Patent Publication Number: US-2005136394-A1

Title: Cell-based assay for identifying peptidase inhibitors

Description:
The present application claims benefit of priority to U.S. Provisional Ser. No. 60/480,625, filed Jun. 23, 2003, the entire contents of which are hereby incorporated by reference. 
    
    
      The government owns rights in the present invention pursuant to NSF CAREER Award #9985479 and NSF MCB #9604669, both from the National Science Foundation. 
    
    
     BACKGROUND OF THE INVENTION  
      1. Field of the Invention  
      The invention relates generally to the fields of microbiology and pathology. More particularly, the invention relates to assays for the rapid identification of peptidase inhibitors.  
      2. Description of Related Art  
      The emerging bioterrorism threat has galvanized the need for rapidly effective treatments against deadly bacterial toxins. Many of the bacterial toxins are endopeptidases that destroy specific essential proteins within host cells. These bacterial toxins include, but are not limited to, botulinum neurotoxin (BONT) and anthrax lethal factor.  
      A traditional method of treating infections of this nature is by administering antibiotics. One of the major impediments in treating bacterial infections in this way is the limited susceptibility of bacteria to various antibiotics. Also, as bacteria reproduce quickly, any that are resistant to the drug used may quickly replace the ones that are killed, thereby further reducing the effectiveness of the drug.  
      Even with effective antibiotic control of infection through antibiotics, the effects of the toxins that have already been produced are not mitigated. Therefore, to counteract the effects of the toxins, another drug or treatment needs to be administered. One such class of drugs are bacterial endopeptidase inhibitors, which prevent the toxins from damaging host cells. However, identifying inhibitors of specific toxins is a time consuming process, requiring many tests and trials before a drug may be produced. For example, certain assays measure of peptidase activity using electrophoretic separation of the cleavage products—a slow and cumbersome approach. Assays using fluorogenic substrates have been developed, and liquid chromatography (HPLC) and mass spectroscopy provide promising new avenues of attack, but to date, these have not provided entirely satisfactory results. Thus, there remains a need for simple and fast methods of identifying and isolating bacterial endopeptidase inhibitors.  
     SUMMARY OF THE INVENTION  
      Thus, in accordance with the present invention, there is provided a method of identifying an endopeptidase inhibitor comprising (a) providing a yeast cell, wherein said cell expresses a polypeptide that is essential to yeast cell viability or growth, wherein said polypeptide comprises or has been modified to comprise a cleavage site for said endopeptidase; (b) contacting said yeast cell and said endopeptidase in the presence of a candidate substance; and (c) assessing the viability and/or growth of said yeast cell, wherein improved viability and/or growth of said yeast cell in the presence of said candidate substance, as compared to viability and/or growth of said yeast cell in the absence of said candidate substance, identifies said candidate substance as a endopeptidase inhibitor. The endopeptidase may be a serine endopeptidase, a cysteine endopeptidase, an aspartic endopeptidase or a metallo endopeptidase, more particularly a bacterial toxin endopeptidase, and more specifically a  Botulinum  neurotoxin (BoNT), wherein said endopeptidase cleavage site is Q/F for BoNTB/LC, and K/A for BoNTC/LC. The modified polypeptide may also comprise a protease binding site.  
      The essential polypeptide may be Snc1 or Snc2 or Sso1 or Sso2. Viability may be measured by standard culture methods, by flow cytometry by selective staining, by the slide viability method, by flocculation test, or by fermentation test. Growth may be measured by assessing incorporation of radioactive nucleotides or by cell counting. The yeast cell may further comprises a null mutation in a functionally redundant homolog of said essential polypeptide. The yeast cell may also further comprise an endopeptidase transgene under the control of an inducible promoter, and contacting comprises growing said yeast cell under conditions that induce said promoter, thereby permitting expression of said endopeptidase in said yeast cell. The inducible promoter may be a yeast inducible promoter (e.g., Gal1, Gal10, GalS, or GalL, and said conditions that induce said promoter comprises culturing said yeast in galactose), or a non-yeast inducible promoter (e.g., a tetracycline-responsive promoter and said conditions that induce said promoter comprises culturing said yeast in tetracycline).  
      The candidate substance may be a peptide or polypeptide and providing said peptide or polypeptide comprises contacting said yeast cell with an expression construct encoding said peptide or polypeptide. The polypeptide may be an antibody or an enzyme. The candidate substance may be an organopharmaceutical or a siRNA.  
      In another embodiment, there is provided a yeast cell that expresses a polypeptide that is essential to yeast cell viability or growth, wherein said polypeptide comprises a heterologous cleavage site for a endopeptidase, and optionally a heterologous endopeptidase binding site. The yeast cell may further comprise a transgene encoding said endopeptidase under the control of an inducible promoter. The inducible promoter is a yeast inducible promoter or a non-yeast inducible promoter. The yeast cell may further comprise a null mutation in a functionally redundant homolog of said polypeptide that comprises said heterologous cleavage site.  
      The present invention, in all of the preceding embodiments, also envisions the use of exopeptidases in analogous assays. Such exopeptidases may have either C-terminal or N-terminal peptidase funtions.  
      It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” 
      Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.  
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.  
       FIG. 1 —Example of yeast cell-based assays using Snc2. The agar plate on the left contains glucose, and the agar plate on the right contains galactose.  
       FIG. 2 —Example of yeast cell-based assays using Sso1/2. The agar plate on the left contains glucose, and the agar plate on the right contains galactose.  
       FIG. 3 —Sequence alignments for Syntaxin and Sso1/2p. Shaded regions show areas of homology (identity or conservative substitution); syntaxin cleavage site indicated by an arrow.  
    
    
     DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS  
      I. The Present Invention  
      The present invention provides a rapid and sensitive system for identifying and isolating pharmaceutically effective compounds that inhibit the proteolytic activity of peptidases (also known as proteases), such as endo- and exopeptidases (endo- and exoproteases) within eukaryotic cells. In a particular embodiment, the assay of the invention makes use of recombinant yeast cells that harbor an endopeptidase that cleaves an essential yeast protein. In certain cases, the yeast cells will have been further engineered to comprise an essential protein that contains a heterologous proteolytic cleavage site for the endopeptidase in question. When expression of endopeptidase is induced, cleavage of the essential protein occurs and cell death ensues. However, inclusion of an appropriate endopeptidase inhibitor in this culture can block cleavage and prevent cell death.  
      This format is readily scalable so that one can screen large numbers of putative inhibitors, such as peptides, siRNA, antisense molecules, small molecules, in a rapid fashion. The assay offers several distinct advantages: (1) positive growth selection is a much more powerful, efficient and economic approach than existing screening procedures; (2) the technology employs function-based assays to isolate toxin inhibitors, which is preferable over the affinity binding-based assays mostly commonly used in inhibitor screening procedures; and (3) a one step cell-based assay not only selects for toxin inhibitors, but eliminates inhibitors that are toxic to yeast, a model eukaryotic cell that is related to human cells. Together, these advantages provide a faster screen for large numbers of candidate substances which are more likely to be effective and safe when applied to animals and humans. The details of this invention are described further in the following pages.  
      II. Peptidases, Endopeptidases and Exopeptidases  
      A peptidase is an enzyme that cleaves a peptide bond. An endopeptidase is any peptidase that catalyzes the cleavage of internal peptide bonds in a polypeptide or protein. Endopeptidases are divided into subclasses on the basis of catalytic mechanism: the serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases, metalloendopeptidases, and other endopeptidases. Exopeptidases cleave proteins near the carboxy- or amino-termini, and thus are termed carboxy- or amino-exopeptidases.  
      There are a substantial number of different peptidases present in cells with differing specificities, so as to require different sequences and/or conformations of the polypeptide as the cleavage site. With hybrid DNA technology, one tries to provide a high level of production of a polypeptide product, which is in addition to the normal cellular products. Where such polypeptide requires processing, the cell may not be able to respond to the increased processing load. However, the mere fact of providing for enhanced genetic capability of producing the peptidase is no assurance that there will be an enhanced or more efficient processing of the peptidase substrate. See U.S. Pat. No. 5,077,204, herein incorporated by reference in its entirety.  
      A. Serine Endopeptidases  
      This class comprises two distinct families. The chymotrypsin family which includes the mammalian enzymes such as chymotrypsin, trypsin or elastase or kallikrein and the substilisin family which include the bacterial enzymes such as subtilisin. The general 3D structure is different in the two families but they have the same active site geometry and then catalysis proceeds via the same mechanism. The serine endopeptidases exhibit different substrate specificities which are related to amino acid substitutions in the various enzyme subsites interacting with the substrate residues. Some enzymes have an extended interaction site with the substrate whereas others have a specificity restricted to the P1 substrate residue.  
      Three residues which form the catalytic triad are essential in the catalytic process, i.e., His 57, Asp 102 and Ser 195 (chymotrypsinogen numbering). The first step in the catalysis is the formation of an acyl enzyme intermediate between the substrate and the essential Serine. Formation of this covalent intermediate proceeds through a negatively charged tetrahedral transition state intermediate and then the peptide bond is cleaved. During the second step or deacylation, the acyl-enzyme intermediate is hydrolyzed by a water molecule to release the peptide and to restore the Ser-hydroxyl of the enzyme. The deacylation which also involves the formation of a tetrahedral transition state intermediate, proceeds through the reverse reaction pathway of acylation. A water molecule is the attacking nucleophile instead of the Ser residue. The His residue provides a general base and accept the OH group of the reactive Ser.  
      B. Cysteine Endopeptidases  
      This family includes the plant proteases such as papain, actinidin or bromelain, several mammalian lysosomal cathepsins, the cytosolic calpains (calcium-activated) as well as several parasitic proteases (e.g.,  Trypanosoma, Schistosoma ). Papain is the archetype and the best studied member of the family. Recent elucidation of the X-ray structure of the Interleukin-1-beta Converting Enzyme has revealed a novel type of fold for cysteine endopeptidases. Like the serine endopeptidases, catalysis proceeds through the formation of a covalent intermediate and involves a cysteine and a histidine residue. The essential Cys25 and His159 (papain numbering) play the same role as Ser195 and His57 respectively. The nucleophile is a thiolate ion rather than a hydroxyl group. The thiolate ion is stabilized through the formation of an ion pair with neighboring imidazolium group of His159. The attacking nucleophile is the thiolate-imidazolium ion pair in both steps and then a water molecule is not required.  
      C. Aspartic Endopeptidases  
      Most of aspartic endopeptidases belong to the pepsin family. The pepsin family includes digestive enzymes such as pepsin and chymosin as well as lysosomal cathepsins D and processing enzymes such as renin, and certain fungal proteases (penicillopepsin, rhizopuspepsin, endothiapepsin). A second family comprises viral endopeptidases such as the protease from the AIDS virus (HIV) also called retropepsin. Crystallographic studies have allowed to show that these enzymes are bilobed molecules with the active site located between two homologous lobes. Each lobe contributes one aspartate residue of the catalytically active diad of aspartates. These two aspartyl residues are in close geometric proximity in the active molecule and one aspartate is ionized whereas the second one is unionized at the optimum pH range of 2-3. Retropepsins, are monomeric, i.e., carry only one catalytic aspartate and then dimerization is required to form an active enzyme.  
      In contrast to serine and cysteine proteases, catalysis by aspartic endopeptidases do not involve a covalent intermediate though a tetrahedral intermediate exists. The nucleophilic attack is achieved by two simultaneous proton transfer: one from a water molecule to the diad of the two carboxyl groups and a second one from the diad to the carbonyl oxygen of the substrate with the concurrent CO—NH bond cleavage. This general acid-base catalysis, which may be called a “push-pull” mechanism leads to the formation of a non-covalent neutral tetrahedral intermediate.  
      D. Metallo Endopeptidases  
      The metallo endopeptidases may be one of the older classes of endopeptidases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures but the great majority of enzymes contain a zinc atom which is catalytically active. In some cases, zinc may be replaced by another metal such as cobalt or nickel without loss of the activity. Bacterial thermolysin has been well characterized and its crystallographic structure indicates that zinc is bound by two histidines and one glutamic acid. Many enzymes contain the sequence HEXXH, which provides two histidine ligands for the zinc whereas the third ligand is either a glutamic acid (thermolysin, neprilysin, alanyl aminopeptidase) or a histidine (astacin). Other families exhibit a distinct mode of binding of the Zn atom. The catalytic mechanism leads to the formation of a non covalent tetrahedral intermediate after the attack of a zinc-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group.  
      E. Bacterial/Toxin Endopeptidases  
      Toxin endopeptidases, usually of bacterial origin, can have a devastating and sometime lethal impact on host organisms. Some of the better known bacterial endopeptidase toxin are listed below in Table 1.  
