WO2000056894A1 - Fungal beta-tubulin genes - Google Patents
Fungal beta-tubulin genes Download PDFInfo
- Publication number
- WO2000056894A1 WO2000056894A1 PCT/US2000/007995 US0007995W WO0056894A1 WO 2000056894 A1 WO2000056894 A1 WO 2000056894A1 US 0007995 W US0007995 W US 0007995W WO 0056894 A1 WO0056894 A1 WO 0056894A1
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- WO
- WIPO (PCT)
- Prior art keywords
- taxol
- amino acid
- nucleotide
- tubulin
- beta
- Prior art date
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
Definitions
- TAXOL ® Bristol-Myers-Squibb
- taxol paclitaxel
- MTs microtubules
- Taxol an antimitotic agent with a new mechanism of action
- Mechanism of action of taxol " Trends Pharmacol Sci 13: 134- 136
- taxol affects spindle function during mitosis, resulting in cell cycle arrest in G2/M phase.
- taxol promotes MT assembly and prevents their disassembly under conditions which would otherwise cause depolymerization (Schiff, et al. 1979. "Promotion of microtubule assembly in vitro by taxol” Nature 277:665-667; and Pamess, J. and Horwitz, S.B.
- Taxol was found originally in the inner bark of pacific yew trees (Taxus brevifoha) by Wani et al (Warn, et al 1971 "Plant antitumor agents VI The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifoha, " J Am Chem Soc 93 2325-2327), and noted to constitute about 0 02% of dry phloem weight The limited resource of yew trees made it advantageous to locate additional sources for taxol
- taxol inhibited nuclear division at low concentrations, indicating that it acts through a mechanism similar to that in mammalian cells
- four ascomycete species were identified as resistant to taxol (IC50 > 50 ⁇ M). This resistance was reported to be due to the reduced ability of fungal microtubules to interact with taxol Taxol was also shown to be unable to stabilize MTs assembled with purified S.
- beta-tubulin genes useful for developing isogenic fungal strains that are either taxol-sensitive or taxol- resistant
- beta-tubulin genes and/or isogenic fungal strains can then be applied to anticancer drug screening and for developing diagnostic tests for tumor sensitivity assays
- the invention is a purified DNA segment encoding a beta-tubulin of the fungal species Pestalotiopsis microspora or a portion thereof
- the DNA segment encodes at least one taxol binding site
- the DNA segment encodes a protein having taxol binding site I and taxol binding site II
- the DNA segment encodes a protein which has taxol binding site I and taxol binding site II and is able to interact with alpha-tubulin
- An exemplary DNA segment comprises at least a portion of SEQ ID NO 1
- Another exemplary DNA segment comprises a portion of SEQ ID NO 1 comprising the nucleotide sequence from nucleotide 75 through nucleotide 167 of SEQ ID NO 1, with or without substitution
- Another exemplary DNA segment comprises a portion of SEQ ID NOJ comprising the nucleotide sequence from nucleotide 708 through nucleotide 764 of SEQ ID NOJ,
- Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 75 to nucleotide 1412 of SEQ ID NOJ wherein at least one nucleotide in the nucleotide sequence is substituted and wherein the taxol binding capacity of the beta-tubulin is not altered.
- Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 75 to nucleotide 1412 of SEQ ID NOJ wherein at least one nucleotide in the nucleotide sequence is substituted and wherein the taxol binding capacity of the beta-tubulin is altered.
- Another exemplary amino acid sequence comprises a portion of SEQ ID NOJ consisting essentially of Amino Acids 1-446 the portion contains at least one amino acid substitution that alters the taxol binding property of the portion.
- Another exemplary amino acid sequence comprises a portion of SEQ ID NOJ consisting essentially of Amino Acids 1-446 the portion contains at least one amino acid substitution that does not alters the taxol binding property of the portion.
- Another exemplary amino acid sequence is substituted with any amino acid which perturbs the three- dimensional structure of the amino acid sequence surrounding Amino Acid 219 as numbered in SEQ ID NOJ.
- the invention is a purified DNA segment encoding a beta-tubulin of the fungal species Pythium ultimum or a portion thereof.
- the DNA segment encodes at least one taxol binding site.
- the DNA segment encodes a protein having taxol binding site I and taxol binding site II.
- the DNA segment encodes a protein which has taxol binding site I and taxol binding site II and is able to interact with alpha- tubulin.
- An exemplary DNA segment comprises at least a portion of SEQ ID NOJ.
- Another exemplary DNA segment comprises a portion of SEQ ID NOJ comprising nucleotide 92 through nucleotide 184, with or without substitution.
- Another exemplary DNA segment comprises a portion of SEQ ID NOJ comprising the nucleotide sequence from nucleotide 725 through nucleotide 781, with or without substitution.
- Another exemplary DNA segment comprises a portion of SEQ ID NOJ comprising the nucleotide sequence from nucleotide 725 through nucleotide 781, wherein either nucleotide 746, nucleotide 747 or nucleotide 748 or mixtures thereof are substituted.
- Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 92 to nucleotide 1429 of SEQ ID NOJ with at least one nucleotide substitution in the nucleotide sequence and wherein the taxol binding capacity of the beta-tubulin is altered.
- the invention is an amino acid sequence comprising at least a portion of a beta-tubulin of the fungal species Pythium ultimum.
- the amino acid sequence comprises at least one taxol binding site.
- the amino acid sequence is a protein having taxol binding site I and taxol binding site II.
- the amino acid sequence has taxol binding site I and taxol binding site II and is able to interact with alpha-tubulin.
- An exemplary amino acid sequence comprises at least a portion of the beta-tubulin as depicted in SEQ ID NO:4.
- Another exemplary amino acid sequence comprises a portion of SEQ ID NO:4 consisting essentially of Amino Acids 1-446 having at least one amino acid substitution that alters the taxol binding property of the portion.
- Another exemplary amino acid sequence comprises a portion of SEQ ID NO:4 consisting essentially of Amino Acids 1-446 having at least one amino acid substitution that does not alter the taxol binding property of the portion.
- Another exemplary amino acid sequence is substituted with any amino acid which perturbs the three-dimensional structure of the amino acid sequence surrounding Amino Acid 219 as numbered in SEQ ID NO:4.
- Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising nucleotide sequence from nucleotide 644 through nucleotide 700, wherein at least one nucleotide in the nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code nucleotide 665 cannot be adenine while nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine.
- Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising the nucleotide sequence from nucleotide 11 to nucleotide 1342 and wherein the DNA segment encodes a beta- tubulin.
- Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising the nucleotide sequence from nucleotide 1 1 to nucleotide 1342, wherein at least one nucleotide in the nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code, nucleotide 665 cannot be adenine while nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine.
- Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising the nucleotide sequence from nucleotide 11 to nucleotide 1342, wherein at least one nucleotide in the nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code, nucleotide 665 cannot be adenine while nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine, and wherein the taxol binding capacity of the beta-tubulin is altered.
- the invention is an amino acid sequence comprising at least a portion of a beta-tubulin of the fungal species Phytophthora cinnamomi as depicted in SEQ ID NO:6.
- An exemplary amino acid sequence comprises a portion of SEQ ID NO:6 comprising Amino Acids 1-31.
- An exemplary amino acid sequence comprises a portion of SEQ ID NO:6 comprising Amino Acids 1-31, having at least one amino acid substituted, providing that when only one amino acid is substituted Amino Acid 24 is not isoleucine.
- Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 6 comprising Amino Acids 212-230.
- Another exemplary amino acid sequence comprises a portion of SEQ ID NO:6 comprising Amino Acids 212-230, having at least one amino acid substituted, providing that when only one amino acid is substituted Amino Acid 219 is not asparagine.
- Another exemplary amino acid sequence comprises a portion of SEQ ID NO:6 comprising Amino Acids 212-230 with an amino acid substitution at Amino Acid 219, wherein the Amino Acid 219 is not substituted with asparagine.
- the invention is a vector comprising a purified DNA segment encoding a beta-tubulin of the fungal species Pythium ultimum or a portion thereof
- the vector comprises a portion encoding at least one taxol binding site
- the invention is a vector comprising a purified DNA segment encoding a beta-tubulin of the fungal species Phytophthora cinnamomi wherein the DNA segment consists essentially of SEQ ID NO 5 or a portion thereof
- the vector comprises a portion encoding at least one taxol binding site
- the invention is a method of determining the taxol binding capacity of a beta-tubulin or beta-tubulin-like compound comprising providing antibodies raised against amino acid sequences comprising at least one taxol binding site of a beta- tubulin from a taxol-resistant Pestalotiopsis microspora, a taxol-sensitive Pythium ultimum, or taxol-sensitive Phytophthora cinnamomi as depicted in SEQ ID NO 6 to form a reagent, such antibodies distinguishing between taxol-binding and non-taxol-binding properties, contacting the beta-tubulin or beta-tubulin-like compound with the reagent, and determining degree of binding between the antibodies in the reagent and the beta-tubulin or beta-tubulin- like compound; whereby binding of antibodies raised against a taxol-resistant Pestalotiopsis microspora to the beta-tubulin or beta-tubulin-like compound indicates taxol resistance and
- the antibodies in the reagent are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol-resistant Pestalotiopsis microspora. In another embodiment, the antibodies in the reagent are raised against an amino acid sequence comprises at least one taxol binding site from SEQ ID NOJ. In another embodiment, the antibodies in the reagent are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol-sensitive Phythium ultimum. In another embodiment, the antibodies in the reagent are raised against an amino acid sequence comprises at least one taxol binding site from SEQ ID NO:4.
- the antibodies in the reagent are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol-sensitive Phytophthora cinnamomi as depicted in SEQ ID NO:6.
- the antibodies in the reagent are raised against an amino acid sequence comprises at least one taxol binding site from SEQ ID NO:678.
- the beta-tubulin or beta-tubulin-like compound are selected from the group consisting of recombinantly expressed protein, exogenously isolated protein, synthetic peptides, and cell cultures.
- the invention is a method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing beta-tubulins with amino acid sequences comprising both taxol binding sites from Pythium ultimum or taxol- sensitive Phytophthora cinnamomi as depicted in SEQ ID NO: 6 in addition to alpha-tubulin from any taxol-sensitive organism to form a reagent; contacting the composition of matter with the reagent; and determining the ability of the composition of matter to promote MT assembly or ability to prevent depolymerization of assembled MTs under depolymerizing conditions;whereby the ability to promote microtubule assembly or prevent depolymerization indicate the possible presence of taxol or taxol-like compounds in the composition of matter.
- the invention is a method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing mycelia of taxol- sensitive Pythium ultimum or a taxol-sensitive Phytophthora cinnamomi which harbors beta- tubulin in SEQ ID NO:6; contacting the composition of matter with the mycelia in the presence of the labeled taxol; and determining the degree of competitive inhibition of binding between the beta-tubulins and the labeled taxol by the composition of matter, whereby the composition of matter is determined to possess taxol or taxol-like compounds if it is able to block labeled taxol binding to the beta-tubulins from the taxol-sensitive Pythium ultimum or Phytophthora cinnamomi.
- the invention is a method of altering the taxol binding property of a recombinantly expressed beta-tubulin or a portion thereof comprising determining the identity of the codon at Amino Acid 219 as numbered in SEQ ID NOJ in the coding region of the vector; and if the codon at Amino Acid 219 codes for any amino acid except threonine, substituting nucleotides in the codon to code for threonine at Amino Acid 219 to alter a non-taxol-binding beta-tubulin or portion thereof to a taxol-binding beta-tubulin or portion thereof, or if the codon at Amino Acid 219 codes for threonine, substituting nucleotides in the codon to code for any amino acid except threonine at Amino Acid 219 to alter a taxol-binding beta-tubulin or portion thereof to a non-taxol-binding beta-tubulin or portion thereof.
- the invention is a method of developing a taxol-sensitive fungal cell from a taxol-resistant fungal cell comprising transforming the non-taxol-sensitive fungal cell by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ; wherein the transformed fungal cell expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
- the invention is a transgenic taxol-sensitive fungal cell transformed by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ, wherein the transformed fungal cell expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
- the invention is a method of developing a taxol-resistant fungal cell from a taxol-sensitive fungal cell comprising transforming the taxol-sensitive fungal cell by introducing a DNA segment encoding non-taxol-binding beta-tubulin wherein the amino acid at Amino Acid 219 as numbered in SEQ ID NOJ is not threonine; wherein the transformed fungal cell over-expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
- the invention is a transgenic taxol-sensitive fungal cell transformed by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ, wherein the transformed fungal cell over-expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
- the invention is a method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing distinguishable taxol- resistant and taxol-sensitive fungal cells; contacting the composition of matter with the fungal cells; and determining the growth of inhibition of the fungal cells; whereby the composition of matter is determined to possess taxol or taxol-like compounds if it is able to inhibit the growth of taxol-sensitive fungal cells but not able to inhibit the growth of taxol- resistant fungal cells.
- the method can be performed wherein the distinguishable taxol- resistant and taxol-sensitive fungal cells consists essentially of transgenic taxol-resistant and taxol-sensitive isogenic fungal cells.
- the method can also be performed with taxol-resistant fungal cells derived from one fungus which is unrelated to the fungi from which the taxol- sensitive fungal cells are derived.
- the invention is a method for controlling the growth of a plant pathogen comprising determining the taxol sensitivity of the plant pathogen; and if the pathogen is determined to be taxol-sensitive, the plant and soil surrounding the plant are treated with a taxol-producing P. microspora.
- the taxol sensitivity of the plant pathogen is determined by identifying Amino Acid 219, wherein the plant is designated as taxol-sensitive if Amino Acid 219 is threonine.
- Fig 1 is a graph depicting the effect of taxol on mycelial growth in P. microspora, P. ultimum, P. cinnamomi and A. klebsiana.
- Fungal mycelia were grown on potato dextrose agar (PDA) plates containing different concentrations of taxol The inhibitory effect of taxol was assessed by colony diameter, and compared to mycelia grown in the absence of taxol Experiments were conducted in duplicate, and data presented are an average of several experiments
- Fig 2 depicts the nucleotide and deduced amino acid sequence of beta-tubulin from P microspora Ne32 cDNA, TUBB-pm Numerals on the left indicate nucleotide position, and numerals on the right indicate amino acid position
- the sequences of the gene-specific primers NETUB5 and NETUB6 are underlined
- the translation initiation codon ATG is underlined
- the translation termination codon is marked by an asterisk
- the putative polyadenylation signal is double underlined
- Fig 3 depicts the nucleotide and deduced amino acid sequence of beta-tubulin from P. ultimum cDNA, TUBB-pu Numerals on the left indicate nucleotide position, and numerals on the right indicate amino acid position
- the sequences of the gene-specific primers WT1L-U and WT1L-L are underlined
- the translation initiation codon ATG is underlined
- the translation termination codon is marked by an asterisk
- the two putative polyadenylation signals are double underlined
- the arrow at nucleotide 1507 indicates the position of the poly (A) tract in the shorter 1537 bp cDNA
- Fig 4 depicts the nucleotide and deduced amino acid sequence of beta-tubulin from P cinnamomi cDNA
- TUBB-pc Numerals on the left indicate nucleotide position, and numerals on the right indicate amino acid position
- the sequences of the gene-specific primers PCBTUB 1U, PCBTUB2U and PCBTUB4L are underlined
- the translation initiation codon ATG is marked by ###
- Fig. 6 A and 6B depict the amino acid sequence alignment of beta-tubulins. The alignment was obtained using the ClustalW alignment program. The amino acid sequence of P. microspora beta-tubulin is shown in its entirety, and residues which differ in other beta-tubulins are shown below. Numerals on the right indicate amino acid positions. Sequences underlined indicate regions important for GTP binding (Amino Acids 63-77), phosphate binding (Amino Acids 140-146), and Mg 2+ binding (Amino Acids 203-206). Amino Acids 1-31 and 212-231 (denoted here as taxol binding region I and II, respectively) are indicated by a line above the sequence.
- Amino Acids Phe270, Leu273 and Ser364 are marked above with #.
- Amino acids which are important for fungal resistance to benzimidazoles (Amino Acids 6, 165, 167, 198, 200 and 241) are marked above by asterisks. Gaps in alignment are indicated by dashes, and the end of each sequence is marked by "$”.
- Genbank accession numbers for beta-tubulins from N. crassa, A. idulans benA, A. klebsiana and human ⁇ 2 are listed in Table I.
- the P. cinnamomi depicted is SEQ ID NO:6.
- Fig. 7A and 7B are graphs depicting the specific binding of [ 3 H]taxol to P. ultimum but not to P. microspora.
- Fig. 7A demonstrates that specific binding of [ 3 Hltaxol to P. ultimum increased as a function of [ H]taxol concentration, while P. microspora showed no or very little specific binding.
- Actively growing fungal cells were incubated with different concentrations of [ 3 H]taxol at room temperature for 2 hours before quenching. Specific binding was calculated as the difference between binding of [ 3 H]taxol in the presence and absence of a 100-fold excess of unlabeled taxol. Specific binding represents 30-70% of the total binding to P.
- Fig 8 depicts the amino acid sequences of the taxol binding region I (Amino Acids
- beta-tubulins from different organisms
- the amino acid sequences of the taxol binding regions I and II for pig beta-tubulin are shown in their entirety and residues which differ are shown for other beta-tubulins
- the taxol sensitivity of each organism is indicated, "s" for sensitive and “r” for resistant Amino Acids 15-25 and 212-222, which have been shown to be involved in taxol binding by both cross-linking and electron crystallography, are marked with asterisks
- the taxol binding region II of A are marked with asterisks The taxol binding region II of A.
- klebsiana is between Amino Acids 211-230 due to a gap in its sequence
- Pig beta-tubulin is described by Nogales, et al (Nogales, et al 1999 Nature 391 199-203), and Genbank accession numbers for other sequences are listed in Table I
- the sequence for P. cinnamomi presented herein is depicted in SEQ ID NO 6
- One aspect of the present invention is an isolated gene comprising an open reading frame coding for the protein beta-tubulin or a portion thereof
- the corresponding cDNA have been isolated and characterized for taxol-resistant Pestalotiopsis microspora Ne32, taxol-sensitive Pythium ultimum, and taxol-sensitive Pythium cinnamomi
- the nucleotide and deduced amino acid sequences of beta-tubulin for Pestalotiopsis microspora Ne32 are given in SEQ ID NO 1 and SEQ ID NO 2, respectively, for Pythium ultimum, in SEQ ID NO 3 and SEQ ID NO 4, respectively, and for Pythium cinnamomi, in SEQ ID NO 5 and SEQ ID NO 6, respectively
- SEQ ID NO 219 of beta-tubulin As numbered in SEQ ID NO 2, SEQ ID NO 4, or SEQ ID NO:
- the present invention is the beta-tubulin protein or protein fragments encoded by the novel genes disclosed herein. Since the P. ultimum and P. cinnamomi beta-tubulin proteins of the present invention are capable of binding taxol, proteins and protein fragments comprising taxol-binding sites derived from the genes coding for beta-tubulin described herein can be produced by heterologous expression in E. coli and other systems, purified by standard procedures, and used in an in vitro assay for detecting taxol and taxol-like substances by using methods well known in the art (Schiff, et al. 1979. Nature 277:665-667).
- beta-tubulin proteins of the present invention can be used to screen plant or fungal extracts as well as synthetic compounds for taxol or taxol-like substances as possible anticancer drugs.
- Beta-tubulins produced by making specific amino acid substitutions, deletions, or alterations can be used as experimental tools to further determine the molecular basis of taxol binding to the beta-tubulin protein.
