JP2002541782A - Fungal β-tubulin gene - Google Patents

Abstract

(57) [Summary] Taxol, an anticancer drug, binds to β-tubulin in microtubules (MT) assembled in animal cells and causes cell cycle arrest, whereas the effect of taxol is It is different in fungi. For example, the ascomycete taxol-producing Pestalotiopsis microspora Ne 32 is taxol resistant (IC ₅₀ > 11.7 M), and two oomycete species, Pythium ultimum and Phytophthora cinnamomi, are taxol sensitive (IC ₅₀ 0.1 μM). CDNAs encoding β-tubulin from P. microspora, P. ultimum and P. cinnamomi have been isolated. The deduced amino acid sequences of β-tubulin from P. microspora, P. ultimum and P. cinnamomi are: (1) a binding assay to detect taxol and taxol-like substances; (2) pharmacology of taxol on tumor samples (3) Designing drugs with taxol-like activity by utilizing information about the effect of particular residues on taxol binding; and (4) substituting the residues required for taxol binding It can be used to detect taxol and taxol-like activity by using the taxol-sensitive and taxol-resistant isogenic strains of P. ultimum thus constructed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] TECHNICAL FIELD THE INVENTION The present invention relates, biological affect the composition and to the biological diagnostic reagents and biological experiments reagent.

[0002] Background of the Invention TAXOL (registered trademark) (Bristol-Myers-Squibb) is known as the generic name paclitaxel (hereinafter, referred to as "taxol" herein), with respect to breast cancer and uterine cancer Are complex diterpenoids that exhibit antitumor activity (Rowinsky, EK and Don
ehower, RC 1991, The clinical pharamacology and use of antimicrotubule agents in cancer
r chemotherapeutics) Pharmacol Ther 52: 35-84). Its antitumor activity depends on its ability to bind and stabilize β-tubulin in assembled microtubules (MT). (Manfredi
, JJ and Horwitz, SB 1984 "Taxol: an antimitotic agent with a new mechanism of action"
Pharmacol Ther 25: 83-125; and Horwitz, SB 1992 `` Mechanism of action of taxol '' Trends Pharmacol Sci 13: 134-136)
. In vivo, taxol affects spindle function during mitosis and regulates the cell cycle by G
Stop at 2 / M phase. In vitro, taxols promote MT assembly and prevent their degradation under conditions that would otherwise cause depolymerization (Schiff et al., 1979).
“Promotion of microtubule assembly in vitro by taxol” (Promotion of microt
ubule assembly in vitro by taxol) Nature 277: 665-667; and Palmes, J
And Horwitz, SB 1981 "Taxol binds to polymerized tubulin in vitro" J Cell Biol 91: 4
79-487). The binding site of taxol on β-tubulin has been characterized by photocross-labeling, electron crystallography and mutagenesis and includes several regions of β-tubulin (Rao et al., 1994 `` 3 '-( (p-azidobenzamide)
Taxol photolabels the N-terminal 31 amino acids of β-tubulin '' (3 '-(
p-Azidobenzamido) taxol photolabels the N-terminal 31 amino acids of β-
tubulin) J Biol Chem 269: 3132-3134; Rao et al., 1995 `` Characterization of the taxol binding site on t ''
he microtubule) J Biol Chem 270: 20235-20238; Nogales et al., 1998 "Structure of the αβ-tubulin dimer by electron crystallography."
mer by electron crystallography) Nature 391: 199-203; Nogales et al., 1999.
High-resolution model of the microtubule Cell
And Giannakakou et al., 1997, `` Paclitaxel-resistant human ovarian cancer cells have mutant β, with paclitaxel-resistant human uterine cancer cells having mutant β-tubulin and exhibiting defects in paclitaxel-driven polymerization. '' -tubu
lins that exhibit impaired paclitaxel-driven polymerization) J Biol Chem
272: 17118-17125).

[0003] Taxol was originally discovered by Wani et al. In the endothelium of Taxus brevifolia (Wani et al., 1971 "Vegetable antitumor agent VI Taxus brevifolia (Ta
xus brevifolia), a new anti-leukemia and anti-tumor drug, Taxol. (Plant antitumor agents. VI. The isolation and structure of
taxol, a novel antileukemic and antitumor agent from Taxus brevifolia) J
Am Chem Soc 93: 2325-2327), which has been shown to constitute about 0.02% of the dry phloem weight. The limited resources of the yew tree have made the search for additional sources of taxol useful.

[0004] In 1993, Stierle et al. Reported the isolation of a taxol-producing fungus, Taxomyces andreanae, an endoparasitic plant associated with T. brevifolia (Stierle et al., 1993, Taxols and taxanes by Taxomyces andreanae, an endophytic fungus of the yew tree). Taxol and taxane production by Taxomyces andreanae, an endophytic
fungus of pacific yew) Science 260: 214-216). T. andreanae produces authentic taxol, albeit at very low levels (24-50 ng / L in liquid medium). Recently, additional taxol-producing fungi have been isolated, including various strains of various Pestalotiopsis microspora (Li et al., 1996 ``
Endoparasite Taxol-Producing Fungus from Taxodium distichum ”(Endo
phytic taxol-producing fungi from bald cypress, Taxodium distichum) Micr
obiolog 142: 223-226; Li et al., 1998 `` Induction of taxol production in Periconia sp., an endophytic fungus from Torreya grandifolia '' (The induction of
taxol production in the endophytic fungus-Periconia sp. from Torreya gr
andifolia) J Ind Microbiol Biotechnol 20: 259-264; Strobel et al., 1996 `` Taxol from fungal endophytes and biodiversity issues '' (Taxol from fungal e
(Indophytes and the issue of biodiversity) J Ind Microbiol 17: 417-423; and Strobel et al., 1996 `` Pestalotiopsis mi, an endoparasitic fungus of Taxus wallachiana.
Taxol from crospora '' (Taxol from Pestalotiopsis microspora, an endoph
ytic fungus of Taxus wallachiana) Microbiolog 142: 435-440). One P.micro
spora strain, Ne52, was isolated from the endothelium of Himalaya yew T. wallachiana and
Produces about 50 μg taxol per liter (Strobel et al., 1996.
olog 142: 435-440; and Li et al., 1998. "Stimulation of taxol production in li"
quid culture of Pestalotiopsis microspora) Mycol Res 102: 461-464). Taxol produced from a fungal source has been reported to be identical by microscopy and chromatography to taxol isolated from yew and showed similar pharmacological effects on cancer cell lines (Strobel et al.). , 1996.Microbiolog 142: 435-440
). Taxol production from fungal sources provides a broader source of taxol, reduced production costs, and a means to meet the increasing demand for taxol.

[0005] Taxol is a cell of a wide range of organisms, including mammals, sea urchins, Drosophilia, Xenopus, Physarum, Haemanthus, and Trypanosoma. (Bum et al., 1981. "Taxol, a microtubule-stabilizing drug, inhibits replication of Trypanosoma cruzi.") (Taxol, a microtubule
stabilizing agent, blocks the replication of Trypanosoma cruzi) Proc Na
tl Acad Sci USA 78: 4571-4575; Kellogg et al., 1989. "Proteins in the centrosome, spi."
ndle, and kinetochore of the early Drosophila embryo) J Cell Biol 109: 29
77-2991; and Manfredi, JJ and Horwitz, SB 1984. Pharmacol Th
er 25: 83-125) On the other hand, in fungi, various sensitivities to taxol have been reported. Young et al. Tested taxol toxicity against representative species from various fungal groups (Young et al., 1992. “Anti-fungal properties of taxol and various analogs” (Ant
ifungal properties of taxol and various analogues) Experientia 48: 882-88
Five). In a study by Young et al., The plant pathogens Phythium ultimum and Phytoph
Five oomycete species, including thora capsici, were identified as taxol sensitive (IC ₅₀ 0.4-5.9 μM). In P. capsici, taxol inhibits nuclear division at low concentrations, suggesting that taxol acts by a mechanism similar to that on mammalian cells. In contrast, four ascomycetes species were identified as taxol resistant (IC ₅₀ > 50 μM). This resistance has been reported to be due to a reduced ability of fungal microtubules to interact with taxol. Taxol is also purified S. c.
Inability to stabilize erevisiae tubulin assembled MT (Bames et al., 1992.
"Yeast protein associated with microtubules in vitro and in vivo" (Yeast p
roteins associated with microtubules in vitro and in vivo) Mol Biol Cell
3: 29-47), and only slightly stabilizing the MT of Aspergillus nidulans (Yoon, Y. and Oakley, BR 1995. Purification and characterization of assemblable tubulin from Aspergillus nidulans). of
assembly-competent tubulin from Aspergillus nidulans) Biochem 34: 6373-63
81) is shown.

[0006] Due to the fungal diversity for taxol's anti-cancer properties and taxol susceptibility,
There is a continuing need for the isolation and / or identification of novel β-tubulin genes that are useful for generating isogenic fungal strains that are either taxol sensitive or taxol resistant. These β-tubulin genes and / or isogenic fungal strains can then be applied for the screening of anticancer drugs and for the development of diagnostic tests for tumor susceptibility assays.

[0007] In summary one embodiment of the invention, the present invention is a purified DNA segment, or portion thereof encoding a β- tubulin fungal species Pestalotiopsis microspora. Preferably, the D
The NA segment encodes at least one taxol binding site. For some applications, the DNA segment preferably encodes a protein having taxol binding site I and taxol binding site II. With respect to a DNA segment that encodes a protein that functions as β-tubulin, the DNA segment has a taxol binding site I and a taxol binding site II and encodes a protein that can interact with α-tubulin. A typical DNA segment comprises at least a portion of SEQ ID NO: 1. Another exemplary DNA segment comprises a portion of SEQ ID NO: 1, wherein the portion is nucleotides 75 to nucleotides of SEQ ID NO: 1.
It contains up to 167 nucleotide sequences, substituted or unsubstituted. Another exemplary DNA segment comprises a portion of SEQ ID NO: 1, wherein the portion comprises the nucleotide sequence from nucleotide 708 to nucleotide 764 of SEQ ID NO: 1 with or without substitution. Another exemplary DNA segment comprises a portion of SEQ ID NO: 1, wherein the portion comprises the nucleotide sequence from nucleotide 708 to nucleotide 764 of SEQ ID NO: 1, nucleotide 729, nucleotide 730, or nucleotide 731, or a combination thereof. Has been replaced. Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 75 to nucleotide 1412 of SEQ ID NO: 1 and encodes β-tubulin. Another exemplary DNA segment comprises the nucleotide sequence of SEQ ID NO: 1 from nucleotide 75 to nucleotide 1412, wherein at least one of the nucleotide sequences
Have been substituted and the ability of β-tubulin to bind taxol has not changed. Another exemplary DNA segment comprises the nucleotide sequence of SEQ ID NO: 1 from nucleotide 75 to nucleotide 1412, wherein at least one nucleotide in the nucleotide sequence has been substituted and the ability of β-tubulin to bind taxol has been altered. are doing.

In another aspect, the invention is an amino acid sequence comprising at least a portion of β-tubulin of the fungal species Pestalotiopsis microspora. Preferably, the amino acid sequence comprises at least one taxol binding site. For some uses,
The amino acid sequence is preferably a protein having a taxol binding site I and a taxol binding site II. For amino acid sequences that can function as β-tubulin, the amino acid sequence has a taxol binding site I and a taxol binding site II and is capable of interacting with α-tubulin. A typical amino acid sequence comprises at least a portion of β-tubulin as set forth in SEQ ID NO: 2. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 2, which portion includes amino acids 1-31 with or without substitution. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 2, which includes amino acids 212-230, with or without substitution. Another exemplary amino acid sequence includes a portion of SEQ ID NO: 2, which includes amino acids 212-230 and has an amino acid substitution at amino acid 219. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 2, which portion consists essentially of amino acids 1-446, which portion acts as taxol-resistant β-tubulin. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 2, wherein the portion consists essentially of amino acids 1-446, and wherein at least one alters the taxol binding properties of the portion.
Has amino acid substitutions. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 2, wherein the portion consists essentially of amino acids 1-446 and includes at least one amino acid substitution that does not alter the taxol binding properties of the portion. Another exemplary amino acid sequence has been substituted around amino acid 219 of SEQ ID NO: 2 with any amino acid that disrupts the three-dimensional structure of the amino acid sequence.

In another aspect, the invention is a purified DNA segment encoding β-tubulin of the fungal species Phythium ultimum, or a portion thereof. Preferably, the DNA segment encodes at least one taxol binding site. For some applications, the DNA segment comprises taxol binding site I and taxol binding site II.
It preferably encodes a protein having With respect to a DNA segment that encodes a protein that functions as β-tubulin, the DNA segment has a taxol binding site I and a taxol binding site II and encodes a protein that can interact with α-tubulin. A typical DNA segment comprises at least a portion of SEQ ID NO: 3. Another exemplary DNA segment comprises a portion of SEQ ID NO: 3, which comprises nucleotides 92 to 184, with or without substitution. Another exemplary DNA segment comprises a portion of SEQ ID NO: 3, which comprises, with or without substitution, the nucleotide sequence from nucleotide 725 to nucleotide 781. Another exemplary DNA segment comprises a portion of SEQ ID NO: 3, wherein the portion comprises the nucleotide sequence from nucleotide 725 to nucleotide 781, wherein nucleotide 746, nucleotide 747, or nucleotide 748, or a combination thereof, has been replaced. I have. Another exemplary DNA segment comprises the nucleotide sequence from nucleotide 92 to nucleotide 1429 of SEQ ID NO: 3 and encodes β-tubulin. Another exemplary DNA segment comprises the nucleotide sequence of SEQ ID NO: 3 from nucleotide 92 to nucleotide 1429, wherein at least one nucleotide in the nucleotide sequence has been replaced, and the ability of β-tubulin to bind taxol has been altered. I haven't. Another exemplary DNA segment comprises the nucleotide sequence of SEQ ID NO: 3 from nucleotide 92 to nucleotide 1429, wherein at least one nucleotide in the nucleotide sequence has been replaced, and the ability of β-tubulin to bind taxol has been altered. are doing.

