057 (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 of 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, 22:47 (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 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 mammalian 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 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 carl sber gens is 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 17°C, 23°C, 30°C or 37°C. Some yeast mutants have the same YPD+I/YPD ratio and YNB+l/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 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 mammalian insulin gene. Such 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 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 having an altered response 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 0-6 ethyl guanine and at position four of thymine to yield 0-4 ethyl thymine . Modification at these positions leads to mispairing of 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/m.l 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 μl of ethyl methanesulfonate per ml for 1 hour at 30°C 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^"4 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 μm 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 23°C) . After 24, 48, 72 and 96 hours of incubation, plates were examined for growth. Representative recoveries of cells are presented in Figure 3. Following 72 hours of 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^"6 M) of insulin, and some of the phenotypes observed between the permissive temperatures of 23°C and 30°C could be exacerbated at extreme temperatures of 17°C and 37°C. 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 .

TABLE I

Constituent Final mg/1

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. The results of incubation at various temperatures on rich, complex YPD or defined YNB media are presented in Table II.

TABLE IJ ( CONT ' D .

TABLE JT ( CONT ' D .

TABLE J J ⁽CONT'D. )

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 μm 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, 10^"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.

TABLE III

* MORPHOLOGY OF SELECTED INSULIN-DEPENDENT MUTANTS

Mutants defined as "Class I", i.e. having a growth rate score of 2 or higher for YNB+I/YNB Mutant strains are maintained by freezing using the following protocol. 200 μl of a previously frozen stock or stock YPD+I plate stored at 4°C for less than one month, is inoculated into 200 ml of YPD media with insulin. The inoculated media is put on a shaker at 30°C 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 is 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 μl 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 -80°C. Yeast strains 2.1D2, 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) , ATCC 74190 (2.2C5) and ATCC 74191 (2.4B1) , respectively. Strain 2.1D2 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. EXAMPLE 2 : Use of yeasts as a secretagogue sensing 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. 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 (YΝBM) , 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. 2. Assay of cAMP level following glucose exposure Cells of strain S288c (stored for less than one month at 4°C) 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 30°C with shaking at 250 rpm on a New Brunswick Scientific G76 water bath to a density of 3 x 10⁶/ l 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 250 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 30°C 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 YNBM 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 μm 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 μl washes of 1 M formic 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 -20°C.

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 -20°C. 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: L-lysine 0.164 mM, L-isoleucine 0.229 mM, L-leucine 0.229 mM, L-cysteine 1.240 mM, L-glutamic Acid 0.591 mM, L-glutamine 0.684 mM, L-ornithine 0.119 mM, L-arginine 0.095 mM, L-histidine 0.129 mM, L-methionine 0.134 mM, L-tryptophan 0.098 mM, jS-aminoisobutyric acid, 0.0-10 mM. All steps of the cAMP extraction were carried out as described above. 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 μM (with 110 mM) were added; into the second set of 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. Refeeding with 2-deoxyglucose generates a 2- to 3-fold rise in cAMP in the starved cells. 3-0- methylglucopyranoside and galactose do not alter cAMP levels.

Amino acids recognized as secretagogues in mammalian 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 hours of culture. L-glutamine and L-glutamate do not promote cAMP pulses, but these amino acids effectively stimulate 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 activity of the ILP is greatest just at commencement of exponential growth of the cells . 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 10A-G and 11A-D, respectively, and summarized in Table IV-Summary of nutrients.

TABLE IV: SUMMARY OF NUTRIENTS

GLUCOSE AND CONCENTRATION cAMP GROWTH BUDDING ANALOGS TRANSIENT RESPONSE KINETICS

Glucose (fed continuously) 110 mM

Glucose (starved, refed) 110 mM t t

3-O-methylgluco- pyranoside 110 mM (with 110 mM glucose) ND

3-O-methylgluco- pyranoside 110 mM t

2-deoxy-D-glucose 114 mM (with 110 mM glucose) ND 4- J, i i, 2-deoxy-D-glucose 114 mM

AMINO ACIDS AND ANALOGS

^• / ^• i i i

TABLE IV (CONT'D.

CONCENTRATION

L-histidine L-methionine L-tryptophan -aminoisobutyric acid 0.193 mM

OTHER NUTRIENTS

Oleic acid 0.070 mM ft

Guanine 0.1 mM i i ft

Adenine 0.108 mM

Uracil 0.18 mM

Galactose 100 mM •if •if Ψ i i i

Potassium phosphate^f 7 mM ND ND

Ammonium sulfate 38 mM ND ND Insulin (with 100 mM glucose) 10^"7 M ft

SULFONYLUREAS

Tolbutamide (with 110 mM glucose) 0.025 mM

Tolbutamide (with 5 mM glucose) 0.025 mM

Chlorpropamide (with 110 M glucose) 0.025 mM

TABLE IV (CONT'D.)

