CQVSTΠTJΠVE PSEUDOHYΓHAL GROWTH YEAST MUTANTS

Statement of Government Rights

The present invention was made with the support of the National Institute of Health under Contract No. 5R29G 3925405. The Government has certain rights to this invention.

Background of the Invention

Several fungal organisms are dimorphic, i.e., capable of existing in two forms. Such dimorphic fungi exhibit distinct morphologies in response to specific cellular signals. Typically, dimorphic fungi display either an egg- shaped, unicellular, yeast-like form, or a filamentous, mold-like form having attached and elongated cells. One example of such a dimorphism exists in the fungus Ustilago maydis in which haploid sporidia exhibit a yeast-like morphology. Such haploid sporidia may fuse to form an elongated dikaryon filamentous form if they bear distinct alleles at both the a and b compatibility loci. In contrast to the unicellular form, the filamentous form of Ustilago maydis, for example, causes corn smut.

A second well characterized example of dimorphism occurs in Candida albicans. This species of fungus exhibits a basic dimorphism between a budding yeast and a filamentous hyphal form. Several signals have been implicated in the switch between these two forms, including temperature, pH, nutrients, and exposure to serum factors. Mutants of C. albicans are known which are locked in either the yeast form or the hyphal form. Exploiting these observations for any useful purpose by classical genetic analysis is difficult, however. This is at least in part because C. albicans has only been observed as a diploid, and a sexual cycle has not been identified. Saccharomyces cerevisiae (S. cerevisiae), also known as brewer's yeast or baker's yeast, also is a dimorphic species capable of displaying an egg-shaped yeast-like form and a filamentous mold-like form. Unfortunately, however, laboratory isolates of the fungus present a great

variability in their ability to display this dimorphic characteristic. In this organism, nitrogen starvation in the presence of glucose is a natural inducer of the formation of the filamentous form, which is more appropriately termed pseudohyphae. See C.J. Gimeno et al., Cell. £&, 1078 (1992). Stimulation of a signal transduction pathway referred to as RAS 2 facilitates this pseudohyphal response, i.e., the formation of a filamentous form.

S. cerevisiae pseudohyphal cells have an elongated morphology, and stay attached to each other presumably by their cell wall. Furthermore, a unipolar budding pattern occurs in which daughter cells bud, and rebud, away from their mother cell in the great majority of the cell divisions. The result is a filamentous, mold-like structure growing away from the center of the colony. Of particular note is that the pseudohyphal form of S. cerevisiae forage deeply into agar media, possibly as a result of degrading polysaccharides into energy producing monosaccharides. Thus, the pseudohyphal form of S. cerevisiae could be used in the fermentation of complex polysaccharides for the production of ethanol, for example. Unfortunately, however, the wild type S. cerevisiae only undergoes the pseudohyphal response in near-starvation conditions. Exploiting these observations could lead to significant utility in commercial fermentation applications.

Summary

The present invention provides a genetically modified S. cerevisiae yeast strain containing a constitutive pseudohyphal growth mutant gene, wherein the yeast strain exhibits constitutive pseudohyphal growth. Also provided is a constitutive pseudohyphal growth mutant gene capable of causing constitutive pseudohyphal growth on S. cerevisiae. Preferably, the constitutive pseudohyphal growth mutant gene is a deletion allele elml::URA3, an insertion allele elml::HIS3, or a missense allele elml-R117. The present invention also provides an isolated DNA sequence capable of controlling pseudohyphal growth in S. cerevisiae. The isolated DNA sequence

also codes for a Ser/Thr protein kinase, which is involved in the control of pseudohyphal growth.

The present invention also provides a method of identifying constitutive pseudohyphal growth mutant genes in a yeast strain comprising: mutagenizing the yeast strain; visually identifying mutant yeast strains having elongated cells; breeding the mutant yeast strains into defined genetic backgrounds; forming a hybrid diploid strain using the mutant yeast strains having a defined genetic background; and examining the hybrid diploid strain for pseudohyphal growth characteristics. This method could be used in any of a variety of yeast strains, such as S. cerevisiae, Ustilago maydis, and C. albicans, for example. Preferably, the yeast strain is S. cerevisiae. Any known method can be used to mutagenize the yeast, i.e., treat the cells with a mutagenic agent. Preferably, the method used is a chemical or irradiative method. More preferably, it is a chemical method. The present invention is also directed to a method of regulating cellular dimorphism through the use of constitutive pseudohyphal growth genes. In this way, the present invention can be used in controlling pathogenic transformation in fungi. The present invention is also directed to a method of cloning constitutive pseudohyphal growth genes using the foraging characteristic as a genetic marker.

Brief Description of the Drawings

Figure 1. Plasmid pA2 suppresses the phenotype caused by the constitutive pseudohyphal growth mutant gene elml. Strains were cultured in SDC liquid medium supplemented according to the auxotrophic requirements. Cells were photographed while in exponential growth using a phase contrast microscope. (A): Strain αl04Wl, bearing elml-1. (B): Strain A2 which was obtained by transformation of l04Wl with the suppressor plasmid pA2; uracil was omitted from the medium. (C): The A2 strain was cultured in liquid medium containing uracil, allowing plasmid loss. The culture was spread on a plate while still in the presence of uracil. About 5% of the isolated colonies displayed a mutant morphology. Furthermore, the wild type

looking colonies were uracil independent, while the mutant colonies required uracil indicating they had lost the pA2 plasmid. A representative uracil dependent segregant is shown.

Figure 2. Restriction Map of the ELMl Region. (A):

Delineation of a genomic region suppressing the elml-1 phenotype. The restriction maps of inserts from several plasmids are aligned, and the suppressing ability of the corresponding plasmid is indicated. Restriction sites are shown for EcoRI, Pstl, Sail, Xbal, BglH, and HindlTI. Sites in parenthesis are located in the multiple cloning region of the vector. Plasmids pAl to pA4 were selected from a genomic library, based on their ability to suppress the elml-1 defect of strain αl04Wl. Inserts in pE104/STl and pE104/ST3 are the 1.8 kb Pstl-SacI fragment and the 1.4 kb Hindϋl-EcoRI fragment from pA2, respectively (Sad and Hindu! are located in the multiple cloning region of the vector). (B) Map of ELMl and disrupted alleles. The region common to the inserts of pAl to pA4 is shown in the middle diagram. The ELMl coding sequence is marked by the solid arrow. The upper and lower diagrams show the structure of the deletion allele elml::URA3 and the insertion allele elml::MS3. Figure 3. Nucleotide sequence (SEQ ID NO: 1) of the ELMl locus and predicted arnino acid sequence (SEQ ID NO:2) of Elmlp (protein kinase). Only the sense strand is shown. The coding region is translated below the nucleotide sequence. The location of several restriction enzyme recognition sites are indicated for comparison to Figure 2. Figure 4. The suppressor gene resides at the ELMl locus. The suppressor locus, presumably ELMl, had been tagged by the URA3 marker in wild type strain aWΩ (see Experimental Procedures). This strain was mated with the elml-1 strain l04Wl and meiosis was induced in the resulting diploid. Four spores from a single tetrad were separated and allowed to germinate. The resulting haploid strains were respread on YPD plates, cultured for 16 hours, then photographed in situ using an inverted microscope. Uracil requirement was also scored. This tetrad is representative of the thirty

tetrads analyzed from this cross. (A) and (D): Wild type morphology, uracil independent. (B and C): Mutant morphology, uracil dependent. The mutant colonies also are representative of the original collection of mutants obtained by visual screen of the mutagenized D273-10B/A1 strain.

Figure 5. Elmlp (SEQ ID NO:2) is homologous to Ser Thr protein kinases. The deduced amino acid sequence of Elmlp is aligned with that of the protein kinase Cdc28p (SEQ ID NO:3) (disclosed in AT. Lδcrincz et al., Nature. 307. 183-185 (1984)) and the relevant domain of the bovine cAMP dependent protein kinase catalytic subunit, α form (cAPK) (SEQ ID NO:4) (disclosed in S. Shoji et al., Biochemistry. 22, 3702-3709 (1983)). Identical residues are boxed and gaps are represented by dashes. Residues nearly invariant among protein kinases (disclosed in S.K. Hanks et al., Science. 241. 42-51 (1988)) are indicated by stars. Figure 6. Phenotype Caused by elm 1 Deficiency in Inbred and

Hybrid Diploid Yeast Strains. The null alleles elml::URA3 or elml::MS3 were introduced in various strains by homologous recombination, and diploids homozygous for elml deficiency were obtained by mating the appropriate strains. Cells were cultured for 16 hours on a YPD dish and photographed with a regular microscope equipped with Nomarski optics. (A): WWΔelml (W303 background). (B): NN/ lml (NY13 background); the insert shows the elml phenotype in the haploid strain aNΔelml (NY13 background). (C): ∑∑ lml (∑1278b background). (D): Hybrid N∑Λslml. (E): Hybrid NW elml. (F): Hybrid ∑W ml. Colony morphology also was recorded in situ using an inverted microscope. (G): Hybrid ∑WΔslml, which is also representative of ∑∑^lml, NN ml, N∑- lml and NWΔelml observed under the same conditions. (H): Inbred WWΔelml displaying some enlarged, round cells.

