Patent Publication Number: US-5525496-A

Title: DNA sequence encoding sterol Δ14 reductase

Description:
This is a continuation of application Ser. No. 08/107,347 filed on Aug. 16, 1993, now abandoned. 
    
    
     TECHNICAL FIELD OF THE INVENTION 
     This invention relates to the identification of a Saccharomyces cerevisiae gene encoding sterol Δ14 reductase. 
     BACKGROUND OF THE INVENTION 
     Sterols are steroid alcohols of vegetable and animal origin. Ergosterol is the principal membrane sterol of fungi. It is structurally similar to its animal counterpart, cholesterol, and its higher plant counterparts, stigmasterol and sitosterol. Though the biosynthesis of ergosterol in fungi involves steps distinct from the other sterols, the pathways in different organisms share several common steps. The lanosterol 14α-demethylation steps in cholesterol and ergosterol formation in animals and fungi, as well as the obtusifoliol 14Δ-demethylation in stigmasterol and sitosterol biosynthesis in plants, both lead to the formation of a double bond between carbons 14 and 15 of the sterol ring. This double bond is then reduced by sterol Δ14 reductase activity. The enzyme is located in the microsomal fraction in pig liver, yeast and Zea mays, and requires NADPH as an electron donor (Marcireau, C., et al., Curt. Genet. 22: 267-272 (1992)). 
     Genetic studies of ergosterol biosynthesis mainly have been carried out in Saccharomyces cerevisiae (Paltauf, F., et al., in Jones, E. W., et al., eds., The Molecular and Cellular Biology of the Yeast Saccharomyces, Gene Expression, Cold Spring Harbor Laboratory Press, 1992, pages 434-437). In yeast, ergosterol affects membrane fluidity and permeability and plays an essential role in the yeast cell cycle. 
     A number of mutations in the yeast ergosterol biosynthetic pathway have been isolated either by reverse genetic approaches or by selection for mutations producing polyene resistance, and many of the genes have been identified. Toward the end of the pathway, sterol Δ14 reductase, Δ8-Δ7 isomerase, and C-24(28) reductase catalyze steps in the conversion of lanosterol to ergosterol. After ignosterol is reduced by sterol Δ14 reductase, which eliminates a double bond in the D ring of the molecule, the sterol is demethylated and rearranged to fecosterol, which is then isomerized by sterol Δ8-Δ7 isomerase. The sterol is then desaturated in two positions and its side chain is reduced by C-24(28) reductase. Some of the genes encoding the enzymes have been identified and named as follows (Paultauf, et al., cited above, Lorenz, T., and Parks, L. W., DNA and Cell Biol. 11: 685-692 (1992), and Example 1 below): 
     
         ______________________________________                                    
Enzyme             Gene                                                   
______________________________________                                    
Δ14 reductase                                                       
                    ERG24                                                 
C-24 methyl transferase                                                   
                   ERG6                                                   
Δ8 - Δ7 isomerase                                             
                   ERG2                                                   
C-24(28) reductase ERG4                                                   
______________________________________                                    
 
    
     Based on the accumulation of intermediates following fungicide treatment, morpholine fungicidal compounds such as tridemorph and fenpropimorph have been reported to be inhibitors of sterol 14 reductase and sterol Δ8-Δ7 isomerase (Baloch, R. and Mercer, I., Phytochemistry 26: 663-668 (1987)). However, it recently has been found that the sterol Δ8-Δ7 isomerase gene is not essential for viability in S. cerevisiae (Ashman, W. H., et al., Lipids 26: 628-632 (1991)), suggesting that the killing effect of morpholine fungicides may be primarily the result of sterol Δ14 reductase inhibition. 
     It has also been shown that the C-24 methyl transferase gene (ERG6) is not essential for viability in S. cerevisiae (Gaber, R. F., et al., Mol. Cell. Biol. 9: 3447-3456 (1989)). Mutant cells exhibit normal vegetative growth, but they differ from the wildtype in a number of respects, including drug supersensitivity, presumably due to alterations in membrane function (ibid.). Drug super-sensitivity has been observed in other yeast mutants, including one denoted YGL022 which encodes a putative transport protein (Chen, W., et al., Yeast 7: 305-308 (1991)). 
     SUMMARY OF THE INVENTION 
     The objects of the invention are to identify a gene encoding sterol Δ14 reductase, to elucidate the primary structure of the enzyme encoded by the gene, and to investigate the relationship of the structure to other polypeptides, especially other enzymes in the sterol biosynthetic pathway. The sterol Δ14 reductase gene and enzyme are useful in devising screening tests to identify sterol biosynthesis inhibitors that are potential fungicides for a wide variety of agricultural, medical, and veterinary applications. 
     These and other objects are accomplished by the present invention, which provides a gene encoding sterol Δ14  reductase, the polypeptide primary structure it encodes, and the relationship of the structure to other polypeptides. Also provided are RNA sequences corresponding to the DNA sequence of the gene, biologically functional plasmids or vectors comprising the DNA or RNA sequence, and procaryotic or eucaryotic host cells transformed or transfected with the plasmid or vector in a manner allowing the host cell to express the polypeptide. 
     A DNA sequence encoding Saccharomyces cerevisiae sterol Δ14 reductase is cloned by selecting strains carrying sequences on a 2μ based vector for resistance to a morpholine fungicide such as fenpropimorph, fenpropidin, or tridimorph. Fenpropimorph is preferred. When fenpropimorph is employed, four distinct plasmid inserts which produce morpholine resistance are obtained, denoted pML99, pML100, pML101 and pML103, which are useful in screens of sterol biosynthesis inhibition. One of these, pML100, is characterized and sequenced, and the putative amino acid sequence of the polypeptide encoded by the open reading frame is determined (SEQ ID NO 1). 
    
    
     DESCRIPTION OF THE FIGURES 
     FIG. 1 shows restriction maps of four plasmid inserts recovered via selection for fenpropimorph resistance in Saccharomyces cerevisiae as described in Example 1. Selected restriction enzyme digestion sites are shown for each insert. 
     FIG. 2 shows fenpropimorph resistance of subclones of pML100, a plasmid containing the cloned sterol Δ14 reductase gene. 
     FIG. 3 depicts a comparison of the amino acid sequence derived from the major open reading frame of the pML100 sequence encoding S. cerevisiae sterol Δ14 reductase (SEQ ID NO 1) with three homologous sequences: the chicken nuclear lamin B receptor (SEQ ID NO 2, Worman, H. J., et al., J. Cell Biology 111: 1535-1542 (1990)), the Saccharomyces cerevisiae YGL022 sequence (SEQ ID NO 3, Chen, et al., cited above), and the Schizosaccharomyces pombe stsl gene (SEQ ID NO 4, Shimanuki, M., et al., Mol. Biol. Cell 3: 263-273 (1992)). The figure employs standard one-letter nomenclature for the amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The Saccharomyces cerevisiae gene encoding sterol Δ14 reductase is cloned by selecting strains carrying sequences on a 2μ based vector for resistance to a morpholine fungicide. One of the plasmids so obtained is characterized and sequenced to obtain the primary structure of sterol Δ14 reductase. 
     By &#34;morpholine fungicide&#34; is meant any morpholine and structurally related piperidine compound having large ring N-substituents such as dodemorph, tridemorph, aldimorph, fenpropimorph, amorolfine, and fenpropidin which are employed as fungicides. Mixtures of morpholine fungicides may also be employed. Fenpropimorph is employed in one embodiment. 
     In morpholine screenings of S. cerevisiae strains carrying DNA sequences on a 2μ vector, plasmid inserts which produce morpholine resistance are recovered. For example, where fenpropimorph is the morpholine employed, four plasmids denoted pML99, pML100, pML101 and pML103 are recovered. Although fenpropimorph is reported to inhibit the enzymes sterol Δ14 reductase and Δ8-Δ7 isomerase, none of the inserts exhibit restriction maps resembling ERG2, the gene encoding Δ8-Δ7 isomerase. In addition, a 2μ plasmid carrying the ERG2 sequence does not produce fenpropimorph resistance. 
     Plasmid pML100 produces fenpropimorph resistance consistently when tested in a number of different genetic backgrounds. Tests with a panel of fungicides indicate that pML100 produces significant resistance only to the morpholine fungicides fenpropimorph, tridemorph, fenpropidin, and azasterol, compounds which have a shared site of action, the enzyme sterol Δ14 reductase. No increase in resistance is seen to a variety of other fungicides which are not sterol Δ14 reductase inhibitors, suggesting that pML100 encodes a function specific to sterol Δ14 reductase activity. Other investigators report that selection for fenpropidin or fenpropimorph resistance in other S. cerevisiae strains produce plasmids exhibiting properties similar to pML100 (Lorenz and Parks, cited above, and Marcireau, C., et al., cited above). 
