WO2000078994A1 - Compositions and methods for the quantification of sterol biosynthetic flux - Google Patents
Compositions and methods for the quantification of sterol biosynthetic flux Download PDFInfo
- Publication number
- WO2000078994A1 WO2000078994A1 PCT/US2000/017190 US0017190W WO0078994A1 WO 2000078994 A1 WO2000078994 A1 WO 2000078994A1 US 0017190 W US0017190 W US 0017190W WO 0078994 A1 WO0078994 A1 WO 0078994A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- yeast cells
- test compound
- growth
- trna
- growth media
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- 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
-
- 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
Definitions
- This invention generally relates to a novel assay for the screening of compounds that are agonistic or antagonistic to the mevalonate pathway and sterol and cholesterol synthesis.
- this assay incorporates colorimetric, growth, and imrnunological methods for high throughput screening of compounds.
- BACKGROUND A finely tuned mechanism regulates the biosynthesis of mevalonate, the precursor of isoprenoid groups that are incorporated into more than a dozen classes of end products. These include: sterols, especially cholesterol, involved in membrane structure; haem A and ubiquinone, involved with electron transport; dolichol, required for glycoprotein synthesis; isopenentyladenine, present in some transfer RNAs; and intercellular messengers, such as cytokines in plants, farnesylated mating factors in fungi, juvenile hormones in insects and steroid hormones in animals.
- sterols especially cholesterol, involved in membrane structure
- haem A and ubiquinone involved with electron transport
- dolichol required for glycoprotein synthesis
- isopenentyladenine present in some transfer RNAs
- intercellular messengers such as cytokines in plants, farnesylated mating factors in fungi, juvenile hormones in insects and steroid hormones in animals.
- HMG-CoA reductase catalyzes the rate-limiting step of the mevalonate pathway (Voet and Voet, Biochemistn' Wiley, New York, 1990), therefore, inhibitors of this reductase have been developed for administration to patients with hypercholesterolemia in an attempt to lower their blood cholesterol levels (Endo, et al , "Oxygenated cholesterols as hgands for cytosohc-nuclear tumor promoter binding protein yakkasteroids" Biochem Biophys Res Commun 194 1529-35, 1993).
- Zaragozic acid inhibits the enzyme activity of squalene synthase which is the first step of the pathway committed solely to sterol biosynthesis, but appears not to be currently used in the clinical setting.
- Zaragozic acid D3 inhibits farnesyl-protein transferase and, therefore, protein prenylation as well (Tammoto et al , "Inhibitory activity to protein prenylation and antifungal activity of zaragozic acid D3, a potent inhibitor of squalene synthase produced by the fungus, Molhsia sp SANK
- a screen for compounds affecting various steps of the mevalonate pathway could identify potential therapeutics for treatment of hypercholesterolemia and other pathological conditions associated with sterol metabolism in addition to compounds which may inhibit oncogene protein prenylation and farnesynelation.
- the availability of a screen for flux through the sterol pathway could be useful for predicting undesirable side effects of drugs designed to treat other illnesses.
- compounds that regulate the mevalonate pathway may have uses beyond medicine such as agriculture and pest control. Therefore, what is needed is an efficient, flexible, high-throughput assay to screen for agents that are agonistic or antagonistic to mevalonate pathway function.
- the present invention relates to an assay designed to detect flux in the melvonate pathway.
- the assay is a plate based assay incorporating the yeast strain Saccharomyces cerevisiae .
- the modification of tRNA by Mod5p is in competition with flux through the mevalonate pathway. This competition results from both Mod5p and Erg20p using the same substrate, dimethylallyl-PP ( Figure 1).
- Mod5p catalyzes the transfer of an isopetenyl moiety to an adenosine generating i A at position 37 of some tRNAs. This modification affects the function of the tRNA in translation and may be measured by monitoring nonsense suppression.
- yeast growth on particular media can provide an index for both increases and decreases in mevalonate pathway flux.
- the assays contemplated by the present invention are efficient, inexpensive and provide new methods for screening drugs that alter the clinically significant mevalonate pathway . It is not intended that the present invention be limited to the identification of compounds for only to a specific therapeutic application.
- compounds that regulate the mevalonate pathway may be beneficial in: (1) screening for new drugs to treat hyperlipidemias and other disorders in sterol metabolism such as Addison's disease and Cushing's syndrome; (2) screening for drugs that inhibit the function of famesylated and prenylated oncogene products; (3) monitoring other drugs for possible side effects in sterol metabolism; (4) identification of yeast mutants with altered sterol metabolism; (5) screening for agents that alter plant physiological processes such as, for example, photosynthesis, cell growth, respiration, architecture and defense against pathogen attack (e.g., antibiotics and antifungals).
- the present invention relates to a flexible, high throughput screen for agents that are agonistic or antagonistic to mevalonate pathway function.
- the present invention is not limited to any particular high throughput assay. Many high throughput assays are contemplated by the present invention.
- the present invention contemplates visually scoring plates.
- the present invention contemplates culturing cells in microtiter plates, performing the assay, lysing the cells and generating a readout of said cell lysates via a spectrophotometer.
- the present invention contemplates reading the cells in a flow cytometer to detect changes in cell color and in cell growth.
- the present invention contemplates measuring (in one example by scintillation) radiation generated by H 3 -tritium as an index to determine cell growth.
- the assay may be performed manually or with the use of automation and robotics for any or all steps in the aforementioned procedures.
- the present invention relates to yeast strains engineered to provide detectable read-outs for compounds that are agonistic or antagonistic to the mevalonate pathway.
- the yeast strains are ALB1 (genotype: MATamod5-M2 SUP7 ade2-l canI-100 leu2-3, -112 lysl-1 lys2-l trpl ura3-l), ALB8 (genotype: MAT SUP7 canI-100 ade2-l leu2-3, -112 lysl-1 lys2-l trpl mod5::TRPl ura3-l::MOD5) and
- the present invention be limited to the screening of any particular compound or class of compounds. Proteins, lipids, carbohydrates, glycoproteins, lipoproteins, synthetic compounds, compounds contained in combinatorial i libraries or compounds and agents already being used as therapeutics may be screened by the present assay. Moreover, the screening of known therapeutics according to the present invention will reveal, heretofore, unknown side effects associated with the modulation of the mevalonate pathway. It is not intended the present invention be limited to any particular protocol to quantitate or qualitate the assay. In selected embodiments, for example, the assay output may be measured visually, colormetrically, fluorescently, by immunoblot, by radio- immunoassay or by HPLC.
- anti- i 6 A antibody is used to detect i 6 A production in cells treated with a compound.
- a Western blot is used to detect the presence of i 6 A. Quantitation is not limited to any particular method.
- Quantitation may be made by densitometer, chemiluminescence, and radiography.
- the present invention contemplates a composition comprising yeast with the relevant genotype of: SUP7 ade2-l canl-100 leu2-3 mod5-M2 and designated ALB1.
- the present invention further contemplates a composition wherein the yeast ALB1 is a strain of Saccharomyces cerevisiae.
- the present invention contemplates a composition comprising yeast with the relevant genotype of: SUP7 canl-100 ade2-l leu2-3 mod5::TRPl ura3-l::MOD5 and designated ALB8.
- the present invention contemplates a composition wherein the yeast ALB8 is a strain of Saccharomyces cerevisiae.
- the present invention contemplates a composition comprising the yeast strain ALB1 wherein the genotype further comprises:
- MATamod5-M2 SUP7 ade2-l canl-100 leu2-3, -112 lysl-1 lys2-l trpl ura3-l.
- the present invention contemplates a composition wherein the yeast ALB1 is a strain of Sacchoromyces cerevisiae.
- the present invention contemplates a composition comprising yeast with the relevant genotype of:: MATaSUP7 canl-100 ade2-l leu2-3, -112 lysl-1 lys2-l trpl mod5::TRPl ura3-l::MOD5 and designated ALB8.
- the present invention contemplates a composition of claim 7 wherein the yeast is Sacchoromyces cerevisiae.
