WO2002064797A2 - Mitochondrial topoisomerase i - Google Patents

Abstract

An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding mitochondrial topoisomerase I (mt topo I), a 5' fragment thereof, or a variant mt topo I, an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding mt topo I, a 5' fragment thereof, or a variant mt topo I, a vector comprising such an isolated or purified nucleic acid molecule, a cell comprising such a vector, an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding mt topo I or a variant mt topo I, a conjugate comprising such an isolated or purified polypeptide molecule and a cell-surface targeting moiety, a hybridoma cell line that produces a monoclonal antibody that is specific for an aforementioned isolated or purified polypeptide molecule, the monoclonal antibody produced by the hybridoma cell line, a polyclonal antiserum raised against an aforementioned isolated or purified polypeptide molecule, a method of altering the level of mt topo I in a cell, and a method of identifying an inhibitor or an activator of mt topo I.

Description

MITOCHONDRIAL TOPOISOMERASE I

FIELD OF THE INVENTION This invention pertains to an isolated or purified nucleic acid molecule, a vector comprising such a nucleic acid molecule, a cell comprising such a vector, an isolated or purified polypeptide, a conjugate comprising such a polypeptide and a targeting moiety, a hybridoma cell line, a monoclonal antibody, a polyclonal antiserum, and related methods of use.

BACKGROUND OF THE INVENTION

DNA topoisomerases are essential enzymes for regulating the structure of DNA. These enzymes are found in all living organisms. So far, only nuclear topoisomerases have been identified in mammalian cells: one type I topoisomerase, two type II topoisomerases, and two type III topoisomerases. No mitochondrial topoisomerase has been identified as of yet in mammalian cells.

DNA topoisomerases are among the most common targets for chemotherapy. Anticancer drugs, such as etoposide, doxorubicin and mitoxantrone, target type II topoisomerases in humans. Type I topoisomerases in humans are targeted by camptothecin derivatives in the treatment of cancer of the colon, lung and ovary. Antibacterial drugs, such as quinolones, more specifically norfloxacin, ciprofloxacin and their derivatives, poison type II topoisomerases in bacteria (otherwise referred to as gyrase and Topo IV enzymes).

In view of the above, it is an object of the present invention to provide the nucleotide and amino acid sequences of a mitochondrial topoisomerase I. This and other objects and advantages of the present invention, as well as additional inventive features, will become apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION The present invention provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a mitochondrial topoisomerase I (mt topo I) or a fragment thereof. The isolated or purified nucleic acid molecule can (i) encode the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 4) or (ii) consist essentially of the nucleotide sequence of SEQ ID NO: 1 (or SEQ ID NO: 3) or a fragment thereof. Also provided is an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a variant mt topo I. The present invention also provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding mt topo I or a fragment thereof. The isolated or purified nucleic acid molecule can (i) be complementary to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 4) or (ii) be complementary to the nucleotide sequence of SEQ ID NO: 1 (or SEQ ID NO: 3) or a fragment thereof. Also provided is an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding a variant mt topo I. The present invention further provides an isolated or purified DNA molecule consisting essentially of the genomic sequence of mt topo I or a fragment thereof. Preferably, the fragment comprises at least the first 15 contiguous nucleotides from exon 1 of mt topo I, particularly when the fragment is derived from a human mt topo I genomic sequence. Also provided by the present invention is a vector comprising one of the above- described isolated or purified nucleic acid molecules and a composition comprising the same. Further provided is a cell comprising such a vector.

An isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding mt topo I, which is optionally glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated or converted into an acid addition salt, is also provided by the present invention. The isolated or purified polypeptide molecule can consist essentially of the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 4). Also provided is an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a variant mt topo I. Further provided is a conjugate comprising an above-described isolated or purified polypeptide molecule and a targeting moiety. The targeting moiety can be an antibody or an antigenically reactive fragment thereof.

Still further provided is a hybridoma cell line that produces a monoclonal antibody that is specific for an above-described isolated or purified polypeptide molecule. Thus, the monoclonal antibody produced by the hybridoma cell line is also provided as is a polyclonal antiserum raised against an above-described isolated or purified polypeptide molecule.

Yet still further provided is a method of altering the level of mt topo I in a cell. The method comprises contacting a cell with (i) an above-described isolated or purified nucleic acid, (ii) a vector comprising or encoding an antisense molecule that is specific for mt topo I, (iii) a vector comprising or encoding a ribozyme that is specific for mt topo I, (iv) an above-described polypeptide, or (v) an above-described conjugate, whereupon the level of mt topo I in the cell is altered.