     A chromosomal disruption of the sequence producing morpholine resistance results in ergosterol auxotrophy and the build-up of ignosterol, the sterol Δ14 reductase substrate. The DNA sequence which produces this activity is obtained (SEQ ID 1), which contains an open reading frame encoding an integral membrane protein, consistent with an enzyme catalyzing a reaction in the ergosterol biosynthesis pathway. 
     Thus, this invention provides a purified and isolated DNA sequence encoding Saccharomyces cerevisiae sterol Δ14 reductase. Because of the degeneracy of the genetic code, a variety of codon change combinations can be selected to form DNA that encodes sterol Δ14 reductase, so that any nucleotide deletion(s), addition(s), or point mutation(s) that result in a DNA encoding sterol Δ14 reductase are encompassed by this invention. Since certain codons are more efficient for polypeptide expression in certain types of organisms, the selection of gene alterations to yield DNA material that codes for the enzyme are preferably those that yield the most efficient expression in the type of organism which is to serve as the host of the recombinant vector. Altered codon selection may also depend upon vector construction considerations. 
     DNA which encodes Δ14 reductase may be natural, recombinant or synthetic. Thus, DNA of the invention may be isolated from yeast strains or constructed from oligonucleotides using conventional methods. Also encompassed are DNA sequences homologous or closely related to complementary DNA described herein, namely DNA sequences which hybridize, particularly under stringent conditions that result in pairing only between nucleic acid fragments that have a high frequency of complementary base sequences, to DNA encoding sterol Δ14 reductase particularly described herein, and RNA corresponding thereto. In addition to these sequences, DNA encompassed by this invention may contain additional sequences, depending upon vector construction sequences, that facilitate expression of the gene. 
     As described above, DNA encoding the sterol Δ14 reductase of this invention, or RNA corresponding thereto, are useful when introduced into a vector or plasmid, and the recombinant plasmid used to transform microbial host organisms such as S. cerevisiae. Other host cells such as E. coli may be employed in some embodiments. Especially useful in some embodiments are S. cerevisiae cells into which the gene has been introduced at high copy. This invention thus also provides novel, biologically functional RNA and DNA vectors and plasmids incorporating RNA and DNA sequences describing the reductase generated by standard means. Culture of host organisms stably transformed or transfected with such vectors or plasmids under conditions facilitative of large scale expression of the exogenous, vector-borne DNA or RNA sequences and isolation of the desired polypeptides from the growth medium, cellular lysates, or cellular membrane fractions are also provided. 
     The present invention provides for the total and/or partial manufacture of DNA sequences coding for sterol Δ14 reductase, and including such advantageous characteristics as incorporation of codons preferred for expression by selected hosts, provision of sites of cleavage by restriction by endonuclease enzymes, and provision of additional initial, terminal or intermediate DNA sequences which facilitate construction of readily expressed vectors. 
     DNA (and RNA) sequences of this invention code for all sequences useful in securing expression in procaryotic or eucaryotic host cells of polypeptide products having at least a part of the primary structural conformation, and one or more of the biological properties of sterol Δ14 reductase which are comprehended by: (a) the DNA sequence encoding sterol Δ14 reductase; (b) DNA sequences which hybridize to DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b) above. Specifically comprehended are genomic DNA sequences encoding allelic variant forms of the enzyme, and sequences encoding RNA, fragments thereof, and analogues wherein RNA or DNA sequences may incorporate codons facilitating transcription or RNA replication host cells. 
     Particularly useful are S. cerevisiae strains into which has been introduced a DNA sequence of this invention, particularly those having multiple copies of the gene. Such strains are useful in screens for sterol Δ14 reductase inhibition such as those described in copending U.S. application Ser. No. 08/107,348 filed concurrently with this application and incorporated in its entirety by reference. In an example screen, test samples are added to a yeast culture of a transformed strain such as a strain transformed with pML100, and to a corresponding control culture which does not have the introduced gene. Positive samples are identified after incubation by observation that growth inhibition in the culture having no introduced reductase gene exceeds growth in the corresponding culture having the introduced gene. In preferred embodiments, a known inhibitor of sterol Δ14 reductase is employed for comparison purposes in both cultures of the screen. 
     Other plasmids that produce morpholine resistance such as pML99, pML101, and pML103 described above are also useful in other screens for compounds that affect sterol biosynthesis, including screens for sterol Δ14 reductase inhibitors such as those described above. As set out more fully hereinafter, in initial genetic analyses, these plasmids show differences from pML100 in their interactions with ergosterol biosynthesis mutations. Hence, these plasmids are useful with screens such as those described above except that different yeast strains are employed. Alternatively, screening results with these plasmids can be used in combination with screening tests using pML100. 
     This invention also provides the polypeptide encoded by the sterol Δ14 reductase sequences of this invention, e.g., the polypeptide encoded by the open reading frame set out in SEQ ID NO 1. Correspondingly, the invention provides for manufacture (and development by site specific mutagenesis of cDNA and genomic DNA) of DNA sequences coding for microbial expression of sterol Δ14 reductase which differ from the forms specifically described herein in terms of identity or location of one or more amino acid residues (i.e., deletion and/or substitution analogues wherein one or more residues are added to a terminal or medial portion of the polypeptide), and which share the biological properties of the enzyme. In embodiments involving the microbial expression of polypeptides provided by the invention, isolation and purification employ standard methodology including, for example, preparative chromatographic separations and immunological separations, including monoclonal and/or polyclonal antibody preparations. 
     The sequence of pML100 exhibits partial homology to three other previously reported genes as follows (see FIG. 3): the chicken nuclear lamin B receptor (SEQ ID NO 2, Worman, H. J., et al., cited above; 101 out of 419 amino acids), the S. cerevisiae YGL022 sequence (SEQ ID NO 3, Chen, et al., cited above; 95 out of 473 amino acids), and the Schizosaccharomyces pombe stsl gene (SEQ ID NO 4,  Shimanuki, M., et al., cited above; 92 out of 453 amino acids). The phenotypes of strains carrying stsl and YGL022 mutations are consistent with the hypothesis that these mutations produce lesions in erogsterol biosynthesis. The S. pombe stsl +  gene and the S. cerevisiae YGL022 sequence have been reported to encode putative transport proteins which produce drug resistance by pumping compounds out of the cell. Mutations in these genes produce super-sensitivity to a wide variety of compounds. Drug super-sensitivity is also a phenotype associated with ergosterol biosynthesis mutations such as erg6 (Gaber, R. F., et al., Mol. Cell. Biol. 9: 3447-3456 (1989)), erg2 and erg3, presumably due to alterations in membrane function. 
     Physiological studies with an stsl mutant strain and a YGL022 disruption strain more particularly described hereafter and complementation studies with YGL022 provide direct proof that stsl +  and YGL022 encode a function in sterol biosynthesis identified as ERG4, sterol C-24(28) reductase, in S. cerevisiae. Thus, the enzymes sterol Δ14 reductase and sterol C-24(28) reductase are related enzymes, with the former catalyzing the reduction of a double bond in the D ring and the latter catalyzing the reduction of a double bond in the side chain of the sterol. 
     The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard. Fenpropimorph, fenpropidin and tridemorph are purchased from Crescent Chemical Company, Inc., Hauppauge, N.Y. Synthetic dextrose (SD) media contains 0.7% yeast nitrogen base without amino acids, 2% dextrose and 2% agar. Yeast extract, peptone and dextrose (YEPD) media contains 1% yeast extract, 2% peptone, 2% dextrose and 2% agar. 
     EXAMPLE 1 
     This example describes the cloning and sequencing of the Saccharomyces cerevisiae gene encoding sterol Δ 14 reductase. The gene is isolated and cloned by selecting strains carrying sequences on a 2μ based vector for resistance to the morpholine fungicide, fenpropimorph, to obtain a plasmid which is shown to carry the structural gene based upon the phenotype of gene disruption strains. 