               TABLE 1                          Bacterial Endopeptidases                                             Substrate               Mode of   Target   Homolog in       Organism/Toxin   Action   (Cleavage Site)   Yeast   Disease                 B. anthracis /lethal   Metalloprotease   MAPKK1/MAPKK2   MEKK   Anthrax       factor       (multiple)   Family         C. botulinum /   Zinc-   SNAP-25   SEC9   Botulism       neurotxin A   metalloprotease   (ANQ/RAT)         C. botulinum /   Zinc-   VAMP/synaptobrevin   Snc1, Snc2   Botulism       neurotxin B   metalloprotease   (ASQ/FET)         C. botulinum /   Zinc-   Syntaxin   Sso1, Sso2   Botulism       neurotxin C   metalloprotease   (TKK/AVK)         C. botulinum /   Zinc-   VAMP/synaptobrevin   Snc1, Snc2   Botulism       neurotxin D   metalloprotease   (DQK/LSE)         C. botulinum /   Zinc-   SNAP-25   SEC9   Botulism       neurotxin E   metalloprotease   (IDR/IME)         C. botulinum /   Zinc-   VAMP/synaptobrevin   Snc1, Snc2   Botulism       neurotxin F   metalloprotease   (RDQ/KLS)         C. botulinum /   Zinc-   VAMP/synaptobrevin   Snc1, Snc2   Botulism       neurotxin G   metalloprotease   (TSA/AKL)         Yersinia  virulence   Cysteine   Unknown   Unknown   Plague       factor YopJ   protease         Yersinia  virulence   Cysteine   Prenylated cysteine   Unknown   Plague       factor YopT   protease         Salmonella  virulence   unknown   Unknown   Unknown   Salmonellosis       factor AvrA         Clostridium     Zinc-   VAMP/synaptobrevin   Snc1, Snc2   Tetanus         tetani /tetanus toxin   metalloprotease   (ASQ/FET)                  
 
      The  C. botulinum  neurotoxins (BoNTs, serotypes A-G) and the  C. tetani  tetanus neurotoxin (TeNT) are two examples of bacterial toxins that are endopeptidases. BoNTs are most commonly associated with infant and food-borne botulism and exist in nature as large complexes comprised of the neurotoxin and one or more associated proteins believed to provide protection and stability to the toxin molecule while in the gut. TeNT, which is synthesized from vegetative  C. tetani  in wounds, does not appear to form complexes with any other protein components.  
      The BoNTs and TeNT are either plasmid encoded (TeNT, BoNTs/A, G, and possibly B) or bacteriophage encoded (BoNTs/C, D, E, F), and the neurotoxins are synthesized as inactive polypeptides of 150 kDa (44). BoNTs and TeNT are released from lysed bacterial cells and then activated by the proteolytic cleavage of an exposed loop in the neurotoxin polypeptide. Each active neurotoxin molecule consists of a heavy (100 kDa) and light chain (50 kDa) linked by a single interchain disulphide bond. The heavy chains of both the BoNTs and TeNT contain two domains: a region necessary for toxin translocation located in the N-terminal half of the molecule, and a cell-binding domain located within the C-terminus of the heavy chain. The light chains of both the BoNTs and TeNT contain zinc-binding motifs required for the zinc-dependent protease activities of the molecules.  
      The cellular targets of the BoNTs and TeNT are a group of proteins required for docking and fusion of synaptic vesicles to presynaptic plasma membranes and therefore essential for the release of neurotransmitters. The BoNTs bind to receptors on the presynaptic membrane of motor neurons associated with the peripheral nervous system. Proteolysis of target proteins in these neurons inhibits the release of acetylcholine, thereby preventing muscle contraction. BoNTs/B, D, F, and G cleave the vesicle-associated membrane protein and synaptobrevin, BoNT/A and E target the synaptosomal-associated protein SNAP-25, and BoNT/C hydrolyzes syntaxin and SNAP-25. TeNT affects the central nervous system and does so by entering two types of neurons. TeNT initially binds to receptors on the presynaptic membrane of motor neurons but then migrates by retrograde vesicular transport to the spinal cord, where the neurotoxin can enter inhibitory interneurons. Cleavage of the vesicle-associated membrane protein and synaptobrevin in these neurons disrupts the release of glycine and gamma-amino-butyric acid, which, in turn, induces muscle contraction. The contrasting clinical manifestations of BoNT or TeNT intoxication (flaccid and spastic paralysis, respectively) are the direct result of the specific neurons affected and the type of neurotransmitters blocked.  
      Of particular interest is BoNT/LC (serotype C), and specifically BoNTC/LC (as compared to other LC serotypes). First, BoNTC/LC poses a particularly significant bioterror threat because it has a long half-life inside human neuronal cells. Second, an in vitro assay for BoNTC/LC does not currently exist, probably because this LC protease appears to require membranes to function. In the neuronal cell environment, BoNTC/LC cleaves syntaxin, a membrane protein required for synaptic vesicle fusion to the presynaptic membrane. The yeast  Saccharomyces cerevisiae  has two functionally redundant homologs of syntaxin, Ssolp and Sso2. Sso1p and Sso2p perform the same required step in the fusion of secretory vesicles to the plasma membrane of yeast, indicating syntaxin exhibits functional similarities to Ssolp and Sso2p. As can be seen in  FIG. 3 , syntaxin exhibits strong sequence similarity to Ssolp and Sso2p, particularly at the syntaxin cleavage site (indicated by an arrow).  
      Other examples include the  Yersinia  virulence factors YopJ and YopT, as well as  Salmonella  AvrA.  
      F. Exopeptidases  
      Exopeptidases act only near the ends of polypeptide chains, and those acting at a free N-terminus liberate a single amino-acid residue (aminopeptidases), or a dipeptide or a tripeptide (dipeptidyl-peptidases and tripeptidyl-peptidases). The exopeptidases acting at a free C-terminus liberate a single residue (carboxypeptidases) or a dipeptide (peptidyl-dipeptidases). The carboxypeptidases are allocated to three groups on the basis of catalytic mechanism: the serine-type carboxypeptidases, the metallocarboxypeptidases and the cysteine-type carboxypeptidases. Other exopeptidases are specific for dipeptides (dipeptidases), or remove terminal residues that are substituted, cyclized or linked by isopeptide bonds (peptide linkages other than those of α-carboxyl to α-amino groups) (σ peptidases).  
      III. Candidate Endopeptidase Inhibitors  
      A. Known Inhibitors  
      Over 100 naturally-occurring protein protease inhibitors have been identified so far, thereby demonstrating the likelihood of finding additional endopeptidase inhibitors. They have been isolated in a variety of organisms from bacteria to animals and plants. They behave as tight-binding reversible or pseudo-irreversible inhibitors of proteases preventing substrate access to the active site through steric hindrance. Their size are also extremely variable from 50 residues (e.g., BPTI: Bovine Pancreatic Trypsin Inhibitor) to up to 400 residues (e.g., α-1PI: α-1 Endopeptidase Inhibitor). They are strictly class-specific except proteins of the alpha-macroglobulin family (e.g., α-2 macroglobulin) which bind and inhibit most proteases through a molecular trap mechanism.  
      Serine protease inhibitors have been the most studied protein inhibitors up to know and recently a considerable advance has been made in the study of the natural inhibitors of cysteine proteases (cystatins). Some other endopeptidase inhibitors include Amastatin, E-64, Antipain, Elastatinal, APMSF, Leupeptin, Bestatin, Pepstatin, Benzamidine, 1,10-Phenanthroline, Chymostatin, Phosphoramidon, 3,4-dichloroisocoumarin, TLCK, DFP and TPCK.  
      B. Natural and Synthetic Inhibitors  
      As used herein, the term “candidate inhibitor” refers to any molecule that may potentially reduce endopeptidase cleavage. The candidate may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to compounds which interact naturally with endopeptidases. Creating and examining the action of such molecules is known as “rational drug design,” and include making predictions relating to the structure of the target molecules and the candidate substance.  
      The goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a molecule like an endopeptidase, and then design a molecule for its ability to interact with these polypeptides. Alternatively, one could design a partially functional fragment of these polypeptides (binding, but no activity), thereby creating a competitive inhibitor. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.  
      It also is possible to use antibodies to ascertain the structure of a target compound. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.  
      On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to “brute force” the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries (e.g., peptide libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds.  
      Candidate compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds.  
      Yet further, the candidate substance may be a known antibiotic. The term “antibiotics” as used herein is defined as a substance that inhibits the growth of microorganisms without equivalent damage to the host. Yet further, it is within the scope of the present invention to synthesis or produce analogs of known antibiotics. These analogs may have been altered, for example site-directed mutagenesis, to exhibit increased antimicrobial activity.  
      It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.  
      IV. Yeast  
      Yeast are unicellular fungi whose mechanisms of cell-cycle control are remarkably similar to that of humans. The precise classification is a field that uses the characteristics of the cell, ascospore and colony. Physiological characteristics are also used to identify species. One of the more well known characteristics is the ability to ferment sugars for the production of ethanol. Budding yeasts are true fungi of the phylum  Ascomycetes , class  Hemiascomycetes . The true yeasts are separated into one main order  Saccharomycetales . Yeasts are characterized by a wide dispersion of natural habitats, and are common on plant leaves and flowers, soil and salt water. Yeasts are also found on the skin surfaces and in the intestinal tracts of warm-blooded animals, where they may live symbiotically or as parasites.  
      Yeasts multiply as single cells that divide by budding (e.g.,  Saccharomyces ) or direct division (fission, e.g.,  Schizosaccharomyces ), or they may grow as simple irregular filaments (mycelium). In sexual reproduction most yeasts form asci, which contain up to eight haploid ascospores. These ascospores may fuse with adjoining nuclei and multiply through vegetative division or, as with certain yeasts, fuse with other ascospores.  
      The awesome power of yeast genetics is partially due to the ability to quickly map a phenotype producing gene to a region of the  S. cerevisiae  genome. For the past two decades,  S. cerevisiae  has been the model system for much of molecular genetic research because the basic cellular mechanics of replication, recombination, cell division and metabolism are generally conserved between yeast and larger eukaryotes, including mammals. It is also a straightforward matter to engineer yeast cells to express a variety of heterologous constructs, and to do so in a controlled fashion.  
      A. Yeast Cultures  
      Some yeast varieties reproduce almost as rapidly as bacteria and have a genome size less than 1% that of a mammal. They are amenable to rapid molecular genetic manipulation, whereby genes can be deleted, replaced, or altered. They also have the unusual ability to proliferate in a haploid state, in which only a single copy of each gene is present in the cell. This makes it easy to isolate and study mutations that inactivate a gene as one avoids the complication of having a second copy of the gene in the cell.  
      The process of culturing yeast strains involves isolation of a single yeast cell, maintenance of yeast cultures, and the propagation of the yeast until an amount sufficient for pitching is obtained. Pure yeast cultures are obtained from a number of sources such as commercial distributors or culture collections. Various procedures are used to collect pure cultures, including culturing from a single colony, a single cell, or a mixture of isolated cells and colonies.  
      The objective of propagation is to produce large quantities of yeast with known characteristics in as short a time as possible. One method is a batch system of propagation, starting with a few milliliters of stock culture and scaling up until a desired quantity of yeast has been realized. Scale-up introduces actively growing cells to a fresh supply of nutrients in order to produce a crop of yeast in the optimum physiological state.  
      Yeast cells that may be used in accordance with the present invention include, but are not limited to,  Saccharomyses  species (e.g.,  S. cerevisiae; S. carlsbergensis ),  Schizosaccharomyces  species (e.g.,  S. pombi ),  Pichia  species (e.g.,  P. pastoris ),  Hansenula  species (e.g.,  H. polymorpha ),  Kluyveromyces  species (e.g.,  K. lactis ),  Yarrowia  species (e.g.,  Y. lipolytica ). However, virtually any yeast cell genus can be engineered for sensitivity to bacterial toxins as described herein.  
      B. Yeast Viability and Growth  
      The adoption of means to enhance vector stability increases the yield of the expression product from a culture. Many vectors adapted for cloning in yeast include genetic markers to insure growth of transformed yeast cells under selection pressure. Host cell cultures containing such vectors may contain large numbers of untransformed segregants when grown under nonselective conditions, especially when grown to high cell densities. Therefore, it is advantageous to employ expression vectors which do not require growth under selection conditions, in order to permit growth to high densities and to minimize the proportion of untransformed segregants.  
      Vectors which contain a substantial portion of the naturally-occurring two circle plasmid are able to replicate stably with minimal segregation of untransformed cells, even at high cell densities, when transformed into host strains previously lacking two micron circles. Such host strains are termed “circle zero” strains. Additionally, the rate of cell growth at low cell densities may be enhanced by incorporating regulatory control over the promoter such that the expression of the S-protein coding region is minimized in dilute cultures such as early to middle log phase, then turned on for maximum expression at high cell densities. Such a control strategy increases the efficiency of cell growth in the fermentation process and further reduces the frequency of segregation of untransformed cells.  
