WO1994025607A1 - Method of assaying for drugs which restore function of mutant cftr protein - Google Patents

Method of assaying for drugs which restore function of mutant cftr protein Download PDF

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WO1994025607A1
WO1994025607A1 PCT/US1994/004379 US9404379W WO9425607A1 WO 1994025607 A1 WO1994025607 A1 WO 1994025607A1 US 9404379 W US9404379 W US 9404379W WO 9425607 A1 WO9425607 A1 WO 9425607A1
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ser
thr
lys
gly
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PCT/US1994/004379
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French (fr)
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John L. Teem
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University Of Iowa Research Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics
    • G01N2800/382—Cystic fibrosis

Definitions

  • Cystic fibrosis is a human genetic disease of the secretory epithelia. Although the survival rate of those suffering with cystic fibrosis has improved in recent years, the median age for patient survival is still only about twenty five to thirty years despite intensive supportive and prophylactic treatment.
  • United States Patent No. 5,100,647 to Agus, et al discloses a method for treating cystic fibrosis by administration of the compound sparteine (dodecahydro-7, 14methano-2H, 6H-di-pyrido [1,2-a: l',2'-e] [1,5] diazocine), acting as a direct exogenous activator of chloride conductants in epithelial airways.
  • United States Patent No. 5,179,001 to Young, et al discloses a method of treating pulmonary complications associated with cystic fibrosis caused by the gram negative bacterium Pseudomonas aeruqinosa.
  • 4,826,679 to Roy relates to an oral composition for alleviating digestive manifestation in persons afflicted with cystic fibrosis comprising a therapeutic amount of taurine.
  • cystic fibrosis comprising a therapeutic amount of taurine.
  • One method of gene therapy proposed is United States Patent No. 5,149,797 disclosing a method of site specific alteration of RNA and production of encoded polypeptides.
  • This invention is drawn to correcting the abnormal mRNA present in individual cells, cleaving the mRNA by site directed RNAase followed by introduction of the appropriate oligoribonucleotide followed by endogenous RNA ligase and thus production of a wild-type mRNA encoding a normal protein product which then may be translated to produce the correct protein.
  • Cystic fibrosis is characterized at the genetic level by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.
  • the CFTR protein is a member of a superfamily of structurally related membrane proteins named the traffic ATPase or ATP-binding cassette (ABC) transporters.
  • Other members of this superfamily include the multi-drug resistance (MDR) or P-glycoprotein, bovine adenyl cyclase, the yeast STE6 protein, as well as several bacterial amino acid transport proteins (Riordan, et al, 1989; Hyde, et al, 1990).
  • MDR multi-drug resistance
  • bovine adenyl cyclase the yeast STE6 protein
  • the principle distinguishing feature of this superfamily of proteins is a highly conserved nucleotide-binding domain (NBD) .
  • CFTR is a protein of approximately 1480 amino acids consisting of two repeated motifs, each comprising 6 transmembrane segments and a nucleotide-binding domain. The two motifs are separated by a large polar R-domain containing multiple potential phosphorylation sites.
  • CFTR is a Cl ⁇ channel regulated by phosphorylation and by cytosolic ATP (Anderson, et al, "Nucleoside Triphos- phates are Required to Open CFTR Chloride Channel" Cell,67,775-784) .
  • the two nucleotide-binding domains present in CFTR play a central role in the function of CFTR Cl " channels; they interact directly with ATP to open the CFTR Cl ⁇ channel and ATP hydrolysis may be required for this effect.
  • the importance of the NBD's is further emphasized by the large number of cystic fibrosis associated mutations that have been found in the domains. The most common cystic fibrosis associated mutation, accounting for approximately 68% of the cystic fibrosis chromosomes is deletion of a phenylalanine at position 508 ( ⁇ F508) in the middle of NBD1.
  • This mutation is characterized by a loss of apical membrane Cl ⁇ channel activity in the epithelial cells leading to chronic progressive disease of the respiratory system.
  • the ⁇ F508 mutation causes loss of apical membrane Cl ⁇ channel activity affecting the CFTR protein in two ways.
  • First, the mutation causes defective processing and hence mislocalization of the mutant protein. This is presumed to occur from misfolding of the mutant protein so that it fails to exit from the endoplasmic reticulum and progress to the Golgi complex in the apical membrane.
  • the resulting Cl " transport defect associated with the ⁇ F508 is thus largely a consequence of the absence of functional CFTR at the plasma membrane.
  • Second the ⁇ F508 mutation affects the function of the Cl ⁇ channel. Single channel analysis of CFTR ⁇ F508 suggests that although the mutant protein is functional, it has an open state probability (P 0 ) that is one third that of wild-type CFTR.
  • CFTR ⁇ F508 Defective trafficking of CFTR ⁇ F508 can be partially reversed if cells are grown at a lower temperature, indicating that mutant protein is still functional.
  • CFTR ⁇ F508 is produced at a reduced temperature (less than 37°C) some of the mutant protein transists the Golgi complex normally and is localized correctly in the plasma membrane.
  • a reduced temperature less than 37°C
  • CFTR ⁇ F508 retains partial function
  • a possible therapy for cystic fibrosis may involve pharmacologic intervention designed to correct the processing defects so that more CFTR ⁇ F508 reaches the plasma membrane.
  • Potential pharmacologic intervention would represent a non gene-therapy approach for the treatment of cystic fibrosis.
  • Yet another object of the invention is to provide a method for identifying potentially useful compounds for further useful study in the treatment of disease by means of a yeast mating assay.
  • This invention relates to a method for screening treatments which restore the function of mutant CFTR by simple means of a yeast cell mating assay.
  • chimeric genetic sequences which encode the yeast a-factor transporter STE6 and CFTR or another member of the related superfamily such as P-glycoprotein are constructed whereby a segment of the CFTR genetic sequence is exchanged for a corresponding segment of the STE6 genetic sequence to produce a product which will complement a yeast ste6 ⁇ mutation but is non ⁇ functional upon introduction of a desired mutation into the CFTR sequence.
  • the resulting chimeras are then transformed into a yeast strain which has the STE6 gene deleted (ste6 ⁇ ).
  • the yeast strain is then exposed to the desired drug or treatment to be screened and the strain is assayed for the presence of restoration of mating, which indicates a functional chimera.
  • the invention discloses several chimeric sequences useful for screening for utility of treatments and reversing the effects of the CFTR ⁇ F508 mutation which is associated with cystic fibrosis.
  • Figure 1 represents the amino acid sequence alignment of NBDl of STE6 and CFTR.
  • Figure 2 depicts the STE6-CFTR chimeric constructs and their mating efficiency.
  • Figure 3 is a graphical depiction of a yeast assay according to the present invention which presents a potential cystic fibrosis treatment.
  • Figure 4a-g is the gene sequence for chimera
  • Figure 5a-g is the gene sequence for chimera
  • Figure 6a-g is the gene sequence for chimera
  • the STE6 gene of Saccharomyces cerevisiae encodes an integral membrane protein that functions to transport the peptide a-factor out of the cell during mating.
  • yeast Saccharomyces cerevisiae "Functional expression of human MDRl in yeast Saccharomyces cerevisiae.” Proc. Natl . Acad. Sch . , USA89, 2303-2306. Deletion of the STE6 gene from yeast (ste6 ⁇ ) results in a sterile phenotype.
  • chimeric STE6 genes were constructed in which segments of the NBDl of STE6 were substituted by the analagous NBDl sequences from CFTR.
  • FIG. 1 represents an amino acid sequence alignment of the NBD for proteins CFTR and STE6.
  • the CFTR amino acid sequence is depicted on top illustrating amino acids 442 through 578; the corresponding analagous STE6 amino acid sequence is depicted below illustrating amino acids 376 through 536.
  • Corresponding amino acids are indicated by solid vertical bars and give an indication of potential junctions for the genetic constructs.
  • Table 1 indicates the portion of CFTR amino acids sequence inserted into STE6 for the different hybrids.
  • the HI STE6-CFTR chimeric gene was constructed with a DNA sequence coding for the entire NBDl of STE6 (from N377 to A535) replaced by the corresponding region of CFTR (D443 to Y557).
  • H2 replaced the amino terminal half of NBD
  • H6 replaced the carboxy terminal half.
  • the remaining constructs were variations between these two extremes.
  • See Figure 2. The construction of plasmids with these sequences used methods which are well known to those of skill in the art however the following description is included merely for purposes of illustration and is not intended to limit in any way the invention.
  • Plasmid RFG416 (a gift from Rick Gaber, Northwestern University) is a single copy CEN plasmid with the selectable marker URA3 and the pUC19 polylinker region.
  • a 6.5 kb Sall-Sacl fragment containing the STE6 gene was subcloned from STE6-2 ⁇ (a gift from John McGrath, Massachusetts Institute of Technology) into the Sail and Sacl sites of the vector RFG416 to produce plasmid JTS6.
  • the yeast TRPl gene (on a 0.85 kb BgLll-EcoRl DNA fragment) was inserted at nucleotide position 1356 of STE6 NBDl, resulting in JTS6T.
  • a 441 bp DNA fragment containing the CFTR NBDl region was synthesized using two STE6-CFTR oligonucleotide primers (primer 1, 5'CCTTCGGAAGCAGTCCTGAAAGATAT-3' ; primer 2, 5'-GATGAACAATATCTAGGTATCCAAAA-3' . and CFTR cDNA template DNA in a polymerase chain reaction (PCR) (Ho, et al., 1989). PCR reactions were performed with a Temp-Tronic thermal cycler (Barnstead/Thermolyne) .
  • PCR polymerase chain reaction
  • Oligonucleotide primers 1 and 2 encoded fusion junctions of STE6 L375 to CFTR K442 and CFTR 1578 to STE6 D537, respectively, found in the Hi STE6-CFTR hybrid gene. The CFTR portion of each primer is underlined.
  • STE6 DNA flanking NBDl was added to each end of the 441 bp fragment by PCR, resulting in a 867 bp DNA fragment consisting of 419 bp of CFTR DNA (encoding CFTR amino acids K442-L578) that is flanked at the 5' end by 168 bp of STE6 DNA (encoding STE6 amino acids K319-L375) and 280 bp of STE6 DNA at the 3' end (encoding STE6 amino acids D537-G640).
  • Plasmid construction of HI was performed by cotransformation (Ito, et al, 1983) of yeast strain JPY201 with the 867 bp DNA fragment and 3 ⁇ g of plasmid JTS6T and selection of trans- formants on SD-URA (yeast nitrogen base supplemented with all amino acids except uracil) . Homologous recombination between the STE6 DNA sequences at each end of the 867 bp DNA fragment with the STE6 gene on the plasmid results in the targeted integration (Orr-Weaver, et al, 1981) of the CFTR sequences into NBD1 of STE6 on plasmid JTS6T and the consequent loss of the TRP1 gene.
  • Recombinants containing the desired STE6-CFTR hybrid gene were identified as trp auxoptrophs at a frequency of about 1% among the transformants. Plasmid DNA was prepared from trp transformants and the structure of the STE6-CFTR gene was confirmed by DNA sequencing analysis.
  • STE6-CFTR hybrid genes H2-H6 were similarly constructed using the following oligonucleotide primers for constructing the appropriate STE6CFTR junctions; STE6 Q440/CFTR F494, 5'- CACCGTCGTAGAACAGTTTTCCTGGATTA-3' ; CFTR G509/STE6 S457, 5'-CCGAATCTGTTGAACCAAAGATGATATTT-3' ; CFTR G550/STE6 G509, 5'-TTGTTGTTGCCCGCCACTCAGT3TGATTC-3' ; CFTR R560/STE6 A519, 5' ATCTCTGATGAATGCTCTTGCTAAA- GAAAT-3 ' .
  • the resulting plasmids were then transformed into a yeast strain with the STE6 gene deleted (ste6 ⁇ ) to test for complementation by means of a yeast mating assay.
  • JPY201 MATa ste6 ⁇ :HIS3, gal2, ura3-52, lys2-801, trpl, leu2-3,112, his3 ⁇ 200
  • 22-2D MATa, ura352, leu2-3, 112,trpl
  • JPY201 contains a STE6 deletion (including NBDl and extending beyond the termination codon) and replacement with the yeast HIS3 gene (McGrath and Varshavsky, 1989). Quantitative mating assays were performed as in Trueheart, et al (1987).
  • Transformants of JPY201 containing each STE6- CFTR chimera were grown to log phase in 0.1% glucose SD-URA media. From each culture, 3 x 10 6 cells were mixed with an equal number of 22-2D cells grown in YPD media and collected by filtration onto a Millipore filter, which was then placed upon a YPD plate for 4 hours at 30°C. Cells were resuspended, sonicated briefly, and plated from serial dilutions onto SD+LEU/TRP (yeast nitrogen base supplemented with leucine and tryptophan) . Diploid colonies were counted after 3 days at 30°C.
  • SD+LEU/TRP yeast nitrogen base supplemented with leucine and tryptophan
  • Control strains STE6 wild-type and ste6 ⁇ consisted of JPY201 transformed with plasmids JTS6 and JTS6T, respectively.
  • JPY201 transformants containing STE6-CFTR chimeras were grown as patches on SD-URA media and then replica printed to YPD media on a lawn of 22-2D cells. Following incubation at 30°C for 8 hr, the plate containing the mating cells was replica printed to SD-LEU/TRP and incubated 3 days at 30°C to allow growth of diploid colonies.
  • a decrease in mating efficiency (40%) was observed with the strain containing the H4- ⁇ F508 chimera relative to the H4 control. (Sequence I.D. No. 1).
  • the ⁇ F508 mutation lowered mating efficiency by 80 fold in the H5- ⁇ F508 chimera as compared with H5 (Sequence I.D. No. 3).
  • the H6- ⁇ F508 chimera displayed a comparable decrease in mating as compared to H6 (Sequence I.D. No.
  • the portion of CFTR most promising for substitution into STE6 include the amino acid identity locations between a portion of NBDl from amino acid 444 up to amino acid 509 through 577. As earlier seen, the segment from 443 to 557 was inactive as was the portion from 443 to 508. Preferred ranges include from amino acid 494 to 577 including: sequence I.D. No. 1, CFTR amino acid F494-1546; Sequence I.D. No. 3, F494-L558; Sequence I.D. No. 5 CFTR amino acid F494-Y577.
  • sequence I.D. No. 1, CFTR amino acid F494-1546 including: sequence I.D. No. 1, CFTR amino acid F494-1546; Sequence I.D. No. 3, F494-L558; Sequence I.D. No. 5 CFTR amino acid F494-Y577.
  • a yeast strain transformed to contain the discussed chimeric sequences will be used in a yeast mating assay to identify compounds which will correct the affect of the mutation as indicated by an increase in mating.
  • Such an assay may be widely varied by those of skill in the art.
  • the trans ⁇ formed strain will be combined with a strain of the alpha mating type on a suspension of yeast cells spread on the surface of agar medium in the petri dish. These two strains would be unable to mate due to the ⁇ F508 mutation.
  • a compound is then introduced to the medium.
  • a plant screening method may be performed by placing a small disc of leaf material removed by means of a paper punch from a plant and placed on the surface of the agar media. Plant compounds will diffuse from the leaf and interact with the yeast strains.
  • Another method would be to use a filter paper which has been soaked in the desired chemical and then placed on the yeast suspension. Further, other desired mutations can be introduced into the CFTR region to attempt to identify a revertant mutation and a corresponding protein for further study. It is to be understood that any form of exposing the yeast strain to the desired treatment may be used including modifying external conditions such as temperature.
  • the mating plate is then incubated for 24 hours to allow yeast cell mating to occur. If a compound corrects the molecular defect associated with H5- ⁇ F508 transporter then a-factor is transported allowing mating between the two strains, resulting in diploid cells. The mating plate is then replica printed to a selective media plate to allow growth of diploid colonies.
  • strain A could be - MATa, STE6::HIS3, ura3-52, lys2,trpl-298, his3-200, leu2- 3,112 ⁇ plasmid H5- ⁇ F508, URA3, CEN>; strain B - MAT ⁇ , ura3-52, trpl, leu2-3,112.
  • strain A and B could not grow on this media (strain A has the lys2 mutation and strain B has the ura3-52 mutation; these mutations prevent them from going on media without lysine and uracil respectively), however diploids produced as a result of the fusion between strain A and B can grow (diploids contain the genomes of both haploids and consequently has the complementing URA3 and LYS2 wild-type allele).
  • yeast diploid cells in the area surrounding the treatment disc would be a strong indicator of presence of compound(s) which correct the ⁇ F508 defect.
  • a number of yeast mating type assays may be developed easily by those of skill in the art and the foregoing description is not intended to limit in any way the invention.
  • the R553-L558 amino acid region of CFTR is of great interest in CFTR and other trafficking ATPase's as it is directly adjacent to the conserved LSGGQ linker sequence of NBDl, postulated to function as a transducer of signals between the hydrophobic domains and the NBD's (Mimura, et al, 1991). It was hypothesized that a possible treatment to correct the F508 mutation could be interaction of the region of the NBD polypeptide containing F508, with the R553-L558 region. The only difference between the H5 chimera and the H4 chimera is 6 amino acids in the R553-L558 region.
  • This region is also not present in the H3 chimera in which the ⁇ F508 mutation had no effect. Possibly there is an interaction between the two regions of the NBD required for correct folding or function (or both) of the NBD.
  • the method of the present invention was used to test whether a mutation in the region R553-L558 might suppress the adverse effects of the ⁇ F508 in NBDl.
  • the H5- ⁇ F508 construct was mutagenized in the R553-L558 region in vitro by site-specific oligonucleotide mutagenesis (Ho, et al, "Site- Directed Mutagenesis by Overlap Extension Using the Polymerase Chain Reaction Gene” 77,51-59 (1989).
  • the mutagenesis method was designed to introduce random amino acid substitutions at single codon positions within the R553-L558 region of the H5- ⁇ F508 plasmid. The procedure allowed 20 possible substitutions at each amino acid position within R553-L558.
  • the mutagenized DNA was transformed into yeast, and transformant colonies were analyzed by cell mating assays on petri dishes to identify colonies with a mating efficiency higher than the unmutagenized H5- ⁇ F508 control. Two colonies identified displayed higher efficiencies of mating within the first 20 mutant transformants analyzed.
  • These yeast transformants each contained an H5- ⁇ F508 plasmid with a mutation at amino acid R553 of CFTR; in one case, R553 was replaced by methionine (H5- ⁇ F508/R553M) , and in the other plasmid, R553 was replaced by glutamine (H5- ⁇ F508/R553Q) .
  • the relative mating efficiency of the ⁇ F508 revertant strains are shown in Table 2, with results expressed relative to the original H5 strain.
  • the mating efficiency of the H5- ⁇ F508 yeast strain is approximately 1% of the H5 strain, yeast containing the H5- ⁇ F508/R553Q and H5- ⁇ F508/R553M plasmids mated at 3% and 32%, respectively.
  • the revertant mutations therefore, partially suppress the ⁇ F508 mating defect.
  • the R553M mutation alone had little effect on H5 (H5-R553M); when this mutant was transformed into yeast, no further increase in mating efficiency was observed as compared with yeast containing H5 (Table 2).
  • mutations were found which suppress the effects of the ⁇ F508 mutation. TABLE 2
  • the R553Q and R553M mutations were introduced into CFTR ⁇ F508 cDNA and mammalian cells were transfected with these con ⁇ structs to determine whether the revertant mutations would correct the defect in cAMP-regulated Cl ⁇ transport of CFTR ⁇ F508.
  • the revertants R553Q and R553M were tested for their effects on mislocalization and the channel gating of CFTR ⁇ F508. The identified revertants modified both of these effects. Mutation of R553 to methionine partially corrected the processing of CFTR ⁇ F508 as assessed by three criteria; it produced fully glycosylated protein as indicated by electrophoretic separation on SDS polyacrylamide gel; it increased the appearance of the mutant protein in the plasma membrane as determined immunocytochemically; and it produced functional channels in the plasma membrane when measured by the SPQ halide efflux assay.
  • CFTR ⁇ F508 containing the R553Q and R553M mutations would be detected only if the processing defect of CFTR ⁇ F508 was suppressed.
  • the glycosylation patterns of CFTR and the various mutants expressed in HeLa cells were examined.
  • CFTR is a glycoprotein that undergoes progressive glycosylation, resulting in three bands that migrate at different rates on an SDS-polyacrylamide gel.
  • Band A is the most rapidly migrating and represents the nascent, unglycosylated protein; band B has an intermediate rate of migration and a pattern of core glycosylation consistent with processing in the endoplasmic reticulum; band C migrates most slowly and has a pattern of mature glycosylation consistent with processing in the Golgi complex.
  • CFTR ⁇ F508 was only presented as the unglycosylated band A and the core glycosylated band B protein, consistent with its failure to traverse the Golgi complex and reach the plasma membrane.
  • CFTR/R553Q and CFTR ⁇ F508/R553Q formed functional Cl ⁇ channels.
  • Inside-out membrane patches from either mutant showed no Cl ⁇ channel activity under basal conditions.
  • PKA protein kinase A
  • ATP protein kinase A
  • ATP protein kinase A
  • Single- channel events were recorded from +60 V to -120 mV in increments of 20 mV.
  • YEAST MATING ASSAY WITH PLANT EXTRACT SCREENING A suspension of yeast cells (strains A and B) was spread upon the surface of agar media in a petri dish.
  • the suspension consisted of equal proportions of two haploid yeast strains.
  • Yeast strains A and B are of the a and ⁇ mating types respectively, and can mate to form yeast diploids.
  • Strain A contains the STE6/CFTR hybrid a-factor transporter gene H5- ⁇ F508 (Teem, et al, 1993).
  • the hybrid a-factor transporter encoded by the H5- ⁇ F508 gene is defective as a result of the ⁇ F508 mutation.
  • Strain A is therefore unable to transport the mating pheromone a-factor efficiently. Since the transport of the a-factor pheromone is required for yeast cell mating, mating between strains A and B is inefficient.
  • the agar media is one rich in nutrients (nonselective for the growth of strains A and B) necessary for the optimal mating of yeast.
  • An extract prepared from the plant genus Grindelia was prepared by grinding fresh Grindelia leaves in a mixture of ethanol and water (1:1). This extract contains the fraction of plant material soluble in a solution of 1:1 ethanol and water.
  • Small filter paper discs were prepared using a standard paper punch and Whatman filter paper #2.
  • a filter paper disc containing the Grindelia extract was prepared by adding 10 ⁇ l. of extract to the disc, and then allowing the disc to air dry. Additional extract was added in 10 ⁇ l aliquots, allowing the disc to dry after each application until a total of 30 ⁇ l. was added to the disc. The disc was then placed upon the surface of the media, on top of the yeast cells.
  • the mating plate was incubated for 24 hours at 30°C to allow yeast cell mating to occur.
  • the mating plate was replica printed to a selective media plate that is incubated at 30°C for three days to allow the growth of diploid colonies.
  • strains A and B cannot grow on this media (strain A has the lys2 mutation and strain B has the ura3-52 mutation: these mutations prevent them from growing on media without lysine and uracil respectively).
  • diploids produced as a result of the fusion between strain A and B can grow (diploids contain the genomes of both haploids and thus contain the complementing URA3 and LYS2 wild-type alleles) .
  • Production of yeast diploid cells in the area surrounding a leaf disc would be a strong indication that the leaf disc contained a compound which corrects the ⁇ F508 defect.
  • Diploids cells were easily detected by replica printing the yeast cells from the mating plate, onto another petri dish plate containing media which is selective for the growth of the diploid cells. This media will allow the diploid yeast cells to grow and form colonies, while preventing the growth of the haploid (unmated) strain A and B yeast cells.
  • the frequency of diploid colonies produced on the selective plate is an indirect measurement of the ability of strain A to transport a-factor and mate.
  • a high density of yeast colonies on the selective media plate occurred in the location corresponding to the position where the filter paper disc containing the Grindelia extract occurred on the mating plate. This is clearly distinguishable from the low level of diploid colonies which are produced by the mating of the two yeast strains in the absence of a chemical compound that corrects the ⁇ F508 defect. (See Figure 3. )
  • ADDRESSEE Zarley, McKee, Thomte, Voorhees & Sease
  • ORGANISM Saccharomyces cerevisiae
  • GGC AGA GTG TTC GAT CTA CTA TCA GTT TTC GTG GCT AAT GGG TCA CAT 192 Gly Arg Val Phe Asp Leu Leu Ser Val Phe Val Ala Asn Gly Ser His 50 55 60
  • GTT GCT GCA AAC GCT GGG ATC TTG AGA TTT TTG ACG TTG ACT ATG TTT 816 Val Ala Ala Asn Ala Gly He Leu Arg Phe Leu Thr Leu Thr Met Phe 260 265 270
  • MOLECULE TYPE DNA (geno ic)
  • ORGANISM Saccharomyces cerevisiae
  • GGC AGA GTG TTC GAT CTA CTA TCA GTT TTC GTG GCT AAT GGG TCA CAT 192 Gly Arg Val Phe Asp Leu Leu Ser Val Phe Val Ala Asn Gly Ser His 50 55 60
  • GTT GCT GCA AAC GCT GGG ATC TTG AGA TTT TTG ACG TTG ACT ATG TTT 816 Val Ala Ala Asn Ala Gly He Leu Arg Phe Leu Thr Leu Thr Met Phe 260 265 270
  • AAA CAA CAA AAG AAC ACC GAA AGT TCA ACA GGG CCA CAA CTT CTG AGC 2064 Lys Gin Gin Lys Asn Thr Glu Ser Ser Thr Gly Pro Gin Leu Leu Ser 675 680 685
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Saccharomyces cerevisiae