Figure 7. The mutant genes elm2 and elm3 cause constitutive pseudohyphal growth. Diploid strains homozygous for either elm2-l

(a/αELM2) or elm3-l (a/αElm3) were cultured on a YPD plate for 16 hours, then photographed in situ using an inverted microscope or at higher

magnification with a regular microscope equipped with Nomarski optics. (A) and (B): a/oElm2. (C) and (D): a/αElm3.

Figure 8. Constitutive Pseudohyphae Forage Extensively in Agar Medium. (A): Diploid strains homozygous for either elml, elm2, or elm3, as well as two wild type control strains, were cultured for four days on a YPD plate, then photographed. Strains are 1) ∑W (wild type), 2) ∑ Δelml, 3) NWΔelml, 4) a/o_Elm2, 5) a/ocElir , 6) NW (wild type). The elml strains are congenic with the wild type controls. (B): The plate was extensively washed under running tap water and photographed again. Cells invading the agar could not be washed off.

Figure 9. Loss of ELMl function causes constitutive pseudohyphal growth. Single cells of the indicated strains were isolated on a YPD plate using a micromanipulator, and incubated at 30°C. The developing clones were photographed at the indicated times thereafter using an inverted microscope. N∑^elml/elml is homozygous for the deletion allele elml::URA3, whereas N∑ is homozygous for the wild type allele ELMl; otherwise the two strains are genetically identical. Both strains are Fl hybrid diploids formed by mating haploids of the NY13 and ∑1278b backgrounds.

Detailed Description of the Invention

Saccharomyces cerevisiae (S. cerevisiae) grows either as a unicellular, egg-shaped, yeast form or as a filamentous mold-like form, which is referred to as pseudohyphae. Although the yeast form usually prevails, pseudohyphal growth may occur during nitrogen starvation in the wild type S. cerevisiae strain. A general approach has been developed that allows for the isolation of genes involved in this dimorphic transition. An isolated wild type gene, referred to herein as ELMl (ELongated Morphology), is capable of coding for a novel protein kinase homolog, which is required for the yeast morphology. The present invention is based on the discovery that deletion of the wild type gene ELMl causes constitutive pseudohyphal morphology. Herein, "deletion" refers to the removal of the majority of the coding region

of ELMl, or other forms of inactivation of ELMl including: insertion of a foreign DNA sequence within its coding region; or changing a specific nucleotide sequence, such as converting the lysine codon at position 117 to an arginine codon. Furthermore, additional mutations of the wild type gene ELMl and other specific genes, such as those referred to herein as elml, elm2, and elm3, cause constitutive pseudohyphal growth. This is evidenced by mutant strains forming chains of connected and elongated cells that grow invasively into semisolid media, e.g., agar. It is believed that this occurs as a result of degradation of polysaccharides into energy-rich monosaccharides. Thus, the present invention can be used in controlling pathogenic transformations in fungi. This is important in control of the prevalent human pathogen C. albicans, which can cause systemic infection when growing in the hyphal form. Such infections are frequent and life- threatening in immunosuppressed patients such as those with AIDS or undergoing chemotherapy treatment for cancer. Control of plant pathogens such as U. maydis also is possible, because prevention of the hyphal form precludes pathogenicity. Furthermore, the present invention can be used to produce yeast that can degrade polysaccharides, feasibly even cellulose, in fermentation processes. Thus, for example, bulk ethanol could be prepared from corn silage or other agricultural plant byproducts using constitutive pseudohyphal S. cerevisiae strains. Such strains could also be used for production of alcoholic beverages using various cellulose sources as the substrate for fermentation.

Constitutive pseudohyphal growth mutant genes can be obtained by chemical mutagenesis of a wild type S. cerevisiae strain, e.g., the strain containing the wild type ELMl gene. Cells are treated with the mutagenic agent, then individual cells are separated on agar medium and allowed to form colonies. These are screened visually for the presence of elongated cells protruding from the body of the colony. Subsequent analysis of the mutant cells and their genetic properties can identify specific mutant genes that cause constitutive pseudohyphal growth.

The major characteristics imparted to yeast strains as a result of the incorporation of these mutant genes are as follows. Cells are elongated, growth occurs predominantly at the pole of the cell 180° opposite to its connection with its mother cell, and cell separation is delayed. This results in 5 formation of expanded, branched chains of cells that grow outward from the center of a colony. These mutations are named genetically elm (ELongated Morphology). Herein, a constitutive pseudohyphal growth mutant gene is referred to when this term is used in lower case letters. In contrast, the wild type gene is referred to when this term is used in upper case letters.

10 Examples of three particularly effective mutant genes are referred to herein as elml, elm2 and elm3.

A "constitutive pseudohyphal growth mutant gene" is used herein to refer to a gene that imparts filamentous pseudohyphal growth and polysaccharide degradation to a yeast strain in which the gene is incorporated.

15 Preferably and advantageously the mutant genes impart constitutive pseudohyphal growth, including polysaccharide degradation, under substantially all yeast-growing conditions. Such a genetically modified yeast strain is referred to herein as a "constitutive pseudohyphal growth mutant yeast strain."

20 In contrast to wild type S. cerevisiae, which only converts to the pseudohyphal form in near-starvation conditions, the genetically modified form described herein undergoes filamentous, mold-like growth to form elongated cells, and polysaccharide degradation under substantially all yeast- growing conditions. That is, the pseudohyphal mutant strains can degrade

25 polysaccharides, as evidenced by their growing into agar as opposed to growing on the surface of agar, on nitrogen-rich media, on carbon-rich media, on liquid or solid media, etc., and under all temperatures capable of effecting yeast growth (typically about 15-37°C). Although not intended to be limiting to the claims of the present invention, it is believed that the mutant S.

1 301 cerevisiae strains described herein grow into agar media as a result of the excretion of a digestive enzyme capable of degrading polysaccharides, such as for example, a glycohydrolase.

In addition to chemical mutagenesis of the wild type S. cerevisiae strain, a constitutive pseudohyphal growth mutant yeast strain can obtained by incorporating a constitutive pseudohyphal growth mutant gene into an inbred diploid yeast strain or hybrid diploid yeast strain. Examples of inbred diploid yeast strains include, but are not limited to, ∑∑ and W303 (Table 1). Examples of hybrid diploid yeast strains include, but are not limited to, NW and ∑W (Table 1). Preferably, the mutant gene is incorporated into a hybrid diploid yeast strain. The use of hybrid diploid yeast strains imparts greater filamentous growth to the genetically altered yeast.

Strain-dependent variability in the morphology caused by the elm mutations is to be expected, considering that great variability in competence for natural pseudohyphal growth has been reported among S. cerevisiae laboratory isolated. See, for example, C.J. Gimeno, et al., Cell. 68, 1077-1090 (1992). Presumably, pseudohyphal growth is less efficient in several inbred genetic backgrounds (which are expected to be homozygous at all genetic loci), owing to specific defects in genes required for this differentiation state. These defects could become fixed in particular reference strains, since there is no selection against such mutations in the laboratory environment. Expression of the pseudohyphal state in such defective backgrounds, owing to an elm mutation, would then result in an aberrant phenotype composed of defective pseudohyphae. In hybrid diploids formed by crossing two independently maintained laboratory isolates, defects impairing pseudohyphal growth most likely are heterozygous, leading to a behavior closer to normal. Loss of ELMl function in three different inbred diploid backgrounds can lead to three different phenotypes, with various degrees of pseudohyphal growth. In contrast, ELMl loss in three different hybrid diploid backgrounds causes identical phenotypes, which closely resemble healthy pseudohyphal growth. Similarly, the most demonstrative pseudohyphal phenotypes caused by elm2 or elm3 are observed in hybrid backgrounds.

Pseudohyphal growth has not been reported for haploid strains of S. cerevisiae. The original elm mutants, however, are obtained by mutagenesis of an haploid strain. Although the axial budding pattern of haploids is inappropriate for pseudohyphal growth, the elm mutations always cause cell elongation even in haploid strains, allowing identification of the mutants. Several haploid elm mutants also display a unipolar budding pattern typical of diploid pseudohyphae.

The invention has been described with reference to various specific and preferred embodiments and will be further described by reference to the following detailed examples. It is understood, however, that there are many extensions, variations, and modifications on the basic theme of the present invention beyond that shown in the examples and detailed description, which are within the spirit and scope of the present invention.