     Isolation and characterization of morpholine resistance plasmids. Morpholine and structurally related piperidine fungicides reportedly inhibit sterol Δ14 reductase and sterol Δ8 to Δ7 isomerase (Baloch and Mercer, cited above). The growth of S. cerevisiae strain Y294, genotype MATα, leu2-3,112, ura3-52, his3Δ, trpl, Gal +  (Brugge, J. S., et al., Mol. Cell. Biol. 7: 2180-2187 (1987)), in SD medium supplemented with leucine, tryptophan, uracil and histidine is inhibited by 20 μg/ml of the morpholine fungicide fenpropimorph and 50 μg/ml of the morpholine fungicide tridemorph. Fenpropimorph is used for subsequent selection experiments because of its slightly greater potency. 
     When Y294 cells are plated onto 20 μg/ml of fenpropimorph in SD media supplemented with leucine, tryptophan, uracil and histidine, spontaneous mutants are recovered at the rate of ˜1 per 2.5×10 6  plated cells. When a library of S. cerevisiae sequences in the multicopy vector YEp13 (Nasmyth, K. A., and Tatchell, K., cell 19: 753-764 (1980)) is introduced into strain Y294 and cells are plated on SD media supplemented with tryptophan, uracil, histidine and fenpropimorph, resistant colonies appeared at the rate of ˜1 per 10 4 , suggesting that resistance is produced by library plasmids in some of the colonies. Plasmids are cured from randomly selected resistant colonies by growing the cells in non-selective rich YEPD media and retesting for fenpropimorph resistance. In 13 strains, the plasmid-cured derivative shows sensitivity to 20 μg/ml fenpropimorph while the original plasmid carrying strain retested as fenpropimorph-resistant. 
     DNA is isolated from these 13 strains and plasmid DNA is recovered by E. coli transformation. Five different types of plasmid DNA are identified following an examination of restriction enzyme digestion patterns using standard methods (FIG. 1). Seven strains carry one plasmid type, pML99, which has an insert of approximately 5.5 kb. Two additional strains carry a second plasmid type, pML100, which has an insert of approximately 5.6 kb. A third plasmid type, pML101, is found in two strains and carries an insert of approximately 5.5 kb. Two additional plasmid types are each recovered from a single strain and named pML102 (˜7.5 kb insert) and pML103 (˜5.1 kb insert). One representative plasmid of each type is selected and subjected to extensive restriction enzyme analysis, which indicates that the insert from plasmid pML101 is contained within the insert from pML102 so that a total of four unique sequences are recovered in this selection. Restriction enzyme digestion maps of the four different insert sequences are shown in FIG. 1. 
     A panel of fungicides representing a variety of chemical structures and mechanisms of action listed in Table 1 is tested by disk diffusion assay against strains carrying each of these plasmids in a YEp13 vector control. All five strains show similar levels of sensitivity to all of the tested compounds with the exception of the morpholines, fenpropidin, fenpropimorph and tridemorph, and azasterol. These compounds are less active on the strains carrying the four plasmids recovered by selection for fenpropimorph resistance. Consistent with agar dilution sensitivity results, fenpropimorph is more active by disk diffusion than tridemorph. These results suggest that the cloned sequences encode functions specific to the activity of morpholines and related compounds and do not carry genes which produce general fungicide resistance, e.g., by altering cell permeability. 
     
                       TABLE 1                                                     
______________________________________                                    
Fungicides Used For Plasmid Characterization                              
Compound   Target                                                         
______________________________________                                    
amphotericin B                                                            
           plasma membrance (polyene)                                     
cerulenin  fatty acid biosynthesis                                        
haloprogin respiration                                                    
ketoconazole                                                              
           ergosterol biosynthesis (lanosterol 14α-                 
           demethylase)                                                   
miconazole ergosterol biosynthesis (lanosterol 14α-                 
           demethylase)                                                   
dinaconazole                                                              
           ergosterol biosynthesis (lanosterol 14α-                 
           demethylase)                                                   
econazole  ergosterol biosynthesis (lanosterol 14α-                 
           demethylase)                                                   
fepropimorph                                                              
           ergosterol biosynthesis (sterol Δ14 reductase/           
           Δ8 - Δ7 isomerase)                                 
tridemorph ergosterol biosynthesis (sterol Δ14 reductase/           
           Δ8 - Δ7 isomerase)                                 
azasterol  ergosterol biosynthesis (sterol Δ14 reductase)           
tolnaftate ergosterol biosynthesis (squalene mono-                        
           oxygenase)                                                     
U18666A    ergosterol biosynthesis (squalene cyclase)                     
cycloheximide                                                             
           protein biosynthesis                                           
polyoxin D chitin biosynthesis (cell wall)                                
nikkomycin chitin biosynthesis (cell wall)                                
nocodazole microtubule assembly                                           
benomyl    microtubule assembly                                           
maneb      multi-target                                                   
metalaxyl  rRNA biosynthesis                                              
vinclozoline                                                              
           lipid peroxidation                                             
kanamycin  mitochondria                                                   
tunicamycin                                                               
           glycoprotein biosynthesis                                      
carboxin   succinate dehydrogenase                                        
antimycin  respiration                                                    
5-fluorocytosine                                                          
           nucleotide metabolism                                          
cyanobutarate                                                             
           microtubule assembly (herbicide)                               
glyphosate aromatic amino acid biosynthesis (herbicide)                   
phosphinothricin                                                          
           glutamine biosynthesis (herbicide)                             
aminotriazole                                                             
           histidine biosynthesis (herbicide)                             
sulfometuron                                                              
           branched chain amino acid biosynthesis                         
methyl     (herbicide)                                                    
pendimethalin                                                             
           microtubule assembly (herbicide)                               
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     The library employed for the selection is prepared using DNA isolated from strain AB320 (genotype HO, ade2-1, lys2-1, trp5-2, leu2-1, canl-100, ura3-1 and/or ural-1, met4-1, Nasmyth and Tatchell, cited above). When tested, strain AB320 is found to be slightly more sensitive to fenpropimorph than strain Y294, suggesting that the cloned sequences are likely to be producing resistance as the result of gene dosage effects. 
     Morpholine resistance in strains transformed with multi-copy ERG2 (sterol Δ8-Δ7 isomerase) plasmids. One gene that would be expected to produce morpholine resistance at high copy is ERG2, which encodes a reported morpholine target, Δ8-Δ7 isomerase. This gene was recently cloned by the complementation of a polyene resistance mutation (Ashman, cited above). The published ERG2 restriction map is different from the restriction maps of the four sequences recovered by morpholine resistance selection. Since it is possible that the ERG2 sequence is missed in the morpholine resistance screen, this gene is introduced into S. cerevisiae strain Y294 on the 2μ based plasmid, pML104, constructed by subcloning the ERG2 gene on a 2.1 kb HindIII fragment from plasmid PIU406 (Ashman, et al., cited above) into the HindIII site of plasmid YEp351. This strain shows no increase in fenpropimorph resistance relative to YEp351-transformed control strain. Plasmid pML104 does, however, produce nystatin sensitivity when introduced into the erg2 mutant strain WAO (Ashman, et al., cited above), demonstrating that plasmid pML104 carries a functional ERG2 gene. Sterol Δ8-Δ7 isomerase may not over-express when present on a 2μ based, multi-copy plasmid, or the enzyme may not be a morpholine target in S. cerevisiae. 
     Characterization of fenpropimorph resistance plasmid pML100. The four fenpropimorph-resistance plasmids pML99, pML100, pML101, and pML103 are transformed into three ergosterol pathway mutant strains, erg2 (denoted WAO, genotype MATa, his7-2, leu2-3,112, ura3-52, erg2-3, Ashman, et al., cited above); erg3 (denoted XML39-1d, geno-type MATa, leu2-3,112, erg3-2); and erg6 (denoted XML40-1c, genotype MATa, leu2-3,112, ga12, erg6-5). Morpholine sensitivity is determined by disk diffusion assay on appropriately supplemented SD medium using tridemorph and fenpropimorph. A zone size difference of greater than 3 mm performed in duplicate is recorded as resistance. The ergosterol pathway mutant strains vary in absolute level of morpholine sensitivity, and all resistance and sensitivity determinations are reported relative to vector (YEp13)-transformed control strains. The results are tabulated in Table 2. Only plasmid pML100 transformants are consistently fenpropimorph-resistant in all genetic backgrounds. 