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Abstract

A method of identifying potentially useful treatments which restore the function of CFTR or other related protein mutations is disclosed. The method involves creation of yeast STE6-CFTR chimeric sequences which upon introduction of a desired mutation inhibit expression of the yeast STE6 gene. A yeast assay (STE6Δ) transformed to include the hybrid chimeras is then exposed to a potential treatment for the mutation. A treatment which restores the function of the STE6 gene and thereby allows mating of the yeast strain is indicative of a potential remedial treatment for the mutation in this selected protein.

Description

Title: METHOD OF ASSAYING FOR DRUGS WHICH RESTORE FUNCTION OF MUTANT CFTR PROTEIN
BACKGROUND OF THE INVENTION
Cystic fibrosis is a human genetic disease of the secretory epithelia. Although the survival rate of those suffering with cystic fibrosis has improved in recent years, the median age for patient survival is still only about twenty five to thirty years despite intensive supportive and prophylactic treatment. Today cystic fibrosis remains the most common congenital disease among Caucasians, where it has a prevalence of about one in two thousand live births, and is uniformly fatal. Nearly all patients suffering from the disease develop chronic progressive disease of the respiratory system, the most common cause of death being pulmonary disease. Also, in the majority of cases, pancreatic dysfunction occurs; hepatobiliary and genitourinary diseases are also frequent. Because of the multi¬ system clinical manifestations of the disease, current methods of treatment for the disease have focused on therapeutic approaches to reduce the symptoms of cystic fibrosis.
United States Patent No. 5,100,647 to Agus, et al, discloses a method for treating cystic fibrosis by administration of the compound sparteine (dodecahydro-7, 14methano-2H, 6H-di-pyrido [1,2-a: l',2'-e] [1,5] diazocine), acting as a direct exogenous activator of chloride conductants in epithelial airways. United States Patent No. 5,179,001 to Young, et al, discloses a method of treating pulmonary complications associated with cystic fibrosis caused by the gram negative bacterium Pseudomonas aeruqinosa. United States Patent No. 4,826,679 to Roy relates to an oral composition for alleviating digestive manifestation in persons afflicted with cystic fibrosis comprising a therapeutic amount of taurine. Despite much advancement in the treatment of the symptoms of cystic fibrosis very little has been done to effectively "cure" the disease at a genetic level. One method of gene therapy proposed is United States Patent No. 5,149,797 disclosing a method of site specific alteration of RNA and production of encoded polypeptides. This invention is drawn to correcting the abnormal mRNA present in individual cells, cleaving the mRNA by site directed RNAase followed by introduction of the appropriate oligoribonucleotide followed by endogenous RNA ligase and thus production of a wild-type mRNA encoding a normal protein product which then may be translated to produce the correct protein.
Cystic fibrosis is characterized at the genetic level by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The CFTR protein is a member of a superfamily of structurally related membrane proteins named the traffic ATPase or ATP-binding cassette (ABC) transporters. Other members of this superfamily include the multi-drug resistance (MDR) or P-glycoprotein, bovine adenyl cyclase, the yeast STE6 protein, as well as several bacterial amino acid transport proteins (Riordan, et al, 1989; Hyde, et al, 1990). The principle distinguishing feature of this superfamily of proteins is a highly conserved nucleotide-binding domain (NBD) .
CFTR is a protein of approximately 1480 amino acids consisting of two repeated motifs, each comprising 6 transmembrane segments and a nucleotide-binding domain. The two motifs are separated by a large polar R-domain containing multiple potential phosphorylation sites.
Many members of the ABC transport superfamily transport small molecules across biological membranes in an ATP dependent manner. However, CFTR is a Cl~ channel regulated by phosphorylation and by cytosolic ATP (Anderson, et al, "Nucleoside Triphos- phates are Required to Open CFTR Chloride Channel" Cell,67,775-784) . The two nucleotide-binding domains present in CFTR play a central role in the function of CFTR Cl" channels; they interact directly with ATP to open the CFTR Cl~ channel and ATP hydrolysis may be required for this effect. The importance of the NBD's is further emphasized by the large number of cystic fibrosis associated mutations that have been found in the domains. The most common cystic fibrosis associated mutation, accounting for approximately 68% of the cystic fibrosis chromosomes is deletion of a phenylalanine at position 508 (ΔF508) in the middle of NBD1.
This mutation is characterized by a loss of apical membrane Cl~ channel activity in the epithelial cells leading to chronic progressive disease of the respiratory system. The ΔF508 mutation causes loss of apical membrane Cl~ channel activity affecting the CFTR protein in two ways. First, the mutation causes defective processing and hence mislocalization of the mutant protein. This is presumed to occur from misfolding of the mutant protein so that it fails to exit from the endoplasmic reticulum and progress to the Golgi complex in the apical membrane. The resulting Cl" transport defect associated with the ΔF508 is thus largely a consequence of the absence of functional CFTR at the plasma membrane. Second the ΔF508 mutation affects the function of the Cl~ channel. Single channel analysis of CFTR ΔF508 suggests that although the mutant protein is functional, it has an open state probability (P0) that is one third that of wild-type CFTR.
Defective trafficking of CFTR ΔF508 can be partially reversed if cells are grown at a lower temperature, indicating that mutant protein is still functional. When CFTR ΔF508 is produced at a reduced temperature (less than 37°C) some of the mutant protein transists the Golgi complex normally and is localized correctly in the plasma membrane. (See Denning, G.M. ; Anderson, M.P.; ara, J. ; Marshalt, J.; Smith, A.E.; and Welsh, M.J.); "Processing of Mutant CFTR (ΔF508) is Temperature Sensitive." Nature,358,761-764. Since CFTR ΔF508 retains partial function, a possible therapy for cystic fibrosis may involve pharmacologic intervention designed to correct the processing defects so that more CFTR ΔF508 reaches the plasma membrane. Potential pharmacologic intervention would represent a non gene-therapy approach for the treatment of cystic fibrosis.
As can be seen, a great need exists in the art for methods of treating cystic fibrosis that do not concentrate solely on the symptoms produced by the disease, but rather treat the disease at a cellular level by attempting to restore function to the mutant product. In light of the scientific potential for pharmacologic treatments, a method for identification of new candidate drugs for cystic fibrosis treatment is warranted. This is complicated, however, by the fact that a screening method to identify treatments that reverse the CFTR ΔF508 CI~ transport defect in mammalian cells is impractical; large numbers of chemical compounds cannot easily be screened for an activity that restores the cAMP-stimulated Cl~ transport of mutant CFTR.
It is an object of this invention to provide an in vitro method of screening pharmaceutical compositions, and further mutations for their ability to restore function to defective ABC transport proteins resulting from mutations such as CFTR ΔF508, by simple means of a yeast mating assay.
It is another object of the invention to construct chimeric gene sequences which encode the yeast STE6 gene and the CFTR gene for screening of treatments which will restore function to the mutant proteins.
It is another object of the invention to construct chimeric gene sequences which encode the yeast STE6 gene and the P-glycoprotein gene (or other ABC transporter genes) for screening treatments which will inhibit the function of the chimeric protein.
Yet another object of the invention is to provide a method for identifying potentially useful compounds for further useful study in the treatment of disease by means of a yeast mating assay.
Other objects of the invention will become obvious from the description of the invention which follows. SUMMARY OF THE INVENTION
This invention relates to a method for screening treatments which restore the function of mutant CFTR by simple means of a yeast cell mating assay. According to the present invention, chimeric genetic sequences which encode the yeast a-factor transporter STE6 and CFTR or another member of the related superfamily such as P-glycoprotein are constructed whereby a segment of the CFTR genetic sequence is exchanged for a corresponding segment of the STE6 genetic sequence to produce a product which will complement a yeast ste6Δ mutation but is non¬ functional upon introduction of a desired mutation into the CFTR sequence.
The resulting chimeras are then transformed into a yeast strain which has the STE6 gene deleted (ste6Δ). The yeast strain is then exposed to the desired drug or treatment to be screened and the strain is assayed for the presence of restoration of mating, which indicates a functional chimera. The invention discloses several chimeric sequences useful for screening for utility of treatments and reversing the effects of the CFTR ΔF508 mutation which is associated with cystic fibrosis.
DETAILED DESCRIPTION OF THE DRAWINGS
Figure 1 represents the amino acid sequence alignment of NBDl of STE6 and CFTR.
Figure 2 depicts the STE6-CFTR chimeric constructs and their mating efficiency.
Figure 3 is a graphical depiction of a yeast assay according to the present invention which presents a potential cystic fibrosis treatment. Figure 4a-g is the gene sequence for chimera
H4
Figure 5a-g is the gene sequence for chimera
H5,
Figure 6a-g is the gene sequence for chimera
H6.
DETAILED DESCRIPTION OF THE INVENTION
The STE6 gene of Saccharomyces cerevisiae encodes an integral membrane protein that functions to transport the peptide a-factor out of the cell during mating. (Kuchler and Thorner, 1992), "Functional expression of human MDRl in yeast Saccharomyces cerevisiae." Proc. Natl . Acad. Sch . , USA89, 2303-2306. Deletion of the STE6 gene from yeast (ste6Δ) results in a sterile phenotype. According to the present invention, chimeric STE6 genes were constructed in which segments of the NBDl of STE6 were substituted by the analagous NBDl sequences from CFTR.
Selection for sites of exchange was accomplished by comparing the known amino acid sequences of CFTR and STE6. The CFTR and STE6 amino acid sequences were aligned by use of the "BEST FIT" algorithm for the comparison of DNA and amino acid sequences obtained from the University of Wisconsin and found in the GCG Sequence Analysis Package. Figure 1 represents an amino acid sequence alignment of the NBD for proteins CFTR and STE6. The CFTR amino acid sequence is depicted on top illustrating amino acids 442 through 578; the corresponding analagous STE6 amino acid sequence is depicted below illustrating amino acids 376 through 536. Corresponding amino acids are indicated by solid vertical bars and give an indication of potential junctions for the genetic constructs.
Table 1 indicates the portion of CFTR amino acids sequence inserted into STE6 for the different hybrids.
TABLE 1
PORTION OF CFTR HYBRID IDENTIFIER AMINO ACID SEQUENCE
HI D443-Y577
H2 D443-F508
H3 F494-F508
H4 F494-I546
H5 F494-L558
H6 F494-Y577
The HI STE6-CFTR chimeric gene was constructed with a DNA sequence coding for the entire NBDl of STE6 (from N377 to A535) replaced by the corresponding region of CFTR (D443 to Y557). (See Figure 1.) H2 replaced the amino terminal half of NBD and H6 replaced the carboxy terminal half. The remaining constructs were variations between these two extremes. (See Figure 2.) The construction of plasmids with these sequences used methods which are well known to those of skill in the art however the following description is included merely for purposes of illustration and is not intended to limit in any way the invention.
PLASMID CONSTRUCTIONS Plasmid RFG416 (a gift from Rick Gaber, Northwestern University) is a single copy CEN plasmid with the selectable marker URA3 and the pUC19 polylinker region. A 6.5 kb Sall-Sacl fragment containing the STE6 gene was subcloned from STE6-2μ (a gift from John McGrath, Massachusetts Institute of Technology) into the Sail and Sacl sites of the vector RFG416 to produce plasmid JTS6. The yeast TRPl gene (on a 0.85 kb BgLll-EcoRl DNA fragment) was inserted at nucleotide position 1356 of STE6 NBDl, resulting in JTS6T.
To construct the HI STE6-CFTR hybrid plasmid, a 441 bp DNA fragment containing the CFTR NBDl region was synthesized using two STE6-CFTR oligonucleotide primers (primer 1, 5'CCTTCGGAAGCAGTCCTGAAAGATAT-3' ; primer 2, 5'-GATGAACAATATCTAGGTATCCAAAA-3' . and CFTR cDNA template DNA in a polymerase chain reaction (PCR) (Ho, et al., 1989). PCR reactions were performed with a Temp-Tronic thermal cycler (Barnstead/Thermolyne) . Oligonucleotide primers 1 and 2 encoded fusion junctions of STE6 L375 to CFTR K442 and CFTR 1578 to STE6 D537, respectively, found in the Hi STE6-CFTR hybrid gene. The CFTR portion of each primer is underlined. STE6 DNA flanking NBDl was added to each end of the 441 bp fragment by PCR, resulting in a 867 bp DNA fragment consisting of 419 bp of CFTR DNA (encoding CFTR amino acids K442-L578) that is flanked at the 5' end by 168 bp of STE6 DNA (encoding STE6 amino acids K319-L375) and 280 bp of STE6 DNA at the 3' end (encoding STE6 amino acids D537-G640). Plasmid construction of HI was performed by cotransformation (Ito, et al, 1983) of yeast strain JPY201 with the 867 bp DNA fragment and 3 μg of plasmid JTS6T and selection of trans- formants on SD-URA (yeast nitrogen base supplemented with all amino acids except uracil) . Homologous recombination between the STE6 DNA sequences at each end of the 867 bp DNA fragment with the STE6 gene on the plasmid results in the targeted integration (Orr-Weaver, et al, 1981) of the CFTR sequences into NBD1 of STE6 on plasmid JTS6T and the consequent loss of the TRP1 gene. Recombinants containing the desired STE6-CFTR hybrid gene were identified as trp auxoptrophs at a frequency of about 1% among the transformants. Plasmid DNA was prepared from trp transformants and the structure of the STE6-CFTR gene was confirmed by DNA sequencing analysis. STE6-CFTR hybrid genes H2-H6 were similarly constructed using the following oligonucleotide primers for constructing the appropriate STE6CFTR junctions; STE6 Q440/CFTR F494, 5'- CACCGTCGTAGAACAGTTTTCCTGGATTA-3' ; CFTR G509/STE6 S457, 5'-CCGAATCTGTTGAACCAAAGATGATATTT-3' ; CFTR G550/STE6 G509, 5'-TTGTTGTTGCCCGCCACTCAGT3TGATTC-3' ; CFTR R560/STE6 A519, 5' ATCTCTGATGAATGCTCTTGCTAAA- GAAAT-3 ' .
The gene sequences for H4, H5, and H6 are included in Figures 4-6..
The resulting plasmids were then transformed into a yeast strain with the STE6 gene deleted (ste6Δ) to test for complementation by means of a yeast mating assay.
YEAST CELL MATING ASSAY Several types of variations of assays may be used and are commonly known to those of skill in the art. The following description is not intended to limit in any way the invention.