Experimental Procedures

Strains. Media and Genetic Methods

Yeast strains used in this study are described in Table 1 and were cultured at 30°C unless specified otherwise. The following media were used: YPD (1% yeast extract, 2% peptone, 2% glucose); YPAD (YPD supplemented with 40 mg/1 adenine); SD (2% glucose, 0.7% yeast nitrogen base without amino acids, supplemented as required with leucine, tryptophan, histidine, lysine, methionine, uracil and adenine at 20 mg/1 each); SDC (SD supplemented with 0.5% casaminoacids in addition to the auxotrophy requirements); sporulation medium (1% potassium acetate, 0.05% glucose, 0.1% yeast extract); SLAHD (nitrogen starvation media described by C.J. Gimeno et al., Cell. 68, 1077-1090 (1992), which is incorporated herein by reference). Solid media for yeast contained 2% agar.

Standard genetic methods were used for complementation analysis, mating, and tetrad dissection as disclosed in F. Sherman et al., Methods in Yeast Genetics. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, (1986), which is incorporated herein by reference. In those instances where auxotrophic markers were not available for selection of

a diploid from a cross, isolated colonies of potential diploids were selected based on their increased growth rate relative to the haploids parents. In all instances diploidy was verified by the ability of the selected strains to sporulate.

∑1278b Background (M. Grenson et al., Biochem. Biophys. Acta. 127. 325- 338 (1966)):

MB758-5B MATa ura3 Siddiqui and Brandriss, Mol.

Cell Bio

& 4634-4641 (1988)

MB758-6B MATα ura3 Obtained in an analogous manner to that disclosed in Siddiqui and Brandriss (1988)

MB810-3C MATa lys2 MB810-5A MATα lys2

ΣΣ MATa/MATα ura3/ura3 Iys2/lys2 Mating of segregants from

MB758-5B x MB810-5A

7TΔelml/+ MATa/MATα ura3/ura3 Iys2/lys2 Integrative transformation of elml::URA3/+ ∑∑ a∑ lml MATa ura3 lys2 elml::URA3 Segregant from ∑∑_ lml/+ α∑^elml MATα ura3 lys2 elml::URA3 Segregant from ∑∑ elml/+

∑∑ lml MATa/MATα ura3/ura3 1ys2/lys2 a∑ lml x α∑^elml elml::URA3/elml::URA3

W303 Background:

W303 MATa/MATα ura3/ura3 Ieu2/leu2 J. Wallis, Cell, 58, 409-419 his3/his3 tφl/tφl ade2/ade2 (1989)

W303-1A MATa ura3 leu2 his3 t l ade2 Meiotic product of W303

W303-1B MATα ura3 leu2 his3 tφl ade2 Meiotic product of W303 WWΔ MATa/MATα ura3/ura3 Integrative elmlH/+ Ieu2/leu2 transformation of W303 his3/his3 tφl/tφl ade2/ade2 elml::HIS3/+ aWΔelmlH MATa ura3 leu2 his3 tφl ade2 Segregant from elml::HIS3 WWΔelmlH/+

WWΔ MATa/MATα ura3/uτa3 Integrative transformation of elmlU/+ Ieu2/leu2 W303 his3/his3 tφl/tφl ade2/ade2 elml::URA3/+ aWΔslmlU MATa ura3 leu2 his3 tφl ade2 Segregant from elml::URA3 WWΔelmlU/+ αWΔelmlU MATα ura3 leu2 his3 tφl ade2 Segregant from elml::URA3 WWΔelmlU/+

WWΔelml MATa/MATα ura3/ura3 aWΔelmlH x αWΔelmlU

Ieu2/leu2 his3/his3 tφl/tφl ade2/ade2 elml::URA3/elml::HIS3 aWΩ MATa ura3 leu2 his3 tφl ade2 Integrative transformation of ELM1ΩURA3 W303-1A αWΔcdc55 MATα ura3 leu2 his3 tφl ade2 Integrative transformation of cdc55::LEU2-2 W303-1B

Defined Hybrid Backgrounds:

NWΔelml MATa/MATα ura3/ura3 aNΔelml x αWΔelmlU leu2/+ his3/+ frpl/+ ade2/+ elml::URA3/elml::URA3

∑WΔelml MATa/MATα ura3/ura3 a∑≥lml x αWΔelmlU leu2/+ his3/+ frpl/+ ade2/+ lys2/+ elml::URA3/elml::URA3

N∑ lml MATa/MATα ura3/ura3 lys2/+ aNΔelml x α∑Δelml elml::URA3/elml::URA3

∑WΔcdc55/+ MATa/MATα ura3/+ MB810-3C x αWΔcdc55 leu2/+ his3/+ trpl/+ ade2/+ lys2/+ cdc55::LEU2-2/+

NW MATa/MATα ura3/ura3 NY13 x W303-lB leu2/+ his3/+ trpl/+ ade2/+

∑W MATa/MATα ura3/+ MB810-3C x W303-1B leu2/+ his3/+ tφl/+ ade2/+ lys2/+

Other Backgrounds:

D273-10B/ MATα metό A. Tzagoloff, FEBS left.. 65, Al 391-396 (1976)

E104 MATα met6 elml-1 Mutagenesis of D273- 10B/A1 αl04Wl MATα ura3 ade2 his3 leu2 Segregant from El 04 x elml-1 W303-1A E124 MATα met6 elm2-l Mutagenesis of D273- 10B/A1 al24Wla MATa leu2 tφl met6 elm2-l Segregant from E124 x W303-1A al24Wlb MATa ade2 leu2 met6 elm2-l Segregant from E124 x W303-1A αl24∑2 MATα tφl lys2 elm2-l Segregant from second backcross of al24Wla to ∑1278b background a/αElm2 MATa/MATα ade2/+ al24Wlb x αl24∑2 leu2/+ met6/+ trpl/+ lys2/+ elm2-l/elm2-l

E130 MATα met6 elm3-l Mutagenesis of D273- 10B/A1 al30Wla MATα ura3 leu2 his3 elm3-l Segregant from El 30 x W303-1A

al30Wlb MATa ade2 leu2 elm3-l Segregant from El 30 x W303-1A αl30∑2 MATαura3 lys2 elm3-l Segregant from second backcross of al30Wla x ∑l278b background a/αElm3 MATa/MATα ade2/+ leu2/+ al30Wlb x αl30∑2 met6/+ tφl/+ lys2/+ elm3-l/elm3-l

ELMl Gene Isolation

Genes capable of restoring normal appearance, i.e., a normal moφhology, to an elml-1 mutant strain were selected from a yeast genomic library obtained from Francois Lacroute (Centre de Genetique Moleculaire du CNRS, Gif-sur-Yvette, France). The vector used, pFL38, was derived from the pUC19 bacterial vector. pFL38 contains in addition the URA3 selectable marker as well as a centromeric sequence causing maintenance at low copy number in yeast. The genomic inserts were obtained by partial Sau3A digestion (average size 3kb) of chromosomal DNA from wild type S. cerevisiae, and were ligated to the BamHJ site of pFL38.

The elml-1 mutant strain αl04Wl was cultured in 100 ml of YPAD medium and transformed with 50 μg of plasmid library DNA using a scaled up version of the lithium transformation procedure, as disclosed in F. Ausubel et al, Current Protocols in Molecular Biology. John Wiley and Sons, NY (1989), which is incoφorated herein by reference. Immediately after transformation, the cells were resuspended in 6 ml of lOmM Tris-HCl pH 7.5, lmM EDTA (TE) buffer. Two ml of the cell suspension were added to each of three tubes containing 15 ml of the liquid medium SDC supplemented with histidine, leucine, tryptophan and adenine, but lacking uracil. The total number of uracil-independent transformants, 8 x 10 ⁴ , was estimated from a small aliquot of the TE suspension spread directly on selective dishes. The liquid cultures were incubated at 30°C with gentle shaking for three days. An aliquot of each saturated culture (5 μl) was inoculated into 5 ml of fresh SDC medium which was again grown to saturation. The dilution procedure was repeated several times in a row, every three or four days. At various times,

samples from saturated liquid cultures were also spread on selective plates and moφhology of individual colonies was scored. More than 50% of the colonies from the second or third cycle of liquid cultures displayed wild type moφhology. Isolated wild type colonies were selected for further analysis. As a control, the αl04Wl strain was also transformed with the pFL38 vector devoid of insert. These control cells never reverted to wild type, even after five cycles of liquid cultures.

DNA Manipulations and Allele Construction DNA manipulations were performed by standard procedures as disclosed in F.M Ausubel, Current Protocols in Molecular Biology, New York: Greene Publishing Associates and Wiley-Interscience (1989); and J. Sambrook et al., Molecular Cloning. A Laboratory Animal. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1989), which are incoφorated herein by reference. Plasmid pUCl 18E is a modified version of pUCl 18 in which the multiple cloning site was replaced by a unique EcoRI site. See J. Vieira et al., Methods Enzymo 153. 3-11 (1987), which is incoφorated herein by reference. In each instance where a strain was constructed by gene replacement, Southern analysis (as disclosed in E. Southern, J. Mol. Biol.. 98. 503-517 (1975), which is incorporated herein by reference) of the transformant was performed to confirm that integration by homologous recombination had occurred as expected.

