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WO2000001809A1 - Production de taxol par constitution d'adn extrachromosomique dans le champignon pestalotiopsis - Google Patents

Production de taxol par constitution d'adn extrachromosomique dans le champignon pestalotiopsis Download PDF

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WO2000001809A1
WO2000001809A1 PCT/US1999/015135 US9915135W WO0001809A1 WO 2000001809 A1 WO2000001809 A1 WO 2000001809A1 US 9915135 W US9915135 W US 9915135W WO 0001809 A1 WO0001809 A1 WO 0001809A1
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dna
cell
pestalotiopsis
telomerase
extrachromosomal
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PCT/US1999/015135
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English (en)
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David M. Long
Eric D. Smidansky
Gary A. Strobel
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Research And Development Institute, Inc.
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Priority to AU49685/99A priority Critical patent/AU4968599A/en
Priority to EP99933683A priority patent/EP1092016A1/fr
Priority to JP2000558199A priority patent/JP2002519058A/ja
Priority to CA002336122A priority patent/CA2336122A1/fr
Priority to KR1020017000034A priority patent/KR20010083085A/ko
Publication of WO2000001809A1 publication Critical patent/WO2000001809A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • 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
    • C12N15/102—Mutagenizing nucleic acids
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    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms

Definitions

  • the present invention pertains, in general, to the generation of extrachromosomal DNAs.
  • the present invention pertains to extrachromosomal DNAs produced by introducing DNA into a Pestalotiopsis sp. cell, wherein such introduced DNA codes for a product which changes the level of taxol production.
  • telomere sequences DNA into the macronucleus results in apparent random linearization and the addition of paramecium-type telomere sequences to the ends of the DNA (Gilley et al, 1988).
  • the introduced linear DNA is directly modified by the addition of telomeric repeats and the resultant molecules replicate efficiently.
  • Telomeric sequences were also added de novo to linearized Cryptococcus neoformans transforming plasmids.
  • C. neoformans is a basidiomycete with the teleomorph Filobasidiella neoformans.
  • the added telomeric sequences are simple repeats of the octanucleotide AGGGGGTT (Edman et al., 1992).
  • Recovered plasmids showed increased transformation efficiencies in the supercoiled state (up to 200 transformants per ⁇ g) and in the linear state (up to 90,000 transformant per ⁇ g). While vectors derived from the plasmids produced via this process should prove useful for introducing genes back into C. neoformans (see, e.g., Varma et al, 1994), the fact that this organism is a heterothallic basidiomycetous yeast that causes meningeal and systemic infections in humans, its utility for all applied genetic uses will be limited. The fungal rearrangement of normally integrative plasmids results in the creation of linear self-replicating plasmids in Fusarium oxysporum (Powell and Kistler, 1990).
  • the rearrangement results in the addition of fungal DNA, including the telomere sequence TTAGGG, to plasmid termini at a frequency much lower than in Cryptococcus and Pestalotiopsis.
  • the linear extrachromosomal DNA in F. oxysporum undergoes partial duplication of the transforming DNA.
  • a DNA sequence containing the repeated telomeric sequence flanked by a region of twofold symmetry consisting primarily of pUC12 DNA was shown to produce autonomous replication and enhance transformation in several species at relatively low frequencies. For example, a transformation of approximately 300-3,000 transformants per ⁇ g of vector was obtained for F.
  • F. oxysporum has the following taxonomic lineage: Eukaryota; Fungi; Ascomycota; Euascomycetes; Pyrenomycetes; Hypocreales; Nectriaceae; mitosporic Nectriaceae; and Fusarium (National Center for Biotechnology Information (NCBI) Taxonomy Database).
  • H. capsulatum having the teleomorph Ajellmyces capsulatus, has the following taxonomic lineage: Eukaryota; Fungi; Ascomycota; Euascomycetes; Plectomycetes; Onygenales; Onygenaseae; and Ajellomyces (National Center for Biotechnology Information (NCBI) Taxonomy Database). Similar to the situation with C.
  • Histoplasma capsulatum is a dimorphic pathogenic fungus that is a major cause of respiratory and systemic mycosis in mammals will limit its utility in mammalian research.
  • DNA has been previously reported in the taxonomically diverse species Paramecium tetraurelia, Cryptococcus neoformans, Fusarium oxysporum and Histoplasma capsulatum.
  • the actual de novo process which occurs in each of these organisms differs in various ways.
  • the plasmids produced in P. tetraurelia and C. neoformans are approximately the same size or smaller than the input DNA, while the plasmids are larger in F. oxysporum and H. capsulatum, due at least in part to duplication of the input sequences.
  • each of these organisms either adds telomeric sequences which are not useful for mammalian transformation (P. tetraurelia, C. neoformans), are dangerous mammalian pathogens (C. neoformans, H. capsulatum), or add the telomeric repeats at a relatively low efficiency (F. oxysporum).
  • telomeric sequences which are not useful for mammalian transformation
  • C. neoformans are dangerous mammalian pathogens
  • F. oxysporum add the telomeric repeats at a relatively low efficiency
  • Pestalotiopsis microspora is a filamentous fungus that lives as an endophyte in the inner bark of certain trees, including the Himalayan yew tree (Taxus wallachiana) (Strobel et al., 1996); bald cypress (Taxodium distichum) (Li et al., 1996); and Torreya taxifolia, a rare tree with a close taxonomic relationship with Taxus brevifolia (Lee et al, 1995).
  • P. microspora has an endophytic-patho logic relationship with T. taxifolia. While the fungus can reside in the inner bark of symptomless trees, certain physiological and/or environmental factors appear to trigger the fungus into producing the phytotoxins pestalopyrone, hydroxypestalopyrone, and pestaloside.
  • Pestalotiopsis sp. Both P. microspora and its yew tree hosts produce taxol, a diterpenoid secondary metabolite with demonstrated efficacy against certain human cancers (Georg et al., 1994). Cultures of Pestalotiopsis sp. have also been shown to produce a number of different compounds in addition to taxol, including a glucoamylase which digests starch (U.S. Patent Number 5,604,128), non-peptidic endothelian antagonists (Ogawa et al., 1995), a highly branched galactomannan with anti-diabetic activity (Kiho et al., 1997); and mycarosyl macro lide antibiotics (U.S. Patent Number 3,784,447). Although a teleomorph of P. microspora has not been observed, several other species within the Pestalotiopsis genus form ascospores under appropriate conditions (Nag Raj, 1993).
  • Pestalotiopsis species include P. funerea IFO 5427 (SANK 15174); P. microspora IFO 31056 and CP-4; P. asaciae IFO 31054; P. crassiusla IFO 31055; P. neglecta (SANK 13390; FERM BP-3501); and P. royenae (ATCC11816) (see, e.g., U.S. Patent Number 3,784,447; U.S. Patent Number 5,604,128; and Li et al, 1996).
  • taxanes The group of complex terpene-type compounds known as taxanes and taxane- related compounds have proven to have important anti-cancer properties. Examples of such compounds include taxol, baccatin and cephalomannine. Taxol, whose generic name as a drug is paclitaxel, is found in the stem bark of the
  • Taxol is a naturally occurring diterpenoid which has great potential as an anti-cancer drug, and which has shown activity in several tumor systems. Taxol was first isolated and its structure reported by Wani et al. (1971). Taxol is currently on the market. It had demonstrated efficacy with manageable side effects in 30 to 35% of cases of ovarian cancer and 56% of cases of metastatic breast cancer.
  • taxol The biological activity of taxol is related to its effect on cell division. Microtubules form during the mitotic spindle during cell division. Taxol interferes with depolymerization of the tubulin forming the microtubules of the mitotic spindle, which is essential for cell division to take place. Thus, taxol causes cell division to stop.
  • the mechanism of action of taxol is unique since it promotes the formation of tubulin polymers, whereas other anti-cancer drugs, such as vinblastine andieristine, prevent microtubule formation.
