WO1997031104A2

WO1997031104A2 - Candida albicans profilin gene

Info

Publication number: WO1997031104A2
Application number: PCT/US1997/003798
Authority: WO
Inventors: Jessica A. Gorman; Darin B. Ostrander
Original assignee: Bristol-Myers Squibb Company
Priority date: 1996-02-22
Filing date: 1997-02-20
Publication date: 1997-08-28
Also published as: WO1997031104A3; AU2204397A

Abstract

An isolated nucleic acid molecule encoding C. albicans profilin. Preferably, the C. albicans profilin has the amino acid sequence of SEQ. ID. NO.: 2 and the nucleotide sequence of SEQ. ID. NO.: 1. The invention also concerns a nucleic acid molecule having a sequence complementary to the above sequences of 5' and 3' flanking regions thereof. The invention further concerns nucleic acid vectors comprising a DNA sequence coding for C. albicans profilin, host cells containing such vectors, and polypeptides comprising C. albicans profilin. Preferably, the polypeptide is full-length C. albicans profilin or recombinantly produced C. albicans profilin. The present invention also concerns methods for detecting nucleic acids coding for C. albicans profilin and for detecting anti-fungal agents that target C. albicans profilin.

Description

CANDIDA ALBICANS PROFILIN GENE

Field of the Invention

The present invention relates to a gene, PFY1, involved in phospholipid hydrolysis in the yeast Candida albicans, and more particularly to the identification, isolation and cloning of this gene. This invention also relates to a method of using this gene to screen for compounds with antifungal activity.

Background of the Invention

Profilin is a ubiquitous 15 kDa eukaryotic actin and phosphotidylinositol-4,5-bisphosphate- (PIP₂) binding protein. Machesky & Pollard (1993). Trends Cell Biol. 3: 381-5. In mammalian cells, the protein functions by binding PIP₂, which inhibits the phospholipid hydrolysis by phospholipase C (PLC) in resting cells. Goldschmidt-

Clermont et a]. (1990), Science 247: 1575-8. When cells divide following activation of tyrosine kinase receptors, PLC is phosphorylated and thereby activated to hydrolyze PIP2 even in the presence of profilin. Goldschmidt-Clermont et al. (1991), Science 251: 1231-3. This hydrolysis releases the known second messengers inositol triphosphate (IP3) and diacylglycerol (DAG) and profilin. Profilin moves from the plasma membrane to the cytosol, where it binds to actin to catalyze the rearrangement of the cytoskeleton. Goldschmidt-Clermont & Jamey (1991), £eJl 66: 419-21. Profilin catalyzes release of adenosine diphosphate (ADP) from monomeric or globular (G-) actin. Mockrin gt aL (1980), Biochem. 19: 5359-62; Goldschmidt-Clermont et al. (1991). T. Cell Biol. 113: 1081-9. This activity dramatically increases G-actin's exchange of ADP for ATP, which activates actin for polymerization into microfilaments and facilitates reorganization of the cytoskeleton. Carlier (1989), Int. Rev. Cytol. 115: 139-70. Profilin therefore represents an important component of the eukaryotic cell division cycle.

Evidence suggests that profilin is important in the growth cycle of yeasts. Null mutation in the Saccharomyces cerevisiae profilin gene is lethal in many S. cerevisiae strain backgrounds. Magdolen et a]. (1988), Mol. Cell. Biol. 8: 5108-15. In other S. cerevisiae strains, null mutations produce a strain with highly abnormal morphology and severely retarded growth rates. Haarer et aj. (1990), T. Cell Biol. 110: 105-14. While a role in phospholipid hydrolysis has not been shown, PIP₂ -dependent translocation of profilin from the plasma membrane into the cytoplasm has been demonstrated. Ostrander _t _\. (1995), I- Biol. Chem 46: 27045- 50. S_. cerevisiae profilin has also been shown to interact with the RAS/adenylate cyclase pathway. Vojtek et al. (1991), Cell 66: 497-505. The dimorphic yeast Candida albicans is a major opportunistic human pathogen and represents a leading cause of death among immuno-compromised persons. Odds (1988), Candida and Candidosis (2nd ed.), Bailliere Tindall, London. In the past, treatment of Candidiasis has involved drugs with severely toxic side effects. Martin et aj. (1994), Antimicrob. Agents and Chemother. 38: 13-22. Recently, less toxic compounds have become available, but strains of pathogenic Candida species that are resistant to these antifungal agents are already being observed. Rex et al. (1995), Antimicrob. Agent Chemother. 9: 1-8. Therefore, there is a desperate need for new antifungal agents. The recent development of high-throughput screens for the isolation of such agents presents an opportunity for meeting this need. Gorman (1992) in New Approaches for Antifungal Drugs (Fernandes, ed.), Birkhauser, Boston, 143-54.

Profilin thus represents an important new target for antifungal drugs. Summary of the Invention

The present invention concerns an isolated nucleic acid molecule encoding C. albicans profilin. Preferably, the C_. albicans profilin has the amino acid sequence of SEQ. ID. NO.: 2. The inventors also prefer that the nucleic acid molecule has the nucleotide sequence of SEQ. ID. NO.: 1 (GenBank Accession No. 37834).

The present invention also concerns a nucleic acid molecule having a sequence complementary to the above sequences and 5' or 3' flanking regions thereof. The present invention further concerns nucleic acid vectors comprising a DNA sequence coding for C. albicans profilin, host cells containing such vectors, and polypeptides comprising C. albicans profilin. Preferably, the polypeptide is full-length C. albicans profilin or C. albicans profilin recombinantly produced as described hereinafter. The present invention also concerns methods for detecting nucleic acids coding for Q. albicans profilin and for detecting anti-fungal agents that target C. albicans profilin.

Description of the Drawings Figures IA, IB, and IC show a comparison of the amino acid sequences of profilin genes from various organisms. Regions of high homology used to create degenerate oligodeoxynucleotides (listed below with translations) are underlined. Organisms are listed in order of overall similarity to the S. cerevisiae profilin protein. Abbreviations for each organism appear below, followed by the literature reference reporting each sequence and its sequence identification number.