The insertion allele elml::HIS3 was constructed as follows. The 1.4 kb EcoRI fragment of the genomic insert in pAl was subcloned in plasmid pUCl 18E, resulting in plasmid pELMl/ST13. The yeast HIS3 gene (which is disclosed in K. Struhl, Nucleic Acid Res. 13, 8587-8601 (1985), incoφorated herein by reference) was available as a 1.7 kb genomic BamHI fragment cloned in pUCl 18, in the orientation such that a 1.2 kb Pstl fragment containing the entire HIS3 promotor and coding region could be excised. This fragment was subcloned at the unique Pstl site of pELMl/ST13, present at ELMl codon 94, forming pElml::HIS3. A 2.6 kb

EcoRI fragment from pELMl::HJS3 was used for transformation of the his3/his3 diploid strain W303 to histidine prototrophy.

The null allele elml::URA3 was prepared as follows. The 2.8 kb genomic insert of pAl was excised as a Sacl-Sall fragment, and subcloned in pBLUESCRIPT SK+ (Stratagene Cloning System, La Jolla, CA). The resultant plasmid, pELMl/ST16, was digested at the unique Pstl and BgUJ sites, removing ELMl codons 94 to 487. A 1.2 kb Hindu! fragment of yeast DNA bearing the URA3 gene (disclosed in MD. Rose, et al., Gene. 2_ 113- 124 (1984), which is incoφorated herein by reference) was inserted in pELMl/ST16 in place of the deleted sequence. The resulting plasmid, pElml::URA3, was digested with Xbal, generating a 2.9 kb fragment used for DNA transformation of various ur 3 strains to uracil prototrophy.

The chromosomal ELMl locus was tagged with a genetic marker as follows. The insert of the suppressing plasmid pA2 was ligated into the integrative plasmid YIp352 (disclosed in J.E. Hill et al., Yeast 2, 163-167 (1986), which is incoφorated herein by reference) as a 2.4 kb BamHI-SacI fragment. The resulting plasmid, pELMlΩURA3, was linearized by digestion at its unique BgUI site located within ELMl. The linearized plasmid was used to transform wild type strain W303-1A to uracil prototrophy. This type of integration results in duplication of ELMl, both copies being functional and separated from each other by the YIp352 linear plasmid which bears the URA3 marker.

The null allele cdc55::LEU2-2, similar to the cdc55::LEU2 allele described by AM Healy et al, Mol. Cell. Biol.. U, 5767-5780 (1991), which is incoφorated herein by reference, was constructed as follows. The 0.3 kb PvuII fragment from pUCl 19, which bears the multiple cloning region, was replaced by the 2.3 kb PvuII fragment of plasmid YCpHN (AM Healy et al, 1991), which bears CDC55. In the resulting plasmid, the 1.3 kb EcoRI fragment (CDC55 codons 93-526) was replaced by a BamHJ linker, forming p55/ST2. A 3.0 kb Bglll fragment from plasmid YEpl3, containing the LEU2 gene, was inserted at the unique BamHI site of p55/ST2 resulting in

pcdc55::LEU2. The 4.2 kb PvuII fragment of pcdc55::LEU2 was used to transform leu2 strains to leucine prototrophy.

Preparation of the Mutagenic Gene elml-Rlll The missense allele elml-R117 was constructed as follows. The

1177 bp Pstl-BglΩ. fragment from pAl was cloned in pUCl 19. The lysine codon AAG specifying Elmlp residue 117 was changed to the arginine codon CCG by oligonucleotide-directed site-specific mutagenesis. The nucleotide sequence of the entire Pstl-BgM genomic fragment was determined to ensure no other base substitutions occurred during the mutagenesis procedure, elml- R117 was formed by using the Pstl-BglΩ. fragment containing the lysine to arginine mutation to replace the equivalent region of the wile type ELMl sequence in plasmid pELMl/ST16. The 2.8 kb Sacl-Sa fragment from the resulting plasmid was cloned in the centromeric vector pRS315 {257} to form YCpelml. The control plasmid YCpELMl was formed by cloning the 2.8 kb Sc l-SdR. fragment from ρELMl/ST16 in pRS315.

The lysine residue of Elmlp position 117 is conserved in all protein kinases, and is known to be absolutely required for the catalytic mechanism of cAMP dependent protein kinase. In all instances examined, conservative substitution of this lysine by an arginine inactivated the protein kinase activity. Thus, if Elmlp codes for a protein kinase, men an arginine to lysine substitution at position 117 is expected to inactivate the catalytic activity. This mutant allele, termed elml-R117 was constructed and introduced as part of centromeric plasmid YCpelml into strain aWΔelmlU, which bears the deletion allele elml::URA3. YCpelml failed to restore normal cell and colony moφhology to aWΔelmlU, however, the moφhologic defect was corrected by control plasmid YCpELMl (which differs from YCpelml only at two nucleotides within codon 117.). Thus, Lysll7 is essential for activity of Elmlp. The nucleotide sequence of elml-R117 is identical in nucleotide sequence to ELMl with the exception that the sequence AA at positions 649- 650 is changed to the sequence CG. These substitutions result in replacement

of the lysine residue at arnino acid position 117 with an arginine residue (protein sequence is deduced from nucleotide sequence, and has not been confirmed directly). elm-R117 causes constitutive pseudohyphal growth.

Moφhological Analyses

Colony moφhology was examined using an inverted microscope, observing the cells through the gear. Higher magnifications of the cells were obtained from liquid cultures, or by resuspending cells from an agar dish in a drop of water, and examining the suspensions on a slide using phase contrast or Nomarski optics.

Nucleotide Sequence Accession Number

The nucleotide sequence of the isolated ELMl gene has been assigned GenBank/EMBL accession number M81258. The nucleotide sequence and predicted arnino acid sequence of the gene product Elmlp were analyzed and compared to the available databases using the Sequence Analysis

Software Package of the Genetics Computer Group (Madison, WI). See, J.

Devereux et al. Nucleic Acids Res.. 12, 387-395 (1984) for a comprehensive set of sequence analysis programs for the VAX

Experimental Results

Isolation of Mutants with a Constitutive Elongated Moφhology

Wild type strain D273-10B/A1 (strains used in this study are described in Table 1) was moderately mutagenized by exposure to ethyl methanesulfonate (15% survival), then plated for single colonies on YPD medium. After incubation for two to four days at 22°C, colony moφhology was examined directly on the surface of the agar using an inverted microscope. Moφhological mutants were identified by an irregular colony shape and the presence of elongated cells extending outward from the colony; roughly 1% of the mutagenized colonies had such a moφhology. This example describes five mutants from a collection of sixty moφhologically altered strains obtained by this procedure.

Each strain in the study group contains a single recessive mutation that causes cell elongation. Diploids formed by mating the mutants to reference strain W303-1A had no detectable moφhologic abnormality or growth defect on YPD or SD medium. Meiotic progeny of these diploids that displayed the cell elongation phenotype (outcross progeny) were collected and backcrossed successively at least five times to the unmutagenized parent strain D273-10B/A1. The cell elongation and wild type phenotypes segregated consistently at a 2:2 ration in at least 30 tetrads, indicating that in each instance the cell elongation phenotype is a single-gene trait. In the outcross and early rounds of backcrossing considerable variation was observed in the severity of the phenotype, both in the degree of cell elongation and in the growth rate (data not shown). Decreased growth rate was observed only in moφhologically abnormal progeny, and thus was a result of the same mutation that affects cell shape. In the later rounds of backcrossing, however, a uniform moφhologic phenotype was observed for all progeny of each mutant, and no significant difference was detected between the growth rates of the mutant progeny and the wild type parent (data not shown). Thus, certain aspects of the phenotype caused by these mutations apparently depend on the specific genetic background. Complementation and allelism tests determined three different genes were identified by the five moφhological mutants in the study group. Complementation groups were assigned by analyzing the moφhology of diploids formed in reciprocal crosses between the original mutants and their backcross progeny. Three groups were identified, two with two members each and one with a single representative (Table 2). Allelism tests were performed by observing haploid progeny from the diploids obtained in the complementation group analysis. Diploids with elongated cell moφhology always produced tetrads comprising only mutant progeny (30 tetrads analyzed for each cross). Conversely, all diploids with a wild type moφhology produced both mutant and wild type progeny in the ratio expected for independent assortment of unlinked genes. Thus, the three complementation groups represent three distinct gene loci, tentatively named ELMl, ELM2, and

ELM3 (ELongated Moφhology). Similar analysis, including allelism tests, showed ELM2 and ELM3 are distinct from five cell division cycle genes (CDC) known to cause cell elongation, namely CDC3, CDCIO, CDCll, CDC12, and CDC55 (which is disclosed in L. Hartwell, Exptl. Cell Res.. 69. 265-276 (1971) and A. Healy et al, Mol. Cell. Bio 11. 5767-5780 (1971)). ELMl was shown to be different from any of these CDC genes by its unique nucleotide sequence (see below).

TABLE 2. Complementation matrix ³

MAT arent ^b MATα parent 104D5 105D5 102D5 130D5 156D5

+

^a The indicated strains were mated and diploids were selected based on complementing auxotrophies. "-" indicates the diploid had a mutant moφhologic phenotype, and "+" indicates the diploid had wild type or near-wild type moφhology. ^b MATα parents are progeny of the fifth backcross to D273-10B/A1. The original mutants were outcrossed to W303-1A prior to the backcrosses. These strains all contain a leu2 auxotrophic marker; 102D5 also contains a his3 marker.

Cloning of ELMl

The wild type ELMl gene was selected from a genomic library based on its ability to complement the growth defect caused by elml-1 in strain αl04Wl. This strain bears the wxύ marker allowing selection of URA3 plasmids. The elml-1 mutation of αl04Wl causes elongated moφhology, dumpiness, and a reduced growth rate (Figure IA). When DNA was stained with DAPI, a few very elongated cells seemed to bear several nuclei, suggesting that cytokinesis was impaired (data not shown). Cell viability was high despite the severely abnormal appearance of αl04Wl, and the phenotype was stable when the strain was maintained routinely on stock plates. A yeast genomic library based in the centromeric (low copy number) plasmid pFL38 was introduced into the αl04Wl cells, and transformants were inoculated en masse in liquid medium lacking uracil. Absence of uracil from

the medium maintained a selection for transforming plasmids, and growth in liquid culture presumably would allow relatively rapidly growing revertants to overtake cells still suffering from the reduced doubling time associated with the mutant phenotype. Indeed, after about 30 generations, the majority of cells in the liquid cultures displayed wild type moφhology. Liquid cultures were spread on agar medium lacking uracil, and four apparently reverted yeast colonies, named Al to A4, were further characterized (Figure IB shows clone A2).

The reverted phenotype was caused by the plasmids because plasmid loss during mitosis resulted in reappearance of the mutant phenotype. Figure 1C shows a derivative of clone A2 which presumably lost its plasmid. The four plasmids present in the reverted yeast clones were recovered, produced in E. coli, and named pAl to pA4. Restriction enzyme mapping of the plasmids showed they all contain distinct genomic inserts that share an overlapping 2.1 kb sequence (Figure 2). This common region was entirely sequenced and a single long open reading frame was observed, covering 1689 bp. Reintroducing plasmids bearing the entirety of this open reading frame into the elml-1 strain αl04Wl restored wild type moφhology. Subclones containing only parts of the coding region, however, failed to restore wild type moφhology (Figure 2). Thus, this open reading frame corresponds to a yeast gene capable of suppressing the elml-1 phenotype.

The "next door insertion" strategy, which is disclosed in R. Rothstein, Meth. EnzymoL 194. 281-301 (1991), and incoφorated herein by reference, indicated the cloned suppressor gene is the wild type allele of ELMl. The insert of pA2 was subcloned in the integrative plasmid YIp352. The entire plasmid was then integrated in the genome of W303-1A by homologous recombination near the suppressing locus. Thus, in the resulting strain aWΩthe suppressing locus is tagged by the URA3 marker. As expected, this strain displays wild type cell and colony moφhology. The elml-1 strain αl04Wl was mated to aWΩ and meiosis was induced in the resulting heterozygous diploid. Thirty tetrads were dissected, and in every cases, two spore-derived colonies had a wild type moφhology and were uracil

independent, whereas two spore-derived colonies displayed obvious moφhological abnormalities and were uracil dependent (Figure 3). Thus, the cloned suppressor gene marked by URA3 and the mutation elml-1 reside at the same genetic locus.

ELMl Codes for a Putative Novel Protein Kinase

The nucleotide sequence of ELMl (Figure 3) (SEQ ID NO:l) revealed an open reading frame coding for 563 aminoacyl residues (Figure 5) (SEQ ID NOS:2,3,4). The predicted protein (Elmlp) sequence was used in a computer assisted search for related proteins. No close relative was detected, but significant homology was observed with several protein kinases. When Elmlp was compared with the available sequences of protein kinases, it appeared roughly equally diverged from all Ser/Thr kinases. Figure 5 shows, as an example, Elmlp aligned with the CDC28 gene product Cdc28p (also known as p34 or histone kinase) and the catalytical region from the bovine cAMP dependent protein kinase cAPK. In this comparison, Elmlp is 23.7% identical to Cdc28p and 23.1% identical to cAPK while these two reference sequences are 23.1% identical to each other. High conservation is observed in particular regions. For example, from residue 245 to 280, Elmlp is more than 45%) identical to either cAPK or Cdc28p. In addition, the 15 invariant residues found in almost every protein kinases are also conserved in Elmlp. Thus, Elmlp bears a protein kinase catalytic domain, spanning approximately residues 90 to 400. The arnino and carboxy terminal regions of Elmlp, where no significant homology has been detected, may provide regulatory functions. Two subdomains have been described in protein kinases, that display different consensus sequences in enzymes specific for either tyrosine or serine/threonine. See, S.K. Hanks et al. Science. 241. 42-51 (1988). At this first subdomain (residues 259-264), Elmlp bears DIKPSN (SEQ ID NO:5) which fits best the Ser/Thr kinase consensus DLKPEN (SEQ ID NO:6) as opposed to the tyrosine kinase signature sequence DLAARN (SEQ ID NO:7) or DLRAAN SEQ ID NO: 8). Likewise, at the second subdomain (residues 309-317), the Elmlp sequence GTPAFIAPE (SEQ ID NO:9) matches the

consensus G-T/S-X-X-F/Y-X-A-P-E (SEQ ID NO: 10) for Ser/Thr specificity and is diverged from the tyrosine kinase consensus P-I/V-W-T/M-A-P-E (SEQ ID NO:l 1). Thus, Elmlp defines a novel branch in the Ser/Thr protein kinase family.

Inactivation of ELMl Causes a Pseudohyphal Moφhology

The W303 outcross progeny from the original El 04 mutant showed an unusual variability in the severity of the elml-1 phenotype (data not shown), even though a single mutation was known to cause the moφhological defect. This suggested that the genetic background influences the elml phenotype. To test this hypothesis, ELMl was inactivated directly in several laboratory strains using the gene replacement technique disclosed in R Rothstein, Meth. EnzymoL 194. 281-301 (1991), which is incoφorated herein by reference, and the phenotypes were compared. Two different disrupted alleles were constructed, namely elml::URA3 and elml::HIS3, and were integrated by homologous recombination at the ELMl locus of various strains. In the elml::URA3 allele most of the ELMl coding sequence is replaced by the URA3 gene, while ELMl coding sequence is disrupted by HIS3 in the elml::HIS3 allele (see Figure 2). Replacement of ELMl by either construct caused the same elongated moφhology phenotype in haploid strains of the W303 background. Furthermore, diploids formed by mating elml-1 strains to either elml::URA3 or elml::HIS3 strains also displayed the mutant phenotype, confirming that the disruptions of ELMl are allelic with elml-1 (data not shown). Inbred diploid strains deficient for ELMl were obtained in the

