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WO1998038331A1 - Interactions du gene skn7 et utilisation dudit gene dans des procedes de dosage - Google Patents

Interactions du gene skn7 et utilisation dudit gene dans des procedes de dosage Download PDF

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Publication number
WO1998038331A1
WO1998038331A1 PCT/GB1998/000643 GB9800643W WO9838331A1 WO 1998038331 A1 WO1998038331 A1 WO 1998038331A1 GB 9800643 W GB9800643 W GB 9800643W WO 9838331 A1 WO9838331 A1 WO 9838331A1
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Prior art keywords
skn7
polypeptide
binding
yapl
gene
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PCT/GB1998/000643
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English (en)
Inventor
Leland Herries Johnston
Brian Alexander Morgan
Desmond Colum Raitt
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Medical Research Council
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Priority to CA002281983A priority Critical patent/CA2281983A1/fr
Priority to AU66292/98A priority patent/AU6629298A/en
Priority to EP98908211A priority patent/EP0975793A1/fr
Priority to JP53744398A priority patent/JP2001512983A/ja
Publication of WO1998038331A1 publication Critical patent/WO1998038331A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to assay methods and means, and substances identified using assays.
  • the present invention is based on demonstion that the yeast polypeptide Skn7 acts as a transcription factor and that this is in cooperation with other cellular factors including Yapl and Hsfl.
  • Experimental evidence disclosed herein indicates that Skn7 up-regulates expression of genes involved in response to stress by binding a specific nucleotide sequence motif.
  • Substances which disrupt the function of Skn7, by interfering with its interaction with Yapl, Hsfl, or its interaction with the specific nucleotide sequence motif may be used as anti- fungal agents.
  • Skn7 was identified originally as a gene which when over- expressed could alleviate a particular defect in the cell wall of the budding yeast, suggesting a role in intracellular biochemical pathways that control cell wall structure (Brown et al . , (1993) J " . Bacteriol 175: 6908-6915). Krems et al . (1996) Curr. Genet . 29: 327-334) observed that strains lacking the Skn7 gene are extremely sensitive to oxidative stress, indicating a role in the cellular response to this toxicity. This is consistent with the previous results in that stress such as heat shock leads to a specific arrest of yeast cells in the Gl phase of the cell cycle and cells respond to some stresses by altering cell wall structure.
  • the invention provides an assay for a putative inhibitor of fungal growth comprising: a) bringing into contact Skn7 polypeptide and a putative inhibitor compound under conditions where the Skn7 polypeptide, in the absence of inhibitor, is capable of binding to a Skn7-polypeptide-specific nucleotide sequence; b) providing a nucleic acid molecule which includes a Skn7- polypeptide-specific binding nucleotide sequence to which the polypeptide is capable of binding to activate transcription of a sequence operably linked to the Skn7- polypeptide-specific binding nucleotide sequence; and c) measuring the degree of inhibition of binding of Skn7 to the Skn7-polypeptide-specific binding nucleotide sequence or transcriptional activation caused by said inhibitor compound .
  • the transcription factor Yapl binds to the TRX2 promoter with ablation of either the Skn7 or Yapl genes abolishing the stress-dependent induction of TRX2 and TRR1. While not wishing to be bound by any one particular theory, the evidence suggests that Skn7 and Yapl bind to different sites of the TRX2 and TRR1 promoters, and bring about a conformational change to the promoter which is dependent upon an interaction between the two transcription factors.
  • the invention provides an assay for a putative inhibitor of fungal growth comprising: a) bringing into contact a Skn7 polypeptide " and a Yapl polypeptide and a putative inhibitor compound under conditions where the Skn7 polypeptide, in the absence of inhibitor, is capable of binding a Skn7-polypeptide-specific nucleotide sequence and cooperating with Yapl to activate transcription; b) providing a nucleic acid molecule which includes a Skn7- polypeptide-specific binding nucleotide sequence to which the Skn7 polypeptide is capable of binding to activate transcription of a sequence operably linked to the Skn7- polypeptide-specific binding nucleotide sequence; and c) measuring the degree of inhibition of interaction of Skn7 polypeptide and Yapl polypeptide or of Skn7 polypeptide and the Skn7-polypeptide-specific binding nucleotide sequence or transcriptional activation caused by said inhibitor compound.
  • Skn7 interacts with the yeast Heat Shock Factor, Hsflp (referred to herein as Hsfl) , and that the direct physical interaction between these two proteins provides a maximal response to oxidative stress in yeast cells.
  • Hsflp yeast Heat Shock Factor
  • an assay for a putative inhibitor of fungal growth comprising: a) bringing into contact a Skn7 polypeptide, and a Hsfl polypeptide and a putative inhibitor compound under conditions where the Skn7 polypeptide, in the absence of inhibitor, is capable of binding to the Hsfl polypeptide; and b) measuring the degree of inhibition of binding between the Skn7 and Hsfl polypeptides caused by said inhibitor compound .
  • A Quantitation of RNA relative to actin from a Northern blot analysis of RNA isolated from different mid-log yeast strains prior to (tracks 1, 3, 5, 7, 9) and following treatment with lmM hydrogen peroxide for 1 hour at 25°C (tracks 2, 4, 6, 8, 10) . Strains used were W303-la (tracks 1 and 2) , skn7 ⁇ (tracks 3 and 4) , W303-lb (tracks 5 and 6) , yapl ⁇ (tracks 7 and 8) , and skn7 ⁇ yapl ⁇ (tracks 9 and 10) .
  • B Comparison of potential Yapl binding sites (Kuge and Jones, 1994) with a possible Yapl binding site in the TRR1 promoter.
  • Skn7 is required for induction by tetra-butyl -hydrogen peroxide of LacZ fused to the TRX2 promoter.
  • Mid-log cultures of W303-la, skn7 ⁇ and yapl ⁇ strains, transformed with the TRXLACZ plasmid (Kuge and Jones, 1994) were treated with tetra-butyl-hydrogen peroxide for 1 hour, ⁇ - galactosidase assays were performed on the untreated and treated cultures.
  • Fig.5. Skn7 binding region in the TRX2 promoter. Schematic diagram of the TRX2 promoter and the probes used to identify the Skn7 binding. Near match MCB and SCB elements are indicated. Probes are described in the Materials and Methods.
  • Fig.6 Alignment of Skn7 DNA binding domain, amino acids 87-150 (SEQ ID NO: 18) with DNA binding domains of S . cerevisiae Hsflp (SEQ ID N0.19), the fisson yeast Heat Shock Factor (Hsflp sp, SEQ ID NO: 20), and the human Heat Shock Factor 2 (Hsf2hs, SEQ ID NO: 21) Highly conserved residues which may directly contact the DNA and have diverged in Skn7 are indicated by * .
  • Fig.7 Map of Skn7 showing the HSF DNA binding domain, the potential coiled-coil structure, the homology to the bacterial response regulator domain and a region rich in gluatmine that is commonly found in eukaryotic transcription factors.
  • the present invention is concerned with the provision of assays, in particular assays for substances which inhibit interaction between Skn7 polypeptide and the specific nucleotide sequence motif it binds to. Further assays are for substances which inhibit interaction between Skn7 and Yapl polypeptides or Skn7 and Hsfl polypeptides. Substances which inhibit either or both of these interactions may be used in inhibit the response of a fungal cell to stress, and are thus anti -fungal agents.
  • the invention provides compounds obtainable by the above-described assays, for example peptide compounds or other small molecules.
  • Peptide compounds may be based on the portions of Skn7 polypeptide which interact with the Yapl or Hsfl polypeptides, or the corresponding portions of said polypeptides which interact with each Skn7.
  • Skn7 and Yapl polypeptides may be used in attacking any of a wide range of fungi, including ascomycete fungi and the others mentioned herein, such as, for example, fungi responsible for conditions such as Candidiasis, farmers' Lung, Cyrptococcosis and opportunistic fungal infections, e.g. as are prevalent in immuno-compromised individuals, such as transplant patients and AIDS sufferers,.
  • Skn7 polypeptide includes a polypeptide including the amino acid sequence for Saccharomyces cerevisiae wild-type Skn7 shown in Brown et al . , 1993, and Morgan et al . , 1995b and variants thereof (which may be naturally occurring or synthetic) , as discussed below, in particular showing a characteristic of S . cerevisiae Skn7 polypeptide, such as binding to the specific nucleotide sequence motif bound by S . cerevisiae Skn7, discussed below, or a variant thereof, transcription factor activity, particularly ability to activate transcription in cooperation with Yapl polypeptide, and/or a role in activation of a cellular response to stress, such as oxidative stress and/or heat shock.
  • Preferred Skn7 polypeptides have a DNA binding domain which includes a sequence of amino acids with at least about 60%, or 70%, or 80%, or 90%, or 95% identity with the following amino acid sequence, which is 80% identical in the homologue/analogue in the fission yeast S.pombe (as we have determined by comparison of the S . cerevisiae Skn7 polypeptide sequence with sequence information available to the public in the Fission Yeast Genome Sequencing Project, Sanger Centre, Cambridge, UK): LPNHFKHSNFASFVRQLNKYDFHKV (SEQ ID NO:l).
  • Variants include homologues and analogues which are likely to exist in all fungi, as evidenced by the very close structural identity, close sequence identity in the DNA binding domain, and high level of homology of the homologues/analogues in S. pombe and S . cerevisiae, which yeast are not closely related.
  • the S.pombe protein has the Hsfl-related DNA binding domain found in Skn7, a receiver domain and also a coiled-coil region and a glutamine-rich domain that occur in Skn7.