     
                       TABLE 2                                                     
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Plasmid Phenotype in Ergosterol Pathway Mutant Strains                    
         Ergosterol                                                       
Strain   Genotype   Plasmid   Morpholine Resistance                       
______________________________________                                    
Y294     ERG+       YEp13     -                                           
Y294     ERG+       pML99     +                                           
Y294     ERG+       pML100    +                                           
Y294     ERG+       pML101    +                                           
Y294     ERG+       pML103    +                                           
WAO      erg2       YEp13     -                                           
WAO      erg2       pML99     -                                           
WAO      erg2       pML100    +                                           
WAO      erg2       pML101    -                                           
WAO      erg2       pML103    -                                           
XML39-1d erg3       YEp13     -                                           
XML39-1d erg3       pML99     +                                           
XML39-1d erg3       pML100    +                                           
XML39-1d erg3       pML101    -                                           
XML39-1d erg3       pML103    +/-*                                        
XML40-1c erg6       YEp13     -                                           
XML40-1c erg6       pML99     +                                           
XML40-1c erg6       pML100    +                                           
XML40-1c erg6       pML101    -                                           
XML40-1c erg6       pML103    +/-*                                        
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 *-Resistance was observed with fenpropimorph but not tridemorph.         
 
    
     Resistance is also seen with other morpholine anti-fungals (tridemorph and fenpropidin) and azasterol, all of which are reported to be inhibitors of sterol Δ14 reductase. However, no increase is seen to a variety of other fungicides which are not sterol Δ14 reductase inhibitors. Since resistance occurs only to sterol Δ14 reductase inhibitors and is seen for such inhibitors from two different chemical classes, it is likely that pML100 encodes a function specific to sterol Δ14 reductase activity. 
     Subclones of the pML100 insert are prepared in the yeast shuttle vector YEp352, transformed into yeast strain Y294, and tested for fenpropimorph resistance. As shown in FIG. 2, the fenpropimorph resistance region is limited to a 2.5 kb SphI/XbaI fragment located near one side of the insert/vector border. 
     Plasmid pML106, which contains this fragment in vector YEp352, is cleaved with BglII, which cuts once at a site near the middle of the SphI/XbaI fragment. A 3.0 kb BglII fragment containing the S. cerevisiae LEU2 gene is isolated from plasmid YEp13 and ligated into this BglII cite, producing plasmid pML108. The disrupted 5.5 kb SphI/XbaI fragment containing the LEU2 gene is isolated from plasmid pML108 and used to transform S. cerevisiae strain YPH501 to leucine prototrophy. Transformants in which the 5.5 kb SphI/XbaI fragment replaced the 2.5 kb SphI/XbaI fragment in one chromosomal homologue are identified by Southern analysis. 
     Tetrads from one such transformant (strain YPH501-2-1) are dissected and the spores germinated under anaerobic conditions on YEPD medium supplemented with Tween® 80 (500 μg/ml) and ergosterol (20 μg/ml). Strain YPH501-2-1 shows low (approximately 50%) spore viability, and no tetrads are recovered. This is found to be a property of strain YPH501 which showed a similar low level of spore viability when spores from the host strain are germinated anaerobically. By random spore analysis, 15 of 32 segregants are both Leu +  and obligate anaerobes, suggesting that the disruption has produced a genetic lesion in sterol biosynthesis. (The remaining 17 segregants are Leu -  and grow aerobically.) 
     One such obligate anaerobe segregant, denoted YPH501-2-1-3C, is analyzed for sterol content. The strain is grown anaerobically on YEPD medium containing ergosterol (5 μg/ml) to facilitate sterol uptake. After one day, the cells are harvested, washed in saline, resuspended in YEPD medium with no added sterol and grown for an additional 2 days to deplete cellular sterol. After 3 days, sterols are extracted from stationary phase yeast cells into n-heptane and analyzed by ultraviolet (UV) between 200 and 300 nm, gas chromatography (GC) and gas chromatographymass spectrometry (GC-MS). GC-MC analyses are performed on a Hewlett Packard (HP) 5980 instrument using a 30 meter×0.25 mm HP-5 column with a 25 micron film thickness. The column temperature is programmed from 280° C. to 300° C. with the initial temperature maintained for 2 minutes and increased at 3° C./minute. The final temperature is held for 6 minutes. The mass spectrometer is operated in the electron impact ionization mode at 70 eV. High pressure liquid chromatography (HPLC) analyses are performed using a reverse phase column (2.1×100 mm) packed with 5 micron spherical C18 bonded silica. Sterol samples are dissolved in a methanol:ethyl acetate (1:1) mixture and eluted from HPLC with 95% acetonitrile in water at 1 ml/minute. The detection wavelength is 270 nm. 
     UV analysis demonstrates a 250 nm broad peak indicative of a sterol containing a conjugated double bond system involving C-8(9) and C-14(15). GC analyses indicate a major peak with the relative retention time of 1.30 consistent with ignosterol (ergosta-8,14-dien-3β-ol, molecular weight 398), the sterol Δ14 reductase substrate. GC-MS analysis confirms that the major sterol accumulating in this disrupted strain is ignosterol. Small amounts of lanosterol, approximately 5%, are also observed, consistent with a block in the sterol pathway downstream of lanosterol and affecting the reduction of the C-14 double bond. The accumulation of ignosterol indicates a genetic lesion in sterol Δ14 reductase activity. 
     DNA sequence analysis of plasmid pML100. DNA sequences are performed using an Applied Biosystems automatic DNA sequencer from Applied Biosystems, Inc., Foster City, Calif. 94404, following the manufacturer&#39;s directions. Dye primers and dye terminators are used as appropriate for the insert to be sequenced. Oligonucleotides used for sequencing with dye terminators are synthesized using an Applied Biosystems oligonucleotide synthesizer according to the manufacturer&#39;s directions. 
     The DNA sequence of the 2.5 kb SphI/XbaI fragment of plasmid pML100 is set out in the Sequence Listing section hereinafter as SEQ ID NO 1. An open reading frame of 1314 base pairs is identified starting at an ATG codon at position 419 within the sequence. No other open reading frame of significant size is present within this fragment. Upstream of this ATG codon is an AT-rich sequence (66%), typical of many functionally expressed S. cerevisiae genes. This open reading frame encodes a 438 amino acid, 50.5 kilo-dalton basic (pI=9.2), presumptive integral membrane protein which, based upon hydropathy analysis using a computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence (Kyte, J., and Doolittle, R. F., J. Mol. Biol. 157: 105-132 (1982)), contains 8 or 9 putative transmembrane domains. 
     EXAMPLE 2 
     The open reading frame of the plasmid pML100 DNA sequence of Example 1 is compared to other sequences in this example. 
     The sequence is compared with sequences deposited in the Genbank® DNA sequence data base. Three sequences show partial homology: the chicken nuclear lamin B receptor (SEQ ID NO 2, Worman, H. J., et al., cited above; 101 out of 419 amino acids), the S. cerevisiae YGL022 sequence (SEQ ID NO 3, Chen, et al., cited above; 95 out of 473 amino acids) and the S. pombe stsl gene (SEQ ID NO 4, Shimanuki, M., et al., cited above; 92 out of 453 amino acids). A comparison of the amino acid sequences of the three yeast genes is shown in FIG. 3. A certain amount of sequence similarity is seen along the entire length of the three sequences and is particularly pronounced at the carboxy termini of these polypeptides. 
     EXAMPLE 3 
     The physiological characteristics of mutant phenotypes Schizosaccharomyces pombe stsl and Saccharomyces cerevisiae YGL022 found to be somewhat homologous to the pML100 sequence as described in Example 2 are characterized in this example. 
     S. pombe strains HM123 and JY6(stsl + ) and 111-1A (stsl) are obtained and analyzed for their sterol profiles. UV, GC-MC and HPLC analyses are carried out as described in Example 1 above; proton nuclear magnetic resonance (NMR) analysis on samples dissolved in d 6  -acetone is obtained on a Brunker AMX300 MHz spectrometer, Brunker Instruments, Inc., Billerica, Mass. 01821. As set out in the data summarized in Table 3 below, while the wild-type strains accumulate ergosterol and small amounts of lanosterol and perhaps 24-methylene-dihydrolanosterol (molecular weight 440), the mutant strain acccumulated principally the tetraene, ergosta-5,7,22,24(28)-tetraen-3-β-ol. 
     The conversion of the tetraene to ergosterol is considered to be the last step in ergosterol biosynthesis and the gene encoding this enzymatic step has been designated ERG4 in S. cerevisiae as discussed above. Whereas ergosterol gives absorption maxima at 262, 271, 282 and 293 nm, the precursor 24(28)-ergosterol gives absorption maxima at 232 nm reflecting the presence of a conjugated double bond system in the sterol side chain. The identity of the tetraene is confirmed by GC-MS and NMR. 