      Briefly, yeast may be transfected with an expression vector expressing an essential polypeptide that has been engineered to include an endopeptidase cleavage site. Rates of growth in liquid medium of transformed yeast may be measured in the presence of galactose, which induces expression. Viability is a measure of yeast&#39;s ability to ferment. Yeast viability is determined by the standard-culture method, flow cytometry by selective staining, or by more advanced methods such as the Slide Viability Method, flocculation tests, and fermentation tests.  
      The standard slide-culture method of determining viability of yeasts has three steps: perform a hemacytometer count on a suspension of cells, plate a measured quantity on a wort gelatin medium, and then incubate and count the resultant colonies. However, this method may be inaccurate due to cell clumping and the death of cells during preparation.  
      Methylene blue remains an industry standard for viability assessment. It has also been suggested that methylene violet might provide a more accurate and reproducible assessment of viability than does methylene blue because of impurities in the latter. Other stains that may be used include fluorophore dyes, such as oxonol (DiBAC), 1-anilino-8-naphtalene-sulfonic acid (MgANS), berberine, Sytox Orange, propidium iodide, FUN1, and other conventional brightfield dyes. For the most part, fluorophore staining has been perceived to be less subjective to the operator compared with brightfield dye staining because of the lack of intermediate color variations.  
      C. Yeast Promoters  
      Useful yeast promoters for the conditional expression of toxic peptidases include those directing expression of metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Vectors and promoters suitable for use in yeast expression are further described in EP 73,675A, herein incorporated by reference in its entirety. Other examples of strong yeast promoters are the alcohol dehydrogenase, lactase and triosephosphate isomerase promoters  
      For expression of yeast genes in yeast, to determine the effects of mutations, it is generally best to use the gene&#39;s promoter in a CEN plasmid so expression is similar to the wild-type gene. However, there are a variety of promoters to choose from for various purposes. One such promoter is the Gal 1,10 promoter, which is inducible by galactose. It is frequently valuable to be able to turn expression of the gene on and off so one can follow the time dependent effects of expression.  
      The Gal 1 gene and Gal 10 gene are adjacent and transcribed in opposite directions from the same promoter region. The regulatory region containing the UAS sequences can be cut out on a DdeI Sau3A fragment and placed upstream of any other gene to confer galactose inducible expression and glucose repression. The PGK, GPD and ADH1 promoters are high expression constitutive promoters (PGK=phosphoglycerate kinase, GPD=glyceraldehyde 3 phosphate dehydrogenase, ADH1=alcohol dehydrogenase). The ADH2 promoter is glucose repressible and it is strongly transcribed on non-fermentable carbon sources (similar to GAL 1 or 10) except not inducible by galactose. The CUPI promoter is the metalothionein gene promoter. It is activated by copper or silver ions added to the medium. The CUP 1 gene is one of a few yeast genes that is present in yeast in more than one copy. Depending on the strain, there can be up to eight copies of this gene. The PHO5 promoter is a secreted gene coding for an acid phosphatase. It is induced by low or no phosphate in the medium. The phosphatase is secreted in the chance it will be able to free up some phosphate from the surroundings. When phosphate is present, no PHO5 message can be found. When it is absent, it is turned on strongly.  
      D. Non-Yeast Inducible Promoters  
      The identity of tissue-specific promoters or elements is well known to those of skill in the art. Nonlimiting examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), DIA dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), and human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996) and the Tet-On™ and Tet-Off™ Systems from Clontech. Additional inducible promoters are discussed Table 2, below.  
               TABLE 2                          Inducible Elements                         Element   Inducer   References               MT II   Phorbol Ester (TFA)   Palmiter et al., 1982; Haslinger           Heavy metals   et al., 1985; Searle et al., 1985;               Stuart et al., 1985; Imagawa               et al., 1987, Karin et al., 1987;               Angel et al., 1987b; McNeall               et al., 1989       MMTV (mouse mammary   Glucocorticoids   Huang et al., 1981; Lee et al.,       tumor virus)       1981; Majors et al., 1983;               Chandler et al., 1983; Lee et al.,               1984; Ponta et al., 1985; Sakai               et al., 1988       β-Interferon   Poly(rI)x   Tavernier et al., 1983           Poly(rc)       Adenovirus 5 E2   E1A   Imperiale et al., 1984       Collagenase   Phorbol Ester (TPA)   Angel et al., 1987a       Stromelysin   Phorbol Ester (TPA)   Angel et al., 1987b       SV40   Phorbol Ester (TPA)   Angel et al., 1987b       Murine MX Gene   Interferon, Newcastle   Hug et al., 1988           Disease Virus       GRP78 Gene   A23187   Resendez et al., 1988       α-2-Macroglobulin   IL-6   Kunz et al., 1989       Vimentin   Serum   Rittling et al., 1989       MHC Class I Gene H-2κb   Interferon   Blanar et al., 1989       HSP70   E1A, SV40 Large T   Taylor et al., 1989, 1990a, 1990b           Antigen       Proliferin   Phorbol Ester-TPA   Mordacq et al., 1989       Tumor Necrosis Factor α   PMA   Hensel et al., 1989       Thyroid Stimulating   Thyroid Hormone   Chatterjee et al., 1989       Hormone α Gene                  
 
      E. Yeast Transformation Protocols  
      A variety of approaches are available for transforming yeast cells and include electroporation, lithium acetate and protoplasting. In certain embodiments of the present invention, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Pat. No. 5,384,253, incorporated herein by reference). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.  
      Protoplast fusion has been used to overcome sexual barriers that prevent genetically unrelated strains from mating (Svoboda, 1976), thus facilitating the total or partial exchange of genetic components (Provost et al., 1978; Wilson et al., 1982; Perez et al., 1984; Spencer et al., 1985; Pina et al., 1986; Skala et al., 1988; Janderova et al., 1990; Gupthar, 1992; Molnar and Sipiczki, 1993). The process relies on cell wall digestion followed by fusion with, e.g., polyethylene glycol (Kao and Michayluk, 1974) and the protoplast adhesion promoter, Ca 2+  have been exploited in yeast fusion experiments (van Solingen and van der Plaat, 1977; Svoboda, 1978; Wilson et al., 1982; Pina et al., 1986). Other workers report “an enhancement of the protoplast fusion rate” using electro-fusion techniques instead of polyethylene glycol (Weber et al., 1981; Halfmann et al., 1982). The action of polyethylene glycol is not specific. It catalyses the aggregation of protoplasts between the same or different species.  
      The fusion process may be summarized as follows: (i) random aggregation of protoplasts into clumps of various sizes (Anne and Peberdy, 1975; Sarachek and Rhoads, 1981); (ii) conversion of the aggregates into syncytia (“chimaeric protoplast fusion product”) by dissolution of membranes and merging of cytoplasmic contents (Ahkong et al., 1975a; Gumpert, 1980; Svoboda, 1981; Sarachek and Rhoads, 1981; Klinner and Bottcher, 1984); (iii) membrane re-organisation (Ahkong et al., 1975a; Gumpert, 1980) and fusion of nuclei within heterokaryons (Sarachek and Rhoads, 1981; Klinner and Bottcher, 1984).  
      Another approach uses electroporation. Cells are first grown to a density of about 1×10 7 /ml (OD595 ca. 0.5) in minimal medium (transformation frequency is not harmed by growth until early stationary phase (OD595=1.5)). Cells are harvested by spinning at 3000 rpm for 5 minutes at 20° C., followed by washing once in ice-cold water and harvesting; a second time in ice-cold 1M sorbitol. It has been reported (Suga and Hatakeyama, 2001), that 15 min incubation of these cells in the presence of DTT at 25 mM increases electrocompetence. The final resuspension is in ice-cold 1M sorbitol at a density of 1-5×10 9 /ml. Forty ul of the cell suspension are added to chilled eppendorfs containing the DNA for transformation (100 ng) and incubated on ice for 5 minutes.  
      The electroporator may be set as follows: (a) 1.5 kV, 200 ohms, 25 uF (Biorad); (b) 1.5 kV, 132 ohms, 40 uF (Jensen/Flowgen). Cells and DNA are transferred to a pre-chilled cuvette and pulsed; 0.9 ml of ice-cold 1M sorbitol is then immediately added to the cuvette; the cell suspension is then returned to the eppendorf and placed on ice while other electroporations are carried out. Cells are plated as soon as possible onto minimal selective medium. Transformants should appear in 4-6 days at 32° C. The following lithium acetate protocol is derived from Okazaki et al. (1990), High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of  Schizosaccharomyces pombe . Cells are grown in a 150 ml culture in minimal medium to a density of 0.5-1×10 7  cells/ml (OD595=0.2-0.5). Media with low glucose, or MB media (see Okazaki et al.), in which the cells are less happy, may increase transformation efficiency. Cells are harvested at 3000 rpm for 5 minutes at room temperature, then washed in 40 ml of sterile water and spun down as before. The cells are resuspend at 1×10 9  cells/ml in 0.1 M lithium acatate (adjusted to pH 4.9 with acetic acid) and dispensed in 100 ul aliquots into eppendorf tubes. Incubation is at 30° C. (25° C. for ts mutants) for 60-120 min. Cells will sediment at this stage. One ug of plasmid DNA in 15 ul TE (pH 7.5) is added to each tube and mix by gentle vortexing, completely resuspending cells sedimented during the incubation. The tubes should not be allowed to cool down at this stage. 290 μl of 50% (w/v) PEG 4000 prewarmed at 30° C. (25° C. for ts mutants) is added. Next, mix by gentle vortexing and incubate at 30° C. (25° C. for ts mutants) for 60 minutes. The tubes are heat shocked at 43° C. for 15 minutes, followed by cooling to room temperature for 10 minutes. The tubes are then centrifuged at 5000 rpm for 2 minutes in an eppendorf centrifuge. The supernatant is carefully removed by aspiration. Cells are resuspend in 1 ml of ½ YE broth by pipetting up and down with a pipetman P1000, transferred to a 50 ml flask and diluted with 9 ml of ½ YE. The cells are incubated with shaking at 32° C. (25° C. for ts mutants) for 60 minutes or longer. Aliquots of less than 0.3 ml are plated onto minimal plates. If necessary, cells are centrifuged at this stage and resuspended in 1 ml of media to spread more cells on a plate.  
      F. Yeast Essential Genes  
      An “essential” yeast gene is defined as one that is imperative for the vegetative life cycle of a yeast cell grown on rich YPD media at 30° C. Over 800 essential yeast genes have been identified thus far. At present 16-18% of all yeast genes are essential for growth by the following definition. This number is probably an underestimation due to the huge number of gene families and the fact that many non-essential genes might become essential once functionally redundant genes have been deleted. This phenotype is termed synthetic lethality. The following table lists yeast essential genes which may be modified in accordance with the present invention.  