The yeast strains JPY201 (MATa ste6Δ:HIS3, gal2, ura3-52, lys2-801, trpl, leu2-3,112, his3 Δ200) and 22-2D (MATa, ura352, leu2-3, 112,trpl) were used for all mating experiments. JPY201 contains a STE6 deletion (including NBDl and extending beyond the termination codon) and replacement with the yeast HIS3 gene (McGrath and Varshavsky, 1989). Quantitative mating assays were performed as in Trueheart, et al (1987).
Transformants of JPY201 containing each STE6- CFTR chimera were grown to log phase in 0.1% glucose SD-URA media. From each culture, 3 x 106 cells were mixed with an equal number of 22-2D cells grown in YPD media and collected by filtration onto a Millipore filter, which was then placed upon a YPD plate for 4 hours at 30°C. Cells were resuspended, sonicated briefly, and plated from serial dilutions onto SD+LEU/TRP (yeast nitrogen base supplemented with leucine and tryptophan) . Diploid colonies were counted after 3 days at 30°C. Control strains STE6 wild-type and ste6Δ consisted of JPY201 transformed with plasmids JTS6 and JTS6T, respectively. For qualitative petri dish mating assays, JPY201 transformants containing STE6-CFTR chimeras were grown as patches on SD-URA media and then replica printed to YPD media on a lawn of 22-2D cells. Following incubation at 30°C for 8 hr, the plate containing the mating cells was replica printed to SD-LEU/TRP and incubated 3 days at 30°C to allow growth of diploid colonies.
The complementation results are given in Figure 2. As can be seen yeast transformants containing the Hi sequence were unable to complement the ste6Δ mutation, indicating a nonfunctional STE6 chimera. Similarly, the H2 sequence replacing the amino half only was nonfunctional. See Figures 1 and 2. These results suggest that the amino terminal region of NBDl from CFTR cannot substitute for that of STE6.
In contrast, the STE6-CFTR chimeras containing the central part of NBDl from CFTR (H3 and H4, See Figures 1 and 2) maintained a- actor transport activity comparable with the wild-type STE6 yeast strain. This suggests that amino acid residues between the conserved Walker A and B motifs (Walker, et al, 1982) although different in the NBDl of CFTR and STE6 provide a similar overall structure and function. When the chimeras included progressively larger segments of the carboxy-terminal region of NBDl from CFTR (H5 and H6) mating efficiency was reduced. Yeast transformed with the hybrid gene H5 which contained the substitution of 74 residues of STE6 NBDl (from R441 to 1516) by that from CFTR (from F494 to L558) mated at 12% efficiency of wild- type STE6. Mating efficiency was further reduced in H6 to background levels. However, a low level of mating was observed when the H6 gene construct was expressed on a high copy number plasmid.
INTRODUCTION OF THE ΔF508 MUTATION A previous study has suggested that missense mutations in STE6 that were analogous to CF- associated mutations in NBDl of CFTR result in defective a-factor transport, however, single amino acid deletions analogous to ΔF508 within the central region of STE6 NBDl had no effect on the STE6 function. (Berkower and Michaelis, "Mutational Analysis of the Yeast a-factor Transporter STE6; a Member of the ATP Binding Cassette (ABC Protein Superfamily)," EMBO J 10, 3777-3785 (1991)).
Contrary to this finding, it was found that by incorporating CFTR into NBDl of the STE6 gene and also including the ΔF508 mutation within the CFTR segment, function of the STE6 chimeric gene was inhibited. The chimeric sequence constructs H3, H4, H5, and H6 which demonstrated complementation of the ste6Δ yeast strain were also used to assess the effect of the ΔF508 mutation. The ΔF508 mutation was introduced into H3, H4, H5, and H6 (resulting in H3-ΔF508, H4-ΔF508, H5-ΔF508, and H6-ΔF508 respectively). Figure 2 shows the effect of the ΔF508 mutation on a-factor transport as assessed by the quantitative yeast cell mating assay.
As seen from the results in Figure 2, yeast transformed with the H3-ΔF508 chimera mated with an efficiency equal to the H3 control (no F508 mutation). A decrease in mating efficiency (40%) was observed with the strain containing the H4-ΔF508 chimera relative to the H4 control. (Sequence I.D. No. 1). The ΔF508 mutation lowered mating efficiency by 80 fold in the H5-ΔF508 chimera as compared with H5 (Sequence I.D. No. 3). The H6-ΔF508 chimera displayed a comparable decrease in mating as compared to H6 (Sequence I.D. No. 5) efficiency due to the ΔF508 mutation when expressed on a high copy number plasmid, (and relative to the H6 chimera gene also on high copy number plasmid vector) . For use in accordance with the present invention, expression of the H6-ΔF508 gene on a high copy number plasmid would be required.
Based on the foregoing junction analysis, the portion of CFTR most promising for substitution into STE6 include the amino acid identity locations between a portion of NBDl from amino acid 444 up to amino acid 509 through 577. As earlier seen, the segment from 443 to 557 was inactive as was the portion from 443 to 508. Preferred ranges include from amino acid 494 to 577 including: sequence I.D. No. 1, CFTR amino acid F494-1546; Sequence I.D. No. 3, F494-L558; Sequence I.D. No. 5 CFTR amino acid F494-Y577. Several areas of identity between the two aligned sequences as demonstrated in Figure 1 exist within this range of CFTR amino acid substitutions and will represent other chimeric sequences useful for the present invention.
YEAST MATING ASSAY TO IDENTIFY COMPOUNDS THAT CORRECT THE ΔF508 DEFECT
In accordance with the present invention, a yeast strain transformed to contain the discussed chimeric sequences will be used in a yeast mating assay to identify compounds which will correct the affect of the mutation as indicated by an increase in mating. Such an assay may be widely varied by those of skill in the art. Generally, the trans¬ formed strain will be combined with a strain of the alpha mating type on a suspension of yeast cells spread on the surface of agar medium in the petri dish. These two strains would be unable to mate due to the ΔF508 mutation.
A compound is then introduced to the medium. For example a plant screening method may be performed by placing a small disc of leaf material removed by means of a paper punch from a plant and placed on the surface of the agar media. Plant compounds will diffuse from the leaf and interact with the yeast strains.
Another method would be to use a filter paper which has been soaked in the desired chemical and then placed on the yeast suspension. Further, other desired mutations can be introduced into the CFTR region to attempt to identify a revertant mutation and a corresponding protein for further study. It is to be understood that any form of exposing the yeast strain to the desired treatment may be used including modifying external conditions such as temperature.
The mating plate is then incubated for 24 hours to allow yeast cell mating to occur. If a compound corrects the molecular defect associated with H5-ΔF508 transporter then a-factor is transported allowing mating between the two strains, resulting in diploid cells. The mating plate is then replica printed to a selective media plate to allow growth of diploid colonies.
For example strain A could be - MATa, STE6::HIS3, ura3-52, lys2,trpl-298, his3-200, leu2- 3,112 <plasmid H5-ΔF508, URA3, CEN>; strain B - MATα, ura3-52, trpl, leu2-3,112.
The selective media used would then be lacking lysine and uracil. Consequently strains A and B could not grow on this media (strain A has the lys2 mutation and strain B has the ura3-52 mutation; these mutations prevent them from going on media without lysine and uracil respectively), however diploids produced as a result of the fusion between strain A and B can grow (diploids contain the genomes of both haploids and consequently has the complementing URA3 and LYS2 wild-type allele).
Production of yeast diploid cells in the area surrounding the treatment disc would be a strong indicator of presence of compound(s) which correct the ΔF508 defect. A number of yeast mating type assays may be developed easily by those of skill in the art and the foregoing description is not intended to limit in any way the invention. EXAMPLE 1
REVERTANTS OF ΔF508 MATING DEFECT IN YEAST (MUTATION IN R553-L558 REGION TO SUPPRESS ΔF508 IN NBDl)
The R553-L558 amino acid region of CFTR is of great interest in CFTR and other trafficking ATPase's as it is directly adjacent to the conserved LSGGQ linker sequence of NBDl, postulated to function as a transducer of signals between the hydrophobic domains and the NBD's (Mimura, et al, 1991). It was hypothesized that a possible treatment to correct the F508 mutation could be interaction of the region of the NBD polypeptide containing F508, with the R553-L558 region. The only difference between the H5 chimera and the H4 chimera is 6 amino acids in the R553-L558 region. This region is also not present in the H3 chimera in which the ΔF508 mutation had no effect. Possibly there is an interaction between the two regions of the NBD required for correct folding or function (or both) of the NBD. Thus the method of the present invention was used to test whether a mutation in the region R553-L558 might suppress the adverse effects of the ΔF508 in NBDl.
To isolate revertants of the H5-ΔF508 mating defect, the H5-ΔF508 construct was mutagenized in the R553-L558 region in vitro by site-specific oligonucleotide mutagenesis (Ho, et al, "Site- Directed Mutagenesis by Overlap Extension Using the Polymerase Chain Reaction Gene" 77,51-59 (1989). The mutagenesis method was designed to introduce random amino acid substitutions at single codon positions within the R553-L558 region of the H5- ΔF508 plasmid. The procedure allowed 20 possible substitutions at each amino acid position within R553-L558. The mutagenized DNA was transformed into yeast, and transformant colonies were analyzed by cell mating assays on petri dishes to identify colonies with a mating efficiency higher than the unmutagenized H5-ΔF508 control. Two colonies identified displayed higher efficiencies of mating within the first 20 mutant transformants analyzed. These yeast transformants each contained an H5-ΔF508 plasmid with a mutation at amino acid R553 of CFTR; in one case, R553 was replaced by methionine (H5- ΔF508/R553M) , and in the other plasmid, R553 was replaced by glutamine (H5-ΔF508/R553Q) . It is possible that other mutations within the R553-L558 region of the H5-F508 plasmid could also result in increased mating efficiency; however, the R553Q and R553M mutants were analyzed in greater detail without further mutagenesis of the R553-L558 region.
The relative mating efficiency of the ΔF508 revertant strains are shown in Table 2, with results expressed relative to the original H5 strain. Whereas the mating efficiency of the H5-ΔF508 yeast strain is approximately 1% of the H5 strain, yeast containing the H5-ΔF508/R553Q and H5-ΔF508/R553M plasmids mated at 3% and 32%, respectively. The revertant mutations, therefore, partially suppress the ΔF508 mating defect. The R553M mutation alone had little effect on H5 (H5-R553M); when this mutant was transformed into yeast, no further increase in mating efficiency was observed as compared with yeast containing H5 (Table 2). In accordance with this invention, mutations were found which suppress the effects of the ΔF508 mutation. TABLE 2
Relative Mating Efficiency of ΔF508 Reverants
Mating Efficiency Genotype Relative to H5 (%)
H5-R553M 77.4 = 3.7
H5-ΔF508/R553M 34.2 = 7.8
H5-ΔF508/R553Q 3.2 = 0.8
H5-ΔF508 1.1 = 0.5
Mating efficiencies were determined by quantitative mating assays and are expressed as a percentage relative to H5 (100%). Data are means = SEM.
EXAMPLE 2
TESTING OF THE R553M and R553Q MUTATIONS IN THE MAMMALIAN SYSTEM
To determine whether the mutations which corrected the ΔF508 mutation would also correct the defect in human CFTR ΔF508, the R553Q and R553M mutations were introduced into CFTR ΔF508 cDNA and mammalian cells were transfected with these con¬ structs to determine whether the revertant mutations would correct the defect in cAMP-regulated Cl~ transport of CFTR ΔF508.
The revertants R553Q and R553M were tested for their effects on mislocalization and the channel gating of CFTR ΔF508. The identified revertants modified both of these effects. Mutation of R553 to methionine partially corrected the processing of CFTR ΔF508 as assessed by three criteria; it produced fully glycosylated protein as indicated by electrophoretic separation on SDS polyacrylamide gel; it increased the appearance of the mutant protein in the plasma membrane as determined immunocytochemically; and it produced functional channels in the plasma membrane when measured by the SPQ halide efflux assay. These results demonstrate the functional utility of the yeast model system for identifying revertants of CF-associated mutations of the NBDl of CFTR and its use in drug screening for pharmacologic compounds that correct the mating defect. The mutations discovered could be used to identify peptides which may be of utility in reversing the effects of the ΔF508 mutation.
EXAMPLE 3
R553Q AND R553M SUPPRESS THE CFTR ΔF508 ANION TRANSPORT DEFECT
The effect of the R553Q and R553M mutations on CFTR function was determined by a cAMP-stimulated halide efflux using the halide-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) (Illsley and Verkman, 1987). Expression of CFTR cDNA containing either the R553Q or the R553M mutation alone (without the ΔF508 mutation) in HeLa cells generated cAMP-stimulated halide efflux like wild- type CFTR. Cells expressing the ΔF508 allele in this recombinant system showed little, if any, cAMP- stimulated halide efflux. However, when the mutations R553Q and R553M were introduced into CFTR ΔF508 (CFTR ΔF508/R553Q and CFTR ΔF508/R553M, respectively), cAMP-dependent anion permeability was restored. These results indicate that the CFTR Cl" channel defect observed with the ΔF508 mutant could be suppressed by either R553 mutation.
Cl~ transport by CFTR ΔF508 containing the R553Q and R553M mutations would be detected only if the processing defect of CFTR ΔF508 was suppressed. To determine further whether the suppressor mutations correct the processing defect associated with the ΔF508 mutation, the glycosylation patterns of CFTR and the various mutants expressed in HeLa cells were examined. CFTR is a glycoprotein that undergoes progressive glycosylation, resulting in three bands that migrate at different rates on an SDS-polyacrylamide gel. Band A is the most rapidly migrating and represents the nascent, unglycosylated protein; band B has an intermediate rate of migration and a pattern of core glycosylation consistent with processing in the endoplasmic reticulum; band C migrates most slowly and has a pattern of mature glycosylation consistent with processing in the Golgi complex. CFTR ΔF508 was only presented as the unglycosylated band A and the core glycosylated band B protein, consistent with its failure to traverse the Golgi complex and reach the plasma membrane.
We first examined the glycosylation state of CFTR in HeLa cells expressing CFTR cDNA containing either the R553Q and R553M mutations alone (without the ΔF508 mutation) . Band C was present in cells expressing wild-type CFTR and also mutant CFTR containing either the R553Q or R553M mutation. In contrast, only bands A and B were present in cells expressing CFTR ΔF508. Thus, the R553Q and R553M mutations alone do not affect the glycosylation of CFTR.
In cells transfected with the CFTR ΔF508/R553M, a small increase in band C was detectable as compared with CFTR ΔF508. This result was observed in three separate experiments. Band C was not able to be consistently detected in cells expressing CFTR ΔF508/R553Q possibly owing to limitations in the sensitivity of the assay. However, a detectable increase in Band C with CFTR ΔF508/R553M as compared with CFTR ΔF508 occurred.