- the present invention contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media formulated to allow scoring of nonsense suppression in yeast, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p, or its homolog, as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a gene with a nonsense mutation and a suppressor tRNA gene coding for a tRNA modified with isopentenyl adenosine by Mod5 or its homolog; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.
- the present invention also contemplates a measuring of step which comprises examining the color of said yeast cells of said treated portion.
- the present invention contemplates a measuring of step which comprises comparing said treated portion with said untreated portion, wherein said untreated portion is exposed to said growth media in the absence of said test compound.
- the present invention contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking adenine, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise an ADE gene having a nonsense mutation and a gene coding for a nonsense suppressor tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.
- the present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking adenine, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise an ADE gene having a nonsense mutation and a SUP7 gene coding for a tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.
- the present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking arginine and comprising a canavanine salt, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a CAN1 gene having a nonsense mutation
- the present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking arginine and comprising a canavanine salt, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a CANl gene having a nonsense mutation and a SUP7 gene coding for a tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion: and c) measuring for growth of said treated portion.
- the present invention contemplates a method wherein said gene coding for said nonsense suppressor tRNA is selected from the group consisting of SUP7 and SUP11.
- the present invention further contemplates a method comprising: a) providing i) one or more compounds, ii) a first yeast cell line designated ALB1; ii) a second yeast cell line designated ALB8; b) contacting a portion of said cells from i) said first yeast cell line and ii) said second yeast cell line, with said one or more said compounds, so as to create treated portions and untreated portions of cells; and, c) comparing said treated cells with said untreated cells.
- the method of comparison of said treated or untreated cells may be by color and cell growth (i.e., the amount of cell division).
- the present invention further contemplates a method comprising: a) providing i) one or more compounds and ii) a yeast cell line selected from a group consisting of yeast strains designated ALB1 and ALB8; b) contacting a portion of said cells from said yeast cell line with said one or more said compounds, so as to create treated portions and untreated portions of cells; and, c) i) comparing said treated cells with said untreated cells.
- the present invention further contemplates the comparison of treated and untreated cells by color and by cell growth. DESCRIPTION OF THE FIGURES
- Figure 1 shows the mevalonate pathway
- Figure 2 shows the level of isopentenylated tRNA found in ALB 1 over- expressing ERG20 is substantially reduced.
- RNA Low molecular weight RNA was prepared from ALB 1 (mod5-M2) with each of the candidate genes or vector alone. ALB8 (MODS) or MD14A (modS-1). The RNAs were resolved on polyacrylamide gels, transferred to membranes and probed with anti- isopentenyl adenosine antibody (upper panel) or radiolabeled oligonucleotide complementary to mature tRNA Tyr (lower panel).
- Figure 3 presents a model of competition between i 6 A modification of tRNA and sterol biosynthesis.
- Figure 4 presents selected mutations introduced into the MODS gene and oligonucleo tides. See, Gillman et al., "MOD 5 translation initiation sites determine N6- isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA", " Mol Cell
- Figure 5 presents two yeast strains that present limited cytosolic levels of Mod5p.
- Cells with the mod5-M2KR6 allele (T8-ID with YCfmod5-M2KR6 as projected in Fig. 5) have a very small cytosolic pool of Mod5p and the cells are unable to grow in the absence of lysine.
- Figure 6 presents a restriction map of YEpMOD5(7.0) and subclones YEpMOD5(1.8) and YEpMOD5(1.9). Numbers in parentheses indicate size of insert. See, Dihanich, et al., "Isolation and characterization of MOD 5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs o ⁇ Saccharomyces cerevisiae" Mol Cell Biol 7: 177-184, 1987).
- Gene shall be defined as the genetic makeup of an organism as encoded in its DNA.
- Relevant genotype shall be defined as specific genes that, if expressed, have impact on the investigation under way.
- a "nonsense suppressor tRNA" is meant to indicate one of several known suppressor tRNAs.
- SUP7-1 codes for an altered tRNA that is one of several efficient tyrosine-inserting UAA suppressors.
- the LYS2 gene encodes an enzyme involved in the biosynthesis of lysine. Mutations in this gene prevent growth on media lacking exogenous lysine.
- the term "scoring of nonsense suppression in yeast” refers to determining the level of suppression due to translation though the nonsense codon in mRNA relative to a control sample wherein said control sample comprises reduced or absent translation through the nonsense codon in mRNA because of differences in suppressor tRNA.
- cells with the mod5-M2 allele strain ALB1 projected in Fig.
- M2KR6 as projected in Fig. 5 have a very small cytosolic pool of Mod5p and the cells are unable to grow in the absence of lysine.
- Yeast strain "ALB1" is defined as a substantially pure population of yeast with the genotype of MATamod5-M2 SUP7 ade2-l canl-100 leu2-3, -112 lysl-1 lys2-l trpl ura3-l and the relevant genotype of: SUP7 ade2-l canl-100 leu2-3-112 modS-M2.
- the yeast may be of the species Sacchoromyces cerevisiae.
- Yeast strain "ALB8" shall be defined as a substantially pure population of yeast with the genotype of:: MATaSUP7 canl-100 ade2-l leu2-3, -112 lysl-1 lys2-l trpl modS::TRPl ura3-l::MOD5.
- the yeast may be of the species Sacchoromyces cerevisiae.
- Yeast strain "T8-1D” shall be defined as a substantially pure population of yeast with the genotype of:: MATa SUP11 ade2-l leu2-3, -112 modS-1 lys2-l his4-519 ura 3-1.
- the yeast may be of the species Sacchoromyces cerevisiae.
- CANl refers to a sequence encoding an arginine permease that allows the uptake of the arginine analog canavanme Canavanme interferes with the process of translation and cells cannot grow in its presence.
- ADE2 refers to a sequence which encodes an enzyme involved in the synthesis of adenine Cells with ade2-l turn red in color and fail to grow on defined medium lacking adenme (Ade), ⁇ hereas cells producing functional Ade2p can grow on such medium (Ade+) and are white Cells with the ade2-l allele and sufficient ⁇ 6A- modified suppressor tRNA can grow in the absence of exogenous adenine and generate white colonies on ⁇ ch medium. ⁇ hereas cells with insufficient ⁇ 6A-mod ⁇ fied tRNA are unable to grow in the absence of exogenous adenme and generate red colonies on ⁇ ch medium. Cells with intermediate levels of 16A modified tRNA have intermediate phenotypes in colony color and intermediate rates of growth m the absence of exogenous adenine.
- substantially deficient adenine growth media refers to a culture media that, in one embodiment, has less than 20mg /L of adenme while in a preferred emodiment has less than 5mg/L of adenine
- Wild type shall be defined as the genomic makeup of an organism (the genotype) before modifications ha ⁇ e been made. In other words, it is the parent strain of the organism.
- binding interaction when used in relation to RNA shall be defined as the ability of two or more proteins to bind to each other (e g , to produce an aggregate)
- the present invention makes no limit on the st ⁇ ngency of the binding interaction so long as the interaction can be detected by methods known to those practiced m the art (e.g., by Western blot, coimmunoprec ⁇ itation, spectrophotometry, colo ⁇ met ⁇ c assay, etc.).
- the term "homology" when used in relation to proteins refers to a degree of similarity There may be partial homology or complete homology ( ⁇ e , identity)
- a partially similar sequence is one that may partially inhibit a similar sequence from performing its function (e g , enzymatic, binding, etc) in vivo or in vitro and is referred to using the functional term "substantially homologous"
- the inhibition function of the ⁇ 0 substantially similar sequence may be examined using an enzymatic assay, a binding assay or other assay designed to measure the particular function of the substantially similar protein.
- a substantially homologous proteins may compete for or interfer with a homolog and inhibit its function (e.g., the binding or enzymatic function). This is not to say that conditions of low stringency are such that non-specific interaction is permitted; low stringency conditions require that the interaction be a specific (i.e., selective) interaction.
- Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 » H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ug/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE.