In another aspect, the invention is an amino acid sequence comprising at least a portion of β-tubulin of the fungal species Phythium ultimum. Preferably, the amino acid sequence comprises at least one taxol binding site. For some applications, the amino acid sequence is preferably a protein having taxol binding site I and taxol binding site II. For amino acid sequences that can function as β-tubulin, the amino acid sequence has a taxol binding site I and a taxol binding site II and is capable of interacting with α-tubulin. A typical amino acid sequence comprises at least a portion of β-tubulin as set forth in SEQ ID NO: 4. Another exemplary amino acid sequence includes a portion of SEQ ID NO: 4, wherein the portion includes amino acids 1-31 with or without substitution. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 4, which portion includes amino acids 212-230, with or without substitution. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 4, wherein the portion comprises amino acids 212-23.
Including 0 and having an amino acid substitution at amino acid 219. Another typical amino acid sequence is
It comprises a portion of SEQ ID NO: 4, which portion consists essentially of amino acids 1-446 and acts as a taxol-sensitive β-tubulin. Another typical amino acid sequence is
4 comprising a portion of SEQ ID NO: 4, wherein the portion consists essentially of amino acids 1-446 and has at least one amino acid substitution that alters the taxol binding properties of the portion. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 4, wherein the portion consists essentially of amino acids 1-446 and includes at least one amino acid substitution that does not alter the taxol binding properties of the portion. Another exemplary amino acid sequence has been substituted around amino acid 219 of SEQ ID NO: 4 with any amino acid that disrupts the three-dimensional structure of the amino acid sequence.

In another aspect, the invention is a purified DNA segment encoding β-tubulin of the fungal species Phytophthora cinnamomi, or a portion thereof, wherein the segment consists essentially of at least a portion of SEQ ID NO: 5. A typical DNA segment comprises a portion of SEQ ID NO: 5, which portion comprises the nucleotide sequence from nucleotide 11 to nucleotide 103. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5, wherein the portion comprises a nucleotide sequence from nucleotide 11 to nucleotide 103, wherein at least one nucleotide has been replaced in the nucleotide sequence, If substitution of a nucleotide changes only one amino acid code, provided that nucleotide 81 cannot be adenine when nucleotide 81 is thymine and nucleotide 82 is adenine, cytosine, or thymine. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5, which portion comprises a nucleotide sequence from nucleotide 644 to nucleotide 700. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5 and comprises a nucleotide sequence from nucleotide 644 to nucleotide 700, wherein at least one nucleotide has been substituted in the nucleotide sequence, provided that the nucleotide substitution has been made. Is 1
If only one amino acid code is changed, provided that nucleotide 666 is adenine and nucleotide 665 cannot be adenine when nucleotide 667 is cytosine or thymine. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5, which portion comprises a nucleotide sequence from nucleotide 11 to nucleotide 1342 and encodes β-tubulin. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5, wherein the portion is from nucleotide 11 to nucleotide 1342.
And wherein at least one nucleotide in the nucleotide sequence has been replaced, but wherein the nucleotide substitution changes only one amino acid code, nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine. , Provided that nucleotide 665 cannot be adenine. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5, wherein the portion comprises a nucleotide sequence from nucleotide 11 to nucleotide 1342, wherein at least one nucleotide has been replaced in the nucleotide sequence, If the nucleotide substitution changes only one amino acid code, provided that nucleotide 666 cannot be adenine when nucleotide 666 is adenine and nucleotide 667 is cytosine or thymine, this substitution results in β-tubulin Taxol binding ability does not change. Another exemplary DNA segment comprises a portion of SEQ ID NO: 5, wherein the portion comprises the nucleotide sequence from nucleotides 11 to 1342, wherein at least one nucleotide has been replaced in the nucleotide sequence, except that the nucleotide If the substitution changes only one amino acid code, provided that nucleotide 666 is adenine and that nucleotide 665 cannot be adenine when nucleotide 667 is cytosine or thymine, and that the β-tube Alters the ability of phosphorus to bind taxol.

In another embodiment, the present invention provides a fungal species Phytophthora cinnam as set forth in SEQ ID NO: 6.
2 is an amino acid sequence containing at least a portion of omi β-tubulin. A typical amino acid sequence comprises a portion of SEQ ID NO: 6, wherein the portion comprises amino acids 1-31. A typical amino acid sequence comprises a portion of SEQ ID NO: 6, wherein the portion is amino acid 1
And at least one substituted amino acid, provided that it cannot be isoleucine if amino acid 24 is the only substituted amino acid. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 6,
The portion comprises amino acids 212-230. Another exemplary amino acid sequence is SEQ ID NO: 6
Which comprises amino acids 212-230 and has at least one substituted amino acid, provided that when amino acid 219 is the only substituted amino acid, it cannot be asparagine It is a condition. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 6, which includes amino acids 212-230, has an amino acid substitution at amino acid 219, and amino acid 219 is not substituted with asparagine. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 6, which portion consists essentially of amino acids 1-446 and acts as a taxol-sensitive β-tubulin. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 6, wherein the portion consists essentially of amino acids 1 to 446, wherein the portion has at least one amino acid substitution, with the proviso that amino acid 219 is unique. If it is a substituted amino acid, provided that it cannot be asparagine, and this amino acid substitution alters the taxol binding properties of the portion. Another exemplary amino acid sequence comprises a portion of SEQ ID NO: 6, wherein the portion has at least one amino acid substitution, provided that amino acid 219 is the only substituted amino acid, And the amino acid substitution does not alter the taxol binding properties of the portion. Another typical amino acid sequence is amino acid 21
9 has been replaced by any amino acid other than asparagine that disrupts the three-dimensional structure of the amino acid sequence around amino acid 219.

In another aspect, the invention is a vector comprising a purified DNA segment encoding β-tubulin of a 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 β-tubulin of a fungal species Phythium ultimum, or a portion thereof. Preferably, the vector comprises a portion encoding at least one taxol binding site.

[0015] In another embodiment, the invention is a vector encoding a β-tubulin of the fungal species Phytophthora cinnamomi and comprising a purified DNA segment consisting 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 embodiment, the present invention is a method for measuring the taxol-binding ability of β-tubulin or a β-tubulin-like compound, wherein the taxol-resistant Pestalotiopsis microspora of SEQ ID NO: 6, a taxol-sensitive Pythium ultimum, Alternatively, an antibody against an amino acid sequence containing at least one taxol-binding site of β-tubulin derived from taxol-resistant Phytophthora cinnamomi, which distinguishes taxol-binding and non-taxol-binding characteristics, is prepared; Contacting the reagent with a tubulin or β-tubulin-like compound; and determining the extent of binding of the antibody in the reagent to β-tubulin or β-tubulin-like compound; thereby Taxol-resistant Pestalotiopsis microspora
Binding of antibodies to β-tubulin or β-tubulin-like compounds to taxol resistance, and binding of antibodies specifically recognizing taxol binding properties to taxol sensitivity; specificity to taxol non-binding properties This is a method in which the binding of an antibody that specifically recognizes taxol sensitivity. In one embodiment, the antibody in the reagent induces an amino acid sequence comprising at least one taxol binding site of taxol-resistant β-tubulin from Pestalotiopsis microspora. In another embodiment, the antibody in the reagent is directed against an amino acid sequence comprising at least one taxol binding site of SEQ ID NO: 2. In another embodiment, the antibody in the reagent is directed against an amino acid sequence comprising at least one taxol binding site of β-tubulin from taxol-sensitive Phythium ultimum. In another embodiment, the antibody in the reagent is directed against an amino acid sequence comprising at least one taxol binding site of SEQ ID NO: 4. In another embodiment, the antibody in the reagent is a taxol-sensitive Phytophthora cinnamomi-derived β
-Directed against an amino acid sequence containing at least one taxol binding site of tubulin. In another embodiment, the antibody in the reagent is directed against an amino acid sequence comprising at least one taxol binding site of SEQ ID NO: 678. In this method, the β-tubulin or β-tubulin-like compound is selected from the group consisting of a recombinantly expressed protein, an exogenously isolated protein, a synthetic peptide, and a cell culture.

In another embodiment, the present invention is a method of screening a substance composition by the presence of taxol or a taxol-like compound, wherein in addition to α-tubulin from any taxol-sensitive organism, the method is set forth in SEQ ID NO: 6. A β-tubulin having an amino acid sequence containing both taxol binding sites from Phythium ultimum or taxol-sensitive Phytophthora cinnamomi is prepared to prepare a reagent; contacting the substance composition with the reagent; and Measuring the ability of a substance composition to promote the assembly of, or inhibit the dissociation of, MT under dissociation conditions; thereby, the ability of the substance composition to promote the assembly of, or inhibit the dissociation of, the MT The method is to indicate the possible presence of taxol or a taxol-like compound in an object.

In another embodiment, the invention is a method of screening a composition of matter for the presence of taxol or a taxol-like compound, the method comprising a taxol-sensitive Phythium ultimum carrying β-tubulin of SEQ ID NO: 6. Or preparing a mycelium of taxol-sensitive Phytophthora cinnamomi; contacting the composition of the substance with the mycelium in the presence of labeled taxol; and the composition of the substance competitively binding β-tubulin to labeled taxol. Measuring the degree of inhibition; including, if able to block the binding of labeled taxol to β-tubulin from taxol-sensitive Phythium ultimum or Phytophthora cinnamomi,
The composition of matter is determined to carry taxol or a taxol-like compound.

In another embodiment, the invention is a method of altering the taxol binding properties of a recombinantly expressed β-tubulin or a portion thereof, the method comprising the steps of SEQ ID NO: 1 in the coding region of the vector. Determining the identity of the codon at amino acid 219;
Then, when the codon at amino acid 219 encodes any amino acid other than threonine, the nucleotide in the codon is substituted so that amino acid 219 encodes threonine, and the non-taxol-linked β-tubulin or a part thereof is replaced with
Change to taxol-bound β-tubulin or a portion thereof, or
When the codon at amino acid 219 encodes a threonine, the nucleotides in the codon are substituted such that amino acid 219 encodes any amino acid other than threonine to reduce taxol-linked β-tubulin or a portion thereof to non-taxol. Changing to bound β-tubulin or a portion thereof.

[0020] In another embodiment, the invention is a method of making a taxol-sensitive fungal cell from a taxol-resistant fungal cell, the method comprising a taxol comprising threonine at amino acid number 219 numbered in SEQ ID NO: 2. DN encoding bound β-tubulin
Transforming a non-taxol-sensitive fungal cell by introducing an A segment, wherein the transformed fungal cell, when exposed to expressible conditions, has a suitable constitutive or inducible promoter. Express the DNA segment under control.

In another embodiment, the present invention has been transformed by introducing a DNA segment encoding taxol-linked β-tubulin comprising threonine at amino acid 219 numbered in SEQ ID NO: 2 and expressing A transgenic taxol-sensitive fungal cell that expresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to possible conditions.

In another embodiment, the present invention is a method of producing a taxol-resistant fungal cell from a taxol-sensitive fungal cell, wherein the amino acid at amino acid 219 numbered in SEQ ID NO: 2 is not threonine. Transforming a taxol-sensitive fungal cell by introducing a DNA segment encoding a non-taxol-linked β-tubulin, wherein the transformed fungal cell, when exposed to expressible conditions, Under the control of various constitutive or inducible promoters.
Overexpress DNA segments.

In another embodiment, the present invention has been transformed by introducing a DNA segment encoding taxol-linked β-tubulin comprising threonine at amino acid 219 numbered in SEQ ID NO: 2 and expressing A transgenic taxol-sensitive fungal cell that overexpresses the DNA segment under the control of a suitable constitutive or inducible promoter when exposed to possible conditions.

In another aspect, the invention is a method of screening a composition of matter for the presence of taxol or a taxol-like compound, the method comprising providing distinguishable taxol-resistant and taxol-sensitive fungal cells. Contacting a composition of a substance with the fungal cell; and measuring the inhibition of growth of the fungal cell; wherein the composition of the substance can inhibit the growth of a taxol-sensitive fungal cell;
If the growth of taxol-resistant fungal cells cannot be suppressed, it is determined that the composition carries taxol or a taxol-like compound. The method comprises essentially transgenic taxol-resistant and taxol-sensitive fungal cells comprising taxol-resistant and taxol-sensitive isogenic transgenic fungal cells. It can be performed when it consists of fungal cells. The method can also be performed with taxol-resistant fungal cells derived from a fungus independent of the fungus from which the taxol-sensitive fungal cells are derived.