CONCENTRATION cAMP GROWTH BUDDING TRANSIENT RESPONSE KINETICS

Chlorpropamide (with 5 mM glucose) 0.025 mM

Glyburide (with 110 mM glucose) 2.5 μM

Glyburide (with 5 mM glucose) 2.5 μM

to co increased response decreased or inhibited response no change in response

ND not done

EXAMPLE 3 : Monitoring of phosphotyrosine content of total veast proteins

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 bu t f ew have de f i ne d ro 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 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 po be (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 negatively 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 30°C 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 KH₂P0₄. 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 KH₂P0₄ and 0.125 mCi/MI ²P0₄. 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. IN NAOH and scintillation counted. Figure 12 shows TCA precipitable counts measured from actively growing or phosphate- arrested cultures supplemented with (A) 100% KH₂P0₄, (B) 25% KH₂P0₄, (C) 10% KH₂P0₄ and (D) KH₂P0₄-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 μl aliquots of cells were harvested and resuspended in citrate buffer-based fixative. Cells treated with 100 μg/ml RNase A and 5 μg/ml propidium iodide were analyzed 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 (A) 100% KH₂P0₄ and (B) 10% KH₂P0₄.

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 ³²P0₄ in a final volume of 2.5 ml. Cells were incubated for 90 minutes at 30°C, 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% polyacry-lamide gel at 150V until the dye front migrated 1.5 cm into the separating gel. Pieces (1 cm²) of the gel cut behind the 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 YNB 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 4°C onto nitrocellulose (0.22 μm; 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 proliferated twelve to thirteen generations in phosphate-deficient conditions and displayed a slightly greater specific 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 through exponential phase without phosphate restriction (1.6 fold less) . Expression of low affinity, high K_m phosphate transporters in phosphate unlimited cells, as opposed to expression of high affinity, low K_m 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 unlimited, exponential phase cells at the higher concentrations of exogenous phosphate (FIGS. 12A and 12B) than in cells 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 0.lx standard medium phosphate induced proliferation in 9% of phosphate-limited cells (FIG. 13B) . 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 Gl phase, were stimulated with 0.lx 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., 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., lj):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., 9_9:387 (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 contaminants, 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 phosphate yielded 2.1 x 10^"7 moles of 3' -UMP and 1.2 x 10^"7 moles of phosphotyrosine. In exponential cells (5 x 10⁸) , restricted for phosphate, 1 x 10^"7 moles of 3' -UMP and 1.1 x 10^*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, 2:1311 (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 contaminants 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 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 0. 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 of 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 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 buffer and the proteins analyzed by electrophoresis [(Laemmli, Nature, 227:680 (1970)] . This experiment revealed extensive proteolytic digestion in stationary phase cells absent in logarithmic phase cells. Significant, 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 of 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 complex nitrogen substrates (e.g. peptone) and certain 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 agents

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 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.

Claims

CLAIMS :

1. A mutant yeast or fungi strain which possesses different growth, morphology or viability properties in the presence of insulin than the parent strain from which it was derived.

2. The mutant yeast or fungi of claim 1, which is of the class Ascomycetes.

3. The .mutant yeast strain of claim 1, which is strain 2.1D2, identified as ATCC 74189.

4. The mutant yeast strain of claim 1, which is strain 2.2C5, identified as ATCC 74190.

5. The mutant yeast strain of claim 1, which is strain 2.4B1, identified as ATCC 74191.

6. The mutant yeast strain of claim 1, having at least one of the following additional characteristics: a response to an insulin secretagogue is altered compared with said response observed in the strain from which it was derived; transport of an insulin secretagogue is altered compared with transport of said secretagogue observed in wild-type yeast; secretion of an insulin-like peptide is altered compared with secretion of said 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 fully active endogenous insulin- like peptide; and does not produce one or more functional effectors in the insulin sensing/signaling system.

7. A yeast strain which grows better than Saccharomyces cerevisiae strain S288c in the presence of insulin at a concentration of 10^"°M at a temperature between 17° to 37°C.

8. A method for making 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.

9. 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.

10. A method 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; ii) 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) .

11. A method for detecting an insulin secretagogue which comprises: i) culturing cells of a mutant yeast strain as described in claim 1, wherein a response to said secretagogue is altered so as to be an exaggerated respons.e, 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. 12. 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 of claim 1; and analyzing basal status of said components; and adding an agent to said yeast; and measuring the status of said components; and comprising said status with said basal status.