- High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 ⁇ 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
- the art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions.
- factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions
- Stringency when used in reference to nucleic acid hybridization typically occurs in a range from about T m -5°C (5°C below the T m of the probe) to about 20°C to 25°C below T m . As will be understood by those of skill in the art. a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under “stringent conditions" a nucleic acid sequence of interest will hybridize to its exact complement and closely related sequences.
- purified or “to purify” refers to the removal of contaminants from a sample.
- the present invention contemplates purified compositions (e.g., the ALB 1 and ALB8 stains of S. cerevisiae).
- substantially purified refers to the removal of a significant portion of the contaminants of a sample to the extent that the substance of interest is recognizable by techniques known to those skilled in the art as the most abundant substance in the mixture.
- portion when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein.
- the fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
- the present invention contemplates "functional portions” of a protein. Such portions are “functional” if they contain a binding region (i.e., a region having affinity for another molecule) and such binding can take place (i.e., the binding region functions, albeit with perhaps lower affinity than that observed for the full-length protein). Such “functional portions” of the gene product are typically greater than 50 amino acids in length, and more typically greater than 100 amino acids in length. “Functional portions” may also be “conserved portions” of the protein.
- the alignment of the yeast and human gene products permit one skilled in the art to select conserved portions of the protein (i.e., those portions in common between yeast and man) as well as unconserved portions (i.e., those portions unique to either yeast or man).
- the present invention contemplates conserved portions 20 amino acids in length or greater, and more typically greater than 50 amino acids in length.
- portion when in reference to an oligonucleotide sequence (as in “a portion of a given sequence”) refers to fragments of that sequence.
- the fragments may range in size from four base residues to the entire oligonucleotide sequence minus one base. More typically, such portions are 15 nucleotides in length or greater. Again, such portions may be conserved portions (see Figure 8). On the other hand, such portions may be unique portions of the gene.
- Compound refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function.
- Compounds comprise both known and potential therapeutic compounds.
- a compound can be determined to have therapeutic potential by screening, e.g., using the screening methods of the present invention.
- a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
- "Antibody” shall be defined as a glycoprotein produced by B cells that binds with high specificity to the agent (usually, but not always, a peptide), or a structurally similar agent (e.g., i 6 A), that generated its production (e.g., the i 6 A antibody).
- Antibodies may be produced by any of the known methodologies [Current Protocols in Immunology (1998) John Wiley and Sons, Inc., N.Y.] and may be either polyclonal or monoclonal.
- Antigen shall be defined as a protein, glycoprotein, lipoprotein, lipid or other substance that is reactive with an antibody specific for a portion of the molecule.
- immunoprecipitate refers to the use of antibody to take an antigen out of solution by precipitation.
- affinity purification refers to the use of an antibody to separate its antigen or a portion thereof from a mixture of other molecules because of affinity for the antigen.
- Immunofluorescence is a staining technique used to identify, mark, label, visualize or make readily apparent by procedures known to those practiced in the art, where a ligand (usually an antibody) is bound to a receptor (usually an antigen) and such ligand, if an antibody, is conjugated to a fluorescent molecule, or the ligand is then bound by an antibody specific for the ligand, and said antibody is conjugated to a fluorescent molecule, where said fluorescent molecule can be visualized with the appropriate instrument (e.g., a fluorescent microscope).
- Staining shall be defined as any number of processes known to those in the field that are used to better visualize, distinguish or identify a specific component(s) and/or feature(s) of a cell or cells.
- RIA or "radioimmunoassay” shall be defined as an assay wherein antibody is used to detect antigen and thereafter quantitated with radioisotopes.
- Immunoabsorbant and “immunoaffmity” shall be defined as binding antigen with antibody reactive to said antigen. The antibody or the antigen may be bound to a solid or semi-solid substrate.
- “Western blot” shall be defined as an assay comprising antigen that is detected by antibodies reactive to the antigen after the antigen has been at least partially separated from contaminants by, for example, electrophoresis.
- “In operable combination”, “in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
- “Heterologous DNA” sequence refers to a nucleotide sequence which is not endogenous to the cell into which it is introduced.
- Heterologous DNA includes a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
- Heterologous DNA also includes a nucleotide sequence which is naturally found in the cell into which it is introduced and which contains some modification relative to the naturally-occurring sequence.
- Morphology shall be defined as the visual appearance of a cell or organism when viewed with the eye, a light microscope, a confocal microscope or an electronmicroscope, as appropriate. Morphology shall include, but is not limited to, cell shape, cell appearance and cell color.
- Patient shall be defined as a human or other animal, such as a guinea pig or mouse and the like.
- This invention generally relates to a novel assay for the screening of compounds that are agonistic or antagonistic to the mevalonate pathway and sterol and cholesterol synthesis.
- this assay incorporates colorimetric, growth, biochemical (e.g., HPLC) and immunological methods for high through-put screening of compounds.
- Baker's yeast Saccharomyces cerevisiae are single celled eukaryotes used extensively as model organisms. This model system provides for the facile manipulations of yeast genes, availability of the complete genomic sequence of baker's yeast, and identification of numerous yeast genes which directly correlate with known human disease genes.
- One fundamental discovery that has become clear from the use of this model system is that many physiologically significant regulatory pathways and proteins are highly conserved among all eukaryotes.
- mammalian homologues of many yeast proteins are known to be important in regulating cell physiology, including the mevalonate pathway. Therefore, these model systems have been effectively utilized for the rapid identification and functional characterization of compounds that may be of use as therapeutics in higher eukaryotes.
- yeast is well suited as a model system not only for the elucidation of basic cell biology but also for large-scale screening of compounds which specifically target cell growth.
- Saccharomyces cerevisiae protein Mod5p catalyzes the addition of an isopentenyl group to adenosine ( ⁇ 6 A) at position 37 of the anticodon loop of some tRNAs (Kline, et al, "N6-(delta-2- ⁇ sopentenyl) adenosine.
- Mod5p-I is translated starting at codon one of the MODS ORF and is localized to mitochond ⁇ a and the cytoplasm.
- Mod5p-II translated from codon twelve of the MODS ORF, is located in the nucleus and the cytoplasm (Gillman, et al., "MODS translation initiation sites determine N6- ⁇ sopentenyladenosme modification of mitochond ⁇ al and cytoplasmic tRNA" Mol Cell Biol 1 1.2382-2390, 1991 ; Boguta, et al., "Subcellular locations of MODS proteins: mapping of sequences sufficient for targeting to mitochond ⁇ a and demonstration that mitochond ⁇ al and nuclear isoforms commingle in the cytosol" Mol Cell Biol 14.2298-2306. 1994)
- Erg20p causes a decrease in ⁇ 6 A tRNA-mediated suppression m yeast and an approximately 70% decrease m ⁇ 6 A modification of tRNA. These effects are most likely due to the loss of Mod5p substrate to Erg20p.
- the data demonstrate the dependence of tRNA processing upon changes in components of the sterol pathway. The data also indicate that, at least in yeast, Erg20p may also catalyze a rate-limiting step m sterol biosynthesis. Hence, the tRNA and the sterol biosynthetic pathways are in apparent competition and a delicate balance m protein levels is required to maintain proper functioning of translation and mevalonate metabolism
- the ⁇ 6 A modification promotes the efficiency of SUP7o ⁇ SUP11 tRNA in cytosolic suppression of UAA nonsense mutations by the insertion of tyrosme (Laten, et al , "Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae" Nucleic Acids Res 5.4329-4342, 1978)
- Cells possessing only Mod5p-I have limiting cytosolic amounts of lsozyme and changes m the subcellular dist ⁇ bution and/or the activity of this isozyme alter nonsense suppression.
- the first category includes genes that affect nonsense suppression via alteration of the protein synthetic machinery.
- Applicants' studies suggest that the yeast gene product encoded by YDL219w may function in protein synthesis and that the translation elongation factor EFl- ⁇ may function in translational proofreading. SAL6 and ARC1, with previously established effects on protein synthesis, were also recovered.