  • the present invention may be applied to any fungus, including pathogens such as Cryptococcus neoformans, Candida albicans and Aspergillus and others including those mentioned above.
  • the homologue or analogue of Skn7 of any of these organisms may be used in the present invention.
  • Yapl polypeptide includes a polypeptide including the amino acid sequence for Saccharomyces cerevisiae wild-type Yapl shown in Moye-Rowley et al . (1989) Genes and Dev. 3: 283- 292, and variants, thereof (which may be synthetic or naturally occurring) , as discussed below, in particular showing a characteristic of S . cerevisiae Yapl polypeptide, such as binding to its specific nucleotide sequence motif, or a variant thereof, ability to activate transcription in cooperation with Skn7 polypeptide, and/or a role in activation of a cellular response to stress, such as oxidative stress and/or heat shock.
  • Preferred Yapl polypeptides may include a DNA binding domain with at least about 50%, 60%, 70%, 80%, 85%, 88%, 90% or 95% identity with that of S . cerevisiae Yapl.
  • Papl the analogue of Yapl in fission yeast, has in its DNA binding domain a region of 88% identity with the S . cerevisiae Yapl and 50% identity over the 50 C-terminal amino acids (Toda et al . , (1991) Genes Dev. 5: 60-73) .
  • Hsfl polypeptide includes a polypeptide including the amino acid sequence for Saccharomyces cerevisiae wild-type Hsfl shown in Wiederrecht et al , 1988 and variants thereof (which may be synthetic or naturally occurring) , as discussed below, in particular showing a characteristic of S . cerevisiae Hsfl polypeptide, such as binding to the Skn7 ' polypeptide and/or cooperating with it in the induction of heat shock gene expression in response to oxidative stress.
  • Preferred Hsfl polypeptides may include a DNA binding domain with at least about 50%, 60%, 70%, 80%, 85%, 88%, 90% or 95% identity with that of S. cerevisiae Hsfl. This DNA binding domain is shown in Figure 6.
  • Variants of the above-described polypeptides may be mutants, such as temperature sensitive mutants, alleles such as sequence variants of the proteins described above from S . cerevisiae which demonstrate a substantially similar phenotype, homologues and analogues, such as found in other species, or synthetic variants and derivatives, which retain the functions of the above described proteins to the extent necessary for the paricular assay format being utilised.
  • mutants such as temperature sensitive mutants, alleles such as sequence variants of the proteins described above from S . cerevisiae which demonstrate a substantially similar phenotype, homologues and analogues, such as found in other species, or synthetic variants and derivatives, which retain the functions of the above described proteins to the extent necessary for the paricular assay format being utilised.
  • Yapl polypeptide or Hsfl polypeptide employed in various aspects and embodiments of the present invention may include an amino acid sequence which differs by one or more amino acid residues from the wild-type amino acid sequence, by one or more (e.g. from 1 to 20, such as 2 , 3, 5 or 10) of addition, insertion, deletion and substitution, preferably substitution, of one or more amino acids.
  • variants, derivatives, alleles, mutants and homologues e.g. from other organisms, are included.
  • the amino acid sequence of a variant shares homology with the S. cerevisiae Skn7, Yapl or Hsfl sequences, as the case may be.
  • the homology is a degree of amino acid identity of at least about 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or more preferably at least about 90% or 95% identity.
  • Variants also include fragments of wild type or variant Skn7 , Yapl and Hsfl proteins, since it is not necessary to use the entire proteins for assays of the invention. Fragments may be any suitable size, for example from 20 to 300 amino acids, for example from 100 to 200 amino acids.
  • Fragments may be generated and used in any suitable way known to those of skill in the art. Suitable ways of generating fragments include, but are not limited to, recombinant expression of a fragment from encoding DNA. Such fragments may be generated by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Small fragments (up to about 20 or 30 amino acids) may also be generated using peptide synthesis methods which are well known in the art.
  • Fragments include those which are an "active portion", which means a peptide which is less than said full length polypeptide, but which retains biological activity.
  • active portion which means a peptide which is less than said full length polypeptide, but which retains biological activity.
  • Skn7 polypeptide this is the ability to interact with Yapl or Hsfl polypeptides and/or the specific nucleotide sequence
  • Yapl and Hsfl polypeptides the ability to interact with Skn7 polypeptide.
  • Such portions may be used to interfere with interaction between Skn7 polypeptide and Yapl or Hsfl polypeptides and/or Skn7 polypeptide binding to its specific nucleic acid motif, with anti-fungal potential.
  • the Skn7 , Yapl .and Hsfl polypeptides and their variants, including fragments, may also comprise additional sequences, usually located at the N- and/or C-termini, which are useful for the provision of the assays described herein.
  • the additional sequences may comprise a polyhistidine or epitope (e.g. HA) tag to serve as a tag for pull -down or similar assays.
  • the additional sequences may alternatively be a functional domain, such as a domain for use in a two-hybrid assay.
  • the domain may be a marker domain, such as a beta-galactosidase, chloramphenicol acetyl transferase, luciferase or green fluorescent protein sequence.
  • homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine . Similarity may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J " . Mol . Biol . 215: 403-10, which is in standard use in the art.
  • This program may also be used to determine DNA homology.
  • Amino acid identity may also be determined by reference to the Smith-Waterman algorithm, currently used by the United States Patent and Trademark Office.
  • Homology, i.e. identity may be over the full-length of the relevant polypeptide or may more preferably be over a contiguous sequence of about 15, 20, 25, 30, 40, 50 or more amino acids, compared with the relevant wild-type amino acid sequence .
  • polypeptide may be provided in free from it may also be used in the form of a fusion protein linked to a marker or reporter protein.
  • Assays of the invention maybe conducted in the following ways, which are provided by way of illustration and are not limiting:
  • a two-hybrid assay comprises the expression in a host cell of the the two proteins, one being a fusion protein comprising a DNA binding domain (DBD) , such as the yeast GAL4 binding domain, and the other being a fusion protein comprising an activation domain, such as that from GAL4 or VP16.
  • DBD DNA binding domain
  • the host cell will carry a reporter gene construct with a promoter comprising a DNA binding element compatible with the DBD.
  • the reporter gene may be a reporter gene such as chloramphenical acetyl transferase, luciferase, green fluorescent protein (GFP) and ⁇ - galactosidase, with luciferase being particularly preferred.
  • Two-hybrid assays may be in accordance with those disclosed by Fields and Song, 1989, Nature 340; 245-246.
  • the DNA binding domain (DBD) and the transcriptional activation domain (TAD) of the yeast GAL4 transcription factor are fused to the first and second molecules respectively whose interaction is to be investigated.
  • a functional GAL4 transcription factor is restored only when two molecules of interest interact.
  • interaction of the molecules may be measured by the use of a reporter gene operably linked to a GAL4 DNA binding site which is capable of activating transcription of said reporter gene.
  • two hybrid assays may be performed in the presence of a potential modulator compound and the effect of the modulator will be reflected in the change in transcription level of the reporter gene construct compared to the transcription level in the absence of a modulator.
  • Host cells in which the two-hybrid assay may be conducted include mammalian, insect, yeast and bacterial cells, with mammalian and yeast cells (such as S. cerivisiae and S .pombe) being particularly preferred.
  • the Skn7 or Yapl polypeptide may be fused to a heterologous DNA binding domain such as that of the yeast transcription factor GAL 4.
  • the GAL 4 transcription factor includes two functional domains. These domains are the DNA binding domain (DBD) and the transcriptional activation domain (TAD) .
  • DBD DNA binding domain
  • TAD transcriptional activation domain
  • a functional GAL 4 transcription factor is restored only when two proteins of interest interact.
  • interaction of the proteins may be measured by the use of a reporter gene probably linked to a GAL 4 DNA binding site which is capable of activating transcription of said reporter gene.
  • the Yapl polypeptide may be replaced by the Hsfl polypeptide.
  • the interaction between the Skn7 and Yapl or Hsfl polypeptides may be studied in vi tro in a "pull -down" format by labelling one with a detectable label and bringing it into contact with the other which has been immobilised on a solid support, or carries a tag allowing it to be immobilised.
  • Suitable detectable labels include 35 S-methionine which may be incorporated into recombinantly produced Skn7 and/or Yapl and/or Hsfl polypeptides.
  • the recombinantly produced Skn7 and/or Yapl and/or Hsfl polypeptide may also be expressed as a fusion protein containing an epitope which can be labelled with an antibody.
  • the protein which is, or is to be, immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se .
  • a preferred in vi tro interaction may utilise a fusion protein including glutathione-S-transferase (GST) . This may be immobilized on glutathione agarose beads.
  • the putative inhibitor compound in an in vi tro assay format of the type described above can be assayed by determining its ability to diminish the amount of labelled Skn7 , Yapl or Hsfl polypeptide which binds to the immobilized GST-Yapl or GST-Hsfl polypeptide on the one hand or GST-Skn7 polypeptide on the other, as the case may be. This may be determined by fractionating the glutathione-agarose beads by SDS- polyacrylamide gel electrophoresis. Alternatively, the beads may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter.
  • such an assay according to the present invention may also take the form of an in vivo assay wherein the interaction is studied in by way of immunoprecipitation of Skn7 or one of Yapl or Hsfl.
  • the amount of the Yapl or Hsfl, or both, in Skn7 immunoprecipitates, or vice versa, may be examined by any suitable means, for example Western blotting of the immunoprecipitate and probing with the appropriate antibody.