               TABLE 3                          Yeast Essential Genes                         ORF   Name   Description               YOL038w   PRE6   20S proteasome subunit (alpha4)       YJL001w   PRE3   20S proteasome subunit (beta1)       YOR157c   PUP1   20S proteasome subunit (beta2)       YER094c   PUP3   20S proteasome subunit (beta3)       YPR103w   PRE2   20S proteasome subunit (beta5)       YOR362c   PRE10   20S proteasome subunit C1 (alpha7)       YER012w   PRE1   20S proteasome subunit C11 (beta4)       YML092c   PRE8   20S proteasome subunit Y7 (alpha2)       YGL011c   SCL1   20S proteasome subunit YC7ALPHA/Y8 (alpha1)       YGR253c   PUP2   20S proteasome subunit (alpha5)       YMR314w   PRE5   20S proteasome subunit (alpha6)       YBL041w   PRE7   20S proteasome subunit (beta6)       YFR050c   PRE4   20S proteasome subunit (beta7)       YDL007w   RPT2   26S proteasome regulatory subunit       YDR394w   RPT3   26S proteasome regulatory subunit       YER021w   RPN3   26S proteasome regulatory subunit       YFR004w   RPN11   26S proteasome regulatory subunit       YFR052w   RPN12   26S proteasome regulatory subunit       YGL048c   RPT6   26S proteasome regulatory subunit       YHR027c   RPN1   26S proteasome regulatory subunit       YIL075c   RPN2   26S proteasome regulatory subunit       YKL145w   RPT1   26S proteasome regulatory subunit       YOR259c   RPT4   26S proteasome regulatory subunit       YGR195w   SKI6   3′-&gt;5′ exoribonuclease required for 3′ end formation of 5.8S rRNA       YHR069c   RRP4   3′-&gt;5′ exoribonuclease required for 3′ end formation of 5.8S rRNA       YLR100w   ERG27   3-keto sterol reductase       YBR265w   TSC10   3-ketosphinganine reductase       YOL040c   RPS15   40S small subunit ribosomal protein       YOR048c   RAT1   5′-3′ exoribonuclease       YHR183w   GND1   6-phosphogluconate dehydrogenase       YLR075w   RPL10   60S large subunit ribosomal protein       YLR029c   RPL15A   60s large subunit ribosomal protein L15.e.c12       YOR063w   RPL3   60S large subunit ribosomal protein L3.e       YGL030w   RPL30   60S large subunit ribosomal protein L30.e       YBL092w   RPL32   60S large subunit ribosomal protein L32.e       YPL131w   RPL5   60S large subunit ribosomal protein L5.e       YJL085w   EXO70   70 kDa exocyst component protein       YPL028w   ERG10   acetyl-CoA C-acetyltransferase, cytosolic       YNR016c   ACC1   acetyl-CoA carboxylase       YLR340w   RPP0   acidic ribosomal protein L10.e       YFL039c   ACT1   actin       YBR211c   AME1   actin related protein       YJR065c   ARP3   actin related protein       YIR006c   PAN1   actin-cytoskeleton assembly protein       YDL029w   ARP2   actin-like protein       YJL081c   ARP4   actin-related protein       YMR033w   ARP9   Actin-related protein       YOL052c   SPE2   adenosylmethionine decarboxylase precursor       YJL005w   CYR1   adenylate cyclase       YOR335c   ALA1   alanyl-tRNA synthetase, cytosolic       YBR126c   TPS1   alpha,alpha-trehalose-phosphate synthase, 56 KD subunit       YML085c   TUB1   alpha-1 tubulin       YDR341c       arginyl-tRNA synthetase, cytosolic       YBR234c   ARC40   ARP2/3 protein complex subunit, 40 kilodalton       YKL112w   ABF1   ARS-binding factor       YHR019c   DED81   asparaginyl-tRNA-synthetase       YLL018c   DPS1   aspartyl-tRNA synthetase, cytosolic       YMR309c   NIP1   associated with 40s ribosomal subunit       YLR298c   YHC1   associated with the U1 snRNP complex       YGR013w   SNU71   associated with U1 snRNP, no counterpart in mammalian U1               snRNP       YMR301c   ATM1   ATP-binding cassette transporter protein, mitochondrial       YBR142w   MAK5   ATP-dependent RNA helicase       YGL171w   ROK1   ATP-dependent RNA helicase       YOR204w   DED1   ATP-dependent RNA helicase       YFL002c   SPB4   ATP-dependent RNA helicase of DEAH box family       YIL048w   NEO1   ATPase whose overproduction confers neomycin resistance       YKL004w   AUR1   aureobasidin-resistance protein       YMR308c   PSE1   beta karyopherin       YBR110w   ALG1   beta-mannosyltransferase       YFL037w   TUB2   beta-tubulin       YDL141w   BPL1   biotin holocarboxylase synthetase       YLR116w   MSL5   branch point bridging protein       YNL280c   ERG24   C-14 sterol reductase       YGL001c   ERG26   C-3 sterol dehydrogenase (C-4 decarboxylase)       YGR060w   ERG25   C-4 sterol methyl oxidase       YBR109c   CMD1   calmodulin       YJL033w   HCA4   can suppress the U14 snoRNA rRNA processing function       YPL204w   HRR25   casein kinase I, ser/thr/tyr protein kinase       YFL029c   CAK1   cdk-activating protein kinase       YPR113w   PIS1   CDP diacylglycerol-inositol 3-phosphatidyltransferase       YER026c   CHO1   CDP-diacylglycerol serine O-phosphatidyltransferase       YBR136w   MEC1   cell cycle checkpoint protein       YDR499w   LCD1   cell cycle checkpoint protein       YDR113c   PDS1   cell cycle regulator       YOR373w   NUD1   cell cycle regulatory protein       YBR202w   CDC47   cell division control protein       YDL220c   CDC13   cell division control protein       YDR168w   CDC37   cell division control protein       YDR182w   CDC1   cell division control protein       YFL009w   CDC4   cell division control protein       YGL116w   CDC20   cell division control protein       YJL194w   CDC6   cell division control protein       YLR274w   CDC46   cell division control protein       YLR314c   CDC3   cell division control protein       YNL188w   KAR1   cell division control protein       YPL255w   BBP1   cell division control protein       YOL123w   HRP1   CF Ib (RNA3′ Cleavage factor Ib)       YJL142w   CCT2   chaperonin of the TCP1 ring complex, cytosolic       YIL014w   CCT3   chaperonin of the TCP1 ring complex, cytosolic       YOR020c   HSP10   chaperonin, mitochondrial       YFL008w   SMC1   chromosome segregation protein       YFR031c   SMC2   chromosome segregation protein       YLR115w   CFT2   cleavage and polyadenylation specificity factor, part of CF II       YOR250c   CLP1   cleavage/polyadenylation factor IA subunit       YDL145c   COP1   coatomer complex alpha chain of secretory pathway vesicles       YDR238c   SEC26   coatomer complex beta chain of secretory pathway vesicles       YGL137w   SEC27   coatomer complex beta′ chain (beta′-cop) of secretory pathway               vesicles       YFR051c   RET2   coatomer complex delta chain       YNL287w   SEC21   coatomer complex gamma chain (gamma-COP) of secretory               pathway vesicles       YLL050c   COF1   cofilin, actin binding and severing protein       YGL029w   CGR1   Coiled-coil protein, may play a role in ribosome biogenesis       YMR288w   HSH155   component of a multiprotein splicing factor       YDL143w   CCT4   component of chaperonin-containing T-complex       YDR212w   TCP1   component of chaperonin-containing T-complex       YDR188w   CCT6   component of chaperonin-containing T-complex (zeta subunit)       YIL109c   SEC24   component of COPII coat of ER-Golgi vesicles       YDR170c   SEC7   component of non-clathrin vesicle coat       YDR228c   PCF11   component of pre-mRNA 3′-end processing factor CF I       YGL044c   RNA15   component of pre-mRNA 3′-end processing factor CF I       YMR061w   RNA14   component of pre-mRNA 3′-end processing factor CF I       YJR093c   FIP1   component of pre-mRNA polyadenylation factor PF I       YLR277c   YSH1   component of pre-mRNA polyadenylation factor PF I       YPR056w   TFB4   component of RNA polymerase transcription initiation TFIIH               factor       YPR034w   ARP7   component of SWI-SNF global transcription activator complex and               RSC chromatin remodeling complex       YCR042c   TSM1   component of TFIID complex       YHR099w   TRA1   component of the Ada-Spt transcriptional regulatory complex       YLR127c   APC2   component of the anaphase promoting complex       YOR249c   APC5   component of the anaphase-promoting complex       YDL195w   SEC31   component of the COPII coat of ER-golgi vesicles       YKL049c   CSE4   component of the core centromere       YPL011c   TAF47   component of the TBP-associated protein complex       YMR028w   TAP42   component of the Tor signaling pathway       YHR148w   IMP3   component of the U3 small nucleolar ribonucleoprotein       YJR002w   MPP10   component of the U3 small nucleolar ribonucleoprotein       YNL075w   IMP4   component of the U3 small nucleolar ribonucleoprotein YDL132w       CDC53       controls G1/S transition       YNL232w   CSL4   core component of the 3′-5′ exosome       YOR206w   NOC2   crucial for intranuclear movement of ribosomal precursor particles       YBR135w   CKS1   cyclin-dependent kinases regulatory subunit       YBR160w   CDC28   cyclin-dependent protein kinase       YDL108w   KIN28   cyclin-dependent ser/thr protein kinase       YNL247w       cysteinyl-tRNA synthetase       YHR007c   ERG11   cytochrome P450 lanosterol 14a-demethylase       YER023w   PRO3   delta 1-pyrroline-5-carboxylate reductase       YHR068w   DYS1   deoxyhypusine synthase       YAL034w-a   MTW1   determining metaphase spindle length       YMR113w   FOL3   dihydrofolate synthetase       YNL256w   FOL1   Dihydroneopterin aldolase, dihydro-6-hydroxymethylpterin               pyrophosphokinase, dihydropteroate synthetase       YJR016c   ILV3   dihydroxy-acid dehydratase       YIL144w   TID3   DMC1P interacting protein       YHR164c   DNA2   DNA helicase       YIL143c   SSL2   DNA helicase       YER171w   RAD3   DNA helicase/ATPase       YJL173c   RFA3   DNA replication factor A, 13 KD subunit       YNL312w   RFA2   DNA replication factor A, 36 kDa subunit       YAR007c   RFA1   DNA replication factor A, 69 KD subunit       YOLO094c   RFC4   DNA replication factor C, 37 kDa subunit       YBR087w   RFC5   DNA replication factor C, 40 KD subunit       YNL290w   RFC3   DNA replication factor C, 40 kDa subunit       YJR068w   RFC2   DNA replication factor C, 41 KD subunit       YOR217w   RFC1   DNA replication factor C, 95 KD subunit       YNL088w   TOP2   DNA topoisomerase II (ATP-hydrolysing)       YNL216w   RAP1   DNA-binding protein with repressor and activator activity       YKL144c   RPC25   DNA-direcred RNA polymerase III, 25 KD subunit       YKL045w   PRI2   DNA-directed DNA polymerase alpha, 58 KD subunit (DNA               primase)       YIR008c   PRI1   DNA-directed DNA polymerase alpha 48 kDa subunit (DNA               primase)       YNL102w   POL1   DNA-directed DNA polymerase alpha, 180 KD subunit       YBL035c   POL12   DNA-directed DNA polymerase alpha, 70 KD subunit       YJR006w   HYS2   DNA-directed DNA polymerase delta, 55 KD subunit       YDL102w   CDC2   DNA-directed DNA polymerase delta, catalytic 125 KD subunit       YNL262w   POL2   DNA-directed DNA polymerase epsilon, catalytic subunit A       YPR175w   DPB2   DNA-directed DNA polymerase epsilon, subunit B       YOR210w   RPB10   DNA-directed polymerase I, II, III 8.3 subunit       YPR010c   RPA135   DNA-directed RNA polymerase I, 135 KD subunit       YOR341w   RPA190   DNA-directed RNA polymerase I, 190 KD alpha subunit       YOR340c   RPA43   DNA-directed RNA polymerase I, 36 KD subunit       YOR224c   RPB8   DNA-directed RNA polymerase I, II, III 16 KD subunit       YPR187w   RPO26   DNA-directed RNA polymerase I, II, III 18 KD subunit       YBR154c   RPB5   DNA-directed RNA polymerase I, II, III 25 KD subunit       YPR110c   RPC40   DNA-directed RNA polymerase I, III 40 KD subunit       YNL113w   RPC19   DNA-directed RNA polymerase I, III 16 KD subunit       YDR308c   SRBY   DNA-directed RNA polymerase II holoenzyme and kornberg&#39;s               mediator (SRB) subcomplex subunit       YER022w   SRB4   DNA-directed RNA polymerase II holoenzyme and Kornberg&#39;s               mediator (SRB) subcomplex subunit       YLR071c   RGR1   DNA-directed RNA polymerase II holoenzyme subunit       YOL005c   RPB11   DNA-directed RNA polymerase II subunit, 13.6 kD       YBR253w   SRB6   DNA-directed RNA polymerase II suppressor protein       YOR151c   RPB2   DNA-directed RNA polymerase II, 140 kDa chain       YDR404c   RPB7   DNA-directed RNA polymerase II, 19 KD subunit       YDL140c   RPO21   DNA-directed RNA polymerase II, 215 KD subunit       YOR207c   RET1   DNA-directed RNA polymerase III, 130 KD subunit       YOR116c   RPO31   DNA-directed RNA polymerase III, 160 KD subunit       YNL151c   RPC31   DNA-directed RNA polymerase III, 31 KD subunit       YNR003c   RPC34   DNA-directed RNA polymerase III, 34 KD subunit       YDL150w   RPC53   DNA-directed RNA polymerase III, 47 KD subunit       YPR190c   RPC82   DNA-directed RNA polymerase III, 82 KD subunit       YHR143w-a   RPC10   DNA-directed RNA polymerases I, II, III 7.7 KD subunit       YIL021w   RPB3   DNA-directed RNA-polymerase II, 45 kDa       YMR013c   SEC59   dolichol kinase       YPR183w   DPM1   dolichyl-phosphate beta-D-mannosyltransferase       YLR129w   DIP2   DOM34P-interacting protein       YMR239c   RNT1   double-stranded ribonuclease       YOL066c   RIB2   DRAP deaminase       YJR057w   CDC8   dTMP kinase       YFR028c   CDC14   