- the second category includes ERG20, involved in sterol biosynthesis.
- the mevalonate pathway generates sterols, prenylated proteins, heme A, dolichol, ubiquinone and the substrate required for isopentenylation of tRNAs ( Figure 1).
- Both Mod5p and Erg20p use the same substrate, dimethylallyl pyrophosphate (DMAPP).
- Sal ⁇ p/Ppqlp is a serine-threonine protein phosphatase very similar to mammalian phosphatase PP1 (Vincent, et al., "The yeast translational allosuppressor, SAL6: a new member of the PPl-like phosphatase family with a long serine-rich N-terminal extension” Genetics 138:597-607, 1994; Chen, et al., "PPQ, a novel protein phosphatase containing a Ser + Asn-rich amino-terminal domain, is involved in the regulation of protein synthesis" Eur J Biochem 218:689-699, 1993).
- Yeast cells containing a disrupted PPQ1 gene exhibit a slowed translation rate and hypersensitivity to inhibitors of protein synthesis (Chen, et al., "PPQ, a novel protein phosphatase containing a Ser + Asn-rich amino-terminal domain, is involved in the regulation of protein synthesis” Eur J Biochem 218:689-699, 1993).
- Tef4p is the gamma subunit of elongation factor 1 (EF-1).
- Eukaryotic EF-1 functions in delivering incoming tRNAs to the ribosome and is composed of at least three subunits.
- EF-l ⁇ binds GTP and then the proper aminoacyl-tRNA and positions the aminoacyl tRNA in the ribosomal A site.
- GTP is hydrolyzed to GDP and EF-l ⁇ catalyzes the exchange of GDP for GTP to restore EF-l ⁇ to its initial state
- EF-l ⁇ catalyzes the exchange of GDP for GTP to restore EF-l ⁇ to its initial state
- yeast EF-1 serves a similar function as the bacterial counterpart, it is unlikely that the ⁇ subunit participates in this function as extra copies of the genes encoding EF-l ⁇ decrease fidelity (i.e., they enhance suppression of nonsense mutations; Song, et al., "Elongation factor EF-1 alpha gene dosage alters translational fidelity in Saccharomyces cerevisiae" Mol Cell Biol 9:4571-4575, 1989).
- Tef4p reduces nonsense suppression and therefore it is possible that it is the EFl- ⁇ subunit that functions in EF-1 proofreading.
- ORF YDL219w is predicted to code for a 150 amino acid protein with no significant homology to any characterized protein. However two lines of evidence indicate that this protein may function in the translation process.
- the gene possesses an intron (Saccharomyces Genome Database, http://genome-www.stanford.edu/cgi- bin dbrun).
- introns are rare in yeast other than for approximately half of the genes encoding ribosomal proteins (Woolford and Wamer, in "The molecular and cellular biology of the yeast Saccharomyces: Genomic dynamics, protein synthesis and energetics" eds. Broach, et al. [Cold Spring Harbor Lab Press, Plainview, NY] Vol. 1, pp.
- Arclp or G4pl, was originally discovered as a quadruplex nucleic acid binding protein (Frantz and Gilbert, "A novel yeast gene product, G4pl , with a specific affinity for quadruplex nucleic acids” J Biol Chem 270:20692-20697, 1995). Subsequently, Arclp was demonstrated to bind tRNA and to complex with methionyl and glutamyl tRNA synthetases (Simos, et al., "The yeast protein Arclp binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases" EMBO J 15:5437-5448, 1996.
- yeast protein Arclp binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases” EMBO J 15:5437-5448, 1996) uncovered ARC1 by synthetic interactions with Loslp, the yeast tRNA exportin (Arts, et al, "Identification of a nuclear export receptor for tRNA” Curr Biol 8:305-314, 1998; Sarkar and Hopper, "tRNA nuclear export in Saccharomyces cerevisiae: in situ hybridization analysis” Mol Biol Cell 9:3041-3055, 1998).
- FIG. 4 presents the nucleotide sequence of the MODS gene from two nucleotides 5' of the SnaBI site upstream of the open reading frame codon 14, followed by the sequence in the area of the first Nsil site.
- the DNA sequences of oligonucleotides Ml and M2 are shown below the corresponding wild-type (WT) sequence. Restriction endoculease recognition sites are underlined and indicated above the sites. Encoded amino acids are in three-letter code below codons. The first two methionine codons are indicated by capital letters.
- the M2 oligonucleotide encodes the only amino acid change in MOD5 (Met to He); all other changes become part of the 5' noncoding portion of the gene. Arrows show positions of 5' ends of MODS transcripts.
- the mevalonate pathway generates sterols, prenylated proteins, heme A, dolichol, ubiquinone and isopentenyl tRNAs.
- Inhibitors of HMG-CoA reductase, catalyzing a rate-limiting step of the pathway have been developed for administration to patients with hypercholesterolemia (Goldstein and Brown, "Regulation of the mevalonate pathway” Nature 343:425-430, 1990.
- the "statin” class e.g., compactin and lovastatin
- reductase inhibitors are safe and effective in lowering total cholesterol levels and preventing the progression and reducing the occurrence of coronary disease events (Endo,
- Zaragozic acid inhibits squalene synthetase catalzying the first step of the pathway committed solely to sterol biosynthesis, but appears not to be currently used in the clinical setting.
- a novel type of zaragozic acid, ZAD3 also inhibits farnesyl protein transferase and, therefore, protein prenylation as well (Tanimoto, et al, "Inhibitory activity to protein prenylation and antifungal activity of zaragozic acid D3, a potent inhibitor of squalene synthase produced by the fungus,
- the substance may be increasing the flow of DMAPP through the mevalonate pathway and decreasing tRNA modification.
- the mevalonate pathway produces isoprenoids that are vital for diverse cellular functions, ranging from cholesterol synthesis to growth control.
- Several mechanisms for feedback regulation of low-density-lipoprotein receptors and of two enzymes involved in mevalonate biosynthesis ensure the production of sufficient mevalonate for several end- products. Manipulation of this regulatory system could be useful in treating certain forms of cancer as well as heart disease.
- a finely tuned metabolic feedback mechanism involving transcriptional and post-transcriptional inputs maintains a pool of mevalonate pathway intermediates essential for cell survival in addition to maintaining cholesterol homeostasis.
- Cholesterol the bulk end product of the pathway in sterologenic tissues, exerts transcriptional control on sequential activities in the mevalonate pathway, principally those catalyzed by HMG CoA synthetase, HMG CoA reductase and farnesyl pyrophosphate synthase.
- HMG CoA reductase is the rate-limiting enzyme of mevalonate biosynthesis as well as the most highly regulated enzyme in this series.
- HGM CoA reductase integrates activities at the transcriptional, post- transcriptional and post-translational levels.
- Sterol-mediated regulation of transcription is mediated through the sterol regulatory element (SRE), a promoter-enhancer octanucleotide sequence located in the 5' flanking region of the reductase gene.
- SRE sterol regulatory element
- Bile cholesterol for example, consists of unesterfied cholesterol already solubilized in mixed bile salts (BS) phospholipids (PL) micelles. Exogenous cholesterol is not water soluble and is not absorbed an in FA ester. Pancreatic cholesterol esterase, activated by BS. is capable of splitting cholesterol ester into the free sterol and fatty acid (FA), but this is a rather slow process compared to the hydrolysis of triglycerides (TG). Free cholesterol is dissolved in the oil phase and into micelles. When the oil phase disappears, cholesterol precipitates slowing down the rate of absorption.
- FA free sterol and fatty acid
- micellar solubilization may explain the relatively poor absorption of exogenous cholesterol in healthy subjects, but it does not explain why endogenous cholesterol is poorly absorbed also. It is most likely that cholesterol absorption from micelles slows down considerably when monoglycerides (MG), FA, and PL have disappeared from the micelle because their rate of absorption is much faster and BS micelles alone are poor detergents.