"Sc" refers to Saccharomyces cerevisiae (yeast). Magdolen gt al. (1988), Mol- Cell. Biol. 8: 5108-15. Sc profilin is SEQ. ID. NO.: 9.

"Pp" refers to Physarum polycephalum a and p (acellular slime mold). Binette et al. (1990), DNA Cell. Biol. 9: 323-34. Ppa profilin is SEQ. ID. NO.: 10; Ppp, SEQ. ID. NO. 14. "Ac" refers to Acanthamoeba castellanii I and II (amoeba). Ampe fit al. (1985), Biol. Chem. 260: 834-40. "Aci" and "Acii" refer to the two known Ac profilins. Aci profilin is SEQ. ID. NO.: 13; Acii, SEQ. ID. NO.: 11. "Dm" refers to Drosophila melanogaster (fruit fly). Cooley ei al-

(1992), £ell 69: 173-84. Dm profilin is SEQ. ID. NO.: 12.

"Dd" refers to Dictyostelium discoideum I and II (cellular slime mold). Haugwitz et aj. (1991), I. Cell Science 100: 481-9. "Ddi" and "Ddii" refer to the two known Dd profilins. Ddi profilin is SEQ. ID. NO.: 15; Ddii, SEQ. ID. NO.: 16.

"Susp" refers to Strongylocentrotus purpuratus (sea urchin). Smith et al. (1992), Mol. Biol. Cell 3: 403-14. Susp profilin is SEQ. ID. NO.: 17.

"Sd" refers to Clypeaster japonicus (sand dollar). Takagi et aj. (1990), Eur. I. Biochem 192: 777-81. Sd profilin is SEQ. ID. NO.: 18.

"Bv" refers to Betula verrucosa (plant). Valenta et al (1991), Science 253: 557-60. Bv profilin is SEQ. ID. NO.: 19.

"Suac" refers to Anthocidaris crassispina (sea urchin). Takagi et aj. (1990), Eur. I. Biochem 192: 777-81. Suac profilin is SEQ. ID. NO.: 20. "Tp" refers to Tetrahymena pyriformis (protozoa). Edamatsu et al

(1991), Biochem. Biophys. Res. Comm. 175: 543-50. Tp profilin is SEQ. ID. NO.: 21.

"Hs" refers to Homo sapiens (human). Kwiatkowski & Bruns (1988), I. BM- Chem. 263: 5910-15. Hs profilin is SEQ. ID. NO.: 22. "Mm" refers to Mus musculus (mouse). Widada et al. (1989),

Nucleic Acids Res. 17: 2855. Mm profilin is SEQ. ID. NO.: 23.

"Prof" refers to the consensus sequence. Capital letters represent complete conservation; lower-case letters, conservative changes and greater than 80% identity for the residue. The consensus sequence is SEQ. ID. NO.: 24. Nonstandard abbreviations for nucleotides appearing in the foregoing sequences have the following meanings.

"K" refers to G or T.

"N" refers to any of the four nucleotides. "R" refers to A or G.

"S" refers to C or G.

"W" refers to A or T.

"Y" refers to C or T.

Figure 2 shows degenerate oligonucleotides matching highly homologous regions of profilin proteins. The forward primer (SEQ. ID. NO.: 5) appears at the upper left; the corresponding expressed amino acid sequence (SEQ. ID. NO.: 6) appears at the lower left. The antisense reverse primer (SEQ. ID. NO.: 7) appears at the upper right; the amino acid sequence (SEQ. ID. NO.: 8) expressed from the corresponding sense strand appears at the lower right. The nonstandard abbreviations for nucleotides are as defined for Figure 1.

Figure 3 shows the nucleotide (SEQ. ID. NO.: 1) and predicted amino acid (SEQ. ID. NO.: 2) sequences of the C albicans profilin gene PFY1. Underlining denotes the regions recognized by the degenerate oligodeoxynucleotides. The Apal site used to reconstruct the full-length gene and the Ncol site used to subclone the gene are in bold type.

Figure 4 shows a western analysis of total protein isolated from various profilin-expressing strains using antisera to purified C. albicans profilin. Lane 1, . albicans strain B-792; lane 2, pfylΔ S. cerevisiae strain SD017 containing S. cerevisiae PFY1 on an expression vector; lane 3, SD017 containing £. albicans PFY1 on an expression vector. Approximate molecular weights are indicated from markers; the predicted size of the cross-reacting species is 14 kDa. Detailed Description of the Invention Definition of terms

The following definitions apply to the terms as used throughout this specification, unless otherwise limited in specific instances: "C. albicans profilin" refers to proteins or polypeptides present in

Candida albicans and capable of binding actin and PIP2 and having at the N-terminus a profilin signature sequence of the formula

SEQ. ID. NO. 4: A1-A2-A3-W-A4-A5-A6-A7-A8- (SEQ. ID. NO. 3) wherein: A¹ is absent or is a natural amino acid residue;

A² seryl, threonyl, or alanyl; A³ is absent or is a natural amino acid residue; A⁴ is aspartyl, glutamyl, asparaginyl, glutaminyl, or histidyl; A⁵ is a natural amino acid residue; A⁶ is isoleucyl or tyrosyl;

A⁷ is a natural amino acid residue; A⁸ is aspartyl, glutamyl, or glutaminyl. For a description of the profilin signature sequence, see Sohn et aL (1994), Bioessays 16: 465-72. "Control region" refers to a nucleotide sequence that regulates expression of a nucleic acid or any subunit thereof, including but not limited to any promoter, silencer, enhancer, splice site, transcriptional initiation element, transcriptional termination signal, polyadenylation signal, translational control element, translational start site, translational termination site, and message stability element. Such control regions may reside 5' or 3' to the coding region or in introns interrupting the coding region.

"Isogenic" refers to strains that have identical genomes but may have different plasmids. "Multi-copy plasmid" refers to a plasmid having 10 to 30 copies present in a cell.