dual specificity phosphatase       YBR252w   DUT1   dUTP pyrophosphatase precursor       YDR390c   UBA2   E1-like (ubiquitin-activating) enzyme       YKL210w   UBA1   E1-like (ubiquitin-activating) enzyme       YDL064w   UBC9   E2 ubiquitin-conjugating enzyme       YDR054c   CDC34   E2 ubiquitin-conjugating enzyme       YBR247c   ENP1   effects N-glycosylation       YBL040c   ERD2   ER lumen protein-retaining receptor       YDR086c   SSS1   ER protein-translocase complex subunit       YLR378c   SEC61   ER protein-translocation complex subunit       YOR254c   SEC63   ER protein-translocation complex subunit       YPL094c   SEC62   ER protein-translocation complex subunit       YFL017c   GNA1   essential acetyltransferase       YDR331w   GPI8   essential for GPI anchor attachment       YGR113w   DAM1   essential mitotic spindle pole protein       YGL061c   DUO1   essential mitotic spindle protein       YIL026c   IRR1   essential protein       YDR473c   PRP3   essential splicing factor       YEL020w-a   TIM9   essential subunit of the TIM22-complex for mitochondrial protein               import       YOR319w   HSH49   essential yeast splicing factor       YDR172w   SUP35   eukaryotic peptide chain release factor GTP-binding subunit       YBR102c   EXO84   exocyst protein essential for secretion       YPL169c   MEX67   factor for nuclear mRNA export       YHR190w   ERG9   farnesyl-diphosphate farnesyltransferase       YJL167w   ERG20   farnesyl-pyrophosphate synthetase       YPL231w   FAS2   fatty-acyl-CoA synthase, alpha chain       YKL182w   FAS1   fatty-acyl-CoA synthase, beta chain       YDL014w   NOP1   fibrillarin       YDL045c   FAD1   flavin adenine dinucleotide (FAD) synthetase       YMR203w   TOM40   forms the hydrophilic channel of the mitochondrial import pore for               preproteins       YPR180w   AOS1   forms together with       UBA2P       a heterodimeric activating enzyme for SMT3P       YKL060c   FBA1   fructose-bisphosphate aldolase       YDR397c   NCB2   functional homolog of human NC2beta/Dr1       YLR212c   TUB4   gamma tubulin       YER136w   GDI1   GDP dissociation inhibitor       YGL225w   GOG5   GDP-mannose transporter into the lumen of the Golgi       YGL097w   SRM1   GDP/GTP exchange factor for GSP1P/GSP2P       YLR310c   CDC25   GDP/GTP exchange factor for RAS1P and RAS2P       YGL207w   SPT16   general chromatin factor       YJL031c   BET4   geranylgeranyl transferase, alpha chain       YGL155w   CDC43   geranylgeranyltransferase beta subunit       YOR370c   MRS6   geranylgeranyltransferase regulatory subunit       YPR176c   BET2   geranylgeranyltransferase type II beta subunit       YDR300c   PRO1   glutamate 5-kinase       YPR035w   GLN1   glutamate-ammonia ligase       YOR168w   GLN4   glutaminyl-tRNA synthetase       YBR121c   GRS1   glycine-tRNA ligase       YGR172c   YIP1   golgi membrane protein       YDR302w   GPI11   GPI11-protein involved in glycosylphosphatidylinositol (GPI)               biosynthesis       YGR267c   FOL2   GTP cyclohydrolase I       YHR005c   GPA1   GTP-binding protein alpha subunit of the pheromone pathway       YLR229c   CDC42   GTP-binding protein of RAS superfamily       YPL218w   SAR1   GTP-binding protein of the ARF family       YFL038c   YPT1   GTP-binding protein of the rab family       YFL005w   SEC4   GTP-binding protein of the ras superfamily       YLR293c   GSP1   GTP-binding protein of the ras superfamily       YML064c   TEM1   GTP-binding protein of the RAS superfamily       YPR165w   RHO1   GTP-binding protein of the rho subfamily of ras-like proteins       YAL041w   CDC24   GTP/GDP exchange factor for       CDC42P   YMR235c   RNA1 GTPase activating protein       YDR454c   GUK1   guanylate kinase       YHR026w   PPA1   H+-ATPase 23 KD subunit, vacuolar       YGL008c   PMA1   H+-transporting P-type ATPase, major isoform, plasma membrane       YDR420w   HKR1   Hansenula MraKII k9 killer toxin-resistance protein       YNL007c   SIS1   heat shock protein       YOR232w   MGE1   heat shock protein - chaperone       YLR259c   HSP60   heat shock protein - chaperone, mitochondrial       YGL073w   HSF1   heat shock transcription factor       YER125w   RSP5   hect domain E3 ubiquitin-protein ligase       YMR290c   HAS1   helicase associated with       SET1P   YPR033c   HTS1 histidine-tRNA ligase, mitochondrial       YOR244w   ESA1   histone acetyltransferase       YJR139c   HOM6   homoserine dehydrogenase       YBR153w   RIB7   HTP reductase       YDR189w   SLY1   hydrophilic suppressor of YPT1 and member of the SEC1P family       YDL139c   SCM3   hypothetical protein       YDR016c   DAD1   hypothetical protein       YDR396w       hypothetical protein       YGL098w       hypothetical protein       YGR128c       hypothetical protein       YGR251w       hypothetical protein       YHR083w       hypothetical protein       YIR010w       hypothetical protein       YJR023c       hypothetical protein       YLR007w       hypothetical protein       YLR033w   RSC58   hypothetical protein       YLR112w       hypothetical protein       YLR132c       hypothetical protein       YLR145w       hypothetical protein       YMR298w       hypothetical protein       YNL150w       hypothetical protein       YNL158w       hypothetical protein       YNL258c       hypothetical protein       YOL026c       hypothetical protein       YPL012w       hypothetical protein       YGL238w   CSE1   importin-beta-like protein       YBR011c   IPP1   inorganic pyrophosphatase, cytoplasmic       YML104c   MDM1   intermediate filament protein       YDL058w   USO1   intracellular protein transport protein       YLL011w   SOF1   involved in 18S pre-rRNA production       YKL021c   MAK11   involved in cell growth and replication of M1 dsRNA virus       YKR063c   LAS1   involved in cell morphogenesis, cytoskeletal regulation and bud               formation       YGR099w   TEL2   involved in controlling telomere length and position effect       YPL242c   IQG1   involved in cytokinesis, has similarity to mammalian IQGAP               proteins       YJL090c   DPB11   involved in DNA replication and S-phase checkpoint       YLR336c   SGD1   involved in HOG pathway       YDR021w   FAL1   involved in maturation of 18S rRNA       YGR158c   MTR3   involved in mRNA transport       YJL050w   MTR4   involved in nucleocytoplasmic transport of mRNA       YOR148c   SPP2   involved in pre-mRNA processing       YLR197w   SIK1   involved in pre-rRNA processing       YCL031c   RRP7   involved in pre-rRNA processing and ribosome assembly       YBR257w   POP4   involved in processing of tRNAs and rRNAs       YDR087c   RRP1   involved in processing rRNA precursor species to mature rRNAs       YNL282w   POP3   involved in processsing of tRNAs and rRNAs       YDR299w   BFR2   involved in protein transport steps at the Brefeldin A blocks       YMR001c   CDC5   involved in regulation of DNA replication       YNL251c   NRD1   involved in regulation of nuclear pre-mRNA abundance       YIL046w   MET30   involved in regulation of sulfur assimilation genes and cell cycle               progression       YGL201c   MCM6   involved in replication       YGR095c   RRP46   involved in rRNA processing       YDL153c   SAS10   involved in silencing       YDR180w   SCC2   involved in sister chromatid cohesion       YFR027w   ECO1   involved in sister chromatid cohesion during replication       YGL120c   PRP43   involved in spliceosome disassembly       YKR068c   BET3   involved in targeting and fusion of ER to golgi transport vesicles       YML077w   BET5   involved in targeting and fusion of ER to golgi transport vesicles       YDR082w   STN1   involved in telomere length regulation       YPL117c   IDI1   isopentenyl-diphosphate delta-isomerase       YNL189w   SRP1   karyopherin-alpha or importin       YLR347c   KAP95   karyopherin-beta       YGR140w   CBF2   kinetochore protein complex CBF3, 110 KD subunit       YDR328c   SKP1   kinetochore protein complex CBF3, subunit D       YMR168c   CEP3   kinetochore protein complex, 71 KD subunit       YMR094w   CTF13   kinetochore protein complex, CBF3, 58 KD subunit       YHR072w   ERG7   lanosterol synthase       YPL160w   CDC60   leucine-tRNA ligase, cytosolic       YDR037w   KRS1   lysyl-tRNA synthetase, cytosolic       YDL055c   PSA1   mannose-1-phosphate guanyltransferase       YER003c   PMI40   mannose-6-phosphate isomerase       YGL065c   ALG2   mannosyltransferase       YHR066w   SSF1   mating protein       YKR008w   RSC4   member of RSC complex, which remodels the structure of               chromatin       YPR019w   CDC54   member of the CDC46P/MCM2P/MCM3P family       YBL023c   MCM2   member of the MCM2P, MCM3P, CDC46P family       YMR208w   ERG12   mevalonate kinase       YNR043w   MVD1   mevalonate pyrophosphate decarboxylase       YDL126c   CDC48   microsomal protein of CDC48/PAS1/SEC18 family of ATPases       YJL042w   MHP1   microtubule-associated protein       YOR272w   YTM1   microtubule-interacting protein       YGR029w   ERV1   mitochondrial biogenesis and regulation of cell cycle       YJR045c   SSC1   mitochondrial heat shock protein 70-related protein       YIL022w   TIM44   mitochondrial inner membrane import receptor subunit       YJL143w   TIM17   mitochondrial inner membrane import translocase subunit       YNR017w   MAS6   mitochondrial inner membrane import translocase subunit       YNL131w   TOM22   mitochondrial outer membrane import receptor complex subunit       YLR163c   MAS1   mitochondrial processing peptidase       YDR376w   ARH1   mitochondrial protein with similarity to human adrenodoxin               reductase and ferredoxin-NADP+ reductase       YDL003w   MCD1   Mitotic Chromosome Determinant       YBL034c   STU1   mitotic spindle protein       YBR156c   SLI15   Mitotic spindle protein involved in chromosome segregation       YGL018c   JAC1   molecular chaperone       YGR255c   COQ6   monooxygenase       YBR236c   ABD1   mRNA cap methyltransferase       YGL130w   CEG1   mRNA guanylyltransferase (mRNA capping enzyme, alpha               subunit)       YER165w   PAB1   mRNA polyadenylate-binding protein       YKL186c   MTR2   mRNA transport protein       YPL085w   SEC16   multidomain vesicle coat protein       YMR200w   ROT1   mutant suppresses TOR2 mutation       YJL153c   INO1   myo-inositol-1-phosphate synthase       YGL106w   MLC1   MYO2P light chain       YOR326w   MYO2   myosin heavy chain       YMR281w   GPI12   N-acetylglucosaminyl phosphatidylinositol deacetylase       YPL076w   GPI2   N-acetylglucosaminyl-phosphatidylinositol biosynthetic protein       YPL175w   SPT14   N-acetylglucosaminyltransferase       YGR147c   NAT2   N-acetyltransferase for N-terminal methionine       YLR195c   NMT1   N-myristoyltransferase       YDR120c   TRM1   N2,N2-dimethylguanine tRNA methyltransferase       YLR457c   NBP1   NAP1P-binding protein       YDR464w   SPP41   negative regulator of PRP3 and PRP4 gene expression       YPR168w   NUT2   negative transcription regulator from artifical reporters       YER127w   LCP5   NGG1P interacting protein       YLL036c   PRP19   non-snRNP sliceosome component required for DNA repair       YHR170w   NMD3   nonsense-mediated mRNA decay protein       YDL148c   NOP14   nuclear and nucleolar protein with possible role in ribosome               biogenesis       YBL020w   RFT1   nuclear division protein       YJR112w   NNF1   nuclear envelope protein       YML031w   NDC1   nuclear envelope protein       YGR218w   CRM1   nuclear export factor, exportin       YJL034w   KAR2   nuclear fusion protein       YPL124w   NIP29   nuclear import protein       YGL122c   NAB2   nuclear poly(A)-binding protein       YFR002w   NIC96   nuclear pore protein       YGL092w   NUP145   nuclear pore protein       YGL172w   NUP49   nuclear pore protein       YGR119c   NUP57   nuclear pore protein       YIL115c   NUP159   nuclear pore protein       YJL041w   NSP1   nuclear pore protein       YJL061w   NUP82   nuclear pore protein       YCR093w   CDC39   nuclear protein       YBR170c   NPL4   nuclear protein localization factor and ER translocation component       YBR167c   POP7   nuclear RNase P subunit       YER009w   NTF2   nuclear transport factor       YAL025c   MAK16   nuclear viral propagation protein       YDR432w   NPL3   nucleolar protein       YNL061w   NOP2   nucleolar protein       YPL043w   NOP4   nucleolar protein       YOL144w   NOP8   nucleolar protein required for 60S ribosome biogenesis       YDL208w   NHP2   nucleolar rRNA processing protein       YHR072w-a   NOP10   nucleolar rRNA processing protein       YHR089c   GAR1   nucleolar rRNA processing protein       YJL039c   NUP192   nucleoporin localize at the inner site of the nuclear membrane       YGL091c   NBP35   nucleotide-binding protein       YEL002c   WBP1   oligosaccharyl transferase beta subunit precursor       YGL022w   STT3   oligosaccharyl transferase subunit       YMR149w   SWP1   oligosaccharyltransferase delta subunit       YOR103c   OST2   oligosaccharyltransferase epsilon subunit       YJL002c   OST1   oligosaccharyltransferase, alpha subunit       YML065w   