- Chijiwa and Linscheer's (Chijiwa and Linscheer, "Effect of intraluminal pH on cholesterol and oleic acid absorption from micellar solutions in rat" Am J Physiol 246:G492-G499, 1984) observation that absorption rates of cholesterol are much higher at the lower pH of the duodenum and proximal jejunum than at the higher pH in the distal half of the small bowel suggests that this may be a contributing factor.
- cholesterol is essential for the intestinal excretion mechanism of cholesterol.
- Cholesterol is also the substrate for BS synthesis, but reabsorption of BS is highly efficient and the amount of BS excreted in feces is not the main excretory pathway of cholesterol.
- the present invention makes possible the ability to screen for compounds that may have chemotherapeutic use without the need to run complex genetic experiments. It also allows for the screening of compounds that are agonistic or antagonistic to a pathway known to influence tumor growth and survival. Mutant ras oncogenes and alterations in the mitogenic signaling pathways that they regulate are associated with a wide variety of solid tumors and leukemias for which existing chemotherapeutics have limited utility.
- the present invention is also useful for the screening of compounds that may be beneficial in the control of obesity.
- Metabolic flux in the mevalonic acid pathway depends on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which regulates the first part of the pathway and the overall synthesis of cholesterol (Goldstein and Brown. "Regulation of the mevalonate pathway” Nature 343:425-430, 1990).
- HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A
- GGPP is over expressed in multiple tissues of the ob/ob mouse. It is also expressed in increased amounts during adipocyte differentiation in culture. Mammalian GGPP synthase catalyzes the synthesis of FPP and GGPP, the two isoprenoids that take part in protein prenylation.
- this enzyme by itself or in combination with FPP synthase, serves as a favored point for regulation of prenyl group production for protein prenylation.
- This protein appears highly regulated during adipogenesis and is overexpressed in an animal model of obesity.
- compounds that regulate the mevalonate pathway may be useful in the regulation of downstream products of the pathway that influence adipogenesis and obesity.
- DMAPP dimethylallyl pyrophosphate
- i°A isopentenyladenasine
- the mevalonate pathway in plants is responsible for the production of compounds that are necessary for photosynthesis (carotenoids, chlorophylls, phylloquinone, plastoquinone), respiration (ubiquinone, cytochrome a), membrane architecture (sterols and triterpenoids), regulation of growth and development (gibberellic acids, abscisic acid, brassinosteriods, certain cytokinins), defense against pathogen attack (numerous isoprenoid phytoalexins), exchange of signals and prenylation of proteins (allowing for membrane targeting and cell cycle regulation) (Bach, "Some new aspects of isoprenoid biosynthesis in plants - A review" Lipids 30: 191-202, 1995).
- the present invention is useful for the screening of compounds that may be beneficial as regulators of photosynthesis, membrane integrity, plant growth (e.g., fertilizers) and protection (e.g., insecticides, fungicides, antimicrobials and antivirals).
- Fungi are single celled living forms of life which inhabit the land, air and waters of the earth. They are more highly developed than bacteria and viruses. It is estimated that there are over 500,000 different species. Fungi have existed on earth hundreds of ay millions of years and, quite remarkably, have experienced little genetic change during that period of time. Viable fungi can grow from spores which have been dormant for thousands of years, such as has been observed in spores which were found in Egyptian tombs. Single fungal cells can only be seen under the microscope, but a colony of these cells makes a visible presence in the form of mushrooms, toad stools and molds on food or elsewhere.
- fungi In between these extremes are fungal infections associated with diseases such as diabetes, cancer as well as conditions which include intra- and inter-specific cross infections.
- yeast and molds Two groups of fungi that are of medical importance are yeast and molds. Both are nearly ubiquitous and species of both can cause infections in mammals. Common ailments attributed to fungi are yeast infections and various fungal skin infections. More severe medical conditions can occur if internal infections arise, e.g., after surgery.
- mycotoxins are biologically active substances that have evolved as a protective mechanism for the fungus. Some mycotoxins have become useful to humans. Antibiotics, such as penicillin, are examples. However, many mycotoxins can be harmful or deadly. Fungal infections, therefore, are not only limited to the site of infection, but, through the production of mycotoxins, can adversely affect the entire organism. For example, mycotoxins have been linked to cancer. Therefore, even if the fungal infection is cleared, the patient may still suffer long term consequences as a result of the infection.
- This invention contemplates the screening of compounds that can be useful as anti-fungal agents for both animals and plants.
- the desired compound would have to be toxic to the fungus without causing undue negative effects on the patient.
- the invention by virtual of the ubiquitousness of the mevalonate pathway, provides a screen where compounds that are potential antifungal therapeutics can be identified. Additionally, the present invention will be useful in the identification of compounds that may be used to eliminate yeast as an infectious agent.
- Oligonucleotides can be synthesized on an Applied BioSystems oligonucleotide synthesizer [for details see Sinha et al., Nucleic Acids Res. 12:4539 (1984)], according to specifications provided by the manufacturer. Complementary oligonucleotides are annealed by heating them to 90°C in a solution of 10 mM Tris-HCl buffer (pH 8.0) containing NaCl (200 mM) and then allowing them to cool slowly to room temperature.
- duplex DNA is purified from native polyacrylamide (15% w/v) gels.
- the band corresponding to double-stranded DNA is excised and soaked overnight in 0.30 M sodium acetate buffer (pH 5.0) containing EDTA (1 mM). After soaking, the supernatant is extracted with phenol/chloroform ( 1/1 v/v) and precipitated with ethanol.
- DNA substrates are radiolabeled on their 5'-OH group by treatment with
- the present invention relates to the screening of compounds that are agonistic or antagonistic to the mevalonate pathway. Such compounds may be further screened for effectiveness in regulating the production of various end products of the mevalonate pathway.
- the regulation of these products may be instrumental in the control or treatment of clinical disease states.
- such compounds may regulate vascular tone and endothelial function including vascular smooth muscle proliferation, plaque stabilization associated with atherosclerotic lesions and associated cholesterol regulation, prevention of glomerular injury in renal disease, treatment of malignancies and prevention of rejection in organ transplant.
- the current class of drugs, the statins, used for the control of cholesterol levels exert a physiological effect via regulation of the mevalonate pathway. However, these drugs do not all perform equally in terms of modulating cholesterol levels or in other therapeutic properties. In this regard, new drugs or classes of drugs may be more effective for the various therapeutic purposes listed here or for other therapeutic applications.
- the present invention is not limited to any particular mechanism or embodiment.
- the i 6 A modification of tRNA catalyzed by Mod5p promotes the efficiency of SUP7 or SUP11 tRNAs in the suppression of UAA nonsense mutations by insertion of tyrosine at positions in the growing peptide corresponding to UAA stop codons (Laten et al, "Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae" Nucleic Acids Res. 5:4329-4342).
- the extent to which this nonsense suppression occurs during translation in the cytoplasm can be used as an indication of the amount of Mod5p and isopentenylated tRNA in the cytoplasm.
- the canl-100, ade2-l, and lys2-l alleles contain UAA nonsense mutations.
- CANl encodes an arginine permease that allows the uptake of the arginine analog canavanine (Whelan et al, "The CANl locus of Saccharomyces cerevisiae: fine-structure analysis and forward mutation rates” Genetics 91 :35-51, 1979). Canavanine interferes with the process of translation and cells can not grow in its presence. Therefore, cells with wild-type CANl are sensitive to canavanine (Can s ), but cells with the mutant canl-
- Cells with i 6 A completely modified SUP7 or SUPll tRNAs can not grow in the presence of canavanine, whereas cells lacking i 6 A on these tRNAs are able to grow in the presence of this drug.
- Cells with intermediate levels of i A show intermediate levels of growth in the presence of the drug.
- ADE2 encodes an enzyme involved in the synthesis of adenine.