In another embodiment, the present invention is a method of protecting the growth of a phytopathogenic bacterium, the method comprising determining taxol susceptibility of the phytopathogenic bacterium, Taxol-producing P.microspo
Includes processing with ra. In a typical method, taxol susceptibility of a plant pathogen is determined by identifying amino acid 219, and if amino acid 219 is threonine, the plant is designated as taxol susceptible.

[0026] DETAILED DESCRIPTION One aspect of the present invention is an isolated DNA comprising an open reading frame or a portion thereof encoding a β- tubulin protein. Corresponding cDN
A is taxol-resistant Pestalotiopsis microspora Ne32, taxol-sensitive Pythi
um ultimum and taxol-sensitive Pythium cinnamomi have been isolated and characterized. The nucleotide sequence and the deduced amino acid sequence of β-tubulin derived from Pestalotiopsis microspora Ne32 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, by Py
Those derived from thium ultimum are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, by Pythium cin
Those derived from namomi are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. By characterizing the taxol sensitivity of β-tubulin encoded by the gene of the invention,
The identity of amino acid 219 of β-tubulin numbered in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 was found to be an indicator of taxol sensitivity. The presence of threonine ("Thr219") at amino acid 219 indicates taxol sensitivity, whereas asparagine ("Asn219") or glutamine ("Gl
n219 ”) indicates taxol resistance.

[0027] In another aspect, the invention is a β-tubulin protein or protein fragment encoded by the novel genes disclosed herein. Pu of the present invention
Because ltimum and P. cinnamomi β-tubulin proteins can bind to taxol, proteins and protein fragments containing a taxol binding site derived from the β-tubulin-encoding gene described herein are E. coli and Produced by heterologous expression in other systems, purified by standard methods, and used in in vitro assays to detect taxol and taxol-like substances using methods known in the art (Schiff et al., 1979.Nature 277: 665-667). Can be used. For example, the β-tubulin proteins of the invention can be used to screen plant or fungal extracts, as well as synthetic compounds, for taxol or taxol-like substances as potential anticancer agents. Β-tubulin generated by substituting, deleting or modifying specific amino acids can be used as an experimental tool to further investigate the molecular basis for taxol binding to β-tubulin protein.

In another embodiment, an antibody (polyclonal or monoclonal) to all or a portion of β-tubulin of the invention may be used to determine whether a composition of matter has taxol binding properties. it can. In one method, antibodies capable of binding to taxol-sensitive β-tubulin and / or taxol-resistant β-tubulin are purified by in situ hybridization (Ausbel et al., 1997).
Current Protocols in Molecular Biology, John Wiley & Sons), Elisa, or contact a composition of matter prepared for a Western blot. Visualization of antibody-antigen binding is mediated by any method known in the art, for example, a colorimetric method using a radiolabeled or fluorescently labeled secondary antibody, or peroxidase and / or alkaline phosphatase (Harlow , ED and Lane, D. 1988. An
tibodies: A Laboratory Manual). For example, an antibody against a portion of SEQ ID NO: 4 that includes amino acid 219 binds to β-tubulin having a threonine at amino acid 219 but does not bind to β-tubulin having a different amino acid at amino acid 219 . As such, detectable binding indicates the presence of threonine at amino acid 219, and therefore sensitivity to taxol. This type of assay is useful for screening compositions of various substances for the presence of β-tubulin, which include living substances, such as plants or microorganisms, or plant material, patient samples, or compound live. Includes non-living substances such as rallies.

In another embodiment, the present invention relates to taxol analogs that mimic the interaction between taxol and β-tubulin based on the identification of specific amino acid sequences in β-tubulin that correspond to taxol binding and taxol sensitivity. A method of designing the body or other compounds. Previously reported three-dimensional structures (Nogales et al., 1998.
e 391: 199-203) can be used to develop and optimize antitumor and antifungal compounds with respect to amino acid 219 and surrounding regions. Further, using such information, it is also possible to prepare a mutant β-tubulin having a modified taxol sensitivity by substituting an amino acid at a specific position of the β-tubulin protein.

In another aspect, the invention is a method of making a fungal isogenic strain using a gene of the invention. This method allows taxol-related pharmacological studies,
Allows you to do against a known technical background. In addition, the present invention provides a method for screening plant extracts, fungal extracts, extracts from other organisms, and synthetic compounds for taxol-like substances as potential anti-cancer agents, using these isogenic fungal strains (on the other hand). Are taxol-sensitive and the other is taxol-resistant). The present invention also provides for the screening of plant extracts, fungal extracts, extracts from other organisms, and synthetic compounds for taxol-like substances as potential anti-cancer agents, using two unrelated fungal strains ( One is taxol-sensitive and the other is taxol-resistant).

The features of the genes and proteins of the present invention are described in the following examples. It is understood that these examples are illustrative of the invention and are intended to illustrate the invention, but should not be construed as limiting the scope of the invention in any way. The structural features of the invention can be modified without departing from the scope of the invention.

Example 1 Different Taxol Sensitivity in Selected Fungi Taxol sensitivity was examined for fungal strains used for the isolation of the β-tubulin cDNA of the present invention.

The Pestalotiopsis microspora strain Ne32 (previously disclosed in US Pat. No. 5,861,302) was licensed from Montana State University. Pyt
hium ultimum (ATCC 26083), Achlya klebsiana (ATCC 52605), and Pythium cin
Namomi (ATCC 200982) was purchased from the 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 tested. For comparison, A. is an oomycete very close to P. ultimum.
klebsiana was also included in these experiments. As shown in Fig. 1, P. microspora
Was very resistant to taxol, up to 11.7 μM. In comparison, A
klebsiana was moderately sensitive and its growth was inhibited by up to 40% against 11.7 μM taxol. Finally, P. ultimum and P. cinnamomi were found to be the most sensitive of these four species. The growth of P. ultimum and P. cinnamomi was suppressed even at low taxol concentrations (IC ₅₀ 0.1 μM). This sensitivity is comparable to taxol levels (0.25 μM) that inhibit Hela cell division (Schiff et al.,
1979. Nature 277: 665-667).

Example 2: Isolation of β-tubulin cDNA sequence The β-tubulin cDNA sequence was isolated from P. microspora Ne32, P. ultimum, and P. ci
For nnamomi, it was determined from RNA isolated from fungal mycelium. Automated dideoxynucleotide sequencing was performed by a contracting laboratory. Sequence comparisons were performed on the Internet site of the National Center for Biotechnology Information using the BLAST program. Amino acid sequence alignments were performed using the ClustalW program, and other analyzes were performed using the Mac Vector program.

To isolate β-tubulin cDNA sequences from P. microspora Ne32 and P. ultimum, four degenerate primers were designed with conserved motifs in the fungal β-tubulin amino acid sequence. The forward degenerate primer BTUB1 (
5'-CTGGGCYAAGGGYCAYTACACYGAG-3 '; SEQ ID NO: 7), amino acid residue Trp-Ala-Lys-Gly
-His-Tyr-Thr-Glu (that is, corresponding to WAKGHYTE in the one-letter code amino acid code; SEQ ID NO: 8); reverse primer BTUB2 (5'-CGAAGAARTGRARNCGRGGGAARGG-3 '; SEQ ID NO: 9), amino acid residue Pro-Phe- Corresponds to Pro-Arg-Leu-His-Phe-Phe (i.e., PFPRLHFF; SEQ ID NO: 10 in single letter amino acid code); forward primer BTUB3 (5'-CGAGCCYTACAACGCYACYCT-3 '; SEQ ID NO: 11), amino acid residue Glu -Pro-T
yr-Asn-Ala-Thr-Leu (ie, EPYNATL in one letter code amino acid code; SEQ ID NO: 12); and reverse primer BUTB4 (5'-CTCGTTCATGTTRSWCTCRGCC
TC-3 '; SEQ ID NO: 13), and designed to correspond to the amino acid residue Glu-Ala-Glu-Ser-Asn-Met-Asn-Asp (that is, EAESNMND; SEQ ID NO: 14 in the one-letter code). All primers were made by contracting laboratories according to our specifications.

To isolate β-tubulin cDNA from P. microspora Ne32, using 5 μg of total RNA from hyphae grown for 5 days, using the primer BTUB4 in a 20 μl reaction in the first strand cDNA was synthesized. A polymerase chain reaction (PCR) reaction was performed using 1 μl of the cDNA product as a template. The cycle program consisted of 8 cycles at an annealing temperature of 52 ° C., followed by 22 cycles at an annealing temperature of 62 ° C. The degenerate primers BTUB3 and BTUB4 produced a 0.8 kb amplification product, while BTUB2 and BTUB3 produced a 0.3 kb product. Geneclean the desired band
(BIO 101, Vista, CA), gel purification and pPCR2.1 vector (Invitrogen; San
Diego CA) and transformed E. coli XL1-Blue cells. The insert was sequenced and the gene-specific forward primer NETUB5 (5'-GGGTGTCACCACTTGCTTGCGT
TT-3 ′; SEQ ID NO: 15) and reverse primer NETUB6 (5′-TCGAGTTTCCGACGAAAG
TGGACGA-3 ′; SEQ ID NO: 16) was synthesized. Marathon c to obtain full-length clones
The DNA library was prepared using 1 μg of mRNA by the manufacturer (Clontech, Palo Alto, CA).
It was constructed according to the instructions. Dilute 1 μl of this library 250-fold and 5 μl
A cDNA end rapid amplification (RACE) reaction was performed according to the manufacturer's recommended PCR cycling program. For 5'RACE, library adapter primer AP1 (Clon
tech) and primer NETUB6 produced a 1.3 kb product. At 3'RACE,
A 1.0 kb product was generated with primers AP1 and NETUB5. The desired band was gel purified and cloned into the pPCRII-TOPO vector (Invitrogen, Carlsbad, CA). A region of 1 to 1105 bp derived from the 5 ′ RACE product was ligated to a region between 1106 to 1668 bp derived from the 3 ′ RACE product at an internal BamHI site to prepare a complex cDNA (FIG. 2). P. microspo
The resulting composite cDNA from ra had a total length of 1668 bp and was named TUBB-pm. The nucleotide sequence and the deduced amino acid sequence are similarly shown in FIG. 2 in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. This cDNA encodes a protein of 446 amino acids and has a calculated molecular weight of 49,832 and a pI of 4.6. This protein contains a 74 nucleotide 5 'untranslated region (UTR) followed by a 229 nucleotide 3' UTR followed by a 24 nucleotide poly (A) tail. Sequence AATAA (nucleotides 1539-1 of SEQ ID NO: 1)
543) is an animal and viral polyadenylation signal AATAAA (Proudfoot, NJ
And Brownlee, GG 1976 "3 'non-coding region sequence of eukaryotic messenger RNA" (
3'Non-coding region sequences in eukaryotic messenger RNA), Nature 263: 2
11-214) was located 103 bp upstream of the poly (A) region with the closest similarity.

To isolate β-tubulin cDNA from P. ultimum, oligo dT-primers (GibcoBRL, Gaithers) were used with 5 μg of total RNA from hyphae grown for 6 days.
burg, MD) in a 20 μl reaction to synthesize first strand cDNA. A polymerase chain reaction (PCR) reaction was performed using 2 μl of the cDNA product as a template and a cycle program similar to the above. 1.0kb with degenerate primers BTUB1 and BTUB4
BTUB1 and BTUB2 produced a 0.5 kb product. The desired band was gel purified and ligated into the pPCR2.1 vector. The insert was sequenced and the gene-specific forward primer WT1L-U (5'-CTATCATGTGCACGTACTCGGT
GTGC-3 ′; SEQ ID NO: 17) and reverse primer WT1L-L (5′-CTGGGACGGTCAAAGCA
CGGTACTGC-3 ′; SEQ ID NO: 18). First for 5'RACE
Strand cDNA was synthesized using primer WT1L-L as a template in a PCR reaction. 0.95 kb product from primers WT1L-L and Cap-Switch (Clontech), Advantag
It was prepared using the e-GC cDNA PCR kit (Clontech). For 3'RACE, first strand c
DNA was synthesized using CapFinder cDNA library construction kit (Clontech). Using the obtained cDNA as a template, two types of PCR products of 1.0 kb and 1.1 kb were produced from the primers WT1L-U and CDS / 3 '(Clontech), respectively. The PCR fragment was gel purified and cloned into the pPCR2.1 vector. In P. ultimum, there were two types of isolated tubulin cDNA, one composite cDNA was 1650 bp in length and the other was 1537 bp. These two cDNAs differ only in the position where the poly (A) tail is added. A region between 1 to 824 bp from the 5 'RACE product was ligated to a region between 825 to 1650 bp from the 1.1 kb 3' RACE product at an internal MfeI site, and a 1650 bp composite cDNA
And named TUBB-pu. The nucleotide sequence and deduced amino acid sequence are similarly shown in FIG. 3 in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. This cD
NA encodes a protein of 446 amino acids with a calculated molecular weight of 50,047 and a pI of 4.6. This protein has a 91 nucleotide 5'UTR and a 199 nucleotide 3'UTR.
And a subsequent 19 nucleotide poly (A) region. Two incomplete polyadenylation signals were experimentally identified. 57 bp upstream of the poly (A) region of the 1537 bp cDNA
ATATA (nucleotides 1445-1449 of SEQ ID NO: 3) and AATATT (nucleotides 1546-1551 of SEQ ID NO: 3) 80 bp upstream of the poly (A) region of the 1650 bp cDNA. these
The size of the cDNA was in good agreement with the size of the transcript in Northern analysis, as shown below, indicating that they were complete.