  • Pestalotiopsis microspora isolated from the bark, phloem and/or xylem of the Himalayan yew ⁇ Taxus wallachiana) and the bald cypress (Taxodium distichum) produces taxol in significant but not commercially viable amounts (Strobel et al, 1996; Li et al, 1996)).
  • the taxol biosynthetic pathway in yew plants is beginning to be elucidated
  • microspora the properties of the extrachromosomal DNAs formed by this modification, as well as the use of such a process in genetic research and for the production of transgenic organisms, including transgenic mammals.
  • methods of producing taxol based on our surprising discovery that P. microspora produces extrachromosomal DNA from DNA introduced into its cells.
  • compositions and methods useful for producing extrachromosomal DNA More specifically, the present invention provides compositions and methods based on the unexpected discovery that the Pestalotiopsis fungi add one or more telomeric repeats to the ends of exogenous DNA.
  • This invention provides methods of adding one or more telomeric repeats to exogenous DNA by introducing exogenous DNA into a Pestalotiopsis cell.
  • the exogenous DNA can consist of telomeric or non-telomeric DNA.
  • the exogenous DNA can be either circular, linear or mulitmeric DNA.
  • the exogenous DNA codes for a product which changes the level of taxol production, such as one or more enzymes or enzyme subunits responsible for taxol biosynthesis.
  • the present invention provides methods of increasing taxol production on a cellular, tissue, organ or whole-organism basis.
  • This invention also provides methods of generating extrachromosomal DNA by introducing exogenous DNA into a Pestalotiopsis cell, wherein such DNA codes for a product which changes the level of taxanes, such as DNA which codes for taxol biosynthesis.
  • Extrachromosomal DNA produced using the procedures of this invention can be isolated and used to transform a prokaryotic or eukaryotic cell.
  • the extrachromosomal DNAs produced by the procedures of this invention can be used to transform a Pestalotiopsis cell or other eukaryotic cell with one or more genes coding for an enzymatic step in taxane biosynthesis, including the production of taxol and related diterpenoids.
  • This invention further provides methods of generating a replicable nucleic acid element by introducing exogenous DNA into a Pestalotiopsis cell, wherein the exogenous DNA codes for a product which changes the level of taxol production, such as enzymes or enzyme subunits which catalyze one or more steps in taxol biosynthesis. Therefore, the procedures of the present invention can be used to transform a Pestalotiopsis cell with genes related to cellular taxol production. Alternatively, the replicable nucleic acid elements can be isolated and used to transform other eukaryotic cells with genes related to cellular taxol production.
  • This invention also provides methods of adding one or more telomeric repeats to exogenous DNA wherein the method comprises introducing the exogenous DNA into a cell of an ascomycete, with the proviso that the ascomycete is not Fusarium oxysporum.
  • One particular embodiment of the present invention comprises introducing exogenous DNA into a Pestalotiopsis cell; permitting one or more telomeric repeats to be added to the exogenous DNA to produce extrachromosomal DNA; extracting the extrachromosomal DNA from the transformed Pestalotiopsis cell; and introducing the extracted extrachromosomal DNA into a second cell.
  • the exogenous DNA utilized in the invention can be either from any Pestalotiopsis sp. or be non-Pestalotiopsis DNA.
  • the second cell can be either a different Pestalotiopsis cell or other eukaryotic cell.
  • DNA of particular interest is DNA which codes for a product which changes the level of taxol production.
  • the present invention involves introducing exogenous DNA coding for enzymes or enzyme subunits involved in taxol biosynthesis into a Pestalotiopsis cell and thereby increasing production of taxol by the transformed Pestalotiopsis cell.
  • the present invention involves introducing exogenous DNA coding for enzymes or enzyme subunits involved in taxol biosynthesis into a Pestalotiopsis cell and thereby increasing production of taxol by the transformed Pestalotiopsis cell.
  • the present invention involves introducing exogenous DNA coding for enzymes or enzyme subunits involved in taxol biosynthesis into a
  • Pestalotiopsis cell extracting the extrachromosomal DNA's produced by such an method, and then introducing the extracted extrachromosomal DNA's into a yew plant cell, thereby increasing production of taxol by the transformed yew cells.
  • the taxol-related gene(s) can be isolated from either Pestalotiopsis or a tree species which produces taxol, such as the Pacific yew.
  • genes coding for taxol biosynthesis include those coding for the enzymes or enzyme subunits responsible for the cyclization of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),l l(12)-diene; the cytochrome P450-catalyzed hydroxylation of this olefin to taxa-4(20),l l(12)-dien-5 alpha-ol; and the acetyl CoA-dependent conversion of the alcohol to the corresponding acetate ester.
  • DNA sequences useful in the present invention include those coding for taxadiene synthase, taxadiene-5-hydroxylase, acetyl-coenzyme A, or those coding for any one of the additional oxidation steps in taxol biosynthesis.
  • the present invention also provides telomerase enzymes or enzyme subunits isolated and purified from Pestalotiopsis; DNA sequences coding for such telomerase enzymes or enzyme subunits; and a recombinant DNA construct comprising the RNA sequence of such telomerase enzymes or enzyme subunits.
  • the present invention also provides methods of producing stable DNA fragments by adding one or more telomeric repeats to the ends of the DNA using telomerase isolated from Pestalotiopsis.
  • the present invention further provides artificial chromosomes comprising the
  • the artificial chromosomes of the present invention can further comprise additional genes of interest, wherein those additional genes code for one or more enzymes or enzyme subunits responsible for taxol biosynthesis.
  • Particular embodiments of the present invention provide artificial chromosomes which include one or more genes coding for taxadiene synthase, taxadiene- 5-hydroxylase and acetyl-coenzyme A.
  • the present invention also provides host cells transformed with a recombinant nucleic acid comprising an oligonucleotide having a contiguous sequence of at least 25 nucleotides in a sequence complementary or identical to a Pestalotiopsis genomic DNA sequence encoding the RNA component of Pestalotiopsis telomerase.
  • the recombinant nucleic acid can further comprise a promoter positioned to drive the transcription of an RNA having a sequence complementary to the oligonucleotide.
  • the host cells utilized in this aspect of the invention include eukaryotic cells other than those of Pestalotiopsis.
  • the present invention also provides methods for producing the RNA component of Pestalotiopsis telomerase comprising the step of culturing a prokaryotic or a eukaryotic host cell transformed with a recombinant nucleic acid comprising a promoter positioned to drive the transcription of a DNA sequence encoding an RNA component oi Pestalotiopsis telomerase.
  • the invention also provides methods for producing a recombinant Pestalotiopsis telomerase enzyme, said method comprising transforming a prokaryotic or a eukaryotic host cell capable of expressing protein components of telomerase, with a recombinant nucleic acid comprising a promoter positioned to drive the transcription of a DNA sequence encoding the RNA component of Pestalotiopsis telomerase, said recombinant nucleic acid functioning to produce the oligonucleotide in the host cell, and culturing said host cells transformed with said vector under conditions such that the protein components and RNA component are expressed and assemble to form an active telomerase molecule capable of adding sequences to telomeres of chromosomal DNA.
  • This invention also provides the RNA component of, as well as the gene for the RNA component of, P. microspora telomerase in substantially pure form, as well as nucleic acids comprising all or at least a useful portion of the nucleotide sequence of the
  • RNA component of P. microspora telomerase The present invention also provides RNA component nucleic acids from other species, which nucleic acids share substantial homo logy with the RNA component of P. microspora telomerase.
  • nucleic acids of the invention include nucleic acids with sequences complementary to the RNA component; nucleic acids with sequences related to but distinct from nucleotide sequences of the RNA component and which interact with the RNA component or the gene for the RNA component or the protein components of P. microspora telomerase in a useful way; and nucleic acids that do not share significant sequence homology or complementarity to the RNA component or the gene for the RNA component but act on the RNA component in a desired and useful way.