  • the in vivo assay may be performed in a cell line such as a yeast strain in which Skn7 polypeptide and/or Yapl or Hsfl polypeptides are expressed from a vector introduced into the cell.
  • a cell line such as a yeast strain in which Skn7 polypeptide and/or Yapl or Hsfl polypeptides are expressed from a vector introduced into the cell.
  • the assay of the invention relates to the interaction of Skn7 with Yapl
  • the assay is conducted in the presence of DNA with one or more binding sites for each factor.
  • the interaction of the two polypeptides may be examined by the use of electrophoretic mobility shift assays, for example as described in the accompanying examples.
  • DNA comprising Skn7 and/or Hsfl binding sites may be included in assays which measure the interaction between these two factors.
  • the binding of Skn7 to a specific nucleotide sequence may be utilised to provide further assays of the invention, by assaying for antagonists of such binding.
  • a nucleotide sequence comprising the specific nucleotide sequence may be immobilised and Skn7 together with a putative inhibitor compound may be brought into contact with the sequence, and the degree of binding measured, for example by labelling the Skn7 and detecting the amout of labelled Skn7 bound to the immobilised sequence.
  • the Skn7 may be immobilised and the amount of congnate sequence, optionally labelled, which binds to it in the presence or absence of inhibitor may be determined.
  • DNA binding assays of the type described above may be conducted in the presence or absence of an additional factor, particularly Yapl or Hsfl, which interacts with Skn7 in binding to a specific nucleotide sequence.
  • a further way of identifying interaction with of Skn7 with its binding sequence is a bandshift assay, such as the EMSA assay described in the accompanying examples .
  • a reporter gene construct including a
  • Skn7-polypeptide-specific binding nucleotide sequence operably linked to a reporter gene may be introduced into an expression system such as a cell or cell free expression system together with an expression vector or vectors capable of expressing Skn7 or one of Yapl or Hsfl.
  • Two or more Skn7 binding sites may be present in the nucleic acid construct and this may enhance sensitivity of the assay.
  • the expression of the reporter gene may be determined in the presence and absence of the putative inhibitor, such that a reduction in expression of the reporter gene indicates a putative inhibition of the interaction of Skn7 with Yapl or Hsfl, as the case may be.
  • one or more Yapl-polypeptide-specific binding nucleotide sequences may be included in the construct .
  • the reporter gene may be any suitable reporter gene used in the art.
  • reporter genes include reporter genes mentioned above, for example -galactosidase or luciferase.
  • the expression vector (s) will include DNA encoding Skn7 and/or one of Yapl and Hsfl polypeptide operably linked to a promoter capable of expressing the gene in the host cell .
  • Suitable promoters include yeast promoters such as GAL or ADH promoters .
  • the cell lines used in assays of the invention may be used to achieve transient expression, although in a further aspect of the invention cells which are stably transfected with constructs which express Skn7 polypeptide and, where required, Yapl or Hsfl polypeptide may also be generated. Means to generate stably transformed cell lines are well known in the art and such means may be used here .
  • a construct capable of expressing this protein may also be introduced into the cell operably linked to a suitable promoter.
  • Suitable host cells include bacteria, mammalian cells and yeast, and baculovirus systems.
  • a common, preferred bacterial host is E. coli .
  • Preferred for performance of aspects of the present invention are yeast cells.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al . eds., John Wiley & Sons, 1992.
  • a further aspect of the invention provides cells which comprise an expression construct comprising a Skn7 polypeptide- encoding sequence operably linked to a heterologous promoter, together with an expression construct comprising one of a Yapl or Hsfl polypeptide-encoding sequence operably linked to a heterologous promoter.
  • the two constructs may be present on separate vectors, or the same vector.
  • the cells may further comprise a reporter gene which comprises a promoter capable of being transcriptionally activated by the presence of Skn7 polypeptide when in the presence of one of Yapl and Hsfl polypeptides.
  • the reporter gene is generally one with an easily assayable expression product, and in any event is one which is heterologous to the promoter.
  • Inhibitor Compounds The amount of putative inhibitor compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01. to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM.
  • Inhibitor compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants which contain several characterised or uncharacterised components may also be used. Inhibitor compounds may be provided by way of libraries of compounds may by combinatorial chemistry.
  • a further class of putative inhibitor compounds can be derived from Skn7 polypeptide, Yapl polypeptide or Hsfl polypeptide. Peptide fragments of from 5 to 40 amino acids, for example from 6 to 10 amino acids from the region of the relevant polypeptide responsible for interaction between these proteins, or interaction with nucleic acid, may be tested for their ability to disrupt such interaction.
  • Antibodies directed to the site of interaction in either protein form a further class of putative inhibitor compounds.
  • Candidate inhibitor antibodies may be characterised and their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for disrupting the interaction.
  • Antibodies may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof . Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . , 1992, Nature 357: 80-82). Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.
  • an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage o.r filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • the library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or fragments) , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
  • Antibodies according to the present invention may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any binding substance having a binding domain with the required specificity. Thus the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.
  • Example antibody fragments capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.
  • a hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs) , of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP184187A, GB 2188638A or EP-A-0239400. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A- 0125023.
  • Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells, eukaryotic or prokaryotic, containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression.
  • the invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.
  • candidate inhibitor compounds may be based on modelling the 3 -dimensional structure of Skn7 polypeptide and/or Yapl polypeptide and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.
  • An inhibitor compound identified using the present invention has therapeutic potential .
  • Budding yeast that are deleted for Skn7 are alive but compromised in their response to stress. Invading pathogenic organisms will respond to attack by the host defences through their own defence systems, the intracellular stress response. Neutralising Skn7 function will render the pathogen more sensitive to bodily defences.
  • the conservation of Skn7 in fungi provides indication that any substance identified with the requisite inhibitor activity will be of therapeutic value against a wide spectrum of fungal pathogens.
  • drugs active against Skn7 function should not harm human cells.
  • Anti-fungal treatment is useful against Candidiasis, farmers' Lung, Cyrptococcosis and opportunistic fungal infections, e.g. as are prevalent in immuno-compromised individuals, such as transplant patients and AIDS sufferers.
  • Inhibitor compounds may also be used in combination with any other anti-fungal compounds, e.g. azole compounds such as fluconazole.
  • the assay of the invention when conducted in vivo, need not measure the degree of inhibition of binding or transcriptional activation caused by the inhibitor compound being tested. Instead the effect on fungal cell growth and/or viability be measured. It may be that such a modified assay is run in parallel or subsequent to the main assay of the invention in order to confirm that any effect on cell growth and/or viability is as a result of the inhibition of binding or transcriptional activation caused by said inhibitor compound and not merely a general toxic effect.
  • a 23 nucleotide sequence within the TRX2 promter has been identifed as a binding sequence for Skn7 polypeptide.
  • This sequence is identifed in Figure 5 at nucleotides -164 to -142 of the TRX2 promoter. This sequence comprises: 5' TTTCCAGCCAGCCGAAAGAGGGA (SEQ ID NO : 2 ) .
  • a further binding sequence for Skn7 is present in the 26 bp sequence from the HSE2 region of the SSA1 promoter (see examples) .
  • This has the sequence: 5' TGCATTTTCCAGAACGTTCCATCGGC (SEQ ID NO: 3)
  • the minimal sequence to which Skn7 binds may be identified by using synthetic oligonucleotides of these regions as competitor DNA in a gel mobility shift assay. Systematic mutation of the nucleotide sequences will define key residues. In another approach, randomly generated oligonucleotides may be passed over immobilised, pure Skn7 protein. Elution " of bound oligonucleotides followed by DNA sequencing will directly reveal the sequences .
  • the invention thus provides in a further aspect an oligonucleotide which consists essentially the sequences SEQ ID NO: 2 and SEQ ID NO: 3, or has a sequence which is a mutant, variant, derivative or homologue sequence by way of addition, deletion, substitution and/or insertion of one or more base pairs, which retains Skn7 binding, preferably a shorter sequence than these 23 and 26 base pair sequences shown.
  • a variant, mutant, derivative or homologue may have at least about 50%, 60%, 70%, 80%, 90% or 95% homology with the sequences shown.
  • the core triplet GAA appears to be a potential core recognition sequence.
  • preferred oligonucleotides of the invention comprise derivative of SEQ ID NO: 2 or SEQ ID NO: 3 which comprise this core, are at least 15, preferably 18, nucleotides in length, and are at least 75% homologous and preferably no more than three, more preferably no more than two nucleotides different from either of the above sequences when aligned to the GAA core.
  • the invention also provides a promoter construct, able to activate transcription of an operably linked sequence, including SEQ ID NO: 2 or SEQ ID NO: 3 or a said mutant, variant, derivative or homologue thereof, other than a naturally occurring promoter sequence.
  • an Skn7-binding site may be used in construction of a promoter that contains one or more other regulatory motifs, transcription factor elements, and promoter elements to produce a promoter which contains a heterologous Skn7-polypeptide-binding site.
  • the invention further provides a nucleic acid construct which ⁇ U t to ⁇ >
  • the gene may be transcribed into mRNA which may be translated into a peptide or polypeptide product which may be detected and preferably quantitated following expression.
  • a gene whose encoded product may be assayed following expression is termed a "reporter gene", i.e. a gene which "reports" on promoter activity.
  • a reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product.
  • a detectable signal preferably a visually detectable signal, such as a coloured product.
  • Many examples are known, including -galactosidase and luciferase.
  • jS-galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectrophotometer to measure absorbance . Fluorescence, for example that produced as a result of luciferase activity, may be quantitated using a spectrophotometer.
  • Radioactive assays may be used, for instance using chloramphenicol acetyl - transferase, which may also be used in non-radioactive assays.
  • the presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof.
  • the binding molecule may be labelled directly or indirectly using any standard technique.
  • a substance able to down-regulate expression of the promoter may be sought .
  • a method of screening for ability of a substance to modulate activity of a promoter may comprise contacting an expression system, such as a host cell, containing a nucleic acid construct as herein disclosed with a test or candidate substance and determining expression of the heterologous gene.
  • excipient vehicle or carrier, and optionally other ingredients .
  • Suitable mimetics of peptide fragments of Skn7 polypeptide or Yapl or Hsfl polypeptides which interfere with interaction between these polypeptides or with binding of Skn7 polypeptide to its specific nucleic acid motif.
  • the term "functional mimetic” means a substance which may not contain an active portion of the relevant amino acid sequence, and probably is not a peptide at all, but which retains the relevant interfering activity. The design and screening of candidate mimetics is described in detail below.
  • a substance identified using the present invention may be peptide or non-peptide in nature.
  • Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimick of the substance (particularly if a peptide) may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
  • a range of sources e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modelling process.
  • the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • Mimetics of substances identified as having ability to interfere with the interaction of Skn7 polypeptide with its binding site motif and/or its interaction with Yapl or Hsfl polypeptide in a screening method as disclosed herein are included within the scope of the present invention.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practioners and other medical doctors .
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions according to the present invention may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • a polypeptide, peptide or other substance able to interfere with the interaction of Skn7 polypeptide with its binding site motif and/or its interaction with Yapl polypeptide or Hsfl polypeptide according to the present invention may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment .
  • a kit may include instructions for use.
  • EXAMPLE 1 This example shows that deletion of the bacterial two component response regulator homologue Skn7 results in sensitivity of yeast to oxidizing agents indicating that Skn7 is involved in the response to this type of stress.
  • Skn7 regulates the induction of two genes, TRX2 , encoding thioredoxin, and a gene encoding thioredoxin reductase.
  • TRX2 is already known to be induced by oxidative stress dependent on the Yapl protein, an API-like transcription factor responsible for the induction of gene expression in response to various stresses .
  • the thioredoxin reductase gene has not previously been shown to be activated by oxidative stress and, significantly, we find that it too is regulated by Yapl.
  • the control of at least TRX2 by Skn7 is a direct mechanism as Skn7 binds to the TRX2 gene promoter in vitro. This shows Skn7 to be a transcription factor, at present the only such eukaryotic two component signalling protein. Our data further suggests that Skn7 and Yapl co-operate on the TRX2 promoter, to induce transcription in response to oxidative stress.
  • the data of Example 1 may also be found in Morgan et al , 1997, published after the priority date of the present application, whose contents are incorporated herein by reference .
  • ROS Reactive oxygen species
  • Yapl and Yap2 are members of the c-jun family of proteins which constitute part of the API transcription factor of higher eukaryotes. Both Yapl and Yap2 contain a basic leucine zipper domain (bZIP) adjacent to a DNA binding domain located at the N-terminus of the proteins. Null mutants of both YAP1 and YAP2 result in sensitivity of S. cerevisiae to the oxidizing agent hydrogen peroxide (Stephen et al . , 1995) .
  • bZIP basic leucine zipper domain
  • Yapl binds directly to the promoter, and regulates the expression of the TRX2 gene (Kuge and Jones, 1994) encoding thioredoxin which acts in the oxidative stress response to reduce protein disulphides. Yapl also regulates GSH1, encoding glutamyl cysteine synthetase, in response to hydrogen peroxide .
  • GSH1 encoding glutamyl cysteine synthetase
  • yeast mounts a complex defence to oxidative stress involving several different transcription factors and signalling pathways.
  • SKN7 has been implicated in the regulation of cell wall biosynthesis and the cell cycle (Brown et al., 1993, 1994; Morgan et al . , 1995b).
  • Overexpression of SKN7 suppresses the cell wall defect associated with mutation of the KRE9 gene (Brown et al . , 1993) and additionally, the growth defect associated with deletion of PKC1 (Brown et al .
  • CACGAAAA an SCB element
  • ACGCGT an MCB element
  • SKN7 has been shown to be due to the absence of Gl cyclin expression.
  • High copy SKN7 was shown to restore Gl cyclin expression through the MCB and SCB elements present in the cyclin promoters (Morgan et al . , 1995b) .
  • SKN7 does not appear to bind directly to MCB and SCB elements (Morgan et al . , 1995b) and is more likely regulating a MCB/SCB binding factor other than MBF and SBF.
  • the sequence of SKN7 revealed homology to the DNA binding domain of heat shock factor (HSF1) (Brown et al . , 1993; Morgan et al . , 1995b) hence it is possible that SKN7 binds to a sequence related to the HSE elements that HSF1 recognises .
  • HSF1 heat shock factor
  • the Skn7 protein contains a potential receiver domain found in the two component signal transduction family of proteins in prokaryotes (Brown et al . , 1993; Morgan et al . , 1995b). These signal transduction systems are a common method of detecting and responding to the environment in bacteria (for reviews see Bourret et al . , 1991; Parkinson, 1993).
  • the first component a homodimer histidine kinase present in the cell membrane, detects the signal, and phosphorylates a conserved histidine residue on its partner. This phosphate is then transferred to a conserved aspartic acid residue within the 120 amino acid receiver domain of the second component, the response regulator protein.
  • Response regulator proteins are generally transcription factors that are activated by the phosphorylation. In eukaryotes only a few potential two component signal transduction proteins have been identified (reviewed in Morgan et al . , 1995a) . In no case, including Skn7, have genes regulated by these systems been identified.
  • Saccharomyces cerevisiae genetic screens have identified one histidine kinase, Slnl, and two response regulator proteins Sskl and Skn7.
  • Slnl and Sskl were shown to act in the regulation of the response of yeast to osmolarity by regulating the Hogl MAP kinase pathway (Maeda et al . , 1994, 1995).
  • deletion of SKN7 does not result in any osmolarity defect.
  • the only stress defect known for skn7 mutants is in oxidative stress as mentioned above, although the role of Skn7 in the OSR remains unclear.
  • Skn7 ⁇ strains are sensitive to a range of oxidizing agents skn7 ⁇ strains are sensitive to hydrogen peroxide (Krems et al . , 1996) . We have confirmed this result and find that skn7 ⁇ strains are sensitive to a range of oxidizing agents including t-butyl-hydrogen peroxide, cadmium, menadione but not significantly sensitive to diamide.
  • the Skn7 protein is required for the cellular response to a variety of free radicals. Deletion of the YAP1 gene also results in sensitivity of yeast cells to many of these agents (Kuge and Jones, 1994) .
  • SKN7 The sensitivity of yapl ⁇ strains has been shown to be due to the role of the Yapl protein in the induction of several genes which function in the OSR.
  • deletion of the SKN7 gene does not enhance the sensitivity of a yapl ⁇ strain to diamide, hydrogen peroxide, cadmium or menadione (Krems et al . , 1996; our unpubl . obs . ) suggesting that SKN7 and YAP1 are epistatic, functioning in the same pathway.
  • Skn7 affects the expression of a, set of genes also regulated by Yapl in response to oxidative stress.
  • Skn7 is required for the induction of thioredoxin and thioredoxin reductase gene expression by the oxidative stress response
  • Expression of the TRX2 gene is induced in response to several oxidizing agents including hydrogen peroxide and this induction is under the control of the Yapl protein which binds the TRX2 promoter (Kuge and Jones, 1994) .
  • Yapl protein which binds the TRX2 promoter
  • deletion of the SKN7 gene mimics the deletion of the YAP1 gene.
  • TRX2 induction is almost abolished but once again residual induction is observed.
  • the skn7 ⁇ yapl ⁇ double mutant is no more defective in TRX2 induction than either single mutant, consistent with the genetic epistasis described above.
  • the residual induction of TRX2 that occurs even in the skn7 ⁇ yapl ⁇ double mutant suggests the existence of a further minor induction mechanism.
  • the important result is that skn7 ⁇ and yapl ⁇ mutants appear to have identical phenotypes with respect to TRX2 induction consistent with their functioning in the same pathway.
  • TRR1 thioredoxin reductase
  • the Yapl protein also binds directly to the TRX2 promoter at two sites termed site 1 and site 2 (Kuge and Jones, 1994; Figure 5) .
  • site 1 and site 2 Two bands in addition to band 1, bands 3 and 4, are sensitive to the presence of the YAP1 gene.
  • To determine whether all these Yapl -dependent bands contain the Yapl protein various crude extracts were mixed with a polyclonal antibody against Yapl and the effect on the bands examined. The wild type extracts clearly show a prominent supershifted band and essentially recreate the pattern obtained from yapl ⁇ extracts. Hence all the Yapl-dependent bands contain the Yapl protein.
  • band 1 contains at least the Skn7 and Yapl proteins
  • band 2 contains Skn7
  • bands 3 and 4 contains Yapl.
  • Another important conclusion from this data is that significant binding of Skn7 and Yapl proteins can be detected in the absence of oxidative stress and, furthermore, that Skn7 can bind in the absence of the YAP1 gene and vice versa.
  • the probes Trx ⁇ l, Trx ⁇ 3 , Trx ⁇ 4 , WTMCB, and MUTMCB (see Methods) containing the Skn7 binding site gave a single band which was not detectable in skn7 ⁇ extracts. In contrast, no Skn7 sensitive band could be detected with probes Trx ⁇ 2 and Trx ⁇ 6.
  • Skn7 binds within a 23 nucleotide region between the Yapl binding site 2 (Kuge and Jones 1994) and the potential TATA sequence.
  • the potential DNA binding domain of Skn7 has homology to HSFl and whilst the 23 nucleotide sequence does not contain a complete HSE element, there is some limited homology to one.
  • nucleotides are also some limited homology to an SCB element. Mutation of the CG to TA within this lowers Skn7 binding to this region 20-fold, as determined by comparing the binding of Skn7 to the Trx ⁇ 4 and Trx ⁇ 5 probes using EMSA. However, the addition of a large molar excess of a fragment from the HO gene promoter containing SCB elements did not compete with Skn7 binding. Thus, although the CG nucleotides are important for Skn7 binding, this sequence is unlikely to constitute a functional SCB element.
  • Extracts were prepared from the cells following 1 mM H 2 0 2 treatment for 1 hour at 25 * C. This is at a time when maximal induction of the TRX2 and TRR1 genes had been observed.
  • the extracts were mixed with a probe from the TRX2 promoter. No obvious effect on the binding of the Yapl or Skn7 proteins was observed under these conditions. This implies that, at least in vitro, DNA binding per se is not the major regulatory step in the induction of gene expression and that another mechanism is involved. In addition, no new bands were evident suggesting that the induction does not involve the binding of a new protein to the TRX2 promoter.
  • CEN plasmids containing either the wild type SKN7 gene or with a D427N mutation were introduced into the skn7 ⁇ strain and the sensitivity to hydrogen peroxide tested.
  • a 20 ml of a fresh overnight culture of the strains indicated were streaked radially on a YPD plate and allowed to dry.
  • a filter disc saturated with hydrogen peroxide was placed in the centre of the plate. Following incubation at 30 'C the extent of inhibition of growth of each strain from the disc was measured.
  • pBAMl contains wild type Skn7 and pBAM2 contains Skn7 D427N.
  • Both plasmids clearly behave identically, they fully complemented the sensitivity of the skn7 ⁇ strain to hydrogen peroxide .
  • the same strains were grown in minimal medium and treated with ImM hydrogen peroxide for 1 hour.
  • Northern hybridisation revealed that Skn7 ⁇ 427N behaves identically to wild type and TRX2 expression is induced normally.
  • the CEN plasmids were introduced into the skn7 ⁇ yapl ⁇ strain.
  • the receiver domain as a whole is essential for the response to oxidative stress.
  • a C-terminal truncation of Skn7 missing amino acids 353-623 containing the receiver domain and the glutamine rich region, was isolated by transposon mutagenesis (Morgan et al . , 1996). This construct was unable to rescue the sensitivity of skn7 ⁇ to hydrogen peroxide (data not shown) . Nonetheless this truncated protein, which contains the domain with homology to the HSFl DNA binding domain, was still able to bind the TRX2 promoter.
  • the receiver domain may play a structural role that is necessary for the oxidative stress response but phosphorylation of Asp 427 seems unimportant .
  • the Yapl transcription factor is required for the OSR through the induction of gene expression (rev. in Moradas-Ferreira et al., 1996) .
  • Deletion of the SKN7 gene also results in the increased sensitivity of yeast to oxidative stress.
  • response regulator proteins like Skn7 in bacteria are transcription factors it seemed likely that Skn7 , in addition to Yapl, was also required for the induction of gene expression.
  • TRX2 and TRR1 two genes normally induced by oxidative stress, TRX2 and TRR1 , require the SKN7 gene for induction.
  • deletion of the SKN7 gene has a less deleterious effect than deletion of the YAP1 gene for sensitivity to the agent and for both TRX2 and TRR1 induction.
  • the double delete combination in this case behaves identically to the deletion of YAP1 alone.
  • Yapl binding increases detectably in response to diamide in contrast to Skn7. This increase in Yapl binding occurs even in the absence of Skn7. Thus there must be some separation in function of the two proteins and some activation of Yapl which is Skn7 independent . This is not observed with hydrogen peroxide treatment and indicates that Yapl is regulated differently by these treatments.
  • Slnl histidine kinase in budding yeast
  • Slnl histidine kinase in control of Skn7.
  • Deletion of SKN7 does not lead to osmotic sensitivity, although whether mutation of SLN1 results in increased sensitivity to oxidative stress is not yet clear.
  • Slnl appears to be constitutively active, the kinase activity becoming inactivated in high osmolarity.
  • Sskl its cognate response regulator, Sskl
  • Sskl its cognate response regulator
  • Sskl its cognate response regulator
  • Skn7 activity If Slnl controlled Skn7 activity and responded to oxidative stress as it does to osmotic stress, this might lead to dephosphorylation of the Skn7 receiver domain by analogy with Sskl. Since the Asp427 is not essential for regulation of OSR genes, this could direct Skn7 to these genes rather than cell wall or cyclin genes. Note, however, that Skn7 is also phosphorylated on serine and/or threonine residues (Brown et al . , 1994) so that control of the protein could involve alternative pathways without any histidine kinase involvement. Some form of modification of Skn7 may be required for its DNA binding activity, as we were unable to detect binding to the TRX2 promoter of bacterially produced Skn7.
  • Skn7 regulates at least two groups of genes, those requiring phosphorylation of Asp427, which include cell wall and Gl cyclin genes, and the OSR genes which do not require the phosphorylation.
  • the role of Skn7 in regulation of this disparate group of genes remains obscure.
  • the functional receiver domain argues that the protein transduces signals rather than acting as a non-specific transcription factor.
  • the common feature to the genes mentioned above might be an involvement in stress.
  • skn7 ⁇ strains are lethal in combination with pkcl ⁇ , the protein kinase C gene (Brown et al . , 1994; Morgan et al . , 1995b).
  • PKC1 regulates a MAP kinase pathway that responds to hypotonic stress (rev. in Herskowitz, 1995) and that regulates the synthesis of cell wall genes (Igual et al . , 1996) .
  • Skn7 DNA binding domain which is, related to that of the heat shock factor. Indeed we have recently found that skn7 ⁇ strains are sensitive to specific heat stresses. Concerning the Gl cyclin genes, certain stresses lead to a cell cycle delay in Gl (Rowley et al . , 1993) and we are currently exploring the possibility that Skn7-induced cyclin expression is necessary for recovery from this arrest.
  • strains of S. cerevisiae used in this study were as follows : W303-la (a ade2-l trpl-1 canl-100 leu2-3,112 his3-ll ura3), skn7 ⁇ (a ade2-l trpl-1 canl-100 leu2-3,112 his3-ll ura3 skn7::HIS3), W303-lb (a ade2-l trpl-1 canl-100 leu2-3,112 his3-ll ura3) , yapl ⁇ (a ade2-l trpl-1 canl-100 leu2-3,112 his3-ll ura3 yapl::TRPl), skn7 ⁇ yapl ⁇ (a ade2-l trpl-1 canl-11 leu2-3,112 his3-ll ura3 skn7::HIS3 yapl::TRPl).
  • the skn7 ⁇ yapl ⁇ strain was constructed by disrupting the SKN7 gene in the yapl ⁇ strain. This was performed by transforming a restriction fragment carrying a HIS3 insertion in the wild type SKN7 gene into the his3- yapl ⁇ strain and selecting for his-t- (Morgan et al . , 1995) . Possible double mutants were then tested by PCR to confirm the disruption.
  • the yeast transformations were performed by a derivation of the lithium acetate technique described previously (Schiestl and Gietz, 1989).
  • /3-galactosidase assays were performed on mid-log phase cells as described previously (Guarente, 1983) . Activities are given in OD units at 420 nm per minute per mg of protein. Values represent the averages for duplicate samples in three independent experiments.
  • Sstl-Xbal fragment was isolated from plasmids pB-BRYl and pB-BRYl Asn427 respectively (Morgan et al . , 1995b) and ligated with the URA3 CEN vector YCplac33 (Gietz and Sugino, 1988) digested with Sstl-Xbal to create plasmids pBAMl (wild type SKN7) and pBAM2 (skn7 D427N) respectively.
  • the C-terminal truncation derivative of Skn7 was constructed by transposition of a derivative of the bacterial transposon TnlOOO (Morgan et al., 1996) into the plasmid YEp24/BRYl (Morgan et al . , 1995b).
  • the probes for the TRX2 and TRR1 genes were obtained by PCR from the yeast genome using gene-specific oligonucleotides.
  • Probes were obtained by PCR using the combinations of these oligonucleotides described below. PCR was performed on the TRXLACZ plasmid described in Figure 2. The following oligonucleotides were used to obtain probes 1/2, oligonucleotides Shift 1 and Shift 2; Trx ⁇ l, oligonucleotides Trx ⁇ l and Shift 2; Trx ⁇ 2 , oligonucleotides Trx ⁇ 2 and Shift 2 ; Trx ⁇ 3, oligonucleotides Trx ⁇ 3 and Shift 1; Trx ⁇ 4 , oligonucleotides Trx ⁇ 4 and Shift 2; Trx ⁇ 5, oligonucleotides
  • Yeast cell extracts were prepared from midlog cultures. Cell pellets were vortexed for 8 x 30s with glass beads in 100-200ml of 200 mM Tris-HCl pH 8.0 , 10 mM MgCl 2 , 10% glycerol and protease inhibitor mix (PI mix- 100 ⁇ g/ml phenylmethyl sulfonyl fluoride, 2 ⁇ g/ml aprotinin, 2 ⁇ g/ml leupeptin, 2 ⁇ g/ml pepstatin A, 50 ⁇ g/ml TLCK and 100 ⁇ g/ml TPCK) . After centrifugation, protein concentrations were determined and extracts were stored at -70 * C.
  • EMSAs of DNA-protein complexes were conducted as follows. 7 ⁇ g protein extract was incubated with 0.5 ng 32 P 5' -end labelled DNA fragment in 10 ml of 25 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM EDTA, 7 mM MgCl 2 , 10% glycerol, PI mix, 1 mg/ l 3- [ (3 -cholamidopropyl) dimethyl- ammonio] -1-propanesulfonate (CHAPS) and 50 ng/ml poly(dI:dC) at room temp for 15 min and on ice for a further 15 min. Competition experiments included a 50-fold molar excess of unlabelled competitor DNA over labelled probe.
  • Hsflp The yeast Heat Shock Factor, Hsflp, is also central to the induction of another set of stress-inducible genes, namely the heat shock genes.
  • These two regulatory trans-activators, Hsflp and Skn7p share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, known to mediate protein-protein interactions.
  • Hsflp and Skn7p interact both genetically and physically.
  • Skn7p can bind to the same regulatory sequences as Hsflp, namely Heat Shock Elements, and that strains deleted for the SKN7 gene and containing a temperature-sensitive mutation in HSFl are hypersensitive to oxidative stress.
  • Oxygen in the form of superoxide anion (02-) , hydroxyl ion (0H-) , and hydrogen peroxide, cause damage to nucleic acids, cell membranes and proteins (Halliwell and Gutteridge, 1984; Halliwell, 1994) .
  • Yeast in common with all other organisms, have evolved protective mechanisms to survive in the presence of these byproducts of aerobic metabolism and can mount distinct adaptive responses to different sources of oxidative stress (reviewed in Ruis and Schuller, 1995; Jamieson, 1992) .
  • the Cu,Zn-linked superoxide dismutase encoded by the S0D1 gene, detoxifies superoxide anion to hydrogen peroxide and catalase, encoded by the cytosolic CTT1 gene, can catalyse the breakdown of H 2 0 2 .
  • Other free radical scavengers in the cell include glutathione, ascorbic acid and thioredoxin.
  • the Skn7p protein contains a region with a high degree of homology to the receiver domain of response regulator proteins found in bacterial two-component signal transduction systems (Brown et al . , 1993; Morgan et al . , 1995).
  • a membrane bound sensor histidine kinase can phosphorylate a conserved aspartate residue within the receiver domain of its cognate response regulator (reviewed in Parkinson, 1993; Stock et al . , 1989) .
  • This phospho-aspartate form of the response regulator can then carry out a function appropriate to the incoming signal, usually the transcriptional activation of a specific set of genes.
  • Skn7 ⁇ cells are sensitive to acute heat stress. Deletion of the SKN7 gene does not confer a heat shock sensitive phenotype when cells are shifted from 25 'to 37 'C. (Morgan et al . , 1997) . However, given the high degree of homology between the DNA binding domains of Skn7p and Hsflp, we investigated the effect of a skn7 ⁇ mutation on cell viability under acute heat shock at 51 'C. Mid-log cultures of W303-la and isogenic skn7 ⁇ cells were grown in YPD at 25°C and an aliquot shifted to 51°C.
  • the SSA1 gene encodes a major isoform of the yeast Hsp70 protein which is abundant under non-stressed conditions and is strongly induced by heat shock (Craig et al . , 1985) .
  • the plasmid pZJHSE2-137 contains a heat shock element, HSE2 , from the SSA1 promoter fused to the ⁇ -galactosidase coding sequence (Slater and Craig, 1987) .
  • This HSE2 sequence is responsible for the majority of both basal and heat shock induced expression of SSA1 (Slater & Craig, 1987) .
  • ⁇ - galactosidase assays were carried out on wild type W303-la and isogenic skn7 ⁇ cells containing the reporter plasmid following treatment for one hour with hydroperoxide. In wild type cells this resulted in an eight-fold induction of -galactosidase activity (Table 1) . In marked contrast, in the skn7 ⁇ strain induction in response to oxidative stress was abolished.
  • Wild type and isogenic skn7 ⁇ cells containing pZJHSE2-137 were assayed for /3-galactosidase levels before (-) and after (+) the addition of butyl hydroperoxide to 0.6 mM for one hour.
  • cells were grown at 25 * C and transferred to 37 * C for one hour.
  • ⁇ -galactosidase activity is expressed as ⁇ OD420/min/mg protein. Values are averages of duplicate samples from two independent experiments.
  • Skn7p can specifically bind the HSE2 element from the SSA1 promoter.
  • the DNA-binding domain of Skn7p (residues 87-150) is highly homologous to that of Hsflp) .
  • electrophoretic mobility shift assays with E.coli expressed 6His-Skn7p.
  • the 6His-Skn7 fusion protein was purified on a Ni2+-NTA agarose affinity column (see Materials and Methods) and added to a 26bp probe derived from the 137bp HSE2 region of the SSA1 promoter. Electrophoretic moblity shift assays were performed using E.
  • the 6His-Skn7 fusion protein clearly bound the HSE2 sequence.
  • polyclonal antiserum to the protein was added to the reaction mix.
  • the retarded complex formed by the HSE2 probe and 6His-Skn7 protein was super-shifted by antibody to Skn7p whereas no effect was observed by the addition of pre-immune serum at the same concentration .
  • Hsflp and Skn7p have previously been shown to play a role in the activation of stress-responsive gene expression under conditions of free radical stress (Liu and Thiele, 1996; Krems et al., 1996; Morgan et al . , 1997).
  • HSFl is essential so that a temperature-sensitive allele, hsfl-m3 (Smith and Yaffee, 1991) , was used.
  • Transforming strain DR20-2b with a CEN version of the SKN7 gene restores growth at 34 * C, indicating that the increased temperature-sensitivity of DR20- 2b is due specifically to the deletion of the SKN7 gene rather than genetic background effects.
  • deletion of SKN7 exacerbates the growth defect of the hsfl ts mutant allele.
  • the hsfl ts strain was transformed with either the vector YexH-SKN7, which expresses high levels of Skn7p under the control of a galactose inducible promoter, or the empty vector alone.
  • the plasmid YexH-SKN7 had previously been shown to rescue the hydroperoxide sensitivity of a skn7 ⁇ strain, thus confirming the functionality of the fusion.
  • the hsfl ts cells expressing high levels of Skn7p displayed strong growth at 35.5 * C, where the hsfl ts strain containing the empty vector alone could not form colonies (Fig. 4B) .
  • over expression of SKN7 failed to rescue the hsfl ts strain at 37 * C which is not surprising given the pleiotropic nature of the hsfl-m3 allele (Smith and Yaffe, 1991) .
  • the D427N version of the SKN7 gene was also found to reverse the hypersensitivity of DR20-2b to hydroperoxide. Furthermore, the D427N mutated form of SKN7 also restored HSE2-LacZ expression in skn7 ⁇ suggesting that phosphorylation of D427 is not required for Skn7p function through heat shock elements. In contrast, Skn7D427N fails to activate CLN1 and CLN2 expression in a swi4tsswi6 ⁇ background (Morgan et al . , 1995) or rescue the cell wall assembly defect of the kre9 ⁇ mutant (Brown et al . , 1994) .
  • HSP82 and SSA1 are induced two-fold after 45 minutes in hydroperoxide. However, this increased expression does not occur in the isogenic skn7 ⁇ strain. The more dramatic ten-fold induction of HSP12 in W303-la is also abolished by the deletion of the SKN7 gene.
  • the hsfl ts mutation at the semi-permissive temperature of 30 * C has the effect of lowering the level of HSP82 and SSA1 expression, relative to the HSFl strain, without dramatically affecting the overall induction of these genes by hydroperoxide.
  • the hsfl ts lesion virtually eliminates the six-fold induction of HSP12 by hydroperoxide observed in the HSFl parental strain.
  • the double mutant strain DR20-2b was examined, a pronounced additive effect of the skn7 ⁇ and hsfl ts mutations was apparent. The peroxide- induction of all three heat shock genes tested was essentially abolished.
  • skn7 deletion had no effect on heat shock induction of SSA1, HSP26, and HSP104, their induction by hydroproxide was significantly reduced.
  • SKN7 is therefore specifically required for the oxidative stress induction of heat shock genes and is not required for their heat shock-mediated induction. This is in accord with Table 1 and our original observations that skn7 ⁇ cells show no increased sensitivity upon a temperature shift from 25 * C to 37 "C when compared to the isogenic wild type strain (Morgan et al . , 1997).
  • Skn7p and Hsflp have previously been shown to play important roles in the cellular response to oxidative stress.
  • the response regulator Skn7 cooperates with the yeast AP-1 homologue Yapl on the promoter of the thioredoxin gene, TRX2 , and activates transcription of the gene in the presence of hydrogen peroxide (Morgan et al . , 1997).
  • Hsflp-dependent activation of the CUP1 metallothionein gene occurs in yeast cells treated with the superoxide generator menadione
  • Hsflp and Skn7 Physical and Genetic interactions between Hsflp and Skn7.
  • a genetic interaction between Hsflp and Skn7p was first indicated by the lowered restrictive temperature of a hsfl ts skn7 ⁇ strain relative to the hsfl ts strain alone.
  • the growth defect of the hsfl s strain could be partially suppressed by high copy expression of the SKN7 gene. That this suppression is partial indicates that Skn7 can fulfil some but not all of the functions of Hsflp in the cell. This is not surprising given the pleiotropic nature of the hsfl-m3 mutation (Smith and Yaffee, 1991) .
  • Skn7 is required to activate heat shock gene expression specifically in response to hydroperoxide
  • Preliminary studies using a SSAl-LacZ fusion construct indicated that Skn7 was required for HSE-mediated LacZ induction in response to hydroperoxide but was not required for the heat shock induction of the reporter (Table 1) .
  • Northern analysis of heat shock gene expression in wild type and skn7 ⁇ cells confirmed that SKN7 was not required for induction of these genes in response to heat shock. For example, when cells are shifted from 25 * C to 37 * C, the heat shock induction of HSP104 in skn7 ⁇ cells was identical to that in the isogenic wild type strain.
  • HOG1 pathway is itself regulated by the SLN1 histidine kinase, which, as the only such protein in S. cerevisiae, may well act upstream of the Skn7 response regulator.
  • the HOG1 pathway is thought only to be involved in adapting to changes in extracellular osmolarity (Sch ⁇ ller et al .
  • the other region of structural homology between these two proteins lies between residue 222 and 257 of the Skn7 protein.
  • This stretch contains five heptad repeats, with hydrophobic residues at positions 1 and 4, and polar residues elsewhere in the repeat units, characteristic of regions which form coiled- coil structures (reviewed in Lupas, 1996) .
  • Hsflp contains six heptad repeats which have been shown to mediate trimerisation of the protein through the formation of triple-stranded a- helical coiled coils (Sorger and Nelson, 1989; Peteranderl and Nelson, 1992, Rabindran et al . , 1993).
  • coiled-coils are also known to mediate hetero- and homodimerisation, for example, of the yeast GCN4 member of the bZIP transcription factor family (Harbury et al . , 1993).
  • yeast strains used were as follows: W303-la (a ade2-l trpl-
  • MYY290 is a ura3 derivative of strain AH216 (a leu2 his3 phoC phoE)
  • MYY385 a leu2 his3 ura3 phoC phoE hsfl-m3 (Smith and Yaffe, 1991)
  • DR20-2b was obtained as a haploid HIS+ and temperature- sensitive spore clone from a cross of MYY385 and W303 skn7 ⁇ .
  • Minimal and rich medium for yeast propagation has been described previously (Sherman et al . , 1986) .
  • ⁇ -Galactosidase assays The vector pZJHSE2-137 (a gift from E. Craig) containing a region of the SSA1 promoter inserted into the 2 ⁇ m based LacZ fusion plasmid pLG660 was transformed into W303-la and an isogenic skn7 ⁇ strain. Transformants were grown to mid-log phase in selective medium at 30 * C and harvested before or after the addition of butyl hydroperoxide to 0.6mM for one hour. For the heat shock experiment, cells were initially grown in selective minimal medium at 25 ⁇ C and cells harvested before and one hour after the culture was shifted to 37 "C, Cell extracts were prepared as described previously (Guarente, 1983) .
  • the CEN-SKN7 and D427N-SKN7 plasmids were constructed by inserting a 3.8Kb Xbal-Sacl fragment of either pBAMl or pBAM2 (Morgan et al . , 1997), containing the entire SKN7 coding and promoter regions, into the multiple cloning site of YCplaclll (Gietz and Sugino, 1988).
  • pAKS80 (YEpHSFl) contains a 3.9Kb Eco RI fragment of the S. cerevisiae HSFl gene in YEplacl95.
  • YexH is a modified derivative of the 2 ⁇ m-based galactose-inducible expression plasmid pEMBLYex4 (Murray, 1987) in which a six histidine tag sequence was inserted upstream of the multiple cloning site to allow the N-terminal tagging of inserted genes.
  • YexH-SKN7 was constructed by inserting a 2 Kb Bam HI fragment containing the SKN7 coding region into the Bam HI cloning site of the vector. DNA sequencing confirmed the reading frame and the construct allows galactose-inducible expression of 6His- SKN7.
  • RNA analysis Northern hybridisation was as previously described (White et al . , 1986) . In all cases, probes for hybridisation to the heat shock genes used in this study were derived from PCR amplification of an internal fragment of the coding sequence of the gene.
  • the internal control used for mRNA quantitation in hydroperoxide treated cells was RPB4 (Choder, 1993) ; for heat shock experiments the actin gene was used.
  • Yeast cells were broken by vortexing with glass beads for 5-x30 seconds with 30 second intervals on ice in breakage buffer: 150mM NaCl, 50mM Tris-HCl pH7.5 , ImM MgC12 , 1% NP40, 10% glycerol, ImM EDTA, lOmM BaF, 50mM -glycerol phosphate.
  • a protease inhibitor mix was added to a final concentration of lOO ⁇ g/ml phenylmethyl sulfonyl fluoride, 2 ⁇ g/ml leupeptin, 2 ⁇ g/ml pepstain A, 50 ⁇ g/ml TLCK and 100 ⁇ g/mlTPCK.
  • Cleared lysates were prepared by centrifugation for 20 minutes at 18K rpm (Beckman SS34 rotor) and lmg of whole cell extract, initially pre-cleared with protein A Sepharose beads (Pharmacia) was incubated at 4°C with l ⁇ l polyclonal antiserum to the Skn7p under constant mixing for 1 hour.
  • Protein A Sepharose beads (approx. 50 ⁇ l of a 50% suspension in breakage buffer) were then added and mixing was allowed to continue for a further hour at 4°C. The beads were then harvested and washed four times in breakage buffer containing 200mM NaCl, followed by one wash in the same buffer with 50mM NaCl, and finally resuspended in an equal volume of 2X SDS sample buffer. Proteins were separated by SDS-PAGE through 6% acrylamide gels and transferred to nitrocellulose membranes via semi-dry transfer prior to ECL (Amersham) Western analysis with polyclonal antiserum to yeast Hsflp. ECL was performed in accordance with manufacturers guidelines and membranes were exposed to X-ograph XB-200 film for between 30 seconds and 5 minutes.
  • lmg cell extract prepared as above (with the omission of EDTA and MgCl 2 in the breakage buffer) was added to 200 ⁇ l Ni 2+ -NTA resin (50% slurry) equilibrated in breakage buffer. After incubation with mixing at 4 * C for one hour, the resin was washed four times in wash buffer (200mM NaCl, 50mM Tris-HCl, 1% NP40) , and once in wash buffer containing 50mM NaCl. Beads were then boiled for two minutes in 2X sample buffer and the supernatant subject to SDS-PAGE as described above.
  • Cells were harvested by centrifugation, washed in cold distilled water, resuspended in 2 -5ml breakage buffer (150mM NaCl, 25mM Tris pH7.5, 10% glycerol, 0.5% Nonident P-40) .
  • breakage buffer 150mM NaCl, 25mM Tris pH7.5, 10% glycerol, 0.5% Nonident P-40
  • lysozyme was added at lmg/ml and PMSF at ImM.
  • the cell suspension was incubated on ice for 30 minutes and then passed twice through a chilled French press chamber.
  • the clarified supernatant was then incubated with 5ml of a 50% slurry of Ni 2+ - NTA resin, equilibrated in binding buffer (250mM NaCl, 50mM Tris-HCl pH 7.5, 15mM immidazole) , and allowed to mix at 4 * C for one hour before preparing a 5ml column.
  • binding buffer 250mM NaCl, 50mM Tris-HCl pH 7.5, 15mM immidazole
  • tagged protein was eluted by a step gradient of binding buffer containing 50mM, lOOmM and 250mM immidazole.
  • Bradford protein assays Biorad were carried out on 0.5ml fractions and DNA-binding activity assayed by gel mobility shift assay.
  • Mobility shift assays have been described elsewhere (Lowndes et al . , 1991) .
  • 6His-Skn7 protein was incubated with 0.5ng (1x105 cpm) 32P 5' -end labelled double-stranded oligonucleotides of the following sequence: HSE2-5' tcgaTTTTCCAGAACGTTCCATCGGC (SEQ ID NO: 16); MUT HSE2-5' tcgaTTTTCCAAAACGTTTCATCGGC (SEQ ID NO: 17).
  • Binding reactions in 25mM Tris-HCl pH 7.5, lOOmM NaCl, ImM EDTA, 7mM MgCl2, 10% glycerol, protease mix as above and l ⁇ g poly(dl.dC) were incubated at room temperature for 15 minutes and on ice for a further 20 minutes. Protein-DNA complexes were resolved on a 4% non-denaturing polyacrylamide gel (37.5:1) by electrophoresis at 200V in 0.5X TBE buffer for 2 hours. Gels were dried on to Whatman 3MM paper and exposed to Kodak X-OMAT AR film over night at -20 * C.
  • Mbpl is a DNA binding protein which acts as a heterodimer in c ⁇ njuction with the regulatory protein, Swi6 (Breeden and Nasmyth, 1987; Lowndes et al , 1992 Koch et al 1993) .
  • Swi6 the regulatory protein
  • Mbpl binds principally to MCB elements but also to SCB elements.
  • Mbpl is involved in the regulation of transcription of DNA synthetic genes in the yeast S . cerevisiae as well as CLN1 and CLN2 which are expressed in late Gl .
  • Mbpl is a DNA binding protein which acts as a heterodimer in c ⁇ njuction with the regulatory protein, Swi6 (Breeden and Nasmyth, 1987; Lowndes et al , 1992 Koch et al 1993) .
  • Mbpl binds principally to MCB elements but also to SCB elements.