ORC1   origin recognition complex, 104 KD subunit       YHR118c   ORC6   origin recognition complex, 50 KD subunit       YNL261w   ORC5   origin recognition complex, 50 kDa subunit       YPR162c   ORC4   origin recognition complex, 56 KD subunit       YLL004w   ORC3   origin recognition complex, 62 kDa subunit       YBR060c   ORC2   origin recognition complex, 72 kDa subunit       YBR070c   SAT2   osmotolerance protein       YCR057c   PWP2   periodic tryptophan protein       YDR329c   PEX3   peroxisomal assembly protein - peroxin       YLR060w   FRS1   phenylalanyl-tRNA synthetase, alpha subunit, cytosolic       YKL203c   TOR2   phosphatidylinositol 3-kinase       YNL267w   PIK1   phosphatidylinositol 4-kinase       YMR079w   SEC14   phosphatidylinositol(PI)/phosphatidylcholine(PC) transfer protein       YLR305c   STT4   phosphatidylinositol-4-kinase       YDR208w   MSS4   phosphatidylinositol-4-phosphate 5-kinase       YEL058w   PCM1   phosphoacetylglucosamine mutase       YKL152c   GPM1   phosphoglycerate mutase       YFL045c   SEC53   phosphomannomutase       YMR220w   ERG8   phosphomevalonate kinase       YDL235c   YPD1   phosphorelay intermediate between SLN1P and SSK1P       YKR025w   RPC37   Pol III transcription       YKR002w   PAP1   poly(A) polymerase       YPL190c   NAB3   polyadenylated RNA-binding protein       YNL317w   PFS2   polyadenylation factor I subunit 2 required for mRNA 3′-end               processing, bridges two mRNA 3′-end processing factors       YJL025w   RRN7   polymerase I specific transcription initiation factor       YLR430w   SEN1   positive effector of tRNA-splicing endonuclease       YDR301w   CFT1   pre-mRNA 3′-end processing factor CF II       YBR237w   PRP5   pre-mRNA processing RNA-helicase       YAL032c   PRP45   pre-mRNA splicing factor       YDL043c   PRP11   pre-mRNA splicing factor       YJL203w   PRP21   pre-mRNA splicing factor       YML046w   PRP39   pre-mRNA splicing factor       YMR268c   PRP24   pre-mRNA splicing factor       YDL030w   PRP9   pre-mRNA splicing factor (snRNA-associated protein)       YDR088c   SLU7   pre-mRNA splicing factor affecting 3′ splice site choice       YDR243c   PRP28   pre-mRNA splicing factor RNA helicase of DEAD box family       YGR091w   PRP31   pre-mRNA splicing protein       YLR223c   IFH1   pre-rRNA processing machinery control protein       YAL043c   PTA1   pre-tRNA processing protein/PF I subunit       YMR229c   RRP5   processing of pre-ribosomal RNA       YHR024c   MAS2   processing peptidase, catalytic 53 kDa (alpha) subunit,               mitochondrial       YJR017c   ESS1   processing/termination factor 1       YOR122c   PFY1   profilin       YBR088c   POL30   Proliferating Cell Nuclear Antigen (PCNA)       YPR137w   RRP9   protein associated with the U3 small nucleolar RNA, required for               pre-ribosomal RNA processing       YNL221c   POP1   protein component of ribonuclease P and ribonuclease MRP       YCL043c   PDI1   protein disulfide-isomerase precursor       YKL019w   RAM2   protein farnesyltransferase, alpha subunit       YLL031c   GPI13   protein involved in glycosylphosphatidylinositol biosynthesis       YOL142w   RRP40   protein involved in ribosomal RNA processing, component of the               exosome complex responsible for 3′ end processing and               degradation of many RNA species       YDL017w   CDC7   protein kinase       YOR149c   SMP3   protein kinase C pathway protein       YAR019c   CDC15   protein kinase of the MAP kinase kinase kinase family       YPR107c   YTH1   protein of the 3′ processing complex       YGL075c   MPS2   Protein of the nuclear envelope/endoplasmic reticulum required for               spindle pole body assembly and normal chromosome segregation       YDR060w   MAK21   protein required for 60S ribosomal subunit biogenesis       YOR372c   NDD1   protein required for nuclear division       YER147c   SCC4   protein required for sister chromatid cohesion       YML069w   POB3   protein that binds to DNA polymerase I (PolI)       YDR164c   SEC1   protein transport protein       YIL004c   BET1   protein transport protein       YIL068c   SEC6   protein transport protein       YLR208w   SEC13   protein transport protein       YNL272c   SEC2   protein transport protein       YPR055w   SEC8   protein transport protein       YMR128w   ECM16   putative DEAH-box RNA helicase       YDL098c   SNU23   Putative RNA binding zinc finger protein       YDL031w   DBP10   putative RNA helicase involved in ribosome biogenesis       YLR175w   CBF5   putative rRNA pseuduridine synthase       YAL038w   CDC19   pyruvate kinase       YBR190w       questionable ORF       YDR053w       questionable ORF       YDR355c       questionable ORF       YDR412w       questionable ORF       YGR190c       questionable ORF       YKL036c       questionable ORF       YKL083w       questionable ORF       YLR076c       questionable ORF       YLR101c       questionable ORF       YLR140w       questionable ORF       YLR198c       questionable ORF       YLR317w   KRE34   questionable ORF       YMR290w-a       questionable ORF       YPL142c       questionable ORF       YPL238c       questionable ORF       YPL251w       questionable ORF       YPR136c   FYV15   questionable ORF       YDR002w   YRB1   ran-specific GTPase-activating protein       YLR383w   RHC18   recombination repair protein       YCL017c   NFS1   regulates Iron-Sulfur cluster proteins, cellular Iron uptake, and Iron               distribution       YDR373w   FRQ1   regulator of phosphatidylinositol-4-OH kinase protein       YOR294w   RRS1   regulator of ribosome synthesis       YDR052c   DBF4   regulatory subunit for CDC7P protein kinase       YKL193c   SDS22   regulatory subunit for the mitotic function of type I protein               phosphatase       YEL032w   MCM3   replication initiation protein       YMR213w   CEF1   required during G2/M transition       YNL048w   ALG11   required for asparagine-linked glycosylation       YLR088w   GAA1   required for attachment of GPI anchor onto proteins       YIL106w   MOB1   required for completion of mitosis and maintenance of ploidy       YPL211w   NIP7   required for efficient 60S ribosome subunit biogenesis       YDL015c   TSC13   required for elongation of the very long chain fatty acid (VLCFA)               moiety of sphingolipids       YGL145w   TIP20   required for ER to Golgi transport       YDR166c   SEC5   required for exocytosis       YLR166c   SEC10   required for exocytosis       YGL142c   GPI10   required for Glycosyl Phosphatdyl Inositol synthesis       YHR065c   RRP3   required for maturation of the 35S primary transcript       YLR103c   CDC45   required for minichromosome maintenance and initiation of               chromosomal DNA replication       YPR082c   DIB1   required for mitosis       YKL089w   MIF2   required for normal chromosome segregation and spindle integrity       YGR245c   SDA1   required for normal organization of the actin cytoskeleton; required               for passage through Start       YGL033w   HOP2   required for pairing of homologous chromosomes       YCL052c   PBN1   required for post-translational processing of the protease B               precursor       PRB1P   YOR310c   NOP58 required for pre-18S rRNA processing       YKL172w   EBP2   required for pre-rRNA processing and ribosomal subunit assembly       YHR062c   RPP1   required for processing of tRNA and 35S rRNA       YAL033w   POP5   required for processing of tRNAs and rRNAs       YBL018c   POP8   required for processing of tRNAs and rRNAs       YGR030c   POP6   required for processing of tRNAs and rRNAs       YML130c   ERO1   required for protein disulfide bond formation in the ER       YMR005w   MPT1   required for protein synthesis       YCL054w   SPB1   required for ribosome synthesis, putative methylase       YIL150c   DNA43   required for S-phase initiation or completion       YGR098c   ESP1   required for sister chromatid separation       YJL074c   SMC3   required for structural maintenance of chromosomes       YNL006w   LST8   required for transport of permeases from the golgi to the plasma               membrane       YIR011c   STS1   required for transport of RNA15P from the cytoplasm to the               nucleus       YML091c   RPM2   ribonuclease P precursor, mitochondrial       YER070w   RNR1   ribonucleoside-diphosphate reductase, large subunit       YJL026w   RNR2   ribonucleoside-diphosphate reductase, small subunit       YGR180c   RNR4   ribonucleotide reductase small subunit       YDR064w   RPS13   ribosomal protein       YHL015w   RPS20   ribosomal protein       YOL127w   RPL25   ribosomal protein L23a.e       YPL143w   RPL33A   ribosomal protein L35a.e.c16       YLR185w   RPL37A   ribosomal protein L37.e       YPR043w   RPL43A   ribosomal protein L37a.e       YNL178w   RPS3   ribosomal protein S3.e       YML025c   YML6   ribosomal protein, mitochondrial       YPL228w   CET1   RNA 5′-triphosphatase (mRNA capping enzyme, beta subunit)       YDR381w   YRA1   RNA annealing protein       YDR478w   SNM1   RNA binding protein of RNase MRP       YDL207w   GLE1   RNA export mediator       YOR046c   DBP5   RNA helicase       YLL008w   DRS1   RNA helicase of the DEAD box family       YNR038w   DBP6   RNA helicase required for 60S ribosomal subunit assembly       YKL078w   DHR2   RNA helicase, involved in ribosomal RNA maturation       YER172c   BRR2   RNA helicase-related protein       YKL125w   RRN3   RNA polymerase I specific transcription factor       YBL014c   RRN6   RNA polymerase I specific transcription initiation factor       YML043c   RRN11   RNA polymerase I specific transcription initiation factor       YHR058c   MED6   RNA polymerase II transcriptional regulation mediator       YDR045c   RPC11   RNA polymerase III subunit C11, required for RNA cleavage               activity and transcription termination       YPL007c   TFC8   RNA Polymerase III transcription initiation factor TFIIIC (tau), 60 kDa               subunit       YKR086w   PRP16   RNA-dependent ATPase       YIR015w   RPR2   RNase P subunit       YPL266w   DIM1   rRNA (adenine-N6,N6-)-dimethyltransferase       YCR035c   RRP43   rRNA processing protein       YDL111c   RRP42   rRNA processing protein       YDR280w   RRP45   rRNA processing protein       YDR190c   RVB1   RUVB-like protein       YPL235w   RVB2   RUVB-like protein       YER043c   SAH1   S-adenosyl-L-homocysteine hydrolase       YDR498c   SEC20   secretory pathway protein       YLR078c   BOS1   secretory pathway protein       YHR107c   CDC12   septin       YNL038w   GPI15   sequence and functional homologue of human Pig-H protein       YER133w   GLC7   ser/thr phosphoprotein phosphatase 1, catalytic chain       YBL105c   PKC1   ser/thr protein kinase       YPL209c   IPL1   ser/thr protein kinase       YPR161c   SGV1   ser/thr protein kinase       YHR102w   KIC1   ser/thr protein kinase that interacts with CDC31P       YPL153c   RAD53   ser/thr/tyr protein kinase       YDR062w   LCB2   serine C-palmitoyltransferase subunit       YMR296c   LCB1   serine C-palmitoyltransferase subunit       YHR205w   SCH9   serine/threonine protein kinase involved in stress response and               nutrient-sensing signaling pathway       YDL028c   MPS1   serine/threonine/tyrosine protein kinase       YDR023w   SES1   seryl-tRNA synthetase, cytosolic       YLR066w   SPC3   signal peptidase subunit       YPL210c   SRP72   signal recognition particle protein       YPL243w   SRP68   signal recognition particle protein       YIR022w   SEC11   signal sequence processing protein       YLL003w   SFI1   similarity to Xenopus laevis XCAP-C       YOR119c   RIO1   similarity to a  C. elegans  ZK632.3 protein       YNL132w   KRE33   similarity to  A. ambisexualis  antheridiol steroid receptor       YPL252c   YAH1   similarity to adrenodoxin and ferrodoxin       YLR022c       similarity to  C. elegans  and  M. jannaschii  hypothetical proteins       YDL060w   TSR1   similarity to  C. elegans  hypothetical protein       YKL018w   SWD2   similarity to  C. elegans  hypothetical protein       YKL059c       similarity to  C. elegans  hypothetical protein       YNL313c       similarity to  C. elegans  hypothetical protein       YPR133c   IWS1   similarity to  C. elegans  hypothetical protein       YHR186c       similarity to  C. elegans  hypothetical protein C10C5.6       YKL095w   YJU2   similarity to  C. elegans  hypothetical proteins       YKL088w       similarity to  C. tropicalis  hal3 protein, to C-term. of SIS2P and to               hypothetical protein YOR054c       YBR159w       similarity to human 17-beta-hydroxysteroid dehydrogenase       YLR051c       similarity to human acidic 82 kDa protein       YDR013w       similarity to human hypothetical KIAA0186 protein       YPL217c   BMS1   similarity to human hypothetical protein KIAA0187       YDR398w       similarity to human KIAA0007 gene       YNL240c   NAR1   similarity to human nuclear prelamin A recognition factor       YMR131c   RSA2   similarity to human retinoblastoma-binding protein       YOL010w   RCL1   similarity to