To assess further the ability of suppressor mutations to correct the mislocalization defect of CFTR ΔF508, immunocytochemistry was used to detect CFTR at the cell surface in HeLa cells expressing wild-type or mutant CFTR. An antibody specific to an external epitope in the first extracellular loop of CFTR was used. (Denning, et al, 1992b). Wild-type CFTR is generally detected at the surface of unpermeabilized HeLa cells. In contrast, no CFTR was detectable at the surface of cells expressing CFTR ΔF508. However, when cells expressed CFTR ΔF508/R553M, CFTR was detected at the plasma membrane. For CFTR ΔF508/R553Q, plasma membrane staining of CFTR was weak and variable and could not be demonstrated consistently. Only nonspecific staining was observed with preimmune serum or in the absence of CFTR. These results were consistent with the observation that more CFTR ΔF508/R553M than CFTR ΔF508/R553Q was found in the band C form and suggest that the amount of CFTR ΔF508/R553Q at the plasma membrane is exceedingly low.
SINGLE-CHANNEL ANALYSIS OF CFTRΔF508/R5530 CFTR To determine whether the suppressor mutations altered the single-channel properties of CFTR ΔF508, the revertant mutant CFTR ΔF508/R553Q was expressed in HeLa cells and analyzed with the patch-clamp technique. Single-channel analysis of CFTR ΔF508 indicated that the P0 of the CFTR ΔF508 Cl- channel was reduced as compared with wild-type CFTR. Denning, et al, (1992a) reported that CFTR ΔF508 had a P0 of 0.13 + 0.01 (after incubation at reduced temperature), compared with a value of 0.34 + 0.02 for wild-type, and Dalemans, et al, (1991) reported that the P0 of CFTR ΔF508 Cl" channels ranged from 0.05 to 0.10, compared with a range of 0.22 to 0.35 for wild type.
As expected from the response observed in the SPQ studies, CFTR/R553Q and CFTR ΔF508/R553Q formed functional Cl~ channels. Inside-out membrane patches from either mutant showed no Cl~ channel activity under basal conditions. However, addition of protein kinase A(PKA) and ATP to the cytosolic surface of the membrane patch activated channels in both cases; both agents were required to activate the channels (n = 6 for each mutant). Single- channel events were recorded from +60 V to -120 mV in increments of 20 mV. The channels were Cl~ selective as indicated by the reversal potentials (E = 20 mV:Ecι- = 27 mV) . CFTR/R553Q had a single- channel slope conductance (from -120 mV to 0 mV) of 9.8 + 0.5 pS (n = 7), and CFTR ΔF508/R553Q had a conductance of 10.2 + 0.2 pS (n = 11). The current measured at -100 mV for CFTR/R553Q (-1.1 + 0.1 pA, n = 6) and CFTR ΔF508/R553Q (-1.2 + 0.0 pA, n = 5) was not different from the current measured for wild-type CFTR (-1.1 ± 0.0 pA, n = 7). Thus, these data indicate that neither mutation altered the single-channel conductive properties of the CFTR Cl~ channel, nor did the mutations abolish the regulation by PKA. The results also indicate that, as predicted by the functional analysis of CFTR ΔF508/R553Q by the SPQ halide efflux assay above, CFTR ΔF508/R553Q is localized in the plasma membrane. Following activation with PKA and ATP, we measured the P0 for CFTR/R553Q and CFTR ΔF508/R553Q at different MgATP concentrations. As the concentration of MgATP increased, both CFTR/R553Q and CFTR ΔF508/R553Q spent more time in the open state. The P0 of CFTR/R553Q Cl" channels was similar to that previously reported for wild-type CFTR Cl" channels (Anderson and Welsh, 1992). However, at ImM ATP, the P0 of CFTR ΔF508/R553Q (0.29 + 0.02, n = 7) was greater than that of CFTR ΔF508 Cl" channels (0.13 + 0.01, n = 4). Thus, the R553Q mutation corrected the functional defect in gating of the CFTR ΔF508 Cl" channels.
YEAST MATING ASSAY WITH PLANT EXTRACT SCREENING A suspension of yeast cells (strains A and B) was spread upon the surface of agar media in a petri dish.
The suspension consisted of equal proportions of two haploid yeast strains. Strain A - MATa, STE6::HIS3, ura3-52,lys2,trpl-298, his3-200, leu2-3,112 <plasmid H5-ΔF508, URA3, CEN>; strain B -MAT , ura3-52, trpl, leu2-3,112.
Yeast strains A and B are of the a and α mating types respectively, and can mate to form yeast diploids. Strain A contains the STE6/CFTR hybrid a-factor transporter gene H5-ΔF508 (Teem, et al, 1993). The hybrid a-factor transporter encoded by the H5-ΔF508 gene is defective as a result of the ΔF508 mutation. Strain A is therefore unable to transport the mating pheromone a-factor efficiently. Since the transport of the a-factor pheromone is required for yeast cell mating, mating between strains A and B is inefficient. The agar media is one rich in nutrients (nonselective for the growth of strains A and B) necessary for the optimal mating of yeast.
An extract prepared from the plant genus Grindelia was prepared by grinding fresh Grindelia leaves in a mixture of ethanol and water (1:1). This extract contains the fraction of plant material soluble in a solution of 1:1 ethanol and water. Small filter paper discs were prepared using a standard paper punch and Whatman filter paper #2. A filter paper disc containing the Grindelia extract was prepared by adding 10 μl. of extract to the disc, and then allowing the disc to air dry. Additional extract was added in 10 μl aliquots, allowing the disc to dry after each application until a total of 30 μl. was added to the disc. The disc was then placed upon the surface of the media, on top of the yeast cells.
The mating plate was incubated for 24 hours at 30°C to allow yeast cell mating to occur.
Chemical compounds that diffuse out of the filter paper disc into the agar will come in contact with the mating cells. A component of the Grindelia extract promotes mating of strains A and B.
The mating plate was replica printed to a selective media plate that is incubated at 30°C for three days to allow the growth of diploid colonies.
The selective media is lacking lysine and uracil. Consequently, strains A and B cannot grow on this media (strain A has the lys2 mutation and strain B has the ura3-52 mutation: these mutations prevent them from growing on media without lysine and uracil respectively). However, diploids produced as a result of the fusion between strain A and B can grow (diploids contain the genomes of both haploids and thus contain the complementing URA3 and LYS2 wild-type alleles) . Production of yeast diploid cells in the area surrounding a leaf disc would be a strong indication that the leaf disc contained a compound which corrects the ΔF508 defect. Diploids cells were easily detected by replica printing the yeast cells from the mating plate, onto another petri dish plate containing media which is selective for the growth of the diploid cells. This media will allow the diploid yeast cells to grow and form colonies, while preventing the growth of the haploid (unmated) strain A and B yeast cells. The frequency of diploid colonies produced on the selective plate is an indirect measurement of the ability of strain A to transport a-factor and mate.
A high density of yeast colonies on the selective media plate occurred in the location corresponding to the position where the filter paper disc containing the Grindelia extract occurred on the mating plate. This is clearly distinguishable from the low level of diploid colonies which are produced by the mating of the two yeast strains in the absence of a chemical compound that corrects the ΔF508 defect. (See Figure 3. )
As can be seen in Figure 3 the extract reverses the effect of the ΔF508 mutation indicating its potential for further study.
Thus it can be seen the invention accomplishes all of its objectives. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: University of Iowa Research Foundation.
(ii) TITLE OF INVENTION: Method of Assaying for Drugs which Restore Function of CFTR Mutations
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Zarley, McKee, Thomte, Voorhees & Sease
(B) STREET: 801 Grand Ave. Suite 3200
(C) CITY: Des Moines
(D) STATE: Iowa
(E) COUNTRY: US
(F) ZIP: 50309
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vii) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US94/
(B) FILING DATE: 20-APR-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/052,207
(B) FILING DATE: 04-APR-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Nebel, Heidi S.
(B) REGISTRATION NUMBER: 37,719
(C) REFERENCE/DOCKET NUMBER: Uirf 93-50
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 515-288-3667
(B) TELEFAX: 515-288-1338
(2) INFORMATION FOR SEQ ID Nθ:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3840 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Saccharomyces cerevisiae
(ix) FEATURE:
Figure imgf000029_0001
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1317..1488
(D) OTHER INFORMATION: /note= "Substituted analogous sequence from human CFTR gene"
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:
ATG AAC TTT TTA AGT TTT AAG ACT ACA AAA CAC TAT CAC ATT TTC AGG 48 Met Asn Phe Leu Ser Phe Lys Thr Thr Lys His Tyr His lie Phe Arg 1 5 10 15
TAC GTG AAC ATA CGG AAT GAC TAC AGG CTG TTA ATG ATA ATG ATA ATA 96 Tyr Val Asn lie Arg Asn Asp Tyr Arg Leu Leu Met lie Met lie lie 20 25 30
GGT ACC GTG GCA ACA GGC CTA GTG CCG GCA ATT ACT TCT ATC CTG ACG 144 Gly Thr Val Ala Thr Gly Leu Val Pro Ala lie Thr Ser lie Leu Thr 35 40 45
GGC AGA GTG TTC GAT CTA CTA TCA GTT TTC GTG GCT AAT GGG TCA CAT 192 Gly Arg Val Phe Asp Leu Leu Ser Val Phe Val Ala Asn Gly Ser His 50 55 60
CAA GGT TTG TAT TCC CAA CTA GTA CAG AGG TCA ATG GCA GTA ATG GCA 240 Gin Gly Leu Tyr Ser Gin Leu Val Gin Arg Ser Met Ala Val Met Ala 65 70 75 80
CTT GGT GCG GCT TCT GTG CCA GTA ATG TGG CTT TCT CTA ACA AGT TGG 288 Leu Gly Ala Ala Ser Val Pro Val Met Trp Leu Ser Leu Thr Ser Trp 85 90 95
ATG CAC ATC GGC GAG AGA CAA GGC TTT AGA ATA CGG TCA CAG ATA TTG 336 Met His lie Gly Glu Arg Gin Gly Phe Arg lie Arg Ser Gin lie Leu 100 105 110 GAG GCA TAT TTG GAG GAA AAG CCA ATG GAA TGG TAC GAC AAT AAT GAA 384 Glu Ala Tyr Leu Glu Glu Lys Pro Met Glu Trp Tyr Asp Asn Asn Glu 115 120 125
AAA TTG TTA GGA GAT TTT ACT CAA ATC AAC AGA TGT GTG GAA GAG CTA 432 Lys Leu Leu Gly Asp Phe Thr Gin He Asn Arg Cys Val Glu Glu Leu 130 135 140
AGA TCA AGC TCC GCA GAG GCA TCA GCC ATA ACT TTC CAG AAT TTA GTT 480 Arg Ser Ser Ser Ala Glu Ala Ser Ala He Thr Phe Gin Asn Leu Val 145 150 155 160
GCA ATA TGT GCG CTT CTG GGG ACG TCA TTC TAC TAT TCT TGG TCA TTA 528 Ala He Cys Ala Leu Leu Gly Thr Ser Phe Tyr Tyr Ser Trp Ser Leu 165 170 175
ACT TTA ATT ATT CTT TGC AGC TCT CCA ATA ATC ACA TTT TTT GCA GTG 576 Thr Leu He He Leu Cys Ser Ser Pro He He Thr Phe Phe Ala Val 180 185 190
GTG TTT TCC AGA ATG ATT CAT GTA TAT TCA GAG AAG GAG AAT TCT GAA 624 Val Phe Ser Arg Met He His Val Tyr Ser Glu Lys Glu Asn Ser Glu 195 200 205
ACG AGT AAA GCA GCC CAA TTA CTT ACA TGG TCG ATG AAT GCC GCT CAA 672 Thr Ser Lys Ala Ala Gin Leu Leu Thr Trp Ser Met Asn Ala Ala Gin 210 215 220
TTA GTG AGA TTA TAT TGT ACA CAA CGT CTA GAA AGG AAA AAA TTC AAG 720 Leu Val Arg Leu Tyr Cys Thr Gin Arg Leu Glu Arg Lys Lys Phe Lys 225 230 235 240
GAA ATC ATA CTA AAT TGT AAC ACT TTC TTC ATC AAG AGT TGC TTT TTT 768 Glu He He Leu Asn Cys Asn Thr Phe Phe He Lys Ser Cys Phe Phe 245 250 255
GTT GCT GCA AAC GCT GGG ATC TTG AGA TTT TTG ACG TTG ACT ATG TTT 816 Val Ala Ala Asn Ala Gly He Leu Arg Phe Leu Thr Leu Thr Met Phe 260 265 270
GTA CAG GGA TTC TGG TTT GGT TCC GCA ATG ATC AAA AAG GGC AAG CTG 864 Val Gin Gly Phe Trp Phe Gly Ser Ala Met He Lys Lys Gly Lys Leu 275 280 285
AAC ATT AAC GAT GTA ATC ACT TGC TTC CAT TCA TGC ATT ATG CTG GGC 912 Asn He Asn Asp Val He Thr Cys Phe His Ser Cys He Met Leu Gly 290 295 300
TCG ACT TTA AAT AAT ACA TTA CAC CAA ATA GTT GTT CTT CAA AAA GGC 960 Ser Thr Leu Asn Asn Thr Leu His Gin He Val Val Leu Gin Lys Gly 305 310 315 320 GGA GTG GCT ATG GAA AAA ATC ATG ACT CTA TTA AAA GAT GGA TCC AAG 1008 Gly Val Ala Met Glu Lys He Met Thr Leu Leu Lys Asp Gly Ser Lys 325 330 335
CGA AAT CCT TTA AAT AAA ACT GTA GCC CAC CAA TTT CCA CTA GAT TAT 1056 Arg Asn Pro Leu Asn Lys Thr Val Ala His Gin Phe Pro Leu Asp Tyr 340 345 350
GCC ACC AGT GAT CTA ACA TTT GCT AAT GTT TCG TTT TCT TAT CCA AGC 1104 Ala Thr Ser Asp Leu Thr Phe Ala Asn Val Ser Phe Ser Tyr Pro Ser 355 360 365
AGA CCT TCG GAA GCA GTT TTA AAG AAC GTT AGT TTA AAT TTC TCT GCA 1152 Arg Pro Ser Glu Ala Val Leu Lys Asn Val Ser Leu Asn Phe Ser Ala 370 375 380
GGA CAA TTT ACT TTC ATA GTA GGA AAA TCA GGC TCA GGT AAA TCT ACA 1200 Gly Gin Phe Thr Phe He Val Gly Lys Ser Gly Ser Gly Lys Ser Thr 385 390 395 400
TTA TCC AAC TTA TTA TTA AGG TTC TAC GAT GGC TAT AAT GGA TCG ATA 1248 Leu Ser Asn Leu Leu Leu Arg Phe Tyr Asp Gly Tyr Asn Gly Ser He 405 410 415
TCT ATC AAT GGC CAC AAT ATC CAA ACA ATC GAC CAA AAA TTG CTA ATT 1296 Ser He Asn Gly His Asn He Gin Thr He Asp Gin Lys Leu Leu He 420 425 430
GAA AAT ATC ACC GTC GTA GAA CAG TTT TCC TGG ATT ATG CCT GGC ACC 1344 Glu Asn He Thr Val Val Glu Gin Phe Ser Trp He Met Pro Gly Thr 435 440 445
ATT AAA GAA AAT ATC ATC TTT GGT GTT TCC TAT GAT GAA TAT AGA TAC 1392 He Lys Glu Asn He He Phe Gly Val Ser Tyr Asp Glu Tyr Arg Tyr 450 455 460
AGA AGC GTC ATC AAA GCA TGC CAA CTA GAA GAG GAC ATC TCC AAG TTT 1440 Arg Ser Val He Lys Ala Cys Gin Leu Glu Glu Asp He -Ser Lys Phe 465 470 475 480
GCA GAG AAA GAC AAT ATA GTT CTT GGA GAA GGT GGA ATC ACA CTG AGT 1488 Ala Glu Lys Asp Asn He Val Leu Gly Glu Gly Gly He Thr Leu Ser 485 490 495
GGC GGG CAA CAA CAA AGA GTT GCT ATA GCA CGT GCA TTC ATC AGA GAT 1536 Gly Gly Gin Gin Gin Arg Val Ala He Ala Arg Ala Phe He Arg Asp 500 505 510
ACT CCA ATA TTA TTC TTA GAC GAA GCT GTA TCG GCT CTA GAT ATT GTT 1584 Thr Pro He Leu Phe Leu Asp Glu Ala Val Ser Ala Leu Asp He Val 515 520 525 CAT CGC AAC CTG TTG ATG AAG GCA ATT AGG CAT TGG AGG AAA GGA AAG 1632 His Arg Asn Leu Leu Met Lys Ala He Arg His Trp Arg Lys Gly Lys 530 535 540
ACT ACA ATC ATA TTG ACG CAT GAG TTG AGC CAA ATT GAA TCT GAT GAC 1680 Thr Thr He He Leu Thr His Glu Leu Ser Gin He Glu Ser Asp Asp 545 550 555 560
TAT TTA TAT TTA ATG AAG GAA GGT GAA GTT GTT GAA AGC GGC ACC CAG 1728 Tyr Leu Tyr Leu Met Lys Glu Gly Glu Val Val Glu Ser Gly Thr Gin 565 570 575
TCT GAA CTT CTA GCC GAT CCG ACC ACT ACA TTT AGC ACA TGG TAT CAC 1776 Ser Glu Leu Leu Ala Asp Pro Thr Thr Thr Phe Ser Thr Trp Tyr His 580 585 590
CTA CAG AAT GAC TAC TCT GAT GCG AAA ACT ATT GTA GAT ACA GAG ACT 1824 Leu Gin Asn Asp Tyr Ser Asp Ala Lys Thr He Val Asp Thr Glu Thr 595 600 605
GAA GAA AAA TCT ATA CAC ACT GTG GAA AGT TTT AAC TCT CAA TTG GAA 1872 Glu Glu Lys Ser He His Thr Val Glu Ser Phe Asn Ser Gin Leu Glu 610 615 620
ACA CCA AAA CTT GGA TCA TGC TTA AGT AAT CTG GGA TAT GAT GAG ACA 1920 Thr Pro Lys Leu Gly Ser Cys Leu Ser Asn Leu Gly Tyr Asp Glu Thr 625 630 635 640
GAT CAG TTG TCC TTT TAC GAA GCA ATC TAT CAA AAA AGA TCG AAC GTT 1968 Asp Gin Leu Ser Phe Tyr Glu Ala He Tyr Gin Lys Arg Ser Asn Val 645 650 655
AGA ACA AGA AGG GTT AAA GTT GAA GAG GAA AAT ATT GGG TAT GCA CTA 2016 Arg Thr Arg Arg Val Lys Val Glu Glu Glu Asn He Gly Tyr Ala Leu 660 665 670
AAA CAA CAA AAG AAC ACC GAA AGT TCA ACA GGG CCA CAA. CTT CTG AGC 2064 Lys Gin Gin Lys Asn Thr Glu Ser Ser Thr Gly Pro Gin Leu Leu Ser 675 680 685
ATT ATT CAG ATT ATC AAA AGA ATG ATT AAA AGC ATA AGA TAC AAA AAA 2112 He He Gin He He Lys Arg Met He Lys Ser He Arg Tyr Lys Lys 690 695 700
ATT CTA ATC TTG GGA CTG CTA TGT TCT CTT ATC GCA GGC GCC ACA AAT 2160 He Leu He Leu Gly Leu Leu Cys Ser Leu He Ala Gly Ala Thr Asn 705 710 715 720
CCC GTC TTT TCA TAC ACA TTC AGT TTC TTA CTA GAA GGA ATT GTC CCA 2208 Pro Val Phe Ser Tyr Thr Phe Ser Phe Leu Leu Glu Gly He Val Pro 725 730 735 TCC ACG GAT GGA AAA ACT GGC TCT TCA CAT TAT TTG GCG AAA TGG TCG 2256 Ser Thr Asp Gly Lys Thr Gly Ser Ser His Tyr Leu Ala Lys Trp Ser 740 745 750