W303, NY13 and ∑1278b backgrounds (respectively WWΔelml, NNΔelml and ∑∑^elml). Al three strains presented elongated cells attached to each other, reminiscent of the elml-1 phenotype (Figure 6A, B, C). Strain-specific particularities were observed, however, confirming the elml phenotype is dependent at least in part upon the genetic background. In WWΔelml, growth was slow, cell shape was irregular, cytokinesis was seemingly impaired and some round, enlarged cells were present (Figure 6A, H). In

contrast, NNz lml strain displayed cells very regular in their elongated shape. Neither cytokinesis defects nor enlarged round cells were seen and the growth rate on plates was not significantly reduced when compared to a congenic wild type strain. During exponentional growth in liquid YPD medium, NNΔelml cells stayed attached presumably by their cell wall (Figure 6B). This moφhology, and in particular the budding pattem of NN lml resembles the recently described pseudohyphae of S. cerevisiae: chains of elongated cells which stay attached to each other, where daughter cells bud opposite to their mother, while mother cells rebud near their daughter. The result is an ejφanded, highly branched, mold-like structure as disclosed in C.J. Gimeno, et al, £eU, £8, 1077-1090 (1992). In the haploid background of NY13 the phenotype caused by elml deletion was similar except for the budding pattern which was axial, as expected for haploid cells. See, for example, D. Freifelder, J. BacterioL. 80, 567-568 (1960), and J. Chant et al, Cell, 65, 1203-1212 (1991). In this instance the elongated cells always formed buds near their mother. This resulted in small, star like clumps where each branch is composed of a single elongated cell (Figure 6B insert). The diploid ∑∑ elml strain had a phenotype close to NN ml, except for less uniformity in the shape of individual cells (Figure 6C). The elml phenotype was also analyzed in the three hybrid diploid strains (N∑^elml, ∑ Δelml and NWΔelml) obtained by pairwise mating of haploid elml strains in the W303, NY13, and ∑1278b backgrounds. The phenotype of these three mutant strains was virtually identical, and resembles mostly that of NNΔelml. Cell shape was very regular, and defective cytokinesis was not observed. Time-course examination of single cells on a plate for several generations showed the doubling time of N∑ lml and ∑WΔelml to be approximately 1.5 hours, the same as congenic ELMl/ELMl strains (data not shown). Cell elongation in the hybrids, however, was not as extreme as in the inbred NNΔelml strain. The pseudohyphal budding pattem and formation of branched structures were particularly obvious in all three elml/elml hybrid diploids (Figure 6D, E, F, G). The phenotype depicted by the hybrids, most likely represents the actual

elml phenotype, while the inbreds probably bear some genetic defects responsible for their more or less aberrant phenotype.

elm2 and elm3 Aso Cause Constitutive Pseudohyphal Growth Many other elongated mutants obtained by mutagenesis of D273-

10B/A1 behaved similarly to elml strains in the respect that the W303-1A outcross progeny displayed variable phenotypes, including some with pseudohyphal moφhology. Strains containing elm2 or elm3 mutations were characterized further in this regard. A diploid homozygous for elm2-l, OW∑βlm2-l/elm2-l, was formed in a largely hybrid background by mating a progeny clone from the outcross of the original mutant to W303-1A with one from the second backcross to ∑1278b (Table 1). Pseudohyphal characteristics including cell elongation, cell attachment (after sonication), and formation of expanded, branched chains of cells all were obvious in DW∑βlm2-l/elm2-l (Fig. 7), and the form of these cells was very similar to that of elml deletion mutants in Fl hybrid diploid backgrounds. Growth of single cell clones of the elm2 mutant DDelm2-2/elm2-2 was observed over time on solid YPD medium. This strain exhibited the typical extended, branched chains characteristic of pseudohyphal growth. Direct observation of clonal development showed the doubling time of DDelm2-2/elm2-2 to be essentially the same as the congenic wild type strain DD (data not shown). The same analysis was applied to the elm3 mutant ODelm3-l/elm3-l with similar results, except for more variability in cell length (data not shown). Thus, mutations in ELMl, ELM2 or ELM3 all cause a dimoφhic transition leading to a nearly identical constitutive pseudohyphal growth phenotype.

elml. elm2 and elm3 Mutants Grow Invasively in Agar Media

A distinctive property of the previously described pseudohyphal form of S. cerevisiae is the ability to grow invasively under the surface of an agar medium, referred to herein as "foraging". The foraging capacity of elml, elm2, and elm3 strains was examined by culturing patches of cells for several

days on YPD plates, then scrubbing the surface of the agar with a finger under running tap water to remove the cells from the plate's surface.

Haploid elm2-l, elm2-2, or elm3-l strains in the D273-10B background could not be washed from the plate, whereas the congenic wild type control strain was completely removed (Fig. 8). Observation with an inverted microscope showed most of the cells remaining after washing were located completely under the agar surface, with chains extending up to 5 cell lengths into the medium (data not shown). The ability to forage results from the elm2 or elm3 mutation, because this property consistently co-segregated with the cell elongation phenotype in at least 12 complete tetrads derived from elm2-2/ELM2 or elm3-l/ELM3 heterozygous diploids (data not shown). In the haploid D273-10B background elml-1 and elml-2 mutants also foraged, although to a lesser extent than the congenic elm2 or elm3 strains (data not shown). W∑fielml/telml and NWΔe/w7/Δe/ also exhibited obvious foraging behavior, whereas the congenic wild type control strains were completely or nearly completely removed from the YPD plate by the washing procedure (Fig. 8).

ELM2 and ELM3 Function Affects Pseudohyphal Differentiation in Response to Nitrogen Starvation.

The comprehensive phenotypic resemblance of the pseudohyphal moφhologies caused either by elml, elm2, or elm3 mutations, or by nitrogen starvation of wild type cells, suggested the mutations result in constitutive execution of the differentiation pathway that normally is triggered by nutrient availability. To test this hypothesis the effects of ELMl, ELM2, and ELM3 gene dosage on the ability of a strain to form pseudohyphae in response to nitrogen starvation were examined. Congenic diploid strains were constructed in the D273-10B background that contained either one or two functional copies of each gene to be examined. Al strains displayed typical yeast-like moφhology in nitrogen-rich media such as YPD or SD. On the nitrogen starvation medium SLAHD the homozygous wild type strain DD failed to display pseudohyphal growth even after 14 days on SLAHD medium, which

is typical of most inbred laboratory strains. In contrast, pseudohyphal differentiation was obvious in the congenic strains ODelm2-2/+ and ODelm3- 11+ after three days on SLAHD medium. Moφhologic differentiation of these two strains is dependent on the nutritional environment, because pseudohyphal cells transferred from an SLAHD plate to the nitrogen-rich medium YPD produced clones with typical yeast-like moφhology; these clones again differentiated into pseudohyphae when they were replated on SLAHD. Thus, function of both ELM2 and ELM3 significantly affects the ability to flip a developmental switch in response to nitrogen starvation. This gene dosage effect was not observed for ELMl in strain ODelml-l/+.

The disclosures of all patents, patent applications, patent documents, and publications cited herein are incoφorated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Iowa State University Research Foundation, Inc. Institut Pasteur

Institut National de la Sante et de la Recherche Medicale Myers, Alan M. Madaule, Pascal

(ii) TITLE OF INVENTION: Constitutive Pseudohyphal Growth Yeast Mutants

(iii) NUMBER OF SEQUENCES: 11

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Schweg an, Lundberg & Woessner

(B) STREET: 3500 IDS Center, 80 South Eighth Street

(C) CITY: Minneapolis

(D) STATE: MN

(E) COUNTRY: USA

(F) ZIP: 55402

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/061,636

(B) FILING DATE: 12 MAY 1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Mueting, Ann M. and Raasch, Kevin .

(B) REGISTRATION NUMBER: 33,977 and 35,651

(C) REFERENCE/DOCKET NUMBER: 900.38WO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 612-339-0331

(B) TELEFAX: 612-339-3061

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2105 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GATCCTTCTT GAAGTAGCTA TTAAGTTGTT CGAAATGAAG TAATTATTAA AATAGAAGTA 60