human RNA 3′-terminal phosphate cyclase       YLR002c       similarity to hypothetical  C. elegans  protein       YHR122w       similarity to hypothetical  C. elegans  protein F45G2.a       YJL097w       similarity to hypothetical  C. elegans  protein T15B7.2       YHR188c   GPI16   similarity to hypothetical  C. elegans  proteins F17c11.7       YNL245c       similarity to hypothetical protein CG2843  D. melanogaster         YDR361c   BCP1   similarity to hypothetical protein  S. pombe         YKL033w       similarity to hypothetical protein  S. pombe         YKL052c   ASK1   similarity to hypothetical protein  S. pombe         YDR367w       similarity to hypothetical protein SPAC26H5.13c  S. pombe         YKL014c       similarity to hypothetical protein SPCC14G10.02  S. pombe         YPR105c       similarity to hypothetical protein SPCC338.13  S. pombe         YDR066c       similarity to hypothetical protein       YER139c   YHR036w   similarity to hypothetical protein       YGL247w   YHR088w   RPF1 similarity to hypothetical protein       YNL075w   YLR008c   similarity to hypothetical protein       YNL328c   YDL209c   similarity to hypothetical  S. pombe  protein       YGL047w       similarity to hypothetical  S. pombe  protein       YNL124w       similarity to hypothetical  S. pombe  protein       YLR106c       similarity to Kaposi&#39;s sarcoma-associated herpes-like virus ORF73               homolog gene       YOL133w   HRT1   similarity to Lotus RING-finger protein       YPL093w   NOG1   similarity to  M. jannaschii  GTP-binding protein       YNL207w       similarity to  M. jannaschii  hypothetical protein MJ1073       YGR211w   ZPR1   similarity to  M. musculus  zinc finger protein       ZPR1   YLL034c   similarity to mammalian valosin       YKL189w   HYM1   similarity to mouse hypothetical calcium-binding protein and                 D. melanogaster  Mo25 gene       YOR077w   RTS2   similarity to mouse KIN17 protein       YDL193w       similarity to  N. crassa  hypothetical 32 kDa protein       YJL072c       similarity to PIR: T40665 hypothetical protein SPBC725.13c  S. pombe         YGR046w       similarity to proline transport helper PTH1  C. albicans         YLR009w       similarity to ribosomal protein L24.e.B       YPR112c   MRD1   similarity to RNA-binding proteins       YJR067c   YAE1   similarity to  S. pombe  SPAC2C4.12c putative phosphotransferase       YLL035w   GRC3   similarity to  S. pombe  SPCC830.03 protein of unknown function       YNL308c   KRI1   similarity to  S. pombe  and  C. elegans  hypothetical proteins       YDR434w   GPI17   similarity to  S. pombe  hypothetical protein       YNL026w       similarity to  S. pombe  hypothetical protein       YDR303c   RSC3   similarity to transcriptional regulator proteins       YJL012c   VTC4   similarity to YPL019c and YF1004w       YHR178w   STB5   SIN3 binding protein       YNL263c   YIF1   SLH1P Interacting Factor       YBL026w   LSM2   Sm-like (Lsm) protein       YER112w   LSM4   Sm-like (Lsm) protein       YLR438c-a   LSM3   Sm-like (Lsm) protein       YKL196c   YKT6   SNARE protein for Endoplasmic Reticulum-Golgi transport       YKL006c-a   SFT1   SNARE-like protein       YGR074w   SMD1   snRNA-associated protein       YFR005c   SAD1   SnRNP assembly defective       YFL017w-a   SMX2   snRNP G protein (the homologue of the human Sm-G)       YBR055c   PRP6   snRNP(U4/U6)-associated splicing factor       YDR356w   NUF1   spindle pole body component       YHR172w   SPC97   spindle pole body component       YKL042w   SPC42   spindle pole body component       YNL126w   SPC98   spindle pole body component       YOR257w   CDC31   spindle pole body component, centrin       YDR201w   SPC19   spindle pole body protein       YER018c   SPC25   spindle pole body protein       YKR037c   SPC34   spindle pole body protein       YMR117c   SPC24   spindle pole body protein       YOL069w   NUF2   spindle pole body protein       YOR159c   SME1   spliceosomal snRNA-associated Sm core protein required for               mRNA splicing, also likely associated with telomerase TLC1 RNA       YLR147c   SMD3   spliceosomal snRNA-associated Sm core protein required for pre-               mRNA splicing       YJR022w   LSM8   splicing factor       YKL012w   PRP40   splicing factor       YKL165c   MCD4   sporulation protein       YGR175c   ERG1   squalene monooxygenase       YLR086w   SMC4   Stable Maintenance of Chromosomes       YPL151c   PRP46   strong similarity to  A. thaliana  PRL1 and PRL2 proteins       YJR072c       strong similarity to  C. elegans  hypothetical protein and similarity to               YLR243w       YOL077c   BRX1   strong similarity to  C. elegans  K12H4.3 protein       YLR397c   AFG2   strong similarity to       CDC48   YLR117c   CLF1 strong similarity to  Drosophila  putative cell cycle control               protein crn       YHR169w   DBP8   strong similarity to DRS1P and other probable ATP-dependent               RNA helicases       YDR141c   DOP1   strong similarity to  Emericella nidulans  developmental regulatory               gene, dopey (dopA)       YCL059c   KRR1   strong similarity to fission yeast rev interacting protein mis3       YNR053c       strong similarity to human breast tumor associated autoantigen       YHR020w       strong similarity to human glutamyl-prolyl-tRNA synthetase and               fruit fly multifunctional aminoacyl-tRNA synthetase       YDR091c   RLI1   strong similarity to human RNase L inhibitor and  M. jannaschii                 ABC transporter protein       YOR145c       strong similarity to hypothtical  S. pombe  protein and to               hypothetical  C. elegans  protein       YNL002c   RLP7   strong similarity to mammalian ribosomal L7 proteins       YER036c   KRE30   strong similarity to members of the ABC transporter family       YHR070w   TRM5   strong similarity to  N. crassa  met-10+ protein       YIL003w       strong similarity to NBP35P and human nucleotide-binding protein       YCR072c       strong similarity to  S. pombe  trp-asp repeat containing protein       YLR186w   EMG1   strong similarity to  S. pombe  hypothetical protein C18G6.07C       YAL047c   SPC72   STU2P Interactant       YBL084c   CDC27   subunit of anaphase-promoting complex (cyclosome)       YHR166c   CDC23   subunit of anaphase-promoting complex (cyclosome)       YKL022c   CDC16   subunit of anaphase-promoting complex (cyclosome)       YNL172w   APC1   subunit of anaphase-promoting complex (cyclosome)       YLR272c   YCS4   subunit of condensin protein complex       YDL008w   APC11   subunit of the anaphase promoting complex       YDR118w   APC4   subunit of the anaphase promoting complex       YKL013c   ARC19   subunit of the ARP2/3 complex       YDL097c   RPN6   subunit of the regulatory particle of the proteasome       YDL147w   RPN5   subunit of the regulatory particle of the proteasome       YCR052w   RSC6   subunit of the RSC complex       YFR037c   RSC8   subunit of the RSC complex       YIL126w   STH1   subunit of the RSC complex       YLR321c   SFH1   subunit of the RSC complex       YBR091c   MRS5   subunit of the TIM22-complex       YDL217c   TIM22   subunit of the TIM22-complex       YHR005c-a   MRS11   subunit of the TIM22-complex       YLR316c   TAD3   subunit of tRNA-specific adenosine-34 deaminase       YIR012w   SQT1   suppresses dominant-negative mutants of the ribosomal protein               QSR1       YLR045c   STU2   suppressor of a cs tubulin mutation       YOR329c   SCD5   suppressor of clathrin deficiency       YNL222w   SSU72   suppressor of cs mutant of SUA7       YOR057w   SGT1   suppressor of G2 allele of SKP1       YLR026c   SED5   syntaxin (T-SNARE)       YOR075w   UFE1   syntaxin (T-SNARE) of the ER       YDR416w   SYF1   synthetic lethal with       CDC40   YJR064w   CCT5 T-complex protein 1, epsilon subunit       YML114c   TAF65   TBP Associated Factor 65 KDa       YPL128c   TBF1   telomere TTAGGG repeat-binding factor 1       YKL058w   TOA2   TFIIA subunit (transcription initiation factor), 13.5 kD       YOR194c   TOA1   TFIIA subunit (transcription initiation factor), 32 kD       YPR086w   SUA7   TFIIB subunit (transcription initiation factor), factor E       YBR198c   TAF90   TFIID and SAGA subunit       YDR145w   TAF61   TFIID and SAGA subunit       YDR167w   TAF25   TFIID and SAGA subunit       YGL112c   TAF60   TFIID and SAGA subunit       YMR236w   TAF17   TFIID and SAGA subunit       YGR274c   TAF145   TFIID subunit (TBP-associated factor), 145 kD       YML098w   TAF19   TFIID subunit (TBP-associated factor), 19 kD       YML015c   TAF40   TFIID subunit (TBP-associated factor), 40 KD       YMR227c   TAF67   TFIID subunit (TBP-associated factor), 67 kD       YKR062w   TFA2   TFIIE subunit (transcription initiation factor), 43 KD       YKL028w   TFA1   TFIIE subunit (transcription initiation factor), 66 kD       YMR277w   FCP1   TFIIF interacting component of CTD phosphatase       YGR186w   TFG1   TFIIF subunit (transcription initiation factor), 105 kD       YGR005c   TFG2   TFIIF subunit (transcription initiation factor), 54 kD       YDR311w   TFB1   TFIIH subunit (transcription initiation factor), 75 kD       YPR025c   CCL1   TFIIH subunit (transcription initiation factor), cyclin C component       YLR005w   SSL1   TFIIH subunit (transcription initiation factor), factor B       YDR460w   TFB3   TFIIH subunit (transcription/repair factor)       YPL122c   TFB2   TFIIH subunit (transcription/repair factor)       YPR186c   PZF1   TFIIIA (transcription initiation factor)       YGR246c   BRF1   TFIIIB subunit, 70 kD       YNL039w   TFC5   TFIIIB subunit, 90 kD       YGR047c   TFC4   TFIIIC (transcription initiation factor) subunit, 131 kD       YAL001c   TFC3   TFIIIC (transcription initiation factor) subunit, 138 kD       YOR110w   TFC7   TFIIIC (transcription initiation factor) subunit, 55 kDa       YDR362c   TFC6   TFIIIC (transcription initiation factor) subunit, 91 kD       YBR123c   TFC1   TFIIIC (transcription initiation factor) subunit, 95 kD       YER148w   SPT15   the TATA-binding protein TBP       YOR143c   THI80   thiamin pyrophosphokinase       YIL078w   THS1   threonyl tRNA synthetase, cytosolic       YOR074c   CDC21   thymidylate synthase       YGR116w   SPT6   transcription elongation protein       YML010w   SPT5   transcription elongation protein       YBL093c   ROX3   transcription factor       YBR049c   REB1   transcription factor       YMR043w   MCM1   transcription factor of the MADS box family       YOR174w   MED4   transcription regulation mediator       YPL082c   MOT1   transcriptional accessory protein       YBR193c   MED8   transcriptional regulation mediator       YAL003w   EFB1   translation elongation factor eEF1beta       YLR249w   YEF3   translation elongation factor eEF3       YNL163c   RIA1   translation elongation factor eEF4       YNL244c   SUI1   translation initiation factor 3 (eIF3)       YPR016c   TIF6   translation initiation factor 6 (eIF6)       YMR260c   TIF11   translation initiation factor eIF1a       YPL237w   SUI3   translation initiation factor eIF2 beta subunit       YER025w   GCD11   translation initiation factor eIF2 gamma chain       YJR007w   SUI2   translation initiation factor eIF2, alpha chain       YDR211w   GCD6   translation initiation factor eIF2b epsilon, 81 kDa subunit       YLR291c   GCD7   translation initiation factor eIF2b, 43 kDa subunit       YGR083c   GCD2   translation initiation factor eIF2B, 71 kDa (delta) subunit       YOR260w   GCD1   translation initiation factor eIF2bgamma subunit       YBR079c   RPG1   translation initiation factor eIF3 (p110 subunit)       YDR429c   TIF35   translation initiation factor eIF3 (p33 subunit)       YNL062c   GCD10   translation initiation factor eIF3 RNA-binding subunit       YOR361c   PRT1   translation initiation factor eIF3 subunit       YMR146c   TIF34   translation initiation factor eIF3, p39 subunit       YOL139c   CDC33   translation initiation factor eIF4E       YPR041w   TIF5   translation initiation factor eIF5       YBR143c   SUP45   translational release factor       YJL125c   GCD14   translational repressor of       GCN4   YJL054w   TIM54 translocase for the insertion of proteins into the               mitochondrial inner membrane       YBL050w   SEC17   transport vesicle fusion protein       YDR407c   TRS120   TRAPP subunit of 120 kDa involved in targeting and fusion of ER               to golgi transport vesicles       YMR218c   TRS130   TRAPP subunit of 130 kDa involved in targeting and fusion of ER               to golgi transport vesicles       YBR254c   TRS20   TRAPP subunit of 20 kDa involved in targeting and fusion of ER               to golgi transport vesicles       YDR472w   TRS31   TRAPP subunit of 31 kDa involved in targeting and fusion of ER               to golgi transport vesicles       YOL102c   TPT1   tRNA 2′-phosphotransferase       YJL087c   TRL1   tRNA ligase       YER168c   CCA1   tRNA nucleotidyltransferase       YPL083c   SEN54   tRNA splicing endonuclease alpha subunit       YLR105c   SEN2   tRNA splicing endonuclease beta subunit       