  • one type of useful nucleic acid of the invention is an antisense oligonucleotide, a triple helix-forming oligonucleotide, or other oligonucleotide that can be used in vivo or in vitro to inhibit the activity of P. microspora telomerase.
  • Such oligonucleotides can block telomerase activity in a number of ways, including by preventing transcription of the telomerase gene (for instance, by triple helix formation) or by binding to the RNA component of telomerase in a manner that prevents a functional ribonucleoprotein telomerase from assembling or prevents the RNA component, once assembled into the telomerase enzyme complex, from serving as a template for telomeric DNA synthesis.
  • these oligonucleotides of the invention comprise a specific sequence of from about 10 to about 25 to 200 or more nucleotides that is either identical or complementary to a specific sequence of nucleotides in the RNA component of telomerase or the gene for the RNA component of telomerase.
  • Another type of useful nucleic acid of the invention is a ribozyme able to cleave specifically the RNA component of P. microspora telomerase, rendering the enzyme or enzyme subunits inactive.
  • Yet another type of useful nucleic acid of the invention is a probe or primer that binds specifically to the RNA component of P.
  • useful nucleic acids of the invention include recombinant expression plasmids for producing the nucleic acids of the invention.
  • One especially useful type of such a plasmid is a plasmid used for genetic transformation of animals.
  • the invention provides methods for treating a condition associated with the telomerase activity within a cell or group of cells by contacting the cell(s) with a therapeutically effective amount of an agent that alters telomerase activity in that cell.
  • Such agents include the telomerase RNA component-encoding nucleic acids, triple helix-forming oligonucleotides, antisense oligonucleotides, plasmids, ribozymes, small molecules, other chemical entities.
  • the invention provides pharmaceutical compositions comprising these therapeutic agents together with a pharmaceutically acceptable carrier or salt.
  • the invention provides diagnostic methods for determining the level, amount, or presence of the RNA component of P. microspora telomerase, telomerase, or telomerase activity in a cell, cell population, or tissue sample, or an extract of any of the foregoing.
  • the present invention provides useful reagents for such methods (including the primers and probes noted above), optionally packaged into kit form together with instructions for using the kit to practice the diagnostic method.
  • the present invention provides recombinant P. microspora telomerase preparations and methods for producing such preparations.
  • the present invention provides a recombinant P. microspora telomerase that comprises the protein components of P. microspora telomerase as well as the protein components of telomerase from a species with an RNA component substantially homologous to the RNA component of P. microspora telomerase in association with a recombinant RNA component of the invention.
  • Such recombinant RNA component molecules of the invention include those that differ from naturally occurring RNA component molecules by one or more base substitutions, deletions, or insertions, as well as RNA component molecules identical to a naturally occurring RNA component molecule that are produced in recombinant host cells.
  • the method for producing such recombinant telomerase molecules comprises transforming a prokaryotic or eukaryotic host cell that expresses the protein components of telomerase with a recombinant expression vector that encodes an RNA component molecule of the invention, and culturing said host cells transformed with said vector under conditions such that the protein components and RNA component are expressed and assemble to form an active P.
  • microspora telomerase molecule capable of adding sequences (not necessarily the same sequence added by native telomerase) to telomeres of chromosomal DNA.
  • the invention provides methods for purifying the protein components of P. microspora telomerase as well as the protein components of telomerase from a different species with an RNA component substantially homologous to the RNA component of the Pestalotiopsis telomerase.
  • the present invention also provides methods for isolating and identifying nucleic acids encoding such protein components.
  • the present invention provides purified P. microspora telomerase and purified telomerase of species with an RNA component substantially homologous to the RNA component of P. microspora telomerase, as well as purified nucleic acids that encode one or more components of such telomerase preparations.
  • the present invention also provides pharmaceutical compositions comprising as an active ingredient the protein components of P. microspora telomerase or a nucleic acid that encodes or interacts with a nucleic acid that encodes a protein component of P. microspora telomerase.
  • FIG. 1 Southern blot of undigested total DNA from P. microspora wild-type and transformants. Lane 33, 200 pg each of linear (Lin) and circular (Cir) pDH33. Lane Wt, wild-type total DNA. Lanes 1 through 8, transformant DNA. The position of chromosomal DNA is indicated (Chm, approximately 50 kb). Numbers to the right are molecular size markers (in kb). For lanes 1 through 8, 1.5 ⁇ g of DNA was electrophoresed through 0.6% agarose, transferred to nylon membrane, and hybridized with 32 P-labeled random primed pDH33.
  • Figure 2 Stability of the hygromycin resistant phenotype of P. microspora transformants in the absence of selection. After the indicated number of days of growth on PDA, transformants were transferred to PDA containing 200 ⁇ g/ml hygromycin and colony areas were measured after 7 days (see Materials and Methods). ⁇ , trl4 and ⁇ , trl9, are integrants. T, trlO; O, trl3; A, trl5; and ⁇ jtrH, are transformants with extrachromosomal DNAs. b. The effect of hygromycin concentration on the vegetative growth of P. microspora wild-type and transformants.
  • Colony areas were measured after 7 days growth on PDA containing the indicated concentrations of hygromycin. •, wild-type. ⁇ , trl4 and ⁇ , trl9, integrants. T, trlO; O, trl3; ⁇ , trl5; and ⁇ ,trl7, transformants with extrachromosomal DNAs.
  • Lane 33 200 pg each of linear (Lin) and circular (Cir) pDH33. Lane Wt, wild-type P. microspora total DNA.
  • Figure 4. a. PCR amplification of P. microspora transformant tr3 total DNA.
  • Lane Wt amplification of wild-type total DNA with primer TE2 yielded no visible product.
  • Lane 1 amplification of tr3 total DNA with primer TE2 yielded full-length 5.5 kb extrachromosomal DNA.
  • Lane 2 amplification of tr3 total DNA with primers TE2 and Hyg2 yielded the 2.4 kb 3' fragment of extrachromosomal DNA.
  • Lane 3 amplification of tr3 total DNA with primers TE2 and Hygl yielded the 3.1 kb 5' fragment of extrachromosomal DNA.
  • Lanes 4 and 5 Banl restricted PCR product from lane 1 and pDH33, respectively. Numbers to the right are molecular size markers. b.
  • FIG. 1 Southern blot of P. microspora transformant and wild- type DNA probed with the telomeric probe TE2.
  • Lane 33 10 ng of pDH33.
  • Lane Ca 0.5 ⁇ g Candida albicans DNA (which has a telomeric repeat different from P. microspora).
  • Lane Wt 0.5 ⁇ g wild-type DNA.
  • Lanes 1, 3, 4, and 5, transformant DNAs. Numbers to the right are molecular size markers.
  • Pestalotiopsis has the following taxonomic lineage: Eukaryota; Fungi; Ascomycota; Euascomycetes; Loculoascomycetes; Dothideales; mitosporic Dothideales; Pestalotiopsis.
  • Recent taxonomic evidence indicates that the taxonomic lineage may actually be as follows: Eukaryota; Fungi; Ascomycota; Euascomycetes;. Pyrenomycetes; Xylariales; Amphisphaeriaceae; Pestalosphaeria (see Source Organism description accompanying GenBank Accession No. AF104356).
  • a Pestalotiopsis species includes any fungus that has an 18S ribosomal RNA gene sequence with at least 80% sequence similarity to the
  • Pestalosphaeria sp. NE-32 18S ribosomal RNA gene described by GenBank Accession No. AF104356 More specifically, a Pestalotiopsis species includes any fungus that has an 18S ribosomal RNA gene sequence with at least 85% sequence similarity to the Pestalosphaeria sp. NE-32 18S ribosomal RNA gene described by GenBank Accession No. AF104356. Even more specifically, a Pestalotiopsis species includes any fungus that has an 18S ribosomal RNA gene sequence with at least 90% sequence similarity to the Pestalosphaeria sp. NE-32 18S ribosomal RNA gene described by GenBank Accession No.
