1.ApplicationNumber: US-58190900-A
1.PublishNumber: US-6566048-B1
2.Date Publish: 20030520
3.Inventor: DIXON GRAHAM K
THAIN JOHN L
VINCENT JOHN P
SCHNELL NORBERT F
CHAVDA SUBERNA J

4.Inventor Harmonized: DIXON GRAHAM K(GB)
THAIN JOHN L(GB)
VINCENT JOHN P(GB)
SCHNELL NORBERT F(GB)
CHAVDA SUBERNA J(CA)

5.Country: US
6.Claims:
(en)The Acetyl-COA-carboxylase (ACCase) gene from Candida albicans.

7.Description:
(en)This application is the national phase of international application PCT/GB98/03857 filed Dec. 21, 1998 which designated the U.S.


The present invention relates to Acetyl-COA-carboxylase (ACCase) genes from Candida Albicans ( C. albicans ) and methods for its expression. The invention also relates to novel hybrid organisms for use in such expression methods.
C. albicans is an important fungal pathogen and the most prominent target organism for antifungal research. ACCase is an enzyme of fatty acid biosynthesis and essential for fungal growth and viability. Inhibitors of the ACCase enzyme should therefore be potent antifungals. The ACCase proteins in all organisms are homologous to each other but they also differ significantly in the amino acid sequence. Because selectivity problems (for example fungal versus human) it is extremely important to optimise potential inhibitor leads directly against the target enzyme ( C. albicans ) and not against a homologous but non-identical model protein, for example from Saccharomyces cerevisiae ( S. Cerevisiae ).
We have now successfully cloned the ACCase gene from C. albicans (hereinafter referred to as the C. Albicans ACC1 gene) and elucidated its full length DNA sequence and corresponding polypeptide sequence, as set out in FIGS. 4 and 5 of this application respectively. The coding DNA sequence of the C. Albicans ACC1 gene is 6810 nucleotides in length and the corresponding protein sequence is 2270 amino acids in length. As will be explained below there are two forms of the C. Albicans ACC1 gene, the above numbers relate to the longer version, Met1.
Therefore in a first aspect of the present invention we provide a polynucleotide encoding a C.albicans ACCase gene, in particular the (purified) C. albicans ACC1 gene as set out in FIG. 5 hereinafter. It will be appreciated that the polynucleotide may comprise any of the degenerate codes for a particular amino acid including the use of rare codons. The polynucleotide is conveniently as set out in FIG. 4 . It will be apparent from FIG. 4 that the gene is characterised by the start codons Met1 and Met2 (as indicated by the first and second underlined at codons, hereinafter referred to as atg1 and atg2 respectively). Both forms of the gene starting from Met1 and Met2 respectively are comprised in the present invention. The invention further comprises convenient fragments of any one of the above sequences. Convenient fragments may be defined by restriction endonuclease digests of sequence, suitable fragments include a full length C. Albicans ACC1 gene (starting with Met1 or Met2) flanked by unique Stu1 (5′-end)-NotI (3′-end) restriction sites as detailed in FIG. 6 .
We also provide a polynucleotide probe comprising any one of the above sequences or fragments together with a convenient label or marker, preferably a non-radioactive label or marker. Following procedures well known in the art, the probe may be used to identify corresponding nucleic acid sequences. Such sequences may be comprised in libraries, such as cDNA libraries. We also provide RNA transcripts corresponding to any of the above C. Albicans ACC1 sequences or fragments.
In a further aspect of the invention we provide a C. albicans ACC1 enzyme, especially the ACC1 enzyme having the polypeptide sequence set out in FIG. 5, in isolated and purified form. This is conveniently achieved by expression of the coding DNA sequence of the C. Albicans ACC1 gene set out in FIG. 4, using methods well known in the art (for example as described in the Maniatis cloning manual—Molecular Cloning: A Laboratory Manual, 2 nd Edition 1989, J. Sambrook, E. F. Fritsch & Maniatis). As indicated for FIG. 4 above, the enzyme is characterised by two forms Met1 and Met2. Both form of the enzyme are comprised in the present invention.
The C. Albicans ACC1 enzyme of the present invention is useful as a target in biochemical assays. However, to provide sufficient enzyme for a biochemical assay for C. Albicans ACC1 (for example, for a high throughput screen for enzyme inhibitors) this has to be purified. Two major constraints impair this purification.
1) any new organism will necessitate deviation from published procedures because it will differ in its lysis and protease activity. C. albicans is known to express and secrete many aspartyl proteases.
2) The expression of C. Albicans ACC1 is very low and satisfying purification results can only be achieved if the enzyme is overexpressed.
We have now been able to overcome these problems by controlled overexpression of the C. albicans ACC1 in a Saccharomyces strain. This means that subsequent purification of the enzyme may then for example follow published procedures.
Therefore in a further aspect of the present invention we provide a novel expression system for expression of a C. albicans ACC1 gene which system comprises an S. cerevisiae host strain having a C. albicans ACC1 gene inserted in place of the native ACC1 gene from S. Cerevisiae , whereby the C. albicans ACC1 gene is expressed. Preferred S. cerevisiae strains include JK9-3Daα and its haploid segregants.
The C. albicans ACC1 gene is preferably over-expressed relative to that as may be achieved by a C. albicans wild type strain, ie under the control of its own ACC1 promoter. Whilst we do not wish to be bound by theoretical considerations, we have achieved approximately 14 fold over-expression relative to the wild-type host S. cervisiae strain JK9-3D. This may be achieved by replacing the C. albicans promoter in the expression construct by a stronger and preferably inducible promoter such as the S. cerevisiae GAL1 promoter.
Controlled overexpression is used to improve expression of a C. albicans polypeptide relative to expression under the control of a C. albicans promoter. In addition using procedures outlined in the accompanying examples we have been able to isolate a fully functional C. albicans ACC1 gene as determined by 100% inhibition by SoraphenA.
The novel expression system is conveniently prepared by transformation of a heterozygous ACC1 deletion strain of a convenient S. cerevisiae host by a convenient plasmid comprising the C. albicans ACC1 gene. Transformation is conveniently effected using methods well known in the art of molecular biology (Ito et al. 1983).
The plasmid comprising the C. albicans ACC1 gene and used to transform a convenient S. cerevisiae host represents a further aspect of the invention. Preferred plasmids for insertion of the C. Albicans ACC1 gene include YEp24, pRS316 and pYES2(Invitrogen).
The heterozygous ACC1 deletion strain of a convenient (diploid) S. cerevisiae host is conveniently achieved by disruption preferably using an antibiotic resistance cassette such as the kanamycin resistance cassette described by Wach et al (Yeast, 1994, 10, 1793-1808).
The expression systems of the invention may be used together with, for example cell growth and enzyme isolation procedures identical to or analogous to those described herein, to provide an acetyl-COA-carboxylase (ACCase) gene from C. albicans in sufficient quantity and with sufficient activity for compound screening purposes.
In a further aspect of the invention we provide the use of an acetyl-COA-carboxylase (ACCase) gene from C. albicans in assays to identify inhibitors of the polypeptide. In particular we provide the their use in pharmaceutical or agrochemical research.
As presented above the C. albicans ACC1 enzyme may be used in biochemical assays to identify agents which modulate the activity of the enzyme. The design and implementation of such assays will be evident to the biochemist of ordinary skill. The enzyme may be used to turn over a convenient substrate whilst incorporating/losing a labelled component to define a test system. Test compounds are then introduced into the test system and measurements made to determine their effect on enzyme activity. Particular assays are those used to identify inhibitors of the enzyme useful as antifungal agents. By way of non-limiting example, the activity of the ACC1 enzyme may be determined by (i) following the incorporation (HCO 3 , Acetyl-CoA) or loss (ATP) of a convenient label from the relevant substrate (T. Tanabe et al, Methods in Enzymology, 1981, 71, 5-60; M. Matasuhashi, Methods in Enzymology, 1969, 14, 3-16), (ii) following the release of inorganic phosphate from ATP (P. Lanzetta et al, Anal. Biochem. 1979, 100, 95-97), or (iii) following the oxidation of NADH in a coupled assay, for example using either fatty acid synthetase or pyruvate kinase/lactate dehydrogenase enzymes. Convenient labels include carbon14, tritium, phosphorous32 or 33.
Any convenient test compound(s) or library of test compounds may be used. Particular test compounds include low molecular weight chemical compounds (molecular weight less than 1500 daltons) suitable as pharmaceutical agents for human, animal or plant use.
The enzyme of the invention, and convenient fragments thereof may be used to raise antibodies. Such antibodies have a number of uses which will be evident to the molecular biologist of ordinary skill. Such uses include (i) monitoring enzyme expression, (ii) the development of assays to measure enzyme activity and precipitation of the enzyme.
In addition we provide antisense polynucleotides specific for all or a part of an ACC1 polynucleotide of the invention.