The published β-tubulin cDNA sequence from P. cinnamomi (Weerakoon et al., 1998.
Mycologia 90: 85-95), four types of gene-specific primers were synthesized. The forward primer PCBTUB1U (5′-CAGCGACAACATGAGAGAGCTCG-3 ′; SEQ ID NO: 19) is
Corresponding to the 270 to 292 region of the cDNA sequence, the forward primer PCBTUB2U (5'-CGATGAGG
TCATGTGCCTGGATAA-3 ′; SEQ ID NO: 20) corresponds to the 867 to 890 region; reverse primer PCBTUB3L (5′-AAACGGAGGCACGTGGTGATG-3 ′; SEQ ID NO: 21) corresponds to the 984 to 1005 region, and the reverse primer PCBTUB4L (5′- CGCGTCTATCTCATCCATTCCTCG-3 ′; SEQ ID NO: 22) corresponds to the 1596 to 1619 region.

Using 5 μg of total RNA from P. cinnamomi hypha grown for 5 days,
First strand cDNA was synthesized using the dT-primer in a 20 μl reaction. 1 μl cD
PCR was performed using the NA product as a template. The cycle program is 62
The annealing was performed at an annealing temperature of 30 ° C. for 30 cycles. Primer PCBTUB1U and PC
BTUB4L produced a 1.3 kb amplification product, while PCBTUB2U and PCBTUB4L produced a 0.75 kb product. Primer PCBTUB1U or PCBTUB2U to PCBTUB3L
No amplification product was produced when combined with. Geneclea the desired band
n (BIO101), ligated into pPCR2.1 vector (Invitrogen), and
i XL1-Blue cells were transformed. Four clones (clone number C16-1, 4, 9 and 10) for the 1.3 kb fragment and one clone (clone number C10-5) for the 0.75 kb fragment were bidirectionally sequenced and Found a match. TUBB
the nucleotide sequence and deduced amino acid sequence of a 1.3 kb cDNA designated -pc,
SEQ ID NO: 5 and SEQ ID NO: 6 are also shown in FIG. This cDNA is
Encodes a 444 amino acid β-tubulin protein with a calculated molecular weight of 50 kDa
, PI is 4.7. 10 nucleotides 5 'untranslated region (UTR) and 5 nucleotides 3'
UTR exists.

The sequence encoding β-tubulin of P. cinnamomi has been published previously (Weerakoon et al., 1998. Mycologia 90: 85-95; Genbank accession number U22050) and is disclosed herein. -The deduced amino acid sequence of tubulin was compared with that disclosed by Weerakoon et al. The TUBB-pc cDNA sequence shown in SEQ ID NO: 5 and FIG. 4 is 36 nucleotides (2.
7%) different. Amino acid sequence of β-tubulin deduced from TUBB-pc (SEQ ID NO: 6) and the amino acid sequence published by Weerakoon et al. (“U22050”; SEQ ID NO: 23)
5 is shown in FIG. The two sequences differ by 8 amino acids. Four of these are conservative changes, while the other four are non-conservative changes. There is one change within each taxol binding site. In Taxol binding region I (amino acids 1-31), amino acid V24 in TUBB-pc (where amino acid 24 is valine) is U2
Different from I24 at 2050 (where amino acid 24 is isoleucine). In taxol binding domain II (amino acids 212-23l), T219 (amino acids
The 219 position is threonine) is different from the N219 in U22050 (the amino acid 219 position is asparagine).

The deduced β-tubulin amino acid sequences from P. microspora (SEQ ID NO: 2), P, ultimum (SEQ ID NO: 4) and P. cinnamomi (SEQ ID NO: 6) show the expected characteristics of β-tubulin. As shown, it is the human β2-tubulin (SEQ ID NO: 24), Ne in FIG.
urospora crassa (SEQ ID NO: 25), A. nidulans benA (SEQ ID NO: 26), and A
klebsiana (SEQ ID NO: 27), shown by alignment with β-tubulin. These sequences can be split into an N-terminal domain (amino acids 1-205), an intermediate domain (amino acids 206-381) and a C-terminal domain (Nogales et al., 1998.
ure 391: 199-203). Their N-terminal domain is a conserved motif important for GTP binding [Ala-Ile-Leu-Val-Asp-Leu-Glu-Pro-Gly-Thr-Met-Asp-Ser-Val-Arg, In the amino acid code shown, AILVDLEPGTMDSVR (SEQ ID NO: 28), and Ala-Val-Leu-Val-Asp-Leu-Glu-Pro-Gly-Thr-Met-Asp-Ser-Val-Arg, AVLVDLEPGTMDSVR (SEQ ID NO: 29); 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
Between amino acids 63-77 in SEQ ID NO: 27 and between amino acids 62-76 in SEQ ID NO: 27], a conserved motif important for phosphate binding [Gly-Gly-Gly-Thr-Gly-Ser-Gly, ie one letter 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
Between 140-146 and between amino acids 139-145 in SEQ ID NO: 27], and a conserved motif important for Mg2 ⁺ binding [Asp-Asn-Glu-Ala, i.e. DNEA in the one letter code amino acid code. SEQ ID NO: 31); between amino acids 203 to 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 to 205 in SEQ ID NO: 27] (Linse, K. and Mandelkow, EM 198
8. “The GTP-binding peptide of β-tubin: Positioning by direct photoaffinity labeling and comparison with nucleotide-binding protein”
ulin: Localization by direct photo affinity labeling and comparison with
nucleotide-binding proteins) J Biol Chem 263: 15205-152lO; and Farr,
GW and Stemlicht, H, 1992, `` Site-directed mutagenesis of the GTP-binding domain of β-tubulin ''
β-tubulin) J Mol Biol 227: 307-321). Furthermore, N-terminal Met-Arg-Glu-Ile
That is, in the single letter amino acid code, the MREI (SEQ ID NO: 32) motif has been shown to automatically regulate the stability of β-tubulin mRNA in animal cells (Yen et al., 1988. Autoregulated instability of β-tubulin mRNA by recognition of free amino terminus
-tubulin mRNAs by recognition of the nascent amino terminus of β-tubuli
n) Nature 334: 580-585). This motif and the variant Met-Arg-Glu-Leu (ie, MREL in single letter amino acid code; SEQ ID NO: 33) are present in the fungal β-tubulin shown in FIG. 6 and may function similarly. . The C-terminal domain contains microtubule-associated proteins (MAPS) and motor proteins (Nogales et al., 199).
8 Nature 391: 199-203). The sequence near the C-terminus is highly variable and acidic, a feature common to β-tubulin (Sullivan, KF 1988; `` Structure and utilization of tubulin isoforms '' (Structure and utilization of tubulin isotypes) Ann Rev Ce
ll Biol 4: 687-716).

The amino acid sequences of β-tubulin from different organisms are well conserved and show at least 63% identity. (Oakley. BR 1994. “γ-tubulin” (G
(amma-tubulin) Hyams JS, Lloyd CW (ed.) `` Microtubules '' (Microtubules), Wiley-Li
ss, New York pp. 38-45). Table I shows the percent identity of the β-tubulin amino acid sequences of P. microspora and P. ultimum with β-tubulin from other organisms. Β-tubulin from P. microspora is available from A. flavus, A. nidulans benA
And has the highest identity (93-97%) with filamentous ascomycetes such as N. crassa, and low identity (73-78%) with unicellular ascomycetes and oomycetes. The identity with β-tubulin from non-fungal organisms is also relatively low (78-85%). In contrast, P
ultimum-derived β-tubulin is composed of two oomycetes, A. klebsiana and P. cinnamo.
It has the highest identity (96-97%) with β-tubulin from mi, but shows only limited identity (71-78%) with β-tubulin from Ascomycetes. P. ulti
β-tubulin from mum is also relatively higher than β-tubulin from non-fungal organisms such as the green algae C. reinhardtii, the protozoan T thermophila, the slime mold Physarum polycephalum and various animals. Shows identity (86-93%).

Example 3 Extraction of Genomic DNA and Southern Analysis The taxol-dependent properties of microtubules can be changed by the diversity of the gene encoding β-tubulin. , ult
It was determined whether imum had one or more copies of the β-tubulin gene.

[Table 1]

The mycelium of P. microspora Ne32 or P. ultimum grown for 3 days was collected, and the genomic DNA was transferred to an Elu-Quik Hi-Volume Genomic kit (Schleicher Schuelle, Neene, N.
H). 5 μg of genomic DNA was cut with restriction enzymes at 37 ° C. for 4 hours, separated on a 0.8% agarose gel, and filtered with a nylon filter (Tropix, Bedford, M.D.).
A) and transfer the wet filter to the GS Gene Linker UV chamber (Biorad; Hercules
, CA). Southern blot analysis was performed under stringent conditions by standard methods (Sambrook et al., 1989. Molecular Cloning. A laboratory Manual) Cold Spring Harbor Labo
ratory, Cold Spring Harbor, NY). P. micr by primers NETUB5 and NETUB6
A 413 bp PCR fragment of ospora β-tubulin was generated, while primer WTIL-U was used.
WTIL-L generated a 377 bp PCR fragment of P. ultimum β-tubulin.
The PCR products were gel purified, with ^{α- 32 P dCTP, Ready-to} -Go beads (Pharmacia, Piscata
way, NJ). Probe the Micro Bio-
Purification was performed using a Spin column (Biorad).

In P. microspora, the β-tubulin probe hybridized with a single band from genomic DNA digested with EcoRI, HindIII or SalI, and hybridized with two bands from a sample digested with BamHI. Soy. In P. ultimum, the β-tubulin probe hybridized with a single band from genomic DNA digested with BamHI, SalI, Pvul or Pstl, and hybridized with two bands from a sample digested with EcoRI. did. The size of these fragments is consistent with that expected from the restriction endonuclease map of the corresponding cDNA clone. Since the β-tubulin gene is generally highly conserved, these results
And P. ultimum both have a single copy of the β-tubulin gene, indicating that fungi generally have one or at most a few β-tubulin genes. Consistent: (Neff et al., 1983. "β-
Isolation of the tubulin gene and demonstration of its essential function in vivo '' (Isolation
of the β-tubulin gene from yeast and demonstration of its essential fun
Cell 33: 211-219; Hiraoka et al., 1984. "The NDA3 gene in fission yeast encodes β-tubulin: cold-sensitive nda3 mutations reversibly shift chromosomes during spindle formation and mitosis. (The NDA3 gene of fission yeast en
codes β-tubulin: a cold sensitive nda3 mutation reversibly blocks spind
le formation and chromosome movement in mitosis) Cell 39: 349-358; Orbach
1986. "Cloning and characterization of the β-tubulin gene from a Neurospora crassa benomyl-resistant mutant and its use as a dominant selectable marker" (Cloni
ng and characterization of the gene for β-tubulin from a benomyl-resist
ant mutant of Neurospora crassa and its use as a dominant selectable mar
ker) Mol Cell Biol 6: 2452-2461; Cameron et al., 1990.``Cloning and analysis of β-tubulin gene from protoctist '' (Cloning and analysis of β-tubuli
n Gene from a protoctist) J Biol Chem 265: 15245-15252; and Weerakoon et al., 1998. Mycologia 90: 85-95; May et al., 1987. "The β-tubulin gene of Aspergillus nidulans is usually diversified." Aspergillus nidulans β-tubulin ge
nes are usually divergent) Gene 55: 231-243; Panaccione et al., 1988; `` Colletotrichum graminicola transformed with a homologous or heterologous benomyl resistance gene,
Retains expected pathogenicity to corn '' (Colletotrichum gr
aminicola transformed with homologous and heterologous benomyl-resistanc
e genes retains expected pathogenicity to corn) Mol Plant Microbe Intera
ct 1: 113-120; and Goldman et al., 1993. "A nucleotide substitution in one of the β-tubulin genes of Trichoderma viride confers resistance to the anti-mitotic agent methylbenzimidazol-2-yl-carbamate. (A nucleotide
substitution in one of the β-tubulin genes of Trichoderma viride confer
s resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate
) Mol Gen Genet 240, 73-80). The fact that P. microspora and P. ultimum have only a single copy of the β-tubulin gene indicates that it accounts for the sensitive or resistant nature of the organism to taxol.

Example 4 mRNA Isolation and Northern Analysis The expression level of tubulin mRNA in P. microspora Ne32 and P. ultimum was examined by Northern and PCR analysis.