"Natural amino acid residue" refers to alanyl, arginyl, aspartyl, asparaginyl, cysteinyl, glutamyl, glutaminyl, glycyl, histidyl, isoleucyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, threonyl, tryptophyl, tyrosyl, or valyl.

"Subject nucleic acid" refers to a nucleic acid (RNA or DNA) encoding G albicans profilin.

"Subject sequence" refers to a nucleotide sequence encoding C. albicans profilin.

"Subject polypeptide" and "subject protein" refer to C albicans profilin, whether isolated from a cell, synthesized by PCR, or produced by other means. Use and utility Persons of ordinary skill in the art may use the nucleic acids of the present invention in a variety of ways. For example, one can use the nucleic acids (1) to select DNA for related proteins from cDNA and genomic DNA libraries by hybridization; (2) to amplify cDNA or genomic DNA by polymerase chain reaction (PCR) techniques; or (3) to identify adjacent sequences in the cDNA or genome (e.g., control regions) by hybridization. One may also use the polypeptides and nucleic acids in assays to identify profilin-inhibitory compounds, which would be useful antifungal agents. For example, one could use the nucleic acids to prepare vectors, cells or cell lines used in such assays. Additional methods of using the nucleic acids, polypeptides, expression vectors and host cells are apparent from the present specification. Process of preparation

Nucleic acids

The present invention concerns an isolated nucleic acid molecule comprising a nucleic acid sequence coding for C. albicans profilin. The inventors prefer a nucleic acid having the nucleotide sequence of SEQ. ID. NO.: 1 or a nucleotide sequence complementary thereto. One may obtain the subject nucleic acid and related nucleic acids by a number of methods, including (1) Southern or northern blotting, (2) immunoblotting, (3) chemical synthesis, and (4) PCR amplification. In the first method, one may screen a genomic or cDNA library to identify a DNA encoding the subject protein or related proteins. Typically, one spreads the library clones on agarose plates, transfers them to filter membranes (e.g., nitrocellulose), and treats them with a detectable marker. The genomic library is usually contained in, for example, a yeast-E. coli shuttle vector such as YEplacl95. See Skorski & Heiter (1989), Genetics. 122: 15-27. The cDNA library may be contained in such vectors as λgtlO, λgtll, or lambda ZAP.

Alternatively, one of ordinary skill in the art may use known hybridization techniques on DNA or RNA isolated from yeast cells. See, for example, Kaiser et al (1994), Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. To detect nucleic acids having the subject sequence or related sequences, one typically treats the library or isolated nucleic acid sample with a labeled marker. The labeled marker may be an oligonucleotide (e.g., a labeled cDNA) having a sequence complementary to at least a portion of the subject sequence.

Suitable labels include radioactive ions (e.g., ³²P and ³⁵S) and biotin. One of ordinary skill in the art may add labels to oligonucleotides by known methods (e.g., the random primer method). Depending on the label, one can detect bound marker by various known methods (e.g., autoradiography, spectrophotometry). The presence of bound marker indicates presence of the desired nucleic acid.

In chemical synthesis, one typically synthesizes a series of 50 to 100 base oligonucleotides. Using appropriate terminal restriction sites, one then sequentially ligates these oligonucleotides to form the desired nucleic acid. In PCR, one typically uses a pair of selected DNA primers together with a polymerase to amplify an intervening region of DNA. The primers (usually about 15 to 40 base pairs long) anneal to opposite strands of the target DNA, and the polymerase catalyzes extension of the 3'-termini of the annealed primers. One causes this process to continue by repeated cycles of heat denaturation, thus amplifying the segment defined by the primers. See White, T. J. et al-, Trends Genet. 5, 185-9 (1989). The preferred method uses oligonucleotides corresponding to selected conserved amino acid sequences (shown in Figure 2). The preferred upstream primer is

SEQ. ID. NO. 5: ATGWSNTGGCAANSNTAYRWNGA which corresponds to the conserved amino acid sequence

SEQ. ID. NO. 6: MX*TWQX²YX³D. wherein X¹ is seryl or threonyl, X² is alanyl or seryl, and X³ is threonyl or valyl. The preferred downstream reverse primer is

SEQ. ID. NO. 7: TARTCNSCNARYTKYTCNAC the sense strand of which corresponds to the conserved amino acid sequence

SEQ. ID. NO. 8: VEX LX⁵DY wherein X⁴ is lysyl or glutaminyl and X⁵ is alanyl or glycyl.

Although the inventors determined the preferred SEQ. ID. NO.: 1 experimentally, the term "subject sequence" — and the scope of this invention — includes other sequences, as well. Due to the degeneracy of the genetic code, other DNA sequences encode the polypeptide having SEQ. ID. NO.: 2. Allelic variations also exist, which may change the amino acid sequence or may be silent. One can also modify the subject nucleic acids to prepare various mutations. Such mutations may be silent or may cause deletion, substitution, insertion, inversion or addition of one or more amino acids in the encoded polypeptide. One can make amino acid substitutions, for example, based on similar polarity, charge, solubility, hydrophobicity, hydrophilicity and /or the amphipathic nature of the residues involved. These mutations may modify activity (e.g., higher or lower activity), permit higher levels of production or easier purification, or provide additional restriction endonuclease recognition sites in the nucleic acid. One of ordinary skill in the art can effect such mutations with kits available from commercial vendors (e.g., Amersham Corp., Arlington Heights, IL) and known methods. See, for example, Taylor _t al- (1985), Nucl. Acids Res. 13: 8749- 64; Kunkel (1985), P e. Natl. Acad. So. USA 82: 482-92; Sayers et al (1988), Nucl. Acids Res. 16: 791-800. The present invention encompasses such nucleic acids and resulting polypeptides. Vectors

The present invention further concerns vectors comprising the subject sequence. The vectors preferably include the nucleotide sequence of SEQ. ID. NO.: 1. The inventors further prefer expression vectors comprising one or more control regions operatively linked to the subject sequence.