The invention will now be illustrated but not limited by reference to the following Table, Example, References and Figures wherein:
Table 1 shows the comparative properties of native and recombinant acetyl-CoA carboxylase enzymes
FIG. 1 shows a partial sequence (SEQ ID NO:1) from the C. albicans genome. Underlined regions were used to derive PCR primers, to generate a C. Albicans ACC1 specific probe.
FIG. 2 shows cloned fragments of the C. albicans ACC1 gene isolated from genomic DNA libraries. Arrows indicate extension of the fragment beyond the region displayed.
FIG. 3 shows sequenced XbaI-HinDIII and HinDIII subclones of clone CLS1-b1.
FIG. 4 shows the full DNA sequence (SEQ ID NO:2) of the C. albicans ACC1 gene. The atg start codons for Met1 and Met2 are in lower case and underlined, as is the tag stop codon.
FIG. 5 shows the full protein sequence (SEQ ID NO:3) of the C. albicans ACC1 gene. Putative start codons for Met1 and Met2 are shown in bold.
FIG. 6 shows the generation of a tailored ACC1 gene (minus promoter) for expression under control of the GAL1 promoter in plasmid pYES2. From the initial ACCase gene (line1) the core SacI-BamHI (line3) is modified by the addition of 3′ BamHI-NotI (line2) and 5′ StuI-SacI (different fragments for Met1 and -2 lines 5 and 7 respectively) to generate the final “portable” gene flanked by StuI-NotI (lines 6 and 8).
FIG. 7 shows the results of the in-vitro ACCase enzyme assay set out in the accompanying Example when Soraphen A (a specific inhibitor of the ACCase enzyme) was supplied (X-axis) over the range 0.1 nM-100 μM in the dose response regimen of the assay.