  • a Pestalotiopsis species includes any fungus that has an 18S ribosomal RNA gene sequence with at least 95% sequence similarity to the Pestalosphaeria sp. NE-32 18S ribosomal RNA gene described by GenBank Accession No. AF104356.
  • exogenous DNA refers to any DNA derived or developed outside the Pestalotiopsis cell undergoing transformation or derived or developed outside the Pestalotiopsis cell which has undergone transformation.
  • exogenous DNA includes, but is not limited to, foreign DNA, synthetic DNA, and/or DNA from a different Pestalotiopsis cell than the Pestalotiopsis cell being transformed or which has been transformed.
  • Foreign DNA includes, but is not limited to, any DNA from a genus other than Pestalotiopsis or any DNA from a species other than P. microspora.
  • telomerase enzyme subunit refers to any domain, or region or discrete part of a polypeptide sequence that can be equated with telomerase enzyme function.
  • the present invention is directed to the in vivo addition of terminal telomeric repeats to exogenous DNA during transformation of the taxol- producing filamentous fungus P. microspora.
  • Multiple copies of the sequence 5 '-TTAGGG-3 ' which is the telomeric repeat found in a number of filamentous fungi and in vertebrates (Henderson, 1995), are added to transforming DNA termini in a reaction that produces extrachromosomal DNAs.
  • the DNAs do not change in size, rearrange, or undergo chromosomal integration after six months of growth with selection, but are lost after only 20 days of growth in the absence of selection. No evidence for the presence of extrachromosomal telomeric DNAs in wild- type P. microspora was obtained.
  • Transformation of P. microspora with an in vivo modified DNA amplified from one transformant by PCR is 10- to 50-fold more efficient than with the original unmodified transforming plasmid that lacks telomeric repeats. In addition to transformants harboring extrachromosomal DNAs, approximately 10% are shown to contain chromosomally-integrated sequences.
  • the extrachromosomal DNA characterized to the fullest extent is a 5.5 kb linear molecule composed of a contiguous, apparently unmodified stretch of the transforming plasmid fused directly to terminal telomeric repeats (Figure 4b). No additional fungal sequences were detected in the extrachromosomal DNA and given the present level of structural characterization (23 observed restriction fragments and approximately 1.9 kb sequenced) the presence of these unlikely.
  • Each P. microspora transformant containing extrachromosomal DNAs clearly harbors more than one type of molecule as indicated by Southern blots of transformants showing multiple bands. These are probably different conformations of the same molecule, because if the transformants are ordered according to band size, approximately the same order is obtained whether the smallest or second smallest band is used.
  • different covalent forms of the DNAs are possible, for example concatemers or other multimeric species, they could also be noncovalent topological forms that arise from interactions between telomeres.
  • microspora extrachromosomal DNAs demonstrate the presence of telomeric repeats, and the structural stability of the DNAs implies the presence of fully functional telomeres. Most interesting is the fact that exogenous DNA can apparently be placed under cellular protection and control via addition of partial or complete telomeres.
  • three other fungi are known to add terminal telomeric repeats to transforming DNA. These are C. neoformans, a basidiomycetous yeast (Edman, 1992); H. capsulatum, a dimorphic ascomycete (Woods and Goldman, 1992); and F.
  • telomeres High frequency in vivo addition of telomeric repeats to nontelomeric DNA termini is generally believed to occur only during developmentally programmed processes (Blackburn, 1995). For example, in certain ciliated protozoans, during a specific stage of development the genome undergoes massive fragmentation followed by de novo addition of telomeres to newly formed DNA ends (Coyne et al, 1996). Outside of developmentally regulated processes, however, the de novo addition of telomeres to nontelomeric DNA apparently occurs rarely (Blackburn, 1995; Cooke, 1995; Melek and Shippen, 1996). Herein, we have shown that P.
  • microspora possesses the biochemical mechanisms to add telomeric repeats to nontelomeric exogenous DNA and generate extrachromosomal DNAs at a relatively high rate. It is interesting that the vast majority of fungal species, including the well-studied model organisms, have not been observed to do this (Lemke and Peng, 1995).
  • DNA deoxyribonucleic acid
  • Telomeres are specialized DNA sequences found at the ends of the chromosomes of eukaryotes which function in chromosome protection, positioning, and replication. Telomeres protect linear chromosomes from degradation and fusion to other chromosomes, and are thought to be a site of attachment to the nuclear matrix at times during the cell cycle. As chromosome caps they reduce the formation of damaged and rearranged chromosomes which arise as a consequence of recombination-mediated chromosome fusion events.
  • telomeres consist of tens to thousands of tandem repeats of a telomere motif sequence and associated proteins.
  • the telomeres from all species show the same pattern: a short DNA sequence, one strand G-rich and one C-rich, that is tandemly repeated many times.
  • the repeating telomeric unit found in Tetrahymena is T 2 G 4
  • in the ciliated protozoan Oxytricha it is T 4 G 4
  • yeast in yeast it is T ⁇ -3 G ⁇ -3 .
  • this motif is 5'-d(TTAGGG)-3'. Sequences specific to other species such as plants may be found in Greider et al. (1990).
  • Telomeres of all human chromosomes are composed of variable length arrays of the TTAGGG repeat units with the G-rich strand oriented 5' to 3' towards the telomere.
  • Variant telomere repeat units such as TTGGGG and TGAGGG have been identified but tend to be located at the proximal ends of human telomeres.
  • Methods for detecting and quantitating multiple copies of a repeat sequence, such as a telomere (or centromere) repeat sequence are provided in WO 97/14026.
  • Methods for characterizing variability in telomere DNA by Polymerase Chain Reaction (PCR) are provided in WO 96/12821.
  • telomeres The maintenance of telomeres is required for cells to avoid replicative senescence and to continue to multiply. Chromosomes lose about 50-200 nucleotides of telomeric sequence from their ends per cell division, and the shortening of telomeres may act as a mitotic clock shortening with age both in vitro and in vivo in a replication dependent manner (Harley, 1991). Telomeric sequences can be added back to the chromosome ends, by telomere terminal transferase, also known as telomerase enzyme or simply as telomerase. Methods and compositions for increasing telomere length in normal cells to increase the proliferative capacity of cells and to delay the onset of senescence are provided in U.S. Patent Number 5,686,306.
  • Telomerase is a ribonucleoprotein enzyme that elongates the G-rich strand of chromosomal termini by adding telomeric repeats. This elongation occurs by reverse transcription of a part of the telomerase RNA component, which contains a sequence complementary to the telomere repeat. Following telomerase-catalyzed extension of the G-rich strand, the complementary DNA strand of the telomere is presumably replicated by more conventional means.
  • telomerase is a reverse transcriptase composed of both ribonucleotide acid (RNA) and protein, wherein the RNA molecule functions as the template for the telomeric repeat.
  • RNA ribonucleotide acid
  • the RNA moiety of human telomerase contains the 5'-CCCTAA-3' sequence that may act as the template for de novo synthesis.
  • the enzyme also contains a region that recognizes the guanine rich single strands of a DNA substrate.
  • telomere enzymes of these ciliates synthesize telomeric repeat units distinct from that in mammals.
  • the nucleic acids comprising the RNA of a mammalian telomerase are provided in U.S. Patent No. 5,583,016.
  • telomere activity has been identified in germ line cells and tumor cells but is repressed in differentiated somatic cells. It is now believed that the reactivation of telomerase is an essential step in tumor progression and in the immortalization of cells in culture. It is postulated that inhibition of telomerase in an immortalized cell line or in the malignant condition would cause senescence or cell death.
  • the introduction of synthetic oligonucleotides which mimic telomere motifs has been shown to inhibit the proliferation of immortal cells or cells that express telomerase (U.S. Patent Number 5,643,890).
  • telomere motif TTAGGG exhibited greater cellular uptake and higher inhibition of proliferation than longer oligonucleotides.