- Cells with ade2-l turn red in color, due to accumulation of a metabolic intermediate, and fail to grow on defined media lacking adenine (Ade ⁇ ), while cells producing a functional Ade2p can grow on such media and are white (Ade + ) (Rasse-Messenguy and Fink, "Temperature- sensitive nonsense suppressors in yeast” Genetics 75:459-464, 1973).
- Cells with the ade2-l allele and sufficient i 6 A modified suppressor tRNA can grow in the absence of exogenous adenine and generate white colonies on rich media whereas cells with insufficient i 6 A modified suppressor tRNA are unable to grow in the absence of exogenous adenine and generate red colonies on rich media.
- Cells with intermediate levels of i 6 A modified tRNA have intermediate phenotypes in colony color and intermediate rates of growth in the absence of exogenous adenine.
- LYS2 encodes an enzyme involved in the biosynthesis of lysine. Mutations in this gene prevent growth the on media lacking exogenous lysine.
- the lys2-l nonsense allele can be suppressed by SUP7 or SUPll provided that the encoded nonsense codon suppressing tRNAs possess an i 6 A modification at position 37.
- Cells with no Mod5p lack i 6 A modified tRNA and are unable to grow in the absence of exogenous lysine whereas cells with active cytosolic pools of Mod5p can grow in the absence of this amino acid.
- Cells with intermediate levels of i 6 A modified tRNA have intermediate abilities for growth on media lacking lysine.
- Cells which have the mutant modS-1 allele contain approximately 1.5% of the amount of isopentenyl adenosine present in MODS cells, so little suppression of canl-100, ade 2-1 and lys2-l will occur, making them Can r , Ade " and Lys " .
- Cells with the modS-M2 allele strain ALB1, Figure 2), which contains a mutation at codon 12 preventing the initiation of Mod5p-II translation and allowing only Mod5p-I production, have about 50% of the SUP7 pool modified ( Figure 3) resulting in intermediate levels of suppression.
- Such cells show intermediate growth on canavanine (Can r/s ) and on media lacking adenine (Ade + ⁇ ) and generate pink colonies on rich media.
- Cells with the modS-M2KR6 allele Boguta et al, "Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol" Mol Cell Biol.
- ERG20 In the process of searching for genes that caused decreased cytosolic pools of Mod5p-I, ERG20 was found (Benko et al, "Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression" PNAS 97:61-66, 2000). ERG20 encodes farnesyl diphosphate synthetase. This enzyme catalyzes the formation of geranyl pyrophosphate
- DMAPP dimethylallyl pyrophosphate
- IPP isopentenyl pyrophosphate
- agents to inhibit or enhance flux through the mevalonate pathway provide for facile high-throughput screening of agent banks (e.g., compound libraries, peptide libraries, and the like) to identify antagonists or agonists.
- agent banks e.g., compound libraries, peptide libraries, and the like
- mevalonate pathway antagonists and agonists may be further developed as potential therapeutics and diagnostic or prognostic tools for diverse types of clinical diseases.
- yeast cells of the stains ALB l and ALB8 are cultured, exposed to one or more compounds and color is determined visually. In another embodiment, cells are lysed and color is determined with a spectrophotometer. ii. Growth Assays
- yeast cells of the stains ALB l, ALB8, MD14A, T8-ID are cultured, exposed to one or more compounds and growth is determined visually.
- cells are suspended in a isotonic solution and cell growth is determined with a spectrophotometer or with a flow cytometer.
- antibodies to i 6 A After the generation of antibodies to i 6 A, cells are lysed and then incubated with the antibodies. Antibodies interact with the i 6 A and any associated proteins can then be pulled down with protein-A Sepharose or protein-G Sepharose beads or detected by
- EXAMPLE I Identification of Plasmids Causing Reduced Nonsense Suppression and Sequencing of Inserts of Library Clones.
- the applicants employed the canl-100 and ade2-l alleles in a modS-M2 background (strain ALB 1) to identify plasmids causing alterations in nonsense suppression.
- strains were made as follows: Strain ALBl (MAT ⁇ modS-M2 SUP7 ade2-l canl-100 leu2-3, -112 lysl-1 lys2-l trpl ura3-l) was created from MD14A (Gillman, et al, "MODS translation initiation sites determine N6- isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA" Mol Cell Biol 14:2298-2306, 1994) by the replacement of the genomic modS-1 through a pop- in/pop-out procedure (Rothstein, "Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast” Methods Enzymol 194:281-301, 1991).
- Plasmid YCfmod5-M2 (Gillman, et al, "MODS translation initiation sites determine N6- isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA" Mol Cell Biol 14:2298-2306, 1994) was used as the donor of the M2 mutation.
- CEN4 sequences were removed from YCfrnod5-M2 by a BglU/Smal digestion.
- the Rg II end of the remaining plasmid was filled in using Klenow DNA Polymerase, and then ligated to the S/ ⁇ I blunt end.
- the resulting plasmid was linearized by SnaBl just upstream of the modS-M2 ORF and then transformed into MD14A. Integration of the plasmid was selected for by growth on media lacking uracil. Resulting cells contained two modS alleles: modS-1 and modS-M2.
- Strain ALB8 (MATa SUP7 canl-100 ade2-l leu2-3, -112 lysl-1 lys2-l trpl modSv.TRPl ura3-l::MODS) was created through integration of a plasmid containing MODS at the ura3-l locus of strain MT-8 (Gillman, et al, "MODS translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA" Mol Cell Biol 1 1 : 2382-2390, 1991). Plasmid YCfMOD5 (Gillman, et al,
- Strain SL680-9C (MATa asu9-lsup45-2 ura3-S2 canl-132 leu2-l lys2-l met8-l trpl-1 or trpl-289) (Vincent, et al, "The yeast translational allosuppressor, SAL6: a new member of the PPl-like phosphatase family with a long serine-rich ⁇ -terminal extension" Genetics 138:597-607, 1994) was obtained from S. Liebman.
- Yeast strains were propagated in YEPD media or synthetic complete (SC) media lacking certain nutrients to select for the presence of plasmid and to assay suppression.
- Canavanine sulfate salt (SIGMA) was added to media lacking arginine to a final concentration of 30 ⁇ g/ml.
- Yeast strains were transformed following a modified TRAFO protocol (Gietz, et al, "Improved method for high efficiency transformation of intact yeast cells" Nucleic Acids Res 20: 1425, 1992).
- D ⁇ A library based in YEpl3 ( ⁇ asmyth and Tatchell, "The structure of transposable yeast mating type loci” Cell 19:753-764, 1980)
- a genomic D ⁇ A library based in YEp24 (Carlson and Botstein, et al, "Two differentially regulated mR ⁇ As with different 5' ends encode secreted with intracellular forms of yeast invertase"
- a cD ⁇ A library based in pMac561 a 2 ⁇ m vector with the ADHl promoter (McKnight and McConaughy, "Selection of functional cD ⁇ As by complementation in yeast” Proc Natl Acad Sci USA 80:4412-4416) and a cD ⁇ A library based in pRS316 (Liu, et al, "Construction of a GAL 1 -regulated yeast cD ⁇ A expression library and its application to the identification of genes whose overexpression causes lethality in yeast” Genetics 132:665-673, 1992).
- YEpMOD5 contains the 1.8 kb D ⁇ A fragment encoding wild-type MODS gene and regulatory regions in the YEp24 vector (Dihanich, et al, "Isolation and characterization of MODS, a gene required for isopentenylation of cytoplasmic and mitochondrial tR ⁇ As of Saccharomyces cerevisiae" Mol Cell Biol 7: 177-184, 1987).
- pRH 127-3 a gift from R. Wright, is a 2micron-based plasmid that contains a truncated
- Nucleotide sequences were identified by a BLAST (Altschul, et al, "Basic local alignment search tool" J Mol Biol 215:403-410, 1990) search at the Saccharomyces Genome Database BLAST server (http://genome-www2.stanford.edu/cgi-bin/SGD/nph- blast2sgd7).
- Mod5-M2 is an allele of MODS that encodes normal levels of mitochond ⁇ al but reduced levels of cytosolic Mod5p activity.