  • Mbpl is involved in the regulation of transcription of DNA synthetic genes in the yeast S
  • an assay for a putative inhibitor of fungal growth comprising: a) bringing into contact a Skn7 polypeptide, and a Mbpl polypeptide and a putative inhibitor compound under conditions where the Skn7 polypeptide, in the absence of inhibitor, is capable of binding to the Mbpl polypeptide; and b) measuring the degree of inhibition of binding between the Skn7 and Mbpl polypeptides caused by said inhibitor compound.
  • Mbpl polypeptide includes a polypeptide including the amino acid sequence for Saccharomyces cerevisiae wild-type Hsfl shown in Koch et al and variants thereof (which may be synthetic or naturally occurring) , in particular showing a characteristic of S. cerevisiae Mbpl polypeptide, such as binding to the Skn7 polypeptide and/or cooperating with it in the induction of Mbpl dependent transcription in S. cerevisiae in the absence of Swi6.
  • Variants inlcude mutants such as temperature sensitive mutants, alleles such as sequence variants of the proteins described above from S . cerevisiae which demonstrate a substantially similar phenotype, homologues and analogues, such as found in other species, or synthetic variants and derivatives, which retain the functions of the above described proteins to the extent necessary for the paricular assay format being utilised.
  • Variants and fragment of Mbpl may be defined as above for those of Yapl and Hsfl, with the definitions above applicable mutatis mutandis to Mbpl.
  • variants may include those with at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% amino acid identity to Mbpl of S . cerevisiae . Fragments of Mbpl and its variants, defined as above, may also be used.
  • fragments of Skn7 and its variants which may be used include those in which the RIM region (see below) is retained, together with sufficient flanking sequences such that the interaction with Mbpl is retained.
  • Assays of this aspect of the invention may be in any of the formats described herein for other aspects of the invention, including a two-hybrid assay, a pull -down assay, an immunoprecipitation assay, of a DNA binding assay in the presence of DNA comprising MCB and SCB elements to which the Mbpl binds or a transcription assay.
  • Assay conditions may be as described herein for the above assays of Skn7 with Yapl or Hsfl.
  • the assays may be in vi tro or in vivo as appropriate.
  • Cells which comprise a Skn7 polypeptide-encoding sequence operably linked to a heterologous promoter, together with an expression construct comprising a Mbpl polypeptide-encoding sequence operably linked to a heterologous promoter form a further embodiment of this further aspect of the invention.
  • the two constructs may be present on separate vectors, or the same vector, as described above for Yapl or Hsfl.
  • Mbpl was retained by a GST-Swi6 fusion protein but not by GST alone. More importantly, Mbpl also clearly bound to a GST-Skn7 fusion protein. The Mbpl/Skn7 association is a strong interaction, for it is stable in up to 1M salt.
  • RIM A short region in the Skn7 protein, designated RIM, has been shown to mediate interactions between Skn7 and certain other proteins.
  • the RIM region lies between residues 238 and 261 at the beginning o the coiled-coil domain and deletion of the RIM region is expected to impair any protein-protein interaction involving the coiled-coil domain.
  • the RIM region is also required for at least part of the in vivo function of Skn7.
  • a C-terminal truncation, mission amino-acids 353-623 including the receiver domain and the glutamine-rich region did not suppress the temperature-sensitivity of a swi4 ts swi ⁇ mutant, indicating that the receiver domain is also necessary for in vivo Skn7 function.
  • the RIM region as well as the receiver domain are required for the in vivo interaction with Mbpl.
  • the W303 3HAMBP1 skn7A strain was constructed by disrupting SKN7 in W303 3HAMBP1. This was performed by transforming a restriction fragment carrying skn7 :HIS3 (Morgan et al , 1995b) into W303 3HAMBP1 and selecting His + transformants.
  • plasmid pAB52 was created by ligating the coding region of SKN7 , with BamHI (5') and Spel (3') linkers added by PCR, into pT7linktag, a plasmid which results in SKN7 being under the control of the T7 promoter.
  • pAB53 was constructed by ligating the coding region of SKN7 , with BamHI (5') and Spel (3') linkers added by PCR, into PGEX-KG (Pharmacia) , so that Skn7 can be expressed in E coli as fusion with GST.
  • pAB ⁇ l, pAB63, pAB64 are deletions of pAB53, in which the fusion protein is truncated after residue 247, 473 and 311, respectively.
  • pAB65 is a fusion between GST and the residues 381-623 of SKN7, including the receiver domain.
  • the skn7 ⁇ RIM allele encoding a protein deleted from the residues 238-261, was amplified by PCR from the pGAD skn7 ⁇ RIM plasmid, and inserted into the EcoRI site of pGEX-KG in frame with the GST, thus creating pAB81.
  • the MPB1 open reading frame was amplified by PCR with BamHL sites -added at either end of the gene, and then cloned into pEMBLyex4 (Murray, 1987), to create pGAL-MBPl .
  • pEMBLyex4 Murray, 1987
  • pGAL-MBPl pGAL-MBPl
  • an Ncol -Bglll fragment corresponding to residues 215 to 833, was cloned in frame into pASl-CYH 2 (Harper et al . , 1983), thus creating- pAB75.
  • the plasmid expressing SW16 under the control of the T7 promoter has already been " described (Primig et al . , 1992).
  • Protein extracts from log-phase cultures grown at 30°C in YEPD were prepared and 0.5 mg was immunoprecipitated with 1 ⁇ l of polyclonal antibody directed against Skn7 as previously described (Toyn and Johnston, 1994) .
  • the presence of £HAMbpl in the immunoprecipitates was then assessed by Western blot with the 12CA5 monoclonal antibody (Morgan et al . , 1995).
  • SKN7 a yeast multicopy suppressor of a mutation affecting cell wall b-glucan assembly, encodes a product with domains homologous to prokaryotic two-component regulators and to heat shock transcription factors. J. Bacteriol . , 175, 6908-6915. Brown, J.L., Bussey, H. and Stewart, R.C. (1994) Yeast Skn7p functions in a eukaryotic two-component regulatory pathway. EMBO J. , 13, 5186-5194.
  • a distal heat shock element promotes the rapid response to heat shock of the HSP26 gene in the yeast Saccharomyces cerevisiae. J. Biol. Chem., 268, 7442-7448.
  • Saccharomyces cerevisiae has an inducible response to menadione which differs from that to hydrogen peroxide. J.
  • Cipl The p21 Cdk-interacting protein Cipl is a potent inhibitor of Gl-dependent kinases .
  • the Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae. EMBO J. , 16, 1035- 1044. Morgan, B.A., Bouquin, N. and Johnston, L.H. (1995a)
  • the yeast heat shock transcription factor contains a transcription activation domain whose activity is repressed under non-shock conditions.
  • Hspl04 is required for tolerance to many forms of stress. EMBO J, 11, 2357-2364.
  • Saccharomyces cerevisiae CTT1 gene EMBO J, 13, 4382-4389. Sewell,A.K., Yokoya,F., Yu,W., Miyagawa,T., Muragama,T. and
  • Yeast heat shock factor is an essential binding protein that exhibits temperature dependent phosphorylation. Cell, 54, 855-864.
  • Yeast heat shock factor contains seperable transient and sustained response transcriptional activators.
  • Heat shock factor is regulated differently in yeast and HeLa cells.
  • Saccharomyces cerevisiae AP-1 protein discriminates between oxidative stress elicited by the oxidants H 2 0 2 and diamide. J.
  • Periodic transcription as a means of regulating gene expression during the cell cycle contrasting mode of expression of DNA ligase genes in budding and fission yeast.

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Abstract

L'invention se rapporte à la découverte selon laquelle la protéine de levure Skn7 agit comme facteur de transcription et est impliquée dans la fixation directe sur l'ADN. La transcription par Skn7 comprend la coopération avec d'autres facteurs cellulaires, notamment les protéines Yap1, Hsf1 et Mbp1. La fixation de Skn7 sur des séquences spécifiques d'ADN et son interaction avec d'autres facteurs fournissent des cibles aux dosages qui permettent de cribler les inhibiteurs potentiels de la prolifération fongique.
PCT/GB1998/000643 1997-02-28 1998-03-02 Interactions du gene skn7 et utilisation dudit gene dans des procedes de dosage WO1998038331A1 (fr)

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* Cited by examiner, † Cited by third party
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WO1999052941A3 (fr) * 1998-04-09 1999-12-02 Medical Res Council Domaines polypeptidiques capables de se fixer aux proteines rho et leur utilisation dans des analyses
EP0999284A1 (fr) * 1998-09-11 2000-05-10 Hoechst Marion Roussel Procédé de triage pour des substances antimycotiques utilisant des gènes dérivant de C.albicans, et les gènes eux-mêmes
WO2000015838A3 (fr) * 1998-09-11 2001-02-01 Hoechst Marion Roussel Inc Genes essentiels derives de c. albicans et methode de detection de substances antimycotiques utilisant lesdits genes
WO2000058521A3 (fr) * 1999-03-31 2001-04-05 Rosetta Inpharmatics Inc Methodes d'identification de molecules rapporteurs et cibles a l'aide de profils complets d'expression de genes
US7078169B2 (en) 1999-03-31 2006-07-18 Rosetta Inpharmatics Llc Methods for the identification of inhibitors of an isoprenoid metabolic pathway
WO2011146439A3 (fr) * 2010-05-17 2012-05-10 Worcester Polytechnic Institute Identification d'agents antifongiques qui inhibent iaa ou un membre de la famille yap

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JP2001512983A (ja) 2001-08-28

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