  • Telomere-telomere recombination provides an alternate pathway for telomere maintenance in at least some eukaryotes (Zakian, 1997). Wang et al. (1990) provided evidence for a telomere-telomere recombination process in yeast which involves a gene conversion event that requires little homology, occurs at or near the boundary of telomeric and non-telomeric DNA, and resembles the recombination process involved in bacteriophage T4 DNA replication.
  • Yeast cells which lack a functional estl gene exhibit a continuous decline in the terminal (G 1-3 T) n tract, a progressive increase in the frequency of chromosome loss, and a concomitant increase in the frequency of cell death (Lundblad et al, 1989).
  • ESTl is not a catalytic component of telomerase (Cohn et al, 1995)
  • the same phenotypes are produced by deleting the S. cerevisiae telomerase RNA gene, tlcl (Singer and Gottschling, 1994).
  • tlcl S. cerevisiae telomerase RNA gene
  • yeast cells have a RAD52-dependent bypass pathway by which cells can circumvent a defect in the ESTl -mediated pathway for yeast telomere replication. Most of the surviving cells have very short telomeres but acquire long tandem arrays of subtelomeric repeats by gene conversion. The researchers concluded that "even when the primary pathway for telomer replication is defective, an alternative backup pathway exists that restores sufficient telomere function for continued cell viability.”
  • telomerase RNA gene terl
  • yeast Kluyveromyces lactis results in the gradual loss of telomeric repeats and progressively declining cell growth capability, some cells are able to continuing growing without telomerase.
  • telomere cap-prevented recombination CPR
  • Artificial chromosomes are man-made linear DNA molecules constructed from essential DNA sequence elements that are responsible for the proper replication and partitioning of natural chromosomes (Murray et al, 1983).
  • the essential elements necessary to construct artificial chromosomes include:
  • centromere which is the site of kinetochore assembly and is responsible for the proper distribution of replicated chromosomes at cell division (i.e., mitosis and meiosis);
  • ARS Autonomous Replication Sequences
  • Artificial chromosome vectors can be constructed to include gene sequences capable of producing specific polypeptides, wherein the gene sequences can include extremely long stretches of exogenous DNA.
  • selectable marker genes can also be included in such artificial chromosomes to aid in the selection of transformed cells.
  • artificial chromosome recombinant molecules as vectors solves many of the problems associated with alternative transformation technologies which are used to introduce new DNA into higher eukaryotic cells. Since artificial chromosomes are maintained in the cell nucleus as independently replicating DNA molecules, sequences introduced on such vectors are not subject to the variable expression due to integration position effects. In addition, the delivery of artificial chromosomes to the nucleus of a cell as intact, unbroken, double-stranded DNA molecules with telomeric ends ensures that the introduced DNA can be maintained stably in that form and that rearrangements should not occur.
  • artificial chromosome vectors will be stably maintained in the nucleus through meiosis and will be available to participate in homology-dependent meiotic recombination.
  • Exogenous DNA introduced via artificial chromosome vectors can be delivered to practically any cell without host range limitations, in contrast to some other transformation methods such as the Agrobacterium-mediated DNA transfer systems.
  • Yeast artificial chromosomes are genetically engineered chromosomes that contain the essential DNA sequence elements of Saccharomyces and segments of exogenous DNAs that are much larger than those accepted by conventional cloning vectors.
  • YACs are generated from synthetic minichromosomes that contain a yeast centromere, a replication origin, and fused telomeres.
  • the circular chromosome also contains three marker genes (ml, m2, and m3), which when expressed, allow selection of the cells carrying the plasmid and two specific sites (Burke et al, 1987). These two sites allow specific restriction endonucleases to break the molecule. Cleavage at one site opens the ring, while cleavage at the second site generates centric and acentric fragments with ends that will accept exogenous DNA fragments.
  • an artificial chromosome is generated with a short and a long arm, with the long arm containing the spliced segment of exogenous DNA to be cloned.
  • Such artificial chromosomes are distributed normally during subsequent yeast divisions creating colonies containing the YACs.
  • the ml and m3 markers are expressed, but the damaged M2 is not, allowing religated YACs to be distinguished from unbroken plasmids.
  • T. A. Brown Gene Cloning, Second Edition, Chapman & Hall (1990), U.S. Patent Number 4,889,806 and U.S. Patent Number 5,270,201.
  • telomere sequences of human DNA including the sequence for the human telomere, ranging in size from 50 to 250 kilobases have been cloned into Saccharomyces cerevisiae using YAC vectors ⁇ see, e.g., Riethman et al, 1989; Guerrini et al, 1990).
  • YAC vectors can be constructed according to the methods detailed in U.S. Patent
  • Yeast ARSs have not been found to replicate in filamentous fungi (Fincham, 1989).
  • Mammalian Artificial Chromosomes The controlled construction of mammalian artificial chromosomes (MACs) has been difficult because, with the exception of telomeres, the corresponding essential elements in mammals have not been fully defined.
  • Higher eukaryotes ⁇ e.g., mammals
  • yeast contain repetitive DNA sequences which form a boundary at both sides of the centromere. This highly repetitive DNA interacting with certain proteins, especially in animal chromosomes, creates a genetically inactive zone (heterochromatin) around the centromere.
  • Alpha-satellite (alphoid) DNA forms a family of repeated DNA sequences found in amounts varying from 500 kb to 5 mb at the centromeres of human chromosomes. Alphoid sequences consist of a repeated 171 bp monomer that exhibits chromosome-specific variation in nucleotide sequence and higher order repeat arrangement.
  • U.S. Patent Number 5,288,625 reports that a cell line which contains a dicentric chromosome, one of the centromeres of which contains a segment of human DNA, can be treated so as to isolate the centromere which contains the human DNA on a chromosome away from other mammalian chromosomes.
  • a mouse lung fibroblast cell which contains such a dicentric chromosome wherein the centromere is linked to a dominant selectable marker ⁇ e.g., aminoglycoside-3' phosphotransferease-II
  • the inventor was able to isolate derivative cell lines which stably replicated a chromosome containing only centromeres comprising cloned human DNA.
  • Harrington et al (1997) have constructed stable human artificial chromosomes by cotransfecting large synthetic arrays of alphoid repeats, telomere repeats, and random genomic DNA fragments into human cultured cells.
  • the resultant minichromosomes acquired host sequences by means of either a chromosome truncation event or rescue of an acentric fragment, but in one case minichromosome formation was by a de novo mechanism.
  • the inclusion of uncharacterized genomic DNA in the transfection mixture raises the possibility that sequences other than the transfected alphoid and telomere DNA contributed to chromosome formation.
  • telomere repeats and selectable markers into a 100 kb YAC containing human centromeric DNA.
  • the resultant YAC which has regular repeat sequences of alpha- satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E.
  • the MACs appear to be about 1-5 Mb in size and contain YAC multimers. It is not known whether the MACs are linear or circular.
  • the data from structural analyses of the MACs by FISH and Southern blot hybridization suggest that the introduced YAC DNA itself must have been multimerized by recombination and/or amplification.
  • Transgenic animals are genetically modified animals into which cloned genetic material has been transferred.
  • the cloned genetic material is often referred to as a transgene.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species, including non-animal species, than the species of the target animal.
  • transgenic technology allows investigators to create mammals of virtually any genotype and to assess the consequences of introducing specific exogenous nucleic acid sequences on the physiological and morphological characteristics of the transformed animals.
  • the availability of transgenic animals permits cellular processes to be influenced and examined in a systematic and specific manner not achievable with most other test systems.
  • the development of transgenic animals provides biological and medical scientists with models that are useful in the study of disease. Such animals are also useful for the testing and development of new pharmaceutically active substances.
  • Gene therapy can be used to ameliorate or cure the symptoms of genetically-based diseases.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, biolistics (also called gene particle acceleration or microprojectile bombardment), gene targeting in embryonic stem cells and recombinant viral and retro viral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al, Hypertension 22(4):630-633 (1993); Brenin et al, Surg. Oncol. 6(2)99-110 (1997); Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)).