A method of identifying an inhibitor or an activator of mt topo I is also provided. The method comprises contacting an above-described polypeptide with an agent in vitro and assaying for topoisomerase activity. A decrease in topoisomerase activity after contact of the polypeptide with the agent indicates that the agent is an inhibitor of mt topo I. An increase in topoisomerase activity after contact of the polypeptide with the agent indicates that the agent is an activator of mt topo I.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the nucleotide sequence of human mt topo I cDNA (SEQ ID NO: 1).

Figure 2 represents a plasmid map of a construct comprising mt topo I cDNA used to express mt topo I in E. coli.

Figure 3 is a map of human chromosome 8 showing the position of the gene encoding mt topo I. Figure 4 A is a schematic representation of the exons for the human mt topo I gene

(designated mTopl in the figure). The numbers correspond to the last amino acid residue for each exon.

Figure 4B is a schematic representation of the exons for the human nuclear topoisomerase I (designated topi in the figure). The numbers correspond to the last amino acid residue for each exon.

Figure 5 is a diagram of the three possible alternative splices in the first intron of the human mt topo I gene. The numbers on the top correspond to the amino acid residues for exons 1 and 2. The numbers at the bottom are nucleotide portions for the sequences deposited with GenBank (see Figure 7). Figure 6 is a ClustalW formatted alignment of the mt topo I sequence (top line in each row; SEQ ID NO: 2) with the nuclear topo I sequence (bottom line in each row). Figure 7 represents genomic sequences for human mt topo I. Figure 8 is the nucleotidic sequence of mouse mt topo I cDNA (SEQ ID NO: 3) in which the sequence is set forth from 5' to 3' from left to right, starting at the upper left and ending at the bottom right and in which the codon region is given in capital letters.

Figure 9 is the deduced amino acid sequence encoded by the mouse mt topo I cDNA (SEQ ID NO: 4) in which the sequence is set forth from N-terminus to C-terminus from left to right, starting at the upper left and ending at the bottom right.

Figure 10 presents a partial genomic sequence of mouse mt topo I.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a mt topo I or a fragment thereof. By "isolated" is meant the removal of a nucleic acid from its natural environment. By "purified" is meant that a given nucleic acid, whether one that has been removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and/or amplified under laboratory conditions, has been increased in purity, wherein "purity" is a relative term, not "absolute purity." "Nucleic acid molecule" is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double- stranded and which can contain non-natural or altered nucleotides.

Preferably, the isolated or purified nucleic acid molecule that consists essentially of a nucleotide sequence encoding a mt topo I or a fragment thereof (i) encodes the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO:4) or (ii) consists essentially of the nucleotide sequence of SEQ ID NO: 1 (or SEQ ID NO: 3) or a fragment thereof. Preferably, the fragment is a 5' fragment, such as one comprising at least the first 15 contiguous nucleotides, particularly when the fragment is derived from a human mt topo I nucleic acid molecule.

Also provided is an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a variant mt topo I. The variant comprises one or more insertions, deletions and/or substitutions and, when the variant mt topo I is a variant human mt topo I, the one or more insertions, deletions and/or substitutions are in a region other than that which encodes alanine at amino acid positions 88 and 93, wherein the amino acid positions are given with respect to SEQ ID NO: 2. Desirably, the variant mt topo I does not differ functionally from the corresponding unmodified mt topo I, such as that comprising SEQ ID NO: 2 (or SEQ ID NO: 4). Preferably, the variant mt topo I relaxes DNA, preferably in the presence of divalent metals, e.g., Mg or Ca, at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the corresponding unmodified mt topo I as determined by in vitro assay. The manner in which the assay is carried out is not critical and can be conducted in accordance with methods known in the art.

The present invention also provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding mt topo I or a fragment thereof. Such an isolated or purified nucleic acid molecule preferably (i) is complementary to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 4) or (ii) is complementary to the nucleotide sequence of SEQ ID NO: 1 (or SEQ ID NO: 3) or a fragment thereof. Also provided is an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding a variant mt topo I, which comprises one or more insertions, deletions and/or substitutions and, when the variant mt topo I is a variant human mt topo I, the one or more insertions, deletions and/or substitutions are in a region other than that which encodes alanine at amino acid positions 88 and 93, wherein the amino acid positions are given with respect to SEQ ID NO: 2.