Vectors in the present invention are often in the form of plasmids — circular double-stranded DNA loops not bound to a chromosome. However, the invention includes other forms of vectors that serve equivalent functions and become known in the art subsequently hereto. One may also use the vectors to integrate the subject sequence into a chromosome of an appropriate host cell (e.g., E. coli, S. cerevisiae). Vectors of this invention typically contain control regions for inserted coding regions. Such control regions might include an origin of replication, a promoter, transcription termination sequences, and the like. The vectors may also include other DNA sequences known in the art, for example, stability leader sequences, which provide for stability of the expression product; secretory leader sequences, which provide for secretion of the expression product; sequences that allow expression of the structural gene to be modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium); marking sequences, which can provide phenotypic selection in transformed host cells; restriction sites, which provide sites for cleavage by restriction endonucleases; and sequences which allow expression in various types of hosts, including prokaryotes, yeasts, fungi, plants and higher eukaryotes.

The vectors of this invention are at least capable of directing the replication, and possibly the expression, of the subject sequence. One may insert the subject sequence into a number of known vectors, including baculovirus vectors (e.g., pBlueBac), prokaryotic vectors (e.g., pcDNAII) and yeast vectors (e.g., pYes2). One may also construct suitable vectors using recombinant DNA techniques known in the art. See, for example, Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY. For use in yeast cells, a suitable vector may contain the 2μ (2 micron) element, an autonomously replicating sequence (ARS), a gallO promoter, and any of a number of termination sequences (e.g., gallO). For E. coli cells, a suitable vector may contain the ColEl origin of replication and the lacZ promoter and termination sequences. For baculovirus expression systems, a suitable vector may include the SV40 viral origin of replication and the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter and termination sequences. To aid in selecting cells having the vector, the vector may also have a selectable marker gene. For yeast cells, the marker typically is a wild-type gene that confers a prototrophic phenotype to an auxotrophic mutant host cell. See, for example, Rosenbluh et aj. (1985), Mol. Gen- Genet., 200: 500-2. For E. coli cells, the marker typically confers resistance to an antibiotic (e.g., ampicillin). Host cells

The present invention additionally concerns host cells having an expression vector that comprises the subject sequence. The inventors prefer host cells that have a vector comprising the nucleotide sequence substantially as shown in SEQ. ID. NO.: 1. See, for example, the vector described hereinbelow. The inventors further prefer host cells having a vector with one or more control regions operatively linked to the subject sequence. Suitable host cells include both prokaryotic cells (e.g., E. coli strains HB101, DH5a, XL1 Blue, Y1090 and JM101) and such eukaryotic cells as Spodoptera frugiperda insect cells and S. cerevisiae cells (e.g., strain W303-1A).

One of ordinary skill in the art may introduce expression vectors into host cells by various known methods; for example, electroporation, viral or phage infection, or transformation of spheroplasts or lithium- treated cells. Thereafter, one may culture the host cell under conditions permitting expression of large amounts of the desired polypeptide. One can identify host cells having a vector with the subject sequence by any of several approaches. For example, one might (a) look for the presence of certain marker gene functions (e.g., rescue of auxotrophic mutant cells) as described above; (b) perform a Southern blot using probes complementary to the subject sequence; (c) isolate polyadenylated or total RNA and analyze it by Northern blotting or nuclease protection assay; (d) detect the presence of the subject protein by Western blotting with an antibody thereto; or (e) conduct PCR with primers homologous to the vector (either to the subject sequence or to other portions of the vector to produce DNA of predicted length.

One of ordinary skill in the art may determine the sequences of nucleic acids and vectors of this invention by various known methods; see for example, Sanger e l. (1977), Proc. Natl. Acad. Sci. USA 74: 5463-7, or Maxam-Gilbert (1977), Proc. Natl. Acad. Sci. USA 74: 560-4. Not all expression vectors and control regions will function equally well to express the subject sequence. Neither will all host cells function equally well with the same expression system. However, one of ordinary skill in the art may make a selection among expression vectors, DNA regulatory sequences, and host cells using the guidance provided herein without undue experimentation and without departing from the scope of the present invention. Polypeptides The invention further concerns isolated and purified C_. albicans profilin proteins and polypeptides. The inventors prefer polypeptides having the amino acid sequence of SEQ. ID. NO.: 2. One may obtain the subject polypeptides synthetically; i.e., chemical synthesis from component amino acids by such known methods as the solid phase procedure of Houghton et al. (1985), Proc. Natl. Acad. Sci. 82: 5131-5. One may also obtain them from the host cells described above or from in vitro translation of mRNA having the subject sequence. One may then purify them by such techniques as ion exchange chromatography, gel filtration chromatography and immunoaffinity chromatography.

One of ordinary skill in the art can use the subject polypeptides in a variety of ways. For example, one might prepare polyclonal or monoclonal antibodies capable of binding the polypeptides. In turn, one can use these antibodies to detect the subject polypeptides in a cell sample by, for example, radioimmunoassay, enzyme immunoassay, or immunocytochemistry. One can also use the antibodies in affinity chromatography to isolate and purify polypeptides from various sources. One of ordinary skill in the art can prepare various truncated, substituted, or otherwise modified polypeptides as described previously herein. One can also prepare salts, esters, or precursors of the aforementioned polypeptides. Precursors may, for example, have N- terminal substituents such as methionine, N-formylmethionine and leader sequences. The present invention encompasses all such variations.

Methods for detecting C_. albicans profilin inhibitors The present invention further concerns methods for detecting inhibitors of the subject protein. In this method, one compares zones of growth inhibition of a strain overexpressing or underexpressing the subject sequence and an isogenic control strain in the presence of test substances. An overexpressing strain has the subject sequence on a multi-copy vector or under the control of a strong yeast promoter on a single-copy vector. An underexpressing strain has the subject sequence under the control of a compromised promoter. The control strain includes the same vector but without the subject sequence. An inhibitor is any test substance that inhibits growth of the overexpressing strain less strongly than the control strain. See, for example, Rine et al- (1983), Proc. Natl. Acad. ≤cj. USA 80: 6750-4.