  • biolistics also called gene particle acceleration or microprojectile bombardment
  • knock-out generally refers to mutant organisms which contain a null allele of a specific gene.
  • knock-in generally refers to mutant organisms into which a gene has been inserted through homologous recombination.
  • the knock-in gene may be a mutant form of a gene which replaces the endogenous, wild-type gene.
  • a number of recombinant rodents have been produced, including those which express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV 40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S.
  • Patent No. 5,602,307 possess an bovine growth hormone gene (Clutter et al., Genetics 143(4):1753-1760 (1996)); and are capable of generating a fully human antibody response (McCarthy, The Lancet 349(9049):405 (1997)). While rodents, especially mice and rats, remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al., Mol. Reprod.
  • the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules.
  • Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the recitations in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307. 2. Production of Transgenic Plants
  • Transgenic plants can be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment; viral- mediated transformation; and Agrobacterium-mediated transformation (see, e.g., U.S. Patent Numbers 5,405,765, 5,472,869, 5,538,877, 5,538,880, 5,550,318, 5,641,664, 5,736,369 and 5,736369; Watson et al, Recombinant DNA, Scientific American Books (1992); Hinchee et al., Bio/Tech. 6:915-922 (1988); McCabe et al., Bio/Tech.
  • Genes successfully introduced into plants using recombinant DNA methodologies include, but are not limited to, those coding for the following traits: seed storage proteins, including modified 7S legume seed storage proteins (U.S. Patent Numbers 5,508,468,
  • Genes can be introduced in a site directed fashion using homologous recombination. This can be used in the creation of a transgenic animal, wherein the animal would be mutated, and the phenotype of the mutation could be studied for purposes of drug screening, investigating physiologic processes, developing new products and the like. Papers discussing homologous recombination are discussed in U.S. Patent No. 5,413,923.
  • homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome.
  • the application of homologous recombination to gene therapy depends on the ability to carry out homologous recombination or gene targeting in normal, somatic cells for transplantation.
  • embryonic stem cells or a stem cell line may be obtained.
  • Cells other than embryonic stem cells can be utilized (e.g. hematopoietic stem cells etc.) (See U.S. Patent No. 5,589,369 for more examples).
  • the cells may be grown on an appropriate fibroblast fetal layer or grown in the presence of leukemia inhibiting factor (LIF) and then used.
  • LIF leukemia inhibiting factor
  • the embryonic stem cells may be injected into a blastocyst, that has been previously obtained, to provide a chimeric animal.
  • the main advantage of the embryonic stem cell technique is that the cells transfected with the "transgene" can be tested prior to reimplantation into a female animal for gestation for integration and the effect of the transgenes. By subsequent cross-breeding experiments, animals can be bred which carry the transgene on both chromosomes. If mutations are incorporated into the transgenes which block expression of the normal gene production, the endogenous genes can be eliminated by this technique and functional studies can thus be performed.
  • Methods for intracellularly producing DNA segments by homologous recombination of smaller overlapping DNA fragments and transgenic mammalian cells and whole animals produced by such methods are disclosed in U.S. Patent No. 5,612,205. Cell lines useful for analysis of human homologous interchromosomal recombination are provided in U.S. Patent Application No. 5,554,529.
  • Homologous recombination can also proceed extrachromasomally, which may be of benefit when handling large gene sequences (e.g., larger than 50 kb). Methods of performing extrachromosomal homologous recombination are described in U.S. Patent No. 5,721,367.
  • Nucleic acid molecules of the invention include the nucleotide sequences coding for a Pestalotiopsis telomerase enzyme or a subunit of a Pestalotiopsis telomerase enzyme. Any nucleic acid sequence which specifically hybridizes to such nucleic acid molecules such that the sequence remains stably bound to said nucleic acid molecules under highly stringent or moderately stringent conditions is also encompassed within this invention. Stringent and moderately stringent conditions are those commonly defined and available, such as those defined by Sambrook et al. (1989) or Ausubel et al. (1995). The precise level of stringency is not important, rather, conditions should be selected that provide a clear, detectable signal when specific hybridization has occurred.
  • Hybridization is a function of sequence identity (homology), G+C content of the sequence, buffer salt content, sequence length and duplex melt temperature (T[m]) among other variables.
  • sequence identity identity
  • buffer salt content sequence length
  • duplex melt temperature T[m]
  • the buffer salt concentration and temperature provide useful variables for assessing sequence identity (homology) by hybridization techniques. For example, where there is at least 90 percent homology, hybridization is commonly carried out at 68 ° C in a buffer salt such as 6XSCC diluted from 20XSSC. See Sambrook et al. (1989).
  • the buffer salt utilized for final Southern blot washes can be used at a low concentration, e.g., O.IXSSC and at a relatively high temperature, e.g., 68° C, and two sequences will form a hybrid duplex (hybridize).
  • Use of the above hybridization and washing conditions together are defined as conditions of high stringency or highly stringent conditions.
  • Moderately stringent conditions can be utilized for hybridization where two sequences share at least about 80 percent homology.
  • specific hybridization occurs under conditions in which a high degree of complementarity exists between a nucleic acid comprising the sequence of an isolated sequence and another nucleic acid. With specific hybridization, complementarity will generally be at least about 70%, 75%, 80%, 85%, preferably about 90-100%, or most preferably about 95-100%.
  • homology or identity is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al. Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, S. F. J. Mol. Evol. 36: 290-300(1993), both of which are herein incorporated by reference) which are tailored for sequence similarity searching.
  • the approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance.
  • the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
  • nucleic acids of the present invention can be used in a variety of ways in accordance with the present invention. For example, they can be used as nucleic acid probes to screen other cDNA and genomic DNA libraries so as to select by hybridization other DNA sequences that encode homologous sequences.
  • Contemplated nucleic acid probes could be RNA or DNA labeled with radioactive nucleotides or by non-radioactive methods (for example, biotin). Screening may be done at various stringencies (through manipulation of the hybridization Tm, usually using a combination of ionic strength, temperature and/or presence of formamide) to isolate close or distantly related homologs.
  • the nucleic acids may also be used to generate primers to amplify cDNA or genomic DNA using polymerase chain reaction (PCR) techniques.
  • PCR polymerase chain reaction
  • the nucleic acid sequences of the present invention can also be used to identify adjacent sequences in the genome, for example, flanking sequences and regulatory elements.
  • the nucleic acids may also be used to generate antisense primers or constructs that could be used to modulate the level of gene expression.
  • the amino acid sequence may be used to design and produce specific antibodies. Taxol Biosynthesis
  • the first dedicated step in the biosynthesis of taxol in T. brevifolia is the cyclization of the universal diterpene precursor geranylgeranyl diphosphate to taxa-
  • taxa-4(5),l l(12)-diene synthase also called taxadiene synthase, isolated from the Pacific yew tree has been purified and characterized (Hezari et al, 1995).
  • the sequence specifies an open reading frame of 2586 nucleotides and the complete deduced polypeptide, including a long presumptive plastidial targeting peptide, contains 862 amino acid residues and has a molecular weight of 98,303. Sequence comparisons with monoterpene, sesquiterpene, and diterpene cyclases of plant origin indicate a significant degree of similarity between these enzymes.
  • the taxadiene synthase most closely resembles (46% identify, 67% similarity) abietadiene synthase, a diterpene cyclase from grand fir.
  • Huang et al. report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein.
  • the maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(D ⁇ 3)/p ⁇ T32TX.
  • taxol is naturally produced by yew plant species and by microorganisms such P. microspora. In an effort to produce greater quantities of taxanes, such as taxol, a number of different artificial systems have been utilized.
  • U.S. Patent No. 5,322,779 provides in vitro methods of producing taxol using the fungus Taxomyces andreanae, wherein the fungus is isolated from tissue fragments from a tree of the genus Taxus.
  • U.S. Patent No. 5,310,672 provides methods for the production of taxol and taxol precursors from in vitro propagated cultures of somatic embryos derived from explants of the genus Taxus, especially from T. brevifolia.