AATCATTAAA TGATGCCGCT CAACAGAGGT TATGCCAAAT TAGTATATAG CATGATTTTA 120

CATCACTTTA AACGTATAAT TTGTGAATGA TGAGGTAGCA ACAAATAAAC AATGCAACAG 180

TCTCTAGTCC TATGAACTAA TTIOGCCTTG AAACCCCCCG ATGATACTTC TTTAGGTGTT 240

ACAACTTACT CGCATAGATA TTATTTTTGA CGCCAGGTTA ACAATAATTA CTTAGCATGA 300

ATG TCA CCG CGA CAG CTT ATA CCG ACA TTA ATT CCG GAA TGG GCA

CCA 348

Met Ser Pro Arg Gin Leu lie Pro Thr Leu lie Pro Glu Trp Ala

Pro

1 5 10 15

TTA TCC CAG CAA TCG TGC ATA AGA GAG GAT GAG TTA GAT AGT CCC

CCG 396

Leu Ser Gin Gin Ser Cys lie Arg Glu Asp Glu Leu Asp Ser Pro

Pro

20 25 30

ATA ACG CCT ACG AGC CAG ACA TCT TCA TTT GGT TCT TCT TTT TCT

CAA 444 lie Thr Pro Thr Ser Gin Thr Ser Ser Phe Gly Ser Ser Phe Ser

Gin

35 40 45

CAG AAA CCA ACC TAT AGT ACA ATT ATA GGA GAA AAT ATA CAC ACG

ATC 492

Gin Lys Pro Thr Tyr Ser Thr lie lie Gly Glu Asn lie His Thr

He

50 55 60

CTG GAT GAA ATT CGA CCA TAT GTG AAA AAA ATA ACT GTT AGT GAC

CAA 540

Leu Asp Glu He Arg Pro Tyr Val Lys Lys He Thr Val Ser Asp

Gin

65 70 75

80

GAT AAG AAA ACT ATA AAC CAA TAT AGG CTA GGA GTC TCT GCA GGA

AGT 588

Asp Lys Lys Thr He Asn Gin Tyr Thr Leu Gly Val Ser Ala Gly

SS2T

85 90 95

GGA CAA TTT GGT TAT GTA CGA AAA GCG TAC AGT TCT ACT TTA GGC

AAG 636

Gly Gin Phe Gly Tyr Val Arg Lys Ala Tyr Ser Ser Thr Leu Gly

Lys

100 105 110

GIT GTT GCT GTC AAG ATT ATA CCA AAA AAA CCT TGG AAT GCC CAG

CAA 684

Val Val Ala Val Lys He He Pro Lys Lys Pro Trp Asn Ala Gin

Gin

115 120 125

TAT TCA GTA AAT CAA GTA ATG AGG CAA ATC CAG CTT TGG AAG AGT

AAA 732

Tyr Ser Val Asn Gin Val Met Arg Gin He Gin Leu Trp Lys Ser

Lys

130 135 140

GGA AAA ATA ACG ACA AAT ATG AGT GGT AAT GAG GCT ATG AGA CTT

ATG 780

Gly Lys He Thr Thr Asn Met Ser Gly Asn Glu Ala Met Arg Leu

Met

145 150 155

160

AAT ATC GAA AAA TGT AGG TGG GAA ATT TTT GCG GCT TCA AGA CTT

CGA 828

Asn He Glu Lys Cys Arg Trp Glu He Phe Ala Ala Ser Arg Leu

Arg

165 170 175

AAT AAT GTT CAT ATT GTG CGA CTA ATA GAA TGC TTG GAC TCT CCT

TTC 876

Asn Asn Val His He Val Arg Leu He Glu Cys Leu Asp Ser Pro

Phe

180 185 190

AGC GAA TCT ATC TGG ATA GTC ACT AAT TGG TGC AGC CTT GGT GAA

CTA 924

Ser Glu Ser He Trp He Val Thr Asn Trp Cys Ser Leu Gly Glu

Leu

195 200 205

CAG TGG AAA CGT GAC GAT GAT GAA GAT ATT TTA CCG CAA TGG AAA

AAA 972

Gin Trp Lys Arg Asp Asp Asp Glu Asp He Leu Pro Gin Trp Lys

Lys

210 215 220

ATT GTG ATT TCA AAT TGT AGT GTT TCT ACA TTT GCC AAA AAA ATC

CTG 1020

He Val He Ser Asn Cys Ser Val Ser Thr Phe Ala Lys Lys He

Leu

225 230 235

240

GAG GAT ATG ACA AAA GGG TTG GAA TAT TTG CAT TCT CAG GGT TGT

ATT 1068

Glu Asp Met Thr Lys Gly Leu Glu Tyr Leu His Ser Gin Gly Cys

He

245 250 255

CAT CGT GAT ATC AAA CCG TCC AAT ATT TTA TTG GAT GAA GAA GAA

AAA 1116

His Arg Asp He Lys Pro Ser Asn He Leu Leu Asp Glu Glu Glu

Lys

260 265 270

GTA GCG AAA CTT TCT GAT TTT GGA AGT TGT ATT TTC ACT CCC CAA

TCA 1164

Val Ala Lys Leu Ser Asp Phe Gly Ser Cys He Phe Thr Pro Gin

Ser

275 280 285

TTA CCT TTC AGC GAT GCT AAT TTT GAA GAT TGT TTT CAG AGG GAA

TTG 1212

Leu Pro Phe Ser Asp Ala Asn Phe Glu Asp Cys Phe Gin Arg Glu

Leu

290 295 300

AAC AAA ATT GTT GGT ACT CCG GCA TTT ATT GCA CCA GAG CTA TGT

CAT 1260

Asn Lys He Val Gly Thr Pro Ala Phe He Ala Pro Glu Leu Cys

His

305 310 315

320

TTG GGC AAT TCC AAA AGA GAT TTT GTG ACG GAT GGC TTT AAG TTG

GAT 1308

Leu Gly Asn Ser Lys Arg Asp Phe Val Thr Asp Gly Phe Lys Leu

Asp

325 330 335

ATT TGG TCA TTG GGA GTG ACA CTA TAC TGC TTA CTG TAC AAC GAG

CTG 1356

He Trp Ser Leu Gly Val Thr Leu Tyr Cys Leu Leu Tyr Asn Glu

Leu

340 345 350

CCA TTT TTC GGG GAA AAT GAA TTC GAA ACC TAC CAC AAA ATC ATC

GAA 1404

Pro Phe Phe Gly Glu Asn Glu Phe Glu Thr Tyr His Lys He He

Glu

355 360 365

GTA TCA TTG AGT TCC AAA ATA AAT GGT AAT ACT TTA AAC GAT TTA

GTC 1452

Val Ser Leu Ser Ser Lys He Asn Gly Asn Thr Leu Asn Asp Leu

Val

370 375 380

ATT AAA AGG TTA TTG GAG AAA GAC GTT ACT TTA CGC ATA AGT ATT

CAG 1500

He Lys Arg Leu Leu Glu Lys Asp Val Thr Leu Arg He Ser He

Gin

385 390 395

400

GAT TTA GTA AAG GTT TTG TOG CGT GAC CAG CCC ATA GAT TCT AGG

AAT 1548

Asp Leu Val Lys Val Leu Ser Arg Asp Gin Pro He Asp Ser Arg

Asn

405 410 415

CAC AGT CAA ATT TCA TOG TCC AGT GTG AAC CCC GTA AGA ACG GAA

GGT 1596

His Ser Gin He Ser Ser Ser Ser Val Asn Pro Val Arg Thr Glu

Gly

420 425 430

CCT GTA AGA AGA TTT TTT GGT AGG CTA CTG ACT AAA AAA GGA AAG

AAA 1644

Pro Val Arg Arg Phe Phe Gly Arg Leu Leu Thr Lys Lys Gly Lys

Lys

435 440 445

AAG ACC TCA GGA AAA GGG AAA GAC AAG GTA TTG GTA TCT GCA ACT

AGT 1692

Lys Thr Ser Gly Lys Gly Lys Asp Lys Val Leu Val Ser Ala Thr

Ser

450 455 460

AAA GTA ACA CCT TCG ATA CAT ATC GAC GAG GAA CCG GAT AAA GAA

TGT 1740

Lys Val Thr Pro Ser He His He Asp Glu Glu Pro Asp Lys Glu

Cys

465 470 475

480

TTT TCG ACT ACG GAC CTT AGA TCT TCG CCA GAC TCG AGC GAT TAT

TGT 1788

Phe Ser Thr Thr Asp Leu Arg Ser Ser Pro Asp Ser Ser Asp Tyr

Cys

485 490 495

TCA TCG TTA GGG GAG GAA GCC ATT CAG GTT ACG GAT TTC TTA GAT

ACT 1836

Ser Ser Leu Gly Glu Glu Ala He Gin Val Thr Asp Phe Leu Asp

Thr

500 505 510

TTT TGT AGG TCA AAT GAA AGC TTA CCT AAT TTG ACT GTC AAT AAT

GAT 1884

Phe Cys Arg Ser Asn Glu Ser Leu Pro Asn Leu Thr Val Asn Asn

Asp

515 520 525

AAG CAG AAT TCG GAC ATG AAA ACT GAC AGA AAG CGA GTC ATC CTC

TCA 1932

Lys Gin Asn Ser Asp Met Lys Thr Asp Arg Lys Arg Val He Leu

Ser

530 535 540

TTC GTC ATT GAA AAT CCC AAC ACC TAT CAA AGC CAT GAT AAG ACT

AAA 1980

Phe Val He Glu Asn Pro Asn Thr Tyr Gin Ser His Asp Lys Thr

Lys

545 550 555

560

GAG TTC CCC T AAAGAGAACG GGAACAGAAC CCATATTAAT TGCTCACAGG

2030 Glu Phe Pro

ACAAACCGAG TTCCCCACTA ATGGATAGGA CTGTTGGAAA GCGCACGGTT AATAATTCAG 2090

GGGCTAGAAA GCTTC 2105

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 563 arnino acids

(B) TYPE : arnino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE : protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :

Met Ser Pro Arg Gin Leu He Pro Thr Leu He Pro Glu Trp Ala Pro

1 5 10 15

Leu Ser Gin Gin Ser Cys He Arg Glu Asp Glu Leu Asp Ser Pro Pro

20 25 30

He Thr Pro Thr Ser Gin Thr Ser Ser Phe Gly Ser Ser Phe Ser Gin

35 40 45

Gin Lys Pro Thr Tyr Ser Thr He He Gly Glu Asn He His Thr He

50 55 60

Leu Asp Glu He Arg Pro Tyr Val Lys Lys He Thr Val Ser Asp Gin

65 70 75

80

Asp Lys Lys Thr He Asn Gin Tyr Thr Leu Gly Val Ser Ala Gly Ser

85 90 95

Gly Gin Phe Gly Tyr Val Arg Lys Ala Tyr Ser Ser Thr Leu Gly Lys

100 105 110

Val Val Ala Val Lys He He Pro Lys Lys Pro Trp Asn Ala Gin Gin

115 120 125

Tyr Ser Val Asn Gin Val Met Arg Gin He Gin Leu Trp Lys Ser Lys

130 135 140

Gly Lys He Thr Thr Asn Met Ser Gly Asn Glu Ala Met Arg Leu

Met

145 150 155

160

Asn He Glu Lys Cys Arg Trp Glu He Phe Ala Ala Ser Arg Leu Arg

165 170 175

Asn Asn Val His He Val Arg Leu He Glu Cys Leu Asp Ser Pro Phe

180 185 190

Ser Glu Ser He Trp He Val Thr Asn Trp Cys Ser Leu Gly Glu Leu

195 200 205

Gin Trp Lys Arg Asp Asp Asp Glu Asp He Leu Pro Gin Trp Lys Lys

210 215 220

He Val He Ser Asn Cys Ser Val Ser Thr Phe Ala Lys Lys He

Leu

225 230 235

240

Glu Asp Met Thr Lys Gly Leu Glu Tyr Leu His Ser Gin Gly Cys He

245 250 255

His Arg Asp He Lys Pro Ser Asn He Leu Leu Asp Glu Glu Glu Lys

260 265 270

Val Ala Lys Leu Ser Asp Phe Gly Ser Cys He Phe Thr Pro Gin Ser

275 280 285

Leu Pro Phe Ser Asp Ala Asn Phe Glu Asp Cys Phe Gin Arg Glu Leu

290 295 300

Asn Lys He Val Gly Thr Pro Ala Phe He Ala Pro Glu Leu Cys

His

305 310 315

320

Leu Gly Asn Ser Lys Arg Asp Phe Val Thr Asp Gly Phe Lys Leu Asp

325 330 335

He Trp Ser Leu Gly Val Thr Leu Tyr Cys Leu Leu Tyr Asn Glu Leu

340 345 350

Pro Phe Phe Gly Glu Asn Glu Phe Glu Thr Tyr His Lys He He Glu

355 360 365

Val Ser Leu Ser Ser Lys He Asn Gly Asn Thr Leu Asn Asp Leu Val

370 375 380

He Lys Arg Leu Leu Glu Lys Asp Val Thr Leu Arg He Ser He

Gin

385 390 395

400

Asp Leu Val Lys Val Leu Ser Arg Asp Gin Pro He Asp Ser Arg Asn

405 410 415

His Ser Gin He Ser Ser Ser Ser Val Asn Pro Val Arg Thr Glu Gly

420 425 430

Pro Val Arg Arg Phe Phe Gly Arg Leu Leu Thr Lys Lys Gly Lys Lys

435 440 445

Lys Thr Ser Gly Lys Gly Lys Asp Lys Val Leu Val Ser Ala Thr Ser

450 455 460

Lys Val Thr Pro Ser He His He Asp Glu Glu Pro Asp Lys Glu

Cys

465 470 475

480

Phe Ser Thr Thr Asp Leu Arg Ser Ser Pro Asp Ser Ser Asp Tyr Cys

485 490 495

Ser Ser Leu Gly Glu Glu Ala He Gin Val Thr Asp Phe Leu Asp Thr

500 505 510

Phe Cys Arg Ser Asn Glu 'Ser Leu Pro Asn Leu Thr Val Asn Asn Asp

515 520 525

Lys Gin Asn Ser Asp Met Lys Thr Asp Arg Lys Arg Val He Leu Ser

530 535 540

Phe Val He Glu Asn Pro Asn Thr Tyr Gin Ser His Asp Lys Thr

Lys

545 550 555

560

Glu Phe Pro

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 298 arnino acids

(B) TYPE: arnino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Ser Gly Glu Leu Ala Asn Tyr Lys Arg Leu Glu Lys Val Gly Glu

1 5 10

15

Gly Thr Tyr Gly Val Val Tyr Lys Ala Leu Asp Leu Arg Pro Gly Gin

20 25 30

Gly Gin Arg Val Val Ala Leu Lys Lys He Arg Leu Glu Ser Glu Asp

35 40 45

Glu Gly Val Pro Ser Thr Ala He Arg Glu He Ser Leu Leu Lys Glu

50 55 60

Leu Lys Asp Asp Asn He Val Arg Leu Tyr Asp He Val His Ser Asp

65 70 75

80

Ala His Lys Leu Tyr Leu Val Phe Glu Phe Leu Asp Leu Asp Leu Lys

85 90

95

Arg Tyr Met Glu Gly He Pro Lys Asp Gin Pro Leu Gly Ala Asp He

100 105 110

Val Lys Lys Phe Met Met Gin Leu Cys Lys Gly He Ala Tyr Cys His

115 120 125

Ser His Arg He Leu His Arg Asp Leu Lys Pro Gin Asn Leu Leu He

130 135 140

Asn Lys Asp Gly Asn Leu Lys Leu Gly Asp Phe Gly Leu Ala Arg Ala

145 150 155

160

Phe Gly Val Pro Leu Arg Ala Tyr Thr His Glu He Val Thr Leu Trp

165 170

175

Tyr Arg Ala Pro Glu Val Leu Leu Gly Gly Lys Gin Tyr Ser Thr Gly

180 185 190

Val Asp Thr Trp Ser He Gly Cys He Phe Ala Glu Met Cys Asn Arg

195 200 205

Lys Pro He Phe Ser Gly Asp Ser Glu He Asp Gin He Phe Lys He

210 215 220

Phe Arg Val Leu Gly Thr Pro Asn Glu Ala He Trp Pro Asp He Val

225 230 235

240

Tyr Leu Pro Asp Phe Lys Pro Ser Phe Pro Gin Trp Arg Arg Lys Asp

245 250

255

Leu Ser Gin Val Val Pro Ser Leu Asp Pro Arg Gly He Asp Leu Leu

260 265 270

Asp Lys Leu Leu Ala Tyr Asp Pro He Asn Arg He Ser Ala Arg Arg

275 280 285

Ala Ala He His Pro Tyr Phe Gin Glu Ser 290 295

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 260 arnino acids

(B) TYPE: arnino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Asn Thr Ala His Leu Asp Gin Phe Glu Arg He Lys Thr Leu Gly Thr

1 5 10

15

Gly Ser Phe Gly Arg Val Met Leu Val Lys His Met Glu Thr Gly Asn

20 25 30

His Tyr Ala Met Lys He Leu Asp Lys Gin Lys Val Val Lys Leu Lys

35 40 _, 45

Gin He Glu His Thr Leu Asn Glu Lys Arg He Leu Gin Ala Val Asn

50 55 60

Phe Pro Phe Leu Val Lys Leu Glu Phe Ser Phe Lys Asp Asn Ser Asn

65 70 75

80

Leu Tyr Met Val Met Glu Tyr Val Pro Gly Gly Glu Met Phe Ser His

85 90

95

Leu Arg Arg He Gly Arg Phe Ser Glu Pro His Ala Arg Phe Tyr Ala

100 105 110

Ala Gin He Val Leu Thr Phe Glu Tyr Leu His Ser Leu Asp Leu He

115 120 125

Tyr Arg Asp Leu Lys Pro Glu Asn Leu Leu He Asp Gin Gin Gly Tyr

130 135 140

He Gin Val Thr Asp Phe Gly Phe Ala Lys Arg Val Lys Gly Arg Thr

145 150 155

160

Trp Thr Leu Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu He He Leu

165 170

175

Ser Lys Gly Tyr Asn Lys Ala Val Asp Trp Trp Ala Leu Gly Val Leu

180 185 190

He Tyr Glu Met Ala Ala Gly Tyr Pro Pro Phe Phe Ala Asp Gin Pro

195 200 205

He Gin He Tyr Glu Lys He Val Ser Gly Lys Val Arg Phe Pro Ser

210 215 220

His Phe Ser Ser Asp Leu Lys Asp Leu Leu Arg Asn Leu Leu Gin Val

225 230 235

240

Asp Leu Thr Lys Arg Phe Gly Asn Leu Lys Asp Gly Val Asn Asp He

245 250

255

Lys Asn His Lys 260

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Asp He Lys Pro Ser Asn 1 5

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Asp Leu Lys Pro Glu Asn 1 5

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Asp Leu Ala Ala Arg Asn 1 5

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Asp Leu Arg Ala Ala Asn 1 5

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Gly Thr Pro Ala Phe He Ala Pro Glu 1 5

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Gly Xaa Xaa Xaa Xaa Xaa Ala Pro Glu 1 5

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Pro Xaa Trp Xaa Ala Pro Glu 1 5