Four agar plugs of P. ultimum or P. microspora were added to 40 ml of FM1 medium (5 g soytone, 5 g dextrose, 20 g sucrose and 1 g yeast extract per 1 L medium) or modified M1D (Li et al., 1998. Mycol Res 102: 461-464), and grown without shaking in 250 ml Erlenmeyer flasks at 24 ° C.
Mycelium was harvested, blotted on Whatman filter paper and dried. 1 g of mycelium was ground using a mortar and pestle in the presence of liquid nitrogen to obtain a powder. Add 10 ml of Trizol to powder
(GibcoBRL) was transferred to a Dounce homogenizer and homogenized. Total RNA was isolated according to the manufacturer's instructions. Total RNA was dissolved in diethyl carbonate-treated water at room temperature and stored at -80 ° C. Northern blots were performed under stringent conditions by standard methods (Sambrook et al., 1989. Molecular cloning: laboratory manual) (Molecular
Cloning: A laboratory Manual), Cold Spring Harbor Laboratory, Cold Spri
ng Harbor, NY).

[0048] Northern analysis revealed that levels of β-tubulin transcript in P. microspora were very low (data not shown). Therefore, total RNA from mycelium grown for 2, 5, 6, or 11 days was converted to cDNA and used as a template in a PCR reaction. Using the gene-specific primers NETUB5 and NETUB6, the logarithmic phase (2,
A 413 bp β-tubulin cDNA fragment was amplified from the growing mycelium in the stationary phase (day 5 and 6) or stationary phase (day 11), but not from the template-free control reactions.

In P. ultimum, total RNA was isolated from mycelium grown for 2, 4, 6, 7, 8 and 10 days and used for Northern analysis. The β-tubulin probe hybridized with two transcripts of 1.5 and 1.6 kb, which were the two isolated transcripts.
It matches the size of the cDNA. The 1.6 kb transcript was more numerous.

[0050] Example 5: ^[3 H] in the binding animal systems to fungal cells of Taxol, ^[3 H] using binding to untreated cells of taxol, interaction of taxol and microtubules characteristics (Manfredi et al., 1982; taxol binds to cellular microtubules) J Cell
Biol 94: 688-696). Since P. microspora and P. ultimum different taxol susceptible investigate whether correlated with taxol binding properties of these microtubules, specific binding of the present inventors to ^[3 H] taxol these fungal cells The ability was checked. Total binding of [ ³ H] taxol to fungal cells consists of specific binding to microtubules and non-specific binding to other cellular structures. By binding to ^[3 H] Taxol fungal cells were measured total binding and the value for nonspecific binding in the presence or absence of 100-fold excess of unlabeled taxol. Subsequently, the specific binding of [ ³ H] taxol was calculated as the difference between the total binding and the non-specific binding.

The newly prepared mycelium from P. microspora Ne32 and P. ultintum were
Grow in a bottle with 140 ml of modified M1D at 24 ° C. for 1-2 days. These actively growing mycelium were transferred to a 50 ml conical tube and centrifuged at 7,000 rpm for 5 minutes at room temperature. The mycelium was suspended in 1 ml of residual M1D medium and 1 ml of freshly prepared MID medium. Cells were either untreated or pre-treated with the anti-mitotic drug thiabendazole to disassociate microtubules. For pretreated cells, thiabendazole (in DMSO) was added at the desired concentration and adjusted to the same concentration of DMSO in all samples. The samples were subsequently incubated at room temperature for 3 hours. [ ³ H] taxol (3.7 × 10 ⁷ Bq / ml, Moravek) was then added at the desired concentration, either in the presence or absence of a 100-fold excess of unlabeled taxol. The samples were incubated for 2 hours at room temperature and then quenched on ice. [ ³ H] Taxol binding to P. microspora cells requires Ol% (v / v) to disrupt cell membranes.
Performed in the presence of Triton X-1OO.

The weight of each GFC filter (Whatman; Clifion, NJ) was measured using an analytical balance. For P. ultimum, the mycelium was transferred from the conical tube onto a GFC filter held by 3-piece filter paper (Whatman). 1 conical tube
Washed three times with 0 ml MilliQ water (Millipore, Inc .; Bedford, MA). The mycelium on the GFC filter was washed with 120 ml of MilliQ water. For P. microspora, mycelia were harvested by centrifugation at 5,000 rpm for 5 minutes at room temperature and washed three times with 40 ml of MilliQ water. The mycelium was transferred to a GFC filter, and the conical tube was rinsed with 5 ml of MilliQ water, and then combined with the remaining mycelium. GFC filter overnight in oven, 80
C. and then weighed to determine the dry weight of mycelium. Filter 5
Beckman L under 20 ml Cytoscint (Fisher Scientific; Pittsburgh, PA)
Counted in an S3801 scintillation counter. Specific binding in the presence of 100-fold excess unlabeled taxol and bound in the absence ^[3 H] was calculated as the difference of taxol. Non-specific binding was measured as binding in the presence of a 100-fold excess of unlabeled taxol.

[ ³ H] taxol specifically binds to P. ultimum cells and the amount of specific binding is [ ³ H]
It was found to increase as a function of taxol concentration (FIG. 7A). Furthermore, in cells pretreated with thiabendazole to reduce the amount of assembled microtubules, specific binding of [ ³ H] taxol was reduced in a dose-dependent manner (FIG. 7B). In fact, treatment with 1 mM thiabendazole completely abolished the specific binding of [ ³ H] taxol. These results indicate that taxol can interact with microtubules in P. ultimum, which is consistent with the organism being taxol-sensitive.

On the other hand, in the first experiment, very small amounts of P. microspora^ThreeH]
Specific binding of taxol was found (data not shown). This result shows that taxol and
Either due to poor interaction with P. microspora microtubules, or [ ^Three This is probably due to a membrane barrier that prevents the accumulation of [H] taxol in cells. Animal cells
In this case, 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 xol is a membrane-localized pump that causes drug efflux
(Jachez et al., 1993.
Of Taxol Sensitivity by Induced SDZ PSC 833 and Cyclopeptide SDZ 280-446
(Restoration of taxol sensitivity of multi drug-registant by the cyclo
sporine SDZ PSC 833 and the cyclopeptide SDZ 280-446) J Natl Cancer Ins
t 85: 478-483)). Obtain whether P. microspora has such a system
There is no information available. Non-ionic detergents for animal cells, eg 0.1% (v / v)
 Treatment with NP-40 or Triton X-1OO destroys cell membranes and causes
Most of the soluble proteins, including tubulin, are released but not assembled
Microtubules have been shown to be intact (Schliwa et al., 1981, ed.
Stabilization of cytoplasmic material in cells opened by surfactants, and sets thereof
Stabilization of the cytoplasmic ground ''
 substance in detergent-opened cell and a structural and biochemical ana
lysis of its composition) Proc Natl Acad Sci USA 78: 4329-4333; Duerr et al.,
1981. "In situ molecular analysis of cytoplasmic microtubules: ubiquitous and specific proteins"
(Molecular analysis of cytoplasmic microtubules in situ: ide
ntification of both widespread and specific proteins) Cell 24: 203-222; M
anfredi et al., 1982. J Cell Biol 94: 688-696). Source of low specific binding rate due to low membrane barrier
In the presence of Triton X-lOO to assess whether^ThreeH] Taxol
Was bound to P. microspora cells. Treated with Triton X-1OO (O.l% (v / v))
P. microspora cells have no or very few [^ThreeH] Taxol specific
From binding to 75 nM [^ThreeH] Taxol (FIG. 7A). In addition, cheabendazo
Pretreated with Triton X-100^ThreeH] Taxol specific
No binding was shown (data not shown). These results indicate that taxol is P. mic
cannot or only slightly interacts with the microtubules of rospora
This is consistent with the fact that this organism is taxol resistant.
You. In summary,[^ThreeThe result of binding of [H] taxol is
Shows that the nature of Brin determines the specific taxol sensitivity.

Taxol binds to β-tubulin in assembled MT by
, But its binding site has been characterized by photocross-linking electron crystallography and mutagenesis. The region between amino acids 1-31 and the region between 217-231 were found to crosslink to the C-3 'and C-2 groups of taxol, respectively (Rao et al., 1994. J Biol Chem 269: 3132-3134; And Rao et al., 1995. JB
iol Chem 270: 20235-20238). Recently, the structure of β-tubulin dimer has been analyzed by electron crystallography of zinc-derived tubulin dimer sheets (Nogales
Et al., 1998. Nature 391: 199-203). By modeling the binding of taxol to this structure, the C-3 ′ group of taxol is near amino acids 15-25 of β-tubulin (near the apex of helix H1) and the C-2 group is amino acids 212-222. Near the helix (around the helix H6 and the loop between H6 and H7). The identification of amino acids 15-25 and 217-222 in both cross-linking and electron crystallographic studies indicates that these regions are important for taxol binding. In addition, electron crystallographic models also show that Leu273 of bovine β-tubulin (located in the M-loop) contacts the taxane ring of taxol (Nogales et al., 1998. Nature 391: 199-203).
Furthermore, mutations in human ovary cells at Phe270 or Ala364 in the M40 isoform of β-tubulin result in taxol resistance (Giannakakou et al., 1997.
ol Chem 272: 17118-17125).

Amino acids 270, 273 and 364 (indicated by # in FIG. 6) correspond to the fungal β-types listed in FIG.
As it does not vary between tubulins, it is not responsible for the specific taxol response in these organisms. However, by comparing amino acids 1-31 and 212-231 (defined as taxol binding regions I and II, respectively) of β-tubulin in taxol-resistant or taxol-sensitive organisms, the residuals of taxol interaction are significant. The group turned out. FIG. 8 shows a comparison of the amino acid sequences of taxol binding region I and taxol binding region II for the following animals. pig(
I, SEQ ID NO: 34; II, SEQ ID NO: 35), human β2 (I, SEQ ID NO: 36; II,
SEQ ID NO: 37), Drosophila β1 (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 βl (I, SEQ ID NO: 4)
4; 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. klebsiana (I,
SEQ ID NO: 50; II, SEQ ID NO: 51), P. microspora (I, SEQ ID NO: 52; II
, SEQ ID NO: 53), A. nidulans benA (I, SEQ ID NO: 54; II, SEQ ID NO: 55)
And S. cerevisiae (I, SEQ ID NO: 56; II, SEQ ID NO: 57).

Taxol-sensitive organisms, such as β-tubulin from humans, pigs, Drosophila, Xenopus, Tetrahymena and Physarum, are highly conserved in Taxol binding regions I and II and have amino acids 15-25 and
217-222 (except for a conservative substitution of Drosophila βl at amino acid 23). Β-tubulin from P. ultimum shows only four substitutions compared to the above sequence, but none of them occurred between amino acids 15-25 and 217-222 . This similarity is consistent with the fact that P. ultimum is as taxol-sensitive as the animal organisms described above. Similarly, in agreement with previous biochemical studies of animal tubulin, and the binding data of [ ³ H] taxol to P. ultimum demonstrated herein (FIGS. 7A and 7B), Bind to β-tubulin in the assembled MT of these organisms (Kellogg et al., 1).
989.J Cell Biol 109: 2977-2991; and Manfredi, JJ and Horwitz, SB,
1984. Pharmacol Ther 25: 83-125). Β- from P. ultimum and A. klebsiana
The tubulin sequence contains amino acids 219 in Taxol binding regions I and II.
A. klebsiana is relatively resistant to taxol (I
C ₅₀ > 11.7 μM). This reduction in sensitivity is due, in part, to the fact that A.
And six other β-tubulins derived from P. ultimum and taxol-sensitive organisms contain threonine.

Taxol-resistant organisms such as P. microspora, A. nidulans and S. cerevisia
β-tubulin from e is similar to each other in taxol binding regions I and II, but at seven positions (19, 22, 23, 25, 218) in regions 15-25 and 217-222.
, 219 and 221) differ only in the above sequences. [ ³ H] taxol binding data presented herein (FIGS. 7A and 7B) were obtained from previous biochemical studies (Yoon, Y. and Oak
ley, BR 1995. Biochem 34: 6373-6381; and Bames et al., 1992. Mol Biol Cel.
l 3: 29-47) show that β-tubulin in the assembled MT of these organisms cannot bind to taxol efficiently. These sequences are from A. klebsia
It contains asparagine (or glutamine in the case of S. cerevisiae) at amino acid 219 as found in na, and this substitution contributes, though partially, to reduced taxol sensitivity in these fungi. Other substitutions involved in changes in charge and polarity, such as Lys19 to Ala, Glu22 to Gln, and Val23
The substitution of Thr for Thr also contributes to the taxol-resistant phenotype of these organisms.

The above results indicate that a number of residues including threonine at amino acid 219 are important for β-tubulin binding to taxol. In particular, amino acids
219 plays an important role in determining the taxol binding properties of β-tubulin and, consequently, in determining taxol sensitivity of cells. Β-tubulin from taxol-sensitive species has 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). Taxol sensitivity of P. cinnamomi is consistent with the presence of Thr219 and not Asn219 in TUBB-pc (SEQ ID NO: 6), as previously reported by Weerakoon et al. Asn21 found in P. microspora
The presence of 9 (asparagine at amino acid 219) is consistent with this type of taxol resistance. With the information that the presence of threonine at amino acid 219 in β-tubulin corresponds to taxol binding and taxol sensitivity, taxol analogs or other compounds can be designed that mimic the interaction between taxol and β-tubulin . In addition, such information can also be used to make taxol-resistant mutant β
-Tubulin can be made by replacing threonine with another amino acid residue at amino acid 219.