  • Taxus suspension cultures have been used for the production of taxanes such as taxol (see, e.g., Hezari et al., 1997; U.S. Patent No. 5,665,576).
  • Taxus cuspidata grown in either shake flasks or Wilson-type reactors in which bubbled air provided both agitation and mixing, Pestchanker et al. (1996) obtained taxol titers of 22 mg per liter in 20 days.
  • U.S. Patent No. 5,279,953 provides a method for improved in vivo production of taxanes by inoculation of tissue of the genus Taxus with virulent or avirulent strains of Agrobacterium. Incomplete harvesting of galls permits regeneration on the trees, resulting in a renewable resource. The net yield increase demonstrated was 2 to 6 times normal (4 to 6 times for the galls themselves), did not require special growth hormones or media, did not result in sacrificing the tree, and can be started in the sapling stage.
  • Example 1 Source oi Pestalotiopsis microspora.
  • P. microspora was isolated from the inner bark of a Himalayan yew tree (Taxus wallachiana) near Kathmandu, Nepal as previously described (Strobel et al, 1996). Fungal material for all experiments was derived from a single germinating conidium from an isolate designated NE-32.
  • Plasmid pDH33 is a derivative of pBR322 containing the Aspergillus niger glaA promoter and Aspergillus nidulans trpC terminator fused to the E. coli hygromycin resistance gene (Smith et al, 1990).
  • Plasmid pDH25 is a derivative of pBR322, but has the A. nidulans trpC promoter fused to hygromycin resistance gene (Cullen et al, 1987).
  • Oligonucleotides were synthesized by Operon Technologies (Alameda, CA) or by the Iowa State University DNA Sequencing Facility. DNA sequencing was performed by the Iowa State University DNA Sequencing Facility with an ABI 377 DNA Sequencer (Perkin Elmer).
  • Protoplasts of P. microspora were produced by cell wall digestion of two day old mycelium (grown in potato dextrose broth, PDB) in digest buffer (20 mM K 2 HPO 4 , 50 mM MgSO 4 « 7H 2 O, 0.7 M sorbitol, pH 6.3) containing 20 mg/ml Novozyme 234 (Calbiochem) and 20 mg/ml ⁇ -glucuronidase (Sigma) at 30°C for 90 min with 130 rpm shaking, in a proportion of 2 ml per 100 mg mycelium wet weight.
  • Protoplasts were filtered through glass wool, collected by centrifugation (825g, 7 min, 20°C), washed in digest buffer, resuspended in 0.5 ml transformation buffer (10 mM Tris HC1, 0A M CaCl 2 , 0.7 M sorbitol, pH 7.5), and counted with a hemocytometer.
  • transformants were transferred to PDA containing 200 ⁇ g/ml hygromycin.
  • Results Treatment of P. microspora protoplasts with CaCl 2 and PEG in the presence of plasmid pDH33 typically yielded 50 to 1000 hygromycin-resistant transformants per microgram of pDH33 per 4 x 10 5 regenerating protoplasts. At least 10-fold more transient transformants, which clearly demonstrated resistance but for only one to four days, were also observed. Comparable transformation efficiencies were obtained with pDH25, a similar plasmid but with a different promoter controlling the hygromycin resistance gene.
  • Stability of the transformant phenotype during vegetative growth in the absence of selection was measured in a two step procedure. First, four 3 mm x 3 mm blocks from different regions of the growing margin of P. microspora transformant colonies were transferred to PDA (lacking hygromycin) every 5 days for 35 days, with incubation at 24°C. Second, at the time of each transfer, retention of the transformant phenotype
  • hygromycin resistance was assessed by transferring four blocks of mycelium from the growing margin to PDA containing 0, 100, 200, 400, 600, 800, and 1000 ⁇ g/ml hygromycin and calculating colony areas after 7 days of growth at 24°C.
  • Conidiation was induced by placing blocks of mycelia from P. microspora transformants on carnation leaf agar (1.5% water agar, W/V) containing ⁇ -irradiated carnation leaf pieces (Nelson et al, 1983). After 10-14 days of growth at room temperature, conidia were collected from acervuli and suspended in 0.1% Tween 20 (which prevents conidia from adhering to pipette tips and Eppendorff tubes), and counted with a hemocytometer. The fraction of conidia resistant to hygromycin was measured by comparing the germination rate on PDA with that on PDA containing 200 ⁇ g/ml hygromycin.
  • Phenotypic characterization was undertaken to determine whether the stability of resistance in transformants was consistent with Southern blot evidence for extrachromosomal DNAs.
  • DNA extraction from lyophilized mycelium was accomplished as described by Raeder and Broda (1985).
  • Mycelium for DNA extraction was grown in modified Czapek's medium (2 g NaNO 3 , 1 g KH 2 PO 4 , 500 mg MgSO 4 » 7H 2 O, 500 mg KC1, 10 mg FeSO 4 *7H 2 O, 500 mg yeast extract, and 100 mg sucrose per liter H 2 O with addition of
  • Membranes were prehybridized for 1 h at 68° C in a solution consisting of 6X SSC, 5X Denhardt's reagent (IX Denhardt's reagent is 200 mg Ficoll, 200 mg polyvinylpyrrolidone, and 200 mg bovine serum albumin per liter water), 0.5% SDS (W/V), 8% PEG 6000 (W/V), 100 ⁇ g/ml sheared denatured salmon sperm DNA (Sigma). After prehybridization labeled probe was added to a final concentration of 20 ng/ml and incubation was continued at 68 °C for approximately 18 h.
  • IX Denhardt's reagent is 200 mg Ficoll, 200 mg polyvinylpyrrolidone, and 200 mg bovine serum albumin per liter water
  • 0.5% SDS W/V
  • 8% PEG 6000 W/V
  • 100 ⁇ g/ml sheared denatured salmon sperm DNA Sigma
  • Hybridized membranes were washed twice at room temperature for 15 min in 100 ml 2X SSC/0.1% SDS, washed once at 68 °C in 100 ml 0.1 X SSC/0.1% SDS, and exposed to Hyperfilm-MP (Amersham) with an intensifying screen at -70 °C.
  • Southern blots to detect telomeric sequences employed an 18 base synthetic oligonucleotide probe (TE2, 5 '-(CCCTAA) 3 -3 ') 5' end labeled with 32 P using T4 polynucleotide kinase according to procedures recommended by the supplier (Promega).
  • Hybridized membranes were washed twice for 15 min at room temperature in 100 ml 2X SSC/0.1% SDS and exposed to Hyperfilm-MP (Amersham) with an intensifying screen at -70 °C or to a phosphor screen (Molecular Dynamics).
  • Example 6 Amplification of P. microspora DNA in E. coli.
  • P. microspora transformant total DNA was transformed into SURE strain E. coli cells (Stratagene) by electroporation using an Electro Cell Manipulator 600 electroporator (BTX) according to suggestions from the manufacturer, plated on LB agar (10 g tryptone, 5 g yeast extract, 5 g NaCl, 15 g agar, and 100 ⁇ l 10 NNaOH per liter) containing 50 ⁇ g/ml ampicillin, and incubated overnight at 37 °C. Plasmids from E. coli were prepared by standard alkaline lysis (Sambrook et al, 1989) or with a Wizard Plus SV Miniprep kit (Promega). Example 7. Polymerase Chain Reaction Materials and Methods.
  • PCR amplifications were carried out in a Model 480 DNA Thermal Cycler (Perkin Elmer) in 50 ⁇ l reactions containing 2 mM Mg 2+ and approximately 100 ng of total transformant DNA.
  • Amplifications with primer TE2 (final concentration 0.8 ⁇ M , sequence described above) utilized 2 ⁇ l of ELONGASE Enzyme Mix (Gibco/BRL Life Technologies) and employed temperature cycling parameters of 94°C for 30 sec, followed by 30 cycles of 94°C for 30 sec, 46°C for 30 sec, and 68°C for 10 min.