- cytosolic tRNAs are only partially modified with i 6 A (60%; Gillman, et al, "MODS translation initiation sites determine N6- ⁇ sopentenyladenos ⁇ ne modification of mitochond ⁇ al and cytoplasmic tRNA" Mol Cell Biol 11 :2382-2390, 1991) and further alterations in Mod5p levels are readily assessed by changes in nonsense suppression (Zoladek.
- Cells with wild-type CANl are sensitive to canavanine (Can s ), but cells with the mutant canl-100 are resistant to canavanine (Can 1 ).
- Cells containing SUP7 tRNAs completely modified with i 6 A have functional arginine permease and, therefore, cannot grow in the presence of canavanine, whereas cells lacking ⁇ 6 A on their tRNAs are able to grow in the presence of this drug.
- Cells with intermediate levels of i°A show intermediate levels of growth in the presence of the drug.
- ADE2 encodes an enzyme involved in the synthesis of adenine. Cells with ade2-
- 3A cells producing functional Ade2p can grow on such medium (Ade + ) and are white (Rasse-Messenguy and Fink, "Temperature-sensitive nonsense suppressors in yeast” Genetics 75:459-464, 1973).
- Cells with the ade2-l allele and sufficient i 6 A-modif ⁇ ed suppressor tRNA can grow in the absence of exogenous adenine and generate white colonies on rich medium, whereas cells with insufficient i 6 A-modified tRNA are unable to grow in the absence of exogenous adenine and generate red colonies on rich medium.
- Cells with intermediate levels of i°A modified tRNA have intermediate phenotypes in colony color and intermediate rates of growth in the absence of exogenous adenine.
- Transformants able to grow on canavanine were selected from approximately 1.5 million independent ALB l (relevant genotype: SUP7 ade2-l canl-100 modS-M2) transformed cells.
- ALB l relevant genotype: SUP7 ade2-l canl-100 modS-M2
- One hundred and fifty-eight candidates that exhibited growth on medium containing canavanine were tested for lack of growth on medium lacking adenine to eliminate those gaining canavanine resistance because of additional nonsuppressible mutations in canl-100.
- an assay was performed to determine whether these phenotypes required the presence of the library plasmid. Thirty- three candidates showed dependence of suppression levels upon the presence of a plasmid.
- ERG20 which encodes farnesyl diphosphate synthetase, was found once in each of the Y ⁇ pl3, pMac561, and pRS316 libraries.
- SAL6 cDNA encoding a protein phosphatase, was discovered four times in the pRS316 library.
- ARC ⁇ . 7EE4, and the putative ORF YDL219w were single candidates from the pRS316 library (Table 1).
- the pRS316 library is constructed such that the cloned genes are regulated by the GAL1 promoter. Thus, transcription is induced by galactose and repressed by glucose in the media.
- SUP45 encodes a translation termination factor, and mutation of this gene can result in omnipotent suppression (Stansfield and Tuite, "Polypeptide chain termination in Saccharomyces cerevisiae” Curr Genet 25:385-395, 1994; Stansfield, et al, "The products of the SUP45 (eRFl) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae” EMBO J 14:4365-4373, 1995).
- SL680-9C (relevant genotype sup45-2 asu9-l leu2-l met8-l ura3-S2) is capable of growing on medium lacking methionine because the mutant sup4S permits translation through nonsense mutations such as met8-l (Vincent, et al, "The yeast translational allosuppressor, SAL6: a new member of the PPl-like phosphatase family with a long serine-rich N-terminal extension” Genetics 138:597-607, 1994; Stansfiel 1 d and Tuite, "Polypeptide chain termination in Saccharomyces cerevisiae” Curr Genet 25:385-395, 1994; Stansfield, et al, "The products of the SUP45 (eRFl) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae” EMBO J 14:4365-4373, 1995).
- the asu9 antisuppressor allele serves to weaken the effects of sup45 and permits better growth of cells with both mutations (Vincent, et al, "The yeast translational allosuppressor, SAL6: a new member of the PPl-like phosphatase family with a long serine- ⁇ ch N-terminal extension” Genetics 138:597-607, 1994; Stansfield and Tuite, "Polypeptide chain termination in Saccharomvces cerevisiae” Curr Genet 25:385-395, 1994).
- SL680-9C cells transformed by each of the candidate genes in the pRS316 vector were analyzed for growth on galactose-containing or glucose-containing medium lacking uracil and methionine.
- the data (Table 2) indicate that overexpressed SAL6 and, to a lesser degree, TEF4 and YDL219w cause antisuppression, and, therefore, loss of suppression of the canl-100 and ade2-l in ALBl is likely Mod5p-I-mdependent.
- EXAMPLE 3 Overexpression of ERG20 Results in the Reduction of Isopentenyl Adenosine Present in tRNA.
- DMAPP is the same molecule used by Mod5p as a substrate in the synthesis of i 6 A in tRNA ( Figure 1 ; Brown and Goldstein. "Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth" J Lipid Res 21 :505-517, 1980; Voet and Voet, Biochemistry (Wiley, NY) 1990). It was possible that the large pool of Erg20p produced from the library clones used the DMAPP normally utilized for tRNA modification. This would cause reduced i 6 A-modif ⁇ ed tRNA and, hence, reduced function of these tRNAs in nonsense suppression.
- RNA Low-molecular-weight RNA was prepared from cells with each plasmid and from control strains of ALBl with vector only, ALB8, and MD14A. The RNAs were resolved on urea-polyacrylamide gels, transferred to membranes, and probed with anti-i 6 A antibody ( Figure 2A, upper). Hybridization with a radiolabeled oligonucleotide complementary to part of mature tRNA Tyr was used to assess the relative amount of tRNA in each lane ( Figure 2A, lower). Through densitometric analysis, the amount of i 6 A formed in tRNA of each strain as a fraction of the amount formed in the vector-only control strain was determined ( Figure 2B).
- Applicants introduced a multicopy vector harboring a mutant HMG1 gene, pRH127-3, that causes elevated levels of intermediates of the mevalonate pathway (Donald, et al, "Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzvme A reductase on squalene synthesis in Saccharomyces cerevisiae" Appl Environ Microbiol 63:3341-3344, 1997) into ALBl containing YEpERG20. Applicants found that increased levels of the intermediates also restored nonsense suppression (Table 3). The data support the model that the mevalonate pathway and the i 6 A tRNA modification pathways are in competition.
- RNA was obtained (Hopper, et al, "Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs" Cell 19:741-751, 1980) from overnight log phase cultures of the following strains: ALB8, MD14A and ALB l with the pRS316 library plasmids containing SAL6, ARCl, TEF4, ERG20, YDL219w or no cDNA insert. The RNA concentration was calculated from optical density measurement.
- RNAs were resolved on a 12% polyacrylamide / 7M urea gel. The RNA was transferred to Hybond N+ Nylon Membrane (Amersham) in a Hoefer TE Series
- RNA was fixed onto the membrane by UV crosslinking.
- the membrane was subjected to immunoblotting following the protocol of the Renaissance Western Blot Chemiluminescence Kit (NEN) with slight variation.
- the primary antibody, anti-i 6 A isolated from rabbit prepared by the method of Senapathy and Jacob (Senapathy and Jacob, "Identification and purification of tRNAs containing N6-(delta 2- ⁇ sopentenyl) adenosine using antibodies specific for N6-(delta- isopentenyl) adenosine" J Biol Chem 256: 1 1580-1 1584. 1981), was diluted either 1 :33.3 or 1 :72.5 prior to use.
- the membrane was also utilized for RNA detection using radiolabeled oligonucleotide complementary to a region of mature tRNA Tyr .
- the intensities of the fluorescent and radioactive signals for each specimen on the immunoblots and RNA blots, respectively were determined by densitometry using a Molecular Dynamics laser scanner with the Discovery Series Quantity One computer software (Protein Database. Inc.). For each sample, the ratio of its signal with respect to that of the signal from the ALB l with vector only sample was calculated from the immunoblots (Ratio 1 ) and from the RNA blots (Ratio 2). The average values for Ratio 1 to Ratio 2 for samples from two membranes were calculated.