Example 6: Sensitivity to Microtubule Disassembly Drugs Several MT-disassembly drugs are identified as P. microspora Ne32, P. ultimum and A. klebsi.
The effects on ana growth were investigated. Examples of these drugs include colchicine, colcemide (a derivative of colchicine) and the two benzimidazole drugs nocodazole and thiabendazole.

[0061] Colchicine, colcemide, nocodazole and thiabendazole are available from Sigma Ch.
Emical Company (St. Louis, MO). A stock solution of colchicine was prepared using water and other stock solutions were prepared using DMSO. Insert freshly prepared mycelial agar plugs (6 mm diameter) in the presence or absence of anti-microtubule agents with 1%
(vlv) transferred to a PDA plate containing DMSO. Fungal colonies were grown at 24 ° C. for 24 hours for P. ullimum and 48 hours for P. microspora and A. klebsiana. The growth inhibitory effect of these anti-mitotic agents was measured by colony diameter.

[0062] Biochemical and genetic evidence has shown that these drugs bind to β-tubulin in tubulin dimers and cause MT disassembly (
Davidse, LC and Flach, W. 1978. Interaction of thiabendazole with fugal tubulin. Brochrm Bro
phys Acta 543 82-90; Jung, MK and Oakley. BR 1990. "Identification of an amino acid substitution in the β-tubulin gene of Aspergillus nidulans conferring thiabendazole resistance and benomyl hypersensitivity"
ubstitution in the β-tubulin gene of Aspergillus nidulans that confers
thiabendazole resistance and benomyl supersensitivity) Cell Motil Cytosk
eleton 11: 87-94; Manfredi, JJ and Horwitz. SB 1984. Pharmacol Ther
25: 83-125). Although many fungi are resistant to colchicine (Cameron et al., 1990. J Biol
Chem 265: 15245-15252; Kilmartin, JV 1981.
Purification of yeast tubulin by self-assembly in v
itro) Biochem 20: 3629-3633; and Davidse, LC and Flach, W. 1977.
Methylbenzimidazole is an anti-mitotic agent in Aspergillus nidulans mutants
Differential binding of methyl benzimidazole-2-yl-car as a mechanism of resistance to 2-ylcarbanate
barnate to fungal tubulin as a mechanism of resistance to this antimitot
ic agent in mutant strain of Aspergillus nidulans) J Cell Biol 72: 174-1
93), nocodazole (Kilmartin, JV 1981.Biochem 20: 3629-3633) and thiabendazole (Davidse, LC and Flach, W. 1978.Biochim Biophys Acta 54
3: 82-90).

As shown in Table II, the three fungal species tested here were resistant to colchicine and colcemide (IC ₅₀ > 100 μM) and were sensitive to nocodazole (I
C ₅₀ 2-22 μM). These results are consistent with the studies described above. In contrast, these fungi were differently sensitive to thiabendazole. P. microspor
a was very sensitive (IC ₅₀ 3 μM), while P. ultimum and A. klebsiana were less sensitive (IC ₅₀ 270-350 pM). These results indicate that the biochemical properties of β-tubulin are different in these three fungi.

[Table 2]

Mutations in amino acids 6, 165, 167, 198, 200 and 241 of β-tubulin (indicated by asterisks in FIG. 6) caused thiabendazole and other thiabendazole and other Altered sensitivity to benzimidazole drugs (Thomas et al., 1985; Isolation and characterization of mutations in the β-tubulin gene of Saccharomyces cerevisiae) (Isolation and charac
terization of mutations in the β-tubulin gene of Saccharomyces cerevisi
ae) Genetics 112: 715-734; Orbach et al., 1986 Mol Cell Biol 6: 2452-2461; Jung
Et al., 1992. “Amino acid alterations in the β-tubulin g in Aspergillus nidulans β-tubulin gene conferring benomyl resistance”.
ene of Aspergillus nidulans that coufer benomyl resistance) Cell Motil
Cytoskeleton 22: 170-174; Jung, MK and Oakley, BR 1990. Cell Motil
Cytoskeleton 17: 87-94; Fugimura et al., 1992. (A single amino-acid substitution in the β-tubu
lin gene of Neurospora confers both cabendazim resistance and diethofenc
arb sensitivity) Curr Genet 21: 399-404; and Goldman et al., 1993.Mol Gen
Genet 240: 73-80). The six residues in β-tubulin from these P. microspora are different from other thiabendazole-sensitive species such as N. crassa and A. nidulans benA.
Identical to that found in β-tubulin of origin. In contrast, P. ulti
β-tubulin from mum and A, klebsiana has amino acids 165, 167 and 200
Are different. Substitution of phenylalanine for tyrosine at amino acid 167 confers benzimidazole resistance on N. crassa (Orbach et al., 1986.
l Cell Biol 6: 2452-2461), and the fact that both P. ultimum and A. klebsiana have tyrosine at this position has previously been shown to be responsible for such drug resistance. In summary, the different thiabendazole resistances exhibited by these three fungi were consistent with the comparison of fungal β-tubulin shown in FIG.

Example 7: Antibodies capable of distinguishing between taxol-binding and non-binding β-tubulin
Preparation of monoclonal or polyclonal antibodies can be made against the following antigens
: l) Natural β-tubulin extracted from P. microspora, P. ultimum or P. cinnamomi; 2) P produced from heterologous systems such as E. coli, yeast and insect cells
.beta.-tubulin of microspora, P. ultilnum or P. cinnamomi; and 3) a synthetic peptide comprising SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 and preferably at least one taxol binding region. This antibody is prepared according to standard protocols.
Used to interact with the β-tubulin described above using Elisa or Western blot (Harlow, ED and Lane. D. 1988. “Antibodies; A laboratory Manual”). An antibody that can distinguish between taxol-bound β-tubulin and non-taxol-bound β-tubulin is selected as a reagent.

Particular examples are given in SEQ ID NO: 4 or SEQ ID NO: 6 comprising at least one taxol binding domain, eg, taxol binding domain II comprising Thr219, or taxol binding domain II wherein Thr219 is replaced by Asn219 / Gln219. A polyclonal or monoclonal antibody to the corresponding synthetic peptide is produced.
The ability of these antibodies to interact with β-tubulin is tested in Elisa using standard protocols. Binds to peptides containing Thr219 but Asn / Gln219
An antibody that does not bind to the peptide containing is selected as a specific reagent for the Thr219-containing taxol binding site. On the other hand, an antibody that specifically binds to the Asn219 / Gln219-containing peptide but does not bind to the Thr219-containing peptide is selected as a reagent that specifically recognizes a Thr219-deficient taxol binding site.

Example 8: Screening Assay for Detecting β-Tubulin Several assays can be used to determine whether a composition contains β-tubulin that can bind to taxol. These assays are useful for screening a variety of compositions for the presence of β-tubulin. As used herein, "composition" is intended to include an organism, such as a plant or microorganism, or a non-organism, such as a plant material or a sample from a patient.

The first assay is performed using Northern or Southern hybridization techniques well known in the art (Sambrook et al., “Molecular Cloning: A laboratory Manual”).
Spring Harbor Laboratory, Cold Spring Harbor, New York 1989). Total RNA, mRN
A or genomic DNA is isolated from the composition and separated by electrophoresis. DNA, synthetic oligonucleotide or RNA corresponding to a coding region or a part of β-tubulin comprising at least one taxol binding region (eg derived from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5) is It can be used to synthesize probes labeled with radioisotopes. Hybridization with a probe derived from SEQ ID NO: 1 may indicate β-tubulin, which is likely to have taxol resistance. On the other hand, hybridization with a probe derived from SEQ ID NO: 3 or SEQ ID NO: 5 can indicate β-tubulin that is likely to have taxol sensitivity.

A second assay uses a PCR-based assay according to standard protocols (Sambrook et al., "Molecular Cloning: Laboratory Manual" (Molecula
r Cloning; A laboratory Manual), Cold Spring Harbor Laboratory, Cold Spr
ing Harbor, New York, 1989). Both genomic DNA or cDNA converted from total RNA or mRNA are used as templates in PCR assays. Gene-specific or degenerate primers (such as those derived from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5) corresponding to the coding region of β-tubulin containing at least one taxol binding region are synthesized. Only DNA containing suitable primer sequences is amplified, and all other variants can be suppressed. Amplification of a PCR fragment of the expected size using primers derived from SEQ ID NO: 3 or SEQ ID NO: 5 but not SEQ ID NO: 1 is likely to be taxol-bound β-tubulin Indicates that On the other hand, amplification of a PCR fragment of the expected size using primers derived from SEQ ID NO: 1 but not SEQ ID NO: 3 or SEQ ID NO: 5 may be taxol-unbound β-tubulin It indicates that the property is high.
Subsequent determination of the sequence of β-tubulin and investigation of the presence of the Thr219 residue will be made.

A third assay uses Elisa or Western blot according to standard protocols (Harlow, ED and Lane, D. 1988. Antibodies: A Laboratory Manual). A cell extract of the composition is prepared. Generate polyclonal or monoclonal antibodies using synthetic peptides or natural β-tubulin extracted from P. microspora, P. ultimunl or P. cinnamomi, or produced by heterologous systems, such as E. coli, yeast and insect cells it can. Antibodies can be used in Elisa or Western blots as described above. Antibodies that can distinguish taxol binding from non-taxol binding are used in these assays.

Example 9 Construction of Taxol-Sensitive and Taxol-Resistant Homologous Strains P. ultimum contains a single β-tubulin. In vitro, the β-tubulin gene or cDNA can be modified to change Thr219 to a different residue, such as Asn219 or Gln219. The modified DNA sequence is cloned into a transformation vector and used to transform a wild-type P. ultimum strain by established methods (Balance et al., 1985. Gene 36: 321-331). Homologous recombination occurs between the wild-type β-tubulin gene and the modified β-tubulin in this vector. The transformed fungi are selected on a taxol-containing medium. Select taxol resistant clones,
The β-tubulin cDNAs are sequenced to confirm the absence of Thr219. P. cinn
A taxol-resistant syngeneic strain of amomi is similarly constructed and used in the screening assays described in the Examples below. The only difference between these syngeneic strains is that taxol-sensitive strains can bind to taxol due to the presence of Thr219; taxol-resistant strains cannot bind to taxol due to the absence of Thr219. Such taxol-resistant strains can be used in combination with wild-type taxol-sensitive strains for screening as described in the Examples below.

[0072] Example 10: screening Guassei Several assays for the detection of taxol or taxol-like compounds, it can detect the taxol or taxol-like compounds in the composition. These assays are useful for screening a variety of compositions for taxol or taxol-like compounds. Compositions include organisms such as plants or microorganisms, or non-living organisms such as plant materials, patient samples or compound libraries.

One screening method uses taxol-resistant P. microspora in combination with taxol-sensitive P. ultimum or P. cinnamomi. Taxol inhibits the growth of both P. ultimum by binding to their β-tubulin, while Taxol has no effect on the growth of P. microspora. Taxol does not interact with β-tubulin of P. microspora. P. u
Compositions that can inhibit ltimum but not P. microspora are likely to include taxol or a taxol-like compound.

As described in the above examples, the improved screening method involves P. ultimum
Alternatively, a taxol-sensitive and taxol-resistant syngeneic strain of P. cinnamomi is used. The composition is tested for effects on the growth of both taxol-sensitive strains and taxol-resistant strains. Inhibiting a taxol-sensitive strain but not a taxol-resistant strain indicates the presence of taxol or a taxol-like compound. on the other hand,
Failure to inhibit both taxol-sensitive and taxol-resistant strains indicates the absence of taxol or a taxol-like compound.

The composition is based on the ability to stabilize the assembly of microtubules with respect to the presence of taxol or a taxol-like compound, and the ability to stabilize the assembled microtubules under conditions that cause depolymerization (such as low temperatures). (Schiff et al., 1979;
Horwitz, 1981). The α- and β-tubulin used in these assays can be from the following sources: l) Natural microtubules consisting of β-tubulin and α-tubulin extracted from P. ultimum or P. cinnarnomi ; 2) P. ultimu
m or α-tubulin extracted from P. cinnamomi and derived from other sources, such as
Β-tubulin interacted with bovine α-tubulin; 3) all or part of SEQ ID NO: 4 or SEQ ID NO: 6 produced by a heterologous system, such as E. coli, yeast, insect cells, etc., and P. ultimun, Α-tubulin from any of P. cinnanmomi or other sources. If the composition has the ability to promote the assembly of these MTs and the ability to prevent disassembly under conditions that cause disassembly of the assembled MTs, the composition may contain taxol or a taxol-like compound. Is high. On the other hand, these isolated compounds must not be able to promote MT assembly, and P. microspora
The disassembly of MT consisting of β-tubulin of origin must be prevented.

Another screening method can be based on the competitive inhibition of [ ³ H] taxol binding to MT by taxol or taxol-like compounds in P. ultimum or P. cinnamomi. Specific binding of [ ³ H] taxol to P. ultimum was described in the Examples
Perform as described in 5. The amount of [ ³ H] taxol specifically bound to P. ultimum in the presence of the inhibitor was taken as 100%. The composition is added to the assay mixture and the amount of [ ³ H] taxol specifically bound to P. ultimum in the presence of the composition is determined. [ ³ H]
A decrease in taxol-specific binding indicates that the composition has taxol-like properties. If increasing the concentration of the composition can completely inhibit the binding of [ ³ H] taxol, it indicates that the compound is likely to bind to the same binding site on β-tubulin in MT.