- antibodies polyclonal or monoclonal raised against all or portions of the beta tubulins of the present invention can be used to determine if a composition of matter has taxol binding properties.
- antibodies capable of binding to taxol-sensitive beta-tubulin and/or taxol-resistant beta tubulins are exposed to a composition of matter prepared for in situ hybridization (Ausubel, et al. 1997. Current
- antibodies raised to a portion of SEQ ID NO:4 comprising Amino Acid 219 would bind to a beta-tubulin which had threonine at Amino Acid 219 but would not bind to a beta-tubulin having a different amino acid at Amino Acid 219, so that detectable binding would indicate the presence of threonine at Amino Acid 219, and hence, sensitivity to taxol.
- This type of assay is useful for screening a variety of compositions of matter, including living matter such as plants or microorganisms, or non-living matter such as plant materials, patient samples, or compound libraries for the presence of beta-tubulin.
- the present invention is a method of designing taxol analogs or other compounds which mimic the interaction of taxol with beta-tubulin based on the identification of specific amino acids in the beta-tubulins corresponding to taxol-binding and taxol-sensitivity.
- the previously reported three-dimensional structure (Nogales, et al. 1998. Nature 391 : 199-203) can be applied to developing and optimizing antineoplastic and antifungal compounds with respect to Amino Acid 219 and the surrounding area. Further, such information can also be used to generate mutant beta-tubulins with altered taxol sensitivity by substituting amino acids at specific positions in the beta-tubulin protein.
- the present invention is a method of generating isogenic strains of fungi using a gene of the present invention, which allows studies of taxol related pharmacology to be performed against a known background. Further, the present invention is a method of using these isogenic fungal strains, one of which is taxol sensitive and the other taxol resistant, to screen plant extracts, fungal extracts, extracts from other organisms, and synthetic compounds for taxol-like substances as possible anticancer agents. The present invention is also a method of using two unrelated fungal strains, one of which is taxol sensitive and the other taxol resistant, to screen plant extracts, fungal extracts, extracts from other organisms, and synthetic compounds for taxol-like substances as possible anticancer agents.
- Example 1 Differential taxol sensitivity in selected fungi Taxol sensitivity was established for the fungal strains used in the isolation of the beta-tubulin cDNAs of the present invention.
- Pestalotiopsis microspora strain Ne32 previously disclosed in U.S. Patent No. 5,861,302, was licensed from Montana State University. Pythium ultimum (ATCC 26083), Achlya klebsiana (ATCC 52605), and Pythium cinnamomi (ATCC 200982) were purchased from American Type Culture Collection (Manassas, VA) Taxol was obtained from Sigma Chemical Company (St Louis, MO)
- cinnamomi was inhibited even at low concentrations of taxol (IC50 0 1 ⁇ M) This sensitivity is comparable to the level of taxol (0 25 ⁇ M) that inhibits Hela cell division (Schiff, et al 1979 Nature 277 665-667)
- Beta-tubulin cDNA sequences were determined for P. microspora Ne32, P. ultimum, and P. cinnamomi from RNA isolated from fungal mycelia Automated dideoxynucleotide sequencing was performed by a contracting laboratory Sequence comparison was performed using the BLAST program at the Internet site of the National Center for Biotechnology Information The amino acid sequence alignment was performed using ClustalW program, and other analysis using Mac Vector program
- a forward degenerate primer BTUB 1 5'-CTGGGCYAAGGGYC AYTACACYGAG-3' (SEQ ID NO 7, was designed corresponding to amino acid residues Trp-Ala-Lys-Gly-His-Tyr-Thr-Glu (or WAKGHYTE in single letter amino acid code, SEQ ID NO 8), a reverse primer BTUB2, S'-CGAAGAARTGRARNCGRGGGAARGG-S' (SEQ ID NO 9), corresponding to amino acid residues Pro-Phe-Pro-Arg-Leu-His-Phe-Phe (or
- the resulting composite cDNA from P. microspora was 1668 bp long, designated as TUBB-pm, and its nucleotide and deduced amino acid sequence are shown in SEQ ID NO 1 and SEQ ID NO 2, respectively, as well as Fig 2
- This cDNA encodes a protein of 446 amino acids with a calculated Mr of 49,832 and pi of 4 6 It contains 74 nucleotides in the 5' untranslated region (UTR), and 229 nucleotides in the 3' UTR followed by a 24 nucleotide poly (A) tail
- a sequence AATAA nucleotides 1539- 1543 of SEQ ID NO 1 with the closest similarity to the animal and viral polyadenylation signal AATAAA (Proudfoot, N J and Brownlee, G G 1976 "3' Non-coding region sequences in eukaryotic messenger RNA," Nature 263 211-214) was located 103 bp upstream of the poly (A) tract
- RNA from mycelia grown for six days was used to synthesize first strand cDNA with oligo-dT primer (GibcoBRL, Gaithersburg, MD) in a twenty microliter (20 ⁇ l) reaction
- oligo-dT primer Gaithersburg, MD
- Two microliters (2 ⁇ l) of cDNA product were used as the template in PCR reactions with a cycling program similar to that described above
- Degenerate primers BTUB1 and BTUB4 generated a product of 1 0 kb
- BTUB1 and BTUB2 amplified a product of 0 5 kb
- the desired bands were gel-purified and ligated into the pPCRJ 1 vector Inserts were sequenced and used to design a gene-specific forward primer WT1L-U, 5 -CTAT
- first-strand cDNA was synthesized using primer WT1L-L and used as template in PCR reactions
- Primers WT1L-L and Cap-Switch generated a 0 95 kb product using Advantage- GC cDNA PCR kit (Clontech)
- Second strand cDNA was synthesized using CapFinder cDNA Library Construction kit (Clontech) With the resulting cDNAs as template, primers WTIL-U and CDS/3' (Clontech) generated two PCR products of 1 0 and 1 1 kb, respectively PCR fragments were gel-purified and cloned into the pPCRJ 1 vector In P.
- isolated tubulin cDNAs were of two types, one composite cDNA was 1650 bp long, and the other was 1537 bp long These two cDNAs differ only in the position at which the poly (A) tail has been added
- the region between 1 to 824 bp from the 5' RACE product was ligated at an internal Mfel site with the region between 825 to 1650 bp from the 1 1 kb 3' RACE product to form the composite 1650 bp cDNA designated as TUBB-pu, and its nucleotide and deduced amino acid sequence are shown in SEQ ID NO 3 and SEQ ID NO 4, respectively, as well as Fig 3
- This cDNA encodes a protein of 446 amino acids with a calculated Mr of 50,047 and pi of 4 6 It contains 91 nucleotides in the 5' UTR, and 199 nucleotides in the 3' UTR followed by a 19 nucleotide poly (A) tract Two imperfect polyadenylation signals were tentatively
- RNA from P. cinnamomi mycelia grown for 5 days was used to synthesize first-strand cDNA using oligo-dT primer in a 20 ⁇ l reaction
- One microliter (1 ⁇ l) of the cDNA product was used as template for PCR
- the cycling program comprised 30 cycles with an annealing temperature of 62°C Primer PCBTUBIU and PCBTUB4L generated an amplification product of 1 3 kb, and primer PCBTUB2U and PCBTUB4L generated an amplification product of 0 75 kb Primer PCBTUBIU or PCBTUB2U in combination with PCBTUB3L did not generate any product
- the desired bands were gel-purified using Geneclean (BIO101), ligated into the pPCRJ 1 vector (Invitrogen), and transformed into E.
- TUBB-pc are shown in SEQ ID NO 5 and SEQ ID NO 6, respectively, as well as Fig 4 It encodes a 444 amino acid long beta- tubulin protein, with a calculated Mr of 50 kDa, and a pi of 4 7 There are 10 nucleotides in the 5' untranslated region (UTR), and 5 nucleotides in the 3' UTR
- beta-tubulin from P. microspora SEQ ID NO 2
- P. ultimum SEQ ID NO 4
- P. cinnamomi SEQ ID NO 6
- FIG. 1 The deduced amino acid sequence of beta-tubulin from P. microspora (SEQ ID NO 2), P. ultimum (SEQ ID NO 4), and P. cinnamomi (SEQ ID NO 6) show features expected of beta-tubulin, as shown by an alignment with human ⁇ 2-tubulin (SEQ ID NO 24) and from beta-tubulins from Neurospora crassa (SEQ ID NO 25), A. mdulans benA (SEQ ID NO 26), and A.
- klebsiana depicted in Fig 6
- N-terminal amino Acids 1-205
- intermediate amino Acids 206-381
- C- terminal domains Nogales, et al 1998 Nature 391 199-203
- Their N-terminal domain contains conserved motifs important for GTP binding [Ala-Ile-Leu-Val-Asp-Leu-Glu-Pro- Gly-Thr-Met-Asp-Ser-Val-Arg or AILVDLEPGTMDSVR in single letter amino acid code (SEQ ID NO 28) and Ala-Val-Leu-Val-Asp-Leu-Glu-Pro-Gly-Thr-Met-Asp-Ser-Val-Arg or AVLVDLEPGTMDSVR in single letter amino acid code (SEQ ID NO 29) between Amino Acids 63-77 in SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO
- beta-tubulins from different organisms are well conserved and exhibit at least 63% identity (Oakley, B R 1994 "Gamma-tubulin " In Hyams JS, Lloyd CW (eds) Microtubules. Wiley-Liss, New York, pp 38-45) Table I shows the percentage identity between the beta-tubulin amino acid sequence of P. microspora and P. ultimum with beta-tubulins of other organisms
- the beta-tubulin from P. microspora shares the highest identity (93-97%) with filamentous ascomycetes such as A flavus, A. mdulans benA and N.
- beta-tubulin from P. ultimum shows the highest identity (96-97%) with beta-tubulin from two oomycetes, A. klebsiana and P. cinnamomi, but shares limited identity (71-78%) with beta-tubulin from ascomycetes
- the beta-tubulin from P. ultimum also shows relatively high identity (86-93%) with beta-tubulin from non- fungal organisms such as the green algae C reinhardtn, the protozoa T. thermophila, the slime mold Physarum polycephalum, and various animals
- beta-tubulin a The amino acid sequences of beta-tubulin were retrieved from Genbank or Swiss- Prot. Pairwise identity was performed either with ClustalW or BLAST program.
- ultimum beta-tubulin was generated with primers WT1L-U and WT1L-L.
- PCR products were gel-purified and labeled with ⁇ - 32 P dCTP using Ready-to-Go beads (Pharmacia, Piscataway, NJ) by random priming. Probes were purified with Micro Bio-Spin columns (Biorad).
- the beta-tubulin probe hybridized to a single band from genomic DNA digested with EcoK , Hindlll, or Sail, and two bands from BamHl digested sample.
- the beta-tubulin probe hybridized to a single band from genomic DNA digested with BamHl, Sail, Pvul, or Pstl, and two bands from EcoRI digested sample. The sizes of these fragments match those predicted from the restriction endonuclease map of the corresponding cDNA clones. Since beta-tubulin genes are typically highly conserved, these results show that both P. microspora and P.
- ultimum contain a single copy of the beta- tubulin gene, consistent with previous reports that fungi generally have one, or at most a few, beta-tubulin genes (Neff, et al. 1983. "Isolation of the ⁇ -tubulin gene from yeast and demonstration of its essential function in vivo, " Cell 33:211-219; Hiraoka, et al. 1984. "The NDA3 gene of fission yeast encodes ⁇ -tubulin: a cold sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis," Cell 39:349-358; Orbach, et al. 1986. "Cloning and characterization of the gene for ⁇ -tubulin from a benomyl- resistant mutant o ⁇ Neurospora crassa and its use as a dominant selectable marker," Moi
- RNA from mycelia grown for 2, 5, 6, or 11 days were converted to cDNA, and used as a template in PCR reactions Using gene-specific primers NETUB5 and NETUB6, a 413 bp beta-tubulin cDNA fragment was amplified from mycelia grown in log (2, 5 and 6 days) or stationary phase (11 days), but not from a control reaction that contained no template
- the values of total and nonspecific binding were determined by binding of [ 3 H]taxol to fungal cells in the absence or presence of 100-fold excess unlabeled taxol. The specific binding of [ 3 H]taxol was then calculated as the difference between the amount of total and nonspecific binding.
- Fresh mycelia from P. microspora Ne32, and P. ultimum were grown in 140 milliliters modified MID media in Roux bottles at 24°C for 1-2 days. These actively growing mycelia were transferred to 50 milliliter conical tubes, and centrifuged at 7,000 rpm for 5 minutes at room temperature. Mycelia were suspended in 1 milliliter remaining MID medium and 1 milliliter fresh MID medium. Cells were either untreated or pretreated with the anti-mitotic drug thiabendazole to depolymerize microtubules. In pretreated cells, thiabendazole (in DMSO) was added to desired concentrations, and DMSO was adjusted to the same concentration in all samples. Samples were then incubated at room temperature for 3 hours.
- [ 3 H]taxol (3.7 X 10 7 Bq/ml, Moravek) was added to desired concentrations either in the presence or absence of 100-fold excess unlabeled taxol. Samples were incubated for 2 hours at room temperature, then quenched on ice. [ 3 H]taxol binding to P. microspora cells was performed in the presence of 0.1% (v/v) Triton X-100 to disrupt the cell membrane.
- Each GFC filter (Whatman; Clifton, NJ) was weighed using an analytical balance.
- GFC filters were dried at 80°C in an oven overnight and then weighed to obtain mycelia dry weight. Filters were counted for 5 minutes under 20 milliliters of Cytoscint (Fisher Scientific; Pittsburgh, PA) in a Beckman LS3801 scintillation counter. Specific binding was calculated as the difference between [ 3 H]taxol bound in the presence and absence of a 100-fold excess unlabeled taxol. Nonspecific binding was determined as binding in the presence of 100-fold excess unlabeled taxol.
- [ 3 H]taxol was found to bind specifically to P. ultimum cells, and the amount of specific binding increased as a function of [ 3 H]taxol concentration (Fig. 7A).
- the specific binding of [ 3 H]taxol decreased in a dose-dependent manner (Fig. 7B).
- treatment with 1 mM of thiabendazole completely abolished the specific binding of [ 3 H]taxol.
- Taxol stabilizes MTs by binding to beta-tubulin in assembled MTs, and its binding site has been characterized by photo cross-linking, electron crystallography, and mutagenesis Regions between Amino Acids 1-31 and 217-231 were found to cross-link to the C-3' and C-2 group of taxol, respectively (Rao, et al 1994 JBiol Chem 269 3132-3134, and Rao, et al 1995 JBiol Chem 270 20235-20238) Recently, the structure of the beta- tubulin dimer was solved by electron crystallography of zinc induced sheets of tubulin dimer (Nogales, et al 1998 Nature 391 199-203) Modeling of taxol bound to this structure shows that the C-3' group of taxol is near Amino Acids 15-25 of beta-tubulin (near the top of helix HI), and the C-2 group is near Amino Acids 212-222 (near helix H6 and the loop between H6-H7)
- Beta-tubulins from taxol-sensitive organisms such as human, pig, Drosophila, Xenopus, Tetrahymena and Physarum are highly conserved in taxol binding region I and II, and are identical between Amino Acids 15-25 and 217-222 (except a conserved substitution at Amino Acid 23 in Drosophila ⁇ 1)
- Beta-tubulin from P. ultimum displays only four substitutions compared to the above sequences, none of which occurs between Amino Acids 15-25 and 217-222 This similarity is consistent with the fact that P.
- ultimum like the animal organisms noted above, is taxol-sensitive Also consistent with this, previous biochemical studies of animal tubulins and data of [ 3 H]taxol binding to P. ultimum demonstrated herein (Fig 7A and 7B), show that taxol binds beta-tubulin in assembled MTs of these organisms (Kellogg, et al 1989 J Cell Biol 109 2977-2991, and Manfredi, J J and Horwitz, S B 1984 Pharmacol Ther 25 83-125) Beta-tubulin sequences from P. ultimum and A. klebsiana are identical in taxol binding region I and II except Amino Acid 219, but A.
- klebsiana is relatively resistant to taxol (IC50 > 11 7 ⁇ M) This reduced sensitivity is due in part to the fact that A. klebsiana contains an asparagine at Amino Acid 219, whereas P. ultimum, and six other beta-tubulins from taxol-sensitive organisms, have threonine
- Beta-tubulins from taxol-resistant organisms such as P. microspora, A. mdulans and S. cerevisiae are similar to each other within taxol binding region I and II, but differ from the above discussed sequences in seven positions (19, 22, 23, 25, 218, 219, and 221) within regions 15-25 and 217-222
- the [ 3 H]taxol binding data presented herein (Fig 7A and 7B), together with previous biochemical studies (Yoon, Y and Oakley, B R 1995 Biochem 34 6373-6381, and Barnes, et al 1992 Moi Biol Cell 3 29-47), show that beta-tubulins in assembled MTs of these organisms are unable to efficiently bind taxol
- These sequences contain the asparagine (or glutamine in the case of S.
- Asn219 (asparagine at Amino Acid 219) or Glu219 (glutamine at Amino Acid 219).
- the taxol sensitivity of P. cinnamomi is consistent with the presence of Thr219 in TUBB-pc (SEQ ID NO:6) and not Asn219 as previously reported by Weerakoon et al. The presence of Asn219 (asparagine at Amino Acid 219) found in P.
- microspora is consistent with the taxol resistance of this species Using the information that the presence of threonine at Amino Acid 219 in beta-tubulins corresponds to taxol-binding and taxol-sensitivity, taxol analogs or other compounds can be designed which mimic the interaction of taxol with beta- tubulin. Further, such information can also be used to generate mutant beta-tubulins resistant to taxol by substituting the threonine for another amino acid residue at Amino Acid 219.
- Example 6 Sensitivity to microtubule-depolymerization drugs.
- Colchicine, colcemid, nocodazole, and thiabendazole were obtained from Sigma Chemical Company (St. Louis, MO).
- a stock solution of colchicine was prepared in water, and other stock solutions in DMSO.
- An agar plug (6 mm in diameter) of fresh mycelia was transferred onto PDA plates containing 1 % (v/v) DMSO in the presence or absence of an anti-microtubule agent.
- Fungal colonies were grown at 24°C for 24 hours in the case of P. ultimum or 48 hours in the case of P. microspora and A. klebsiana. The growth inhibitory effect of these anti-mitotic agents was measured by the size of colony diameters.
- beta-tubulin from P. ultimum and A klebsiana differ at Amino Acids 165, 167 and 200 It has been previously shown that a phenylalanine-to- tyrosine change at Amino Acid 167 results in benzimidazole resistance in N. crassa
- Monoclonal or polyclonal antibodies can be raised against the following antigens 1) native beta-tubulins extracted from P. microspora, P. ultimum, or P. cinnamomi, 2) beta- tubulins of P. microspora, P. ultimum, or P. cinnamomi produced from a heterologous system such as E.
- the antibodies are used to interact with the above mentioned beta-tubulins using Elisa or Western blotting using standard protocols (Harlow, E D and Lane, D 1988 Antibodies: A Laboratory Manual)
- the antibodies which could distinguish the taxol binding beta-tubulin from the taxol non-binding beta-tubulin are selected as the reagent
- a specific example is to raise polyclonal or monoclonal antibodies to synthetic peptides corresponding to SEQ ID NO 4 or SEQ ID NO 6 which comprise at least one taxol binding region, for instance containing the taxol-binding region II comprising Thr219 or in which the Thr219 is replaced by Asn219/Gln219
- the ability of these antibodies to interact with beta-tubulin is examined using Elisa using standard protocols
- the antibody which can binds to peptide containing Thr219 but not to peptide containing Asn/Gln 219 is selected as the reagent which is specific for the taxol-binding site containing Thr 219.