EXAMPLE 1
Cloning of the C. albicans ACC1 gene and generation of a heterologous S. cerevisiae expression system:
1) Probe Generation
We used the polymerase chain reaction (PCR) to generate a DNA probe between and including the underlined regions in FIG. 1
2) Identification of Clones from a C. albicans Genomic Library Hybridising to the ACCase Probe
The PCR product was labelled using an “ECL direct nucleic acid labelling and detection kit” (Amersham) as described by the supplier. The PCR product (probe) was then shown to hybridise to S. cerevisiae (weakly) and C. albicans genomic DNA. in a Southern blot procedure (as described Maniatis, 1989). Two genomic DNA libraries (CLS1 and CLS2) of C. albicans (in the yeast- E. coli shuttle plasmids YEp24 and pRS316 respectively, (as described in Sherlock et al. 1994, source: Prof. John Rosamond, Manchester University) were used to isolate fragments hybridising with the probe which was radiolabelled using “Ready To Go” dCTP labelling beads (Pharmacia, as described by the manufacturer). The colony hybridisation was carried out as described by Maniatis (1989). Hybridising colonies were identified, plasmid DNA isolated, purified (Quiagen maxiprep, as described by the supplier) and sequenced (Applied Biosystems, model 377 sequencer) from their junctions with the plasmid. Several fragments carrying partial ACCase gene sequence as well as one full length clone could be identified (FIG. 2 ).
3) Sequencing of the Cloned Gene, Comparison with ACCases from S. cerevisiae , Other Fungi and Higher Eukaryotes (Plants, Mammals, Man)
The bulk of the sequence of the C. albicans ACC1 gene was determined (on both strands) using flanking sequence- or insert sequence-specific primers from defined HinDIII and XbaI-HinDIII subfragments (of clone CLS1-b1) cloned into pUC19 (see FIG. 4 ). The promoter and 5′ coding region absent from this clone was established from CLS2-d1 and the gene's 3′ end from CLS2-13 using insert specific primers. All junctions including the ones between the HinDIII subfragments were verified from the full length clone CLS2-13 (in Yep24. The full length DNA sequence of C. albicans (Ca) ACC1 is shown in FIG. 5 a and the protein translation in FIG. 5 b . The two potential start Methionines, Met1 and Met2 are shown in bold
The protein is homologous to ACCases of other fungi ( S. cerevisiae, S. pombe and U maydis ) and also to the plant ( Brassica napus ), mammalian (sheep, chicken and rat) and human enzymes. Of the two potential start codons of C. albicans ACC1, Met 2 seems the more likely one as the sequence between Met1 and Met2 is unrelated to the other ACCases and indeed to any other protein sequence in the EMBL/Genbank database. The high degree of homology between ACCases of different species and the apparent lack of an identifiable fungal subgroup makes it even more important to use the actual target enzyme (here from the pathogen C. albicans ) as a screening tool to identify specific inhibitors.
4) Generation of a Heterozygous ACC1 Deletion Strain of S. cerevisiae
As ACCase is an essential enzyme, only one allele of a diploid cell can be deleted without loss of survival. One ACC1 gene of a diploid S. cerevisiae strain (JK9-3Daa, Kunz et al. 1993) was therefore disrupted using the kanamycin resistance cassette as described by Wach et al. using the protocol described therein. Sporulation of the heterozygous diploid (ACC1/acc1::KANMx) yields only two viable spores (which are kanamycin-sensitive) showing the essentiality of the ACC1 gene as well as the characteristic arrest phenotype for the two inviable spores (as published by Hallacher et al., 1993).
5) Complementation of a S. cerevisiae ACC1 Deletion with the Cloned Candida Gene, Ca ACC1
The heterozygous ACC1/acc1::KANMx strain was transformed with one full length C. albicans gene (CLS2-13 in Yep24). Expression of the gene from this plasmid will be due to functionality of the Candida ACC1 promoter in the heterologous S. cerevisiae system. Complementation of the knockout was demonstrated by sporulating the diploid transformants. In most cases 3-4 viable (haploid) spores were detected. The analysis of tetrads indicated that kanamycin-resistant colonies were only formed if they also contained the complementing CLS2-13 plasmid, as indicated by the presence of the URA3 transformation marker. This clearly shows that the C. albicans gene fully complements the ACCase function in S. cerevisiae . Therefore the strain generated can be used to screen for inhibitors which are specific for the Candida enzyme in the absence of a background of Saccharomyces enzyme. As demonstrated by its functionality, the heterologous protein folds correctly in the host, S. cerevisiae , where it must also have been correctly biotinylated by the S. cerevisiae machinery (carried out by ACC2, encoding protein-biotin-ligase).
To facilititate purification of C. albicans ACCase, it is beneficial to achieve overexpression of the protein in a suitable host. Therefore the C. albicans promoter was replaced by the stronger and inducible S. cerevisiae GAL1 promoter. As the Candida sequence had revealed two potential start codons (see FIG. 4) for the ACC1 reading frame, both versions were placed under GAL1 control. To generate appropriate restriction sites for cloning, the ACC1 gene was modified via PCR at both ends (see FIG. 6 above). and cloned into plasmid pYES2 (Invitrogen) as a StuI-NotI fragment into HinDIII (fill-in)-NotI sites of the vector. The identity of the PCR-modified gene-parts with the original ones was confirmed by sequencing. Both constructs (Met1 and Met2) complement the S. cerevisiae ACC1 knockout when the cells are grown on galactose but not on glucose (where the GAL1 promoter is switched off). Growth is very poor if the gene is transcribed initiating at Met1, whereas Met2 restores wild type growth rates in S.cerevisiae.
6) Overexpression of the Ca ACCase to Facilitate Protein Purification and use for Screening Purposes
Materials
Growth Media:
Sabouraud Dextrose broth
Yeast peptone dextrose broth (YPD)
Yeast peptone galactose broth (YPGal) (i.e. 2% w/v galactose)
Growth of Cells
Candida albicans B2630 (Janssen Pharmaceutica, Beerse, Belgium) was maintained on Sabouraud dextrose agar slopes at 37° C. which were subcultured biweekly. For the growth of liquid cultures for experiments, C. albicans grown on Sabourauds dextrose agar for 48 h at 37° C. was used to inoculate 50 ml Sabouraud dextrose broth containing 500 μg/l d-biotin. This was incubated for 16 h at 37° C. on a platform shaker (150 rpm). 1.5 ml of this culture was added to each of 24×2 litre conical flasks, each containing 1 litre of Sabouraud dextrose broth containing 500 μg/l d-biotin, giving a final inoculum concentration of approximately 1.5×10 6 cfu ml −1 . The cultures were grown for 9 h, at 37° C. (log phase) with shaking (150 rpm). Cell numbers in liquid culture were determined spectrophotometrically (Philips PU8630 UV/VIS/NIR Spectrophotometer) at 540 nm in a 1 cm path length cuvette. Absorbance was linearly related to cell number up to an OD. of 2.0.
Saccharomyces cerevisiae strains Mey 134 and CLS2-13 were maintained on Yeast peptone dextrose (YPD) agar plates at 30° C., which were subcultured biweekly. For the growth of liquid cultures for experiments, the S. cerevisiae strains were grown on YPD agar for 48 h at 30° C. and were then used to inoculate 50 ml YPD broth containing 500 μg/l d-biotin, which was incubated at 30° C. for 16h on a platform shaker (200 rpm). 2.0 ml of this culture (approx. 4×10 8 cfu/ml) was added to each of 24×2 litre conical flasks, each containing 1 litre of YPD broth containing 500 μg/l d-biotin, giving a final inoculum concentration of approximately 8×10 5 cfu/ml. The cultures were grown for 9 h, at 30° C. (log phase) with shaking (200 rpm). Cell numbers in liquid culture were determined spectrophotometrically (Philips PU8630 UV/VIS/NIR Spectrophotometer) at 540 nm in a 1 cm path length cuvette.