[0077] Screening of the composition for taxol or a taxol-like compound may be performed by one of the methods described above. Preferably, one of the first two methods
The first screening is performed using one of the two. Because they are simple and can handle large samples. The third and fourth methods can be used in subsequent screening.

[0078] Example 11: In the screening assay The diagnostic assay to distinguish taxol sensitivity of sample from the patient, distinguish taxol binding β- tubulin shown in Examples 7 and unbound β- tubulin Use the resulting antibody. Cellular proteins are extracted from tumor samples from patient samples and the presence of either β-tubulin with or without taxol binding is detected.

For example, the taxol binding regions of the taxol-sensitive and taxol-resistant β-tubulin of the invention (such as SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6) are used in diagnostic assays to screen tumors for resistance to taxol. Then, monoclonal or polyclonal antibodies are produced by methods well known in the art (Harlow, ED, and Lane, D. 1988. Antibodies: A Laboratory Manual). For example, a monoclonal antibody probe can be reacted with a patient sample, eg, a tumor specimen, to detect the presence of β-tubulin with or without taxol binding ability. Visualization of antibody-antigen binding may be via any means known in the art.
For example, a colorimetric method using a radiolabeled or fluorescent secondary antibody or a peroxidase and / or phosphatase (Harlow. ED and Lane, D, 1988. “Antibodies: Laboratory Manual” (Antibody: A Laboratory Manual
)). Β-tubulin with taxol binding ability, that is, taxol sensitive β-
Tubulin detection corresponds to a positive response to taxol therapy. Alternatively, the detection of unbound taxol-resistant β-tubulin and / or the absence of taxol-sensitive β-tubulin corresponds to a reduced or lack of response to taxol therapy.

Example 12: Taxol-sensitive pathogenic eggs using P. microspora in plants
Many oomycete containing biocontrol P. ultimum and P. cinnamomi of bacteria, crop damage, a plant pathogen result in serious economic losses. For example, P. ultimum rots legume roots, and P. cinnamomi rots avocado roots (ATCC: Catalog of Filamentous Fungi)
(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 comprises a two-step process: l) examining the phytopathogenic taxol susceptibility, and 2) infecting the taxol-producing P. microspora if the phytopathogenic bacterium is taxol-sensitive. Applied to plants and surrounding soil as a source of anti-proliferative taxol.

The taxol susceptibility 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, the DNA and protein databases are searched to see if a β-tubulin sequence has been reported, and if so, the residue at amino acid 219 from the database. If the pathogen β-tubulin sequence is unknown, the cDNA sequence is isolated and analyzed for amino acid 219 residues. The presence of threonine at amino acid 219 in the pathogen's β-tubulin gene indicates susceptibility to taxol, and therefore the pathogen is taxol-producing P.m.
It is indicated that it can be treated with icrospora. If amino acid 219 is not threonine,
Taxol sensitivity may need to be measured by other techniques, such as growth inhibition by taxol. Other screening methods described herein for determining the presence of taxol-binding β-tubulin can also be used.

P. microspora produces taxol at 50 μg / l (Strobel et al., 1996. Microbiol
142: 435-440), and has previously been published to secrete taxol outside of fungal cells. This taxol concentration inhibits the growth of P. ultimum and P. cinnamomi
(See FIG. 1). For treatment, P. microspora was inoculated into areas of plants or soil infected with taxol-sensitive pathogens. The growth of P. microspora secretes taxol, which eventually inhibits the growth of these taxol-sensitive organisms.

Example 13 Use of Crystal Structure in the Design of Antitumor or Antifungal Agents Using the three-dimensional structure of β-tubulin, a taxol-like compound was rationally designed by methods known in the art. (Ealick et al., 1991; Application of crystallographic and modeling techniques in the design of purine nucleoside phosphorylase inhibitors) (Applicati
on of crystallographic and modeling methods in the design of purine nucl
eoside phosphorylase inhibitors) Science 88: 11540-11544; Rossman et al., 199
1. “Application of crystallography to design antiviral drugs”
y to the design of antiviral agents) Infectious Agents and Disease 1: 3-
lO). In accordance with the teachings of the present invention, applying the knowledge that the protein structure Thr219 plays an important role in taxol binding to taxol-like compounds may also be applied as important to the development of drugs with taxol-like activity. .

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of taxol on the growth of P. microspora, P. ultimum, P. cinnamomi and A. klebsiana bacteria. Mycelium was grown on potato dextrose agar (PDA) 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. The experiments were performed in duplicate and the data shown are the average of several experiments.

FIG. 2 shows the nucleotide sequence of β-tubulin derived from P. microspora Ne32 cDNA and the deduced amino acid sequence TUBB-pm. The numbers on the left indicate nucleotide positions and the numbers on the right indicate amino acid positions. Gene-specific primers NETUB5 and NE
The TUB6 sequence is underlined. The translation initiation codon ATG is underlined, the translation stop codon is indicated by an asterisk, and the putative polyadenylation signal is doubled.

FIG. 3 shows the nucleotide sequence of β-tubulin derived from P. ultimum cDNA and TUBB-pu, which is the deduced amino acid sequence. The numbers on the left indicate nucleotide positions and the numbers on the right indicate amino acid positions. The sequences of the gene-specific primers WT1L-U and WT1L-L are underlined. The translation initiation codon ATG is underlined, the translation stop codon is indicated by an asterisk, and the putative polyadenylation signal is indicated by a double line.

FIG. 4 shows the nucleotide sequence of β-tubulin derived from P. cinnamomi cDNA and the derived amino acid sequence, TUBB-pc. The numbers on the left indicate nucleotide positions and the numbers on the right indicate amino acid positions. Gene-specific primers PCBTUB1U, PC
The sequences of BTUB2U and PCBTUB4L are underlined. The translation start codon ATG is indicated by ###, and the translation stop codon is indicated by an asterisk. TUBB-pc and Weerakoon et al. (Weerakoon et al., 1998.
Isolation and characterization of the single β-tubulin gene in Phyto
phthora cinnamomi) Mycologia 90: 85-95) reported sequence U22050 (Genba
nk accession number) is indicated by a double line.

FIG. 5 shows the amino acid sequence of β-tubulin in P. cinnamomi (“TUBB-pc”) as reported herein and the amino acid sequence described above by Weerakoon et al. (Weerakoon et al., 19).
98. Mycologia 90: 85-95) (“U22050”). P.cinnamomi
Shows the entire deduced amino acid sequence in TUBB-pc and eight residues (amino acids 24, 21) that differ from U22050 (Genbank accession number) reported by Weerakoon et al. (1998).
9, 249, 251-253, 359, and 428) are shown below the TUBB-pc sequence. Amino acids 1-31 and 212-231 (designated herein as taxol binding regions I and II, respectively) are indicated by lines above the sequence.

FIGS. 6A and 6B show amino acid sequence alignments of β-tubulin. This alignment was obtained using the ClustalW alignment program. P.
The entire amino acid sequence of microspora β-tubulin is shown, and the different residues in other β-tubulins are shown below. The numbers on the left indicate nucleotide positions,
Numbers on the right indicate amino acid positions. The underlined sequence is a region important for GTP binding (amino acids 63-77) and a region important for phosphate binding (amino acids 140-14
6), and the region important for Mg ²⁺ binding (amino acids 203-206). Amino acids 1-3
1 and 212-231 (designated herein as taxol binding regions I and II, respectively) are indicated by lines above the sequence. Amino acids Phe270, Leu273 and Ser364 are marked with #. Amino acids important for bacterial resistance to benzimidazole (amino acids 6, 165, 167, 198, 200, and 241) are marked above by an asterisk. Alignment gaps are indicated by dashes and $ is marked at the end of each sequence. N.crassa
Against β-tubulin from A. nidulans benA, A. klebsiana and human β2
The Genbank accession numbers are listed in Table 1. The described P. cinnamomi is SEQ ID NO: 6.

FIGS. 7A and 7B show that [ ³ H] taxol specifically binds to P.ultimum but not P.micro
It is a graph which shows that it does not bind to spora. FIG. 7A shows [ ³ H] against P. ultimum.
Taxol specific binding increases with [ ³ H] taxol concentration, but P.micro
spora indicates no or very little specific binding. Actively growing fungal cells were incubated with different concentrations of [ ³ H] taxol for 2 hours at room temperature before quenching. Specific binding was calculated as the difference in 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.ultimum, while P.microsp
This represents less than 5% of the total binding to ora. [ ³ H] Taxol binding to P. microspora cells was performed in the presence of Triton X-100 (0.1% v / v) to disrupt cell membranes. FIG. 7B shows a dose-dependent decrease in [ ³ H] taxol specific binding to P. ultimum in the presence and absence of the microtubule depolymerizing drug thiabendazole.
Cells were not treated with thiabendazole (0 μM) or treated with different concentrations of thiabendazole for 3 hours at room temperature. The cells were then incubated with 5 nM [ ³ H] taxol for 2 hours in the presence and absence of a 100-fold excess of unlabeled taxol before quenching. Specific binding of [ ³ H] taxol to untreated cells was defined as 100%.
The experiments shown in FIGS. 7A and 7B were performed in duplicate and the data shown are representative of several experiments.

FIG. 8 shows taxol binding region I (amino acids 1-3) of β-tubulin from different organisms.
1) and the amino acid sequences of taxol binding region II (amino acids 212-231). Shows the entire amino acid sequence of taxol binding regions I and II of porcine β-tubulin,
Different residues are shown for other β-tubulins. Taxol sensitivity of each organism is indicated by "s" for sensitivity and "r" for resistance. Amino acids 15-25 and 212-222 are indicated by an asterisk, indicating involvement in taxol binding by both crosslinking and electron crystallography. The taxol binding region II of A. klebsiana is between amino acids 211-230 due to a gap in its sequence. Nogales for porcine β-tubulin
(Nogales et al., 1999. Nature 391: 199-203) and Genbank accession numbers for other sequences are shown in Table I. The sequence for P. cinnamomi represented herein is set forth in SEQ ID NO: 6.

[Procedure amendment]

[Submission date] October 12, 2001 (2001.10.12)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Contents of correction]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/10 A61P 35/00 4H011 35/00 C07K 14/37 4H045 C07K 14/37 C12N 1/13 C12N / 13 C12Q 1/02 C12Q 1/02 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/53 D 33/53 33/566 33/566 C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI) , CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW) EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C R, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 2G045 AA40 BB20 CB21 DA12 DA13 DA14 DA36 FB02 FB03 FB04 4B024 AA01 AA11 BA80 CA04 CA09 DA06 HA14 4B063 QA13 QA18 QQ07 QQ43 QQ61 QQ79 QR32 QR41 QR48 QR62 QR76 QS25 4B06 5 AA57X AA57Y AB01 CA44 CA46 4C084 AA17 MA01 NA14 ZB262 ZB352 ZC412 4H011 AA01 DD04 4H045 AA10 AA20 AA30 BA10 CA15 EA28 FA74

Claims (98)