One may use antisense molecules as inhibitors. See, Toulme & Helene (1988), Gene 72: 51-8; Inouye (1988), Gene 72: 25-34; and Uhlmann & Peyman (1990), Chemical Reviews 90: 543-84. One can design antisense molecules based on genomic DNA and cDNA, corresponding 5' and 3' flanking control regions, other flanking sequences, or intron sequences. Such antisense molecules include antisense oligodeoxyribonucleotides, oligoribonucleotides, oligonucleotide analogues, and the like, and may comprise about 15 to 25 bases or more. Such antisense molecules may bind noncovalently or covalently to the subject nucleic acids. Such binding could, for example, cleave or facilitate cleavage of the subject nucleic acids, increase degradation of nuclear or cytoplasmic mRNA, or inhibit transcription, translation, binding of transactivating factors, or pre-mRNA splicing or processing. All of these effects would decrease expression of the subject sequence and thus make the antisense molecules useful in a method of treatment for fungal infection. Antisense molecules may also contain additional functionalities that increase their stability, activity, transport into and out of cells, and the like. Such additional functionalities may, for example, bind or facilitate binding to target molecules, or cleave or facilitate cleavage of target molecules.

One of ordinary skill in the art could construct vectors that direct the synthesis of antisense DNA or RNA as described previously herein. In this case, the antisense molecule may be much longer. Specific embodiments Results

Cloning of the profilin gene

In order to isolate the gene for G albicans profilin, cross- hybridization to the S. cerevisiae gene was attempted. The S. cerevisiae profilin gene described by Magdolen et al (1988), Mol. Cell. Biol. 8: 5108- 15, was isolated as a PCR product and used as a probe for Southern hybridization to G albicans genomic DNA. No cross-hybridization was found, even under low-stringency conditions, between the S. cerevisiae PFY1 gene and the DNA isolated from the C. albicans strain B792. Therefore, it was concluded that one could not clone the profilin gene from C albicans using simple hybridization. Thus, an alternative approach, using PCR with degenerate oligodeoxynucleotide primers, was employed.

Figure 1 shows an alignment of the predicted amino acid sequences deduced from profilin-encoding genes from various organisms. This alignment was used to select the two regions of high homology. Synthetic degenerate oligodeoxynucleotides that code for these regions (SEQ. ID. NOS. 5 AND 7) were used as primers for PCR with genomic DNA extracted from G albicans strain B792. Two major DNA products of 370 and 540 base pairs were obtained from these reactions. These products were blunt-end ligated into the sequencing plasmid BlueScript (Stratagene). For plasmid amplification the cells were grown on minimal media. DNA sequencing revealed that one of the products contained an open reading frame whose predicted amino acid sequence demonstrated homology with other profilin proteins.

The PCR product was then utilized to select for the full-length gene from the pSS1041 genomic C albicans library by colony hybridization. Seven clones were isolated by this procedure, three of which were unique. All were shown to contain genomic inserts which cross-hybridized to the degenerate PCR product. Restriction mapping also confirmed that the clones contained overlapping regions of genomic DNA. The common region was subcloned, and the nucleotide sequence determined. The region contained the same sequence found in the degenerate PCR product and is shown in Figure 3. However, the sequence terminated just upstream of the open reading frame, so no promoter sequence was present. This was true of all three unique library clones isolated.

In order to obtain the profilin promoter region, PCR was utilized. An oligonucleotide primer corresponding to position 87 of the sequence within the open reading frame in the upstream orientation (Figure 3) was synthesized. This primer was combined with primers homologous to sequences adjacent to the cloning site in the vector pSS1041 in a PCR reaction using DNA from the library as template. A single product was obtained and used to isolate a library clone which contained the promoter region of the profilin gene. Several clones were isolated in this manner, all of which were the same. Sequence analysis revealed that this clone abruptly truncated only fifty nucleotides into the profilin open reading frame.

The gene was termed PFY1, the C_. albicans gene for profilin. The nucleotide sequence predicts a protein of 126 amino acids and 13.8 kDa molecular weight. The sequence is highly homologous to the protein from S. cerevisiae (72% identity, 84% similarity). The inability of the profilin gene from S_. cerevisiae to hybridize with the gene from C_. albicans is explained by the fact that there is only 73% identity between the two genes at the nucleotide level. As with other profilin proteins, the predicted amino acid sequence bears relatively low homology with the protein from distantly related organisms (only 25% identity, 47% similarity with the human protein).

Interestingly, unlike the S . cerevisiae gene, the C. albicans gene does not possess an intron. Southern analysis showed a single hybridizing band with several restriction enzymes (data not shown), suggesting that PFY1 occurs in single copy per haploid genome in C. albicans. Chromosome blot hybridization demonstrated that the gene is located on chromosome III of all C. albicans strains tested (using the numbering scheme in Magee et al. (1988), Mol Cell Biol. 8: 4721-6. No CUG codons, which encode serine as opposed to leucine in G albicans (Santos et al. (1995), Nucleic Acids Res. 23: 1481-6), are found in the PFY1 open reading frame. Northern analysis of total RNA from C. albicans strain B-792 was employed. A single hybridizing band of approximately 700 bases in length was observed.

The cloned S. cerevisiae profilin gene was used to create a pfylΔ strain. Deletion of the PFY1 gene in S. cerevisiae is lethal, so the strain had to be complemented with a S. cerevisiae PFYl-expressing vector.

This vector contains the URA3 marker and, because growth of the strain is dependent on this plasmid, the strain is sensitive to 5-FOA. In addition to the PCR approach described herein, the technique of plasmid shuffle was employed to try to clone the C. albicans gene by complementation. Sikorski et al. (1991). Method Enzymol. 194: 302-18. Numerous attempts at using two different C. albicans genomic libraries to complement this profilin-deficient strain proved unsuccessful. Expression of the profilin gene In order to determine if the cloned gene was functional, a full- length copy of the cloned gene was constructed. Like plasmids containing the S. cerevisiae PFY1 gene without an intron, plasmids containing the full-length Q. albicans PFY1 gene, which does not have an intron, proved to be remarkably unstable in E. coli. It was necessary to select for reconstructions of the full-length gene using minimal bacterial media and growth at 30° C. A plasmid containing a reconstruction of the full-length C_. albicans profilin gene in an S_. cerevisiae shuttle vector was obtained and stably maintained in E. coli using these slow-growth conditions. Its sequence was verified by DNA sequencing.

In order to test if the cloned gene is a functional copy, this plasmid was utilized to transform S. cerevisiae profilin-deficient cells. A haploid pfylΔ strain containing the S. cerevisiae profilin gene on a high-copy URA3 plasmid was transformed with the Q. albicans PFY1 gene and 5- FOA-resistance employed to select for loss of the URA3 S. cerevisiae PFY1 plasmid. No 5-FOA-resistant colonies were obtained. A PFY1 /pfylΔ heterozygous diploid S. cerevisiae strain was also transformed with the plasmid. The strain was sporulated and tetrads dissected, but only two viable colonies per spore were observed. These colonies were verified to contain the full-length C. albicans PFY1 plasmid. These experiments suggest that the gene is not able to complement profilin-deficient S. cerevisiae cells. In order to test whether the C_. albicans promoter is functional in

S_. cerevisiae, the G albicans PFY1 open reading frame was cloned behind the S. cerevisiae GAL1 and MET25 promoters in high-copy shuttle vectors. These plasmid were used to transform both the heterozygous PFY1 /pfylΔ as well as the pfylΔ haploid strains. Transformants were tested for resistance to 5-fluoroorotic acid (5-FOA). No 5-FOA-resistant colonies were observed when plasmid shuffle was attempted with the GALlp /CaPFYl plasmid on glucose media. Likewise, only two viable spores were observed when the heterozygous PFYl/pfylΔ diploid strain containing this plasmid was sporulated on glucose plates. However 5- FOA-resistant colonies were obtained in the former case and four viable spores were observed in the latter case when galactose media was employed. It was further demonstrated that the 5-FOA-resistant colonies contained only the GALlp/CaPFYl plasmid. Likewise it was demonstrated that the profilin-deficient haploid progeny of the heterozygous diploid strain contained the plasmid. These strains were also observed to die when grown on glucose media. This demonstrated that expression of the £ albicans gene is sufficient to rescue the lethal effects of a profilin deletion.

Strains containing the MET25p /CaPFYl plasmid showed complementation under all conditions, but the growth rates of these strains were slow. Growth rates roughly corresponded inversely to the levels of methionine present in the media.

To confirm that complementation was due to expression of the G albicans gene, direct demonstration of presence of protein was provided by western blot analysis (Fig. 4). Although antiserum to S. cerevisiae profilin was shown to recognize the G albicans protein, the antiserum raised with C_. albicans profilin did not cross-react with the S. cerevisiae profilin. However, in strains that were complemented by a C albicans PFYl-expressing plasmid, a 14 kDa band was clearly visible. This is the same size as that seen in Q. albicans extracts and predicted from amino acid translation of the isolated gene. Higher molecular weight cross- reacting species were also observed in protein extracts of both S. cerevisiae and C. albicans. However, presence of these bands is unaffected by expression of the C albicans PFY1 gene. These experiments demonstrate that the C. albicans profilin gene is able to functionally complement S. cerevisiae cells. However, the C albicans PFY1 promoter cannot function in S_. cerevisiae. These experiments show that replacement of a C albicans promoter with an S. cerevisiae promoter is sufficient for expression of this C albicans gene in S. cerevisiae. Methods

Yeast strains and libraries

DNA was isolated from G albicans strains with the Elu-Quik DNA Purification Kit (Schleicher and Schuell). Yeast manipulations employed standard methodology. Kaiser et al. (1994), Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Yeast transformations were performed with a Gene Pulser (Bio-Rad) for electroporation. Becker and Guarente (1991), Methods Enzymol. 194: 182-7; Ostrander and Gorman (1994), Gene 148: 179-85. Hybridization studies utilized standard methodology. Sambrook et al- (1989) Molecular Cloning: A Laboratory Manual (2nd edn.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Chromosome blots were performed as previously described in Thrash-Bingham and Gorman (1992), Curr. Genet. 22: 93-100. The C_. albicans strain utilized for profilin protein isolation was B-

792. Kwon-Chung and Hill (1970), Sabouraudia 8: 48-59. C albicans strains used for cross-hybridization studies were B-311 (ATCC Ace. No. 32354) and B-792. Syverson et aj. (1975), Infect. Immun. 12: 1184-8. The Q. albicans genomic libraries were prepared as follows: in plasmid YEpl3 from strain SC5314— Gillum et al- (1984), Mfll. Gen. Genet. 198: 179-82; in plasmid SS1041 from strain 655 (ATCC Ace. No. 56884), kindly provided by Dr. S. Scherer— Goshorn et al. (1992), Infect Immun. 60: 876-84; in plasmid YSK35 from strain B-792, kindly provided by Dr. Y. Koltin— Fling et al. (1991), Mol. Gen. Genet. 227: 318-29. £. albicans strains used in chromosome blots were clinical isolates described as follows: strain 2023 (ATCC Ace. No. 10261)— MacKinnon et l. (1945), I. Bacteriol. 49: 317-34; A-81Pu— Kwon-Chung and Hill (1970), Sabouraudia 8: 48-59; B-311, B- 792, FC18 (ATCC Ace. No. 62376)— Whelan gt aj. (1981), I. Bacteriol. 145: 896-903; and SC5314 (Bristol-Myers Squibb Culture Collection). An S. cerevisiae PFYl/pfylΔ heterozygous strain, SD012 (ADE1 /adel.his3-ll /his3-Δl.leu2-3.112/leu2-3.112.PFY1 /pfyl ::LEU2.trpl- 1 /trpl-289,ura3-l /ura3-52). was created by transformation of the 3.1 kb EcoRI /Hindlll fragment of plasmid pD07 (described below). This strain produces only two viable spores per tetrad unless a plasmid expressing PFY1 is present. SD012 was transformed with plasmid pD03 (described below) and sporulated in order to give strain SD017 (his3, leu2-3. H2.pfyl::LEU2, trpl. ura3. MATa), a profilin-deficient strain. This strain does not require galactose media to survive, but 5-FOA-sensitive unless another gf^lΔ-complementing plasmid with a marker other than URA3 is present. A srv2-deficient strain, Y1035, was kindly provided by Dr. J. Broach (his3dl.leu2-3.112.1ys2-l.srv2::LEU2.trpl-289.ura3-52. Fedor- Chaiken et al. (1990), Cell 61: 329-40). Plasmids Plasmid constructions employed standard methodology.