- the antibody which specifically binds to the peptide containing Asn219/Gln 219 but not to the peptide containing Thr 219 is selected as the reagent which specifically recognizes taxol binding site devoid of Thr 219.
- Example 8 Screening Assays to Detect Beta-Tubulin in Matter
- compositions of matter contains beta- tubulin capable of binding taxol.
- assays are useful for screening a variety of compositions of matter, including living matter such as plants or microorganisms, or nonliving matter such as plant materials or patient samples for the presence of beta-tubulin.
- the first assay is performed using Northern or Southern hybridization method well known in the art (Sambrook, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
- the total RNA, mRNA or genomic DNA are isolated from the composition of matter and separated by electrophoresis.
- DNA, synthetic oligonucleotide, or RNA corresponding to the coding region or a portion of beta-tubulin (e.g., derived from SEQ ID NOJ, SEQ ID NO: 3 or SEQ ID NO:5) which comprises at least one taxol binding region will be used to synthesize isotopically labeled probes.
- Hybridization with a probe derived from SEQ ID NO: 1 will indicate beta-tubulin with high probability of taxol resistance.
- the hybridization with a probe derived from SEQ ID NOJ or SEQ ID NO: 5 will indicate beta-tubulin with a high probability of taxol sensitivity.
- the second assay is to use a PCR-based assay using standard protocols (Sambrook, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). Both genomic DNA or cDNA converted from total RNA or mRNA are used as template in a PCR assay. Gene-specific or degenerate primers corresponding to the coding region of beta-tubulin (e.g., derived from SEQ ID NO: 1, SEQ ID NOJ or SEQ ID NO:5) which comprises at least one taxol binding region will be synthesized. Only DNA containing the appropriate primer sequences will be amplified, and all other variations will be suppressed.
- beta-tubulin e.g., derived from SEQ ID NO: 1, SEQ ID NOJ or SEQ ID NO:5
- the amplification of PCR fragment of the predicted size using primers derived from SEQ ID NOJ or SEQ ID NO:5 but not from primers derived from SEQ ID NO: 1 will indicate high probability of taxol binding beta-tubulin.
- the amplification of a PCR fragment of the predicted size using primers derived from SEQ ID NO: 1 but not from primers derived from SEQ ID NOJ or SEQ ID NO:5 will indicate high probability of taxol non-binding beta-tubulin.
- the subsequent obtaining of the beta-tubulin sequence and examination of the presence or absence of Thr219 residue will provide further determination.
- the third assay is to use Elisa or Western blotting using standard protocols (Harlow, E.D. and Lane, D. 1988. Antibodies: A Laboratory Manual).
- Cell extracts of the composition of matter are prepared.
- Synthetic peptide, or native beta-tubulins extracted from P. microspora, P. ultimum, or P. cinnamomi, or produced from a heterologous system such as E. coli, yeast, and insect cells will be used to raise polyclonal or monoclonal antibodies.
- the antibodies will be used in the above mentioned Elisa or Western blotting.
- the antibody which recognizes the taxol binding from the non taxol binding is used in these assays.
- Example 9 Construction of Taxol-sensitive and Taxol-resistant Isogenic Strains
- P. ultimum contains a single beta-tubulin.
- its beta-tubulin gene or cDNA can be altered to change the Thr219 to a different residue, for instance to Asn219 or Gln219.
- This altered DNA sequence is cloned into a transformation vector, and used to transform the wild-type strain P. ultimum using established protocols (Balance, et al. 1985. Gene 36:321- 331). Homologous recombination between the wild-type beta-tubulin gene and the modified beta-tubulin in the vector occur. Transformed fungus are selected on media containing taxol.
- the taxol-resistant clones are selected and their beta-tubulin cDNA sequenced to confirm the absence of Thr 219.
- the taxol-resistant isogenic strain of P. cinnamomi is similarly constructed and used in screening assays as described in later examples. The only difference between these isogenic strains is that the taxol-sensitive strain is capable of binding to taxol due to the presence of Thr 219, and the taxol-resistant strain is incapable of binding to taxol due to the absence of Thr 219.
- Such taxol-resistant strains can be used in combination with the wild-type taxol-sensitive strains for screening as described in later examples.
- Example 10 Screening Assays to Detect Taxol or Taxol-like Compounds in Matter
- compositions of matter can be used to detect taxol or taxol-like compounds in a composition of matter. These assays are useful for screening a variety of compositions of matter, including living matter such as plants or microorganisms, or non-living matter such as plant materials, patient samples, or compound libraries for the presence of taxol or taxol-like compounds.
- Taxol inhibits the growth of both P. ultimum by binding to their beta-tubulin, while taxol does not affect the growth of P. microspora since it does not interact with its beta-tubulin.
- a composition of matter which is capable of the inhibition of P. ultimum, but not P. microspora has a high probability of containing taxol-or a taxol-like compound.
- An improved screening method uses taxol-sensitive and taxol-resistant isogenic strains of P. ultimum or P. cinnamomi as described in above example.
- the composition of matter is used to examine its effect on the growth of both the taxol-sensitive as well as the taxol-resistant strains.
- the inhibition of the taxol-sensitive strain but not the taxol-resistant strain indicates the presence of taxol or a taxol-like compound.
- the non- inhibition of both the taxol-sensitive and taxol-resistant strains indicates the absence of taxol or a taxol-like compound.
- composition of matter can be screened for the presence of taxol or taxol-like compounds based on their ability to promote the assembly of microtubules, as well as to stabilize assembled microtubules in conditions such as cold which otherwise cause depolymerization (Schiff, et al. 1979; Horwitz, 1981).
- the alpha- and beta-tubulins used in these assays can be from the following sources. 1) native microtubules consisting of beta- tubulins and alpha-tubulins extracted from P. ultimum or P. cinnamomi; 2) beta-tubulins extracted from P. ultimum or P.
- composition matter has the ability to promote the assembly of these MTs, as well as to prevent depolymerization of assembled MTs in conditions which otherwise cause depolymerization, the composition of matter is likely to contain taxol or a taxol-like compound. Meanwhile, these isolated compounds should be unable to promote the assembly of MTs as well as prevent the depolymerization of MTs which consist of beta-tubulin derived from P. microspora.
- An alternative screening method can be performed based on the competitive inhibition of [ 3 H]taxol binding to MTs in P. ultimum or P. cinnamomi by taxol or taxol-like compounds.
- the specific binding of [ 3 H]taxol to P. ultimum is performed as described in Example 5.
- the amount of [ 3 H]taxol specifically bound to P. ultimum in the absence of inhibitors is considered 100%.
- the composition of matter is added to the assay mixture, and the amount of [ 3 H]taxol specifically bound to P. ultimum in the presence of the composition of matter is measured. Reduction in the [ 3 H]taxol specific binding indicates that the composition of matter possesses taxol-like quality. If increased concentrations of the composition of matter can completely inhibit the [ 3 H]taxol binding, it will indicate that the compound likely binds to the same binding site in the beta-tubulin in MTs.
- compositions of matter for taxol or taxol-like compounds can be performed by one of the above methods.
- one of the first two methods is used for an initial screening, since they are simple to perform and easily handle large amounts of samples.
- the third and fourth method can be used for subsequent screening.
- Example 7 antibodies depicted in Example 7 which could distinguish taxol-binding beta-tubulin from the non-binding beta-tubulin are used.
- Cellular proteins are extracted from a tumor specimen from a patient sample to detect the presence of a beta- tubulin with either taxol-binding or non-binding capabilities.
- the taxol binding regions of taxol-sensitive and taxol-resistant beta-tubulins of the present invention are provided.
- SEQ ID NOJ SEQ ID NO:4, and SEQ ID NO:6
- SEQ ID NOJ SEQ ID NO:4
- SEQ ID NO:6 SEQ ID NO: 6
- monoclonal antibody probes are reacted with a patient sample, such as a tumor specimen, to detect the presence of a beta- tubulins with either taxol-binding or non-binding capabilities.
- Visualization of antibody- antigen binding is mediated through any means known in the art, e.g., secondary radiolabeled or fluorescent antibodies or colorimetric methods using peroxidase and/or alkaline phosphatase (Harlow, E.D. and Lane, D.
- beta-tubulins with taxol-binding capability i.e., taxol-sensitive beta- tubulins
- taxol-binding capability i.e., taxol-sensitive beta- tubulins
- non-binding taxol-resistant beta-tubulins and/or the absence of taxol-sensitive beta-tubulins corresponds to a diminished or lack of response to taxol therapy.
- P. ultimum and P. cinnamomi are plant pathogens which can cause crop damage and result in severe economical loss.
- P. ultimum causes root rot of beans
- P. cinnamomi causes root rot of Avacado (ATCC: Catalogue of Filamentous Fungi, 18th edition, 1991).
- Many of the oomycetes are also taxol-sensitive (Young, et al. 1992. "Antifungal properties of taxol and various analogues," Experientia 48:882-885). Two of these strains, P. ultimum and P. cinnamomi, contain threonine at Amino Acid 219.
- the biocontrol method of the present invention involves a two-step process: 1) the taxol sensitivity of the plant pathogen is determined and 2) if the plant pathogen is taxol- sensitive, a taxol-producing P. microspora is applied to the infected plants and surrounding soil as a source of growth-inhibiting taxol.
- the taxol sensitivity of the plant pathogen is first determined.
- One method of identifying taxol sensitivity is to determine the presence or absence of threonine at Amino Acid 219. If the identity of the pathogen is known, DNA and protein databases are searched to determine whether the beta-tubulin sequence has been reported, if so, the identity of Amino Acid 219 is determined from the database. If the pathogen's beta-tubulin sequence is unavailable, the cDNA sequence is isolated and analyzed to determine the identity of Amino Acid 219. The presence of threonine at Amino Acid 219 in the pathogen's beta-tubulin gene indicates sensitivity to taxol, and thus, the pathogen is designated as treatable by a taxol- producing P. microspora.
- Taxol sensitivity would have to be determined by other means such as taxol growth inhibition.
- Other screening methods presented herein for determining the presence of taxol-binding beta-tubulins can also be used.
- Example 13 Use of Crystal Structures in Design of Antineoplastic or Antifungal Drugs
- beta-tubulins are used to rationally design taxol- like compounds using methods known in the art (Ealick, et al. 1991. "Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors," Science 88: 11540-11544; Rossman, et al. 1991. “Application of crystallography to the design of antiviral agents,” Infectious Agents and Disease 1 :3-10).
- application of the knowledge that Thr219 in the protein structure plays an important role in binding of taxol to taxol-like compounds can be critically applied to the development of drugs having taxol-like activities.
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Abstract
The anticancer drug taxol binds to beta-tubulin in assembled microtubules (MT) and causes cell cycle arrest in animal cells; in contrast, the effect of taxol varies in fungi. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (1C50⊃11.7 M), whereas Pythium ultimum and Phytophthora cinnamomi, two oomycetes, are sensitive to taxol (IC50 0.1 νM). cDNAs encoding beta-tubulin from P.microspora, P. ultimum, and P. cinnamomi were isolated. The deduced amino acid sequences of beta-tubulin from P. microspora, P.ultimum, and P. cinnamomi can be used in (1) binding assays for the detection of taxol and taxol-like substances; (2) diagnostic assays for the pharmacologic efficacy of taxol against a tumor sample; (3) designing drugs with taxol-like activity via application of information regarding the effect of specific residues on taxol binding; and (4) detection of taxol and taxol-like activity via use of taxol-sensitive and taxol-resistant isogenic strains of P.ultimum constructed by substitution of residues necessary for taxol binding.
Description
FUNGAL BETA-TUBULIN GENES
TECHNICAL FIELD OF THE INVENTION
The invention relates to a bio-affecting composition and to a biological diagnostic and experimental agent.
BACKGROUND OF THE INVENTION TAXOL® (Bristol-Myers-Squibb), generically known as paclitaxel (hereinafter referred to as "taxol"), is a complex diterpenoid which has demonstrated anti-tumor activity against breast and ovarian cancer (Rowinsky, E.K. and Donehower, R.C. 1991. "The clinical pharmacology and use of antimicrotubule agents in cancer chemotherapeutics," Pharmacol Ther 52:35-84). Its anti-tumor activity is due to its ability to bind to beta-tubulin in assembled microtubules (MTs) and stabilize them (Manfredi, J.J. and Horwitz, S.B. 1984. "Taxol: an antimitotic agent with a new mechanism of action," Pharmacol Ther 25:83-125; and Horwitz, S.B. 1992. "Mechanism of action of taxol, " Trends Pharmacol Sci 13: 134- 136 In vivo, taxol affects spindle function during mitosis, resulting in cell cycle arrest in G2/M phase. In vitro, taxol promotes MT assembly and prevents their disassembly under conditions which would otherwise cause depolymerization (Schiff, et al. 1979. "Promotion of microtubule assembly in vitro by taxol" Nature 277:665-667; and Pamess, J. and Horwitz, S.B. 1981 "Taxol binds to polymerized tubulin in vitro," JCellBiol 91 :479-487). The taxol binding site on beta-tubulin has been characterized by photo cross-labeling, electron crystallography, and mutagenesis, and involves several regions of beta-tubulin (Rao, et al. 1994. 'J'-(p-Azidobenzamido) taxol photolabels the N-terminal 31 amino acids of β- tubulin," JBiol Chem 269:3132-3134; Rao, et al. 1995. "Characterization of the taxol binding site on the microtubule," JBiol Chem 270:20235-20238; Nogales, et al. 1998. "Structure of the αβ tubulin dimer by electron crystallography," Nature 391 : 199-203; Nogales, et al. 1999. "High-resolution model of the microtubule," Cell 95:79-88; and Giannakakou, et al. 1997. "Paclitaxel-resistant human ovarian cancer cells have mutant β- tubulins that exhibit impaired paclitaxel-driven polymerization," JBiol Chem 272: 17118- 17125).
Taxol was found originally in the inner bark of pacific yew trees (Taxus brevifoha) by Wani et al (Warn, et al 1971 "Plant antitumor agents VI The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifoha, " J Am Chem Soc 93 2325-2327), and noted to constitute about 0 02% of dry phloem weight The limited resource of yew trees made it advantageous to locate additional sources for taxol
In 1993, Stierle et al reported the isolation of a taxol-producing fungus, Taxomyces andreanae, an endophyte associated with T. brevifoha (Stierle, et al 1993 "Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of pacific yew," Science 260 214-216) T andreanae produces authentic taxol, though at very low levels (24-50 ng/liter of liquid culture) Recently, additional taxol-producing fungi have been isolated, including various strains of Pestalotiopsis microspora (Li, et al 1996 "Endophytic taxol- producing fungi from bald cypress, Taxodium distichum, " Microbiolog 142 223-226, Li, et al 1998 "The induction of taxol production in the endophytic fungus-Periconia sp from Torreya grandifoha, " J Ind Microbiol Biotechnol 20 259-264, Strobel, et al 1996 "Taxol from fungal endophytes and the issue of biodiversity," J Ind Microbiol 17 417-423, and
Strobel, et al 1996 "Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana, "Microbiolog 142 435-440) One P. microspora strain, Ne32, was isolated from the inner bark of Himalayan yew T. wallachiana, and produces approximately 50 μg taxol per liter of liquid culture (Strobel, et al 1996 Microbiolog 142 435-440, and Li, et al 1998 "Stimulation of taxol production in liquid culture of Pestalotiopsis microspora, " Mycol Res 102 461-464) The taxol produced from fungal sources has been reported to be spectroscopically and chromatographically identical to taxol isolated from yew trees, and has shown similar pharmacological effects on cancer cell lines (Strobel, et al 1996 Microbiolog 142 435-440) The production of taxol from fungal sources has provided broader resources of taxol, reduced production costs, and a means of meeting the increasing demand for taxol
While taxol has been shown to be toxic to cells from a wide range of organisms including mammals, sea urchin, Drosophila, Xenopus, Physarum, Haemanthus, and Trypanosoma (Baum, et al 1981 "Taxol, a microtubule stabilizing agent, blocks the replication of Trypanosoma cruzi, " Proc Natl Acad Sci USA 78 4571 -4575, Kellogg, et al 1989 "Proteins in the centrosome, spindle, and kinetochore of the early Drosophila
embryo, " J Cell Biol 109 2977-2991, and Manfredi, J J and Horwitz, S B 1984 Pharmacol Ther 25 83-125), variable sensitivity to taxol has been reported in fungi Young et al tested taxol toxicity on representative species from different fungal groups (Young, et al 1992 "Antifungal properties of taxol and various analogues," Experientia 48 882-885) In Young's study, five oomycete species were identified as sensitive to taxol (IC50 0 4-5 9 μM), including the plant pathogens Pythium ultimum and Phytophthora capsici In P. capsici, taxol inhibited nuclear division at low concentrations, indicating that it acts through a mechanism similar to that in mammalian cells In contrast, four ascomycete species were identified as resistant to taxol (IC50 > 50 μM). This resistance was reported to be due to the reduced ability of fungal microtubules to interact with taxol Taxol was also shown to be unable to stabilize MTs assembled with purified S. cerevisiae tubulin (Barnes, et al 1992 "Yeast proteins associated with microtubules in vitro and in vivo, " MolBiol Cell 3 29-47) and only weakly stabilize MTs from Aspergillus mdulans (Yoon, Y and Oakley, B R 1995 "Purification and characterization of assembly-competent tubulin from Aspergillus mdulans," Biochem 34 6373-6381;.
Because of the anticancer properties of taxol and the variability of fungi to taxol sensitivity, there is a continuing need for isolating and/or identifying novel beta-tubulin genes useful for developing isogenic fungal strains that are either taxol-sensitive or taxol- resistant These beta-tubulin genes and/or isogenic fungal strains can then be applied to anticancer drug screening and for developing diagnostic tests for tumor sensitivity assays
SUMMARY OF THE INVENTION
In one aspect, the invention is a purified DNA segment encoding a beta-tubulin of the fungal species Pestalotiopsis microspora or a portion thereof Preferably, the DNA segment encodes at least one taxol binding site For some uses, it is preferable that the DNA segment encodes a protein having taxol binding site I and taxol binding site II For DNA segments that encode proteins which function as beta-tubulins, the DNA segment encodes a protein which has taxol binding site I and taxol binding site II and is able to interact with alpha-tubulin An exemplary DNA segment comprises at least a portion of SEQ ID NO 1 Another exemplary DNA segment comprises a portion of SEQ ID NO 1 comprising the nucleotide sequence from nucleotide 75 through nucleotide 167 of SEQ ID NO 1, with or without substitution Another exemplary DNA segment comprises a portion of SEQ ID
NOJ comprising the nucleotide sequence from nucleotide 708 through nucleotide 764 of SEQ ID NOJ, with or without substitution. Another exemplary DNA segment comprises a portion of SEQ ID NOJ comprising the nucleotide sequence from nucleotide 708 through nucleotide 764 of SEQ ID NOJ, wherein either nucleotide 729, nucleotide 730 or nucleotide 731 or mixtures thereof are substituted. Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 75 to nucleotide 1412 of SEQ ID NOJ wherein the DNA segment encodes a beta-tubulin. Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 75 to nucleotide 1412 of SEQ ID NOJ wherein at least one nucleotide in the nucleotide sequence is substituted and wherein the taxol binding capacity of the beta-tubulin is not altered. Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 75 to nucleotide 1412 of SEQ ID NOJ wherein at least one nucleotide in the nucleotide sequence is substituted and wherein the taxol binding capacity of the beta-tubulin is altered.