[Claims] 1. A purified DNA segment encoding β-tubulin of the fungal species Pestalotiopsis microspora or a part thereof. 2. The DNA segment of claim 1, wherein said portion encodes at least one taxol binding site. 3. The method according to claim 1, wherein said portion is taxol binding site I and taxol binding site I.
The DNA segment according to claim 2, which encodes a protein having I. 4. The DNA segment according to claim 3, wherein said protein is capable of interacting with α-tubulin. 5. The DNA segment according to claim 1, comprising at least a portion of SEQ ID NO: 1. 6. The method of claim 1 wherein said portion is from nucleotide 75 to nucleotide 75 of SEQ ID NO: 1.
The DNA segment according to claim 5, comprising up to 167 nucleotide sequences. 7. The DNA segment according to claim 6, wherein at least one nucleotide in said nucleotide sequence has been replaced. 8. The DNA segment of claim 5, wherein said portion comprises the nucleotide sequence of SEQ ID NO: 1 from nucleotide 708 to nucleotide 764. 9. The DNA segment according to claim 8, wherein at least one nucleotide in said nucleotide sequence has been replaced. 10. The DNA segment of claim 9, wherein nucleotide 729, nucleotide 730, nucleotide 731 or a combination thereof has been substituted. 11. The DNA segment according to claim 5, comprising the nucleotide sequence from nucleotide 75 to nucleotide 1412 of SEQ ID NO: 1 and encoding β-tubulin. 12. The DNA segment according to claim 11, wherein at least one nucleotide in the nucleotide sequence has been substituted, and the β-tubulin has no change in taxol-binding ability. 13. The method according to claim 13, wherein at least one nucleotide in the nucleotide sequence has been substituted, and the β-tubulin has an altered ability to bind taxol.
The DNA segment according to claim 11. 14. An amino acid sequence comprising at least a portion of β-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 capable of interacting with α-tubulin. 18. The amino acid sequence according to claim 14, comprising at least a part of the β-tubulin shown in SEQ ID NO: 2. 19. The method according to claim 19, wherein the portion comprises amino acids 1-31 of SEQ ID NO: 2.
An amino acid sequence according to claim 18. 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 NO: 2. 22. The amino acid sequence according to 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 method of claim 18, wherein the portion consists essentially of amino acids 1-446 of SEQ ID NO: 2, and the portion acts as taxol-resistant β-tubulin.
The amino acid sequence according to 1. 25. The amino acid sequence of claim 24, wherein said portion comprises at least one amino acid substitution that alters the taxol binding properties of said portion. 26. The amino acid sequence of claim 24, wherein said portion comprises at least one amino acid substitution that does not alter the taxol binding properties of said portion. 27. The amino acid sequence of SEQ ID NO.
15. The amino acid sequence according to claim 14, wherein the amino acid sequence is substituted with any amino acid that disrupts a dimensional structure. 28. A purified DNA segment encoding β-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 capable of interacting with α-tubulin. 32. The method of claim 28, comprising at least a portion of SEQ ID NO: 3.
DNA segment according to 1. 33. The DNA segment of claim 32, wherein said portion comprises a nucleotide sequence from nucleotide 92 to nucleotide 184 of SEQ ID NO: 3. 34. The DNA segment of claim 33, wherein at least one nucleotide in said nucleotide sequence has been replaced. 35. The DNA segment of claim 32, wherein said portion comprises the nucleotide sequence of SEQ ID NO: 3 from nucleotide 725 to nucleotide 781. 36. The DNA segment of claim 35, wherein at least one nucleotide in said nucleotide sequence has been replaced. 37. The DNA segment of claim 35, wherein nucleotide 746, nucleotide 747, nucleotide 748, or a combination thereof has been substituted. 38. The DNA segment of claim 32, comprising the nucleotide sequence of SEQ ID NO: 3 from nucleotide 92 to nucleotide 1429 and encoding β-tubulin. 39. The DNA segment according to claim 38, wherein at least one nucleotide in the nucleotide sequence is substituted, and the ability of the β-tubulin to bind taxol is not changed. 40. The method according to claim 40, wherein at least one nucleotide in the nucleotide sequence is substituted, and the ability of the β-tubulin to bind to taxol is changed.
A DNA segment according to claim 38. 41. An amino acid sequence comprising at least a portion of β-tubulin of the fungal species Pythium ultimum. 42. The amino acid sequence of claim 41, comprising 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 capable of interacting with α-tubulin. 45. The amino acid sequence according to claim 41, comprising at least a portion of the β-tubulin shown in SEQ ID NO: 4. 46. The method of claim 46, wherein said portion comprises amino acids 1-31 of SEQ ID NO: 4.
An amino acid sequence according to claim 45. 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 according to 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 portion, consisting essentially of amino acids 1-446 of SEQ ID NO: 4, wherein the portion acts as taxol-sensitive β-tubulin.
6. The amino acid sequence according to 5. 52. The amino acid sequence of claim 51, wherein said portion comprises at least one amino acid substitution that alters the taxol binding properties of said portion. 53. The amino acid sequence of claim 51, wherein said portion comprises at least one amino acid substitution that does not alter the taxol binding properties of said portion. 54. The amino acid sequence of SEQ ID NO.
42. The amino acid sequence according to claim 41, wherein the amino acid sequence is substituted with any amino acid that disrupts a dimensional structure. 55. A purified DNA segment, or a portion thereof, encoding β-tubulin of the fungal species Phytophthora cinnamomi and consisting 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 of SEQ ID NO: 5 from nucleotide 11 to nucleotide 103. 57. At least one nucleotide in the nucleotide sequence has been replaced, provided that nucleotide 81 is a thymine and nucleotide 82 is a nucleotide if the nucleotide substitution changes only one amino acid code. 57. The DNA segment of claim 56, wherein if it is adenine, cytosine or thymine, nucleotide 80 cannot be adenine. 58. The DNA segment of claim 55, wherein said portion comprises a nucleotide sequence from nucleotide 644 to nucleotide 700 of SEQ ID NO: 5. 59. At least one nucleotide in the nucleotide sequence has been substituted, provided that nucleotide substitutions change only one amino acid code, nucleotide 666 is adenine, and nucleotide 667 is substituted. If it is cytosine or thymine, nucleotide 665 cannot be adenine;
A DNA segment according to claim 58. 60. The DNA segment of claim 55, comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 11 to nucleotide 1342, encoding β-tubulin. 61. At least one nucleotide in the nucleotide sequence has been replaced, provided that nucleotide substitutions change only one amino acid code, nucleotide 666 is adenine, and nucleotide 667 is substituted. If it is cytosine or thymine, nucleotide 665 cannot be adenine;
A DNA segment according to claim 60. 62. The DNA segment according to claim 60 or 61, wherein at least one nucleotide in the nucleotide sequence has been substituted, and the β-tubulin has no change in taxol-binding ability. 63. wherein at least one nucleotide in the nucleotide sequence has been replaced and the β-tubulin has an altered taxol binding ability;
A DNA segment according to claim 60 or 61. 64. An amino acid sequence comprising at least a part of β-tubulin of fungal species Phytophthora cinnamomi shown in SEQ ID NO: 6. 65. The portion comprising amino acids 1-31 of SEQ ID NO: 6.
An amino acid sequence according to claim 64. 66. At least one amino acid has been substituted, provided that
When only one amino acid is replaced, amino acid 24 is not isoleucine;
An amino acid sequence according to claim 65. 67. The amino acid sequence of claim 64, wherein said portion comprises amino acids 212-230 of SEQ ID NO: 6. 68. At least one amino acid has been substituted, except that
68. The amino acid sequence of claim 67, wherein amino acid 219 is not asparagine when only one amino acid is replaced. 69. The portion comprising an amino acid substitution at amino acid 219,
65. The amino acid sequence of claim 64, wherein amino acid 219 is not replaced with asparagine. 70. The portion, consisting essentially of amino acids 1-446 of SEQ ID NO: 6, wherein the portion acts as taxol-sensitive β-tubulin.
4. The amino acid sequence according to 4. 71. The portion comprises at least one amino acid substitution, with the proviso that when only one amino acid is replaced, amino acid 219 is not asparagine, and the amino acid substitution reduces the taxol binding properties of the portion. 71. The amino acid sequence of claim 70, which is altered. 72. The portion comprises at least one amino acid substitution, with the proviso that when only one amino acid is replaced, amino acid 219 is not asparagine, and the amino acid substitution reduces the taxol binding properties of the portion. 71. The amino acid sequence of claim 70, which does not change. 73. The amino acid substitution around amino acid 219 that is replaced by any amino acid that disrupts the three-dimensional structure of the amino acid sequence, and where only amino acid 219 is the only substitution, the substituted amino acid is not asparagine. The amino acid sequence according to 1. 74. A vector comprising a purified DNA segment encoding β-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 β-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. The vector comprising a purified DNA segment encoding β-tubulin of the fungal species Phytophthora cinnamomi, or a portion thereof, wherein the DNA segment consists essentially of SEQ ID NO: 5. 79. The vector of claim 78, wherein said portion encodes at least one taxol binding site. 80. A method for measuring the taxol-binding ability of β-tubulin or a β-tubulin-like compound, comprising: taxol-resistant Pestalotiopsis microspora, taxol-sensitive Pythium ultim
um or an antibody directed against an amino acid sequence comprising at least one taxol-binding site of taxol-sensitive β-tubulin derived from taxol-sensitive Phytophtora cinnamomi as shown in SEQ ID NO: 6, which comprises taxol-binding properties and non-taxol-binding properties A reagent is prepared by preparing the antibody for identifying the antibody and β-tubulin, and the degree of binding between the antibody in the reagent and β-tubulin or β-tubulin-like compound is determined. Measuring, wherein the binding of the antibody specifically recognizing taxol binding property shows taxol sensitivity, and the binding of the antibody specifically recognizing taxol non-binding property shows taxol resistance, Method. 81. The method of claim 80, wherein said antibody is directed against an amino acid sequence comprising at least one taxol binding site of β-tubulin from 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 antibody, wherein the antibody is β-derived from taxol-sensitive Pythium ultimum.
81. The method of claim 80, wherein the method is directed against an amino acid sequence comprising at least one taxol binding site of tubulin. 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 antibody, wherein the antibody is taxol-sensitive Phytopht shown in SEQ ID NO: 6.
81. The method of claim 80, wherein the method is directed against an amino acid sequence comprising at least one taxol binding site of β-tubulin from hora cinnamomi. 86. The β-tubulin or β-tubulin-like protein is selected from the group consisting of a recombinantly expressed protein, an exogenously isolated protein, a synthetic peptide and a cell culture. Claims 80, 81, 82, 8
89. The method according to any one of 3, 84 or 85. 87. A method of screening a composition of matter for the presence of taxol or a taxol-like compound, comprising: in addition to α-tubulin from any taxol-sensitive organism, taxol-sensitive Pythium ultimum or SEQ ID NO: 6. Taxol-sensitive Phytophthora ci
β- having an amino acid sequence comprising both taxol binding sites from nnamomi
Preparing tubulin to prepare a reagent, contacting the reagent with the composition of the substance, and inhibiting the ability of the composition of the substance to promote MT assembly or depolymerization of the assembled MT under depolymerization conditions. Measuring the ability to promote the assembly of microtubules or to inhibit depolymerization, the likelihood that taxol or a taxol-like compound is present in the composition of the substance. The method as described above. 88. A method of screening a composition of matter for the presence of taxol or a taxol-like compound, comprising: taxol-sensitive Pythium ultimum or taxol-sensitive Phy represented by SEQ ID NO: 6.
preparing a mycelium of tophthora cinnamomi, contacting the composition of the substance with the mycelium in the presence of labeled taxol, and the composition of the substance competitively inhibits the binding between β-tubulin and labeled taxol Measuring the extent to which the taxol is sensitive to Pythium ultimum or Phytophth
The above method, wherein the composition of the substance is determined to carry taxol or a taxol-like compound if the binding of taxol to β-tubulin derived from ora cinnamomi can be blocked. 89. A method of altering the taxol binding properties of recombinantly expressed β-tubulin or a portion thereof, comprising determining the identity of the codon at amino acid 219 of SEQ ID NO: 2 in the coding region of the vector, When the codon at amino acid 219 encodes any amino acid other than threonine, the nucleotides in the codon are replaced such that amino acid 219 encodes threonine to convert non-taxol-linked β-tubulin or a portion thereof to taxol-linked changing to β-tubulin or a portion thereof, or, if the codon at amino acid 219 encodes a threonine, replacing the nucleotide in the codon such that amino acid 219 encodes any amino acid other than threonine;
Converting taxol-bound β-tubulin or a portion thereof to non-taxol-bound β-tubulin or a portion thereof. 90. A method for preparing taxol-sensitive fungal cells from taxol-resistant fungal cells, comprising introducing a DNA segment encoding taxol-binding β-tubulin comprising threonine at amino acid 219 of SEQ ID NO: 2. Transforming a non-taxol-sensitive fungal cell, wherein the transformed fungal cell, when exposed to expressible conditions, under the control of a suitable constitutive or inducible promoter. The above method of expressing a DNA segment. 91. A transformant which has been transformed by introducing a DNA segment encoding taxol-linked β-tubulin comprising threonine at amino acid 219 of SEQ ID NO: 2, which is suitable when exposed to expressible conditions. A transgenic taxol-sensitive fungal cell that expresses the DNA segment under the control of a constitutive or inducible promoter. 92. A method for preparing a taxol-resistant fungal cell from a taxol-sensitive fungal cell, comprising introducing a DNA segment encoding non-taxol-linked β-tubulin wherein amino acid 219 of SEQ ID NO: 2 is not threonine. Transforming a taxol-sensitive fungal cell, wherein the transformed fungal cell, upon exposure to expressible conditions, transforms the DNA segment under the control of a suitable constitutive or inducible promoter. The above method, which is overexpressed. 93. It has been transformed by introducing a DNA segment encoding taxol-linked β-tubulin comprising threonine at amino acid 219 of SEQ ID NO: 2, which is suitable when exposed to expressible conditions. A transgenic taxol-sensitive fungal cell that overexpresses the DNA segment under the control of a constitutive or inducible promoter. 94. A method of screening a composition of a substance for the presence of taxol or a taxol-like compound, comprising providing a taxol-resistant fungal cell and a taxol-sensitive fungal cell that are identifiable, comprising the composition of the substance and the fungal cell. And measuring the inhibition of the growth of the fungal cell, wherein the composition of the substance can inhibit the growth of taxol-sensitive fungal cells, but cannot inhibit the growth of taxol-resistant fungal cells. Wherein the composition is determined to carry taxol or a taxol-like compound. 95. The method of claim 94, wherein said distinguishable taxol-resistant fungal cells and taxol-sensitive fungal cells consist essentially of transgenic taxol-resistant fungal cells and isogenic taxol-sensitive fungal cells. 96. The method of claim 94, wherein said taxol-resistant fungal cells are derived from a fungus independent of the fungus from which the taxol-sensitive fungal cells are derived. 97. A method for protecting the growth of plant pathogens, comprising determining the taxol sensitivity of the plant pathogen, and determining that the pathogen is taxol-sensitive, the method comprises: Treating with Taxol-producing P. microspora. 98. The method of claim 97, wherein said plant pathogen is taxol sensitive by identifying amino acid 219 and designating said plant as taxol sensitive if amino acid 219 is threonine.