Sambrook et aj. (1989), Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor; Kaiser et al. (1994), Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. DNA was isolated from C albicans strain B792 with the Elu-Quik DNA Purification Kit. Dr. S. Scherer provided the C. albicans genomic library described by Goshorn et al- (1992), Infect. Immun. 60: 876-84.

The original library clone chosen for restriction analysis was approximately 17 kb in length. Because the parent vector has a length of 9,850 base pairs, the insert was approximately seven kb. This insert was excised from the vector by digestion with EcoRI and BamHI. leaving approximately 550 base pairs of parent sequence on the EcoRI side. In addition, the insert was cleaved into 3.0 and 4.5 kb fragments with Hpal. The 3.0 kilobase EcoRI /Hpal fragment included the 550 base pairs of parent vector sequence. This fragment was ligated to a 5100 base pair EcoRI/Hpal fragment of the parent vector pSS1041 described by Goshorn et al- This plasmid, pD059, contained a 2.5 kb genomic fragment that included the Q. albicans PFY1 gene. Plasmid pD059 also included the S_. cerevisiae 2 micron element, the ampicillin resistance gene and the C_. albicans ARS element.

Oligodeoxynucleotides were synthesized on an Model 391 PCR- Mate DNA Synthesizer (Applied Biosystems), and PCR reactions were performed in a DNA Thermal Cycler (Perkin-Elmer Cetus) according to established protocols (Saiki et al., 1988; Compton, 1990). Standard PCR conditions consisted of thirty cycles of 94 °C, 1'; 52 °C, 2'; 72 °C, 3'. Low stringency PCR was performed using thirty-five cycles of 94 °C, 1'; 42 °C, 2'; 72 °C, 5' with a ten-second time extension on each subsequent elongation step. PCR reactions utilized Vent polymerase (New England Biolabs), a 3' → 5' exonuclease active thermal-stable polymerase. Sequence alignment, nucleotide and amino acid sequence analyses and comparisons utilized the Genetics Computer Group Sequence Analysis Software Package (Devereux et al., 1984). Sequencing was performed by the dideoxynucleotide technique (Tabor and Richardson, 1987) using the Sequenase Version 2 DNA Sequencing Kit (United States Biochemical) and synthetic oligodeoxynucleotides as primers.

In order to generate antibodies to S. cerevisiae profilin, an intron- less copy of the gene was generated so that the protein could be over- expressed in E. coli. Plasmid pD02 was constructed as follows: The second exon of the S. cerevisiae PFY1 gene was cloned by PCR using the following primers:

SEQ. ID. NO. 25: 5'-CATGGCAAGCATACACTGATAACTTAA-3' and SEQ. ID. NO. 26: 5'-CTGCATAAATTAGTATTGAACAC-3'. The 390 bp PCR product was cut with Kpnl and Ncol and ligated into a pUC18 (Yanisch-Perron et l., 1985) derivative with an Ncol site in the polylinker. The first four amino acids of the gene (the first exon) were added to the second exon by cutting the previous vector with BamHI and Ncol and ligating the following annealed oligonucleotides: SEQ. ID. NO. 27: 5'-GATCCGTTAACATGT-3' and SEQ. ID. NO. 28: 5 -CATGACATGTTAACG-3'.

The sequence of this intron-less copy of the gene was verified by DNA sequencing. The 400 bp gene was extracted from the plasmid with Asp718I and BamHI and placed behind the GAL1 promoter in plasmid MHlOl (Haffey et al., 1988) cut with BamHI and Hindlll. The ligation was accomplished by filling-in the Asp718I and Hindlll sites and gave plasmid pD03. At first, a few constructions were produced at very low frequency all of which proved to possess point mutations which cause frame-shifts very close to the beginning of the open reading frame. By growing the E. coli cells on minimal media at 30 °C, plasmid pD03 was successfully obtained and maintained.

In order to disrupt PFYl in S. cerevisiae cells, plasmid pD07 was constructed. Plasmid pD02 was linearized with Sail and reclosed, leaving 300 bp of the PFYl open reading frame at the 3' end. The LEU2 gene was then added upstream of this fragment using Sail and Sphl. A region of the PFYl promoter was cloned by PCR using the following primers:

SEQ. ID. NO. 29: 5'-GTGAAGCTTGGACGACGAAGACGAGG-3' and SEQ. ID. NO. 30: 5'-TCGCTGCAGACCGGTTCCTATTAAGTTATC-3'. This fragment was then inserted beside the LEU2 gene using

Hindlll and Pstl. The resulting plasmid could be cut with EcoRI and Hindlll to produce a 3.1 kb PFY1::LEU2 disruption fragment (see above). Plasmid pD056, a clone from the pSS1041 library, was isolated by colony hybridization. The plasmid is approximately 17 kb in length, and, because the parent vector has a length of 9,850 base pairs, the insert is approximately seven kb in length. This insert was excised from the vector by cutting with EcoRI and BamHI, leaving approximately 550 bp of parent sequence on the EcoRI side. This fragment was additionally cleaved into 3.0 and 4.5 kb fragments with Hpal. The 3.0 kb EcoRI /Hpal fragment, which included the 550 base pairs of parent vector sequence, was found to cross-hybridize to the PCR-generated PFYl fragment and was ligated to a 5100 bp EcoRI /Hpal fragment of the parent vector. This plasmid, pD059, which includes the S_. cerevisiae 2 micron element, the ampicillin resistance gene and the Q. albicans ARS element, therefore contains a 2.5 kb genomic fragment containing DNA which cross- hybridizes with . albicans PFYl. This plasmid was used to sequence the £. albicans PFYl gene open reading frame and terminator regions (see text).