In another aspect, the invention is an amino acid sequence comprising at least a portion of a beta-tubulin of the fungal species Pestalotiopsis microspora. Preferably, the amino acid sequence comprises at least one taxol binding site. For some uses, it is preferable that the amino acid sequence is a protein having taxol binding site I and taxol binding site II. For amino acid sequences that can function as beta-tubulins, the amino acid sequence has taxol binding site I and taxol binding site II and is able to interact with alpha- tubulin. An exemplary amino acid sequence comprises at least a portion of the beta-tubulin as depicted in SEQ ID NOJ. Another exemplary amino acid sequence comprises a portion of SEQ ID NOJ comprising Amino Acids 1-31, with or without substitution. Another exemplary amino acid sequence comprises a portion of SEQ ID NOJ comprising Amino Acids 212-230, with or without substitution. Another exemplary amino acid sequence comprises a portion of SEQ ID NOJ comprising Amino Acids 212-230 with an amino acid substitution at Amino Acid 219. Another exemplary amino acid sequence comprises a portion of SEQ ID NOJ consisting essentially of Amino Acids 1-446 wherein the portion behaves as a taxol-resistant beta-tubulin. Another exemplary amino acid sequence comprises a portion of SEQ ID NOJ consisting essentially of Amino Acids 1-446 the portion contains at least one amino acid substitution that alters the taxol binding property of the portion. Another exemplary amino acid sequence comprises a portion of SEQ ID NOJ consisting essentially of Amino Acids 1-446 the portion contains at least one amino acid
substitution that does not alters the taxol binding property of the portion. Another exemplary amino acid sequence is substituted with any amino acid which perturbs the three- dimensional structure of the amino acid sequence surrounding Amino Acid 219 as numbered in SEQ ID NOJ.
In another aspect, the invention is a purified DNA segment encoding a beta-tubulin of the fungal species Pythium ultimum or a portion thereof. Preferably, the DNA segment encodes at least one taxol binding site. For some uses, it is preferable that the DNA segment encodes a protein having taxol binding site I and taxol binding site II. For DNA segments that encode proteins which function as beta-tubulins, the DNA segment encodes a protein which has taxol binding site I and taxol binding site II and is able to interact with alpha- tubulin. An exemplary DNA segment comprises at least a portion of SEQ ID NOJ. Another exemplary DNA segment comprises a portion of SEQ ID NOJ comprising nucleotide 92 through nucleotide 184, with or without substitution. Another exemplary DNA segment comprises a portion of SEQ ID NOJ comprising the nucleotide sequence from nucleotide 725 through nucleotide 781, with or without substitution. Another exemplary DNA segment comprises a portion of SEQ ID NOJ comprising the nucleotide sequence from nucleotide 725 through nucleotide 781, wherein either nucleotide 746, nucleotide 747 or nucleotide 748 or mixtures thereof are substituted. Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 92 to nucleotide 1429 of SEQ ID NOJ, wherein the DNA segment encodes a beta-tubulin. Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 92 to nucleotide 1429 of SEQ ID NOJ with at least one nucleotide substitution in the nucleotide sequence and wherein the taxol binding capacity of the beta-tubulin is not altered. Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 92 to nucleotide 1429 of SEQ ID NOJ with at least one nucleotide substitution in the nucleotide sequence and wherein the taxol binding capacity of the beta-tubulin is altered.
In another aspect, the invention is an amino acid sequence comprising at least a portion of a beta-tubulin of the fungal species Pythium ultimum. Preferably, the amino acid sequence comprises at least one taxol binding site. For some uses, it is preferable that the amino acid sequence is a protein having taxol binding site I and taxol binding site II. For amino acid sequences that can function as beta-tubulins, the amino acid sequence has taxol
binding site I and taxol binding site II and is able to interact with alpha-tubulin. An exemplary amino acid sequence comprises at least a portion of the beta-tubulin as depicted in SEQ ID NO:4. Another exemplary amino acid sequence comprises a portion of SEQ ID NO:4 comprising Amino Acids 1-31, with or without substitution. Another exemplary amino acid sequence comprises a portion of SEQ ID NO:4 comprising Amino Acids 212- 230, with or without substitution Another exemplary amino acid sequence comprises a portion of SEQ ID NO:4 comprising Amino Acids 212-230, wherein the amino acid at Amino Acid 219 is substituted. Another exemplary amino acid sequence comprises a portion of SEQ ID NO:4 consisting essentially of Amino Acids 1-446 and wherein the portion behaves as a taxol-sensitive beta-tubulin. Another exemplary amino acid sequence comprises a portion of SEQ ID NO:4 consisting essentially of Amino Acids 1-446 having at least one amino acid substitution that alters the taxol binding property of the portion. Another exemplary amino acid sequence comprises a portion of SEQ ID NO:4 consisting essentially of Amino Acids 1-446 having at least one amino acid substitution that does not alter the taxol binding property of the portion. Another exemplary amino acid sequence is substituted with any amino acid which perturbs the three-dimensional structure of the amino acid sequence surrounding Amino Acid 219 as numbered in SEQ ID NO:4.
In another aspect, the invention is a purified DNA segment encoding a beta-tubulin of the fungal species Phytophthora cinnamomi or a portion thereof, wherein the DNA segment consists essentially of at least a portion of SEQ ID NO:5. An exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising the nucleotide sequence from nucleotide 11 through nucleotide 103. Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising the nucleotide sequence from nucleotide 11 through nucleotide 103, wherein at least one nucleotide in the nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code nucleotide 80 cannot consist of adenine while nucleotide 81 is thymine and nucleotide 82 is adenine, cytosine or thymine. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5 comprising nucleotide sequence from nucleotide 644 through nucleotide 700. Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising nucleotide sequence from nucleotide 644 through nucleotide 700, wherein at least one nucleotide in the nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code nucleotide 665 cannot be adenine while
nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine. Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising the nucleotide sequence from nucleotide 11 to nucleotide 1342 and wherein the DNA segment encodes a beta- tubulin. Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising the nucleotide sequence from nucleotide 1 1 to nucleotide 1342, wherein at least one nucleotide in the nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code, nucleotide 665 cannot be adenine while nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5 comprising the nucleotide sequence from nucleotide 11 to nucleotide 1342, wherein at least one nucleotide in the nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code, nucleotide 665 cannot be adenine while nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine, and wherein the taxol binding capacity of the beta-tubulin is not altered. Another exemplary DNA segment comprises a portion of SEQ ID NO:5 comprising the nucleotide sequence from nucleotide 11 to nucleotide 1342, wherein at least one nucleotide in the nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code, nucleotide 665 cannot be adenine while nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine, and wherein the taxol binding capacity of the beta-tubulin is altered.
In another aspect, the invention is an amino acid sequence comprising at least a portion of a beta-tubulin of the fungal species Phytophthora cinnamomi as depicted in SEQ ID NO:6. An exemplary amino acid sequence comprises a portion of SEQ ID NO:6 comprising Amino Acids 1-31. An exemplary amino acid sequence comprises a portion of SEQ ID NO:6 comprising Amino Acids 1-31, having at least one amino acid substituted, providing that when only one amino acid is substituted Amino Acid 24 is not isoleucine. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 6 comprising Amino Acids 212-230. Another exemplary amino acid sequence comprises a portion of SEQ ID NO:6 comprising Amino Acids 212-230, having at least one amino acid substituted, providing that when only one amino acid is substituted Amino Acid 219 is not asparagine. Another exemplary amino acid sequence comprises a portion of SEQ ID NO:6 comprising Amino Acids 212-230 with an amino acid substitution at Amino Acid 219, wherein the Amino Acid 219 is not substituted with asparagine. Another exemplary amino acid
sequence comprises a portion of SEQ ID NO 6 consisting essentially of Amino Acids 1-446, wherein the portion behaves as a taxol-sensitive beta-tubulin Another exemplary amino acid sequence comprises a portion of SEQ ID NO 6 consisting essentially of Amino Acids 1-446, wherein the portion contains at least one amino acid substitution, providing that when only one amino acid is substituted Amino Acid 219 is not asparagine, and wherein the amino acid substitution alters the taxol binding property of the portion Another exemplary amino acid sequence comprises a portion of SEQ ID NO 6 consisting essentially of Amino Acids 1-446, wherein the portion contains at least one amino acid substitution, providing that when only one amino acid is substituted Amino Acid 219 is not asparagine, and wherein the amino acid substitution does not alter the taxol binding property of the portion Another exemplary amino acid sequence is substituted at Amino Acid 219 with any amino acid except asparagine which perturbs the three-dimensional structure of the amino acid sequence surrounding Amino Acid 219
In another aspect, the invention is a vector comprising a purified DNA segment encoding a beta-tubulin of the fungal species Pestalotiopsis microspora or a portion thereof Preferably, the vector comprises a portion encoding at least one taxol binding site
In another aspect, the invention is a vector comprising a purified DNA segment encoding a beta-tubulin of the fungal species Pythium ultimum or a portion thereof Preferably, the vector comprises a portion encoding at least one taxol binding site
In another aspect, the invention is a vector comprising a purified DNA segment encoding a beta-tubulin of the fungal species Phytophthora cinnamomi wherein the DNA segment consists essentially of SEQ ID NO 5 or a portion thereof Preferably, the vector comprises a portion encoding at least one taxol binding site
In another aspect, the invention is a method of determining the taxol binding capacity of a beta-tubulin or beta-tubulin-like compound comprising providing antibodies raised against amino acid sequences comprising at least one taxol binding site of a beta- tubulin from a taxol-resistant Pestalotiopsis microspora, a taxol-sensitive Pythium ultimum, or taxol-sensitive Phytophthora cinnamomi as depicted in SEQ ID NO 6 to form a reagent, such antibodies distinguishing between taxol-binding and non-taxol-binding properties, contacting the beta-tubulin or beta-tubulin-like compound with the reagent, and determining
degree of binding between the antibodies in the reagent and the beta-tubulin or beta-tubulin- like compound; whereby binding of antibodies raised against a taxol-resistant Pestalotiopsis microspora to the beta-tubulin or beta-tubulin-like compound indicates taxol resistance and whereby binding of antibodies which specifically recognize taxol-binding properties indicate taxol sensitive; whereby binding of antibodies which specifically recognize taxol-non- binding properties indicate taxol resistance. In one embodiment, the antibodies in the reagent are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol-resistant Pestalotiopsis microspora. In another embodiment, the antibodies in the reagent are raised against an amino acid sequence comprises at least one taxol binding site from SEQ ID NOJ. In another embodiment, the antibodies in the reagent are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol-sensitive Phythium ultimum. In another embodiment, the antibodies in the reagent are raised against an amino acid sequence comprises at least one taxol binding site from SEQ ID NO:4. In another embodiment, the antibodies in the reagent are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol-sensitive Phytophthora cinnamomi as depicted in SEQ ID NO:6. In another embodiment, the antibodies in the reagent are raised against an amino acid sequence comprises at least one taxol binding site from SEQ ID NO:678. In this method, the beta-tubulin or beta-tubulin-like compound are selected from the group consisting of recombinantly expressed protein, exogenously isolated protein, synthetic peptides, and cell cultures.
In another aspect, the invention is a method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing beta-tubulins with amino acid sequences comprising both taxol binding sites from Pythium ultimum or taxol- sensitive Phytophthora cinnamomi as depicted in SEQ ID NO: 6 in addition to alpha-tubulin from any taxol-sensitive organism to form a reagent; contacting the composition of matter with the reagent; and determining the ability of the composition of matter to promote MT assembly or ability to prevent depolymerization of assembled MTs under depolymerizing conditions;whereby the ability to promote microtubule assembly or prevent depolymerization indicate the possible presence of taxol or taxol-like compounds in the composition of matter.
In another aspect, the invention is a method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing mycelia of taxol- sensitive Pythium ultimum or a taxol-sensitive Phytophthora cinnamomi which harbors beta- tubulin in SEQ ID NO:6; contacting the composition of matter with the mycelia in the presence of the labeled taxol; and determining the degree of competitive inhibition of binding between the beta-tubulins and the labeled taxol by the composition of matter, whereby the composition of matter is determined to possess taxol or taxol-like compounds if it is able to block labeled taxol binding to the beta-tubulins from the taxol-sensitive Pythium ultimum or Phytophthora cinnamomi.
In another aspect, the invention is a method of altering the taxol binding property of a recombinantly expressed beta-tubulin or a portion thereof comprising determining the identity of the codon at Amino Acid 219 as numbered in SEQ ID NOJ in the coding region of the vector; and if the codon at Amino Acid 219 codes for any amino acid except threonine, substituting nucleotides in the codon to code for threonine at Amino Acid 219 to alter a non-taxol-binding beta-tubulin or portion thereof to a taxol-binding beta-tubulin or portion thereof, or if the codon at Amino Acid 219 codes for threonine, substituting nucleotides in the codon to code for any amino acid except threonine at Amino Acid 219 to alter a taxol-binding beta-tubulin or portion thereof to a non-taxol-binding beta-tubulin or portion thereof.
In another aspect, the invention is a method of developing a taxol-sensitive fungal cell from a taxol-resistant fungal cell comprising transforming the non-taxol-sensitive fungal cell by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ; wherein the transformed fungal cell expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
In another aspect, the invention is a transgenic taxol-sensitive fungal cell transformed by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ, wherein the transformed fungal cell expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
In another aspect, the invention is a method of developing a taxol-resistant fungal cell from a taxol-sensitive fungal cell comprising transforming the taxol-sensitive fungal cell by introducing a DNA segment encoding non-taxol-binding beta-tubulin wherein the amino acid at Amino Acid 219 as numbered in SEQ ID NOJ is not threonine; wherein the transformed fungal cell over-expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
In another aspect, the invention is a transgenic taxol-sensitive fungal cell transformed by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ, wherein the transformed fungal cell over-expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
In another aspect, the invention is a method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing distinguishable taxol- resistant and taxol-sensitive fungal cells; contacting the composition of matter with the fungal cells; and determining the growth of inhibition of the fungal cells; whereby the composition of matter is determined to possess taxol or taxol-like compounds if it is able to inhibit the growth of taxol-sensitive fungal cells but not able to inhibit the growth of taxol- resistant fungal cells. The method can be performed wherein the distinguishable taxol- resistant and taxol-sensitive fungal cells consists essentially of transgenic taxol-resistant and taxol-sensitive isogenic fungal cells. The method can also be performed with taxol-resistant fungal cells derived from one fungus which is unrelated to the fungi from which the taxol- sensitive fungal cells are derived.
In another aspect, the invention is a method for controlling the growth of a plant pathogen comprising determining the taxol sensitivity of the plant pathogen; and if the pathogen is determined to be taxol-sensitive, the plant and soil surrounding the plant are treated with a taxol-producing P. microspora. In an exemplary method, the taxol sensitivity of the plant pathogen is determined by identifying Amino Acid 219, wherein the plant is designated as taxol-sensitive if Amino Acid 219 is threonine.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig 1 is a graph depicting the effect of taxol on mycelial growth in P. microspora, P. ultimum, P. cinnamomi and A. klebsiana. Fungal mycelia were grown on potato dextrose agar (PDA) plates containing different concentrations of taxol The inhibitory effect of taxol was assessed by colony diameter, and compared to mycelia grown in the absence of taxol Experiments were conducted in duplicate, and data presented are an average of several experiments
Fig 2 depicts the nucleotide and deduced amino acid sequence of beta-tubulin from P microspora Ne32 cDNA, TUBB-pm Numerals on the left indicate nucleotide position, and numerals on the right indicate amino acid position The sequences of the gene-specific primers NETUB5 and NETUB6 are underlined The translation initiation codon ATG is underlined, the translation termination codon is marked by an asterisk, and the putative polyadenylation signal is double underlined
Fig 3 depicts the nucleotide and deduced amino acid sequence of beta-tubulin from P. ultimum cDNA, TUBB-pu Numerals on the left indicate nucleotide position, and numerals on the right indicate amino acid position The sequences of the gene-specific primers WT1L-U and WT1L-L are underlined The translation initiation codon ATG is underlined, the translation termination codon is marked by an asterisk, and the two putative polyadenylation signals are double underlined The arrow at nucleotide 1507 indicates the position of the poly (A) tract in the shorter 1537 bp cDNA
Fig 4 depicts the nucleotide and deduced amino acid sequence of beta-tubulin from P cinnamomi cDNA, TUBB-pc Numerals on the left indicate nucleotide position, and numerals on the right indicate amino acid position The sequences of the gene-specific primers PCBTUB 1U, PCBTUB2U and PCBTUB4L are underlined The translation initiation codon ATG is marked by ###, and the translation termination codon is marked by an asterisk Nucleotides and amino acids which differ between TUBB-pc and the sequence U22050 (Genbank accession number) reported by Weerakoon et al (Weerakoon et al 1998 "Isolation and characterization of the single β-tubulin gene in Phytophthora cinnamomi, " Mycologia 90 85-95) are double underlined
Fig. 5 compares the amino acid sequence of P. cinnamomi beta-tubulins ("TUBB- pc") reported herein and previously by Weerakoon et al. (Weerakoon, et al. 1998. Mycologia 90:85-95) ("U22050"). The deduced amino acid sequence ofR. cinnamomi TUBB-pc is shown in its entirety, and the eight residues (Amino Acids 24, 219, 249, 251-253, 359, and 428) which differ from the U22050 sequence (Genbank accession number) reported by Weerakoon et al. (1998) are shown below the TUBB-pc sequence. Amino Acids 1-31 and 212-231 (denoted herein as taxol binding region I and II, respectively) are indicated by a line above the sequence.
Fig. 6 A and 6B depict the amino acid sequence alignment of beta-tubulins. The alignment was obtained using the ClustalW alignment program. The amino acid sequence of P. microspora beta-tubulin is shown in its entirety, and residues which differ in other beta-tubulins are shown below. Numerals on the right indicate amino acid positions. Sequences underlined indicate regions important for GTP binding (Amino Acids 63-77), phosphate binding (Amino Acids 140-146), and Mg2+ binding (Amino Acids 203-206). Amino Acids 1-31 and 212-231 (denoted here as taxol binding region I and II, respectively) are indicated by a line above the sequence. Amino Acids Phe270, Leu273 and Ser364 are marked above with #. Amino acids which are important for fungal resistance to benzimidazoles (Amino Acids 6, 165, 167, 198, 200 and 241) are marked above by asterisks. Gaps in alignment are indicated by dashes, and the end of each sequence is marked by "$". The Genbank accession numbers for beta-tubulins from N. crassa, A. idulans benA, A. klebsiana and human β2 are listed in Table I. The P. cinnamomi depicted is SEQ ID NO:6.