Saccharomyces cerevisiae strains PNS117a 5C, PNS117b 6A, and PNS120a 6C were maintained on Yeast peptone galactose (YPGal) agar plates at 30° C. which were subcultured biweekly. For the growth of liquid cultures for experiments, the S. cerevisiae strains were grown on YPGal agar for 48 h at 30° C. and were then used to inoculate 50 ml YPGal broth containing 500 μg/l d-biotin and 200 μg/ml kanomycin, which were incubated at 30° C. for 30h on a platform shaker (200 rpm). 2.0 ml of this culture (approx. 4×10 8 cfu/ml) was added to each of 24×2 litre conical flasks, each containing 1 litre of YPGal broth containing 500 μg/l d-biotin and 200 μg/ml kanomycin, giving a final inoculum concentration of approximately 8×10 5 cfu/ml. The cultures were grown for approximately 23h at 30° C. (log phase) with shaking (200 rpm).
Determination of Cell Number
Cell numbers were determined using a standard viable count agar based plating method, using the appropriate agar media.
Preparation of Fungal ACCase Enzyme
Cultures of the appropriate yeast strains were grown to the exponential phase of growth (for Saccharomyces and Candida strains respectively). These were then harvested by centrifugation (4400 g, 10 min, 4° C.), washed twice in 700 ml of 50 mM Tris pH7.5 containing 20% w/v gylcerol, resuspending the cell pellet each time. The final washed pellet was fully resuspended into a thick slurry using 10 to 20ml of buffer (50 mM Tris pH7.5 containing 1 mM EGTA, 1 mM EDTA (disodium salt), 1 mM DTT, 0.25mM Pefabloc hydrochloride, 1 μM Leupeptin hemisulphate, 1 μM Pepstatin A, 0.5 μM Trypsin inhibitor and 20% w/v glycerol). The volume of buffer required was dependent on the total packed cell wet weight. (i.e. 1 ml buffer added per 6 gm of packed wet cell pellet).
The cell paste was homogenised using a pre-cooled Bead-Beater (Biospec Products, Bartlesville, Okla. 74005) with 4×10 second Bursts, allowing 20 second intervals on ice. The preparation was then centrifuged at 31,180 g for 30 minutes. After centrifugation the supernatant was immediately decanted into a container, then aliquoted before snap freezing in liquid nitrogen. The preparation was then stored at −80° C. and was found to be stable for at least 2 months.
All enzyme preparation steps were carried out at +4° C., unless otherwise stated.
In-vitro ACCase Assay
The assay was conducted in 96 well, flat bottomed polystyrene microtitre plates. All test and control samples were tested in duplicate in this assay.
100 μl of the ACCase enzyme preparation (in 50 mM Tris pH7.5 containing 1 mM EGTA, 1 mM EDTA (disodium salt), 1 mM DTT, 0.25 mM Pefabloc hydrochloride, 1 μM Leupeptin hemisulphate, 1 μM Pepstatin A, 0.5 μM Trypsin inhibitor, and 20% w/v glycerol) was added to each well of the microtitre plate. Each well contained either a 3 μl test sample made up in DMSO or 3 μl DMSO alone (NB. Final DMSO concentrations in the assay were 1.48% v/v). The microtitre plates were placed in a water bath maintained at 37° C. 10 μl of [ 14 C] NaHCO 3 containing 9.25 kBq in 378 mM NaHCO 3 was then added to each well. The reaction was initiated by the addition of 100 μl of Acetyl Coenzyme A containing assay buffer (50 mM Tris pH7.5 containing 4.41 mM ATP(disodium salt), 2.1 mM Acetyl Coenzyme A, 2.52 mM DTT, 10.5 mM MgCl 2 , and 0.21% w/v Albumin [Bovine, fraction V]), (removed from ice 5 minutes before use) to each well. The tubes were incubated at 37° C. for 5 minutes. The reaction was then terminated by the addition of 50 μl of 6M HCl to each well. In parallel, a pre-stopped assay control was set up which involved adding the 50 μl of 6M HCl prior to [ 4 C] NaHCO 3 and the assay buffer (No further HCl additions were made to these wells after the 5 minute incubation). The DPM values for the pre-stopped assay were subtracted from the normal assay situation.
After the addition of the stop reagent the plates were left open in the water bath for a further 30 minutes to allow the 14 CO 2 to escape. After this time 150 μl of each reaction mixture were applied onto individual GF/C glass microfibre filter discs and allowed to dry thoroughly before adding scintillation fluid. Radioactivity in the samples was then determined by scintillation counting (Wallac WinSpectral 1414, Turku, Finland).
IC50's were calculated from the data using non-linear regression techniques available in the ORIGIN software package (Microcal Software Inc., Massachusetts, USA). Soraphen A which is a specific inhibitor of ACCase was supplied over the range 0.1 nM-100 μM in the dose response regimen of the assay.
Protein Determination
The total protein concentration of each ACCase preparation used was determined by the Coomasie Blue method (Pierce, Ill., USA), (using 1 cm path length cuvettes read 595 nm (Philips PU8630 UV/VIS/NIR Spectrophotometer).
In-vitro Antifungal Activity
Compounds were tested over a concentration range of 1024-0.00098 μg/ml by a broth-dilution method in microtitre plates using doubling dilutions in YPD or YPGal (both containing 500 μg/l d-biotin). Stock solutions of inhibitors were prepared at 51.2 mg/ml in Dimethyl sulphoxide (DMSO) (final assay concentration of DMSO was 2% v/v). Each Yeast culture was added to the well to give a final 10 4 cfu/well. The plates were incubated at 30° C. for 48h and MIC's determined visually.
Discussion
Expression of ACCase, a biotinylated protein, was monitored by a “biotin-avidin affinity western blot” as described by Hal3lacher et al., 1993. Expression of the C. albicans ACC1 gene from its own promoter from plasmid Yep24 was comparable to that of the S. cerevisiae gene (no overexpression). Expression under control of the GAL1 promoter however, was considerably higher indicating a drastically increased level of biotinylated and therefore fully functional enzyme. Transcription of the gene was fully induced as the cells had to be grown on galactose to be viable. On glucose the GAL1 promoter is completely off, causing the cells to arrest and eventually die due to insufficient supply of ACCase). The S. cerevisiae strain described in this application is a convenient source of the C. albicans enzyme. The engineered strain possesses no residual background ACCase because the gene coding for the S. cerevisiae enzyme had been removed. Congenic versions of such a strain (genetically identical apart from the ACCase gene carried) expressing different ACCases (e.g. the different human (Abu-Elheiga et al. 1995), mammalian (Lopez-Casillas et al., 1988, Takai et al. 1988, Barber et al., 1995)), plant (Schulte et al., 1994) or other fitmgal enzymes (Al-Feel et al., 1992, Saito et al., 1996, Bailey et al., 1995)) can be used as tools for screening. Differences in growth of such strains may be solely dependent on differences in their ACCase activity. Differential growth in the presence of ACCase inhibitors (for example soraphenA or compounds yet to be identified) indicates selectivity of the drug towards one type of the ACCase enzyme.
References
Abu-Elheiga L., Jayakumar A., Baldini A., Chirala S. S., Wakil S. J.; Proc. Natl. Acad. Sci. U.S.A. 92: 4011-4015(1995).
Al-Feel W., Chirala S. S., Wakil S. J.; Proc. Natl. Acad. Sci. U.S.A. 89:4534-4538(1992). Bailey A. M., Keon J. P. R., Owen J., Hargreaves J. A.; Mol. Gen. Genet. 249:191-201(1995). Barber M. C., Travers M. T.; Gene 154:271-275(1995). Haβlacher M., Ivessa A. S., Paltauf F., Kohlwein S. D.; J. Biol. Chem. 268:10946-10952 (1988).
Ito, H., Fukuda, Y., Murata, K., Kimura, A.; J. Bacteriol. 153: 163-168 (1983)
Kunz. J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., Hall, M. N.; Cell 73: 585-596 (1993)
Lopez-Casillas F., Bai D.-H., Luo X., Kong I.-S., Hermodson M. A., Kim K.-H.; Proc. Natl. Acad. Sci. U.S.A. 85:5784-5788(1988).
Maniatis T., Frisch E. F., Sambrook J.; Molecular Cloning, Cold Spring Harbour Laboratory Press (1989)
Saiki R. K., Gelfand D. H., Stoffel S., Sharf S. J., Higuchi R., Hom G. T., Mullis K. B., Erlich H. A.; Science 239: 487-494 (1988)
Saito A., Kazuta Y., Toh H., Kondo H., Tanabe T.; S. pombe ACC1, Submitted (Dec-1996) to Embl/Genbank/Ddbj Data Banks.
Schulte W., Schell J., Toepfer R.; Plant Physiol. 106:793-794(1994).
Sherlock G., Bahman A. M., Mahal A., Shieh J. C., Fewrreira M., Rosamond J.; Mol. Gen. Genet. 245: 716-723.
Takai T., Yokoyama C., Wada K., Tanabe T.; J. Biol. Chem. 263:2651-2657(1988). Wach A., Brachat A., Poehlmann R., Philippseen P.; Yeast 10: 1793-1808 (1994)