The present invention further provides an isolated or purified DNA molecule consisting essentially of the genomic sequence of mt topo I or a fragment thereof. Preferably, the fragment comprises at least the first 15 contiguous nucleotides from exon 1 of mt topo I, particularly when the fragment is derived from a human mt topo I genomic sequence. See Figures 7 and 10.

With respect to the above, one of ordinary skill in the art knows how to generate insertions, deletions and/or substitutions in a given nucleic acid molecule. See, for example, the references cited herein under "Examples." With respect to the above isolated or purified nucleic acid molecules, it is preferred that no insertions, deletions and/or substitutions are introduced into the region encoding amino acids 88, 93 and 559 and those amino acids in the immediate vicinity of amino acids 88, 93 and 559 in the human mt topo I, wherein the amino acid positions are given with respect to SEQ ID NO: 2. It is also preferred that the one or more substitution(s) do(es) not result in a change in an amino acid of mt topo I. Alternatively, and also preferred, is that the one or more substitution(s) result(s) in the substitution of an amino acid with another amino acid of approximately equivalent size, shape and charge. Also with respect to the above, "does not differ functionally from" is intended to mean that the variant mt topo I has activity characteristic of the unmodified mt topo I. In other words, it relaxes DNA. However, the variant mt topo I can be more or less active than the unmodified mt topo I as desired in accordance with the present invention.

An indication that polynucleotide sequences are substantially identical is if two molecules selectively hybridize to each other under stringent conditions. The phrase

"hybridizes to" refers to the selective binding of a single-stranded nucleic acid probe to a single-stranded target DNA or RNA sequence of complementary sequence when the target sequence is present in a preparation of heterogeneous DNA and/or RNA. "Stringent conditions" are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.

For example, under stringent conditions, as that term is understood by one skilled in the art, hybridization is preferably carried out using a standard hybridization buffer at a temperature ranging from about 50°C to about 75°C, even more preferably from about 60°C to about 70°C, and optimally from about 65°C to about 68°C. Alternately, formamide can be included in the hybridization reaction, and the temperature of hybridization can be reduced to preferably from about 35°C to about 45°C, even more preferably from about 40°C to about 45°C, and optimally to about 42°C. Desirably, formamide is included in the hybridization reaction at a concentration of from about 30% to about 50%), preferably from about 35% to about 45%), and optimally at about 40%. Moreover, optionally, the hybridized sequences are washed (if necessary to reduce non-specific binding) under relatively highly stringent conditions, as that term is understood by those skilled in the art. For instance, desirably, the hybridized sequences are washed one or more times using a solution comprising salt and detergent, preferably at a temperature of from about 50°C to about 75°C, even more preferably at from about 60°C to about 70°C, and optimally from about 65°C to about 68°C. Preferably, a salt (e.g., such as sodium chloride) is included in the wash solution at a concentration of from about 0.01 M to about 1.0 M. Optimally, a detergent (e.g., such as sodium dodecyl sulfate) is also included at a concentration of from about 0.01% to about 1.0%.

In view of the above, "stringent conditions" preferably allow for from about 25% to about 5% mismatch, more preferably from about 15% to about 5% mismatch, and most preferably from about 10% to about 5% mismatch. "At least moderately stringent conditions" preferably allow for from about 40% to about 15% mismatch, more preferably from about 30%) to about 15% mismatch, and most preferably from about 20% to about 15% mismatch. "Low stringency conditions" preferably allow for from about 60%) to about 35% mismatch, more preferably from about 50% to about 35% mismatch, and most preferably from about 40% to about 35% mismatch. With respect to the preceding ranges of mismatch, 1% mismatch corresponds to one degree decrease in the melting temperature.

The above isolated or purified nucleic acid molecules also can be characterized in terms of "percentage of sequence identity." In this regard, a given nucleic acid molecule as described above can be compared to a nucleic acid molecule encoding a corresponding gene (i.e., the reference sequence) by optimally aligning the nucleic acid sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences, i.e., the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information, Bethesda, MD), or by inspection. Sequences are typically compared using BESTFIT or BlastN with default parameters. "Substantial sequence identity" means that at least 75%, preferably at least 80%, more preferably at least 90%), and most preferably at least 95% of the sequence of a given nucleic acid molecule is identical to a given reference sequence. Typically, two polypeptides are considered to be substantially similar if at least 40%, preferably at least 60%, more preferably at least 90%), and most preferably at least 95% of the amino acids of which the polypeptides are comprised are identical to or represent conservative substitutions of the amino acids of a given reference sequence. One of ordinary skill in the art will appreciate, however, that two polynucleotide sequences can be substantially different at the nucleic acid level, yet encode substantially similar, if not identical, amino acid sequences, due to the degeneracy of the genetic code. The present invention is intended to encompass such polynucleotide sequences.