Fig. 7A and 7B are graphs depicting the specific binding of [3H]taxol to P. ultimum but not to P. microspora. Fig. 7A demonstrates that specific binding of [3Hltaxol to P. ultimum increased as a function of [ H]taxol concentration, while P. microspora showed no or very little specific binding. Actively growing fungal cells were incubated with different concentrations of [3H]taxol at room temperature for 2 hours before quenching. Specific binding was calculated as the difference between binding of [3H]taxol in the presence and absence of a 100-fold excess of unlabeled taxol. Specific binding represents 30-70% of the total binding to P. ultimum but less than 5% of the total binding to P. microspora. Binding of [3H]taxol to P. microspora cells was performed in the presence of Triton X-100 (0.1%
v/v) to disrupt the cell membrane Fig 7B depicts the reduction of specific binding of [3H]taxol to P. ultimum in the presence and absence of the microtubule-depolymerizing drug thiabendazole in a dose-dependent manner Cells were either not treated (0 μM) or treated with different concentrations of thiabendazole for three hours at room temperature Subsequently, cells were incubated with 5 nM [3H]taxol in the presence or absence of a 100- fold excess of unlabeled taxol for two hours before quenching The specific binding of [3H]taxol to untreated cells was defined as 100% The experiments depicted in Fig 7A and 7B were conducted in duplicate, and data presented are representative of several experiments
Fig 8 depicts the amino acid sequences of the taxol binding region I (Amino Acids
1-31) and II (Amino Acids 212-231) of beta-tubulins from different organisms The amino acid sequences of the taxol binding regions I and II for pig beta-tubulin are shown in their entirety and residues which differ are shown for other beta-tubulins The taxol sensitivity of each organism is indicated, "s" for sensitive and "r" for resistant Amino Acids 15-25 and 212-222, which have been shown to be involved in taxol binding by both cross-linking and electron crystallography, are marked with asterisks The taxol binding region II of A. klebsiana is between Amino Acids 211-230 due to a gap in its sequence Pig beta-tubulin is described by Nogales, et al (Nogales, et al 1999 Nature 391 199-203), and Genbank accession numbers for other sequences are listed in Table I The sequence for P. cinnamomi presented herein is depicted in SEQ ID NO 6
DETAILED DESCRIPTION
One aspect of the present invention is an isolated gene comprising an open reading frame coding for the protein beta-tubulin or a portion thereof The corresponding cDNA have been isolated and characterized for taxol-resistant Pestalotiopsis microspora Ne32, taxol-sensitive Pythium ultimum, and taxol-sensitive Pythium cinnamomi The nucleotide and deduced amino acid sequences of beta-tubulin for Pestalotiopsis microspora Ne32 are given in SEQ ID NO 1 and SEQ ID NO 2, respectively, for Pythium ultimum, in SEQ ID NO 3 and SEQ ID NO 4, respectively, and for Pythium cinnamomi, in SEQ ID NO 5 and SEQ ID NO 6, respectively Through characterization of the taxol sensitivity of the beta- tubulins encoded by the genes of the present invention, it has been found that the identity of Amino Acid 219 of beta-tubulin as numbered in SEQ ID NO 2, SEQ ID NO 4, or SEQ ID
NO:6 is an indicator of taxol sensitivity. The presence of threonine at Amino Acid 219 ("Thr219") indicates taxol sensitivity, while the presence of asparagine ("Asn219") or glutamine ("Gln219") indicate taxol resistance.
In another aspect, the present invention is the beta-tubulin protein or protein fragments encoded by the novel genes disclosed herein. Since the P. ultimum and P. cinnamomi beta-tubulin proteins of the present invention are capable of binding taxol, proteins and protein fragments comprising taxol-binding sites derived from the genes coding for beta-tubulin described herein can be produced by heterologous expression in E. coli and other systems, purified by standard procedures, and used in an in vitro assay for detecting taxol and taxol-like substances by using methods well known in the art (Schiff, et al. 1979. Nature 277:665-667). For example, the beta-tubulin proteins of the present invention can be used to screen plant or fungal extracts as well as synthetic compounds for taxol or taxol-like substances as possible anticancer drugs. Beta-tubulins produced by making specific amino acid substitutions, deletions, or alterations can be used as experimental tools to further determine the molecular basis of taxol binding to the beta-tubulin protein.
In another aspect, antibodies (polyclonal or monoclonal) raised against all or portions of the beta tubulins of the present invention can be used to determine if a composition of matter has taxol binding properties. In one method, antibodies capable of binding to taxol-sensitive beta-tubulin and/or taxol-resistant beta tubulins are exposed to a composition of matter prepared for in situ hybridization (Ausubel, et al. 1997. Current
Protocols in Molecular Biology, John Wiley & Sons), Elisa, or Western blot. Visualization of antibody-antigen binding is mediated through any means known in the art, e.g., secondary radiolabeled or fluorescent antibodies or colorimetric methods using peroxidase and/or alkaline phosphatase (Harlow, E.D. and Lane, D. 1988. Antibodies: A Laboratory Manual). For example, antibodies raised to a portion of SEQ ID NO:4 comprising Amino Acid 219 would bind to a beta-tubulin which had threonine at Amino Acid 219 but would not bind to a beta-tubulin having a different amino acid at Amino Acid 219, so that detectable binding would indicate the presence of threonine at Amino Acid 219, and hence, sensitivity to taxol. This type of assay is useful for screening a variety of compositions of matter, including living matter such as plants or microorganisms, or non-living matter such as plant materials, patient samples, or compound libraries for the presence of beta-tubulin.
In another aspect, the present invention is a method of designing taxol analogs or other compounds which mimic the interaction of taxol with beta-tubulin based on the identification of specific amino acids in the beta-tubulins corresponding to taxol-binding and taxol-sensitivity. The previously reported three-dimensional structure (Nogales, et al. 1998. Nature 391 : 199-203) can be applied to developing and optimizing antineoplastic and antifungal compounds with respect to Amino Acid 219 and the surrounding area. Further, such information can also be used to generate mutant beta-tubulins with altered taxol sensitivity by substituting amino acids at specific positions in the beta-tubulin protein.
In another aspect, the present invention is a method of generating isogenic strains of fungi using a gene of the present invention, which allows studies of taxol related pharmacology to be performed against a known background. Further, the present invention is a method of using these isogenic fungal strains, one of which is taxol sensitive and the other taxol resistant, to screen plant extracts, fungal extracts, extracts from other organisms, and synthetic compounds for taxol-like substances as possible anticancer agents. The present invention is also a method of using two unrelated fungal strains, one of which is taxol sensitive and the other taxol resistant, to screen plant extracts, fungal extracts, extracts from other organisms, and synthetic compounds for taxol-like substances as possible anticancer agents.
The genes and proteins of the present invention are characterized in the following examples. It is to be understood that the examples are exemplary of the invention and are intended to be illustrative of the invention, but are not to be construed to limit the scope of the invention in any way. Modifications may be made in the structural features of the invention without departing from the scope of the invention.
Example 1: Differential taxol sensitivity in selected fungi Taxol sensitivity was established for the fungal strains used in the isolation of the beta-tubulin cDNAs of the present invention.
Pestalotiopsis microspora strain Ne32, previously disclosed in U.S. Patent No. 5,861,302, was licensed from Montana State University. Pythium ultimum (ATCC 26083), Achlya klebsiana (ATCC 52605), and Pythium cinnamomi (ATCC 200982) were purchased
from American Type Culture Collection (Manassas, VA) Taxol was obtained from Sigma Chemical Company (St Louis, MO)
The effect of taxol on the growth of P. microspora Ne32, P. ultimum, and P. cinnamomi was examined For comparison, A. klebsiana, an oomycete closely related to P. ultimum, was also included in these experiments As shown in Fig 1, the growth of P. microspora was highly resistant to taxol up to 11 7 μM By comparison, A klebsiana showed moderate sensitivity, and its growth was reduced by 40% in 11 7 μM taxol Finally, P. ultimum and P. cinnamomi were shown to be the most sensitive of the four species Growth of P. ultimum and P. cinnamomi was inhibited even at low concentrations of taxol (IC50 0 1 μM) This sensitivity is comparable to the level of taxol (0 25 μM) that inhibits Hela cell division (Schiff, et al 1979 Nature 277 665-667)
Example 2: Isolation of β-tubulin cDNA sequences
Beta-tubulin cDNA sequences were determined for P. microspora Ne32, P. ultimum, and P. cinnamomi from RNA isolated from fungal mycelia Automated dideoxynucleotide sequencing was performed by a contracting laboratory Sequence comparison was performed using the BLAST program at the Internet site of the National Center for Biotechnology Information The amino acid sequence alignment was performed using ClustalW program, and other analysis using Mac Vector program
To isolate beta-tubulin cDNA sequences from P. microspora Ne32 and P. ultimum, four degenerate primers were designed according to conserved motifs in fungal beta-tubulin amino acid sequences A forward degenerate primer BTUB 1 , 5'-CTGGGCYAAGGGYC AYTACACYGAG-3' (SEQ ID NO 7, was designed corresponding to amino acid residues Trp-Ala-Lys-Gly-His-Tyr-Thr-Glu (or WAKGHYTE in single letter amino acid code, SEQ ID NO 8), a reverse primer BTUB2, S'-CGAAGAARTGRARNCGRGGGAARGG-S' (SEQ ID NO 9), corresponding to amino acid residues Pro-Phe-Pro-Arg-Leu-His-Phe-Phe (or
PFPRLHFF in single letter amino acid code, SEQ ID NO 10), a forward primer BTUB3, 5'- CGAGCCYTACAACGCYACYCT-3' (SEQ ID NO 11), corresponding to amino acid residues Glu-Pro-Tyr-Asn-Ala-Thr-Leu (or EPYNATL in single letter amino acid code, SEQ ID NO 12), and a reverse primer BTUB4, 5'-CTCGTTCATGTTRSWCTCRGCCTC-3' (SEQ ID NO 13), corresponding to amino acid residues Glu-Ala-Glu-Ser-Asn-Met-Asn-Asp
(or EAESNMND in single letter amino acid code, SEQ ID NO 14) All primers were synthesized by a contracting laboratory according to our specifications
In order to isolate beta-tubulin cDNA from P. microspora Ne32, five micrograms (5 μg) of total RNA from mycelia grown for 5 days was used to synthesize first-strand cDNA using primer BTUB4 in a 20 microliter (20 μl) reaction One microliter (1 μl) of the cDNA product was used as template in Polymerase Chain Reactions (PCR) The cycling program contained 8 cycles of an annealing temperature of 52°C, followed by 22 cycles with an annealing temperature of 62°C Degenerate primers BTUB3 and BTUB4 generated an amplification product of 0 8 kb, whereas BTUB2 and BTUB3 generated a product of 0 3 kb The desired bands were gel-purified using Geneclean (BIO 101, Vista, CA), ligated into the pPCR2 1 vector (Invitrogen, San Diego, CA), and transformed into E coli XL1 -Blue cells Inserts were sequenced and used to synthesize a gene-specific forward primer NETUB5, 5'- GGGTGTCACCACTTGCTTGCGTTT-3' (SEQ ID NO 15), and a reverse primer NETUB6, 5'-TCGAGTTTCCGACGAAAGTGGACGA-3' (SEQ ID NO 16) To obtain full-length clones, a Marathon cDNA Library was constructed using one microgram (1 μg) mRNA according to manufacturer (Clontech, Palo Alto, CA). One microliter (1 μl) of this library was diluted 250-fold, and five microliters (5 μl) were used in Rapid Amplification of cDNA Ends (RACE) reactions using the PCR cycling program recommended by the manufacturer For 5' RACE, library adaptor primer API (Clontech) and primer NETUB6 generated a product of 1 3 kb For 3' RACE, primers API and NETUB5 generated a product of 1 0 kb The desired bands were gel-purified and cloned into the pPCRII-TOPO vector (Invitrogen, Carlsbad, CA) The region between 1 to 1105 bp from the 5' RACE product was ligated at an internal BamHl site with the region between 1 106 to 1668 bp from the 3' RACE product to form the composite cDNA (Fig. 2) The resulting composite cDNA from P. microspora was 1668 bp long, designated as TUBB-pm, and its nucleotide and deduced amino acid sequence are shown in SEQ ID NO 1 and SEQ ID NO 2, respectively, as well as Fig 2 This cDNA encodes a protein of 446 amino acids with a calculated Mr of 49,832 and pi of 4 6 It contains 74 nucleotides in the 5' untranslated region (UTR), and 229 nucleotides in the 3' UTR followed by a 24 nucleotide poly (A) tail A sequence AATAA (nucleotides 1539- 1543 of SEQ ID NO 1) with the closest similarity to the animal and viral polyadenylation signal AATAAA (Proudfoot, N J and Brownlee, G G 1976 "3' Non-coding region
sequences in eukaryotic messenger RNA," Nature 263 211-214) was located 103 bp upstream of the poly (A) tract
In order to isolate beta-tubulin cDNA from P. ultimum, five micrograms (5 μg) of total RNA from mycelia grown for six days was used to synthesize first strand cDNA with oligo-dT primer (GibcoBRL, Gaithersburg, MD) in a twenty microliter (20 μl) reaction Two microliters (2 μl) of cDNA product were used as the template in PCR reactions with a cycling program similar to that described above Degenerate primers BTUB1 and BTUB4 generated a product of 1 0 kb, whereas BTUB1 and BTUB2 amplified a product of 0 5 kb The desired bands were gel-purified and ligated into the pPCRJ 1 vector Inserts were sequenced and used to design a gene-specific forward primer WT1L-U, 5 -CTAT
CATGTGCACGTACTCGGTGTGC-3' (SEQ ID NO 17), and a reverse primer WT1L-L, 5'- CTGGGACGGTCAAAGCACGGTACTGC-3' (SEQ ID NO 18) For 5' RACE, first-strand cDNA was synthesized using primer WT1L-L and used as template in PCR reactions Primers WT1L-L and Cap-Switch (Clontech) generated a 0 95 kb product using Advantage- GC cDNA PCR kit (Clontech) For 3' RACE, first strand cDNA was synthesized using CapFinder cDNA Library Construction kit (Clontech) With the resulting cDNAs as template, primers WTIL-U and CDS/3' (Clontech) generated two PCR products of 1 0 and 1 1 kb, respectively PCR fragments were gel-purified and cloned into the pPCRJ 1 vector In P. ultimum, isolated tubulin cDNAs were of two types, one composite cDNA was 1650 bp long, and the other was 1537 bp long These two cDNAs differ only in the position at which the poly (A) tail has been added The region between 1 to 824 bp from the 5' RACE product was ligated at an internal Mfel site with the region between 825 to 1650 bp from the 1 1 kb 3' RACE product to form the composite 1650 bp cDNA designated as TUBB-pu, and its nucleotide and deduced amino acid sequence are shown in SEQ ID NO 3 and SEQ ID NO 4, respectively, as well as Fig 3 This cDNA encodes a protein of 446 amino acids with a calculated Mr of 50,047 and pi of 4 6 It contains 91 nucleotides in the 5' UTR, and 199 nucleotides in the 3' UTR followed by a 19 nucleotide poly (A) tract Two imperfect polyadenylation signals were tentatively identified, ATATA at 57 bp upstream of poly (A) tract in the 1537 bp cDNA (nucleotides 1445-1449 of SEQ ID NO 3), and AATATT at 80 bp upstream of poly (A) tail in the 1650 bp cDNA (nucleotides 1546-1551 of SEQ ID
NO 3) The sizes of these cDNAs match well with transcript sizes in Northern analysis as shown below, indicating they are complete
Four gene-specific primers were synthesized based on the reported beta-tubulin cDNA sequence from P. cinnamomi (Weerakoon, et al 1998 Mycologia 90 85-95) The forward primer PCBTUBIU (5'-CAGCGACAACATGAGAGAGCTCG-3', SEQ ID NO 19) corresponds to region 270-292 in its cDNA sequence, the forward primer PCBTUB2U (5'- CGATGAGGTC ATGTGCCTGGAT AA-3 ', SEQ ID NO 20) corresponds to region 867- 890, the reverse primer PCBTUB3L (5'-AAACGGAGGCACGTGGTGATG-3', SEQ ID NO 21) corresponds to region 984-1005, the reverse primer PCBTUB4L (5 -CGCGTC TATCTCATCCATTCCTCG-3', SEQ ID NO 22) corresponds to region 1596-1619
Five micrograms (5 μg) of total RNA from P. cinnamomi mycelia grown for 5 days was used to synthesize first-strand cDNA using oligo-dT primer in a 20 μl reaction One microliter (1 μl) of the cDNA product was used as template for PCR The cycling program comprised 30 cycles with an annealing temperature of 62°C Primer PCBTUBIU and PCBTUB4L generated an amplification product of 1 3 kb, and primer PCBTUB2U and PCBTUB4L generated an amplification product of 0 75 kb Primer PCBTUBIU or PCBTUB2U in combination with PCBTUB3L did not generate any product The desired bands were gel-purified using Geneclean (BIO101), ligated into the pPCRJ 1 vector (Invitrogen), and transformed into E. coli XL 1 -Blue cells Four clones of the 1 3 kb fragment (clone # C16-1, 4, 9 and 10) and one clone of the 0 75 kb fragment (clone # C10- 5) were sequenced from both directions and found to conform to the same sequence The nucleotide and deduced amino acid sequence of the 1 3 kb long cDNA, designated as
TUBB-pc, are shown in SEQ ID NO 5 and SEQ ID NO 6, respectively, as well as Fig 4 It encodes a 444 amino acid long beta- tubulin protein, with a calculated Mr of 50 kDa, and a pi of 4 7 There are 10 nucleotides in the 5' untranslated region (UTR), and 5 nucleotides in the 3' UTR
A sequence encoding P. cinnamomi beta-tubulin has been previously reported
(Weerakoon, et al 1998 Mycologia 90 85-95, Genbank accession number U22050), and the deduced amino acid sequence of P. cinnamomi beta-tubulin disclosed herein was compared to that disclosed by Weerakoon et al The TUBB-pc cDNA sequence shown in SEQ ID NO 5 and Fig 4 differs by 36 nucleotides (2 7%) within the coding region from the one reported by Weerakoon et al An alignment of the beta-tubulin amino acid sequences deduced from TUBB-pc (SEQ ID NO 6) and the one previously reported by Weerakoon
("U22050", SEQ ID NO 23) is shown in Fig 5 The two sequences differ by 8 amino acids Four are conserved changes, while the other four are nonconserved changes One change is within each of the taxol-binding sites In the taxol-binding region I (Amino Acids 1-31), Amino Acid V24 (valine at Amino Acid 24) in TUBB-pc differs from 124 (isoleucine at Amino Acid 24) in U22050 In the taxol-binding region II (Amino Acids 212-231), Amino Acid T219 (threonine at Amino Acid 219) in TUBB-pc differs from N219 (asparagine at Amino Acid 219) in U22050
The deduced amino acid sequence of beta-tubulin from P. microspora (SEQ ID NO 2), P. ultimum (SEQ ID NO 4), and P. cinnamomi (SEQ ID NO 6) show features expected of beta-tubulin, as shown by an alignment with human β2-tubulin (SEQ ID NO 24) and from beta-tubulins from Neurospora crassa (SEQ ID NO 25), A. mdulans benA (SEQ ID NO 26), and A. klebsiana (SEQ ID NO 27) depicted in Fig 6 These sequences can be divided into N-terminal (Amino Acids 1-205), intermediate (Amino Acids 206-381) and C- terminal domains (Nogales, et al 1998 Nature 391 199-203) Their N-terminal domain contains conserved motifs important for GTP binding [Ala-Ile-Leu-Val-Asp-Leu-Glu-Pro- Gly-Thr-Met-Asp-Ser-Val-Arg or AILVDLEPGTMDSVR in single letter amino acid code (SEQ ID NO 28) and Ala-Val-Leu-Val-Asp-Leu-Glu-Pro-Gly-Thr-Met-Asp-Ser-Val-Arg or AVLVDLEPGTMDSVR in single letter amino acid code (SEQ ID NO 29) between Amino Acids 63-77 in SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26, and between Amino Acids 62-76 in SEQ ID NO 27], phosphate binding [Gly-Gly-Gly-Thr-Gly-Ser-Gly or GGGTGSG in single letter amino acid code (SEQ ID NO 30) between Amino Acids 140-146 in SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26, and between Amino Acids 139 and 145 in SEQ ID NO 27], and Mg2+ binding (Asp-Asn-Glu-Ala or DNEA in single letter amino acid code (SEQ ID NO 31) between Amino Acids 203-206 in SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 24, SEQ ID NO 25, and SEQ ID NO 26, and between Amino Acids 202-205 in SEQ ID NO 27 ) (Linse, K and Mandelkow, E M 1988 "The GTP-binding peptide of β-tubulin localization by direct photoaffinity labeling and comparison with nucleotide-binding proteins," JBiol Chem 263 15205-15210, and Farr, G W and Stemlicht, H 1992 "Site-directed mutagenesis of the GTP-binding domain of β- tubulin," JMolBiol 227 307-321) In addition, the N-terminal Met-Arg-Glu-Ile (or MREI
in single letter amino acid code, (SEQ ID NO 32) motif has been shown to autoregulate the stability of beta-tubulin mRNA in animal cells (Yen, et al 1988 "Autoregulated instability of β-tubulin mRNAs by recognition of the nascent amino terminus of β-tubulin," Nature 334 580-585) This motif and a variant Met-Arg-Glu-Leu (or MREL in single letter amino acid code, SEQ ID NO 33) are present in the fungal beta-tubulins shown in Fig 6, and they may function similarly The C-terminal domain has been reported to be important for interactions with microtubule-associated proteins (MAPS) and motor proteins (Nogales, et al 1998 Nature 391 199-203) Sequences near the C-terminus are hypervariable and acidic, a common feature of beta-tubulins (Sullivan, K F 1988 "Structure and utilization of tubulin isotypes," Ann Rev CellBiol 4 687-716)
The amino acid sequence of beta-tubulins from different organisms are well conserved and exhibit at least 63% identity (Oakley, B R 1994 "Gamma-tubulin " In Hyams JS, Lloyd CW (eds) Microtubules. Wiley-Liss, New York, pp 38-45) Table I shows the percentage identity between the beta-tubulin amino acid sequence of P. microspora and P. ultimum with beta-tubulins of other organisms The beta-tubulin from P. microspora shares the highest identity (93-97%) with filamentous ascomycetes such as A flavus, A. mdulans benA and N. crassa, and lower identity (73-78%) with single-cell ascomycetes and oomycetes Its identity (78-85%) with beta-tubulin from non-fungal organisms is also relatively low In contrast, beta-tubulin from P. ultimum shows the highest identity (96-97%) with beta-tubulin from two oomycetes, A. klebsiana and P. cinnamomi, but shares limited identity (71-78%) with beta-tubulin from ascomycetes The beta-tubulin from P. ultimum also shows relatively high identity (86-93%) with beta-tubulin from non- fungal organisms such as the green algae C reinhardtn, the protozoa T. thermophila, the slime mold Physarum polycephalum, and various animals
Example 3: Extraction of genomic DΝA and Southern analysis
Since multiplicity within the genes that encode beta-tubulin could affect the taxol- dependent property of microtubules, Southern analysis was used to determine whether P. microspora Νe32 and P. ultimum harbor one or more copies of beta-tubulin gene
Mycelia of P. microspora Ne32 or P. ultimum grown for three days were harvested, and genomic DNA was isolated using Elu-Quik Hi-Volume Genomic kit (Schleicher
Table I: Amino Acid Sequence Identity Between Beta-tubulin from P. microspora or P. ultimum and Other Organisms3
Schuelle, Neene, NH). Five micrograms (5 μg) of genomic DNA was digested with restriction enzymes for 4 hours at 37°C, separated on 0.8% agarose gel, transferred to Nylon filters (Tropix, Bedford, MA), and wet filters were cross-linked using GS Gene Linker UV Chamber (Biorad; Hercules, CA). Southern blotting was performed under stringent conditions according to standard protocols (Sambrook et al. 1989. Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). A 413 bp PCR fragment of P. microspora beta-tubulin was generated with primers NETUB5 and NETUB6, while a 377 bp PCR fragment of P. ultimum beta-tubulin was generated with primers WT1L-U and WT1L-L. PCR products were gel-purified and labeled with α-32P dCTP using Ready-to-Go beads (Pharmacia, Piscataway, NJ) by random priming. Probes were purified with Micro Bio-Spin columns (Biorad).