The promoter region of PFYl was isolated by low-stringency PCR using a primer within the open reading frame in the upstream orientation

SEQ. ID. NO. 31: 5'-TAATGCGTCACCTGCTCTTG-3' (position 87, Fig. 3) in combination with pSS1041 vector-sequence primers flanking the library cloning site:

SEQ. ID. NO. 32: 5'-CCAAATCAATTCCTATTAGT-3' and

SEQ. ID. NO. 33: 5'-CACGATGCGTCCGGCGTAGA-3'. This reaction produced a product of approximately 3.0 kb which was utilized to select library clones by cross-hybridization. A second library clone, pD058, isolated in this manner, is approximately 13 kb in length, containing a genomic insert of approximately 3200 bp. The insert was excised from the library vector by cutting with EcoRI and SphI leaving approximately 550 bp of vector sequence on the EcoRI side and 200 bp on the SphI side in a 4.0 kb fragment. The fragment was subcloned into pBlueScript (Stratagene) to create plasmid ρDO60. This plasmid was used to sequence the promoter region of the profilin gene and was found to contain only about 100 bp of PFYl open reading frame. The full gene was reassembled using the Apal site found near the beginning of the open reading frame. The 900 bp Apal/Smal fragment of pDO60 was ligated to pD059 cut with Apal and Hpal. The resulting plasmid, D067, was found to be extremely unstable in E.. coli unless grown on minimal media agar at 30 °C (see above and text). The reassembled gene was then subcloned into YEpLacll2 (Gietz and Sugino, 1988) using Ncol and Xbal to create plasmid pD068 which was used to test expression of the full-length gene in S_. cerevisiae. This plasmid also required slow growth conditions for selection and maintenance. The open reading frame of the C. albicans profilin gene was amplified by PCR using primers: SEQ. ID. NO. 34: 5'-ATAGAATTCTTATGTCTTGGCAAGCATACA-3'

(position -2, Fig. 3) and SEQ. ID. NO. 35: 5 -CGCAAGCTTGCACGTCCATGGATGCAATGA-3' (position 408, Fig. 3).

This fragment was utilized as a probe in both northern and chromosome blots. An S_. cerevisiae expression vector was created by cutting of this PCR product with EcoRI and Hindlll and ligation into a TRP1 version of the high-copy shuttle vector pYES2 (Invitrogen) to create plasmid pD069, a galactose-inducible, glucose-repressible expression vector. The methionine-repressible expression plasmid pDO70 was created by cutting of the same PCR product with EcoRI and ligation into plasmid RS424Met, a high-copy, TRP1. MET25 promoter shuttle vector (Mumberg et al. (1994), Nucleic Acids Res. 22: 5767-8), cut with EcoRI and Smal. Plasmid MF216, containing the SRV2 (CAPl) gene on a high-copy vector was kindly provided by Dr. J. Broach. Fedor-Chaiken et l. (1990), Cell 61: 329-40.

Protein Purification and Antibody Production In order to demonstrate the presence of profilin in C. albicans cells, the protein was isolated. This was accomplished by the well- established method of poly-L-proline column binding Kaiser et al. (1989), Cell Motil. Cytoskel. 14: 251-62. The only protein observed to be specifically retained by the column was one of 14 kDa apparent molecular weight. This is the predicted mass of profilins. Sohn gt al. (1994), BioEssays 16: 465-72. The protein was also observed to cross-react with antisera generated against S. cerevisiae profilin. Therefore a protein of the predicted molecular weight which cross-reacts with antisera from a closely related yeast species is present in C. albicans.

Anti-S_. cerevisiae profilin antisera production has been described previously. Ostrander et al- (1995), I BJol Chem 46: 27045-50. Anti-C. albicans profilin antisera was prepared in the same manner using protein from strain B-792. Western analyses utilized 10-20% gradient Ready Gels (Bio-Rad), and the Mini-V Transfer and PhotoBlot Chemiluminescent Detection Systems (Gibco-BRL).

Claims

What Is Claimed Is:

1. An isolated nucleic acid molecule encoding a C albicans profilin.

2. The nucleic acid molecule of Claim 1 encoding C. albicans profilin having the amino acid sequence of SEQ. ID. NO.: 2.

3. The nucleic acid molecule of Claim 1 having the nucleic acid sequence of SEQ. ID. NO.: 1.

4. A vector comprising the nucleic acid sequence of the nucleic acid molecule of Claim 1.

5. A method for detecting a nucleic acid sequence coding for C. albicans profilin, which comprises:

(a) contacting the nucleic acid sequence with a detectable marker which binds specifically to at least part of the nucleic acid sequence, and

(b) detecting the marker so bound; wherein the presence of bound marker indicates the presence of the nucleic acid sequence.

6. A prokaryotic or eukaryotic host cell comprising the vector according to Claim 5.

7. A method for producing C_. albicans profilin, which comprises culturing a host cell according to Claim 6 under conditions permitting expression of the protein.

8. An isolated and purified C_. albicans profilin protein.

9. The isolated protein of Claim 8 having the amino acid sequence of SEQ. ID. NO.: 2.

10. A process for detecting an inhibitor of C albicans profilin comprising:

(a) incubating a sample thought to contain an inhibitor of C_. albicans profilin with fungal cells; and

(b) detecting the rate of growth and/or death of the fungal cells; wherein the inhibitor will decrease the rate of growth and /or increase the rate of death of the fungal cells.

11. A nucleic acid molecule isolated from C albicans comprising SEQ. ID. NO. 3.