While the above-described nucleic acid molecules can be isolated or purified, alternatively they can be synthesized. Methods of nucleic acid synthesis are known in the art. See, e.g., the references cited herein under "Examples."

. The above-described nucleic acid molecules can be used, in whole or in part (i.e., as fragments or primers), to identify and isolate corresponding genes from other organisms for use in the context of the present inventive method using conventional means known in the art. See, for example, the references cited herein under "Examples."

In view of the above, the present invention also provides a vector comprising an above-described isolated or purified nucleic acid molecule. A nucleic acid molecule as described above can be cloned into any suitable vector and can be used to transform or transfect any suitable host. The selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references (see, in general, "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987) and the references cited herein under "Examples"). Desirably, the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA. Preferably, the vector comprises regulatory sequences that are specific to the genus of the host. Most preferably, the vector comprises regulatory sequences that are specific to the species of the host. Constructs of vectors, which are circular or linear, can be prepared to contain an entire nucleic acid sequence as described above or a portion thereof ligated to a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived from ColEl, 2 mμ plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, the construct can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable vectors include those designed for propagation and expansion or for expression or both. A preferred cloning vector is selected from the group consisting of the pUC series the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clonetech, Palo Alto, CA). Bacteriophage vectors, such as λGTIO, λGTl 1, λZapII

(Stratagene), λ EMBL4, and λ NM1149, also can be used. Examples of plant expression vectors include pBHOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clonetech, Palo Alto, CA). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clonetech). An expression vector can comprise a native or normative promoter operably linked to an isolated or purified nucleic acid molecule as described above. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the skill in the art. Similarly, the combining of a nucleic acid molecule as described above with a promoter is also within the skill in the art. The present invention not only provides a vector comprising an isolated or purified nucleic acid molecule as described above but also provides a vector comprising or encoding an antisense sequence that hybridizes to or a ribozyme that cleaves an RNA molecule encoding mt topo I. The present invention also provides the antisense molecules, which preferably are at least about 20 nucleotides in length, and the ribozymes, which preferably comprise at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.

Also in view of the above, the present invention provides a host cell comprising an isolated or purified nucleic acid molecule or a vector as described above. Examples of host cells include, but are not limited to, a human cell, a human cell line, adenovirus, adeno- associated virus, Rous sarcoma virus, mouse mammary tumor virus, Epstein bar virus, E. coli, B. subtilis, P. aerugenosa, S. cerevisiae, and N. crassa. E. coli, in particular E. coli TB-1, TG-2, DH5α, XL-Blue MRF' (Stratagene), SA2821 and Y1090 are preferred hosts. The present invention further provides an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding mt topo I, which is optionally glycoslyated, amidated, carboxylated, phosphorylated, esterified, N-acylated or converted into an acid addition salt. Preferably, the isolated or purified polypeptide molecule consists essentially of the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 4). Also provided is an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a variant mt topo I, which comprises an alanine at amino acid position 88 and an alanine at amino acid position 93 (wherein the amino acid positions are given with respect to SEQ ID NO: 2) when the variant mt topo I is a variant human mt topo I, and which is optionally glycoslyated, amidated, carboxylated, phosphorylated, esterified, N- acylated or converted into an acid addition salt. The N-terminal segment of the polypeptide, which preferably comprises about 50 amino acids, more preferably at least 40 amino acids, can be used to target a desired polypeptide into the mitochondrion, such as in the context of a fusion protein. The polypeptide preferably comprises an amino end and a carboxyl end. The polypeptide can comprise D-amino acids, L-amino acids or a mixture of D- and L-amino acids. The D-form of the amino acids, however, is particularly preferred since a polypeptide comprised of D-amino acids is expected to have a greater retention of its biological activity in vivo, given that the D-amino acids are not recognized by naturally occurring proteases. The polypeptide can be prepared by any of a number of conventional techniques. The polypeptide can be isolated or purified from a naturally occurring source or from a recombinant source. For instance, in the case of recombinant polypeptides, a DNA fragment encoding a desired peptide can be subcloned into an appropriate vector using well-known molecular genetic techniques (see, e.g., Maniatis et al., Molecular Cloning: A Laboratorv Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989)). The fragment can be transcribed and the polypeptide subsequently translated in vitro. Commercially available kits can also be employed (e.g., such as manufactured by Clontech, Palo Alto, CA; Amersham Life Sciences, Inc., Arlington Heights, IL; InVitrogen, San Diego, CA, and the like). The polymerase chain reaction optionally can be employed in the manipulation of nucleic acids. In addition, the polypeptide or fragment thereof can be glycosylated in accordance with methods known in the art.