In P. microspora, the beta-tubulin probe hybridized to a single band from genomic DNA digested with EcoK , Hindlll, or Sail, and two bands from BamHl digested sample. In P. ultimum, the beta-tubulin probe hybridized to a single band from genomic DNA digested with BamHl, Sail, Pvul, or Pstl, and two bands from EcoRI digested sample. The sizes of these fragments match those predicted from the restriction endonuclease map of the corresponding cDNA clones. Since beta-tubulin genes are typically highly conserved, these results show that both P. microspora and P. ultimum contain a single copy of the beta- tubulin gene, consistent with previous reports that fungi generally have one, or at most a few, beta-tubulin genes (Neff, et al. 1983. "Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo, " Cell 33:211-219; Hiraoka, et al. 1984. "The NDA3 gene of fission yeast encodes β-tubulin: a cold sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis," Cell 39:349-358; Orbach, et al. 1986. "Cloning and characterization of the gene for β-tubulin from a benomyl- resistant mutant oϊNeurospora crassa and its use as a dominant selectable marker," Moi
CellBiol 6:2452-2461; Cameron et al. 1990. "Cloning and analysis of β-tubulin gene from a protoctist," JBiol Chem 265:15245-15252; and Weerakoon et al. 1998. Mycologia 90:85-95; May, et al. 1987. "Aspergillus nidulans β-tubulin genes are usually divergent," Gene 55:231-243; Panaccione et al. 1988. "Colletotrichum grami icola transformed with homologous and heterologous benomyl-resi stance genes retains expected pathogenicity to corn, " Moi Plant Microbe Interact 1 : 1 13- 120; and Goldman et al. 1993. "A nucleotide substitution in one of the β-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate," Moi Gen Genet 240:73-80). The fact that P. microspora and P. ultimum have only a single copy of the beta-tubulin gene indicates that it is responsible for the sensitive or resistance properties of the organism to taxol.
Example 4: Isolation of mRNA and Northern analysis
Northern and PCR analysis were used to examine the expression level of tubulin mRNAs in P. microspora Ne32 and P. ultimum.
Four agar plugs of P. ultimum or P. microspora were inoculated into 40 ml of FM1 medium (5 grams soytone, 5 grams dextrose, 20 grams sucrose, and 1 gram yeast extract per liter of culture) or modified MID (Li, et al 1998 MycolRes 102 461-464), respectively, and grown at 24°C without shaking in 250 ml Erlenmeyer flasks Mycelia were harvested and blotted dry with Whatman paper One gram (1 g) of mycelia was ground to powder in the presence of liquid nitrogen using mortar and pestle The powder was transferred to a Dounce homogenizer containing 10 ml of Trizol (GibcoBRL) and homogenized Total
RNA was isolated according to the manufacturer's instructions Total RNA was dissolved in diethylcarbonate-treated water at room temperature and stored at -80°C Northern blotting was performed under stringent condition according to standard protocols (Sambrook et al 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)
Northern analysis revealed a very low level of beta-tubulin transcripts in P. microspora (data not shown) Therefore, total RNA from mycelia grown for 2, 5, 6, or 11 days were converted to cDNA, and used as a template in PCR reactions Using gene- specific primers NETUB5 and NETUB6, a 413 bp beta-tubulin cDNA fragment was amplified from mycelia grown in log (2, 5 and 6 days) or stationary phase (11 days), but not from a control reaction that contained no template
In P. ultimum, total RNA was isolated from mycelia grown for 2, 4, 6, 7, 8 and 10 days, and used in Northern analysis The beta-tubulin probe hybridized to two transcripts of 1 5 and 1 6 kb, consistent with the sizes of the two cDNAs isolated The 1 6 kb transcript was present in greater abundance
Example 5: Binding of [3H]taxol to fungal cells
In animal systems, binding of [3H]taxol to intact cells has been used to characterize interactions between taxol and microtubules (Manfredi, et al 1982 "Taxol binds to cellular microtubules," J Cell Biol 94 688-696) To determine whether the differential taxol sensitivity of P. microspora and P. ultimum correlates with the taxol binding properties of
their microtubules, we examined the ability of these fungal cells to specifically bind to [3H]taxol. The total binding of [3H]taxol to fungal cells consists of specific binding to microtubules and nonspecific binding to other cellular structures. The values of total and nonspecific binding were determined by binding of [3H]taxol to fungal cells in the absence or presence of 100-fold excess unlabeled taxol. The specific binding of [3H]taxol was then calculated as the difference between the amount of total and nonspecific binding.
Fresh mycelia from P. microspora Ne32, and P. ultimum were grown in 140 milliliters modified MID media in Roux bottles at 24°C for 1-2 days. These actively growing mycelia were transferred to 50 milliliter conical tubes, and centrifuged at 7,000 rpm for 5 minutes at room temperature. Mycelia were suspended in 1 milliliter remaining MID medium and 1 milliliter fresh MID medium. Cells were either untreated or pretreated with the anti-mitotic drug thiabendazole to depolymerize microtubules. In pretreated cells, thiabendazole (in DMSO) was added to desired concentrations, and DMSO was adjusted to the same concentration in all samples. Samples were then incubated at room temperature for 3 hours. Subsequently, [3H]taxol (3.7 X 107 Bq/ml, Moravek) was added to desired concentrations either in the presence or absence of 100-fold excess unlabeled taxol. Samples were incubated for 2 hours at room temperature, then quenched on ice. [3H]taxol binding to P. microspora cells was performed in the presence of 0.1% (v/v) Triton X-100 to disrupt the cell membrane.
Each GFC filter (Whatman; Clifton, NJ) was weighed using an analytical balance.
For P. ultimum, mycelia were transferred from conical tubes onto GFC filter hold by a 3- piece filter funnel (Whatman). Conical tubes were washed three times with 10 milliliters of MilliQ H2O (Millipore, Inc.; Bedford, MA). Mycelia on GFC filter were washed with 120 milliliters of MilliQ H2O. For P. microspora, mycelia were collected by centrifugation at 5,000 rpm for 5 minutes at room temperature, and washed three times with 40 milliliters of MilliQ H2O. Mycelia were transferred onto GFC filter, and combined with residual mycelia after rinsing conical tubes with 5 milliliters of MilliQ H2O. GFC filters were dried at 80°C in an oven overnight and then weighed to obtain mycelia dry weight. Filters were counted for 5 minutes under 20 milliliters of Cytoscint (Fisher Scientific; Pittsburgh, PA) in a Beckman LS3801 scintillation counter. Specific binding was calculated as the difference between [3H]taxol bound in the presence and absence of a 100-fold excess unlabeled taxol.
Nonspecific binding was determined as binding in the presence of 100-fold excess unlabeled taxol.
[3H]taxol was found to bind specifically to P. ultimum cells, and the amount of specific binding increased as a function of [3H]taxol concentration (Fig. 7A). In addition, in cells pretreated with thiabendazole to reduce the amount of assembled microtubules, the specific binding of [3H]taxol decreased in a dose-dependent manner (Fig. 7B). In fact, treatment with 1 mM of thiabendazole completely abolished the specific binding of [3H]taxol. These results indicate that taxol is able to interact with P. ultimum microtubules, and are consistent with the fact that this organism is sensitive to taxol.
On the other hand, initial experiments showed very low amount of specific binding of [3H]taxol to P. microspora (data not shown). This result could be due to inefficient interactions between taxol and P. microspora microtubules, or alternatively due to a membrane barrier which prevents intracellular accumulation of [3H]taxol. In animal cells, taxol crosses the cell membrane by diffusion due to its hydrophobic character (Manfredi et al. 1982. J Cell Biol 94:688-696). In some cases, resistance to taxol has been associated with P-glycoprotein, a membrane-located pump which causes drug efflux (Jachez et al. 1993. "Restoration of taxol sensitivity of multidrug-resistant cells by the cyclosporine SDZ PSC 833 and the cyclopeptide SDZ 280-446," J Natl Cancer Inst 85:478-483)). There is no information available as to whether P. microspora has such system. It has been shown that treatment of animal cells with nonionic detergents such as 0.1% (v/v) NP-40 or Triton X- 100 disrupt the cell membrane, release most soluble proteins including unassembled tubulins, but leave assembled microtubules intact (Schliwa et al. 1981. "Stabilization of the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition," Proc Natl Acad Sci USA 78:4329-4333; Duerr et al. 1981. "Molecular analysis of cytoplasmic microtubules in situ: identification of both widespread and specific proteins," Cell 24:203-222; Manfredi et al. 1982. J Cell Biol 94:688-696). To evaluate whether a membrane barrier was responsible for the low specific binding, [3H]taxol binding to P. microspora cells in the presence of Triton X-100 was performed. P. microspora cells treated with Triton X-100 (0.1% (v/v)) showed none or very little specific binding of [3H]taxol up to 75 nM [3H]taxol (Fig, 7A). Furthermore, cells pretreated with thiabendazole also showed no specific binding of [3H]taxol in the presence of Triton X-100
(data not shown) These results indicate that taxol is unable to interact or interacts poorly with microtubules of P. microspora, and are consistent with the fact that this organism is resistant to taxol In summary, the [3H]taxol binding results demonstrate that the properties of β-tubulin in these organisms determine their differential sensitivity to taxol
Taxol stabilizes MTs by binding to beta-tubulin in assembled MTs, and its binding site has been characterized by photo cross-linking, electron crystallography, and mutagenesis Regions between Amino Acids 1-31 and 217-231 were found to cross-link to the C-3' and C-2 group of taxol, respectively (Rao, et al 1994 JBiol Chem 269 3132-3134, and Rao, et al 1995 JBiol Chem 270 20235-20238) Recently, the structure of the beta- tubulin dimer was solved by electron crystallography of zinc induced sheets of tubulin dimer (Nogales, et al 1998 Nature 391 199-203) Modeling of taxol bound to this structure shows that the C-3' group of taxol is near Amino Acids 15-25 of beta-tubulin (near the top of helix HI), and the C-2 group is near Amino Acids 212-222 (near helix H6 and the loop between H6-H7) The identification of Amino Acids 15-25 and 217-222 in both cross-linking and electron crystallography studies indicate these regions are important for taxol binding In addition, the electron crystallography model also shows that Leu273 of bovine beta-tubulin (located in the M-loop) contacts the taxane ring of taxol (Nogales, et al 1998 Nature 391 199-203) In addition, mutations at Phe270 or Ala364 in the M40 isotype of beta- tubulin result in taxol resistance in human ovarian cells (Giannakakou, et al 1997 J Biol Chem 272 17118-17125)
Since the Amino Acids 270, 273 and 364 (marked by # in Fig 6) do not differ among the fungal beta-tubulins listed in Fig 6, they are not responsible for the differential taxol response among these organisms However, comparison of Amino Acids 1-31 and 212-231 (defined here as taxol binding region I and II, respectively) from beta-tubulins of organisms that are taxol-resistant or taxol-sensitive reveal residues that are important for taxol interaction Fig 8 provides a comparison of the taxol binding region I and taxol binding region II amino acid sequences for pig (I, SEQ ID NO 34, II, SEQ ID NO 35), human β2 (I, SEQ ID NO 36, II, SEQ ID NO 37), Drosophila βl (I, SEQ ID NO 38, II, SEQ ID NO 39), Xenopus β4 (I, SEQ ID NO 40, II, SEQ ID NO 41), Tetrahymena (I, SEQ ID NO 42, II, SEQ ID NO 43), Physarum β 1 (I, SEQ ID NO 44, II, SEQ ID NO 45), P. ultimum (I, SEQ ID NO 46, II, SEQ ID NO 47), P. cinnamomi (I, SEQ ID NO 48, II, SEQ
ID NO 49) A. klebsιana{l, SEQ ID NO 50, II, SEQ ID NO 51), P. microspora (I, SEQ ID NO 52, II, SEQ ID NO 53), A. mdulans benA (I, SEQ ID NO 54, II, SEQ ID NO 55), and S. cerevisiae (I, SEQ ID NO 56, II, SEQ ID NO 57)
Beta-tubulins from taxol-sensitive organisms such as human, pig, Drosophila, Xenopus, Tetrahymena and Physarum are highly conserved in taxol binding region I and II, and are identical between Amino Acids 15-25 and 217-222 (except a conserved substitution at Amino Acid 23 in Drosophila β 1) Beta-tubulin from P. ultimum displays only four substitutions compared to the above sequences, none of which occurs between Amino Acids 15-25 and 217-222 This similarity is consistent with the fact that P. ultimum, like the animal organisms noted above, is taxol-sensitive Also consistent with this, previous biochemical studies of animal tubulins and data of [3H]taxol binding to P. ultimum demonstrated herein (Fig 7A and 7B), show that taxol binds beta-tubulin in assembled MTs of these organisms (Kellogg, et al 1989 J Cell Biol 109 2977-2991, and Manfredi, J J and Horwitz, S B 1984 Pharmacol Ther 25 83-125) Beta-tubulin sequences from P. ultimum and A. klebsiana are identical in taxol binding region I and II except Amino Acid 219, but A. klebsiana is relatively resistant to taxol (IC50 > 11 7 μM) This reduced sensitivity is due in part to the fact that A. klebsiana contains an asparagine at Amino Acid 219, whereas P. ultimum, and six other beta-tubulins from taxol-sensitive organisms, have threonine
Beta-tubulins from taxol-resistant organisms such as P. microspora, A. mdulans and S. cerevisiae are similar to each other within taxol binding region I and II, but differ from the above discussed sequences in seven positions (19, 22, 23, 25, 218, 219, and 221) within regions 15-25 and 217-222 The [3H]taxol binding data presented herein (Fig 7A and 7B), together with previous biochemical studies (Yoon, Y and Oakley, B R 1995 Biochem 34 6373-6381, and Barnes, et al 1992 Moi Biol Cell 3 29-47), show that beta-tubulins in assembled MTs of these organisms are unable to efficiently bind taxol These sequences contain the asparagine (or glutamine in the case of S. cerevisiae) at Amino Acid 219, as observed in A klebsiana, a substitution that contributes in part to the reduced sensitivity to taxol in these fungi Other substitutions, including which involve differences in charge and polarity such as changes from Lysl9 to Ala, Glu22 to Gin, and Val23 to Thr, also contribute to the taxol resistant phenotype of these organisms
These results indicate that a number of residues including threonine at Amino Acid 219 are important in the binding of beta-tubulin to taxol In particular, Amino Acid 219 plays an important role in determining taxol binding property of beta-tubulin and, consequently, the taxol-sensitivity of cells. Beta-tubulins from taxol-sensitive species have Thr219 (threonine at Amino Acid 219), while those from taxol-resistant species have
Asn219 (asparagine at Amino Acid 219) or Glu219 (glutamine at Amino Acid 219). The taxol sensitivity of P. cinnamomi is consistent with the presence of Thr219 in TUBB-pc (SEQ ID NO:6) and not Asn219 as previously reported by Weerakoon et al. The presence of Asn219 (asparagine at Amino Acid 219) found in P. microspora is consistent with the taxol resistance of this species Using the information that the presence of threonine at Amino Acid 219 in beta-tubulins corresponds to taxol-binding and taxol-sensitivity, taxol analogs or other compounds can be designed which mimic the interaction of taxol with beta- tubulin. Further, such information can also be used to generate mutant beta-tubulins resistant to taxol by substituting the threonine for another amino acid residue at Amino Acid 219.
Example 6: Sensitivity to microtubule-depolymerization drugs.