     GC-MS analysis shows a molecular ion at M/Z 394, 2 atomic mass units less than ergosterol. An ion is present at M/Z 123 which has no counterpart in the spectrum of ergosterol. This ion is proposed to represent the side chain fragment C 9  H 15  indicating the presence of two unsaturations. Consistent with the UV and GC-MS analyses, the structure of the conjugated side chain is substantiated by proton NMR signals indicative of the exomethylene protons (chemical shift 4.71 and 4.73, broad singlets) and signals for H22 (chemical shift 5.52, J 21 ,22= 8.8 Hz, J 22 ,23= 15.8 Hz) and H23 (chemical shift 5.87, J 23 ,22= 15.8 Hz). The chemical shift values for the latter two protons are considerably downfield from their position in ergosterol, which has a chemical shift of about 5.25, reflecting the deshielding effects of the conjugation with the C-24(28) double bond. This provides further evidence that an additional double bond is present in the sterol side chain. 
     Upon transformation of strain TP111-1A with a plasmid pST2SC which contains the wild-type C-24(28) reductase gene, an ergosterol profile is observed. However, when selection pressure is removed such that the plasmid is lost, the tetraene profile is restored, indicating that ergosterol synthesis requires the presence of the plasmid containing the wild-type gene (Table 3). Thus, stsl +  appears to encode a protein in the ergosterol biosynthesis pathway. 
     YGL022 is then characterized. A leaky, S. cerevisiae erg4 mutant strain (Molzahn, S. W., and Woods, R. A., J. Gen Microbiol. 72: 339-348 (1972)) is subjected to sterol analysis as described above. Approximately 35% ergosterol and 60% ergosta-5,7,22,24(28)-tetraen-3β-ol accumulates. When this strain is transformed with plasmid pA-B6.5 carrying the YGL022 sequence (Balzi, E., et al., J. Biol. Chem. 262: 16871-16879 (1987)) and retested, mostly ergosterol is detected (Table 3). 
     To confirm these findings, the open reading frame in YGL022 (in plasmid pA-B6.5) is disrupted by deleting an approximately 0.6 kb SmaI/AccI fragment internal to the open reading frame and replacing this with an approximately 2.2 kb HpaI fragment carrying the LEU2 gene (in a gene disruption construction similar to that described by Chen, et al., cited above). This sequence is released from the plasmid by XhoI and BamHI digestion, and the linear sequence is transformed into a diploid strain XML25 which is then sporulated. Integration of the construction into the YGL022 sequence is confirmed by Southern analysis. 
     Forty-seven tetrads are studied and all show 2:2 segration for leucine prototrophy. Leucine auxotrophic segregants are all wild-type for drug sensitivity while leucine prototrophic segregants show increased sensitivity to cycloheximide. One tetrad is analyzed for ergosterol content. The leucine auxotrophic segregants synthesize ergosterol while the leucine prototrophic segretants do not synthesize ergosterol and accumulate ergosta-5,7,22,-4(28)-tetraen-3β-ol (Table 3). This indicates that the YGL022 sequence is required for sterol C-24(28) reductase activity (ERG4) in S. cerevisiae. 
     
                       TABLE 3                                                     
______________________________________                                    
Sterol Accumulation Patterns                                              
in Wild-Type and erg4 Yeast Strains                                       
                  % Sterol Content                                        
                  Geno-     Ergo- 24(28) Other                            
Species                                                                   
       Strain     type      sterol                                        
                                  Tetraene                                
                                         Sterol                           
______________________________________                                    
S. pombe                                                                  
       HM123      erg4+     86    0      14                               
S. pombe                                                                  
       JY-6       erg4+     94    0      6                                
S. pombe                                                                  
       TP111-1A   erg4      0     94     6                                
S. pombe                                                                  
       TP111-1A   erg4+     100   0      0                                
       (pST2Sc)                                                           
S. pombe                                                                  
       TP111-1A   erg4      0     100    0                                
       cured                                                              
S. cere-                                                                  
       erg4-1A    erg4+/-   29    68     3                                
visiae                                                                    
S. cere-                                                                  
       erg4-1A    ERG4      83    5      12                               
visiae (pA-B6.5)                                                          
S. cere-                                                                  
       erg4-1A    erg+/-    37    57     6                                
visiae cured                                                              
S. cere-                                                                  
       XML25-2-1A erg4Δ                                             
                            0     97     3                                
visiae                                                                    
S. cere-                                                                  
       XML25-2-1B ERG4      91    0      9                                
visiae                                                                    
S. cere-                                                                  
       XML25-2-1C erg4Δ                                             
                            0     98     2                                
visiae                                                                    
S. cere-                                                                  
       XML25-2-1D ERG4      88    0      12                               
visiae                                                                    
______________________________________                                    
 
    
     The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary. 
     BIBLIOGRAPHY 
     Ashman, W. H., et al., Lipids 26: 628-632 (1991). 
     Baloch, R. and Mercer, I., Phytochemistry 26: 663-668 (1987v). 
     Balzi, E., et al., J. Biol. Chem. 262: 16871-16879 (1987). 
     Brugge, J. S., et al., Mol. Cell. Biol. 7: 2180-2187 (1987). 
     Chen, W., et al., Yeast 7: 305-308 (1991). 
     Gaber, R. F., et al., Mol. Cell. Biol. 9: 3447-3456 (1989). 
     Kyte, J., and Doolittle, R. F., J. Mol. Biol. 157: 105-132 (1982). 
     Lorenz, T., and Parks, L. W., DNA and Cell Biol. 11: 685-692 (1992). 
     Marcireau, C., et al., Curr. Genet. 22: 267-272 (1992). 
     Molzahn, S. W., and Woods, R. A., J. Gen Microbiol. 72: 339-348 (1972). 
     Nasmyth, K. A., and Tatchell, K., Cell 19: 753-764 (1980). 
     Paltauf, F., et al., in Jones, E. W., et al., eds., The Molecular and Cellular Biology of the Yeast Saccharomyces, Gene Expression, Cold Spring Harbor Laboratory Press, 1992, pages 415, 418-420, 424-428, and 434-437. 
     Shimanuki, M., et al., Mol. Biol. Cell 3:263-273 (1992). 
     Worman, H. J., et al., J. Cell Biology 111: 1535-1542 (1990). 
     
         __________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 5                                              
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 2528 bases and 438 amino acids                                
(B) TYPE: nucleic acid and amino acid                                     
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE:                                                       
 (A) DESCRIPTION: DNA encoding a polypeptide                              
(v) FRAGMENT TYPE: entire sequence                                        
(vi) IMMEDIATE SOURCE: Saccharomyces cerevisiae                           
clone                                                                     
(ix) FEATURE:                                                             
(D) OTHER INFORMATION: sterol Δ14 reductase gene,                   
translated                                                                
polypeptide and flanking DNA                                              
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                  
ATATATATATACCTCTTGCCAGCAACAGGCCAGTTAT AAGTTAAAATTAA50                     
TATGTGACGCACTTCTGAAACAGTATTGAAACAGTATTGAAACATATGTA100                     
TTACCCGGACTCTGCATGCTCTGTCGTTCATTTTATTTTCACCTAAACGA150                     
AAATCCCGTGAAAAAAATTTAT ATCGCCTTTCGCTCTTTTGTATGTAGGC200                    
ATCATCGGAAATTTGCATTGTGTGAAGGTTGTGCATATAAAGGGTTTTGC250                     
ATAACGGACGTTTTTCACGTACTCCGTCTGAGCATCAAGTGAGGCTTGAG300                     
TTTACGTT TGTTTTTAATAATCAGTTTTCATTCTACTATTTTCTTGCGCA350                    
ATTGCTTATCAGATAGACCTTGTAAACAGCATAGGAGTAAAGACAAATTC400                     
GGTGTAGAGAATAAAAGGATGGTATCAGCTTTGAATCCCAGAACTACAGAG 451                   
MetValSerAlaLeuAsnProArgThrThrGlu                                         
510                                                                       
TTTGAATTTGGTGGGCTGATTGGTGCCTTAGGCATCAGCATAGGG 496                         
PheGluPheGlyGlyLeuIleGlyAlaLeuGlyIleSerIleGly                             
152025                                                                    
CTGCCTGTTTTCACTATCATCTTGAATCAAATGATAAGGCCCGAT541                          
LeuProValPheThrIleIleLeuAsnGlnMetIleArgProAsp                             
303540                                                                    
TATTTTATTAAGGGATTTTTCCAGAATTTCGATATAGTTGAGCTT586                          
TyrP heIleLysGlyPhePheGlnAsnPheAspIleValGluLeu                            
455055                                                                    
TGGAACGGTATCAAGCCATTGCGCTACTATCTGGGCAATCGTGAA631                          
TrpAsnGlyI leLysProLeuArgTyrTyrLeuGlyAsnArgGlu                            
606570                                                                    
TTATGGACTGTCTATTGCCTGTGGTATGGAATACTGGCAGTTTTG676                          
LeuTrpThrValTyrC ysLeuTrpTyrGlyIleLeuAlaValLeu                            
758085                                                                    
GACGTCATTTTACCGGGCAGAGTCATGAAGGGTGTTCAGTTAAGG721                          
AspValIleLeuProGlyArgV alMetLysGlyValGlnLeuArg                            
9095100                                                                   
GATGGTTCGAAGCTTTCGTATAAGATCAATGGAATTGCCATGTCT766                          
AspGlySerLysLeuSerTyrLysIleA snGlyIleAlaMetSer                            
105110115                                                                 
ACAACTTTGGTCTTAGTTTTGGCTATCAGATGGAAATTGACTGAT811                          
ThrThrLeuValLeuValLeuAlaIleArgTrpL ysLeuThrAsp                            
120125130                                                                 
GGACAATTGCCTGAATTGCAATATCTGTATGAAAATCACGTTAGT856                          
GlyGlnLeuProGluLeuGlnTyrLeuTyrGluAsnHisV alSer                            
135140145                                                                 
TTATGCATAATATCTATTTTGTTTTCGTTCTTTTTGGCGACGTAC901                          
LeuCysIleIleSerIleLeuPheSerPhePheLeuAlaThrTyr                             
 150155160                                                                
TGCTATGTTGCCAGCTTCATACCATTGATCTTCAAGAAAAATGGT946                          
CysTyrValAlaSerPheIleProLeuIlePheLysLysAsnGly                             
 165170175                                                                
AATGGCAAAAGGGAAAAGATCTTAGCACTAGGTGGAAATTCAGGA991                          
AsnGlyLysArgGluLysIleLeuAlaLeuGlyGlyAsnSerGly                             
180 185190                                                                
AACATCATTTACGATTGGTTTATTGGTAGAGAACTGAACCCTCGT1036                         
AsnIleIleTyrAspTrpPheIleGlyArgGluLeuAsnProArg                             
195 200205                                                                
CTCGGCCCATTAGATATCAAGATGTTTTCAGAGTTGAGACCCGGC1081                         
LeuGlyProLeuAspIleLysMetPheSerGluLeuArgProGly                             
210 215220                                                                
ATGTTGTTATGGTTACTGATCAATCTTTCCTGTCTGCATCACCAT1126                         
MetLeuLeuTrpLeuLeuIleAsnLeuSerCysLeuHisHisHis                             
225230 235                                                                
TACCTGAAGACTGGTAAAATCAACGATGCATTGGTCTTGGTTAAT1171                         
TyrLeuLysThrGlyLysIleAsnAspAlaLeuValLeuValAsn                             
240245 250                                                                
TTCTCGCAAGGATTTTACATTTTCGATGGAGTACTAAACGAGGAA1216                         
PheSerGlnGlyPheTyrIlePheAspGlyValLeuAsnGluGlu                             
25526026 5                                                                
GGTGTATTAACCATGATGGATATCACTACAGATGGGTTTGGTTTC1261                         
GlyValLeuThrMetMetAspIleThrThrAspGlyPheGlyPhe                             
270275280                                                                 
AT GCTAGCGTTTGGTGACTTAAGTTTAGTTCCATTCACCTACTCA1306                        
MetLeuAlaPheGlyAspLeuSerLeuValProPheThrTyrSer                             
285290295                                                                 
TTACAAG CGCGTTACTTGAGTGTTTCCCCTGTGGAATTGGGATGG1351                        
LeuGlnAlaArgTyrLeuSerValSerProValGluLeuGlyTrp                             
300305310                                                                 
GTGAAAGTTGTC GGTATATTAGCCATAATGTTTTTGGGTTTCCAC1396                        
ValLysValValGlyIleLeuAlaIleMetPheLeuGlyPheHis                             
315320325                                                                 
ATCTTCCACTCGGCAAAT AAGCAAAAATCTGAGTTTAGACAAGGT1441                        
IlePheHisSerAlaAsnLysGlnLysSerGluPheArgGlnGly                             
330335340                                                                 
AAATTAGAAAATCTAAAAAGTAT TCAGACAAAGCGTGGTACAAAG1486                        
LysLeuGluAsnLeuLysSerIleGlnThrLysArgGlyThrLys                             
345350355                                                                 
TTATTATGTGACGGGTGGTGGGCTAAAT CACAGCATATCAATTAC1531                        
LeuLeuCysAspGlyTrpTrpAlaLysSerGlnHisIleAsnTyr                             
360365370                                                                 
TTTGGCGATTGGCTGATTTCATTAAGTTGGTGT TTGGCCACCTGG1576                        
PheGlyAspTrpLeuIleSerLeuSerTrpCysLeuAlaThrTrp                             
375380385                                                                 
TTCCAAACTCCCTTGACATATTACTACTCGTTGTACTTC GCCACG1621                        
PheGlnThrProLeuThrTyrTyrTyrSerLeuTyrPheAlaThr                             
390395400                                                                 
TTGTTATTACACCGTCAACAACGTGATGAGCACAAGTGCCGCCT G1666                        
LeuLeuLeuHisArgGlnGlnArgAspGluHisLysCysArgLeu                             
405410415                                                                 
AAATATGGCGAAAATTGGGAAGAATACGAAAGAAAAGTTCCTTAC 1711                        
LysTyrGlyGluAsnTrpGluGluTyrGluArgLysValProTyr                             
420425430                                                                 
AAGATCATTCCATATGTTTATTAAGTTTTTCTACCACTGCTATTTTCTTCA1762                   
 LysIleIleProTyrValTyr                                                    
435                                                                       
TTATCTATGTATGTGTGTATACATGTTATGTATTGGGTGAGTATGAGGAA1812                    
GAAGAAGAATAACAATTGAAAACGCTGGAAAAATTAAAAGGGGTGGCGGT1862                    
CTATC TATGCAACGCTCCCCTTTTCGTTACATGAACACATCAAACTTGTA1912                   
TATCCTTTGAGTGTTCTTTAATCAAGTCATCTTGGTATTTTAGTAGCGTT1962                    
TCCACTACTTTAGGGACAAATTCAGACCTAACCAATCCATCAAAAGCATC 2012                   
AAACCCTTGCGACAAAATCGGAATATCAGACTCGCCATGCATAAACTCTG2062                    
GAATTTCTAGTTTCCCGTCCGCAAGTATGCCGTCATCATCCTCGTCGTCC2112                    
TTATTAGTATCCAAATTTGTCACTTTGACGTTCATCG ACAACTGTAAGTC2162                   
AAAGTAGCAAATCGCCTTGCCCTTCCTTTGAGATACGTTGGAGTCACCGG2212                    
TGATGCTACTCACCTGGGTTAACTCAATTTTGCTCTTCCCATCAGAGGAA2262                    
ACAGTGGACAAACTCGTTAA TTTACCGTTCAAGTAGTCCTTAGACCAAGG2312                   
TAAGGTGTTTTTATCCACCCAATGCCAGTTATTTGGATTCAAGACAACCA2362                    
TATTTTATCGTAAATGTGTTGTAACTTTCCGATCGTTTCAAACTTTAGTA2412                    
GT AGTTTGATGATTTTGTCCAAAAAGTATTTGCTTAAATTTCAGCTTTTT2462                   
TCTTCTTCATATGTATTTCTTTTTTTCCTCGCTTTCTCTGCCCACTTTTT2512                    
TCTTCTGTCTTCTAGA 2528                                                     
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 419 residues                                                  
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE:                                                       
(A) DESCRIPTION: polypeptide                                              
(ix) FEATURE:                                                             
(A) NAME/KEY: chicken                                                     
nuclear lamin B receptor                                                  
(x) PUBLICATION INFORMATION:                                              
(A) AUTHORS: H.J. Worman, C.D. Evans, and G.                              
Blobel                                                                    
(B) TITLE: (excerpt): The Lamin B Receptor of the                         
Nuclear Envelope Inner Membrane                                           
(C) JOURNAL: Journal of Cell Biology                                      
(D) VOLUME: 111                                                           
(F) PAGES: 1535-1542                                                      
Sequence set out in Figure 5, page 1539                                   
( G) DATE: 1990                                                           
(K) RELEVANT RESIDUES IN SEQ ID NO: 190 to 608                            
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                  
ProGluLysProSerSerLysThrLysGluLeuGluPheGlyGly                             
51015                                                                     
ArgPheGlyThrPheMet LeuMetPhePheLeuProAlaThrVal                            
202530                                                                    
LeuTyrLeuValLeuMetCysLysGlnAspAspProSerLeuMet                             
35 4045                                                                   
AsnPheProProLeuProAlaLeuGluSerLeuTrpGluThrLys                             
505560                                                                    
ValPheGlyValPheLeuLeuTrpPhePhePheGlnA laLeuPhe                            
657075                                                                    
TyrLeuLeuProIleGlyLysValValGluGlyLeuProLeuSer                             
808590                                                                    
 AsnProArgLysProGlnTyrArgIleAsnGlyPheTyrAlaPhe                            
95100105                                                                  
LeuLeuThrAlaAlaAlaIleGlnThrLeuLeuTyrPheGlnPhe                             
 110115120                                                                
GluLeuHisTyrLeuTyrAspHisPheValGlnPheAlaValSer                             
125130135                                                                 
AlaAlaAlaPheSerMet AlaLeuSerIleTyrLeuTyrIleArg                            
140145150                                                                 
SerLeuLysAlaProGluGluAspLeuAlaProGlyGlyAsnSer                             
1551 60165                                                                
GlyTyrLeuValTyrAsnPhePheThrGlyHisGluLeuAsnPro                             
170175180                                                                 
ArgIleGlySerPheAspLeuLysTyrPheCysGluL euArgPro                            
185190195                                                                 
GlyLeuIleGlyTrpValValIleAsnLeuAlaMetLeuLeuAla                             
200205210                                                                 
 GluMetLysIleHisAsnGlnSerMetProSerLeuSerMetIle                            
215220225                                                                 
LeuValAsnSerPheGlnLeuLeuTyrValValAspAlaLeuTrp                             
 230235240                                                                
AsnGluGluAlaValLeuThrThrMetAspIleThrHisAspGly                             
245250255                                                                 
PheGlyPheMetLeuAla PheGlyAspLeuValTrpValProPhe                            
260265270                                                                 
ValTyrSerLeuGlnAlaPheTyrIleValGlyHisProIleAla                             
2752 80285                                                                
IleSerTrpProValAlaAlaAlaIleThrIleLeuAsnCysIle                             
290295300                                                                 
GlyTyrTyrIlePheArgSerAlaAsnSerGlnLysA snAsnPhe                            
305310315                                                                 
ArgArgAsnProAlaAspProLysLeuSerTyrLeuLysValIle                             
320325330                                                                 
 ProThrAlaThrGlyLysGlyLeuLeuValThrGlyTrpTrpGly                            
335340345                                                                 
PheValArgHisProAsnTyrLeuGlyAspIleIleMetAlaLeu                             
 350355360                                                                
AlaTrpSerLeuProCysGlyPheAsnHisIleLeuProTyrPhe                             
365370375                                                                 
TyrValIleTyrPheIle CysLeuLeuValHisArgGluAlaArg                            
380385390                                                                 
AspGluHisHisCysLysLysLysTyrGlyLeuAlaTrpGluArg                             
3954 00405                                                                
TyrCysGlnArgValProTyrThrHisIleSerLeuHisLeu                                
410415                                                                    
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 473 residues                                                  
(B) TYPE: amino acid                                                      
 (C) STRANDEDNESS: single                                                 
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE:                                                       
(A) DESCRIPTION: polypeptide                                              
(ix) FEATURE:                                                             
(A) NAME/KEY: Saccharomyces cerevisiaeYGL022                              
(x) PUBLICATION INFORMATION:                                              
(A) AUTHORS: W. Chen, E. Capieaux, E. Balzi, and A.                       
Goffeau                                                                   
(B) TITLE: The YGL022 Gene Encodes a                                      
Putative Transport Protein                                                
 (C) JOURNAL: Yeast                                                       
(D) VOLUME: 7                                                             
(F) PAGES: 305-308                                                        
Sequence set out in Figure 1, pages 306-307                               
(G) DATE: 1991                                                            
(K) RELEVANT RESIDUES IN SEQ ID NO: open reading frame                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                  
MetAlaLysAspAsnSerGluLysLeuGlnValGlnGlyGluGlu                             
 51015                                                                    
LysLysSerLysGlnProValAsnPheLeuProGlnGlyLysTrp                             
202530                                                                    
LeuLysProAs nGluIleGluTyrGluPheGlyGlyThrThrGly                            
354045                                                                    
ValIleGlyMetLeuIleGlyPheProLeuLeuMetTyrTyrMet                             
50 5560                                                                   
TrpIleCysAlaGluPheTyrHisGlyLysValAlaLeuProLys                             
657075                                                                    
AlaGlyGluSerTrpMetHisPheIleLys HisLeuTyrGlnLeu                            
808590                                                                    
ValLeuGluAsnGlyIleProGluLysTyrAspTrpThrIlePhe                             
95100 105                                                                 
LeuThrPheTrpValPheGlnIleIlePheTyrTyrThrLeuPro                             
110115120                                                                 
GlyIleTrpThrLysGlyGlnProLeuSerHisLeuLysGlyLys                             
 125130135                                                                
GlnLeuProTyrPheCysAsnAlaMetTrpThrLeuTyrValThr                             
140145150                                                                 
ThrThrLeuVa lLeuValLeuHisPheThrAsnLeuPheArgLeu                            
155160165                                                                 
TyrValIleIleAspArgPheGlyArgIleMetThrCysAlaIle                             
170 175180                                                                
IleSerGlyPheAlaPheSerIleIleLeuTyrLeuTrpThrLeu                             
185190195                                                                 
PheIleSerHisAspTyrHisArgMetThr GlyAsnHisLeuTyr                            
200205210                                                                 
AspPhePheMetGlyAlaProLeuAsnProArgTrpGlyIleLeu                             
215220 225                                                                
AspLeuLysMetPhePheGluValArgLeuProTrpPheThrLeu                             
230235240                                                                 
TyrPheIleThrLeuGlyAlaCysLeuLysGlnTrpGluThrTyr                             
 245250255                                                                
GlyTyrValThrProGlnLeuGlyValValMetLeuAlaHisTrp                             
260265270                                                                 
LeuTyrAlaAs nAlaCysAlaLysGlyGluGluLeuIleValPro                            
275280285                                                                 
ThrTrpAspMetAlaTyrGluLysPheGlyPheMetLeuIlePhe                             
290 295300                                                                
TrpAsnIleAlaGlyValProTyrThrTyrCysHisCysThrLeu                             
305310315                                                                 
TyrLeuTyrTyrHisAspProSerGluTyr HisTrpSerThrLeu                            
320325330                                                                 
TyrAsnValSerLeuTyrValValLeuLeuCysAlaTyrTyrPhe                             
335340 345                                                                
PheAspThrAlaAsnAlaGlnLysAsnAlaPheArgLysGlnMet                             
350355360                                                                 
SerGlyAspLysThrValArgLysThrPheProPheLeuProTyr                             
 365370375                                                                
GlnIleLeuLysAsnProLysTyrMetValThrSerAsnGlySer                             
380385390                                                                 
TyrLeuLeuIl eAspGlyTrpTyrThrLeuAlaArgLysIleHis                            
395400405                                                                 
TyrThrAlaAspTrpThrGlnSerLeuValTrpAlaLeuSerCys                             
410 415420                                                                
GlyPheAsnSerValPheProTrpPhePheProValPhePheLeu                             
425430435                                                                 
ValValLeuIleHisArgAlaPheArgAsp GlnAlaLysCysLys                            
440445450                                                                 
ArgLysTyrGlyLysAspTrpAspGluTyrCysLysHisCysPro                             
455460 465                                                                
TyrValPheIleProTyrValPhe                                                  
470                                                                       
(2) INFORMATION FOR SEQ ID NO:4:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 453 residues                                                  
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE:                                                       