TABLE 1



Comparative properties of native and

recombinant acetyl-CoA carboxylase enzymes













Specific






activity





IC50 for
of ACCase



Growth

Soraphen A
preparation


Cell
temperature
Liquid MIC
(nM)
(nmoles


doubling
for ACCase
(μg/ml)
against
product/


time
preparation
for
ACCase
min/mg

Yeast strain
(minutes)
(° C.)
Soraphen A
preparations
protein)











C. albicans

56
37
0.003



B2630

S. cerevisiae Mey
160
30
8

0.641

134


S. cerevisiae

163
30
2
 2.499
3.054

CLS2-13

S. cerevisiae PNS
253
30
2
17.518
7.025

117a 5C

S. cerevisiae PNS
222
30
4
13.083
10.573 

117b 6A

S. cerevisiae PNS
303
30
0.5
ND
0.244

120a 6C

S. cerevisiae PNS
287
30
0.125
ND
ND

120b 1C



Key: ND = not determined
















































3


1
523
DNA
Candida albicans

1
gcacgcttga cggttttcac caaatgcgaa aatatgacca aattgagaat ccgaaaatga 60
atggatagaa gattggttac caactgagaa ataaccccac acattagaag aagaacggaa 120
attcaattca tgtaaagaac caccacttgg tttaaaacct tcaccaggat cttcagaagt 180
aatacgacaa gcagtacaat gtccctttgg tgttggtctt ctttgactaa ccaatgaagt 240
ttctgacttg aattcaaaat caatatcagt agtggtatga ggatcggcac cgcacaaagt 300
tctgatatct ctgattctat gcattggtat acccatagca atttgtaatt gagcagctgg 360
taaattaaca cctgtcacca tttcagtggt tggatgttca acttgcaatc ttgggttcaa 420
ttccaaaaag tagaatttat cttcagcgtg gggagtaaag gtactcaaca gtaccagggg 480
gttacataac caacttattt tacccaatct gactggtgga ttt 523


2
8054
DNA
Candida albicans

2
aatatattgc ttccttttga taggaagtaa ctccgagtgt ttgaatttga tatatgttat 60
tcatatacgt tcaatggctc tcttctatgc tttgtatata ctttcttttg aatagatact 120
catgtaaaga gatttgaaac catattctaa ccaacaaaaa tattgtacgg tataggttag 180
aaaaaaaact ccgtaaggtc cgcttacacg gttaaattga aaacacgtta aaaatatatt 240
tgggtaatgg actaagctat atacagtact caacaaaaat gaaatcaaac acaatgttct 300
ttgggaaatt catttcatgc aactagggtg attctctttc tactatccaa caacgataac 360
cctgcttttg aaaaatcttt tctaaattca aattgatata attcttattt atatattact 420
ttctttttcc catataaccc catttttttt ttggaatcat atttgttttt gatttttgct 480
ttccctttca gtctgaggaa catactaatt acgaacaaca attatacatc caatcttcat 540
ctaacgaatt gattatttac atttattaaa cccttggata caaactgatt acacttttta 600
gttagtttgt tcaattataa gggtattata caacaaagat atcatttaaa gttaaatctc 660
aatctggaat aataaaagta ttcaacactt ttgcttacaa taggtatgtt caaaatcaat 720
tgaagccatc gagataagaa attaagcaaa aacgtttaca attgttgtgt gtgtgttgca 780
gtgtttgaag aagctcgagt gattgctttt cttcggcatc agctgtgttg ggaacatctt 840
gtcgttaaag tttcggagta atattagagt aatggaacga aaaaaacaaa ataaagttct 900
ggaaccacaa agatttgaaa aattgggtag aaacaaaaaa aagacaaagc aggaacccaa 960
caataaatga ataaacactc aaaaactact cacaacaaca acacttattt tcacttgctt 1020
tatttcttcg attttttatg agatgcaaat tatctctaat aaagaatact aactcacttg 1080
tacatagatc gcgtttccta attacaaaac cacaactata tatacctcat cgtcattata 1140
tcccattcaa gaacatattc aagtcattgt taatgtcaga tcaatctcca tctcctagtc 1200
ctagcgattc ccttagctac actacattac atgaaaattt gccatctcat ttcttgggtg 1260
gaaattcagt tttgaatgct gaaccttcta aagtcagaga ctttgtcaga gctcatcaag 1320
gtcatacagt tatttcgaaa attttaattg ccaacaatgg tatagctgca gttaaagaaa 1380
tcagatcagt tagaaaatgg gcttatgaaa catttggtga cgaaaaagcc atacagttta 1440
ccgttatggc cactccagaa gatttggaag ctaatgccga atatattaga atggccgacc 1500
aattcattga agtccctggt ggcaccaata acaataacta tgctaatgtt gatctcattg 1560
tagagatagc agaaagtaca aatgctcatg ccgtttgggc tgggtggggg catgcttcag 1620
agaatccttt gttaccagaa aaattagctg catctcccaa aaaaattatt tttattggtc 1680
ctcctggttc agctatgaga tctttaggtg acaagatttc atctactata gttgctcaac 1740
atgctcaagt accatgtatt ccatggtccg gtactggtgt tgatgaagtg aaaatagacc 1800
cacaaactaa tttggtttct gttgctgatg atatttatgc caaagggtgc tgtactagtc 1860
cagaagatgg tttagaaaaa gccaaaaaaa ttgggttccc agttatgatt aaagcctctg 1920
aaggtggtgg tggtaaaggt attagaaaag ttgatgatga gaaaaacttc attaccttat 1980
acaaccaagc agctaatgaa ataccaggtt ctcctatctt tattatgaag ttagcaggtg 2040
atgccagaca tttagaagtt caattactag cagatcaata cggtactaac atttcccttt 2100
ttggaagaga ttgttccgta caaagaagac accaaaagat tattgaagaa gcaccagtca 2160
ccattgccag aaaggaaact ttccacgaaa tggaaaatgc agcagtcaga ttgggtaaat 2220
tagttggtta tgtatccgct ggtactgttg agtatcttta ctcccacgct gaagataaat 2280
tctacttttt ggaattgaac ccaagattgc aagttgaaca tccaaccact gaaatggtga 2340
caggtgttaa tttaccagct gctcaattac aaattgctat gggtatacca atgcatagaa 2400
tcagagatat cagaactttg tacggtgccg atcctcatac cactactgat attgattttg 2460
aattcaagtc agaaacttca ttggttagtc aaagaagacc aacaccaaag ggacattgta 2520
ctgcttgtcg tattacttct gaagatcctg gtgaaggttt taaaccaagt ggtggttctt 2580
tacatgaatt gaatttccgt tcttcttcta atgtgtgggg ttatttctca gttggtaacc 2640
aatcttctat ccattcattt tcggattctc aatttggtca tattttcgca tttggtgaaa 2700
accgtcaagc ttcaagaaaa catatggttg ttgccttgaa agaattgagt attagaggtg 2760
attttagaac tactgttgag tatttaatca aattgttaga aactccagat ttcgaggata 2820
ataccattac aactggttgg ttggatgaat taatcaccaa aaagttgact gctgaaagac 2880
cagatccaat agttgctgtt gtttgtggag ctgtaaccaa agcacacatc caggctgagg 2940
aagagaaaaa ggaatacatc caatctttgg aaaaaggtca agttcctcac agaaacttat 3000
tgaaaactat tttcccagtt gagtttattt atgaaggtga aagatacaag ttcactgcta 3060
ctaaatcttc agaagataaa tatactttgt tccttaatgg ttctcgttgt gttgttggtg 3120
cacgttcatt gtccgatggt ggtttattgt gtgcattaga tgggaaatca cattctgtct 3180
attggaagga agaggcatct gccactagat tatcagttga tggcaaaact tgtttattag 3240
aagttgaaaa tgatccaaca caattaagaa ctccatctcc aggtaaattg gtcaagtatt 3300
tggttgacag tggtgaacat gttgatgctg gtcaaccata cgctgaagtc gaagttatga 3360
aaatgtgtat gcctttgatt gctcaagaaa atggggtagt gcagttgatt aaacaaccgg 3420
gttccacagt taatgctggt gatatcttgg ccattttggc attggacgat ccatctaagg 3480
tcaaacatgc taaaccattt gaaggtactt taccatctat gggtgagcca aatgttacag 3540
gtactaaacc agcacataaa ttcaatcatt gtgctggtat tttgaaaaac attttggctg 3600
gttatgataa tcaagtgatt ttgaattcta ctttaaagag tcttggtgaa gttttgaaag 3660
acaatgaatt gccatactct gaatggcaac aacaaatttc agctttacac tccagattgc 3720
cacctaaatt ggatgacgga ttgactgcat tggttgaaag aactcaaagt agaggtgctg 3780
aattccctgc tcgtcaaatt ttaaaactca tcaccaaatc aattgctgaa aatggtaatg 3840
atatgttaga agatgttgtt gcaccattgg tttctattgc cacaagttac cagaatggtt 3900
tggttgaaca cgaatacgat tactttgcat ctttgattaa cgaatattat gacgttgaaa 3960
gtttgttttc aggtgaaaat gttagagaag ataatgttat cttgaaatta agagatgaaa 4020
acaaatctga tttgaaaaaa gttattggta ttggtttgtc tcattcacgt gttagtgcca 4080
agaacaattt gattttagct attttggaca tttatgaacc attgttgcaa tccaactcgt 4140
cagttgctgc ctctatcaga gaagctttaa agaacttgtt cattagacct cgtgcttgtg 4200
ccaaagttgc attaaaggca agagaaattt taattcaatg ttctttacct tccatcaagg 4260
aaagatccga tcaattggaa catattttga ggtcatctgt tgttcaaacc tcttatggtg 4320
aaatttttgc taaacataga gaaccaaatt tggaaattat tcgtgaggtt gttgattcca 4380
aacatattgt ttttgatgtg ttggcacaat tcttaatcaa tccagaccca tgggttgcca 4440
ttgctgccgc tgaagtttat gtcagacgtt cataccgtgc ttatgatttg ggtaaaattg 4500
aatatcatgt taatgacaga cttcctattg ttgaatggaa attcaagttg gctaatatgg 4560
gagccgctgg tgtaaacgat gctcaacagg ctgctgctgc cggtggcgat gattcgacat 4620
ctatgaaaca tgcagcttct gtgtctgatt tgacctttgt tgttgattct aaaaccgagc 4680
attccacaag aactggtgtt ttagctccag caagacactt ggatgatgtt gatgaaactc 4740
ttacagctgc attggaacaa ttccaaccag ccgatgctat ttcatttaaa gcaaagggtg 4800
aaactccaga gttattaaat gttttgaata ttgtcattac cagtattgat ggttactccg 4860
atgaaaatga atacttgagc agaattaatg aaatcttgtg cgaatacaaa gaagagttga 4920
tttctgctgg tgttcgtcgt gttacatttg tttttgctca tcaaattggt caatatccta 4980
aatattatac ttttactggt cctgactatg aagaaaacaa ggttattaga cacattgaac 5040
cagctttggc tttccaattg gaattgggaa gattagccaa tttcgatatc aaaccaattt 5100
tcactaacaa cagaaacatc catgtatatg atgcaattgg gaagaatgct ccttctgata 5160
aaagattttt caccagaggg attattagaa ccggtgttct taaagaagac attagcatta 5220
gtgaatattt gattgctgaa tccaacagat taatgaatga tattttggat actttagaag 5280
ttattgacac ttctaattct gatttaaacc atattttcat taacttttcc aatgctttca 5340
atgttcaagc ttcagatgtt gaggctgcct ttggatcatt cttagaaaga tttggtagaa 5400
gattatggag attaagagtt actggtgctg aaattagaat tgtctgtact gatcctcaag 5460
gtacttcgtt cccattgcgt gctatcatta ataatgtttc tggttatgtt gtcaaatcag 5520
aattgtattt ggaagtgaaa aatcctaaag gtgaatgggt tttcaaatcc attggtcatc 5580
ctggttccat gcatttgaga cctatctcaa ctccatatcc agttaaagaa tctttacaac 5640
caaaacgtta caaggctcac aatatgggta ccacttatgt gtatgacttc ccagaattgt 5700
ttcgtcaagc aacaatttca caatggaaaa aatatggcaa aaaagtacca aaagatgttt 5760
tcgtgtcttt agaattgatc actgatgaaa ctgattcctt aatagctgtt gaaagagatc 5820
cgggtgctaa caaaattgga atggttggat tcaaagtcac tgctaaaact cctgaatacc 5880
ctcatggtcg tcaattaatt attgttgcca atgatatcac ccacaagatt ggttcttttg 5940
gtccagaaga agataattat ttcaacaagt gtactgaatt ggccagaaaa ttaggtattc 6000
caagaattta cctttctgca aattcaggtg ctagaattgg tgttgctgag gaattgattc 6060
cattatacca agttgcctgg aatgaagaag ggtctcctga caaaggattc agatacttgt 6120
acttgagtac tgctgctaaa gagtctttag aaaaagatgg taaaagtgac agtgttgtta 6180
ctgaacgtat tgttgaaaaa ggtgaagagc gtcatgtcat taaagctatt attggtgccg 6240
aagatggctt aggggttgaa tgtcttaaag gatcaggttt aattgctggt gccacatcaa 6300
gagcttacaa ggatatattt accatcactt tggtaacttg tagatctgtt ggtattggtg 6360
cttatttggt tagattgggt caaagagcca ttcaaatcga tggtcaacct attattttaa 6420
ctggtgctcc tgctatcaat aaattgttgg gtagagaagt gtattcttcc aatcttcaat 6480
tgggtggtac tcaaatcatg tacaataatg gtgtttctca tttgacagct aatgatgatt 6540
tggctggggt tgaaaaaatt atggaatggt tatcatatgt tccagctaaa cgtggtttac 6600
cagtgccaat tttggaatca gaagattctt gggacagaga tgttgattac tacccaccaa 6660
aacaagaagc ttttgatgtt agatggatga tccaaggtag agaagttgat ggtgaatatg 6720
aatctgggtt atttgataaa gattcattcc aagaaacatt atctggttgg gctaaaggtg 6780
ttgttgttgg tagagcacgt ttgggtggta ttccaattgg tgttattggt gtcgaaacca 6840
gaacagtgga aaacttgatt cctgctgatc cagcaaatcc agactctaca gaaagtttga 6900
ttcaagaagc aggtcaagtg tggtatccta actctgcttt taagacagca caagctataa 6960
atgatttcaa caatggtgaa caattgccat taatgatttt agcaaattgg agaggtttct 7020
ctggtggtca aagagatatg tacaatgaag tcttgaaata tggttcattt attgttgatg 7080
ctttagttga cttcaagcaa cctatcttca cttacattcc accaaatgga gaattgagag 7140
gtggctcttg ggttgttgtt gatccaacca tcaactcaga tatgatggaa atgtatgccg 7200
atgtcgattc gagagctggt gttttggaac cagaaggtat ggttggtatc aaatacagac 7260
gtgataaatt attagcaact atggaaagat tagatccaac ttatggtgaa atgaaagcta 7320
agttaaatga ctcgtcatta tctccagaag aacactcgaa aataagcgcc aaattgtttg 7380
cacgtgaaaa ggctttatta ccaatttatg ctcaaatttc cgttcaattt gctgacttgc 7440
acgatagatc aggtcgtatg ttggccaagg gagttattag aaaggaaatc aaatggactg 7500
atgctagacg tttcttcttc tggagattga gaagaagatt gaacgaggaa tatgttttga 7560
gattgattag tgaacaaatt aaagattcta gcaaattgga aagagttgcc agattgaaga 7620
gttggatgcc aactgttgaa tacgatgatg accaagctgt cagtaactgg attgaagaga 7680
accatgccaa attgcaaaag agagttaatg aattgaaaca agaagtttca agaaccaaga 7740
ttatgagatt attaaaagag gatccaaata gtgcaatttc tgcaatgaaa gactatgttg 7800
aaagattgtc aaaagaagat aaagagaaat tcctcaaggc attgaagtag aagtggtttc 7860
cattaattca actttttaat gacattgaaa gtagtagtag ttgttgtttt ttagatttaa 7920
gtatattata ttatgtaata aattatagaa agtaattata gttttgacgg ttaattgacg 7980
agagtgggaa attggctttt ttgttgctcg tgtgatgaaa cagtgattga cacaaaaaaa 8040
tagacaatga aaac 8054


3
2270
PRT
Candida albicans

3
Met Arg Cys Lys Leu Ser Leu Ile Lys Asn Thr Asn Ser Leu Val His
1 5 10 15
Arg Ser Arg Phe Leu Ile Thr Lys Pro Gln Leu Tyr Ile Pro His Arg
20 25 30
His Tyr Ile Pro Phe Lys Asn Ile Phe Lys Ser Leu Leu Met Ser Asp
35 40 45
Gln Ser Pro Ser Pro Ser Pro Ser Asp Ser Leu Ser Tyr Thr Thr Leu
50 55 60
His Glu Asn Leu Pro Ser His Phe Leu Gly Gly Asn Ser Val Leu Asn
65 70 75 80
Ala Glu Pro Ser Lys Val Arg Asp Phe Val Arg Ala His Gln Gly His
85 90 95
Thr Val Ile Ser Lys Ile Leu Ile Ala Asn Asn Gly Ile Ala Ala Val
100 105 110
Lys Glu Ile Arg Ser Val Arg Lys Trp Ala Tyr Glu Thr Phe Gly Asp
115 120 125
Glu Lys Ala Ile Gln Phe Thr Val Met Ala Thr Pro Glu Asp Leu Glu
130 135 140
Ala Asn Ala Glu Tyr Ile Arg Met Ala Asp Gln Phe Ile Glu Val Pro
145 150 155 160
Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Asp Leu Ile Val Glu
165 170 175
Ile Ala Glu Ser Thr Asn Ala His Ala Val Trp Ala Gly Trp Gly His
180 185 190
Ala Ser Glu Asn Pro Leu Leu Pro Glu Lys Leu Ala Ala Ser Pro Lys
195 200 205
Lys Ile Ile Phe Ile Gly Pro Pro Gly Ser Ala Met Arg Ser Leu Gly
210 215 220
Asp Lys Ile Ser Ser Thr Ile Val Ala Gln His Ala Gln Val Pro Cys
225 230 235 240
Ile Pro Trp Ser Gly Thr Gly Val Asp Glu Val Lys Ile Asp Pro Gln
245 250 255
Thr Asn Leu Val Ser Val Ala Asp Asp Ile Tyr Ala Lys Gly Cys Cys
260 265 270
Thr Ser Pro Glu Asp Gly Leu Glu Lys Ala Lys Lys Ile Gly Phe Pro
275 280 285
Val Met Ile Lys Ala Ser Glu Gly Gly Gly Gly Lys Gly Ile Arg Lys
290 295 300
Val Asp Asp Glu Lys Asn Phe Ile Thr Leu Tyr Asn Gln Ala Ala Asn
305 310 315 320
Glu Ile Pro Gly Ser Pro Ile Phe Ile Met Lys Leu Ala Gly Asp Ala
325 330 335
Arg His Leu Glu Val Gln Leu Leu Ala Asp Gln Tyr Gly Thr Asn Ile
340 345 350
Ser Leu Phe Gly Arg Asp Cys Ser Val Gln Arg Arg His Gln Lys Ile
355 360 365
Ile Glu Glu Ala Pro Val Thr Ile Ala Arg Lys Glu Thr Phe His Glu
370 375 380
Met Glu Asn Ala Ala Val Arg Leu Gly Lys Leu Val Gly Tyr Val Ser
385 390 395 400
Ala Gly Thr Val Glu Tyr Leu Tyr Ser His Ala Glu Asp Lys Phe Tyr
405 410 415
Phe Leu Glu Leu Asn Pro Arg Leu Gln Val Glu His Pro Thr Thr Glu
420 425 430
Met Val Thr Gly Val Asn Leu Pro Ala Ala Gln Leu Gln Ile Ala Met
435 440 445
Gly Ile Pro Met His Arg Ile Arg Asp Ile Arg Thr Leu Tyr Gly Ala
450 455 460
Asp Pro His Thr Thr Thr Asp Ile Asp Phe Glu Phe Lys Ser Glu Thr
465 470 475 480
Ser Leu Val Ser Gln Arg Arg Pro Thr Pro Lys Gly His Cys Thr Ala
485 490 495
Cys Arg Ile Thr Ser Glu Asp Pro Gly Glu Gly Phe Lys Pro Ser Gly
500 505 510
Gly Ser Leu His Glu Leu Asn Phe Arg Ser Ser Ser Asn Val Trp Gly
515 520 525
Tyr Phe Ser Val Gly Asn Gln Ser Ser Ile His Ser Phe Ser Asp Ser
530 535 540
Gln Phe Gly His Ile Phe Ala Phe Gly Glu Asn Arg Gln Ala Ser Arg
545 550 555 560
Lys His Met Val Val Ala Leu Lys Glu Leu Ser Ile Arg Gly Asp Phe
565 570 575
Arg Thr Thr Val Glu Tyr Leu Ile Lys Leu Leu Glu Thr Pro Asp Phe
580 585 590
Glu Asp Asn Thr Ile Thr Thr Gly Trp Leu Asp Glu Leu Ile Thr Lys
595 600 605
Lys Leu Thr Ala Glu Arg Pro Asp Pro Ile Val Ala Val Val Cys Gly
610 615 620
Ala Val Thr Lys Ala His Ile Gln Ala Glu Glu Glu Lys Lys Glu Tyr
625 630 635 640
Ile Gln Ser Leu Glu Lys Gly Gln Val Pro His Arg Asn Leu Leu Lys
645 650 655
Thr Ile Phe Pro Val Glu Phe Ile Tyr Glu Gly Glu Arg Tyr Lys Phe
660 665 670
Thr Ala Thr Lys Ser Ser Glu Asp Lys Tyr Thr Leu Phe Leu Asn Gly
675 680 685
Ser Arg Cys Val Val Gly Ala Arg Ser Leu Ser Asp Gly Gly Leu Leu
690 695 700
Cys Ala Leu Asp Gly Lys Ser His Ser Val Tyr Trp Lys Glu Glu Ala
705 710 715 720
Ser Ala Thr Arg Leu Ser Val Asp Gly Lys Thr Cys Leu Leu Glu Val
725 730 735
Glu Asn Asp Pro Thr Gln Leu Arg Thr Pro Ser Pro Gly Lys Leu Val
740 745 750
Lys Tyr Leu Val Asp Ser Gly Glu His Val Asp Ala Gly Gln Pro Tyr
755 760 765
Ala Glu Val Glu Val Met Lys Met Cys Met Pro Leu Ile Ala Gln Glu
770 775 780
Asn Gly Val Val Gln Leu Ile Lys Gln Pro Gly Ser Thr Val Asn Ala
785 790 795 800
Gly Asp Ile Leu Ala Ile Leu Ala Leu Asp Asp Pro Ser Lys Val Lys
805 810 815
His Ala Lys Pro Phe Glu Gly Thr Leu Pro Ser Met Gly Glu Pro Asn
820 825 830
Val Thr Gly Thr Lys Pro Ala His Lys Phe Asn His Cys Ala Gly Ile
835 840 845
Leu Lys Asn Ile Leu Ala Gly Tyr Asp Asn Gln Val Ile Leu Asn Ser
850 855 860
Thr Leu Lys Ser Leu Gly Glu Val Leu Lys Asp Asn Glu Leu Pro Tyr
865 870 875 880
Ser Glu Trp Gln Gln Gln Ile Ser Ala Leu His Ser Arg Leu Pro Pro
885 890 895
Lys Leu Asp Asp Gly Leu Thr Ala Leu Val Glu Arg Thr Gln Ser Arg
900 905 910
Gly Ala Glu Phe Pro Ala Arg Gln Ile Leu Lys Leu Ile Thr Lys Ser
915 920 925
Ile Ala Glu Asn Gly Asn Asp Met Leu Glu Asp Val Val Ala Pro Leu
930 935 940
Val Ser Ile Ala Thr Ser Tyr Gln Asn Gly Leu Val Glu His Glu Tyr
945 950 955 960
Asp Tyr Phe Ala Ser Leu Ile Asn Glu Tyr Tyr Asp Val Glu Ser Leu
965 970 975
Phe Ser Gly Glu Asn Val Arg Glu Asp Asn Val Ile Leu Lys Leu Arg
980 985 990
Asp Glu Asn Lys Ser Asp Leu Lys Lys Val Ile Gly Ile Gly Leu Ser
995 1000 1005
His Ser Arg Val Ser Ala Lys Asn Asn Leu Ile Leu Ala Ile Leu Asp
1010 1015 1020
Ile Tyr Glu Pro Leu Leu Gln Ser Asn Ser Ser Val Ala Ala Ser Ile
1025 1030 1035 1040
Arg Glu Ala Leu Lys Asn Leu Phe Ile Arg Pro Arg Ala Cys Ala Lys
1045 1050 1055
Val Ala Leu Lys Ala Arg Glu Ile Leu Ile Gln Cys Ser Leu Pro Ser
1060 1065 1070
Ile Lys Glu Arg Ser Asp Gln Leu Glu His Ile Leu Arg Ser Ser Val
1075 1080 1085
Val Gln Thr Ser Tyr Gly Glu Ile Phe Ala Lys His Arg Glu Pro Asn
1090 1095 1100
Leu Glu Ile Ile Arg Glu Val Val Asp Ser Lys His Ile Val Phe Asp
1105 1110 1115 1120
Val Leu Ala Gln Phe Leu Ile Asn Pro Asp Pro Trp Val Ala Ile Ala
1125 1130 1135
Ala Ala Glu Val Tyr Val Arg Arg Ser Tyr Arg Ala Tyr Asp Leu Gly
1140 1145 1150