  • 5'-ATAGCTGCGCCGATGGTTTCTA-3' (SEQ ID. NO: 2) employed 0.2 ⁇ M final concentrations of each primer, 1 ⁇ l of enzyme mix, and a cycling protocol of 94°C for 30 sec followed by 25 cycles of 94°C for 30 sec, 50°C for 30 sec, and 68 °C for 10 min.
  • PCR products were purified using a QIAquick PCR purification kit (Qiagen). Results.
  • a primer (TE2) complementary to the 5 '-TTAGGG-3 ' telomeric sequence was used in PCR amplifications of P. microspora transformant total
  • Banl restriction digests revealed that the full-length PCR product has restriction fragments in common with pDH33 ( Figure 4a, lanes 4 and 5), and demonstrated that the amplified 5' and 3' fragments were in fact fragments of the full-length molecule (data not shown).
  • Transformation of wild-type P. microspora with the PCR-amp lifted extrachromosomal DNA produced 10- to 50-fold more transformants per microgram of DNA than did transformation with pDH33, establishing a functional significance for the terminal telomeric repeats present on the P. microspora transformant extrachromosomal DNA.
  • telomeric repeats are a common feature of extrachromosomal DNAs in different transformants.
  • all extrachromosomal bands in P. microspora transformants that hybridized to a radio labeled pDH33 probe Fig.
  • sequence requirements of P. microspora telomerase were further studied by determining additional nucleotide sequences at the pDH33 -telomeric repeat junctions near the 5' and 3' termini of the PCR amplified full-length extrachromosomal DNA from transformant tr3, using the protocol as set forth in Experiment 7. As shown by the following examples, the sequence requirements of P. microspora telomerase are minimal:
  • Taxol production of the transformed and non-transformed P. microspora cells is compared.
  • the transformed cells display a higher production level of taxol and taxol-related compounds than the non-transformed cells.
  • the extrachromosomal DNAs coding for taxadiene synthase are extracted from the transformed P. microspora cells produced in Example 9 and are used to transform somatic embryos derived from explants of the genus T. brevifolia.
  • Taxol and taxol precursors are produced from in vitro propagated cultures of somatic embryos derived from the transformed and non-transformed explants.
  • the taxane and taxane-related compounds are produced at a higher level in the transformed explants than they are produced in the non-transformed explants.
  • the extrachromosomal DNAs coding for taxadiene synthase are extracted from the transformed P. microspora cells produced in Example 9 and are used to transform cells of the fungus T. andreanae. Taxol and taxol precursors are produced from in vitro propagated cultures of the transformed and non-transformed fungus.
  • taxane and taxane-related compounds are produced at a higher level in the transformed fungus than they are produced in the non-transformed fungus.
  • TRAP "TRAP" Assay for Detection of Telomerase. If the de novo addition of telomeres by P. microspora is by telomerase acvtivity, then one would expect that telomerase activity in protoplasts (the entities typically transformed) would be detectable using the commonly employed TRAP assay method.
  • TRAP assay See, for example, U.S. Patent No. 5,629,154; Piatyszek, M. A. et al. (1995); and Kim, N. W. et al, each of which is incorporated by reference in their entirety herein.
  • telomere assay cell extract is mixed with an oligonucleotide which acts as a substrate for telomerase in the extract. Telomerase extends the oligonucleotide in six nucleotide increments. The products of this reaction are then PCR amplified with the original substrate nucleotide, and a second ohgnucleotide complementary to the telomeric repeats added by telomerase.
  • telomerase activity in a cell free extract of P. microspora consisting of lysed protoplasts, and all other components the same as the published TRAP assay components (see references cited above for details).
  • the protoplasts were prepared using the same methods as those used to prepare protoplasts for transformation.
  • the TRAP assys we carried out, the telomerase acvtivity in P. microspora protoplasts was heat sensitive, and RNAase sensitive, providing evidence that the postive TRAP assay is due to the presence of a ribonucleoprotein such as telomerase.
  • Taxa-4(5),l l(12)-diene Synthase from Pacific Yew (Taxus brevifolia) that Catalyzes the First Committed Step of Taxol Biosynthesis. Archives of Biochemistry and
  • Pestalotiopsis species Bio. Pharm. Bull. 20(2): 118-121.
  • Cyclization of geranylgeranyl diphosphate to taxa- 4(5), 1 l(12)-diene is the committed step of taxol biosynthesis in Pacific Yew. J. Biol.
  • RNA-dependent polymerase motifs in ESTl tentative identification of a protein component of an essential yeast telomere. Cell. 60:529- 530.
  • telomere CPR Cap-prevented recombination between terminal telomeric repeat arrays
  • telomere activity in human cells and tumors by telomeric repeat amplification protocal (TRAP). Methods in Cell Science. 17:1.
  • Saccharomyces telomeres acquire single-strand T-G 1-3 tails late in S phase. Cell 72: 51-60.

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Abstract

L'invention concerne des compositions et des procédés utiles pour la constitution d'ADN extrachromosomique, ledit ADN codant un produit qui modifie le niveau de la production de taxol. Plus précisément, l'invention concerne des compositions et des procédés qui permettent de constituer de l'ADN extrachromosomique en utilisant la découverte inattendue selon laquelle les champignons Pestalotiopsis ajoutent une ou plusieurs répétitions télomériques aux extrémités de l'ADN exogène.
PCT/US1999/015135 1998-07-02 1999-07-02 Production de taxol par constitution d'adn extrachromosomique dans le champignon pestalotiopsis WO2000001809A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU49685/99A AU4968599A (en) 1998-07-02 1999-07-02 Taxol production via generation of extrachromosomal dnas in the fungus (pestalotiopsis)
EP99933683A EP1092016A1 (fr) 1998-07-02 1999-07-02 Production de taxol par constitution d'adn extrachromosomique dans le champignon pestalotiopsis
JP2000558199A JP2002519058A (ja) 1998-07-02 1999-07-02 Ｐｅｓｔａｌｏｔｉｏｐｓｉｓ菌における染色体外ＤＮＡの生成を経るタキソール生産
CA002336122A CA2336122A1 (fr) 1998-07-02 1999-07-02 Production de taxol par constitution d'adn extrachromosomique dans le champignon pestalotiopsis
KR1020017000034A KR20010083085A (ko) 1998-07-02 1999-07-02 진균류 페스탈로티옵시스에서의 염색체외 디엔에이의생성에 의한 탁솔 생산방법

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US9166798P 1998-07-02 1998-07-02
US60/091,667 1998-07-02

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997038571A1 (fr) * 1996-04-15 1997-10-23 Washington State University Research Foundation Compositions et procedes pour la biosynthese du taxol

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997038571A1 (fr) * 1996-04-15 1997-10-23 Washington State University Research Foundation Compositions et procedes pour la biosynthese du taxol

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JAVERZAT J.-P. ET AL.: "Isolation of telomeric DNA from the filamentous fungus Podospora anserina and construction of a self-replicating linear plasmid showing high transformation frequency", NUCL. ACIDS RES., vol. 21, no. 3, 1993, pages 497 - 504, XP002124428 *
LONG D.M. ET AL.: "In vivo addition of telomeric repeats to foreign DNA generates extrachromosomal DNAs in the taxol-producing fungus Pestalotiopsis microspora", FUNGAL GENET. BIOL., vol. 24, no. 3, August 1998 (1998-08-01), pages 335 - 344, XP000856850 *
STROBEL ET AL: "Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana", MICROBIOLOGY, vol. 142, no. 142, 1996, pages 435-440-440, XP002103985, ISSN: 1350-0872 *
XX; 1 July 1997 (1997-07-01), LONG D M, STROBEL G A: "SGER: EFFICIENT EXTRACHROMOSOMAL REPLICATION OF EXOGENOUS DNA BY A FILAMENTOUS FUNGUS", XP002124429, Database accession no. GRANT NO., 9724999 *

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