(A) DESCRIPTION: polypeptide                                              
 (ix) FEATURE:                                                            
(A) NAME/KEY: Schizosaccharomyces pombe sts gene                          
(x) PUBLICATION INFORMATION:                                              
(A) AUTHORS: M. Shimanuki, M. Goebl, M. Yanagida,                         
and T.                                                                    
Toda                                                                      
(B) TITLE: Fission Yeast sts1+Gene                                        
Encodes a                                                                 
Protein Similar to the Chicken Lamin B Receptor                           
(C) JOURNAL: Molecu-                                                      
 lar Biology of the Cell                                                  
(D) VOLUME: 3                                                             
(F) PAGES: 263-273                                                        
Sequence set out in Figure 1, page 264                                    
(G) DATE: 1992                                                            
(K) RELEVANT RESIDUES IN SEQ ID NO: open reading frame                    
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                  
MetLysSerThrValLysLysSerAlaProArgGluPhe GlyGly                            
51015                                                                     
AlaLysGlyAlaLeuAlaIleMetThrGlyPheProCysLeuMet                             
202530                                                                    
Ty rTyrLeuTrpAlaCysSerLysPheAsnAspSerGlnPheIle                            
354045                                                                    
LysProGluSerPheThrIleAlaGlyPheGlnAsnPhePheArg                             
 505560                                                                   
ThrLeuGlyHisTyrIleTyrValGlyAlaTyrProThrArgTyr                             
657075                                                                    
AlaPheLeuValPheTrpSer PheCysIleValGlnAlaValMet                            
808590                                                                    
TyrLeuThrLeuProGlyValArgThrGlnGlyLeuProLeuLys                             
95100 105                                                                 
HisArgAsnAsnGluArgLeuProTyrLeuCysAsnAlaIleTrp                             
110115120                                                                 
SerPheTyrThrThrIleValIleLeuAlaValLeuHis ValThr                            
125130135                                                                 
HisValPheProIleThrThrPheIleAspMetPheGlyProLeu                             
140145150                                                                 
Me tSerValAlaIleIleThrAlaPheValCysThrPheValLeu                            
155160165                                                                 
TyrThrGlyThrLeuLeuPheGlyAspArgLeuPheAspLysPro                             
 170175180                                                                
HisArgLeuSerGlyAsnProIleTyrAspAlaPheMetGlyAla                             
185190195                                                                 
CysLeuAsnProArgLeuGly LysLeuLeuAspPheLysMetPhe                            
200205210                                                                 
PheGluValArgIleProTrpPheIleLeuPhePheIleSerVal                             
215220 225                                                                
GlyAlaAlaValArgGlnTyrGluThrTyrGlyThrValSerPro                             
230235240                                                                 
GlnValLeuPheValCysLeuGlyHisTyrLeuTyrAla AsnAla                            
245250255                                                                 
CysSerLysGlyGluGlnLeuIleValProThrTrpAspMetAla                             
260265270                                                                 
Ty rGluLysPheGlyPheMetLeuIlePheTrpAsnMetAlaGly                            
275280285                                                                 
ValProPheThrTyrSerHisCysThrLeuTyrLeuPheSerHis                             
 290295300                                                                
AspProSerValTyrAsnTrpSerThrGlnTyrThrThrGlyIle                             
305310315                                                                 
TyrValLeuLeuLeuCysCys TyrTyrIlePheAspThrCysAsn                            
320325330                                                                 
GlyGlnLysAsnHisPheArgAsnGlnIleTyrGlyThrGluVal                             
335340 345                                                                
HisArgLysThrPheProGlnLeuProTrpLeuIleIleLysAsn                             
350355360                                                                 
ProThrPheIleArgCysAlaAsnGlyGlyThrLeuLeu ThrSer                            
365370375                                                                 
GlyTrpTyrArgTyrAlaArgLysIleHisTyrThrAlaAspPhe                             
380385390                                                                 
Ph eGlnSerLeuSerTrpAlaLeuIleThrGlyPheGlnSerPro                            
395400405                                                                 
LeuProTyrPheTyrProSerPhePhePheValValLeuValHis                             
 410415420                                                                
ArgValSerArgAspIleLysLysCysLysAlaLysTyrGlyAla                             
425430435                                                                 
AspPheAspGluTyrAspArg IleCysProTyrLeuPheIlePro                            
440445450                                                                 
TyrIlePhe                                                                 
(2) INFORMATION FOR SEQ ID NO:5:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 438 amino acids                                               
(B) TYPE: amino acid                                                      
(C) STRANDEDNESS: single                                                  
 (D) TOPOLOGY: linear                                                     
(ii) MOLECULE TYPE:                                                       
(A) DESCRIPTION: polypeptide                                              
(v) FRAGMENT TYPE: entire sequence                                        
(vi) IMMEDIATE SOURCE: Saccharomyces cerevisiae                           
clone                                                                     
(ix) FEATURE:                                                             
(D) OTHER INFORMATION: translated polypeptide of                          
sterol &#34;14                                                                
reductase gene                                                            
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                  
MetVal SerAlaLeuAsnProArgThrThrGluPheGluPheGly                            
51015                                                                     
GlyLeuIleGlyAlaLeuGlyIleSerIleGlyLeuProValPhe                             
2 02530                                                                   
ThrIleIleLeuAsnGlnMetIleArgProAspTyrPheIleLys                             
354045                                                                    
GlyPhePheGlnAsnPheAs pIleValGluLeuTrpAsnGlyIle                            
505560                                                                    
LysProLeuArgTyrTyrLeuGlyAsnArgGluLeuTrpThrVal                             
65 7075                                                                   
TyrCysLeuTrpTyrGlyIleLeuAlaValLeuAspValIleLeu                             
808590                                                                    
ProGlyArgValMetLysGlyValGlnLeuArgA spGlySerLys                            
95100105                                                                  
LeuSerTyrLysIleAsnGlyIleAlaMetSerThrThrLeuVal                             
110115 120                                                                
LeuValLeuAlaIleArgTrpLysLeuThrAspGlyGlnLeuPro                             
125130135                                                                 
GluLeuGlnTyrLeuTyrGluAsnHisValSerLeuCysIleIle                             
 140145150                                                                
SerIleLeuPheSerPhePheLeuAlaThrTyrCysTyrValAla                             
155160165                                                                 
SerPheIl eProLeuIlePheLysLysAsnGlyAsnGlyLysArg                            
170175180                                                                 
GluLysIleLeuAlaLeuGlyGlyAsnSerGlyAsnIleIleTyr                             
185 190195                                                                
AspTrpPheIleGlyArgGluLeuAsnProArgLeuGlyProLeu                             
200205215                                                                 
AspIleLysMetPheSerGluL euArgProGlyMetLeuLeuTrp                            
215220225                                                                 
LeuLeuIleAsnLeuSerCysLeuHisHisHisTyrLeuLysThr                             
230235 240                                                                
GlyLysIleAsnAspAlaLeuValLeuValAsnPheSerGlnGly                             
245250255                                                                 
PheTyrIlePheAspGlyValLeuAsnGluGluGly ValLeuThr                            
260265270                                                                 
MetMetAspIleThrThrAspGlyPheGlyPheMetLeuAlaPhe                             
275280285                                                                 
GlyAspLeuSerLeuValProPheThrTyrSerLeuGlnAlaArg                             
290295300                                                                 
TyrLeuSerValSerProValGluLeuGlyTrpValLysValVal                             
 305310315                                                                
GlyIleLeuAlaIleMetPheLeuGlyPheHisIlePheHisSer                             
320325330                                                                 
AlaAsnLysGln LysSerGluPheArgGlnGlyLysLeuGluAsn                            
335340345                                                                 
LeuLysSerIleGlnThrLysArgGlyThrLysLeuLeuCysAsp                             
350 355360                                                                
GlyTrpTrpAlaLysSerGlnHisIleAsnTyrPheGlyAspTrp                             
365370375                                                                 
LeuIleSerLeuSerTrpCysLeuAl aThrTrpPheGlnThrPro                            
380385390                                                                 
LeuThrTyrTyrTyrSerLeuTyrPheAlaThrLeuLeuLeuHis                             
395400 405                                                                
ArgGlnGlnArgAspGluHisLysCysArgLeuLysTyrGlyGlu                             
410415420                                                                 
AsnTrpGluGluTyrGluArgLysValProTyrLysIleI lePro                            
425430435                                                                 
TyrValTyr                                                                 
__________________________________________________________________________