The effect of several MT-depolymerization drugs on the growth of P. microspora Ne32, P. ultimum and A. klebsiana was examined. These drugs included colchicine, colcemid (a synthetic derivative of colchicine), and two benzimidazole drugs, nocodazole and thiabendazole.
Colchicine, colcemid, nocodazole, and thiabendazole were obtained from Sigma Chemical Company (St. Louis, MO). A stock solution of colchicine was prepared in water, and other stock solutions in DMSO. An agar plug (6 mm in diameter) of fresh mycelia was transferred onto PDA plates containing 1 % (v/v) DMSO in the presence or absence of an anti-microtubule agent. Fungal colonies were grown at 24°C for 24 hours in the case of P. ultimum or 48 hours in the case of P. microspora and A. klebsiana. The growth inhibitory effect of these anti-mitotic agents was measured by the size of colony diameters.
Biochemical and genetic evidence has shown that these drugs bind to beta-tubulin in the tubulin dimer and cause MT depolymerization (Davidse, L.C. and Flach, W. 1978. "Interaction of thiabendazole with fungal tubulin, " Biochim Biophys Acta 543 :82-90; Jung,
M K and Oakley, B R 1990 "Identification of an amino acid substitution in the β-tubulin gene of Aspergillus mdulans that confers thiabendazole resistance and benomyl supersensitivity," CellMotil Cytoskeleton 11 87-94, Manfredi, J J and Horwitz, S B 1984 Pharmacol Ther 25 83-125) It has been shown that many fungi are resistant to colchicine (Cameron et al 1990 JBiol Chem 265 15245-15252, Kilmartin, J V 1981 "Purification of yeast tubulin by self-assembly in vitro," Biochem 20 3629-3633, and Davidse, L C and Flach, W 1977 "Differential binding of methyl benzimidazole-2-yl-carbarnate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus mdulans, " J Cell Biol 72 174-193), but are sensitive to nocodazole (Kilmartin, J V 1981 Biochem 20 3629-3633) and thiabendazole (Davidse, L C and Flach, W 1978 Biochim BiophysActa 543 82-90)
As shown in Table II, the three fungal species tested herein were resistant to colchicine and colcemid (IC50 100 μM), and were sensitive to nocodazole (IC50 2-22 μM) These results are consistent with the studies noted above In contrast, these fungi were differentially sensitive to thiabendazole P. microspora was highly sensitive (IC50 3 μM), while P. ultimum and A. klebsiana were less sensitive (IC50 270-350 pM) These results demonstrate that the biochemical properties of beta-tubulin differ in these three fungi
Table II: Sensitivity of fungi to microtubule depolymerization drugs
Mutations at Amino Acids 6, 165, 167, 198, 200 and 241 in beta-tubulin (marked by asterisks in Fig 6) result in altered sensitivity to thiabendazole and other benzimidazole drugs in yeast, N. crassa, A. mdulans ben and Trichoderma viride (Thomas, et al 1985 "Isolation and characterization of mutations in the β-tubulin gene of Saccharomyces cerevisiae, " Genetics 112 715-734, Orbach, et al 1986 Moi Cell Biol 6 2452-2461, Jung, et
al 1992 "Amino acid alterations in the β-tubulin gene of Aspergillus mdulans that confer benomyl resistance," CellMotil Cytoskeleton 22 170-174, Jung, M K and Oakley, B R 1990 CellMotil Cytoskeleton 17 87-94, Fugimura, et al 1992 "A single amino-acid substitution in the beta-tubulin gene oϊNeurospora confers both cabendazim resistance and diethofencarb sensitivity," Curr Genet 21 399-404, and Goldman et al 1993 Moi Gen Genet 240 73-80) These six residues in beta-tubulin from P. microspora are identical to those observed in beta-tubulin from other thiabendazole sensitive species such as N. crassa and A. mdulans ben A In contrast, beta-tubulin from P. ultimum and A klebsiana differ at Amino Acids 165, 167 and 200 It has been previously shown that a phenylalanine-to- tyrosine change at Amino Acid 167 results in benzimidazole resistance in N. crassa
(Orbach, et al 1986 Moi Cell Biol 6 2452-2461), and the fact that both P. ultimum and A. klebsiana have a tyrosine at this position accounts for their resistance to such drugs In summary, the differential sensitivity to thiabendazole exhibited by these three fungi is consistent with the comparison of fungal beta-tubulins shown in Fig 6
Example 7: Production of Antibodies Capable of Distinguishing
Taxol-Binding and Non-Binding Beta-tubulins
Monoclonal or polyclonal antibodies can be raised against the following antigens 1) native beta-tubulins extracted from P. microspora, P. ultimum, or P. cinnamomi, 2) beta- tubulins of P. microspora, P. ultimum, or P. cinnamomi produced from a heterologous system such as E. coli, yeast, and insect cells, and 3) synthetic peptide corresponding to the SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6, and preferably comprising at least one taxol binding region The antibodies are used to interact with the above mentioned beta-tubulins using Elisa or Western blotting using standard protocols (Harlow, E D and Lane, D 1988 Antibodies: A Laboratory Manual) The antibodies which could distinguish the taxol binding beta-tubulin from the taxol non-binding beta-tubulin are selected as the reagent
A specific example is to raise polyclonal or monoclonal antibodies to synthetic peptides corresponding to SEQ ID NO 4 or SEQ ID NO 6 which comprise at least one taxol binding region, for instance containing the taxol-binding region II comprising Thr219 or in which the Thr219 is replaced by Asn219/Gln219 The ability of these antibodies to interact with beta-tubulin is examined using Elisa using standard protocols The antibody which can binds to peptide containing Thr219 but not to peptide containing Asn/Gln 219 is selected as
the reagent which is specific for the taxol-binding site containing Thr 219. On the other hand, the antibody which specifically binds to the peptide containing Asn219/Gln 219 but not to the peptide containing Thr 219 is selected as the reagent which specifically recognizes taxol binding site devoid of Thr 219.
Example 8: Screening Assays to Detect Beta-Tubulin in Matter
Several assays can be used to determine if a composition of matter contains beta- tubulin capable of binding taxol. These assays are useful for screening a variety of compositions of matter, including living matter such as plants or microorganisms, or nonliving matter such as plant materials or patient samples for the presence of beta-tubulin.
The first assay is performed using Northern or Southern hybridization method well known in the art (Sambrook, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). The total RNA, mRNA or genomic DNA are isolated from the composition of matter and separated by electrophoresis. DNA, synthetic oligonucleotide, or RNA corresponding to the coding region or a portion of beta-tubulin (e.g., derived from SEQ ID NOJ, SEQ ID NO: 3 or SEQ ID NO:5) which comprises at least one taxol binding region will be used to synthesize isotopically labeled probes. Hybridization with a probe derived from SEQ ID NO: 1 will indicate beta-tubulin with high probability of taxol resistance. On the other hand, the hybridization with a probe derived from SEQ ID NOJ or SEQ ID NO: 5 will indicate beta-tubulin with a high probability of taxol sensitivity.
The second assay is to use a PCR-based assay using standard protocols (Sambrook, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). Both genomic DNA or cDNA converted from total RNA or mRNA are used as template in a PCR assay. Gene-specific or degenerate primers corresponding to the coding region of beta-tubulin (e.g., derived from SEQ ID NO: 1, SEQ ID NOJ or SEQ ID NO:5) which comprises at least one taxol binding region will be synthesized. Only DNA containing the appropriate primer sequences will be amplified, and all other variations will be suppressed. The amplification of PCR fragment of the predicted size using primers derived from SEQ ID NOJ or SEQ ID NO:5 but not from primers derived from SEQ ID NO: 1 will indicate high probability of taxol binding beta-tubulin. On
the other hand, the amplification of a PCR fragment of the predicted size using primers derived from SEQ ID NO: 1 but not from primers derived from SEQ ID NOJ or SEQ ID NO:5 will indicate high probability of taxol non-binding beta-tubulin. The subsequent obtaining of the beta-tubulin sequence and examination of the presence or absence of Thr219 residue will provide further determination.
The third assay is to use Elisa or Western blotting using standard protocols (Harlow, E.D. and Lane, D. 1988. Antibodies: A Laboratory Manual). Cell extracts of the composition of matter are prepared. Synthetic peptide, or native beta-tubulins extracted from P. microspora, P. ultimum, or P. cinnamomi, or produced from a heterologous system such as E. coli, yeast, and insect cells will be used to raise polyclonal or monoclonal antibodies. The antibodies will be used in the above mentioned Elisa or Western blotting. The antibody which recognizes the taxol binding from the non taxol binding is used in these assays.
Example 9: Construction of Taxol-sensitive and Taxol-resistant Isogenic Strains P. ultimum contains a single beta-tubulin. In vitro, its beta-tubulin gene or cDNA can be altered to change the Thr219 to a different residue, for instance to Asn219 or Gln219. This altered DNA sequence is cloned into a transformation vector, and used to transform the wild-type strain P. ultimum using established protocols (Balance, et al. 1985. Gene 36:321- 331). Homologous recombination between the wild-type beta-tubulin gene and the modified beta-tubulin in the vector occur. Transformed fungus are selected on media containing taxol. The taxol-resistant clones are selected and their beta-tubulin cDNA sequenced to confirm the absence of Thr 219. The taxol-resistant isogenic strain of P. cinnamomi is similarly constructed and used in screening assays as described in later examples. The only difference between these isogenic strains is that the taxol-sensitive strain is capable of binding to taxol due to the presence of Thr 219, and the taxol-resistant strain is incapable of binding to taxol due to the absence of Thr 219. Such taxol-resistant strains can be used in combination with the wild-type taxol-sensitive strains for screening as described in later examples.
Example 10: Screening Assays to Detect Taxol or Taxol-like Compounds in Matter
Several assays can be used to detect taxol or taxol-like compounds in a composition of matter. These assays are useful for screening a variety of compositions of matter, including living matter such as plants or microorganisms, or non-living matter such as plant materials, patient samples, or compound libraries for the presence of taxol or taxol-like compounds.
One screening method is to use taxol-resistant P. microspora in combination with the taxol-sensitive P. ultimum or P. cinnamomi. Taxol inhibits the growth of both P. ultimum by binding to their beta-tubulin, while taxol does not affect the growth of P. microspora since it does not interact with its beta-tubulin. A composition of matter which is capable of the inhibition of P. ultimum, but not P. microspora has a high probability of containing taxol-or a taxol-like compound.
An improved screening method uses taxol-sensitive and taxol-resistant isogenic strains of P. ultimum or P. cinnamomi as described in above example. The composition of matter is used to examine its effect on the growth of both the taxol-sensitive as well as the taxol-resistant strains. The inhibition of the taxol-sensitive strain but not the taxol-resistant strain indicates the presence of taxol or a taxol-like compound. On the other hand, the non- inhibition of both the taxol-sensitive and taxol-resistant strains indicates the absence of taxol or a taxol-like compound.
Composition of matter can be screened for the presence of taxol or taxol-like compounds based on their ability to promote the assembly of microtubules, as well as to stabilize assembled microtubules in conditions such as cold which otherwise cause depolymerization (Schiff, et al. 1979; Horwitz, 1981). The alpha- and beta-tubulins used in these assays can be from the following sources. 1) native microtubules consisting of beta- tubulins and alpha-tubulins extracted from P. ultimum or P. cinnamomi; 2) beta-tubulins extracted from P. ultimum or P. cinnamomi and interacted with another source of alpha- tubulin, for example, bovine alpha-tubulin; 3) all or portions of SEQ ID NO:4 or SEQ ID NO: 6 produced from a heterologous system such as E. coli, yeast, insect cells or the like and alpha-tubulin either from P. ultimum, P. cinnamomi or another source. If the composition matter has the ability to promote the assembly of these MTs, as well as to prevent
depolymerization of assembled MTs in conditions which otherwise cause depolymerization, the composition of matter is likely to contain taxol or a taxol-like compound. Meanwhile, these isolated compounds should be unable to promote the assembly of MTs as well as prevent the depolymerization of MTs which consist of beta-tubulin derived from P. microspora.
An alternative screening method can be performed based on the competitive inhibition of [3H]taxol binding to MTs in P. ultimum or P. cinnamomi by taxol or taxol-like compounds. The specific binding of [3H]taxol to P. ultimum is performed as described in Example 5. The amount of [3H]taxol specifically bound to P. ultimum in the absence of inhibitors is considered 100%. The composition of matter is added to the assay mixture, and the amount of [3H]taxol specifically bound to P. ultimum in the presence of the composition of matter is measured. Reduction in the [3H]taxol specific binding indicates that the composition of matter possesses taxol-like quality. If increased concentrations of the composition of matter can completely inhibit the [3H]taxol binding, it will indicate that the compound likely binds to the same binding site in the beta-tubulin in MTs.
The screening of compositions of matter for taxol or taxol-like compounds can be performed by one of the above methods. Preferably, one of the first two methods is used for an initial screening, since they are simple to perform and easily handle large amounts of samples. The third and fourth method can be used for subsequent screening.
Example 11: Screening Assay to Distinguish
Taxol-Sensitivity of A Patient Sample
In this diagonostic assay, antibodies depicted in Example 7 which could distinguish taxol-binding beta-tubulin from the non-binding beta-tubulin are used. Cellular proteins are extracted from a tumor specimen from a patient sample to detect the presence of a beta- tubulin with either taxol-binding or non-binding capabilities.
For example, in a diagnostic assay to screen for tumors resistant to taxol, the taxol binding regions of taxol-sensitive and taxol-resistant beta-tubulins of the present invention
(e.g., SEQ ID NOJ, SEQ ID NO:4, and SEQ ID NO:6) are used to raise monoclonal or polyclonal antibodies using standard methods well known in the art (Harlow, E D. and Lane, D. 1988. Antibodies: A Laboratory Manual). For example, monoclonal antibody probes are
reacted with a patient sample, such as a tumor specimen, to detect the presence of a beta- tubulins with either taxol-binding or non-binding capabilities. Visualization of antibody- antigen binding is mediated through any means known in the art, e.g., secondary radiolabeled or fluorescent antibodies or colorimetric methods using peroxidase and/or alkaline phosphatase (Harlow, E.D. and Lane, D. 1988. Antibodies: A Laboratory Manual). The detection of beta-tubulins with taxol-binding capability, i.e., taxol-sensitive beta- tubulins, corresponds to a positive response to taxol therapy. Alternatively, the detection of non-binding taxol-resistant beta-tubulins and/or the absence of taxol-sensitive beta-tubulins corresponds to a diminished or lack of response to taxol therapy.
Example 12: Biocontrol of Taxol-sensitive Pathogenic
Oomycetes Using P. microspora on Plants
Many oomycetes including P. ultimum and P. cinnamomi are plant pathogens which can cause crop damage and result in severe economical loss. For instance, P. ultimum causes root rot of beans, and P. cinnamomi causes root rot of Avacado (ATCC: Catalogue of Filamentous Fungi, 18th edition, 1991). Many of the oomycetes are also taxol-sensitive (Young, et al. 1992. "Antifungal properties of taxol and various analogues," Experientia 48:882-885). Two of these strains, P. ultimum and P. cinnamomi, contain threonine at Amino Acid 219.
The biocontrol method of the present invention involves a two-step process: 1) the taxol sensitivity of the plant pathogen is determined and 2) if the plant pathogen is taxol- sensitive, a taxol-producing P. microspora is applied to the infected plants and surrounding soil as a source of growth-inhibiting taxol.
The taxol sensitivity of the plant pathogen is first determined. One method of identifying taxol sensitivity is to determine the presence or absence of threonine at Amino Acid 219. If the identity of the pathogen is known, DNA and protein databases are searched to determine whether the beta-tubulin sequence has been reported, if so, the identity of Amino Acid 219 is determined from the database. If the pathogen's beta-tubulin sequence is unavailable, the cDNA sequence is isolated and analyzed to determine the identity of Amino Acid 219. The presence of threonine at Amino Acid 219 in the pathogen's beta-tubulin gene indicates sensitivity to taxol, and thus, the pathogen is designated as treatable by a taxol-
producing P. microspora. If Amino Acid 219 is not threonine, the taxol sensitivity would have to be determined by other means such as taxol growth inhibition. Other screening methods presented herein for determining the presence of taxol-binding beta-tubulins can also be used.
It has been previously reported that P. microspora produces taxol at 50 ug /liter
(Strobel, et al. 1996. Microbiol 142:435-440) and secrets taxol outside of the fungal cell. At this taxol concentration, the growth of P. ultimum and P. cinnamomi is inhibited (see Fig. 1). For treatment, P. microspora is inoculated to the area of plants or soil infected with the taxol-sensitive pathogen. The growth of P. microspora results in the secretion of taxol, which consequently inhibits the growth of these taxol-sensitive organisms.
Example 13: Use of Crystal Structures in Design of Antineoplastic or Antifungal Drugs
The three-dimensional structure of beta-tubulins are used to rationally design taxol- like compounds using methods known in the art (Ealick, et al. 1991. "Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors," Science 88: 11540-11544; Rossman, et al. 1991. "Application of crystallography to the design of antiviral agents," Infectious Agents and Disease 1 :3-10). As taught by the present invention, application of the knowledge that Thr219 in the protein structure plays an important role in binding of taxol to taxol-like compounds can be critically applied to the development of drugs having taxol-like activities.
Claims
1. A purified DNA segment encoding a beta-tubulin of the fungal species Pestalotiopsis microspora or a portion thereof.
2. The DNA segment of Claim 1, wherein said portion encodes at least one taxol binding site.
3. The DNA segment of Claim 2, wherein said portion encodes a protein having taxol binding site I and taxol binding site II.
4. The DNA segment of Claim 3, wherein said protein is able to interact with alpha- tubulin.
5. The DNA segment of Claim 1, wherein said DNA segment comprises at least a portion of SEQ ID NOJ .
6. The DNA segment of Claim 5, wherein said portion comprises the nucleotide sequence from nucleotide 75 through nucleotide 167 of SEQ ID NOJ.
7. The DNA segment of Claim 6, wherein at least one nucleotide in said nucleotide sequence is substituted.
8. The DNA segment of Claim 5, wherein said portion comprises the nucleotide sequence from nucleotide 708 through nucleotide 764 of SEQ ID NOJ
9. The DNA segment of Claim 8, wherein at least one nucleotide in said nucleotide sequence is substituted.
10. The DNA segment of Claim 9, wherein nucleotide 729, nucleotide 730, nucleotide 731 or mixtures thereof are substituted.
11. The DNA segment of Claim 5, comprising the nucleotide sequence from nucleotide 75 to nucleotide 1412 of SEQ ID NOJ, said DNA segment encoding a beta- tubulin.
12. The DNA segment of Claim 11, wherein at least one nucleotide in said nucleotide sequence is substituted and wherein the taxol binding capacity of said beta- tubulin is not altered.
13. The DNA segment of Claim 11, wherein at least one nucleotide in said nucleotide sequence is substituted and wherein the taxol binding capacity of said beta- tubulin is altered.
14. An amino acid sequence comprising at least a portion of a beta-tubulin of the fungal species Pestalotiopsis microspora.
15. The amino acid sequence of Claim 14, wherein said portion comprises at least one taxol binding site.
16. The amino acid sequence of Claim 15, wherein said portion comprises taxol binding site I and taxol binding site II.
17. The amino acid sequence of Claim 16, wherein said portion is able to interact with alpha-tubulin.
18. The amino acid sequence of Claim 14, wherein said amino acid sequence comprises at least a portion of the beta-tubulin as depicted in SEQ ID NOJ.