CTT CTT GTT CTT GGT GTG GCT GCG GCA GAT GGT ATT TTC AAT TTT GCT 2304 Leu Leu Val Leu Gly Val Ala Ala Ala Asp Gly He Phe Asn Phe Ala 755 760 765
AAA GGA TTC CTA TTA GAT TGC TGC AGT GAA TAC TGG GTT ATG GAT CTT 2352 Lys Gly Phe Leu Leu Asp Cys Cys Ser Glu Tyr Trp Val Met Asp Leu 770 775 780
AGA AAT GAA GTT ATG GAA AAA CTG ACG AGA AAG AAT ATG GAC TGG TTT 2400 Arg Asn Glu Val Met Glu Lys Leu Thr Arg Lys Asn Met Asp Trp Phe 785 790 795 800
TCT GGT GAA AAC AAC AAG GCT TCT GAA ATT TCT GCT CTA GTC TTG AAT 2448 Ser Gly Glu Asn Asn Lys Ala Ser Glu He Ser Ala Leu Val Leu Asn 805 810 815
GAT TTG CGA GAT TTG AGG TCT TTG GTC TCT GAA TTT TTG AGT GCA ATG 2496 Asp Leu Arg Asp Leu Arg Ser Leu Val Ser Glu Phe Leu Ser Ala Met 820 825 830
ACT AGT TTC GTT ACC GTA TCA ACG ATT GGA CTA ATT TGG GCG TTA GTA 2544 Thr Ser Phe Val Thr Val Ser Thr He Gly Leu He Trp Ala Leu Val 835 840 845
TCG GGC TGG AAG TTA AGT TTG GTT TGT ATT TCG ATG TTT CCA CTC ATA 2592 Ser Gly Trp Lys Leu Ser Leu Val Cys He Ser Met Phe Pro Leu He 850 855 860
ATT ATA TTT TCA GCA ATA TAT GGA GGC ATT TTA CAA AAA TGC GAA ACA 2640 He He Phe Ser Ala He Tyr Gly Gly He Leu Gin Lys Cys Glu Thr 865 870 875 880
GAT TAT AAG ACA TCT GTT GCT CAG TTA GAA AAC TGC CTG- AC CAG ATT 2688 Asp Tyr Lys Thr Ser Val Ala Gin Leu Glu Asn Cys Leu Tyr Gin He 885 890 895
GTC ACT AAC ATT AAA ACC ATT AAG TGC TTA CAA GCT GAA TTT CAT TTT 2736 Val Thr Asn He Lys Thr He Lys Cys Leu Gin Ala Glu Phe His Phe 900 905 910
CAA TTG ACC TAC CAT GAC TTG AAG ATA AAA ATG CAA CAA ATT GCC TCC 2784 Gin Leu Thr Tyr His Asp Leu Lys He Lys Met Gin Gin He Ala Ser 915 920 925
AAA CGC GCC ATT GCC ACA GGA TTT GGT ATA TCT ATG ACA AAC ATG ATT 2832 Lys Arg Ala He Ala Thr Gly Phe Gly He Ser Met Thr Asn Met He 930 935 940 GTC ATG TGT ATC CAA GCT ATT ATT TAC TAC TAT GGC CTA AAG CTG GTT 2880 Val Met Cys He Gin Ala He He Tyr Tyr Tyr Gly Leu Lys Leu Val 945 950 955 960
ATG ATT CAC GAG TAC ACC TCA AAG GAA ATG TTT ACG ACT TTC ACT TTG 2928 Met He His Glu Tyr Thr Ser Lys Glu Met Phe Thr Thr Phe Thr Leu 965 970 975
TTA TTA TTC ACT ATT ATG TCA TGC ACT AGC CTA GTA AGT CAG ATA CCC 2976 Leu Leu Phe Thr He Met Ser Cys Thr Ser Leu Val Ser Gin He Pro 980 985 990
GAT ATA AGT AGA GGC CAA CGT GCT GCC AGT TGG ATC TAT AGG ATT CTT 3024 Asp He Ser Arg Gly Gin Arg Ala Ala Ser Trp He Tyr Arg He Leu 995 1000 1005
GAT GAA AAG CAT AAT ACC CTA GAG GTT GAA AAC AAT AAT GCT AGA ACA 3072 Asp Glu Lys His Asn Thr Leu Glu Val Glu Asn Asn Asn Ala Arg Thr 1010 1015 1020
GTG GGA ATA GCT GGT CAC ACC TAC CAT GGC AAA GAA AAA AAA CCA ATC 3120 Val Gly He Ala Gly His Thr Tyr His Gly Lys Glu Lys Lys Pro He 1025 1030 1035 1040
GTT TCA ATT CAA AAT TTG ACA TTT GCC TAT CCA TCT GCA CCT ACC GCC 3168 Val Ser He Gin Asn Leu Thr Phe Ala Tyr Pro Ser Ala Pro Thr Ala 1045 1050 1055
TTT GTT TAC AAA AAC ATG AAT TTT GAC ATG TTT TGC GGA CAG ACG TTA 3216 Phe Val Tyr Lys Asn Met Asn Phe Asp Met Phe Cys Gly Gin Thr Leu 1060 1065 1070
GGT ATC ATT GGT GAA TCA GGC ACA GGA AAG TCT ACA CTT GTG CTT TTA 3264 Gly He He Gly Glu Ser Gly Thr Gly Lys Ser Thr Leu Val Leu Leu 1075 1080 1085
TTA ACA AAA CTT TAT AAT TGT GAA GTA GGC AAA ATT AAA ATA GAC GGT 3312 Leu Thr Lys Leu Tyr Asn Cys Glu Val Gly Lys He Lys He Asp Gly 1090 1095 1100
ACG GAC GTA AAT GAC TGG AAT TTG ACA AGT TTA AGA AAA GAA ATT TCA 3360 Thr Asp Val Asn Asp Trp Asn Leu Thr Ser Leu Arg Lys Glu He Ser 1105 1110 1115 1120
GTG GTT GAG CAA AAA CCT TTA TTA TTC AAT GGA ACC ATC AGA GAT AAC 3408 Val Val Glu Gin Lys Pro Leu Leu Phe Asn Gly Thr He Arg Asp Asn 1125 1130 1135
CTA ACT TAT GGT TTA CAA GAT GAA ATA CTT GAA ATT GAA ATG TAT GAT 3456 Leu Thr Tyr Gly Leu Gin Asp Glu He Leu Glu He Glu Met Tyr Asp 1140 1145 1150 GCA TTA AAA TAC GTA GGA ATC CAT GAC TTT GTA ATT TCA TCA CCT CAG 3504 Ala Leu Lys Tyr Val Gly He His Asp Phe Val He Ser Ser Pro Gin 1155 1160 1165
GGC TTG GAT ACA CGT ATT GAT ACA ACT TTA CTG TCA GGT GGA CAA GCG 3552 Gly Leu Asp Thr Arg He Asp Thr Thr Leu Leu Ser Gly Gly Gin Ala 1170 1175 1180
CAA AGG CTT TGC ATA GCC AGA GCA CTT CTG AGA AAA TCA AAA ATT CTG 3600 Gin Arg Leu Cys He Ala Arg Ala Leu Leu Arg Lys Ser Lys He Leu 1185 1190 1195 1200
ATT TTA GAT GAG TGT ACT TCA GCC TTG GAT TCT GTC AGC TCC TCT ATC 3648 He Leu Asp Glu Cys Thr Ser Ala Leu Asp Ser Val Ser Ser Ser He 1205 1210 1215
ATC AAT GAG ATC GTC AAA AAA GGT CCA CCT GCT CTA CTA ACA ATG GTT 3696 He Asn Glu He Val Lys Lys Gly Pro Pro Ala Leu Leu Thr Met Val 1220 1225 1230
ATA ACG CAT AGT GAA CAA ATG ATG AGG TCT TGT AAC TCG ATT GCA GTT 3744 He Thr His Ser Glu Gin Met Met Arg Ser Cys Asn Ser He Ala Val 1235 1240 1245
CTT AAA GAT GGT AAA GTG GTT GAG CGA GGT AAC TTC GAC ACT TTA TAT 3792 Leu Lys Asp Gly Lys Val Val Glu Arg Gly Asn Phe Asp Thr Leu Tyr 1250 1255 1260
AAT AAT CGC GGG GAA TTA TTC CAA ATT GTT TCC AAC CAA AGC AGT TAA 3840 Asn Asn Arg Gly Glu Leu Phe Gin He Val Ser Asn Gin Ser Ser 1265 1270 1275
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1279 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Asn Phe Leu Ser Phe Lys Thr Thr Lys His Tyr His He Phe Arg 1 5 10 15
Tyr Val Asn He Arg Asn Asp Tyr Arg Leu Leu Met He Met He He 20 25 30
Gly Thr Val Ala Thr Gly Leu Val Pro Ala He Thr Ser He Leu Thr 35 40 45 Gly Arg Val Phe Asp Leu Leu Ser Val Phe Val Ala Asn Gly Ser His 50 55 60
Gin Gly Leu Tyr Ser Gin Leu Val Gin Arg Ser Met Ala Val Met Ala 65 70 75 80
Leu Gly Ala Ala Ser Val Pro Val Met Trp Leu Ser Leu Thr Ser Trp 85 90 95
Met His He Gly Glu Arg Gin Gly Phe Arg He Arg Ser Gin He Leu 100 105 110
Glu Ala Tyr Leu Glu Glu Lys Pro Met Glu Trp Tyr Asp Asn Asn Glu 115 120 125
Lys Leu Leu Gly Asp Phe Thr Gin He Asn Arg Cys Val Glu Glu Leu 130 135 140
Arg Ser Ser Ser Ala Glu Ala Ser Ala He Thr Phe Gin Asn Leu Val 145 150 155 160
Ala He Cys Ala Leu Leu Gly Thr Ser Phe Tyr Tyr Ser Trp Ser Leu 165 170 175
Thr Leu He He Leu Cys Ser Ser Pro He He Thr Phe Phe Ala Val 180 185 190
Val Phe Ser Arg Met He His Val Tyr Ser Glu Lys Glu Asn Ser Glu 195 200 205
Thr Ser Lys Ala Ala Gin Leu Leu Thr Trp Ser Met Asn Ala Ala Gin 210 215 220
Leu Val Arg Leu Tyr Cys Thr Gin Arg Leu Glu Arg Lys Lys Phe Lys 225 230 235 240
Glu He He Leu Asn Cys Asn Thr Phe Phe He Lys Ser Cys Phe Phe 245 250 255
Val Ala Ala Asn Ala Gly He Leu Arg Phe Leu Thr Leu Thr Met Phe 260 265 270
Val Gin Gly Phe Trp Phe Gly Ser Ala Met He Lys Lys Gly Lys Leu 275 280 285
Asn He Asn Asp Val He Thr Cys Phe His Ser Cys He Met Leu Gly 290 295 300
Ser Thr Leu Asn Asn Thr Leu His Gin He Val Val Leu Gin Lys Gly 305 310 315 320 Gly Val Ala Met Glu Lys He Met Thr Leu Leu Lys Asp Gly Ser Lys 325 330 335
Arg Asn Pro Leu Asn Lys Thr Val Ala His Gin Phe Pro Leu Asp Tyr 340 345 350
Ala Thr Ser Asp Leu Thr Phe Ala Asn Val Ser Phe Ser Tyr Pro Ser 355 360 365
Arg Pro Ser Glu Ala Val Leu Lys Asn Val Ser Leu Asn Phe Ser Ala 370 375 380
Gly Gin Phe Thr Phe He Val Gly Lys Ser Gly Ser Gly Lys Ser Thr 385 390 395 400
Leu Ser Asn Leu Leu Leu Arg Phe Tyr Asp Gly Tyr Asn Gly Ser He 405 410 415
Ser He Asn Gly His Asn He Gin Thr He Asp Gin Lys Leu Leu He 420 425 430
Glu Asn He Thr Val Val Glu Gin Phe Ser Trp He Met Pro Gly Thr 435 440 445
He Lys Glu Asn He He Phe Gly Val Ser Tyr Asp Glu Tyr Arg Tyr 450 455 460
Arg Ser Val He Lys Ala Cys Gin Leu Glu Glu Asp He Ser Lys Phe 465 470 475 480
Ala Glu Lys Asp Asn He Val Leu Gly Glu Gly Gly He Thr Leu Ser 485 490 495
Gly Gly Gin Gin Gin Arg Val Ala He Ala Arg Ala Phe He Arg Asp 500 505 510
Thr Pro He Leu Phe Leu Asp Glu Ala Val Ser Ala Leu Asp He Val 515 520 ' 525
His Arg Asn Leu Leu Met Lys Ala He Arg His Trp Arg Lys Gly Lys 530 535 540
Thr Thr He He Leu Thr His Glu Leu Ser Gin He Glu Ser Asp Asp 545 550 555 560
Tyr Leu Tyr Leu Met Lys Glu Gly Glu Val Val Glu Ser Gly Thr Gin 565 570 575
Ser Glu Leu Leu Ala Asp Pro Thr Thr Thr Phe Ser Thr Trp Tyr His 580 585 590 Leu Gin Asn Asp Tyr Ser Asp Ala Lys Thr He Val Asp Thr Glu Thr 595 600 605
Glu Glu Lys Ser He His Thr Val Glu Ser Phe Asn Ser Gin Leu Glu 610 615 620
Thr Pro Lys Leu Gly Ser Cys Leu Ser Asn Leu Gly Tyr Asp Glu Thr 625 630 635 640
Asp Gin Leu Ser Phe Tyr Glu Ala He Tyr Gin Lys Arg Ser Asn Val 645 650 655
Arg Thr Arg Arg Val Lys Val Glu Glu Glu Asn He Gly Tyr Ala Leu 660 665 670
Lys Gin Gin Lys Asn Thr Glu Ser Ser Thr Gly Pro Gin Leu Leu Ser 675 680 685
He He Gin He He Lys Arg Met He Lys Ser He Arg Tyr Lys Lys 690 695 700
He Leu He Leu Gly Leu Leu Cys Ser Leu He Ala Gly Ala Thr Asn 705 710 715 720
Pro Val Phe Ser Tyr Thr Phe Ser Phe Leu Leu Glu Gly He Val Pro 725 730 735
Ser Thr Asp Gly Lys Thr Gly Ser Ser His Tyr Leu Ala Lys Trp Ser 740 745 750
Leu Leu Val Leu Gly Val Ala Ala Ala Asp Gly He Phe Asn Phe Ala 755 760 765
Lys Gly Phe Leu Leu Asp Cys Cys Ser Glu Tyr Trp Val Met Asp Leu 770 775 780
Arg Asn Glu Val Met Glu Lys Leu Thr Arg Lys Asn Met Asp Trp Phe 785 790 795 • 800
Ser Gly Glu Asn Asn Lys Ala Ser Glu He Ser Ala Leu Val Leu Asn 805 810 815
Asp Leu Arg Asp Leu Arg Ser Leu Val Ser Glu Phe Leu Ser Ala Met 820 825 830
Thr Ser Phe Val Thr Val Ser Thr He Gly Leu He Trp Ala Leu Val 835 840 845
Ser Gly Trp Lys Leu Ser Leu Val Cys He Ser Met Phe Pro Leu He 850 855 860 He He Phe Ser Ala He Tyr Gly Gly He Leu Gin Lys Cys Glu Thr 865 870 875 880
Asp Tyr Lys Thr Ser Val Ala Gin Leu Glu Asn Cys Leu Tyr Gin He 885 890 895
Val Thr Asn He Lys Thr He Lys Cys Leu Gin Ala Glu Phe His Phe 900 905 910
Gin Leu Thr Tyr His Asp Leu Lys He Lys Met Gin Gin He Ala Ser 915 920 925
Lys Arg Ala He Ala Thr Gly Phe Gly He Ser Met Thr Asn Met He 930 935 940
Val Met Cys He Gin Ala He He Tyr Tyr Tyr Gly Leu Lys Leu Val 945 950 955 960
Met He His Glu Tyr Thr Ser Lys Glu Met Phe Thr Thr Phe Thr Leu 965 970 975
Leu Leu Phe Thr He Met Ser Cys Thr Ser Leu Val Ser Gin He Pro 980 985 990
Asp He Ser Arg Gly Gin Arg Ala Ala Ser Trp He Tyr Arg He Leu 995 1000 1005
Asp Glu Lys His Asn Thr Leu Glu Val Glu Asn Asn Asn Ala Arg Thr 1010 1015 1020
Val Gly He Ala Gly His Thr Tyr His Gly Lys Glu Lys Lys Pro He 1025 1030 1035 1040
Val Ser He Gin Asn Leu Thr Phe Ala Tyr Pro Ser Ala Pro Thr Ala 1045 1050 1055
Phe Val Tyr Lys Asn Met Asn Phe Asp Met Phe Cys Gly Gin Thr Leu 1060 1065 1070
Gly He He Gly Glu Ser Gly Thr Gly Lys Ser Thr Leu Val Leu Leu 1075 1080 1085
Leu Thr Lys Leu Tyr Asn Cys Glu Val Gly Lys He Lys He Asp Gly 1090 1095 1100
Thr Asp Val Asn Asp Trp Asn Leu Thr Ser Leu Arg Lys Glu He Ser 1105 1110 1115 1120
Val Val Glu Gin Lys Pro Leu Leu Phe Asn Gly Thr He Arg Asp Asn 1125 1130 1135 Leu Thr Tyr Gly Leu Gin Asp Glu He Leu Glu He Glu Met Tyr Asp 1140 1145 1150
Ala Leu Lys Tyr Val Gly He His Asp Phe Val He Ser Ser Pro Gin 1155 1160 1165
Gly Leu Asp Thr Arg He Asp Thr Thr Leu Leu Ser Gly Gly Gin Ala 1170 1175 1180
Gin Arg Leu Cys He Ala Arg Ala Leu Leu Arg Lys Ser Lys He Leu 1185 1190 1195 1200
He Leu Asp Glu Cys Thr Ser Ala Leu Asp Ser Val Ser Ser Ser He 1205 1210 1215
He Asn Glu He Val Lys Lys Gly Pro Pro Ala Leu Leu Thr Met Val 1220 1225 1230
He Thr His Ser Glu Gin Met Met Arg Ser Cys Asn Ser He Ala Val 1235 1240 1245
Leu Lys Asp Gly Lys Val Val Glu Arg Gly Asn Phe Asp Thr Leu Tyr 1250 1255 1260
Asn Asn Arg Gly Glu Leu Phe Gin He Val Ser Asn Gin Ser Ser 1265 1270 1275
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3840 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (geno ic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Saccharomyces cerevisiae
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..3840
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1318..1521 (D) OTHER INFORMATION: /note= "Substituted analogous sequence from human CFTR gene"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATG AAC TTT TTA AGT TTT AAG ACT ACA AAA CAC TAT CAC ATT TTC AGG 48 Met Asn Phe Leu Ser Phe Lys Thr Thr Lys His Tyr His He Phe Arg 1 5 10 15
TAC GTG AAC ATA CGG AAT GAC TAC AGG CTG TTA ATG ATA ATG ATA ATA 96 Tyr Val Asn He Arg Asn Asp Tyr Arg Leu Leu Met He Met He He 20 25 30
GGT ACC GTG GCA ACA GGC CTA GTG CCG GCA ATT ACT TCT ATC CTG ACG 144 Gly Thr Val Ala Thr Gly Leu Val Pro Ala He Thr Ser He Leu Thr 35 40 45
GGC AGA GTG TTC GAT CTA CTA TCA GTT TTC GTG GCT AAT GGG TCA CAT 192 Gly Arg Val Phe Asp Leu Leu Ser Val Phe Val Ala Asn Gly Ser His 50 55 60
CAA GGT TTG TAT TCC CAA CTA GTA CAG AGG TCA ATG GCA GTA ATG GCA 240 Gin Gly Leu Tyr Ser Gin Leu Val Gin Arg Ser Met Ala Val Met Ala 65 70 75 80
CTT GGT GCG GCT TCT GTG CCA GTA ATG TGG CTT TCT CTA ACA AGT TGG 288 Leu Gly Ala Ala Ser Val Pro Val Met Trp Leu Ser Leu Thr Ser Trp 85 90 95
ATG CAC ATC GGC GAG AGA CAA GGC TTT AGA ATA CGG TCA CAG ATA TTG 336 Met His He Gly Glu Arg Gin Gly Phe Arg He Arg Ser Gin He Leu 100 105 110
GAG GCA TAT TTG GAG GAA AAG CCA ATG GAA TGG TAC GAC AAT AAT GAA 384 Glu Ala Tyr Leu Glu Glu Lys Pro Met Glu Trp Tyr Asp Asn Asn Glu 115 120 125
AAA TTG TTA GGA GAT TTT ACT CAA ATC AAC AGA TGT GTG GAA GAG CTA 432 Lys Leu Leu Gly Asp Phe Thr Gin He Asn Arg Cys Val Glu Glu Leu 130 135 140
AGA TCA AGC TCC GCA GAG GCA TCA GCC ATA ACT TTC CAG AAT TTA GTT 480 Arg Ser Ser Ser Ala Glu Ala Ser Ala He Thr Phe Gin Asn Leu Val 145 150 155 160
GCA ATA TGT GCG CTT CTG GGG ACG TCA TTC TAC TAT TCT TGG TCA TTA 528 Ala He Cys Ala Leu Leu Gly Thr Ser Phe Tyr Tyr Ser Trp Ser Leu 165 170 175 ACT TTA ATT ATT CTT TGC AGC TCT CCA ATA ATC ACA TTT TTT GCA GTG 576 Thr Leu He He Leu Cys Ser Ser Pro He He Thr Phe Phe Ala Val 180 185 190
GTG TTT TCC AGA ATG ATT CAT GTA TAT TCA GAG AAG GAG AAT TCT GAA 624 Val Phe Ser Arg Met He His Val Tyr Ser Glu Lys Glu Asn Ser Glu 195 200 205
ACG AGT AAA GCA GCC CAA TTA CTT ACA TGG TCG ATG AAT GCC GCT CAA 672 Thr Ser Lys Ala Ala Gin Leu Leu Thr Trp Ser Met Asn Ala Ala Gin 210 215 220
TTA GTG AGA TTA TAT TGT ACA CAA CGT CTA GAA AGG AAA AAA TTC AAG 720 Leu Val Arg Leu Tyr Cys Thr Gin Arg Leu Glu Arg Lys Lys Phe Lys 225 230 235 240
GAA ATC ATA CTA AAT TGT AAC ACT TTC TTC ATC AAG AGT TGC TTT TTT 768 Glu He He Leu Asn Cys Asn Thr Phe Phe He Lys Ser Cys Phe Phe 245 250 255
GTT GCT GCA AAC GCT GGG ATC TTG AGA TTT TTG ACG TTG ACT ATG TTT 816 Val Ala Ala Asn Ala Gly He Leu Arg Phe Leu Thr Leu Thr Met Phe 260 265 270
GTA CAG GGA TTC TGG TTT GGT TCC GCA ATG ATC AAA AAG GGC AAG CTG 864 Val Gin Gly Phe Trp Phe Gly Ser Ala Met He Lys Lys Gly Lys Leu 275 280 285
AAC ATT AAC GAT GTA ATC ACT TGC TTC CAT TCA TGC ATT ATG CTG GGC 912 Asn He Asn Asp Val He Thr Cys Phe His Ser Cys He Met Leu Gly 290 295 300
TCG ACT TTA AAT AAT ACA TTA CAC CAA ATA GTT GTT CTT CAA AAA GGC 960 Ser Thr Leu Asn Asn Thr Leu His Gin He Val Val Leu Gin Lys Gly 305 310 315 320
GGA GTG GCT ATG GAA AAA ATC ATG ACT CTA TTA AAA GAT GGA TCC AAG 1008 Gly Val Ala Met Glu Lys He Met Thr Leu Leu Lys Asp Gly Ser Lys 325 330 ' 335
CGA AAT CCT TTA AAT AAA ACT GTA GCC CAC CAA TTT CCA CTA GAT TAT 1056 Arg Asn Pro Leu Asn Lys Thr Val Ala His Gin Phe Pro Leu Asp Tyr 340 345 350
GCC ACC AGT GAT CTA ACA TTT GCT AAT GTT TCG TTT TCT TAT CCA AGC 1104 Ala Thr Ser Asp Leu Thr Phe Ala Asn Val Ser Phe Ser Tyr Pro Ser 355 360 365
AGA CCT TCG GAA GCA GTT TTA AAG AAC GTT AGT TTA AAT TTC TCT GCA 1152 Arg Pro Ser Glu Ala Val Leu Lys Asn Val Ser Leu Asn Phe Ser Ala 370 375 380 GGA CAA TTT ACT TTC ATA GTA GGA AAA TCA GGC TCA GGT AAA TCT ACA 1200 Gly Gin Phe Thr Phe He Val Gly Lys Ser Gly Ser Gly Lys Ser Thr 385 390 395 400
TTA TCC AAC TTA TTA TTA AGG TTC TAC GAT GGC TAT AAT GGA TCG ATA 1248 Leu Ser Asn Leu Leu Leu Arg Phe Tyr Asp Gly Tyr Asn Gly Ser He 405 410 415
TCT ATC AAT GGC CAC AAT ATC CAA ACA ATC GAC CAA AAA TTG CTA ATT 1296 Ser He Asn Gly His Asn He Gin Thr He Asp Gin Lys Leu Leu He 420 425 430
GAA AAT ATC ACC GTC GTA GAA CAG TTT TCC TGG ATT ATG CCT GGC ACC 1344 Glu Asn He Thr Val Val Glu Gin Phe Ser Trp He Met Pro Gly Thr 435 440 445
ATT AAA GAA AAT ATC ATC TTT GGT GTT TCC TAT GAT GAA TAT AGA TAC 1392 He Lys Glu Asn He He Phe Gly Val Ser Tyr Asp Glu Tyr Arg Tyr 450 455 460
AGA AGC GTC ATC AAA GCA TGC CAA CTA GAA GAG GAC ATC TCC AAG TTT 1440 Arg Ser Val He Lys Ala Cys Gin Leu Glu Glu Asp He Ser Lys Phe 465 470 475 480
GCA GAG AAA GAC AAT ATA GTT CTT GGA GAA GGT GGA ATC ACA CTG AGT 1488 Ala Glu Lys Asp Asn He Val Leu Gly Glu Gly Gly He Thr Leu Ser 485 490 495
GGA GGT CAA CGA GCA AGA ATT TCT TTA GCA AGA GCA TTC ATC AGA GAT 1536 Gly Gly Gin Arg Ala Arg He Ser Leu Ala Arg Ala Phe He Arg Asp 500 505 510
ACT CCA ATA TTA TTC TTA GAC GAA GCT GTA TCG GCT CTA GAT ATT GTT 1584 Thr Pro He Leu Phe Leu Asp Glu Ala Val Ser Ala Leu Asp He Val 515 520 525
CAT CGC AAC CTG TTG ATG AAG GCA ATT AGG CAT TGG AGG AAA GGA AAG 1632 His Arg Asn Leu Leu Met Lys Ala He Arg His Trp Arg Lys Gly Lys 530 535 540
ACT ACA ATC ATA TTG ACG CAT GAG TTG AGC CAA ATT GAA TCT GAT GAC 1680 Thr Thr He He Leu Thr His Glu Leu Ser Gin He Glu Ser Asp Asp 545 550 555 560
TAT TTA TAT TTA ATG AAG GAA GGT GAA GTT GTT GAA AGC GGC ACC CAG 1728 Tyr Leu Tyr Leu Met Lys Glu Gly Glu Val Val Glu Ser Gly Thr Gin 565 570 575
TCT GAA CTT CTA GCC GAT CCG ACC ACT ACA TTT AGC ACA TGG TAT CAC 1776 Ser Glu Leu Leu Ala Asp Pro Thr Thr Thr Phe Ser Thr Trp Tyr His 580 585 590 CTA CAG AAT GAC TAC TCT GAT GCG AAA ACT ATT GTA GAT ACA GAG ACT 1824 Leu Gin Asn Asp Tyr Ser Asp Ala Lys Thr He Val Asp Thr Glu Thr 595 600 605
GAA GAA AAA TCT ATA CAC ACT GTG GAA AGT TTT AAC TCT CAA TTG GAA 1872 Glu Glu Lys Ser He His Thr Val Glu Ser Phe Asn Ser Gin Leu Glu 610 615 620
ACA CCA AAA CTT GGA TCA TGC TTA AGT AAT CTG GGA TAT GAT GAG ACA 1920 Thr Pro Lys Leu Gly Ser Cys Leu Ser Asn Leu Gly Tyr Asp Glu Thr 625 630 635 640
GAT CAG TTG TCC TTT TAC GAA GCA ATC TAT CAA AAA AGA TCG AAC GTT 1968 Asp Gin Leu Ser Phe Tyr Glu Ala He Tyr Gin Lys Arg Ser Asn Val 645 650 655
AGA ACA AGA AGG GTT AAA GTT GAA GAG GAA AAT ATT GGG TAT GCA CTA 2016 Arg Thr Arg Arg Val Lys Val Glu Glu Glu Asn He Gly Tyr Ala Leu 660 665 670
AAA CAA CAA AAG AAC ACC GAA AGT TCA ACA GGG CCA CAA CTT CTG AGC 2064 Lys Gin Gin Lys Asn Thr Glu Ser Ser Thr Gly Pro Gin Leu Leu Ser 675 680 685
ATT ATT CAG ATT ATC AAA AGA ATG ATT AAA AGC ATA AGA TAC AAA AAA 2112 He He Gin He He Lys Arg Met He Lys Ser He Arg Tyr Lys Lys 690 695 700
ATT CTA ATC TTG GGA CTG CTA TGT TCT CTT ATC GCA GGC GCC ACA AAT 2160 He Leu He Leu Gly Leu Leu Cys Ser Leu He Ala Gly Ala Thr Asn 705 710 715 720
CCC GTC TTT TCA TAC ACA TTC AGT TTC TTA CTA GAA GGA ATT GTC CCA 2208 Pro Val Phe Ser Tyr Thr Phe Ser Phe Leu Leu Glu Gly He Val Pro 725 730 735
TCC ACG GAT GGA AAA ACT GGC TCT TCA CAT TAT TTG GCG AAA TGG TCG 2256 Ser Thr Asp Gly Lys Thr Gly Ser Ser His Tyr Leu Ala Lys Trp Ser 740 745 ' 750
CTT CTT GTT CTT GGT GTG GCT GCG GCA GAT GGT ATT TTC AAT TTT GCT 2304 Leu Leu Val Leu Gly Val Ala Ala Ala Asp Gly He Phe Asn Phe Ala 755 760 765
AAA GGA TTC CTA TTA GAT TGC TGC AGT GAA TAC TGG GTT ATG GAT CTT 2352 Lys Gly Phe Leu Leu Asp Cys Cys Ser Glu Tyr Trp Val Met Asp Leu 770 775 780
AGA AAT GAA GTT ATG GAA AAA CTG ACG AGA AAG AAT ATG GAC TGG TTT 2400 Arg Asn Glu Val Met Glu Lys Leu Thr Arg Lys Asn Met Asp Trp Phe 785 790 795 800 TCT GGT GAA AAC AAC AAG GCT TCT GAA ATT TCT GCT CTA GTC TTG AAT 2448 Ser Gly Glu Asn Asn Lys Ala Ser Glu He Ser Ala Leu Val Leu Asn 805 810 815
GAT TTG CGA GAT TTG AGG TCT TTG GTC TCT GAA TTT TTG AGT GCA ATG 2496 Asp Leu Arg Asp Leu Arg Ser Leu Val Ser Glu Phe Leu Ser Ala Met 820 825 830
ACT AGT TTC GTT ACC GTA TCA ACG ATT GGA CTA ATT TGG GCG TTA GTA 2544 Thr Ser Phe Val Thr Val Ser Thr He Gly Leu He Trp Ala Leu Val 835 840 845
TCG GGC TGG AAG TTA AGT TTG GTT TGT ATT TCG ATG TTT CCA CTC ATA 2592 Ser Gly Trp Lys Leu Ser Leu Val Cys He Ser Met Phe Pro Leu He 850 855 860
ATT ATA TTT TCA GCA ATA TAT GGA GGC ATT TTA CAA AAA TGC GAA ACA 2640 He He Phe Ser Ala He Tyr Gly Gly He Leu Gin Lys Cys Glu Thr 865 870 875 880
GAT TAT AAG ACA TCT GTT GCT CAG TTA GAA AAC TGC CTG TAC CAG ATT 2688 Asp Tyr Lys Thr Ser Val Ala Gin Leu Glu Asn Cys Leu Tyr Gin He 885 890 895
GTC ACT AAC ATT AAA ACC ATT AAG TGC TTA CAA GCT GAA TTT CAT TTT 2736 Val Thr Asn He Lys Thr He Lys Cys Leu Gin Ala Glu Phe His Phe 900 905 910
CAA TTG ACC TAC CAT GAC TTG AAG ATA AAA ATG CAA CAA ATT GCC TCC 2784 Gin Leu Thr Tyr His Asp Leu Lys He Lys Met Gin Gin He Ala Ser 915 920 925
AAA CGC GCC ATT GCC ACA GGA TTT GGT ATA TCT ATG ACA AAC ATG ATT 2832 Lys Arg Ala He Ala Thr Gly Phe Gly He Ser Met Thr Asn Met He 930 935 940
GTC ATG TGT ATC CAA GCT ATT ATT TAC TAC TAT GGC CTA AAG CTG GTT 2880 Val Met Cys He Gin Ala He He Tyr Tyr Tyr Gly Leu Lys Leu Val 945 950 955 ' 960
ATG ATT CAC GAG TAC ACC TCA AAG GAA ATG TTT ACG ACT TTC ACT TTG 2928 Met He His Glu Tyr Thr Ser Lys Glu Met Phe Thr Thr Phe Thr Leu 965 970 975
TTA TTA TTC ACT ATT ATG TCA TGC ACT AGC CTA GTA AGT CAG ATA CCC 2976 Leu Leu Phe Thr He Met Ser Cys Thr Ser Leu Val Ser Gin He Pro 980 985 990
GAT ATA AGT AGA GGC CAA CGT GCT GCC AGT TGG ATC TAT AGG ATT CTT 3024 Asp He Ser Arg Gly Gin Arg Ala Ala Ser Trp He Tyr Arg He Leu 995 1000 1005 GAT GAA AAG CAT AAT ACC CTA GAG GTT GAA AAC AAT AAT GCT AGA ACA 3072 Asp Glu Lys His Asn Thr Leu Glu Val Glu Asn Asn Asn Ala Arg Thr 1010 1015 1020
GTG GGA ATA GCT GGT CAC ACC TAC CAT GGC AAA GAA AAA AAA CCA ATC 3120 Val Gly He Ala Gly His Thr Tyr His Gly Lys Glu Lys Lys Pro He 1025 1030 1035 1040
GTT TCA ATT CAA AAT TTG ACA TTT GCC TAT CCA TCT GCA CCT ACC GCC 3168 Val Ser He Gin Asn Leu Thr Phe Ala Tyr Pro Ser Ala Pro Thr Ala 1045 1050 1055
TTT GTT TAC AAA AAC ATG AAT TTT GAC ATG TTT TGC GGA CAG ACG TTA 3216 Phe Val Tyr Lys Asn Met Asn Phe Asp Met Phe Cys Gly Gin Thr Leu 1060 1065 1070
GGT ATC ATT GGT GAA TCA GGC ACA GGA AAG TCT ACA CTT GTG CTT TTA 3264 Gly He He Gly Glu Ser Gly Thr Gly Lys Ser Thr Leu Val Leu Leu 1075 1080 1085
TTA ACA AAA CTT TAT AAT TGT GAA GTA GGC AAA ATT AAA ATA GAC GGT 3312 Leu Thr Lys Leu Tyr Asn Cys Glu Val Gly Lys He Lys He Asp Gly 1090 1095 1100
ACG GAC GTA AAT GAC TGG AAT TTG ACA AGT TTA AGA AAA GAA ATT TCA 3360 Thr Asp Val Asn Asp Trp Asn Leu Thr Ser Leu Arg Lys Glu He Ser 1105 1110 1115 1120
GTG GTT GAG CAA AAA CCT TTA TTA TTC AAT GGA ACC ATC AGA GAT AAC 3408 Val Val Glu Gin Lys Pro Leu Leu Phe Asn Gly Thr He Arg Asp Asn 1125 1130 1135
CTA ACT TAT GGT TTA CAA GAT GAA ATA CTT GAA ATT GAA ATG TAT GAT 3456 Leu Thr Tyr Gly Leu Gin Asp Glu He Leu Glu He Glu Met Tyr Asp 1140 1145 1150
GCA TTA AAA TAC GTA GGA ATC CAT GAC TTT GTA ATT TCA TCA CCT CAG 3504 Ala Leu Lys Tyr Val Gly He His Asp Phe Val He Ser Ser Pro Gin 1155 1160 1165
GGC TTG GAT ACA CGT ATT GAT ACA ACT TTA CTG TCA GGT GGA CAA GCG 3552 Gly Leu Asp Thr Arg He Asp Thr Thr Leu Leu Ser Gly Gly Gin Ala 1170 1175 1180
CAA AGG CTT TGC ATA GCC AGA GCA CTT CTG AGA AAA TCA AAA ATT CTG 3600 Gin Arg Leu Cys He Ala Arg Ala Leu Leu Arg Lys Ser Lys He Leu 1185 1190 1195 1200
ATT TTA GAT GAG TGT ACT TCA GCC TTG GAT TCT GTC AGC TCC TCT ATC 3648 He Leu Asp Glu Cys Thr Ser Ala Leu Asp Ser Val Ser Ser Ser He 1205 1210 1215 ATC AAT GAG ATC GTC AAA AAA GGT CCA CCT GCT CTA CTA ACA ATG GTT 3696 He Asn Glu He Val Lys Lys Gly Pro Pro Ala Leu Leu Thr Met Val 1220 1225 1230
ATA ACG CAT AGT GAA CAA ATG ATG AGG TCT TGT AAC TCG ATT GCA GTT 3744 He Thr His Ser Glu Gin Met Met Arg Ser Cys Asn Ser He Ala Val 1235 1240 1245
CTT AAA GAT GGT AAA GTG GTT GAG CGA GGT AAC TTC GAC ACT TTA TAT 3792 Leu Lys Asp Gly Lys Val Val Glu Arg Gly Asn Phe Asp Thr Leu Tyr 1250 1255 1260
AAT AAT CGC GGG GAA TTA TTC CAA ATT GTT TCC AAC CAA AGC AGT TAA 3840 Asn Asn Arg Gly Glu Leu Phe Gin He Val Ser Asn Gin Ser Ser 1265 1270 1275
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1279 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Asn Phe Leu Ser Phe Lys Thr Thr Lys His Tyr His He Phe Arg 1 5 10 15
Tyr Val Asn He Arg Asn Asp Tyr Arg Leu Leu Met He Met He He 20 25 30
Gly Thr Val Ala Thr Gly Leu Val Pro Ala He Thr Ser He Leu Thr 35 40 45
Gly Arg Val Phe Asp Leu Leu Ser Val Phe Val Ala Asn Gly Ser His 50 55 60
Gin Gly Leu Tyr Ser Gin Leu Val Gin Arg Ser Met Ala Val Met Ala 65 70 75 80
Leu Gly Ala Ala Ser Val Pro Val Met Trp Leu Ser Leu Thr Ser Trp 85 90 95
Met His He Gly Glu Arg Gin Gly Phe Arg He Arg Ser Gin He Leu 100 105 110
Glu Ala Tyr Leu Glu Glu Lys Pro Met Glu Trp Tyr Asp Asn Asn Glu 115 120 125 Lys Leu Leu Gly Asp Phe Thr Gin He Asn Arg Cys Val Glu Glu Leu 130 135 140
Arg Ser Ser Ser Ala Glu Ala Ser Ala He Thr Phe Gin Asn Leu Val 145 150 155 160
Ala He Cys Ala Leu Leu Gly Thr Ser Phe Tyr Tyr Ser Trp Ser Leu 165 170 175
Thr Leu He He Leu Cys Ser Ser Pro He He Thr Phe Phe Ala Val 180 185 190
Val Phe Ser Arg Met He His Val Tyr Ser Glu Lys Glu Asn Ser Glu 195 200 205
Thr Ser Lys Ala Ala Gin Leu Leu Thr Trp Ser Met Asn Ala Ala Gin 210 215 220
Leu Val Arg Leu Tyr Cys Thr Gin Arg Leu Glu Arg Lys Lys Phe Lys 225 230 235 240
Glu He He Leu Asn Cys Asn Thr Phe Phe He Lys Ser Cys Phe Phe 245 250 255
Val Ala Ala Asn Ala Gly He Leu Arg Phe Leu Thr Leu Thr Met Phe 260 265 270
Val Gin Gly Phe Trp Phe Gly Ser Ala Met He Lys Lys Gly Lys Leu 275 280 285
Asn He Asn Asp Val He Thr Cys Phe His Ser Cys He Met Leu Gly 290 ' 295 300
Ser Thr Leu Asn Asn Thr Leu His Gin He Val Val Leu Gin Lys Gly 305 310 315 320
Gly Val Ala Met Glu Lys He Met Thr Leu Leu Lys Asp Gly Ser Lys 325 330 335
Arg Asn Pro Leu Asn Lys Thr Val Ala His Gin Phe Pro Leu Asp Tyr 340 345 350
Ala Thr Ser Asp Leu Thr Phe Ala Asn Val Ser Phe Ser Tyr Pro Ser 355 360 365
Arg Pro Ser Glu Ala Val Leu Lys Asn Val Ser Leu Asn Phe Ser Ala 370 375 380
Gly Gin Phe Thr Phe He Val Gly Lys Ser Gly Ser Gly Lys Ser Thr 385 390 395 400 Leu Ser Asn Leu Leu Leu Arg Phe Tyr Asp Gly Tyr Asn Gly Ser He 405 410 415
Ser He Asn Gly His Asn He Gin Thr He Asp Gin Lys Leu Leu He 420 425 430
Glu Asn He Thr Val Val Glu Gin Phe Ser Trp He Met Pro Gly Thr 435 440 445
He Lys Glu Asn He He Phe Gly Val Ser Tyr Asp Glu Tyr Arg Tyr 450 455 460
Arg Ser Val He Lys Ala Cys Gin Leu Glu Glu Asp He Ser Lys Phe 465 470 475 480
Ala Glu Lys Asp Asn He Val Leu Gly Glu Gly Gly He Thr Leu Ser 485 490 495
Gly Gly Gin Arg Ala Arg He Ser Leu Ala Arg Ala Phe He Arg Asp 500 505 510
Thr Pro He Leu Phe Leu Asp Glu Ala Val Ser Ala Leu Asp He Val 515 520 525
His Arg Asn Leu Leu Met Lys Ala He Arg His Trp Arg Lys Gly Lys 530 535 540
Thr Thr He He Leu Thr His Glu Leu Ser Gin He Glu Ser Asp Asp 545 550 555 560
Tyr Leu Tyr Leu Met Lys Glu Gly Glu Val Val Glu Ser Gly Thr Gin 565 570 575
Ser Glu Leu Leu Ala Asp Pro Thr Thr Thr Phe Ser Thr Trp Tyr His 580 585 590
Leu Gin Asn Asp Tyr Ser Asp Ala Lys Thr He Val Asp Thr Glu Thr 595 600 605
Glu Glu Lys Ser He His Thr Val Glu Ser Phe Asn Ser Gin Leu Glu 610 615 620
Thr Pro Lys Leu Gly Ser Cys Leu Ser Asn Leu Gly Tyr Asp Glu Thr 625 630 635 640
Asp Gin Leu Ser Phe Tyr Glu Ala He Tyr Gin Lys Arg Ser Asn Val 645 650 655
Arg Thr Arg Arg Val Lys Val Glu Glu Glu Asn He Gly Tyr Ala Leu 660 665 670 Lys Gin Gin Lys Asn Thr Glu Ser Ser Thr Gly Pro Gin Leu Leu Ser 675 680 685
He He Gin He He Lys Arg Met He Lys Ser He Arg Tyr Lys Lys 690 695 700
He Leu He Leu Gly Leu Leu Cys Ser Leu He Ala Gly Ala Thr Asn 705 710 715 720
Pro Val Phe Ser Tyr Thr Phe Ser Phe Leu Leu Glu Gly He Val Pro 725 730 735
Ser Thr Asp Gly Lys Thr Gly Ser Ser His Tyr Leu Ala Lys Trp Ser 740 745 750
Leu Leu Val Leu Gly Val Ala Ala Ala Asp Gly He Phe Asn Phe Ala 755 760 765
Lys Gly Phe Leu Leu Asp Cys Cys Ser Glu Tyr Trp Val Met Asp Leu 770 775 780
Arg Asn Glu Val Met Glu Lys Leu Thr Arg Lys Asn Met Asp Trp Phe 785 790 795 800
Ser Gly Glu Asn Asn Lys Ala Ser Glu He Ser Ala Leu Val Leu Asn 805 810 815
Asp Leu Arg Asp Leu Arg Ser Leu Val Ser Glu Phe Leu Ser Ala Met 820 825 830
Thr Ser Phe Val Thr Val Ser Thr He Gly Leu He Trp Ala Leu Val 835 840 845
Ser Gly Trp Lys Leu Ser Leu Val Cys He Ser Met Phe Pro Leu He 850 855 860
He He Phe Ser Ala He Tyr Gly Gly He Leu Gin Lys Cys Glu Thr 865 870 875 880
Asp Tyr Lys Thr Ser Val Ala Gin Leu Glu Asn Cys Leu Tyr Gin He 885 890 895
Val Thr Asn He Lys Thr He Lys Cys Leu Gin Ala Glu Phe His Phe 900 905 910
Gin Leu Thr Tyr His Asp Leu Lys He Lys Met Gin Gin He Ala Ser 915 920 925
Lys Arg Ala He Ala Thr Gly Phe Gly He Ser Met Thr Asn Met He 930 935 940 Val Met Cys He Gin Ala He He Tyr Tyr Tyr Gly Leu Lys Leu Val 945 950 955 960
Met He His Glu Tyr Thr Ser Lys Glu Met Phe Thr Thr Phe Thr Leu 965 970 975
Leu Leu Phe Thr He Met Ser Cys Thr Ser Leu Val Ser Gin He Pro 980 985 990
Asp He Ser Arg Gly Gin Arg Ala Ala Ser Trp He Tyr Arg He Leu 995 1000 1005
Asp Glu Lys His Asn Thr Leu Glu Val Glu Asn Asn Asn Ala Arg Thr 1010 1015 1020
Val Gly He Ala Gly His Thr Tyr His Gly Lys Glu Lys Lys Pro He 1025 1030 1035 1040
Val Ser He Gin Asn Leu Thr Phe Ala Tyr Pro Ser Ala Pro Thr Ala 1045 1050 1055
Phe Val Tyr Lys Asn Met Asn Phe Asp Met Phe Cys Gly Gin Thr Leu 1060 1065 1070
Gly He He Gly Glu Ser Gly Thr Gly Lys Ser Thr Leu Val Leu Leu 1075 1080 1085
Leu Thr Lys Leu Tyr Asn Cys Glu Val Gly Lys He Lys He Asp Gly 1090 1095 1100
Thr Asp Val Asn Asp Trp Asn Leu Thr Ser Leu Arg Lys Glu He Ser 1105 1110 1115 1120
Val Val Glu Gin Lys Pro Leu Leu Phe Asn Gly Thr He Arg Asp Asn 1125 1130 1135
Leu Thr Tyr Gly Leu Gin Asp Glu He Leu Glu He Glu Met Tyr Asp 1140 1145 1150
Ala Leu Lys Tyr Val Gly He His Asp Phe Val He Ser Ser Pro Gin 1155 1160 1165
Gly Leu Asp Thr Arg He Asp Thr Thr Leu Leu Ser Gly Gly Gin Ala 1170 1175 1180
Gin Arg Leu Cys He Ala Arg Ala Leu Leu Arg Lys Ser Lys He Leu 1185 1190 1195 1200
He Leu Asp Glu Cys Thr Ser Ala Leu Asp Ser Val Ser Ser Ser He 1205 1210 1215 He Asn Glu He Val Lys Lys Gly Pro Pro Ala Leu Leu Thr Met Val 1220 1225 1230
He Thr His Ser Glu Gin Met Met Arg Ser Cys Asn Ser He Ala Val 1235 1240 1245
Leu Lys Asp Gly Lys Val Val Glu Arg Gly Asn Phe Asp Thr Leu Tyr 1250 1255 1260
Asn Asn Arg Gly Glu Leu Phe Gin He Val Ser Asn Gin Ser Ser 1265 1270 1275
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3786 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Saccharomyces cerevisiae
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..3786
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1264..1524
(D) OTHER INFORMATION: /note= "Substituted analogous sequence from human CFTR gene"-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATG AAC TTT TTA AGT TTT AAG ACT ACA AAA CAC TAT CAC ATT TTC AGG 48 Met Asn Phe Leu Ser Phe Lys Thr Thr Lys His Tyr His He Phe Arg 1 5 10 15
TAC GTG AAC ATA CGG AAT GAC TAC AGG CTG TTA ATG ATA ATG ATA ATA 96 Tyr Val Asn He Arg Asn Asp Tyr Arg Leu Leu Met He Met He He 20 25 30 GGT ACC GTG GCA CTA TCA GTT TTC GTG GCT AAT GGG TCA CAT CAA GGT 144 Gly Thr Val Ala Leu Ser Val Phe Val Ala Asn Gly Ser His Gin Gly 35 40 45
TTG TAT TCC CAA CTA GTA CAG AGG TCA ATG GCA GTA ATG GCA CTT GGT 192 Leu Tyr Ser Gin Leu Val Gin Arg Ser Met Ala Val Met Ala Leu Gly 50 55 60
GCG GCT TCT GTG CCA GTA ATG TGG CTT TCT CTA ACA AGT TGG ATG CAC 240 Ala Ala Ser Val Pro Val Met Trp Leu Ser Leu Thr Ser Trp Met His 65 70 75 80
ATC GGC GAG AGA CAA GGC TTT AGA ATA CGG TCA CAG ATA TTG GAG GCA 288 He Gly Glu Arg Gin Gly Phe Arg He Arg Ser Gin He Leu Glu Ala 85 90 95
TAT TTG GAG GAA AAG CCA ATG GAA TGG TAC GAC AAT AAT GAA AAA TTG 336 Tyr Leu Glu Glu Lys Pro Met Glu Trp Tyr Asp Asn Asn Glu Lys Leu 100 105 110
TTA GGA GAT TTT ACT CAA ATC AAC AGA TGT GTG GAA GAG CTA AGA TCA 384 Leu Gly Asp Phe Thr Gin He Asn Arg Cys Val Glu Glu Leu Arg Ser 115 120 125
AGC TCC GCA GAG GCA TCA GCC ATA ACT TTC CAG AAT TTA GTT GCA ATA 432 Ser Ser Ala Glu Ala Ser Ala He Thr Phe Gin Asn Leu Val Ala He 130 135 140
TGT GCG CTT CTG GGG ACG TCA TTC TAC TAT TCT TGG TCA TTA ACT TTA 480 Cys Ala Leu Leu Gly Thr Ser Phe Tyr Tyr Ser Trp Ser Leu Thr Leu 145 150 155 160
ATT ATT CTT TGC AGC TCT CCA ATA ATC ACA TTT TTT GCA GTG GTG TTT 528 He He Leu Cys Ser Ser Pro He He Thr Phe Phe Ala Val Val Phe 165 170 175
TCC AGA ATG ATT CAT GTA TAT TCA GAG AAG GAG AAT TCT GAA ACG AGT 576 Ser Arg Met He His Val Tyr Ser Glu Lys Glu Asn Ser Glu Thr Ser 180 185 _ 190
AAA GCA GCC CAA TTA CTT ACA TGG TCG ATG AAT GCC GCT CAA TTA GTG 624 Lys Ala Ala Gin Leu Leu Thr Trp Ser Met Asn Ala Ala Gin Leu Val 195 200 205
AGA TTA TAT TGT ACA CAA CGT CTA GAA AGG AAA AAA TTC AAG GAA ATC 672 Arg Leu Tyr Cys Thr Gin Arg Leu Glu Arg Lys Lys Phe Lys Glu He 210 215 220
ATA CTA AAT TGT AAC ACT TTC TTC ATC AAG AGT TGC TTT TTT GTT GCT 720 He Leu Asn Cys Asn Thr Phe Phe He Lys Ser Cys Phe Phe Val Ala 225 230 235 240 GCA AAC GCT GGG ATC TTG AGA TTT TTG ACG TTG ACT ATG TTT GTA CAG 768 Ala Asn Ala Gly He Leu Arg Phe Leu Thr Leu Thr Met Phe Val Gin 245 250 255
GGA TTC TGG TTT GGT TCC GCA ATG ATC AAA AAG GGC AAG CTG AAC ATT 816 Gly Phe Trp Phe Gly Ser Ala Met He Lys Lys Gly Lys Leu Asn He 260 265 270
AAC GAT GTA ATC ACT TGC TTC CAT TCA TGC ATT ATG CTG GGC TCG ACT 864 Asn Asp Val He Thr Cys Phe His Ser Cys He Met Leu Gly Ser Thr 275 280 285
TTA AAT AAT ACA TTA CAC CAA ATA GTT GTT CTT CAA AAA GGC GGA GTG 912 Leu Asn Asn Thr Leu His Gin He Val Val Leu Gin Lys Gly Gly Val 290 295 300
GCT ATG GAA AAA ATC ATG ACT CTA TTA AAA GAT GGA TCC AAG CGA AAT 960 Ala Met Glu Lys He Met Thr Leu Leu Lys Asp Gly Ser Lys Arg Asn 305 310 315 320
CCT TTA AAT AAA ACT GTA GCC CAC CAA TTT CCA CTA GAT TAT GCC ACC 1008 Pro Leu Asn Lys Thr Val Ala His Gin Phe Pro Leu Asp Tyr Ala Thr 325 330 335
AGT GAT CTA ACA TTT GCT AAT GTT TCG TTT TCT TAT CCA AGC AGA CCT 1056 Ser Asp Leu Thr Phe Ala Asn Val Ser Phe Ser Tyr Pro Ser Arg Pro 340 345 350
TCG GAA GCA GTT TTA AAG AAC GTT AGT TTA AAT TTC TCT GCA GGA CAA 1104 Ser Glu Ala Val Leu Lys Asn Val Ser Leu Asn Phe Ser Ala Gly Gin 355 360 365
TTT ACT TTC ATA GTA GGA AAA TCA GGC TCA GGT AAA TCT ACA TTA TCC 1152 Phe Thr Phe He Val Gly Lys Ser Gly Ser Gly Lys Ser Thr Leu Ser 370 375 380
AAC TTA TTA TTA AGG TTC TAC GAT GGC TAT AAT GGA TCG ATA TCT ATC 1200 Asn Leu Leu Leu Arg Phe Tyr Asp Gly Tyr Asn Gly Ser He Ser He 385 390 395 400
AAT GGC CAC AAT ATC CAA ACA ATC GAC CAA AAA TTG CTA ATT GAA AAT 1248 Asn Gly His Asn He Gin Thr He Asp Gin Lys Leu Leu He Glu Asn 405 410 415
ATC ACC GTC GTA GAA CAG TTT TCC TGG ATT ATG CCT GGC ACC ATT AAA 1296 He Thr Val Val Glu Gin Phe Ser Trp He Met Pro Gly Thr He Lys 420 425 430
GAA AAT ATC ATC TTT GGT GTT TCC TAT GAT GAA TAT AGA TAC AGA AGC 1344 Glu Asn He He Phe Gly Val Ser Tyr Asp Glu Tyr Arg Tyr Arg Ser 435 440 445 GTC ATC AAA GCA TGC CAA CTA GAA GAG GAC ATC TCC AAG TTT GCA GAG 1392 Val He Lys Ala Cys Gin Leu Glu Glu Asp He Ser Lys Phe Ala Glu 450 455 460
AAA GAC AAT ATA GTT CTT GGA GAA GGT GGA ATC ACA CTG AGT GGA GGT 1440 Lys Asp Asn He Val Leu Gly Glu Gly Gly He Thr Leu Ser Gly Gly 465 470 475 480
CAA CGA GCA AGA ATT TCT TTA GCA AGA GCA GTA TAC AAA GAT GCT GAT 1488 Gin Arg Ala Arg He Ser Leu Ala Arg Ala Val Tyr Lys Asp Ala Asp 485 490 495
TTG TAT TTA TTA GAC TCT CCT TTT GGA TAC CTA GAT ATT GTT CAT CGC 1536 Leu Tyr Leu Leu Asp Ser Pro Phe Gly Tyr Leu Asp He Val His Arg 500 505 510
AAC CTG TTG ATG AAG GCA ATT AGG CAT TGG AGG AAA GGA AAG ACT ACA 1584 Asn Leu Leu Met Lys Ala He Arg His Trp Arg Lys Gly Lys Thr Thr 515 520 525
ATC ATA TTG ACG CAT GAG TTG AGC CAA ATT GAA TCT GAT GAC TAT TTA 1632 He He Leu Thr His Glu Leu Ser Gin He Glu-Ser Asp Asp Tyr Leu 530 535 540
TAT TTA ATG AAG GAA GGT GAA GTT GTT GAA AGC GGC ACC CAG TCT GAA 1680 Tyr Leu Met Lys Glu Gly Glu Val Val Glu Ser Gly Thr Gin Ser Glu 545 550 555 560
CTT CTA GCC GAT CCG ACC ACT ACA TTT AGC ACA TGG TAT CAC CTA CAG 1728 Leu Leu Ala Asp Pro Thr Thr Thr Phe Ser Thr Trp Tyr His Leu Gin 565 570 575
AAT GAC TAC TCT GAT GCG AAA ACT ATT GTA GAT ACA GAG ACT GAA GAA 1776 Asn Asp Tyr Ser Asp Ala Lys Thr He Val Asp Thr Glu Thr Glu Glu 580 585 590
AAA TCT ATA CAC ACT GTG GAA AGT TTT AAC TCT CAA TTG GAA ACA CCA 1824 Lys Ser He His Thr Val Glu Ser Phe Asn Ser Gin Leu Glu Thr Pro 595 600 605
AAA CTT GGA TCA TGC TTA AGT AAT CTG GGA TAT GAT GAG ACA GAT CAG 1872 Lys Leu Gly Ser Cys Leu Ser Asn Leu Gly Tyr Asp Glu Thr Asp Gin 610 615 620
TTG TCC TTT TAC GAA GCA ATC TAT CAA AAA AGA TCG AAC GTT AGA ACA 1920 Leu Ser Phe Tyr Glu Ala He Tyr Gin Lys Arg Ser Asn Val Arg Thr 625 630 635 640
AGA AGG GTT AAA GTT GAA GAG GAA AAT ATT GGG TAT GCA CTA AAA CAA 1968 Arg Arg Val Lys Val Glu Glu Glu Asn He Gly Tyr Ala Leu Lys Gin 645 650 655 CAA AAG AAC ACC GAA AGT TCA ACA GGG CCA CAA CTT CTG AGC ATT ATT 2016 Gin Lys Asn Thr Glu Ser Ser Thr Gly Pro Gin Leu Leu Ser He He 660 665 670
CAG ATT ATC AAA AGA ATG ATT AAA AGC ATA AGA TAC AAA AAA ATT CTA 2064 Gin He He Lys Arg Met He Lys Ser He Arg Tyr Lys Lys He Leu 675 680 685
ATC TTG GGA CTG CTA TGT TCT CTT ATC GCA GGC GCC ACA AAT CCC GTC 2112 He Leu Gly Leu Leu Cys Ser Leu He Ala Gly Ala Thr Asn Pro Val 690 695 700
TTT TCA TAC ACA TTC AGT TTC TTA CTA GAA GGA ATT GTC CCA TCC ACG 2160 Phe Ser Tyr Thr Phe Ser Phe Leu Leu Glu Gly He Val Pro Ser Thr 705 710 715 720
GAT GGA AAA ACT GGC TCT TCA CAT TAT TTG GCG AAA TGG TCG CTT CTT 2208 Asp Gly Lys Thr Gly Ser Ser His Tyr Leu Ala Lys Trp Ser Leu Leu 725 730 735
GTT CTT GGT GTG GCT GCG GCA GAT GGT ATT TTC AAT TTT GCT AAA GGA 2256 Val Leu Gly Val Ala Ala Ala Asp Gly He Phe Asn Phe Ala Lys Gly 740 745 750
TTC CTA TTA GAT TGC TGC AGT GAA TAC TGG GTT ATG GAT CTT AGA AAT 2304 Phe Leu Leu Asp Cys Cys Ser Glu Tyr Trp Val Met Asp Leu Arg Asn 755 760 765
GAA GTT ATG GAA AAA CTG ACG AGA AAG AAT ATG GAC TGG TTT TCT GGT 2352 Glu Val Met Glu Lys Leu Thr Arg Lys Asn Met Asp Trp Phe Ser Gly 770 775 780
GAA AAC AAC AAG GCT TCT GAA ATT TCT GCT CTA GTC TTG AAT GAT TTG 2400 Glu Asn Asn Lys Ala Ser Glu He Ser Ala Leu Val Leu Asn Asp Leu 785 790 795 800
CGA GAT TTG AGG TCT TTG GTC TCT GAA TTT TTG AGT GCA ATG ACT AGT 2448 Arg Asp Leu Arg Ser Leu Val Ser Glu Phe Leu Ser Ala'Met Thr Ser 805 810 815
TTC GTT ACC GTA TCA ACG ATT GGA CTA ATT TGG GCG TTA GTA TCG GGC 2496 Phe Val Thr Val Ser Thr He Gly Leu He Trp Ala Leu Val Ser Gly 820 825 830
TGG AAG TTA AGT TTG GTT TGT ATT TCG ATG TTT CCA CTC ATA ATT ATA 2544 Trp Lys Leu Ser Leu Val Cys He Ser Met Phe Pro Leu He He He 835 840 845
TTT TCA GCA ATA TAT GGA GGC ATT TTA CAA AAA TGC GAA ACA GAT TAT 2592 Phe Ser Ala He Tyr Gly Gly He Leu Gin Lys Cys Glu Thr Asp Tyr 850 855 860 AAG ACA TCT GTT GCT CAG TTA GAA AAC TGC CTG TAC CAG ATT GTC ACT 2640 Lys Thr Ser Val Ala Gin Leu Glu Asn Cys Leu Tyr Gin He Val Thr 865 870 875 880
AAC ATT AAA ACC ATT AAG TGC TTA CAA GCT GAA TTT CAT TTT CAA TTG 2688 Asn He Lys Thr He Lys Cys Leu Gin Ala Glu Phe His Phe Gin Leu 885 890 895
ACC TAC CAT GAC TTG AAG ATA AAA ATG CAA CAA ATT GCC TCC AAA CGC 2736 Thr Tyr His Asp Leu Lys He Lys Met Gin Gin He Ala Ser Lys Arg 900 905 910
GCC ATT GCC ACA GGA TTT GGT ATA TCT ATG ACA AAC ATG ATT GTC ATG 2784 Ala He Ala Thr Gly Phe Gly He Ser Met Thr Asn Met He Val Met 915 920 925
TGT ATC CAA GCT ATT ATT TAC TAC TAT GGC CTA AAG CTG GTT ATG ATT 2832 Cys He Gin Ala He He Tyr Tyr Tyr Gly Leu Lys Leu Val Met He 930 935 940
CAC GAG TAC ACC TCA AAG GAA ATG TTT ACG ACT TTC ACT TTG TTA TTA 2880 His Glu Tyr Thr Ser Lys Glu Met Phe Thr Thr Phe Thr Leu Leu Leu 945 950 955 960
TTC ACT ATT ATG TCA TGC ACT AGC CTA GTA AGT CAG ATA CCC GAT ATA 2928 Phe Thr He Met Ser Cys Thr Ser Leu Val Ser Gin He Pro Asp He 965 970 975
AGT AGA GGC CAA CGT GCT GCC AGT TGG ATC TAT AGG ATT CTT GAT GAA 2976 Ser Arg Gly Gin Arg Ala Ala Ser Trp He Tyr Arg He Leu Asp Glu 980 985 990
AAG CAT AAT ACC CTA GAG GTT GAA AAC AAT AAT GCT AGA ACA GTG GGA 3024 Lys His Asn Thr Leu Glu Val Glu Asn Asn Asn Ala Arg Thr Val Gly 995 1000 1005
ATA GCT GGT CAC ACC TAC CAT GGC AAA GAA AAA AAA CCA ATC GTT TCA 3072 He Ala Gly His Thr Tyr His Gly Lys Glu Lys Lys Pro He Val Ser 1010 1015 1020
ATT CAA AAT TTG ACA TTT GCC TAT CCA TCT GCA CCT ACC GCC TTT GTT 3120 He Gin Asn Leu Thr Phe Ala Tyr Pro Ser Ala Pro Thr Ala Phe Val 1025 1030 1035 1040
TAC AAA AAC ATG AAT TTT GAC ATG TTT TGC GGA CAG ACG TTA GGT ATC 3168 Tyr Lys Asn Met Asn Phe Asp Met Phe Cys Gly Gin Thr Leu Gly He 1045 1050 1055
ATT GGT GAA TCA GGC ACA GGA AAG TCT ACA CTT GTG CTT TTA TTA ACA 3216 He Gly Glu Ser Gly Thr Gly Lys Ser Thr Leu Val Leu Leu Leu Thr 1060 1065 1070 AAA CTT TAT AAT TGT GAA GTA GGC AAA ATT AAA ATA GAC GGT ACG GAC 3264 Lys Leu Tyr Asn Cys Glu Val Gly Lys He Lys He Asp Gly Thr Asp 1075 1080 1085
GTA AAT GAC TGG AAT TTG ACA AGT TTA AGA AAA GAA ATT TCA GTG GTT 3312 Val Asn Asp Trp Asn Leu Thr Ser Leu Arg Lys Glu He Ser Val Val 1090 1095 1100
GAG CAA AAA CCT TTA TTA TTC AAT GGA ACC ATC AGA GAT AAC CTA ACT 3360 Glu Gin Lys Pro Leu Leu Phe Asn Gly Thr He Arg Asp Asn Leu Thr 1105 1110 1115 1120
TAT GGT TTA CAA GAT GAA ATA CTT GAA ATT GAA ATG TAT GAT GCA TTA 3408 Tyr Gly Leu Gin Asp Glu He Leu Glu He Glu Met Tyr Asp Ala Leu 1125 1130 1135
AAA TAC GTA GGA ATC CAT GAC TTT GTA ATT TCA TCA CCT CAG GGC TTG 3456 Lys Tyr Val Gly He His Asp Phe Val He Ser Ser Pro Gin Gly Leu 1140 1145 1150
GAT ACA CGT ATT GAT ACA ACT TTA CTG TCA GGT GGA CAA GCG CAA AGG 3504 Asp Thr Arg He Asp Thr Thr Leu Leu Ser Gly Gly Gin Ala Gin Arg 1155 1160 1165
CTT TGC ATA GCC AGA GCA CTT CTG AGA AAA TCA AAA ATT CTG ATT TTA 3552 Leu Cys He Ala Arg Ala Leu Leu Arg Lys Ser Lys He Leu He Leu 1170 1175 1180
GAT GAG TGT ACT TCA GCC TTG GAT TCT GTC AGC TCC TCT ATC ATC AAT 3600 Asp Glu Cys Thr Ser Ala Leu Asp Ser Val Ser Ser Ser He He Asn 1185 1190 1195 1200
GAG ATC GTC AAA AAA GGT CCA CCT GCT CTA CTA ACA ATG GTT ATA ACG 3648 Glu He Val Lys Lys Gly Pro Pro Ala Leu Leu Thr Met Val He Thr 1205 1210 1215
CAT AGT GAA CAA ATG ATG AGG TCT TGT AAC TCG ATT GCA GTT CTT AAA 3696 His Ser Glu Gin Met Met Arg Ser Cys Asn Ser He Ala Val Leu Lys 1220 1225 1230
GAT GGT AAA GTG GTT GAG CGA GGT AAC TTC GAC ACT TTA TAT AAT AAT 3744 Asp Gly Lys Val Val Glu Arg Gly Asn Phe Asp Thr Leu Tyr Asn Asn 1235 1240 1245
CGC GGG GAA TTA TTC CAA ATT GTT TCC AAC CAA AGC AGT TAA 3786
Arg Gly Glu Leu Phe Gin He Val Ser Asn Gin Ser Ser 1250 1255 1260
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1261 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Asn Phe Leu Ser Phe Lys Thr Thr Lys His Tyr His He Phe Arg 1 5 10 15
Tyr Val Asn He Arg Asn Asp.Tyr Arg Leu Leu Met He Met He He 20 25 30
Gly Thr Val Ala Leu Ser Val Phe Val Ala Asn Gly Ser His Gin Gly 35 40 45
Leu Tyr Ser Gin Leu Val Gin Arg Ser Met Ala Val Met Ala Leu Gly 50 55 60
Ala Ala Ser Val Pro Val Met Trp Leu Ser Leu Thr Ser Trp Met His 65 70 75 80
He Gly Glu Arg Gin Gly Phe Arg He Arg Ser Gin He Leu Glu Ala 85 90 95
Tyr Leu Glu Glu Lys Pro Met Glu Trp Tyr Asp Asn Asn Glu Lys Leu 100 105 110
Leu Gly Asp Phe Thr Gin He Asn Arg Cys Val Glu Glu Leu Arg Ser 115 120 125
Ser Ser Ala Glu Ala Ser Ala He Thr Phe Gin Asn Leu Val Ala He 130 135 140
Cys Ala Leu Leu Gly Thr Ser Phe Tyr Tyr Ser Trp Ser Leu Thr Leu 145 150 155 160
He He Leu Cys Ser Ser Pro He He Thr Phe Phe Ala Val Val Phe 165 170 175
Ser Arg Met He His Val Tyr Ser Glu Lys Glu Asn Ser Glu Thr Ser 180 185 190
Lys Ala Ala Gin Leu Leu Thr Trp Ser Met Asn Ala Ala Gin Leu Val 195 200 205
Arg Leu Tyr Cys Thr Gin Arg Leu Glu Arg Lys Lys Phe Lys Glu He 210 215 220
He Leu Asn Cys Asn Thr Phe Phe He Lys Ser Cys Phe Phe Val Ala 225 230 235 240 Ala Asn Ala Gly He Leu Arg Phe Leu Thr Leu Thr Met Phe Val Gin 245 250 255
Gly Phe Trp Phe Gly Ser Ala Met He Lys Lys Gly Lys Leu Asn He 260 265 270
Asn Asp Val He Thr Cys Phe His Ser Cys He Met Leu Gly Ser Thr 275 280 285
Leu Asn Asn Thr Leu His Gin He Val Val Leu Gin Lys Gly Gly Val 290 295 300
Ala Met Glu Lys He Met Thr Leu Leu Lys Asp Gly Ser Lys Arg Asn 305 310 315 320
Pro Leu Asn Lys Thr Val Ala His Gin Phe Pro Leu Asp Tyr Ala Thr 325 330 335
Ser Asp Leu Thr Phe Ala Asn Val Ser Phe Ser Tyr Pro Ser Arg Pro 340 345 350
Ser Glu Ala Val Leu Lys Asn Val Ser Leu Asn Phe Ser Ala Gly Gin 355 360 365
Phe Thr Phe He Val Gly Lys Ser Gly Ser Gly Lys Ser Thr Leu Ser 370 375 380
Asn Leu Leu Leu Arg Phe Tyr Asp Gly Tyr Asn Gly Ser He Ser He 385 390 395 400
Asn Gly His Asn He Gin Thr He Asp Gin Lys Leu Leu He Glu Asn 405 410 415
He Thr Val Val Glu Gin Phe Ser Trp He Met Pro Gly Thr He Lys 420 425 430
Glu Asn He He Phe Gly Val Ser Tyr Asp Glu Tyr Arg Tyr Arg Ser 435 440 '445
Val He Lys Ala Cys Gin Leu Glu Glu Asp He Ser Lys Phe Ala Glu 450 455 460
Lys Asp Asn He Val Leu Gly Glu Gly Gly He Thr Leu Ser Gly Gly 465 470 475 480
Gin Arg Ala Arg He Ser Leu Ala Arg Ala Val Tyr Lys Asp Ala Asp 485 490 495
Leu Tyr Leu Leu Asp Ser Pro Phe Gly Tyr Leu Asp He Val His Arg 500 505 510 Asn Leu Leu Met Lys Ala He Arg His Trp Arg Lys Gly Lys Thr Thr 515 520 525
He He Leu Thr His Glu Leu Ser Gin He Glu Ser Asp Asp Tyr Leu 530 535 540
Tyr Leu Met Lys Glu Gly Glu Val Val Glu Ser Gly Thr Gin Ser Glu 545 550 555 560
Leu Leu Ala Asp Pro Thr Thr Thr Phe Ser Thr Trp Tyr His Leu Gin 565 570 575
Asn Asp Tyr Ser Asp Ala Lys Thr He Val Asp Thr Glu Thr Glu Glu 580 585 590
Lys Ser He His Thr Val Glu Ser Phe Asn Ser Gin Leu Glu Thr Pro 595 600 605
Lys Leu Gly Ser Cys Leu Ser Asn Leu Gly Tyr Asp Glu Thr Asp Gin 610 615 620
Leu Ser Phe Tyr Glu Ala He Tyr Gin Lys Arg Ser Asn Val Arg Thr 625 630 635 640
Arg Arg Val Lys Val Glu Glu Glu Asn He Gly Tyr Ala Leu Lys Gin 645 650 655
Gin Lys Asn Thr Glu Ser Ser Thr Gly Pro Gin Leu Leu Ser He He 660 665 670
Gin He He Lys Arg Met He Lys Ser He Arg Tyr Lys Lys He Leu 675 680 685
He Leu Gly Leu Leu Cys Ser Leu He Ala Gly Ala Thr Asn Pro Val 690 695 700
Phe Ser Tyr Thr Phe Ser Phe Leu Leu Glu Gly He V.al Pro Ser Thr 705 710 715 720
Asp Gly Lys Thr Gly Ser Ser His Tyr Leu Ala Lys Trp Ser Leu Leu 725 730 735
Val Leu Gly Val Ala Ala Ala Asp Gly He Phe Asn Phe Ala Lys Gly 740 745 750
Phe Leu Leu Asp Cys Cys Ser Glu Tyr Trp Val Met Asp Leu Arg Asn 755 760 765
Glu Val Met Glu Lys Leu Thr Arg Lys Asn Met Asp Trp Phe Ser Gly 770 775 780 Glu Asn Asn Lys Ala Ser Glu He Ser Ala Leu Val Leu Asn Asp Leu 785 790 795 800
Arg Asp Leu Arg Ser Leu Val Ser Glu Phe Leu Ser Ala Met Thr Ser 805 810 815
Phe Val Thr Val Ser Thr He Gly Leu He Trp Ala Leu Val Ser Gly 820 825 830
Trp Lys Leu Ser Leu Val Cys He Ser Met Phe Pro Leu He He He 835 840 845
Phe Ser Ala He Tyr Gly Gly He Leu Gin Lys Cys Glu Thr Asp Tyr 850 855 860
Lys Thr Ser Val Ala Gin Leu Glu Asn Cys Leu Tyr Gin He Val Thr 865 870 875 880
Asn He Lys Thr He Lys Cys Leu Gin Ala Glu Phe His Phe Gin Leu 885 890 ' 895
Thr Tyr His Asp Leu Lys He Lys Met Gin Gin He Ala Ser Lys Arg 900 905 910
Ala He Ala Thr Gly Phe Gly He Ser Met Thr Asn Met He Val Met 915 920 925
Cys He Gin Ala He He Tyr Tyr Tyr Gly Leu Lys Leu Val Met He 930 935 940
His Glu Tyr Thr Ser Lys Glu Met Phe Thr Thr Phe Thr Leu Leu Leu 945 950 955 960
Phe Thr He Met Ser Cys Thr Ser Leu Val Ser Gin He Pro Asp He 965 970 975
Ser Arg Gly Gin Arg Ala Ala Ser Trp He Tyr Arg He Leu Asp Glu 980 985 990
Lys His Asn Thr Leu Glu Val Glu Asn Asn Asn Ala Arg Thr Val Gly 995 1000 1005
He Ala Gly His Thr Tyr His Gly Lys Glu Lys Lys Pro He Val Ser 1010 1015 1020
He Gin Asn Leu Thr Phe Ala Tyr Pro Ser Ala Pro Thr Ala Phe Val 1025 1030 1035 1040
Tyr Lys Asn Met Asn Phe Asp Met Phe Cys Gly Gin Thr Leu Gly He 1045 1050 1055 He Gly Glu Ser Gly Thr Gly Lys Ser Thr Leu Val Leu Leu Leu Thr 1060 1065 1070
Lys Leu Tyr Asn Cys Glu Val Gly Lys He Lys He Asp Gly Thr Asp 1075 1080 1085
Val Asn Asp Trp Asn Leu Thr Ser Leu Arg Lys Glu He Ser Val Val 1090 1095 1100
Glu Gin Lys Pro Leu Leu Phe Asn Gly Thr He Arg Asp Asn Leu Thr 1105 1110 1115 1120
Tyr Gly Leu Gin Asp Glu He Leu Glu He Glu Met Tyr Asp Ala Leu 1125 1130 1135
Lys Tyr Val Gly He His Asp Phe Val He Ser Ser Pro Gin Gly Leu 1140 1145 1150
Asp Thr Arg He Asp Thr Thr Leu Leu Ser Gly Gly Gin Ala Gin Arg 1155 1160 1165
Leu Cys He Ala Arg Ala Leu Leu Arg Lys Ser Lys He Leu He Leu 1170 1175 1180
Asp Glu Cys Thr Ser Ala Leu Asp Ser Val Ser Ser Ser He He Asn 1185 1190 1195 1200
Glu He Val Lys Lys Gly Pro Pro Ala Leu Leu Thr Met Val He Thr 1205 1210 1215
His Ser Glu Gin Met Met Arg Ser Cys Asn Ser He Ala Val Leu Lys 1220 1225 1230
Asp Gly Lys Val Val Glu Arg Gly Asn Phe Asp Thr Leu Tyr Asn Asn 1235 1240 1245
Arg Gly Glu Leu Phe Gin He Val Ser Asn Gin Ser Ser 1250 1255 1260 ■

Claims

What is claimed is:
1. A method of identifying potential treatments which correct mislocalization and/or misfunction of CFTR resulting from mutations said method comprising: forming a chimeric gene sequence by substituting a portion of a gene sequence which encodes the first nucleotide binding domain of cystic fibrosis conductance regulator with a gene sequence which encodes STE6; said CFTR sequence being a CFTR gene sequence encoding a plurality of amino acids from amino acid 444 to and including amino acid 577, such that introduction of a mutation in the CFTR region inhibits expression of the chimeric STE6 gene producti transforming said chimeric gene sequence into a yeast strain with no functional STE6 gene; introducing said potential treatment to said yeast strain; and assaying for the production of diploid yeast colonies following yeast cell mating.
2. The method of claim 1 wherein said mutation is a deletion of phenylalanine at position 508 of the CFTR gene product.
3. The method of claim 1 wherein said CFTR substitute is the gene sequence which encodes from amino acid 494 through 546 of the CFTR gene product (SEQ ID #1) .
4. The method of claim 1 wherein said CFTR substitution is the gene sequence which encodes from amino acid 494 up to and including amino acid 558 of the CFTR gene product (SEQ ID #3).
5. The method of claim 1 wherein said CFTR substitution is the gene sequence which encodes from amino acid 494 up to and including 577 of the CFTR gene product (SEQ ID #5).
6. The method of claim 3, 4, or 5 wherein said CFTR substitution includes a mutation which causes cystic fibrosis.
7. A chimeric protein comprising a yeast STE6 protein product having substituted therein an analogous portion of CFTR nucleotide binding domain including a plurality of amino acids from amino acid 443 to amino acid 577.
8. The protein of claim 7 wherein said CFTR sequence includes a mutation which results in cystic fibrosis.
9. The protein of claim 7 wherein said amino acid sequence is SEQ ID # 2.
10. The protein of claim 7 wherein said amino acid sequence is SEQ ID # 4.
11. The protein of claim 7 wherein said amino acid sequence is SEQ ID # 6.
12. A chimeric protein sufficiently homologous to that of claim 7 such that introduction of a mutation in the CFTR portion will prevent transport of a-factor out a yeast cell thereby preventing yeast mating.
13. A DNA sequence which encodes the protein of claim 7.
14. A host cell transformed with the DNA sequence of claim 13 in a manner allowing expression of the STE6 gene.
15. A method of screening for potential inhibitors of ABC transporters by the use of STE6 chimeras, said method comprising selecting from the class of related proteins consisting essentially of multi-drug resistance (mdr) or P-glycoprotein, the choroquine resistance transporter (pfmdr) of Plasmodium falciparum, and CFTR; substituting the nucleotide sequence of the STE6 gene with the sequence encoding the analogous portion of said ABC transporter; transforming said chimeric gene sequence into a yeast which is deleted for the STE6 gene; introducing a treatment to said transformed yeast strain; and assaying for the production of yeast diploid colonies following yeast cell mating.
PCT/US1994/004379 1993-04-23 1994-04-20 Method of assaying for drugs which restore function of mutant cftr protein WO1994025607A1 (en)

Priority Applications (1)

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AU69035/94A AU6903594A (en) 1993-04-23 1994-04-20 Method of assaying for drugs which restore function of mutant cftr protein

Applications Claiming Priority (2)

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US5220793A 1993-04-23 1993-04-23
US08/052,207 1993-04-23

Publications (1)

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WO1994025607A1 true WO1994025607A1 (en) 1994-11-10

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Cited By (9)

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US6083954A (en) * 1992-09-29 2000-07-04 Us Health Method of treating cystic fibrosis
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FR2763845A1 (en) * 1997-05-30 1998-12-04 Centre Nat Rech Scient ANTI-CANCER PRODUCTS FOR THE TREATMENT OF MUCOVISCIDOSIS
WO1998053839A3 (en) * 1997-05-30 1999-09-10 Centre Nat Rech Scient Anti-cancer products for treating cystic fibrosis
US6635627B1 (en) 1997-05-30 2003-10-21 Centre National De La Recherche Scientifique (Cnrs) Anti-cancer products for treating cystic fibrosis
US6376521B1 (en) 1998-07-10 2002-04-23 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services A3 adenosine receptor antagonists
WO2001025421A3 (en) * 1999-10-06 2001-08-30 Univ Florida State Res Found Materials and method for detecting interaction of cftr polypeptides
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US7238474B1 (en) 1999-10-06 2007-07-03 Florida State University Research Foundation Materials and methods for detecting interaction of CFTR polypeptides
US8288107B2 (en) 1999-10-06 2012-10-16 Florida State University Research Foundation Materials and methods for detecting interaction of CFTR polypeptides
US7087589B2 (en) 2000-01-14 2006-08-08 The United States Of America As Represented By The Department Of Health And Human Services Methanocarba cycloakyl nucleoside analogues
US7790735B2 (en) 2000-01-14 2010-09-07 The United States Of America As Represented By The Department Of Health And Human Services Methanocarba cycloalkyl nucleoside analogues
US6962813B2 (en) 2001-05-21 2005-11-08 The Brigham And Women's Hospital, Inc. P. aeruginosa mucoid exopolysaccharide specific binding peptides
US7119172B2 (en) 2001-05-21 2006-10-10 The Brigham And Women's Hospital, Inc. P. aeruginosa mucoid exopolysaccharide specific binding peptides
US7230087B2 (en) 2001-05-21 2007-06-12 Beth Israel Deaconess Medical Center, Inc. P. aeruginosa mucoid exopolysaccharide specific binding peptides
US8518957B2 (en) 2009-12-02 2013-08-27 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Methanocarba adenosine derivatives, pharmaceutical compositions, and method of reducing intraocular pressure

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