Lys Ile Glu Tyr His Val Asn Asp Arg Leu Pro Ile Val Glu Trp Lys
1155 1160 1165
Phe Lys Leu Ala Asn Met Gly Ala Ala Gly Val Asn Asp Ala Gln Gln
1170 1175 1180
Ala Ala Ala Ala Gly Gly Asp Asp Ser Thr Ser Met Lys His Ala Ala
1185 1190 1195 1200
Ser Val Ser Asp Leu Thr Phe Val Val Asp Ser Lys Thr Glu His Ser
1205 1210 1215
Thr Arg Thr Gly Val Leu Ala Pro Ala Arg His Leu Asp Asp Val Asp
1220 1225 1230
Glu Thr Leu Thr Ala Ala Leu Glu Gln Phe Gln Pro Ala Asp Ala Ile
1235 1240 1245
Ser Phe Lys Ala Lys Gly Glu Thr Pro Glu Leu Leu Asn Val Leu Asn
1250 1255 1260
Ile Val Ile Thr Ser Ile Asp Gly Tyr Ser Asp Glu Asn Glu Tyr Leu
1265 1270 1275 1280
Ser Arg Ile Asn Glu Ile Leu Cys Glu Tyr Lys Glu Glu Leu Ile Ser
1285 1290 1295
Ala Gly Val Arg Arg Val Thr Phe Val Phe Ala His Gln Ile Gly Gln
1300 1305 1310
Tyr Pro Lys Tyr Tyr Thr Phe Thr Gly Pro Asp Tyr Glu Glu Asn Lys
1315 1320 1325
Val Ile Arg His Ile Glu Pro Ala Leu Ala Phe Gln Leu Glu Leu Gly
1330 1335 1340
Arg Leu Ala Asn Phe Asp Ile Lys Pro Ile Phe Thr Asn Asn Arg Asn
1345 1350 1355 1360
Ile His Val Tyr Asp Ala Ile Gly Lys Asn Ala Pro Ser Asp Lys Arg
1365 1370 1375
Phe Phe Thr Arg Gly Ile Ile Arg Thr Gly Val Leu Lys Glu Asp Ile
1380 1385 1390
Ser Ile Ser Glu Tyr Leu Ile Ala Glu Ser Asn Arg Leu Met Asn Asp
1395 1400 1405
Ile Leu Asp Thr Leu Glu Val Ile Asp Thr Ser Asn Ser Asp Leu Asn
1410 1415 1420
His Ile Phe Ile Asn Phe Ser Asn Ala Phe Asn Val Gln Ala Ser Asp
1425 1430 1435 1440
Val Glu Ala Ala Phe Gly Ser Phe Leu Glu Arg Phe Gly Arg Arg Leu
1445 1450 1455
Trp Arg Leu Arg Val Thr Gly Ala Glu Ile Arg Ile Val Cys Thr Asp
1460 1465 1470
Pro Gln Gly Thr Ser Phe Pro Leu Arg Ala Ile Ile Asn Asn Val Ser
1475 1480 1485
Gly Tyr Val Val Lys Ser Glu Leu Tyr Leu Glu Val Lys Asn Pro Lys
1490 1495 1500
Gly Glu Trp Val Phe Lys Ser Ile Gly His Pro Gly Ser Met His Leu
1505 1510 1515 1520
Arg Pro Ile Ser Thr Pro Tyr Pro Val Lys Glu Ser Leu Gln Pro Lys
1525 1530 1535
Arg Tyr Lys Ala His Asn Met Gly Thr Thr Tyr Val Tyr Asp Phe Pro
1540 1545 1550
Glu Leu Phe Arg Gln Ala Thr Ile Ser Gln Trp Lys Lys Tyr Gly Lys
1555 1560 1565
Lys Val Pro Lys Asp Val Phe Val Ser Leu Glu Leu Ile Thr Asp Glu
1570 1575 1580
Thr Asp Ser Leu Ile Ala Val Glu Arg Asp Pro Gly Ala Asn Lys Ile
1585 1590 1595 1600
Gly Met Val Gly Phe Lys Val Thr Ala Lys Thr Pro Glu Tyr Pro His
1605 1610 1615
Gly Arg Gln Leu Ile Ile Val Ala Asn Asp Ile Thr His Lys Ile Gly
1620 1625 1630
Ser Phe Gly Pro Glu Glu Asp Asn Tyr Phe Asn Lys Cys Thr Glu Leu
1635 1640 1645
Ala Arg Lys Leu Gly Ile Pro Arg Ile Tyr Leu Ser Ala Asn Ser Gly
1650 1655 1660
Ala Arg Ile Gly Val Ala Glu Glu Leu Ile Pro Leu Tyr Gln Val Ala
1665 1670 1675 1680
Trp Asn Glu Glu Gly Ser Pro Asp Lys Gly Phe Arg Tyr Leu Tyr Leu
1685 1690 1695
Ser Thr Ala Ala Lys Glu Ser Leu Glu Lys Asp Gly Lys Ser Asp Ser
1700 1705 1710
Val Val Thr Glu Arg Ile Val Glu Lys Gly Glu Glu Arg His Val Ile
1715 1720 1725
Lys Ala Ile Ile Gly Ala Glu Asp Gly Leu Gly Val Glu Cys Leu Lys
1730 1735 1740
Gly Ser Gly Leu Ile Ala Gly Ala Thr Ser Arg Ala Tyr Lys Asp Ile
1745 1750 1755 1760
Phe Thr Ile Thr Leu Val Thr Cys Arg Ser Val Gly Ile Gly Ala Tyr
1765 1770 1775
Leu Val Arg Leu Gly Gln Arg Ala Ile Gln Ile Asp Gly Gln Pro Ile
1780 1785 1790
Ile Leu Thr Gly Ala Pro Ala Ile Asn Lys Leu Leu Gly Arg Glu Val
1795 1800 1805
Tyr Ser Ser Asn Leu Gln Leu Gly Gly Thr Gln Ile Met Tyr Asn Asn
1810 1815 1820
Gly Val Ser His Leu Thr Ala Asn Asp Asp Leu Ala Gly Val Glu Lys
1825 1830 1835 1840
Ile Met Glu Trp Leu Ser Tyr Val Pro Ala Lys Arg Gly Leu Pro Val
1845 1850 1855
Pro Ile Leu Glu Ser Glu Asp Ser Trp Asp Arg Asp Val Asp Tyr Tyr
1860 1865 1870
Pro Pro Lys Gln Glu Ala Phe Asp Val Arg Trp Met Ile Gln Gly Arg
1875 1880 1885
Glu Val Asp Gly Glu Tyr Glu Ser Gly Leu Phe Asp Lys Asp Ser Phe
1890 1895 1900
Gln Glu Thr Leu Ser Gly Trp Ala Lys Gly Val Val Val Gly Arg Ala
1905 1910 1915 1920
Arg Leu Gly Gly Ile Pro Ile Gly Val Ile Gly Val Glu Thr Arg Thr
1925 1930 1935
Val Glu Asn Leu Ile Pro Ala Asp Pro Ala Asn Pro Asp Ser Thr Glu
1940 1945 1950
Ser Leu Ile Gln Glu Ala Gly Gln Val Trp Tyr Pro Asn Ser Ala Phe
1955 1960 1965
Lys Thr Ala Gln Ala Ile Asn Asp Phe Asn Asn Gly Glu Gln Leu Pro
1970 1975 1980
Leu Met Ile Leu Ala Asn Trp Arg Gly Phe Ser Gly Gly Gln Arg Asp
1985 1990 1995 2000
Met Tyr Asn Glu Val Leu Lys Tyr Gly Ser Phe Ile Val Asp Ala Leu
2005 2010 2015
Val Asp Phe Lys Gln Pro Ile Phe Thr Tyr Ile Pro Pro Asn Gly Glu
2020 2025 2030
Leu Arg Gly Gly Ser Trp Val Val Val Asp Pro Thr Ile Asn Ser Asp
2035 2040 2045
Met Met Glu Met Tyr Ala Asp Val Asp Ser Arg Ala Gly Val Leu Glu
2050 2055 2060
Pro Glu Gly Met Val Gly Ile Lys Tyr Arg Arg Asp Lys Leu Leu Ala
2065 2070 2075 2080
Thr Met Glu Arg Leu Asp Pro Thr Tyr Gly Glu Met Lys Ala Lys Leu
2085 2090 2095
Asn Asp Ser Ser Leu Ser Pro Glu Glu His Ser Lys Ile Ser Ala Lys
2100 2105 2110
Leu Phe Ala Arg Glu Lys Ala Leu Leu Pro Ile Tyr Ala Gln Ile Ser
2115 2120 2125
Val Gln Phe Ala Asp Leu His Asp Arg Ser Gly Arg Met Leu Ala Lys
2130 2135 2140
Gly Val Ile Arg Lys Glu Ile Lys Trp Thr Asp Ala Arg Arg Phe Phe
2145 2150 2155 2160
Phe Trp Arg Leu Arg Arg Arg Leu Asn Glu Glu Tyr Val Leu Arg Leu
2165 2170 2175
Ile Ser Glu Gln Ile Lys Asp Ser Ser Lys Leu Glu Arg Val Ala Arg
2180 2185 2190
Leu Lys Ser Trp Met Pro Thr Val Glu Tyr Asp Asp Asp Gln Ala Val
2195 2200 2205
Ser Asn Trp Ile Glu Glu Asn His Ala Lys Leu Gln Lys Arg Val Asn
2210 2215 2220
Glu Leu Lys Gln Glu Val Ser Arg Thr Lys Ile Met Arg Leu Leu Lys
2225 2230 2235 2240
Glu Asp Pro Asn Ser Ala Ile Ser Ala Met Lys Asp Tyr Val Glu Arg
2245 2250 2255
Leu Ser Lys Glu Asp Lys Glu Lys Phe Leu Lys Ala Leu Lys
2260 2265 2270