19. The amino acid sequence of Claim 18, wherein said portion comprises Amino Acids 1-31 of SEQ ID NOJ.
20. The amino acid sequence of Claim 19 having at least one amino acid substitution.
21. The amino acid sequence of Claim 18, wherein said portion comprises Amino Acids 212-230 of SEQ ID NOJ.
22. The amino acid sequence of Claim 21 having at least one amino acid substitution.
23. The amino acid sequence of Claim 18, wherein said portion comprises an amino acid substitution at Amino Acid 219.
24. The amino acid sequence of Claim 18, wherein said portion consists essentially of Amino Acids 1-446 of SEQ ID NOJ and wherein said portion behaves as a taxol- resistant beta-tubulin.
25. The amino acid sequence of Claim 24, wherein said portion contains at least one amino acid substitution that alters the taxol binding property of said portion.
26. The amino acid sequence of Claim 24, wherein said portion contains at least one amino acid substitution that does not alter the taxol binding property of said portion.
27. The amino acid sequence of Claim 14, wherein said amino acid sequence is substituted with any amino acid which perturbs the three-dimensional structure of said amino acid sequence surrounding Amino Acid 219 as numbered in SEQ ID NOJ.
28. A purified DNA segment encoding a beta-tubulin of the fungal species Pythium ultimum or a portion thereof.
29. The DNA segment of Claim 28, wherein said portion encodes at least one taxol binding site.
30. The DNA segment of Claim 29, wherein said portion encodes a protein having taxol binding site I and taxol binding site II.
31. The DNA segment of Claim 30, wherein said protein is able to interact with alpha-tubulin.
32. The DNA segment of Claim 28, wherein said DNA segment comprises at least a portion of SEQ ID NOJ.
33. The DNA segment of Claim 32, wherein said portion comprises the nucleotide sequence from nucleotide 92 through nucleotide 184 of SEQ ID NOJ.
34. The DNA segment of Claim 33, wherein at least one nucleotide in said nucleotide sequence is substituted.
35. The DNA segment of Claim 32, wherein said portion comprises the nucleotide sequence from nucleotide 725 through nucleotide 781 of SEQ ID NOJ
36. The DNA segment of Claim 35, wherein at least one nucleotide in said nucleotide sequence is substituted.
37. The DNA segment of Claim 35, wherein nucleotide 746, nucleotide 747, nucleotide 748 or mixtures thereof are substituted.
38. The DNA segment of Claim 32, comprising the nucleotide sequence from nucleotide 92 to nucleotide 1429 of SEQ ID NOJ, said DNA segment encoding a beta- tubulin.
39. The DNA segment of Claim 38, wherein at least one nucleotide in said nucleotide sequence is substituted and wherein the taxol binding capacity of said beta- tubulin is not altered.
40. The DNA segment of Claim 38, wherein at least one nucleotide in said nucleotide sequence is substituted and wherein the taxol binding capacity of said beta- tubulin is altered.
41. An amino acid sequence comprising at least a portion of a beta-tubulin of the fungal species Pythium ultimum.
42. The amino acid sequence of Claim 41, wherein said amino acid sequence comprises at least one taxol binding site.
43. The amino acid sequence of Claim 42, wherein said portion comprises taxol binding site I and taxol binding site II.
44. The amino acid sequence of Claim 43, wherein said portion is able to interact with alpha-tubulin.
45. The amino acid sequence of Claim 41, wherein said amino acid sequence comprises at least a portion of the beta-tubulin as depicted in SEQ ID NO:4.
46. The amino acid sequence of Claim 45, wherein said portion comprises Amino Acids 1-31 of SEQ ID NO:4
47. The amino acid sequence of Claim 46, having at least one amino acid substitution.
48. The amino acid sequence of Claim 45, wherein said portion comprises Amino Acids 212-230 of SEQ ID NO:4
49. The amino acid sequence of Claim 48, having at least one amino acid substitution.
50. The amino acid sequence of Claim 45, wherein said portion comprises an amino acid substitution at Amino Acid 219.
51. The amino acid sequence of Claim 45, wherein said portion consists essentially of Amino Acids 1-446 of SEQ ID NO:4 and wherein said portion behaves as a taxol- sensitive beta-tubulin.
52. The amino acid sequence of Claim 51, wherein said portion contains at least one amino acid substitution that alters the taxol binding property of said portion.
53. The amino acid sequence of Claim 51, wherein said portion contains at least one amino acid substitution that does not alter the taxol binding property of said portion.
54. The amino acid sequence of Claim 41, wherein said amino acid sequence is substituted with any amino acid which perturbs the three-dimensional structure of said amino acid sequence surrounding Amino Acid 219 as numbered in SEQ ID NO:4.
55. A purified DNA segment encoding a beta-tubulin of the fungal species Phytophthora cinnamomi or a portion thereof, wherein said DNA segment consists essentially of at least a portion of SEQ ID NO:5.
56. The DNA segment of Claim 55, wherein said portion comprises the nucleotide sequence from nucleotide 11 through nucleotide 103 of SEQ ID NO: 5.
57. The DNA segment of Claim 56, wherein at least one nucleotide in said nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code nucleotide 80 cannot consist of adenine while nucleotide 81 is thymine and nucleotide 82 is adenine, cytosine or thymine.
58. The DNA segment of Claim 55, wherein said portion comprises the nucleotide sequence from nucleotide 644 through nucleotide 700 of SEQ ID NO: 5
59. The DNA segment of Claim 58, wherein at least one nucleotide in said nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code nucleotide 665 cannot be adenine while nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine.
60. The DNA segment of Claim 55, comprising the nucleotide sequence from nucleotide 1 1 to nucleotide 1342 of SEQ ID NO:5, said DNA segment encoding a beta- tubulin.
61. The DNA segment of Claim 60, wherein at least one nucleotide in said nucleotide sequence is substituted, providing that when nucleotide substitution changes only one amino acid code nucleotide 665 cannot be adenine while nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine.
62. The DNA segment of Claim 60 or 61, wherein at least one nucleotide in said nucleotide sequence is substituted, and wherein the taxol binding capacity of said beta- tubulin is not altered.
63. The DNA segment of Claim 60 or 61, wherein at least one nucleotide in said nucleotide sequence is substituted and wherein the taxol binding capacity of said beta- tubulin is altered.
64. An amino acid sequence comprising at least a portion of a beta-tubulin of the fungal species Phytophthora cinnamomi as depicted in SEQ ID NO: 6.
65. The amino acid sequence of Claim 64, wherein said portion comprises Amino Acids 1-31 of SEQ ID NO:6.
66. The amino acid sequence of Claim 65, having at least one amino acid is substituted, providing that when only one amino acid is substituted Amino Acid 24 is not isoleucine.
67. The amino acid sequence of Claim 64, wherein said portion comprises Amino Acids 212-230 of SEQ ID NO:6.
68. The amino acid sequence of Claim 67, having at least one amino acid is substituted, providing that when only one amino acid is substituted Amino Acid 219 is not asparagine.
69. The amino acid sequence of Claim 64, wherein said portion comprises an amino acid substitution at Amino Acid 219, wherein said Amino Acid 219 is not substituted with asparagine.
70. The amino acid sequence of Claim 64, wherein said portion consists essentially of Amino Acids 1-446 of SEQ ID NO:6 and wherein said portion behaves as a taxol- sensitive beta-tubulin.
71. The amino acid sequence of Claim 70, wherein said portion contains at least one amino acid substitution, providing that when only one amino acid is substituted Amino Acid 219 is not asparagine, and wherein said amino acid substitution that alters the taxol binding property of said portion.
72. The amino acid sequence of Claim 70, wherein said portion contains at least one amino acid substitution, providing that when only one amino acid is substituted Amino Acid 219 is not asparagine, and wherein said amino acid substitution does not alter the taxol binding property of said portion.
73. The amino acid sequence of Claim 64, wherein said amino acid sequence is substituted with any amino acid which perturbs the three-dimensional structure of said amino acid sequence surrounding Amino Acid 219 and wherein when only one amino acid is substituted at Amino Acid 219 said substituted amino acid is not asparagine.
74. A vector comprising a purified DNA segment encoding a beta-tubulin of the fungal species Pestalotiopsis microspora or a portion thereof.
75. The vector of Claim 74, wherein said portion encodes at least one taxol binding site.
76. A vector comprising a purified DNA segment encoding a beta-tubulin of the fungal species Pythium ultimum or a portion thereof.
77. The vector of Claim 76, wherein said portion encodes at least one taxol binding site.
78. A vector comprising a purified DNA segment encoding a beta-tubulin of the fungal species Phytophthora cinnamomi wherein said DNA segment consists essentially of SEQ ID No: 5 or a portion thereof.
79. The vector of Claim 78, wherein said portion encodes at least one taxol binding site.
80. A method of determining the taxol binding capacity of a beta-tubulin or beta- tubulin-like compound comprising providing antibodies raised against amino acid sequences comprising at least one taxol binding site of a beta-tubulin from a taxol-resistant Pestalotiopsis microspora, a taxol- sensitive Pythium ultimum, or taxol-sensitive Phytophthora cinnamomi as depicted in SEQ ID NO:6 to form a reagent, wherein said antibodies distinguish between taxol-binding and non-taxol-binding properties; contacting said beta-tubulin with said reagent; and determining degree of binding between said antibodies in said reagent and said beta- tubulin or beta-tubulin-like compound; whereby binding of antibodies which specifically recognize taxol-binding properties indicate taxol sensitive; whereby binding of antibodies which specifically recognize taxol- non-binding properties indicate taxol resistance.
81. The method of Claim 80, wherein said antibodies are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol- resistant Pestalotiopsis microspora.
82 The method of Claim 81, wherein said amino acid sequence comprises at least one taxol binding site from SEQ ID NO 2
83 The method of Claim 80, wherein said antibodies are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol- sensitive Pythium ultimum.
84 The method of Claim 81, wherein said amino acid sequence comprises at least one taxol binding site from SEQ ID NO 4
85 The method of Claim 80, wherein said antibodies are raised against an amino acid sequence comprising at least one taxol binding site of a beta-tubulin from a taxol- sensitive Phytophthora cinnamomi as depicted in SEQ ID NO 6.
86 The method of Claims 80, 81, 82, 83, 84 or 85, wherein said beta-tubulin or beta-tubulin-like protein is selected from the group consisting of recombinantly expressed protein, exogenously isolated protein, synthetic peptides, and cell cultures
87 A method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing beta-tubulins with amino acid sequences comprising both taxol binding sites from taxol-sensitive Pythium ultimum or taxol-sensitive Phytophthora cinnamomi as depicted in SEQ ID NO 6 in addition to alpha-tubulin from any taxol-sensitive organism to form a reagent, contacting said composition of matter with said reagent, and determining the ability of the composition of matter to promote MT assembly or ability to prevent depolymerization of assembled MTs under depolymerizing conditions, whereby the ability to promote microtubule assembly or prevent depolymerization indicate the possible presence of taxol or taxol-like compounds in said composition of matter
88 A method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing mycelia of taxol-sensitive Pythium ultimum or a taxol-sensitive Phytophthora cinnamomi as depicted in SEQ ID NO 6, contacting said composition of matter with said mycelia in the presence of said labeled taxol; and determining degree of competitive inhibition of binding between said beta-tubulins and said labeled taxol by said composition of matter; whereby the composition of matter is determined to possess taxol or taxol-like compounds if it is able to block taxol binding to the beta-tubulins from the taxol-sensitive Pythium ultimum or Phytophthora cinnamomi.
89. A method of altering the taxol binding property of a recombinantly expressed beta-tubulin or a portion thereof comprising determining the identity of the codon at Amino Acid 219 as numbered in SEQ ID NOJ in the coding region of the vector; and if said codon at Amino Acid 219 codes for any amino acid except threonine, substituting nucleotides in said codon to code for threonine at Amino Acid 219 to alter a non-taxol-binding beta-tubulin or portion thereof to a taxol-binding beta-tubulin or portion thereof or if said codon at Amino Acid 219 codes for threonine, substituting nucleotides in said codon to code for any amino acid except threonine at Amino Acid 219 to alter a taxol- binding beta-tubulin or portion thereof to a non-taxol-binding beta-tubulin or portion thereof.
90. A method of developing a taxol-sensitive fungal cell from a taxol-resistant fungal cell comprising transforming said non-taxol-sensitive fungal cell by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ; wherein said transformed fungal cell expresses said DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
91. A transgenic taxol-sensitive fungal cell transformed by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ, wherein said transformed fungal cell expresses said DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
92. A method of developing a taxol-resistant fungal cell from a taxol-sensitive fungal cell comprising transforming said taxol-sensitive fungal cell by introducing a DNA segment encoding non-taxol-binding beta-tubulin wherein the amino acid at Amino Acid 219 as numbered in SEQ ID NOJ is not threonine; wherein said transformed fungal cell over-expresses said DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
93. A transgenic taxol-sensitive fungal cell transformed by introducing a DNA segment encoding taxol-binding beta-tubulin comprising threonine at Amino Acid 219 as numbered in SEQ ID NOJ, wherein said transformed fungal cell over-expresses said DNA segment under the control of a suitable constitutive or inducible promoter when exposed to conditions which permit expression.
94. A method of screening a composition of matter for the presence of taxol or taxol-like compounds comprising providing distinguishable taxol-resistant and taxol-sensitive fungal cells; contacting said composition of matter with said fungal cells; determining the growth inhibition of said fungal cells; whereby the composition of matter is determined to possess taxol or taxol-like compounds if it is able to inhibit the growth of taxol-sensitive fungal cells but not able to inhibit the growth of taxol-resistant fungal cells.
95. The method of Claim 94, wherein said distinguishable taxol-resistant and taxol- sensitive fungal cells consists essentially of transgenic taxol-resistant and taxol-sensitive isogenic fungal cells.
96. The method of Claim 94, wherein said taxol-resistant fungal cells are derived from a fungi which is unrelated to the fungi from which the taxol-sensitive fungal cells are derived.
97. A method for controlling the growth of plant pathogens comprising determining the taxol sensitivity of said plant pathogen; and if said pathogen is determined to be taxol-sensitive, said plant and soil surrounding said plant are treated with a taxol-producing P. microspora.
98. The method of Claim 97, wherein the taxol sensitivity of said plant pathogen is determined by identifying Amino Acid 219, wherein the plant is designated as taxol- sensitive if Amino Acid 219 is threonine.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002367471A CA2367471A1 (en) | 1999-03-23 | 2000-03-23 | Fungal beta-tubulin genes |
| JP2000606753A JP2002541782A (en) | 1999-03-23 | 2000-03-23 | Fungal β-tubulin gene |
| EP00919658A EP1163345A1 (en) | 1999-03-23 | 2000-03-23 | Fungal beta-tubulin genes |
| AU40311/00A AU4031100A (en) | 1999-03-23 | 2000-03-23 | Fungal beta-tubulin genes |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US12571799P | 1999-03-23 | 1999-03-23 | |
| US60/125,717 | 1999-03-23 |
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| WO2000056894A1 true WO2000056894A1 (en) | 2000-09-28 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2000/007995 WO2000056894A1 (en) | 1999-03-23 | 2000-03-23 | Fungal beta-tubulin genes |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1163345A1 (en) |
| JP (1) | JP2002541782A (en) |
| AU (1) | AU4031100A (en) |
| CA (1) | CA2367471A1 (en) |
| WO (1) | WO2000056894A1 (en) |
Cited By (6)
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| WO2001013122A3 (en) * | 1999-08-19 | 2001-06-21 | Tularik Inc | Method for monitoring beta tubulin isotype modification |
| WO2003008965A1 (en) * | 2001-07-20 | 2003-01-30 | Consejo Superior De Investigaciones Científicas | Method of detecting and analysing paclitaxel-mimetic compounds |
| WO2003064617A3 (en) * | 2002-01-30 | 2004-04-01 | Univ Montana State | Pestalotiopsis microsporia isolates and compounds derived therefrom |
| CN104211776A (en) * | 2013-05-03 | 2014-12-17 | 南京工业大学 | Strong secretory signal peptide enhanced small peptide module sequence and application thereof |
| CN106868164A (en) * | 2017-03-23 | 2017-06-20 | 福建省农业科学院植物保护研究所 | A kind of primer and nested PCR detection method for detecting camphor tree phytophthora |
| CN112029887A (en) * | 2020-08-20 | 2020-12-04 | 中国医学科学院药用植物研究所 | Gene, primer, kit and method for detecting fusarium solani carbendazim resistant strain |
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| JP4771755B2 (en) * | 2005-03-28 | 2011-09-14 | アサヒ飲料株式会社 | Oligonucleotide, eukaryotic detection method and identification method using oligonucleotide |
| KR101150749B1 (en) | 2009-11-18 | 2012-06-08 | 주식회사 한국감염관리본부 | A card-type plate for detection of infectious pathogens which is easily attached and detached |
| CN110468233A (en) * | 2019-09-26 | 2019-11-19 | 四川农业大学 | A kind of primer, kit and the detection method of quick detection Machilus nanmu leaf blight |
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2000
- 2000-03-23 EP EP00919658A patent/EP1163345A1/en not_active Withdrawn
- 2000-03-23 AU AU40311/00A patent/AU4031100A/en not_active Abandoned
- 2000-03-23 WO PCT/US2000/007995 patent/WO2000056894A1/en not_active Application Discontinuation
- 2000-03-23 CA CA002367471A patent/CA2367471A1/en not_active Abandoned
- 2000-03-23 JP JP2000606753A patent/JP2002541782A/en active Pending
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001013122A3 (en) * | 1999-08-19 | 2001-06-21 | Tularik Inc | Method for monitoring beta tubulin isotype modification |
| US6306615B1 (en) * | 1999-08-19 | 2001-10-23 | Tularik Inc. | Detection method for monitoring β tubulin isotype specific modification |
| WO2003008965A1 (en) * | 2001-07-20 | 2003-01-30 | Consejo Superior De Investigaciones Científicas | Method of detecting and analysing paclitaxel-mimetic compounds |
| ES2180453A1 (en) * | 2001-07-20 | 2003-02-01 | Consejo Superior Investigacion | Method of detecting and analysing paclitaxel-mimetic compounds |
| US7476511B2 (en) | 2001-07-20 | 2009-01-13 | Jose Manuel Andreu Morales | Method of detecting and analyzing paclitaxel-mimetic compounds |
| WO2003064617A3 (en) * | 2002-01-30 | 2004-04-01 | Univ Montana State | Pestalotiopsis microsporia isolates and compounds derived therefrom |
| US7192939B2 (en) | 2002-01-30 | 2007-03-20 | Montana State University | Pestalotiopsis microsporia isolates and compounds derived therefrom |
| CN104211776A (en) * | 2013-05-03 | 2014-12-17 | 南京工业大学 | Strong secretory signal peptide enhanced small peptide module sequence and application thereof |
| CN106868164A (en) * | 2017-03-23 | 2017-06-20 | 福建省农业科学院植物保护研究所 | A kind of primer and nested PCR detection method for detecting camphor tree phytophthora |
| CN106868164B (en) * | 2017-03-23 | 2020-09-22 | 福建省农业科学院植物保护研究所 | A kind of primer and nested PCR detection method for detecting Phytophthora cinnamomea |
| CN112029887A (en) * | 2020-08-20 | 2020-12-04 | 中国医学科学院药用植物研究所 | Gene, primer, kit and method for detecting fusarium solani carbendazim resistant strain |
Also Published As
| Publication number | Publication date |
|---|---|
| EP1163345A1 (en) | 2001-12-19 |
| CA2367471A1 (en) | 2000-09-28 |
| JP2002541782A (en) | 2002-12-10 |
| AU4031100A (en) | 2000-10-09 |
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