YMR059w   SEN15   tRNA splicing endonuclease delta subunit       YAR008w   SEN34   tRNA splicing endonuclease gamma subunit       YJL035c   TAD2   tRNA-specific adenosine deaminase 2       YLR136c   TIS11   tRNA-specific adenosine deaminase 3       YOL097c   WRS1   tryptophan-tRNA ligase       YIL147c   SLN1   two-component signal transducer       YGR185c   TYS1   tyrosyl-tRNA synthetase       YDR240c   SNU56   U1 snRNA protein, no counterpart in mammalian snRNP       YDR235w   PRP42   U1 snRNP associated protein, required for pre-mRNA splicing       YMR240c   CUS1   U2 snRNP protein       YPR178w   PRP4   U4/U6 snRNP 52 KD protein       YHR165c   PRP8   U5 snRNP protein, pre-mRNA splicing factor       YKL173w   SNU114   U5 snRNP-specific protein       YGR048w   UFD1   ubiquitin fusion degradation protein       YDR510w   SMT3   ubiquitin-like protein       YPL020c   ULP1   Ub1-specific protease       YBR243c   ALG7   UDP-N-acetylglucosamine-1-phosphate transferase       YKL024c   URA6   uridine-monophosphate kinase       YKL035w   UGP1   UTP-glucose-1-phosphate uridylyltransferase       YMR197c   VTI1   v-SNARE: involved in Golgi retrograde protein traffic       YGR094w   VAS1   valyl-tRNA synthetase       YBR080c   SEC18   vesicular-fusion protein, functional homolog of NSF       YMR049c   ERB1   weak similarity to  A. thaliana  PRL1 protein       YOR353c       weak similarity to adenylate cyclases       YLR064w       weak similarity to Anopheles NADH-ubiquinone oxidoreductase,               chain 4       YLR010c       weak similarity to Aquifex aeolicus adenylosuccinate synthetase       YHR074w   QNS1   weak similarity to  B. subtilis  spore outgrowth factor B       YMR211w       weak similarity to beta tubulins       YJL010c       weak similarity to  C. elegans  hypothetical protei ZK792.5       YJL104w   MIA1   weak similarity to  C. elegans  hypothetical protein F45G2.c       YGR280c       weak similarity to CBF5P       YJL011c       weak similarity to chicken hypothetical protein       YHR085w       weak similarity to fruit fly brahma transcriptional activator       YNL110c       weak similarity to fruit fly RNA-binding protein       YPL126w   NAN1   weak similarity to fruit fly TFIID subunit p85       YHR040w       weak similarity to HIT1P       YJR136c       weak similarity to human 3′,5′-cyclic-GMP phosphodiesterase       YJL091c       weak similarity to human G protein-coupled receptor       YOR056c       weak similarity to human phosphorylation regulatory protein HP-               10       YLL037w       weak similarity to human platelet-activating factor receptor       YGL108c       weak similarity to hypotetical  S. pombe  protein       YJR012c       weak similarity to hypothetical protein B17C10.80  N. crassa         YNL260c       weak similarity to hypothetical protein  S. pombe         YPL233w       weak similarity to hypothetical protein  S. pombe         YJR041c       weak similarity to hypothetical protein SPAC2G11.02  S. pombe         YJR141w       weak similarity to hypothetical protein SPBC1734.10c  S. pombe         YOR004w       weak similarity to hypothetical protein       YDR339c   YBR168w   weak similarity to hypothetical protein       YLR324w   YDR339c   weak similarity to hypothetical protein       YOR004w   YDR413c   weak similarity to NADH dehydrogenase       YHR052w       weak similarity to  P. yoelii  rhoptry protein       YBL004w       weak similarity to Papaya ringspot virus polyprotein       YOR281c   PLP2   weak similarity to phosducins       YGR156w   PTI1   weak similarity to PIR: A40220 cleavage stimulation factor 64 K               chain - human       YHR197w       weak similarity to PIR: T22172 hypothetical protein F44E5.2  C. elegans         YGR198w       weak similarity to PIR: T38996 hypothetical protein SPAC637.04                 S. pombe         YLR143w       weak similarity to  Pyrococcus horikoshii  hypothetical protein               PHBJ017       YDL166c       weak similarity to  Pyrococcus horikoshii  hypothetical protein               PHBJ019       YNL182c       weak similarity to  S. pombe  hypothetical protein       YDR365c       weak similarity to  Streptococcus  M protein YBR155w       CNS1       weak similarity to stress-induced STI1P       YCR054c   CTR86   weak similarity to THR4P       YJR046w   TAH11   weak similarity to  Xenopus vimentin  4       YHR196w       weak similarity to YDR398w       YGL113w   SLD3   weak similarity to YOR165w                  
 
      G. Yeast Codon Bias  
      To obtain optimal expression of a heterologous peptidase in yeast cells, nucleic acids encoding peptidases will be designed and synthesized according to yeast codon preference. For example, the inventors have synthesized the gene that encodes light-chain of BoNT/B. Clostridial DNA contains a high content of adenine and thymine, which can terminate transcription in yeast. Without changing the amino acid sequence of the light-chain, the construct eliminates A/T rich stretches and rare yeast condons. The resulting peptidase encoded by the synthetic gene efficiently cleaves the recombinant substrate in yeast cells, causing cell death. The following table, derived from Bennetzen &amp; Hall (1982), lists yeast codon preferences.  
               TABLE 4                          YEAST CODON PREFERENCES                             Codon   Preferred Triplet                                             Ala   GCU, GCC                           Ser   UCU, UCC                       Thr   ACU, ACC                       Val   GUU, GUC                       Ile   AUU, AUC                       Asp   GAC                       Phe   UUC                       Tyr   UAC                       Cys   UGU                       Asn   AAC                       His   CAC                       Arg   AGA                       Glu   GAA                       Leu   UUG                       Lys   AAG                       Gly   GGU                       Gln   CAA                       Pro   CCA                       Met   AUG                       Trp   UGG                      
 
 V. Assays 
 
      As discussed above, the yeast cell system of the present invention is designed such that cleavage of the recombinant protein substrate by the heterologous peptidase causes cell death. Conditional (or regulated) expression of the heterologous peptidase permits growth of the yeast host cell without cell death. Inclusion of an inhibitor of the heterologous peptidase, under conditions supporting peptidase expression, aborts the enzymatic activity of the peptidase and permits proliferation of the yeast cells. One can introduce a large number of biological peptides, proteins, small molecules, or intracellular recombinant antibodies in yeast cells bearing the heterologous peptidase and the recombinant protein substrate to directly and rapidly select/identify specific peptidase inhibitors that permit yeast cell growth.  
      A. Genetic Selection  
      A DNA library encoding potential protein inhibitors can be transformed in yeast cells with high frequency (at 10 4 -10 5  transformants/microgram plasmid DNA). Transformants are plated on agar plates containing an inducer of the peptidase gene, and an amino acid drop-out for the selection of plasmid marker. Most yeast transformants are not able to grow on galactose containing plates since the heterologous peptidase is expressed, and those not transformed will additionally not grow because of the absence of the plasmid. However, the presence of a plasmid-borne peptidase inhibitor in a yeast transformant will lead to cell growth and formation of a colony. The plasmid DNA can be recovered using standard DNA purification procedure, and the DNA sequence of the inhibitor can be determined through DNA sequencing, if not previously known.  
      B. High-Throughput Screen (HTS)  
      Small molecule peptide and chemical inhibitors can be identified by using clear bottom multi-well plates (currently, 96- and 384-well plates are commercially available). Yeast cells are diluted and distributed equally in each well in the presence of yeast growth media containing galactose. Compounds are distributed to each well and yeast cell growth is monitored by visual inspection or measured with a multi-well plate reader (at A 600 ). The presence of a toxin inhibitor will lead to yeast cell growth and increased turbidity in a well. This HTS assay is a standard practice and has been successfully employed in the identification of small molecule inhibitors of process distinct from ours (see Hughes, 2002). To overcome the potentially limiting factor of cell penetration, one can enhance cell permeability of the yeast cells by treating with specific chemicals such as polymixin B (Boguslaski, 1985), or using yeast strain that carries a cell wall mutation (Brendel, 1976). Also contemplated are yeast cells that are impaired in multidrug efflux (Wolfger et al., 2001).  
     VI. EXAMPLES  
      The following examples are included to further illustrate various aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques and/or compositions discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.  
     Example 1  
      A method is presented which allows one to directly select for intracellular inhibitors of the light chain (LC) peptidase of botulinum neurotoxin (serotype B) BoNTB and other bacterial toxins. A yeast mutant that would be susceptible to the lethal effects of intracellular BoNTB/LC was generated. This toxin is an endopeptidase that cleaves a specific QF peptide bond in synaptobrevin (Sb), a neuronal cell protein that is required for vesicle fusion to the presynaptic membrane. Yeast ( Saccharomyces cerevisiae ) possess two functionally redundant Sb homologs, Snc1 and Snc2, that are essential for secretory vesicle fusion to the plasma membrane. Snc1/2 are structurally and functionally related to Sb; however Snc1/2 lack the QF sequence that is recognized by BoNTB/LC. Therefore, whether a Snc2 protein that contains a portion of Sb (with the QF sequence) could be rendered inactive by the expression of BoNTB/LC in yeast cells was investigated.  
      Two yeast strains that lack Snc1 were constructed. Growth of the first mutant is dependent on expression of Snc2. Growth of the second Δsnc1 mutant is dependent on the expression of a Snc2/Sb/Snc2 fusion. As was shown, both of these strains can grow when BoNTB/LC expression is repressed by the regulatable GAL1 promoter. The left-hand side of  FIG. 1  depicts an agar plate containing glucose. In contrast, derepression of the GAL1 promoter was lethal to cells that expressed the Snc2/Sb/Snc2 fusion, which were grown in an agar plate containing glucose, but not to cells that expressed the Snc2 protein, which were grown in an agar plate containing galactose and shown in the right-had side of  FIG. 1 .  
      This yeast cell based assay provides a powerful tool with which to directly select for intracellular inhibitors of BoNTBLC. Yeast expression libraries of scFv (single chain fragment variable) antibodies may be introduced into yeast that contain the Snc2/Sb/Snc2 fusion, selecting for growth in the presence of galactose.  
     Example 2  
      In a second embodiment, the inventors synthesized the gene corresponding to BoNTC/LC, eliminating A/Trich stretches without changing the amino acid sequence. The gene was then placed under control of the GAL1 promotor, which can be regulated in yeast: ON in the presence of galactose and OFF in the presence of glucose. The GAL1-BoNTC/LC construct and a control plasmid vector lacking GAL1-BoNTC/LC were then introduced into yeast cells that expressed either Sso1p or Sso2p. As shown in  FIG. 2 , the vector control and GAL1-BoNTC/LC were not lethal to yeast cells that were grown in the presence of glucose (left hand dish). In addition, the vector did not interfere with cell growth in the presence of galactose (right hand dish). Importantly, the GAL1-BoNTC/LC construct strongly inhibited cell growth in the presence of galactose (right hand panel). These data demonstrate that BoNTC/LC exerts a severe growth defect on yeast that express either Sso1p or Sso2p.  
      A clue to the substrate specificity of BoNTC/LC may already exist. A careful examination of  FIG. 2  (right hand dish) shows the Sso1p strain grows slightly better than the Sso2p strain in the presence of BoNTC/LC. One explanation for this result could be that BoNTC/LC cleaves Sso2p somewhat better than Sso1p. In support of this idea, Sso2p exhibits slightly stronger similarity to syntaxin at the cleavage site as compared to Sso1p ( FIG. 3 ). In particular, syntaxin, Sso1p, and Sso2p have Lys, Asp, Asn, respectively, at the P2 position. These differences result in a change in amino acid charge from positive to negative for Sso1p, but only a change of positive to polar for Sso2p.  
      All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention as defined by the appended claims.  
     VII. REFERENCES  
      The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference: 
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