- the data show that increased levels of enzyme activity of the sterol biosynthetic pathway can be assessed indirectly simply by colony color and / or growth on particular medium because changes in nonsense expression occur. Thus, additional mutations and / or drugs that affect the pathway could be identified by the screening protocol described.
- EXAMPLE 4 Automated assays.
- yeast cells are cultured as described above in microtiter plates and contact with compounds in order to assess the ability of the compound to modulate the mevalonate pathway. After culture, the cells are lysed in situ and the microtiter plates are analyzed with a manual or automated spectrophotographic plate reader. Attachment of the reader to a computer will allow for the near instantaneous recording and analysis of the data.
- Another experiment will utilize automated flow cytometry.
- whole cells are analyzed with an automatic sampling flow cytometer calibrated to detect color and / or emitted fluorescence. Additionally, the relative number of cells between samples will be determined to measure cell growth. In any of these examples robotics will be used to analyze a large number of samples quickly.
- Downstream products of the mevalonate pathway may be both characterized and quantitated.
- effect of the compounds on the mevalonate pathway may be confirmed by analysis of the increased or decreased production of downstream mevalonate pathway products.
- analyses are well known in the art and include radioimmunoassays (RIA) and high-pressure liquid chromatography (HPLC). In this way, the effected downstream pathway or pathways will be determined.
- the present invention provides novel materials and methods that will permit the rapid screening of compounds for their agonistic or antagonistic effect on the mevalonate pathway.
- the present invention provides novel materials and methods for the determination of existing or promising therapeutic compounds that may have agonistic or antagonistic side effects on the mevalonate pathway.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Ecology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU58833/00A AU5883300A (en) | 1999-06-23 | 2000-06-22 | Compositions and methods for the quantification of sterol biosynthetic flux |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14151699P | 1999-06-23 | 1999-06-23 | |
US60/141,516 | 1999-06-23 | ||
US19969900P | 2000-04-26 | 2000-04-26 | |
US60/199,699 | 2000-04-26 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2000078994A1 true WO2000078994A1 (en) | 2000-12-28 |
WO2000078994A9 WO2000078994A9 (en) | 2002-06-13 |
Family
ID=26839188
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/017190 WO2000078994A1 (en) | 1999-06-23 | 2000-06-22 | Compositions and methods for the quantification of sterol biosynthetic flux |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU5883300A (en) |
WO (1) | WO2000078994A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7217506B2 (en) | 2002-07-01 | 2007-05-15 | Tibotec Pharmaceuticals, Ltd. | Mutational profiles in HIV-1 protease correlated with phenotypic protease inhibitor resistance |
US7473524B2 (en) | 2002-07-01 | 2009-01-06 | Hilde Azijn | Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance |
US8563236B2 (en) | 1999-05-28 | 2013-10-22 | Virco, N.V. | Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance |
US8574831B2 (en) | 2000-10-20 | 2013-11-05 | Virco N.V. | Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance |
-
2000
- 2000-06-22 WO PCT/US2000/017190 patent/WO2000078994A1/en active Application Filing
- 2000-06-22 AU AU58833/00A patent/AU5883300A/en not_active Abandoned
Non-Patent Citations (2)
Title |
---|
SCHAFER ET AL.: "Genetic and pharmacological suppression of oncogenic mutations in RAS genes of yeast and humans", SCIENCE,, vol. 245, 28 July 1989 (1989-07-28), pages 379 - 385, XP002931174 * |
ZOLADEK ET AL.: "Mutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis in mitochondrial delivery", MOLECULAR AND CELLULAR BIOLOGY,, vol. 15, no. 12, December 1995 (1995-12-01), pages 6884 - 6894, XP002931175 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8563236B2 (en) | 1999-05-28 | 2013-10-22 | Virco, N.V. | Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance |
US8574831B2 (en) | 2000-10-20 | 2013-11-05 | Virco N.V. | Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance |
US7217506B2 (en) | 2002-07-01 | 2007-05-15 | Tibotec Pharmaceuticals, Ltd. | Mutational profiles in HIV-1 protease correlated with phenotypic protease inhibitor resistance |
US7473524B2 (en) | 2002-07-01 | 2009-01-06 | Hilde Azijn | Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance |
US8076062B2 (en) | 2002-07-01 | 2011-12-13 | Tibotec Pharmaceuticals Ltd. | Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance |
US8592161B2 (en) | 2002-07-01 | 2013-11-26 | Janssen R&D Ireland | Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance |
Also Published As
Publication number | Publication date |
---|---|
AU5883300A (en) | 2001-01-09 |
WO2000078994A9 (en) | 2002-06-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Bahn et al. | Increased high‐affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1‐dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans | |
Guo et al. | Proteomic analysis of SUMO4 substrates in HEK293 cells under serum starvation-induced stress | |
Ivey et al. | Positive Regulation of Adenylyl Cyclase Activity by a GαiHomolog inNeurospora crassa | |
Brust et al. | Cyclophilin D links programmed cell death and organismal aging in Podospora anserina | |
Chen et al. | Contribution of peroxisomal docking machinery to mycotoxin biosynthesis, pathogenicity and pexophagy in the plant pathogenic fungus Fusarium graminearum | |
US6803193B1 (en) | Methods to identify modulators of the mevalonate pathway in sterol synthesis | |
Nemoto et al. | The roles of stress-activated Sty1 and Gcn2 kinases and of the protooncoprotein homologue Int6/eIF3e in responses to endogenous oxidative stress during histidine starvation | |
Dinamarco et al. | The roles played by Aspergillus nidulans apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase (AifA) and NADH-ubiquinone oxidoreductases (NdeA-B and NdiA) in farnesol resistance | |
Kong et al. | FgPex3, a peroxisome biogenesis factor, is involved in regulating vegetative growth, conidiation, sexual development, and virulence in Fusarium graminearum | |
Silva et al. | Genetic interactions between Aspergillus fumigatus basic leucine zipper (bZIP) transcription factors AtfA, AtfB, AtfC, and AtfD | |
Amarsaikhan et al. | Proteomic profiling of the antifungal drug response of Aspergillus fumigatus to voriconazole | |
Kröber et al. | The transcriptional regulators SteA and StuA contribute to keratin degradation and sexual reproduction of the dermatophyte Arthroderma benhamiae | |
Dudley et al. | Developmental regulation of the plant mitochondrial matrix located HSP70 chaperone and its role in protein import | |
WO2000078994A1 (en) | Compositions and methods for the quantification of sterol biosynthetic flux | |
Zheng et al. | Glc7p protein phosphatase inhibits expression of glutamine-fructose-6-phosphate transaminase from GFA1 | |
Brabbs et al. | Isolation and characterization of bleomycin-resistant clones of CHO cells | |
Fracella et al. | Differential HSC70 expression during asexual development of Neurospora crassa | |
PT626454E (en) | FUNGUS POLAR FODDER SEARCH FOR FUNGICIDES | |
Davis et al. | Nonsense mutations of the ornithine decarboxylase structural gene of Neurospora crassa | |
Al-Ouqaili et al. | Detection of aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus isolated from some clinical and environmental sources by HPLC and PCR techniques | |
US11324796B2 (en) | Method for regulating lipolysis and fatty acid oxidative metabolism | |
Arellano et al. | An Antimicrobial peptide induces FIG1-dependent cell death during cell cycle arrest in yeast | |
Park et al. | Antifungal effect with apoptotic mechanism (s) of Styraxjaponoside C | |
RU2662358C2 (en) | Cell based quality control bioassays for nutriceutical and medicinal products | |
Nguyen Van | Antagonistic regulation by global transcription factors Tup1p, and Cyc8p of Flo11 and Flo11-dependent phenotypes in wild yeast |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1/6-6/6, DRAWINGS, REPLACED BY NEW PAGES 1/9-9/9; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |