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WO1997015676A2 - Gene de la topoisomerase i de candida - Google Patents

Gene de la topoisomerase i de candida Download PDF

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Publication number
WO1997015676A2
WO1997015676A2 PCT/US1996/017291 US9617291W WO9715676A2 WO 1997015676 A2 WO1997015676 A2 WO 1997015676A2 US 9617291 W US9617291 W US 9617291W WO 9715676 A2 WO9715676 A2 WO 9715676A2
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Prior art keywords
sequence
cell
polynucleotide
seq
albicans
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PCT/US1996/017291
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English (en)
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WO1997015676A3 (fr
Inventor
Jennifer M. Fostel
Kellie M. Giles
Alison Taylor
Thomas P. Mcgonigal
Aparna V. Sarthy
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Abbott Laboratories
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Publication of WO1997015676A2 publication Critical patent/WO1997015676A2/fr
Publication of WO1997015676A3 publication Critical patent/WO1997015676A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)

Definitions

  • Candida albicans (C. albicans) is an opportunistic pathogen which causes systemic candidiasis in immunocompromised patients.
  • the patient pool at risk includes cancer patients undergoing chemotherapy, organ transplant recipients, patients infected with HIV, and other patients without a healthy immune response.
  • This population is increasing in number annually, making infections by Candida spp. and other fungi an increasingly important clinical challenge (recently reviewed by Kerridge, 1995). It has been estimated that the majority of mycotic infections occurring in cancer patients are caused by Candida albicans and other species of Candida (Kerridge, 1995; Abi-Said and Anaissie, 1995), and that the excess mortality that can be attributed to candidemia is 38% (Wey et al. , 1988).
  • DNA topoisomerases are enzymes which modulate the topological structure of DNA and are classified into two classes based primarily on their mode of cleaving DNA. Type I DNA topoisomerases introduce a transient break in one strand of DNA, pass another strand through the nick and change the linking number (i.e. the number of times the DNA backbone strands intertwine) by one unit.
  • Type II topoisomerases introduce concerted breaks in both DNA strands, pass another double-stranded DNA segment through the gap and change the linking number by two. This difference in mechanism allows type II topoisomerases to catenate and decatenate covalently closed double-stranded DNA rings (Hsieh and Brutlag, 1980; Kreuzer and Cozzarelli, 1980), and also to knot and unknot covalently closed double-stranded circles (Liu et al. 1980; Hsieh, 1983).
  • the type I topoisomerases on the other hand, cannot catenate or decatenate double- stranded DNA unless a nick or gap is present in one of the circles (Tse and Wang 1980;
  • topoisomerases participate in cellular processes which are associated with the separation of complementary DNA strands (i.e. replication, transcription, recombination and repair). Topoisomerases are also required for the segregation of daughter chromosomes during cytokinesis. Despite the important function these enzymes perform, type I topoisomerases are considered to be non-essential enzymes in that disruption or inhibition of their catalytic activity does not lead to cell death. (It has been proposed that functional topoisomerase FI activity may replace or substitute for lost topoisomerase I activity). Thus, selective inhibition of the topoisomerase I catalytic activity of a particular fungal pathogen would not necessarily cure a host of that pathogen. Although type Ll topoisomerases could prove to be effective antifungal targets, to date, no compounds have been identified which selectively target the cleavage complex formed by the topoisomerase II enzymes of fungal origin.
  • cleavage complex formed between a type I topoisomerase and DNA.
  • a cleavage complex is characterized by a transient association between the topoisomerase I and DNA during which time the enzyme performs various catalytic functions (such as binding to and nicking a particular region of the DNA, passing a DNA segment through or around the break to change the linking number and ultimately resealing (i.e. repairing) the nick).
  • Stabilizing agents are those chemical compounds which promote cell death through some manner of interaction with a topoisomerase I/DNA cleavage complex, although the nature of this interaction and the mechanism by which the agent induces cell death is unknown.
  • Nitiss et al. have demonstrated the fungicidal effect of camptothecin (which stabilizes the topoisomerase I cleavage complex o ⁇ Saccharomyces cerevisiae (Nitiss and Wang, 1988) and etoposide and amsacrine (which stabilize the topoisomerase II cleavage complex of Saccharomyces cerevisiae (Nitiss, 1992).
  • Eng et al. have also demonstrated this effect in strains of S.
  • enzyme or enzyme fragments may be desired to generate polyclonal or monoclonal antibodies. Such antibodies may then be used in affinity chromatography to facilitate purification of the enzyme. Large quantities of enzyme or enzyme fragments may also be desired to perform X-ray crystallography and other analytical studies.
  • the present invention solves these problems by identifying, isolating and expressing a type I topoisomerase gene from C. albicans in an expression system suitable for large-scale production.
  • identification and isolation of a C. albicans type I topoisomerase gene allows for its expression in a variety of systems which facilitate purification of a C. albicans topoisomerase I encoded therefrom.
  • a C. albicans type I topoisomerase gene may also used in a assay system for identifying compounds which inhibit the growth of fungi.
  • the present invention provides an isolated and purified single- or double-stranded polynucleotide, typically DNA, having a nucleotide sequence comprising a nucleotide sequence selected from the group consisting of (a) the sense sequence of SEQ ID NO: l from about nucleotide position 474 to about nucleotide position 2807; (b) a sequence complementary to the sequence of (a); (c) sequences that, when expressed, encode a polypeptide encoded by a sequence of (a); and d) analogous sequences that hybridize under stringent conditions to the sequences of (a) or (b).
  • a nucleotide sequence selected from the group consisting of (a) the sense sequence of SEQ ID NO: l from about nucleotide position 474 to about nucleotide position 2807; (b) a sequence complementary to the sequence of (a); (c) sequences that, when expressed, encode a polypeptide encoded by a sequence of (a); and d) analogous
  • a polynucleotide of the present invention may also comprise the sense sequence of SEQ ID NO: 1 from about nucleotide position 345 to about nucleotide position 2807 as well as the longer counte ⁇ arts of (b), (c) and (d) above.
  • a preferred polynucleotide is a DNA molecule, ln another embodiment, the polynucleotide is an RNA molecule.
  • the present invention provides specific isolated and purified single- or double-stranded fragments of the above mentioned polynucleotide including the fragment counte ⁇ arts of (b), (c) and (d) above.
  • a DNA molecule of the present invention is contained in an expression vector.
  • the expression vector preferably further comprises an enhancer-promoter operatively linked to the polynucleotide.
  • the DNA molecule in the vector is one of the preferred sequences or fragments mentioned above.
  • a topoisomerase I of the present invention is a recombinant topoisomerase 1 having the amino acid residue sequence of SEQ LD NO:2 or its subset SEQ
  • a preferred C. albicans topoisomerase I has about 821 or fewer amino acid residues and comprises the amino acid residue sequence of SEQ ID NO:3. Also useful are the shorter polypeptides encoded by the DNA fragments mentioned above.
  • the present invention provides a process of making C. albicans topoisomerase I comprising transforming a host cell with an expression vector that comprises a polynucleotide of the present invention, maintaining the transformed cell for a period of time sufficient for expression of the topoisomerase I, and recovering the topoisomerase I.
  • the host cell is an eukaryotic host cell such as a mammalian, yeast or fungal cell, or a bacterial cell. Especially preferred host cells are Candida spp., S. cerevisiae and E. coli.
  • the present invention also provides a topoisomerase I made by a process of this invention. A preferred such topoisomerase I is recombinant C. albicans topoisomerase I.
  • the present invention provides for a host cell transformed with a polynucleotide or expression vector of this invention.
  • the host cell is a yeast or fungal cell such as S. cerevisiae or Candida spp. or a bacterial cell such as E. coli.
  • the present invention further provides a method for identifying compounds having the ability to kill or inhibit the growth of fungal strains.
  • FIG. 1 shows the amino acid sequence used to design sets of degenerate oligonucleotide primers for isolating a 127 base pair (bp) C. albicans DNA fragment with homology to eukaryotic topoisomerase I genes.
  • the Hu sequence represents amino acids 589-634 (D'A ⁇ a et al.
  • the 5c sequence represents amino acids 516-560 (Thrash, et al. , 1985)
  • the Sp sequence represents amino acids 561-605 (Uemura, et al., 1987)
  • the Dm sequence represents amino acids 81 1-856 (Hsieh.et al. , 1992) of each species' respective topoisomerase I.
  • FIG. 2 shows a comparison of the sequence of the 127 bp C. albicans DNA fragment to nucleotides 2043-2180 of the Saccharomyces cerevisiae topoisomerase I gene.
  • FIG. 3 shows the predicted alignment of the polypeptide encoded by the 127 bp C. albicans DNA fragment with the amino acid sequences of genes encoding type I topoisomerases from other eukaryotic species.
  • the Ca sequence represents the amino acids encoded by the insert in plasmid pKGl
  • Sc represents amino acids 514-561 (Thrash et al., 1985)
  • Sp represents amino acids 559-606 (Uemura et al. , 1987)
  • Hu represents amino acids 587-635 (D'A ⁇ a et al. , 1988)
  • Um represents amino acids 557-616 (Gerhold et al. , 1994)
  • Mm represents amino acids 589-637 (Baumgartner et al.
  • XI represents amino acids 643- 691 (Pandit and Sternglanz, 1992)
  • Dm represents amino acids 809-858 (Hsieh et al., 1992)
  • At represents amino acids 733-780 (Kieber and Signer, 1990) of each species respective topoisomerase I.
  • FIG. 4 shows the amino acid sequence used to design a set of degenerate oligonucleotide primers for isolating a 1044 bp C. albicans DNA fragment with homology to eukaryotic topoisomerase I genes.
  • Hu represents amino acids 273-282
  • Mm represents amino acids 275-284
  • Dm represents amino acids 496-505
  • Sc represents amino acids 201 -210.
  • FIG. 5 shows the sequence of the cloned 1044 bp C. albicans DNA fragment.
  • FIG. 6(a-j) shows the complete double-stranded sequence of C. albicans topoisomerase I gene including upstream and downstream gene sequences and the predicted polypeptide encoded therefrom.
  • FIG. 7(a-b) shows the comparative homology in amino acids between C. albicans topoisomerase 1 and human topoisomerase I (d'A ⁇ a, et al. , 1988).
  • FIG. 8 shows the nucleotide sequence of an adapter DNA which carries the 5' and 3' ends of the C. albicans topoisomerase I gene.
  • FIG. 9 shows a schematic diagram of the construction of plasmid pVT100-UCT7-7. Detailed Description of the Invention
  • the present invention provides isolated and purified polynucleotides that encode a type I topoisomerase from Candida albicans, fragments thereof, expression vectors containing those polynucleotides, host cells transformed with those expression vectors, a process of making C. albicans topoisomerase I using those polynucleotides and vectors, and isolated and purified recombinant C. albicans topoisomerase I and polypeptide fragments thereof.
  • the present invention also provides a method for identifying compounds having antifungal activity using polynucleotides that encode a type 1 topoisomerase from C. albicans.
  • the present invention provides an isolated and purified polynucleotide that encodes a type I topoisomerase from the pathogenic fungus, Candida albicans (C. albicans).
  • TOPI the abbreviation "TOPI” is used to designate a gene or polynucleotide sequence encoding a type I topoisomerase
  • topo I is used to designate a polypeptide or an amino acid sequence encoded by a TOPI .
  • a polynucleotide of the present invention that encodes C. albicans topo I is an isolated and purified polynucleotide having a nucleotide sequence which comprises a nucleotide sequence selected from the group consisting of
  • a preferred polynucleotide is a DNA molecule.
  • the polynucleotide is an RNA molecule.
  • the nucleotide sequence and deduced amino acid residue sequence of Candida albicans topo 1 are set forth in SEQ ID NO: 1 and SEQ ID NO:2 shown in FIG. 6.
  • the nucleotide sequence of SEQ LD NO: 1 is a full-length DNA clone of the gene encoding C. albicans topo I and is intended to represent both the sense strand (shown on top) and its complement (i.e. the strand shown below the top strand in FIG. 6).
  • Ln eukaryotic cells translation of RNA initiates at an AUG codon coding for methionine (which, in the C. albicans topo I shown in FIG. 6, corresponds to nucleic acid position 474).
  • SEQ LD NO: l contains two "coding regions" (hereinafter CR1 and CR2) which, in FIG. 6, correspond to nucleotide positions 345-2807 and 474-2807, respectively, either of which may encode the native topo I enzyme.
  • SEQ ID NO:2 is the deduced amino acid residue sequence encoded from CR1 and represents a polypeptide which is 821 amino acids in length. In FIG. 6, SEQ ID NO:2 starts with the amino glutamic acid (E) at position -43 and ends with the amino acid phenylalanine (F) at position 821.
  • SEQ ID NO:3 is the deduced amino acid sequence encoded from CR2 and represents a polypeptide which is 778 amino acids in length.
  • SEQ ID NO:3 begins with methionine (M) at position +1 and ends with pheylalanine (F) at position 821.
  • a polynucleotide of the present invention is also an isolated and purified single- or double-stranded polynucleotide having a nucleotide sequence which comprises a nucleotide sequence selected from the group consisting of (a) the sense sequence of SEQ ID
  • the present invention also contemplates analogous DNA sequences which hybridize under stringent hybridization conditions to the DNA sequences set forth above.
  • Stringent hybridization conditions are well-known in the art and define a degree of sequence identity greater than about 80%-90%.
  • the modifier "analogous” refers to those nucleotide sequences that encode polypeptides having only conservative differences and which retain the conventional characteristics and activities of a topoisomerase I; eg. nicking DNA, changing the DNA's linking number and resealing the nick.
  • the present invention also contemplates naturally-occurring allelic variations and mutations of the DNA sequences set forth above so long as those variations and mutations code, on expression, for a topo I of this invention as set forth hereinafter.
  • DNA and RNA molecules that can code for the same polypeptides as those encoded by CR 1 , CR2 and fragments thereof.
  • the present invention contemplates those other DNA and RNA molecules which, on expression, encode for the polypeptide of SEQ ID NO:2, its subset SEQ ID NO:3 or fragments thereof. Having identified the amino acid residue sequence encoded by C. albicans TOPI , and with knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein, which molecules are characterized simply by a change in a codon for a particular amino acid, are within the scope of this invention.
  • codons constitute triplet sequences of nucleotides in mRNA molecules and as such, are characterized by the base uracil (U) in place of base thymidine (T) (which is present in DNA molecules).
  • U base uracil
  • T base thymidine
  • a simple change in a codon for the same amino acid residue within a polynucleotide will not change the structure of the encoded polypeptide.
  • SEQ LD NO: 1 see FIG. 6
  • a TCA codon for serine i.e. UCA in mRNA
  • C. albicans the CUG codon has been shown to encode serine rather than leucine (see Santos and Tiute, 1995), although this phenomenon is not seen in all Candida species or in other fungi (such as 5. cerevisiae).
  • a recombinant C. albicans TOPI of the present invention expressed in a C. albicans strain would most likely encode a topo I having a serine residue at amino acid position 407 (Xaa) in SEQ ID NO:2 and 321 in SEQ LD NO:3 (corresponding to the CUG at nucleotide position 1434 and amino acid "X" at position 321 in SEQ LD NO: l ).
  • albicans TOPI in another Candida or other fungal species could result in placement of a leucine residue at this same position.
  • the present invention contemplates a topo I having either a serine or leucine residue at the Xaa position depending on the expression system used.
  • a polynucleotide of the present invention can also be an RNA molecule.
  • a RNA molecule contemplated by the present invention is complementary to or hybridizes under stringent conditions to any of the DNA sequences set forth above. Exemplary and preferred
  • RNA molecules are mRNA molecules that encode a topo I of this invention.
  • the present invention also contemplates oiigonucleotides from about 15 to about 50 nucleotides in length, which oiigonucleotides serve as primers and hybridization probes for the screening of DNA libraries and the identification of DNA or RNA molecules that encode C. albicans topo I.
  • primers and probes are characterized in that they will hybridize to polynucleotide sequences encoding topo I.
  • An oligonucleotide probe or primer contains a nucleotide sequence of at least 15 nucleotides that is identical to or complementary to a contiguous sequence of a topoisomerase I polynucleotide of the present invention.
  • an oligonucleotide probe is 25 nucleotides in length, at least 15 of those nucleotides are identical or complementary to a sequence of contiguous nucleotides of a TOPI of the present invention.
  • Exemplary TOPI polynucleotides of the present invention are set forth above.
  • An oligonucleotide primer or probe of the present invention can be prepared using standard procedures well-known in the art. A preferred method of polynucleotide synthesis is via cyanoethyl phosphoramidite chemistry. A detailed description of the preparation, isolation and purification of polynucleotides encoding C albicans topo I is set forth in the Examples below.
  • the present invention provides a recombinant type I topoisomerase from C albicans.
  • a C. albicans topo I of the present invention is a polypeptide of about 821 or fewer amino acid residues.
  • forms of type I topoisomerases have been identified in various species with from 314 to 972 amino acid residues.
  • the various forms of topo 1 are characterized by a moderate degree of sequence identity.
  • the identity between human and C. albicans enzyme is 45% at the amino acid level.
  • a C. albicans topo I is an isolated and purified polypeptide of about 821 or less amino acid residues, comprising at least one of the following amino acid residue sequences: a) from residue position 1 to residue position 150 of SEQ ID NO:3; b) from residue position 297 to residue position 529 of SEQ ID NO:3; c) from residue position 573 to residue position 717 of SEQ LD NO:3; and d) from residue position 728 to residue position 778 of SEQ ID NO:3.
  • a topo I of the present invention comprises two or more of the above sequences. Most preferably, the topo I has all of the above sequences.
  • a topo I of the present invention is a recombinant topo I from C. albicans.
  • a recombinant topo I from C. albicans.
  • eukaryotic polypeptides are known to begin with a methionine residue
  • the present invention also contemplates a recombinant topo I encoded by the complete ORF of C. albicans TOPI .
  • recombinant C. albicans topo I can be defined as a polypeptide of about 821 or less amino acid residues comprising the amino acid residue sequence of SEQ ID NO:2.
  • a preferred recombinant C. albicans topo I has the amino acid residue sequence of SEQ ID NO:3.
  • the present invention also contemplates amino acid residue sequences that are substantially duplicative of the sequences set forth herein such that those sequences demonstrate like biological activity when compared to disclosed sequences.
  • Such contemplated sequences include those sequences characterized by a minimal change in amino acid residue sequence or type (e.g., conservatively-substituted sequences) which insubstantial change does not alter the fundamental nature and biological activity of C. albicans topo I. It is well known in the art that modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide.
  • amino acids can be substituted for other amino acids in a given polypeptide without any appreciable loss of function, ln making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the Uke.
  • hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (- 1.0); Met (- 1.3); Val (- 1.5); Leu (- 1.8); He (- 1.8); Tyr (-2.3); Phe (-2.5); and T ⁇ (-3.4). It is expected that an amino acid residue can be substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0) and still result in a biologically equivalent polypeptide.
  • substitutions can be made on the basis of similarity in hydropathic index.
  • Each amino acid residue has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • Those hydropathic index values are: He (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+ 1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); T ⁇ (-0.9); Tyr (- 1.3); Pro (- 1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).
  • a value of within plus or minus 2.0 is preferred.
  • a topo I of the present invention has numerous uses.
  • such a polypeptide can be used in assays which identify compounds that stabilize the cleavage complex of topo I and DNA.
  • a topo I polypeptide can also be used to design compounds that interact with the cleavage complex of topo I and DNA. More specifically, the topo I or a portion of the topo I can be crystallized and used for designing drugs that stabilize the cleavage complex formed between the topo I and DNA.
  • a topo I of the present invention or fragments thereof may be used to produce antibodies that immunoreact specifically with C. albicans topo I. Such antibodies may then be used to isolate and purify C. albicans topo I.
  • Means for producing antibodies are well known in the art.
  • An antibody directed against C. albicans topo 1 can be a polyclonal or a monoclonal antibody.
  • Antibodies against C. albicans topo I can be prepared by immunizing an animal with a topo I polypeptide of the present invention. Means for immunizing animals for the production of antibodies are well-known in the art.
  • a mammal can be injected with an inoculum that includes a polypeptide as described herein above.
  • the polypeptide can be included in an inoculum alone or conjugated to a carrier protein such as BSA or keyhole limpet hemocyanin (KLH).
  • the polypeptide can be suspended, as is well- known in the art, in an adjuvant to enhance the immunogenicity of the polypeptide.
  • Sera containing immunologically active antibodies are then produced from the blood or other fluid (eg. ascites) of such immunized animals using standard procedures well-known in the art.
  • the identification of antibodies that immunoreact specifically with C. albicans topo I is made by exposing sera suspected of containing such antibodies to a polypeptide of the present invention to form a conjugate between antibodies and the polypeptide. The existence of the conjugate is then determined using standard procedures well-known in the art.
  • a topo I polypeptide of the present invention or fragments thereof may also be used to prepare monoclonal antibodies against topo I and used as a screening assay to identify such monoclonal antibodies.
  • Monoclonal antibodies are produced from hybridomas prepared in accordance with standard techniques such as that described by Kohler et al.. Briefly, a suitable mammal (e.g., BALB/c mouse) is immunized by injection with a polypeptide of the present invention. After a predetermined period of time, splenocytes are removed from the mouse and suspended in a cell culture medium. The splenocytes are then fused with an immortal cell line to form a hybridoma. The formed hyridomas are grown in cell culture and screened for their ability to produce a monoclonal antibody against topo I. Screening of the cell culture medium is made with a polypeptide of the present invention.
  • the present invention provides a process of making C. albicans topo I.
  • a suitable host cell is transformed with a polynucleotide of the present invention.
  • the transformed cell is maintained for a period of time sufficient for expression of the topo I; the topo I is then recovered.
  • a polynucleotide that encodes the desired polypeptide is placed into an expression vector suitable for a given host cell.
  • That vector can be a viral vector, phage or plasmid and may be one which either replicates autonomously within the host cell or integrates into the host cell chromosome.
  • a host cell used to produce topo I is an eukaryotic host cell and an expression vector is an eukaryotic expression vector (i.e., a vector capable of directing expression in a eukaryotic cell).
  • eukaryotic expression vectors are well known in the art.
  • An especially preferred eukaryotic host cell is an 5. cerevisiae or C. albicans.
  • a preferred expression vector is a vector capable of directing expression in 5. cerevisiae or C. albicans.
  • the host cell is a bacterial cell.
  • An especially preferred bacterial cell is an E. coli.
  • a preferred expression vector is a vector capable of directing expression in E. coli.
  • a polynucleotide of an expression vector of the present invention is preferably operatively associated or linked with an enhancer-promoter.
  • a promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins. That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes.
  • promoter includes what is refe ⁇ ed to in the art as an upstream promoter region or a promoter of a generalized RNA polymerase transcription unit.
  • Preferred promoters are those which are operative in bacteria such as a lac promoter. Even more preferred promoters are those which are operative in yeast or fungi.
  • Especially prefened promoters are an ADH 1 or
  • An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene).
  • a major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer.
  • an enhancer can function when located at variable distances from a transcription start site so long as the promoter is present.
  • enhancer-promoter means a composite unit that contains both enhancer and promoter elements.
  • An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product.
  • operatively linked or its grammatical equivalent means that a regulatory sequence element (e.g. an enhancer-promoter or transcription-terminating region) is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter.
  • a regulatory sequence element e.g. an enhancer-promoter or transcription-terminating region
  • Means for operatively linking an enhancer-promoter to a coding sequence are well-known in the art.
  • An enhancer-promoter used in an expression vector of the present invention can be any enhancer-promoter that drives expression in a host cell.
  • an enhancer- promoter with well-known properties, the level of expression can be optimized. For example, selection of an enhancer-promoter that is active in specifically transformed cells permits tissue or cell specific expression of the desired product. Still further, selection of an enhancer-promoter that is regulated in response to a specific physiological signal can permit inducible expression.
  • a coding sequence of an expression vector is operatively linked to a transcription- terminating region.
  • RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs.
  • DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription.
  • Those DNA sequences are refe ⁇ ed to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).
  • Enhancer- promoters and transcription-terminating regions are well-known in the art. The selection of a particular enhancer-promoter or transcription-terminating region will depend, as is also well-known in the art, on the cell to be transformed.
  • the present invention also comtemplates the use of expression vectors which facilitate purification of a desired polypeptide.
  • a gene encoding the desired polypeptide may be cloned into an expression vector which, when expressed, produces the polypeptide fused or linked to a chemical or biological tag.
  • a tag may be any chemical or biological compound or fragment thereof capable of binding to a specific substrate or receptor.
  • tags serve to facilitate purfication of a fusion product (i.e. tag/polypeptide) via specific binding of the tag portion to its receptor or substrate.
  • the tag is fused to the polypeptide in a manner that permits it to be cleaved from the polypeptide after purification.
  • C C.
  • albicans TOPI could be cloned into a pGEX vector (Pharmacia Biotech, Inc., Piscataway, New Jersey) which, when placed in a suitable host, would express a fusion protein of C. albicans topo I and glutathione S-transferase (GST).
  • pGEX vector Pharmacia Biotech, Inc., Piscataway, New Jersey
  • the fusion protein could then be purified by affinity chromatography using glutathione sepharose 4B (which binds to the GST portion of the fusion product) and the topo I enzyme cleaved from the GST tag using a site-specific protease, the recognition sequence of which is located upstream from the topo I.
  • glutathione sepharose 4B which binds to the GST portion of the fusion product
  • topo I enzyme cleaved from the GST tag using a site-specific protease, the recognition sequence of which is located upstream from the topo I.
  • Other commercially available tags could also be used and are well-known to those of ordinary skill in the art.
  • the present invention also contemplates a host cell transformed with a polynucleotide or expression vector of this invention.
  • a host cell transformed with a polynucleotide or expression vector of this invention.
  • Means for transforming cells and polynucleotides and expression vectors used to transform host cells are set forth above.
  • the host cell is an eukaryotic host cell such as a yeast or fungal cell or a prokaryotic cell such as an E. coli.
  • the present invention provides a method for identifying antifungal compounds and, more specifically, antifungal compounds which act as stabilizing agents of a C. albicans topo I and DNA cleavage complex.
  • a "cleavage complex”, as used herein, refers to a transient association between a topoisomerase and DNA during which time the enzyme performs various catalytic functions (such as binding to and nicking a particular region of the DNA, passing a DNA segment through or around the break to change the linking number, and ultimately resealing (i.e. repairing) the nick).
  • stabilizing agent refers to a chemical compound which inhibits cell growth or promotes cell death through some manner of interaction with a topoisomerase I/DNA cleavage complex.
  • stabilize or “stabilization” when used herein and in reference to a stabilizing agent and a cleavage complex, refers to that interaction of the stabilizing agent with the cleavage complex which leads to significant inhibition of cell growth and reproduction or, preferably, cell death.
  • the chemical compound camptothecin stabilizes the topo I/DNA cleavage complex of Saccharomyces cerevisiae , inducing fungal death. Notwithstanding this observation, the nature of the interaction between the stabilizing agent and the cleavage complex and the mechanism by which the agent induces cell death is unknown.
  • the present invention provides an assay method for identifying stabilizing agents of a C. albicans topo I/DNA cleavage complex.
  • Assays for identifying stabilizing agents may be carried out both in whole-cell preparations and in ex vivo cell-free systems.
  • the assay target is a C. albicans topoisomerase 1/DNA cleavage complex, wherein the C. albicans topoisomerase I is a C. albicans topo I of the present invention.
  • the assay methods of the present invention will be suitable for both small- and large-scale screening of test compounds, as well as in quantitative assays such as serial dilution studies wherein the target topo I/DNA cleavage complex is exposed to a range of test compound concentrations.
  • the target is an intracellular topo I DNA cleavage complex and the entire, living fungal cell is exposed to the test compound under conditions normally suitable for growth.
  • conditions including essential nutrients, optimal temperatures and other parameters, depend upon the particular fungal strain being used and are well-known in the art.
  • Stabilization of the topo I/DNA cleavage complex may be determined by observing the cell culture ' s growth or lack thereof; such observation may be made visually, by optical densitometric or other light abso ⁇ tion/scattering means, or by yet other suitable means, whether manual or automated.
  • an observed lack of cell growth may be due to stabilization of the target topo I/DNA cleavage complex or due to an entirely different effect of the test compound so that further evaluation is required to establish the mechanism of action and to determine whether the test compound is a specific stabilizing agent of the cleavage complex. Accordingly, and in a prefe ⁇ ed embodiment of the present invention, the method may be performed as a paired-cell assay in a manner similar to that described by
  • each test compound is separately tested against two different fungal strains, one having greater topo I activity than the other.
  • strains are isogenic to each other with respect to this particular trait, meaning that they are equivalent genetically except in their expression of TOPI .
  • isogenic strains may differ with respect to the TOPI gene by having a copy of a TOPI gene not found in the other strain or by having multiple copies of a TOPI gene wherein the other strain has only one copy).
  • those cells of a strain having greater topo I activity will be more susceptible to the lethal effects of a stabilizing agent than an isogenic strain of cells having lower topo I activity and thus will have comparatively greater mortality or inhibition of cell growth.
  • one strain of cells may be altered by any manner of chemical or genetic modification to produce a modified strain having differential topoisomerase I activity relative to a second strain.
  • cells may be chemically modified by exposure to mutagenic agents (such as carcinogenic compounds or UV radiation) to produce a modified strain which underexpresses TOPI relative to the unmodified parent.
  • mutagenic agents such as carcinogenic compounds or UV radiation
  • a chemically modified strain could be further modified by genetic manipulation (to remove extraneous genetic changes by performing crosses to the parent strain and recovering progeny having reduced topo I) for use in a paired-cell assay; in such a case, the parent (i.e. without the additional chemical modification) would serve as an isogenic control.
  • a TOPI gene may be chemically modified ex vivo by exposure to a mutagenic agent and then reintroduced to a cell to produce a modified cell which expresses altered topo I activity relative to its unmodified parent.
  • the activity of a topo I can be modified in vivo by the presence of a DNA binding agent (such as ethidium bromide) which can be introduced into the culture medium of one strain to alter the topo I activity of that strain relative to the same strain grown without the binding agent.
  • a DNA binding agent such as ethidium bromide
  • the initial condition of the cells is not critical to the practice of a paired-cell assay; one need only be able to modify the cells in some manner that is not lethal and which generates a measurable difference in topo I activity.
  • a strain may also be genetically manipulated to alter the production of topo I in a particular cell. For example, a strain may be modified to either produce or over-produce topo I by cloning a C.
  • Suitable expression vectors and host cells are those previously described in Section LV above.
  • Other prefe ⁇ ed expression vectors are the non-integrative double- ARS shuttle vectors described by Pla et al. (1995) which are able to autonomously replicate in C. albicans.
  • both the modified strain (harboring an expression vector comprising a TOPI gene of the present invention) and the isogenic parental strain (harboring the expression vector only) would be exposed simultaneously to a compound of interest under essentially identical growth conditions.
  • a compound which induces death of the modified strain or which inhibits that strain's growth would be a putative stabilizing agent.
  • a strain may be genetically modified to reduce the activity of a native topo I by any means known to disrupt a TOPI coding sequence.
  • a cloned C. albicans TOPI of the present invention can be used to disrupt or inactivate a native TOPI within a host strain (i.e. in vivo).
  • a selectable marker into the chromosomal copy of a yeast or fungal TOPI may be accomplished by first cloning the selectable marker gene into a single site within a coding sequence of C. albicans TOPI (i.e. CR 1 or CR2) or a fragment thereof.
  • Selectable marker genes are those which confer upon a cell the ability to grow under a particular set of growth conditions (i.e. in selective medium). Representative examples of selective marker genes include but are not limited to genes which confer drug resistance, such as G418 and genes which confer upon a cell the ability to grow in the absence of an exogenously supplied essential nutrient.
  • a fragment of a coding region when used, it should be of a such a length that the marker gene, after cloning, is flanked on each side by a sequence of the coding region that is at least about 250 base pairs in length.
  • the cloned fragment is then transformed into the targeted host cell to induce recombination into the chromosomal copy, and then grown on selective media to identify recombinant cells. As a consequence, functional topo I will not be produced by that particular cell.
  • a non-functional topo I may be generated by genetically altering a chromosomal copy of a TOPI through the process of gene replacement.
  • Gene replacement may be accomplished by specifically altering the DNA sequence of a C. albicans TOPI (so as to render the topo I encoded therefrom non-functional), subcloning the altered sequence into a suitable vector capable of recombining in the yeast or fungal host, and introducing the vector containing the subcloned sequence into the appropriate host so that exchange of the wild-type allele with the mutated one will occur.
  • the steps involved in gene replacement employ standard recombinant DNA techniques and are well known to those of ordinary skill in the art.
  • both the modified strain and the parental strain would be exposed to a compound or compounds of interest under conditions suitable for growth.
  • the parental strain would be most susceptible to a stabilizing agent and would die or show inhibited growth as a result of exposure to the compound.
  • any techniques for directly or indirectly measuring topo I protein or topo I activity are suitable for establishing differential topo I production between a modified strain and its parent.
  • quantitative analysis of topo I may be performed by preparing lysates of cells, partially purifying the topo I from the lysates and measuring topo I activity ex vivo (cf. Fostel et al. , 1992).
  • a modified strain is generated by the introduction of a multicopy plasmid containing a TOPI
  • the actual copy number of the plasmid in the modified strain may be quantified.
  • a modified strain and its parent may be preliminarily tested for sensitivity to a known stabilizing agent such as camptothecin.
  • the technique selected may vary depending upon the manner in which the modification is achieved but such techniques are either well known to those of ordinary skill in the art or are taught in the present specification.
  • Genomic DNA was isolated from C. albicans strain ATCC 10321 , by the method of Polaina and Adam (1991 ). Cells were grown in YEPD broth ( 1 % Bacto yeast extract, 2%
  • Two sets of degenerate primers for PCR were selected based on conserved regions in other eukaryotic type 1 topoisomerases (Hsieh etal., 1992) as shown in FIG. 1. New sites for restriction endonucleases Xba I and Bam HI were also introduced into the primers to facilitate subcloning.
  • the primer sets used were:
  • the PCR reaction was ca ⁇ ied out using reagents from Perkin Elmer (Norwalk. CN), according to the manufacturer's recommendations, using 2 ⁇ g genomic C. albicans DNA and approximately 1 nmole of each of SEQ ID NO:4 and SEQ ID NO:5 (synthesized by
  • the DNA band of interest was excised from the gel using the "crush and soak method" (Sambrook et al. , 1982). Briefly, the fragment of gel containing the DNA of interest was placed in a tube, crushed with a sterile pipette, and incubated at 37°C for 16 hours in elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 0.1 % SDS, 1 mM EDTA, pH 8.0).
  • elution buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 0.1 % SDS, 1 mM EDTA, pH 8.0.
  • the excised DNA was incubated with the Klenow fragment of DNA polymerase I (Boehringer Mannheim, Indianapolis, IN) in the presence of the four nucleotide triphosphates to fill in single-stranded termini, then extracted with phenol/chloroform and precipitated with ethanol.
  • Plasmid pBSK+ (Stratagene GmbH, Heidelberg, Germany) was cut with EcoRV (GIBCO BRL, Gaithersburg, MD). This and other restriction digests were performed in the manufacturer's recommended reaction buffer, and unless otherwise indicated, all restriction and modifying enzymes were obtained from this same source. After digestion, the plasmid DNA was extracted with phenol/chloroform and precipitated with ethanol.
  • the excised DNA was ligated to the plasmid using T4 DNA ligase in the manufacturer's reaction buffer, at 17° C for 16 hours.
  • the resulting DNA was used to transform competent Maximum Efficiency DH5 ⁇ F'IQ E. coli (GLBCO BRL,
  • the sequence of the inserts was determined using commercial primers from Stratagene.
  • the primers 5'-AATTAACCCTCACTAAAGGG-3' SEQ ID NO: 6
  • 5'-GTAATACGACTCACTATAGGGC-3' SEQ ID NO:7 are homologous to the T3 and T7 promoter sequences respectively, in the pBSK+ plasmid.
  • Primers were end-labeled with ⁇ 32p using polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN) in the buffer provided by the manufacturer, then extended with Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN) using dideoxy-sequencing reagents and the method obtained from Promega (Madison, WI). The sequence of the insert from one clone designated 6AB was determined and is given in FIG. 2 (see "Ca").
  • This insert sequence was also compared to a yeast TOPI , i.e. to nucleotides 2034-2180 of the Saccharomyces cerevisiae TOPI (see sequence "5c” in FIG. 2) using the BESTFIT program (GCG, Wisconsin Software package, Madison WI) and the DNA Strider 1.2 program (C.
  • the pBSK+ plasmid with the 6AB fragment inserted was renamed pKG l .
  • a BLAST search (GCG, Wisconsin Software package, Madison WI) of DNA sequences in the GENEMBL database identified other topoisomerase genes as those sequences with the highest score (i.e. having the greatest percent homology to the 6AB insert).
  • An alignment of the predicted C. albicans protein sequence encoded by 6AB with other homologous regions of type I topoisomerases from other species is shown in FIG. 3.
  • Hybridization to C. albicans genomic DNA using the method of Southern ( 1975) revealed the presence of a single copy sequence homologous to pKGl .
  • SEQ ID NO:8 A new set of degenerate primers, based on the sequence 5'-AARAAYTTYTTYAARGA-3' (SEQ LD NO:8) was constructed for the pu ⁇ ose of synthesizing an additional portion of the C. albicans topo I gene via PCR.
  • the design of SEQ ID NO:8 was based on anticipated homology to other eukaryotic type I topoisomerases as shown in FIG. 4.
  • 5'-AGCCACCGTTCTATTGGCAGC-3' (SEQ LD NO:9) derived from the 6AB sequence, were used in a PCR reaction with genomic C. albicans DNA as template.
  • the reaction was ca ⁇ ied out using reagents from Perkin Elmer, according to the manufacturer's recommendations, with 2 ⁇ g genomic C. albicans DNA and approximately 1 nmole of each primer.
  • the reactions were incubated at 45° C for 2 minutes, raised to 72° C over a one minute interval, held at 72° C for 3 minutes, then 94° for 1 minute. This pattern was repeated for 50 cycles.
  • a DNA species having the anticipated size of approximately 1 kb was produced.
  • the PCR products were eiectrophoresed on an 0.8% low melt agarose gel (LMPA,
  • the 1044 bp sequence was used as a probe for a search of the GENEMBL (European Molecular Biology Organization) sequence database using the BLAST routine (GCG).
  • BLAST uses the algorithm of Altschul et al. (J. Mol. Biol. 215: 403-410 (1990)) to search the database entries for similarity to the probe sequence. The results showed that the highest scoring hits of the insert sequence were to other eukaryotic type I topoisomerases, suggesting that the 1044 bp fragment was indeed part of a C. albicans topoisomerase I gene.
  • C. albicans genomic DNA was prepared according to Olson et al. ( 1979). Cells were grown overnight in 500 mL of Mock YEPD liquid medium (purchased from BIO101 , Vista, CA) from a 3 mL inoculum. The culture was divided into four equal volumes, harvested by centrifugation and washed with water two times. Approximately 6 g wet weight of cells were harvested in total. The 1.5 g aliquots of cells were then resuspended in 4 mL of SCE
  • the lysates were layered on top of 50/20/15% sucrose solutions which also contained 0.8 M NaCl, 20 mM Tris-HCL pH 8.0, and 10 mM EDTA. Gradients were centrifuged at 26,000 ⁇ m for 2.5 hours at 20°C using a Beckman SW28 rotor. DNA was collected from the top of the 50% sucrose pad, extracted by rolling very gently with phenol/chloroform for 20 minutes, dialyzed against TE buffer (lOmM Tris HCl pH 7.5, 5.0 mM EDTA) and then concentrated against solid sucrose.
  • TE buffer lOmM Tris HCl pH 7.5, 5.0 mM EDTA
  • C. albicans genomic DNA was used by Stratagene (La Jolla, CA) to construct a genomic DNA library in ⁇ FLX®D (Stratagene, La Jolla, CA).
  • the ⁇ FLX®II vector accepts inserts of size 9-20 kb, so assuming an average insert size of 15 kb, approximately 1000 clones would be expected to contain one genome equivalent of C. albicans DNA (genome size 15 megabases).
  • This range of insert size was also considered to be the most useful size for isolation of a full length gene product from C. albicans, as the topoisomerase I gene would be expected to span at least 2.5 kb, assuming similarity to the 5. cerevisiae TOPI gene.
  • albicans genomic DNA was partially digested with Sau3A and size-selected by gel electrophoresis. It was then ligated into the ⁇ FLX® ⁇ vector following digestion with Xho I and treatment to a partial fill-in reaction, then grown in a P2 host (XL-1 Blue MRA(P2)). Both unamplified and lx amplified libraries prepared according to this procedure were obtained from Stratagene.
  • the probe was prepared for hybridization by first digesting pATlOO with PstI and SphI .
  • the digested DNA was eiectrophoresed on an 0.8% low melt agarose gel (LMPA, GIBCO BRL, Gaithersburg, MD), the 1 kb band was excised and labeled with ⁇ 3 2 P-dNTP using a Mega Prime labeling kit obtained from Amersham (Arlington Heights, LL).
  • plaques from a total of approximately 45 were purified by one round of plaque purification using the labeled probe derived from pATlOO. Two of the plaques yielding the strongest hybridization signal, #3 and #26, were arbitrarily selected for a third round of plaque purification following which DNA was prepared from clone #26 using a Wizard Lambda DNA Purification kit from Promega (Madison, WI).
  • New sequencing primers were prepared to the 5' and 3' ends of the pATlOO insert in order to read out from each end of the sequence into the remainder of the gene. Sequencing by the dideoxy method of Sanger et al. (1977) was performed on the lambda templates directly, with the use of a fmol DNA sequencing kit from Promega. As new sequence was determined, new primers were synthesized and used to 'walk' along the clones to obtain a complete open reading frame (ORF). Sequencing on the opposite strand using another set of primers confirmed the sequence. A complete sequence was considered to be obtained following translation of the ORFs and comparison to 5. cerevisiae and Schizosaccharomyces pombe topo 1 gene sequences. TRANSLATE, BESTFIT, PILEUP and BLAST analyses (GCG, Wisconsin Software package, UW Madison, WL) were used in this determination. The primers used for the sequence determination are given in Table 3, and are highlighted in bold type in FIG. 6.
  • SEQ ID NO:24 and 25 have a one base deletion with respect to the actual sequence shown in FIG. 6.
  • the primers were initally designed from partial sequence infomation and the presence of two "T"s in the actual sequence (positions 2875 and 2932 in FIG. 6) were unknown when the primers were constructed.
  • SEQ ID NO: l The sequence of the complete open reading frame and su ⁇ ounding genomic sequences determined is given in SEQ ID NO: l shown in FIG. 6. Primers used for DNA sequencing are denoted in FIG 6. in bold letters. Potential TATA and CAAT sequences which may be involved in transeriptional initiation are italicized.
  • the PILEUP routine of the GCG software package was used to compare the predicted amino acid sequence encoded by the C. albicans TOPI gene with that of topoisomerase I from 5. cerevisiae (Thrash et al. , 1985), S. pombe (Uemura et al. , 1987) and human cells (D'A ⁇ a et ai , 1988).
  • the predicted amino acid sequence determined for C. albicans topo I shows 62% overall identity to the predicted amino acid sequence of 5. cerevisiae topo I and 45% overall identity to the predicted amino acid sequence of the human topo I.
  • the 5' amino terminal domain of the C. albicans enzyme may provide a peptide (or nucleic acid ) marker specific for Candida species.
  • the predicted amino acid sequence of the C. albicans topoisomerase I also differs from the human amino acid sequence in two regions which are associated with camptothecin-resistance in mutants of topoisomerase I from human or 5. cerevisiae (Fujimori et al. , 1994; Levin et al.
  • camptothecin has been shown to stabilize the topoisomerase I DNA cleavage complex, and this stabilization is thought to be the mode of action of this cytotoxic and fungicidal compound. Regions of the topoisomerase protein which are co ⁇ elated with resistance to camptothecin may be important targets for fungal specific topoisomerase stabilizing agents.
  • the active site of other type I topoisomerases contains a TYR residue which becomes covalently linked to DNA when the catalytic reaction is inte ⁇ upted (Eng et al. , 1989, Lynn et al. , 1989).
  • the short amino acid sequence su ⁇ ounding this residue i.e. SK-X - NY
  • the C. albicans topo 1 gene was subcloned into an 5. cerevisiae expression vector using standard methods of recombinant DNA technology according to the schematic outline shown in FIG. 9.
  • the C . albicans topo I gene was excised from the lambda clone #26 and moved into the pVTIOO-U 5. cerevisiae expression vector (Gold et al. , 1987), which allows the C. albicans gene to be under the control of the 5. cerevisiae alcohol dehydrogenase 1 (ADH l ) promoter.
  • the majority of the C. albicans topoisomerase I gene was excised by cutting the lambda clone #26 with Xba I and Pvu II. This DNA fragment is missing short fragments of the 5' and 3' ends of the open reading frame, so an adapter DNA containing the missing sequences from the 5' and 3' ends of the gene was synthesized.
  • the sequence of the adapter is given in FIG
  • the first step in the construction was to insert the adapter DNA into pVTIOO-U.
  • pVTIOO-U was digested with HinDIIl and BamHI and eiectrophoresed on an 0.8% low melting point agarose gel (LMPA, GIBCO BRL. Gaithersburg, MD).
  • LMPA low melting point agarose gel
  • the agarose slice containing the DNA was melted, added to ligation mix containing the hybridized adapter oiigonucleotides (see FIG. 8) with T4 DNA ligase and incubated at 16°C for 24 hours.
  • Competent DH5 ⁇ E. coli (GIBCO BRL, Gaithersburg, MD) were then transformed with the reaction mix.
  • Plasmid DNA was isolated as described by Sambrook et al. ( 1989), and restricted with EcoRI to identify clones containing the adapter DNA. Clone pVT100-UCT7 contained the adapter DNA by this criterion.
  • the next step in the construction was to insert the C. albicans gene in the adapter in pVT100-UCT7.
  • pVT100-UCT7 was cut with Xba I and Pvu II and eiectrophoresed on an 0.8% LMPA gel.
  • Lambda clone #26 ca ⁇ ying the C. albicans insert was prepared as described by Sambrook et al. ( 1989), and also restricted with Xba 1 and Pvu II.
  • the lambda DNA was eiectrophoresed on LMPA, and the agarose containing a 2.3 kb band excised and ligated with T4 DNA ligase at 16° C for 24 hours to the cut pVTlOO- UCT7 DNA.
  • the ligation mixture was used to transform competent DH5 ⁇ E. coli (GIBCO BRL, Gaithersburg, MD). Plasmid DNA was prepared from several clones as before and digested with Xba I and Pvu Ll to verify the presence of the 2.3 kb fragment of C. albicans DNA. Clone pVT100-UCT7-7, containing this fragment, was selected for further analysis.
  • a culture of E. coli DH5 ⁇ which contains pVT100-UCT7-7 was deposited under the terms of the Budapest Treaty with the Agricultural Research Culture Collection, Peoria, Dlinois, on
  • DNA from clone pVT100-UCT7-7 was prepared from a 100 mL culture of E. coli using a Wizard Maxi-prep kit obtained from Promega (Madison, WI) and used to transform 5.
  • cerevisiae strains JN362a tl and JN394 tl using the method of Schiestl and Gietz (1989). Strains were obtained from Dr. John Nitiss, Children's Hospital, Los Angeles; genotypes are as given below: JN362a tl : MATa ura3-52, leu2, t ⁇ l , his7, ade l , topi : :LEU2 ISE2 and
  • JN394 tl MATa ura3-52, leu2, txpl , his7, adel , top l ::LEU2 rad52::TRPl ISE2.
  • the ISE mutation alters cell permeability thereby allowing the strain to be measured for sensitivity to topoisomerase agents such as camptothecin (Nitiss and Wang, 1988).
  • the topI::LEU 2 mutation results in the elimination of functional topo I activity.
  • Approximately 5 ⁇ g of pVT100-UCT7-7 DNA was used for each transformation with 50 ⁇ g sonicated denatured salmon sperm DNA (obtained from Dr. Robert Simmer, Abbott Laboratories) added as ca ⁇ ier.
  • the sensitivity of the transformed cells to camptothecin was determined as one measure of topoisomerase activity. These cells have been shown to be sensitive to camptothecin only when they contain a functional topoisomerase (Nitiss and Wang. 1988).
  • strains JN362 tl and JN 394 tl either alone or ca ⁇ ying only the pVTIOO-U plasmid (i.e. the expression vector lacking a TOPI gene), were essentially resistant to camptothecin as evidenced by MIC50 values of greater than 100 ⁇ g/mL. ln contrast, strain JN394 tl carrying either the C. albicans TOPI gene (in pVT100-UCT7-7), the 5.
  • strain JN394 (having a wild-type level of topo I) was shown to have an MIC50 of 1.56 ⁇ g/mL.
  • JN 362a tl >100.0 > 100.0 > 100.0* 10.0 0.2
  • the lysate was recovered and centrifuged at 12.000 ⁇ m in a Sorvall SS34 rotor for 40 minutes at 6°C.
  • the supematant was recovered, and incubated for 140 minutes at 4°C with hydroxylapatite resin.
  • the resin was recovered by centrifugation in an IEC refrigerated centrifuge at 4000 ⁇ m using a type 224 rotor for 6 minutes.
  • the resin was washed twice with buffer G plus 0.3 M KPO 4 , pH 7.5 (buffer G is 0.1 mM EDTA, 10% glycerol) and collected as before.
  • topoisomerase activity was eluted from the resin by incubating at room temperature for 10 minutes in the presence of buffer G plus 0.7 M KPO4 and removing the resin by centrifugation. The eluate was dialyzed overnight at 4°C vs. 50% glycerol, 100 mM KCI, 50 mM Tris pH 7.5, 0.1 mM EDTA, 0.1 mM DTT, 1 mM PMSF and stored at -20°C.
  • the protein content of each lysate was determined using the method of Bradford (1976) using reagents obtained from Bio-Rad (Hercules, CA), and is given in Table 5.
  • Topoisomerase activity was determined using standard conditions (Fostel et al. , 1992). Briefly, dilutions of each lysate were made in a dilution buffer containing bovine serum albumin (BSA; molecular biology grade, obtained from GIBCO BRL, Gaithersberg, MD) in buffer G with 15 mM KPO4 pH 7.5.
  • BSA bovine serum albumin

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Abstract

On décrit un polynucléotide qu'on a isolé et purifié et qui code la topoisomérase de type I de Candida albicans, de même que des procédés permettant de recombiner cette topoisomérase à l'aide de ces polynucléotides et de cellules hôtes transformées avec ces derniers. On décrit aussi un procédé d'identification des composés qui inhibent la croissance des cellules fongiques grâce à ce polynucléotide.
PCT/US1996/017291 1995-10-27 1996-10-28 Gene de la topoisomerase i de candida WO1997015676A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0835303A4 (fr) * 1995-06-07 2000-09-13 Univ Jefferson Agents antifongiques et leurs procedes d'identification et d'utlisation
WO2001025407A3 (fr) * 1999-10-05 2001-11-29 Us Health Tyrosine-adn- phosphodiesterases (tdp) et polypeptides, acides nucleiques, vecteurs, cellules hotes produisant du tdp, anticorps apparentes et procedes d'utilisation
WO2001021839A3 (fr) * 1999-09-24 2002-01-17 Upjohn Co Profils d'expression des genes
US6833373B1 (en) 1998-12-23 2004-12-21 G.D. Searle & Co. Method of using an integrin antagonist and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia
US6858598B1 (en) 1998-12-23 2005-02-22 G. D. Searle & Co. Method of using a matrix metalloproteinase inhibitor and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia
US7087736B1 (en) 1999-10-05 2006-08-08 The United States Of America As Represented By The Department Of Health And Human Services Tyrosine DNA phosphodiesterases (TDP) and related polypeptides nucleic acids vectors TDP producing host cells antibodies and methods of use

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5070192A (en) * 1988-03-23 1991-12-03 The Johns Hopkins University Cloned human topoisomerase i: cdna expression, and use for autoantibody detection
CA2223484A1 (fr) * 1995-06-07 1996-12-19 Thomas Jefferson University Agents antifongiques et leurs procedes d'identification et d'utlisation

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0835303A4 (fr) * 1995-06-07 2000-09-13 Univ Jefferson Agents antifongiques et leurs procedes d'identification et d'utlisation
US6833373B1 (en) 1998-12-23 2004-12-21 G.D. Searle & Co. Method of using an integrin antagonist and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia
US6858598B1 (en) 1998-12-23 2005-02-22 G. D. Searle & Co. Method of using a matrix metalloproteinase inhibitor and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia
WO2001021839A3 (fr) * 1999-09-24 2002-01-17 Upjohn Co Profils d'expression des genes
WO2001025407A3 (fr) * 1999-10-05 2001-11-29 Us Health Tyrosine-adn- phosphodiesterases (tdp) et polypeptides, acides nucleiques, vecteurs, cellules hotes produisant du tdp, anticorps apparentes et procedes d'utilisation
US7087736B1 (en) 1999-10-05 2006-08-08 The United States Of America As Represented By The Department Of Health And Human Services Tyrosine DNA phosphodiesterases (TDP) and related polypeptides nucleic acids vectors TDP producing host cells antibodies and methods of use

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