Alterations of the native amino acid sequence to produce variant polypeptides can be done by a variety of means known to those skilled in the art. For instance, site-specific mutations can be introduced by ligating into an expression vector a synthesized oligonucleotide comprising the modified site. Alternately, oligonucleotide-directed site- specific mutagenesis procedures can be used such as disclosed in Walder et al., Gene, 42, 133 (1986); Bauer et al., Gene, 37, 73 (1985); Craik, Biotechniques. 12-19 (January 1995); and U.S. Patents Nos. 4,518,584 and 4,737,462.

With respect to the above isolated or purified polypeptides, it is preferred that no insertions, deletions and/or substitutions are introduced into the region of amino acids 88, 93 and 559 and those amino acids in the immediate vicinity of amino acids 88, 93 and 559 in the human mt topo I polypeptide, wherein the amino acid positions are given with respect to SEQ ID NO: 2.

Any appropriate expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratorv Manual (Elsevior, NY: 1985)) and corresponding suitable host can be employed for production of recombinant polypeptides. Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomonas, Salmonella, mammalian or insect host cell systems including baculovirus systems (e.g., as described by Luckow et al., Bio/Technology, 6, 47 (1988)), and established cell lines such as the COS-7, C127, 3T3, CHO, HeLa, BHK cell line, and the like. The ordinary skilled artisan is, of course, aware that the choice of expression host has ramifications for the type of polypeptide produced. For instance the glycosylation of polypeptides produced in yeast or mammalian cells (e.g., COS-7 cells) will differ from that of polypeptides produced in bacterial cells such as Escherichia coli.

Alternately, the polypeptide (including the variant peptides) can be synthesized using standard peptide synthesizing techniques well-known to those of skill in the art (e.g., as summarized in Bodanszky, Principles of Peptide Synthesis, (Springer- Verlag, Heidelberg: 1984)). In particular, the polypeptide can be synthesized using the procedure of solid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc, 85, 2149-54 (1963); Barany et al., Int. J. Peptide Protein Res.. 30, 705-739 (1987); and U.S. Patent No. 5,424,398). If desired, this can be done using an automated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished by, for example, acid treatment at reduced temperature. The polypeptide-containing mixture can then be extracted, for instance, with dimethyl ether, to remove non-peptidic organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w/v acetic acid). Following the synthesis of the polypeptide, further purification (e.g., using high performance liquid chromatography (HPLC)) optionally can be done in order to eliminate any incomplete polypeptides or free amino acids. Amino acid and/or HPLC analysis can be performed on the synthesized polypeptide to validate its identity. For other applications according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, either by chemical conjugation, or through genetic means, such as are known to those skilled in the art.

If desired, the polypeptides of the invention (including variant polypeptides) can be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the polypeptides of the invention. The polypeptides also can be modified to create polypeptide derivatives by forming covalent or noncovalent complexes with other moieties in accordance with methods known in the art. Covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the polypeptides, or at the N- or C-terminus. Thus, in this regard, the present invention also provides a conjugate comprising an above-described isolated or purified polypeptide molecule or fragment thereof and a targeting moiety. Preferably, the targeting moiety is an antibody or an antigenically reactive fragment thereof. Alternatively, the targeting moiety can be a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a colorimetric or fiuorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). Methods of conjugation are known in the art. In addition, conjugate kits are commercially available. The present invention also provides a composition comprising a pharmaceutically acceptable carrier and either (i) an above-described isolated or purified nucleic acid molecule or fragment thereof, (ii) an above-described vector, (iii) an above-described polypeptide molecule, or (iv) an above-described conjugate comprising an above-described isolated or purified polypeptide molecule and a targeting moiety. Pharmaceutically acceptable carriers are well-known in the art, and are readily available. The choice of carrier will be determined in part by the particular route of administration and whether a nucleic acid molecule or a polypeptide molecule (or conjugate thereof) is being administered. Accordingly, there is a wide variety of suitable formulations for use in the context of the present invention, and the invention expressly provide a pharmaceutical composition that comprises an active agent of the invention and a pharmaceutically acceptable carrier therefor. The following methods and carriers are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluent, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth. Pastilles can comprise the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients/carriers as are known in the art.

An active agent of the present invention, either alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellents, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit- dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

Additionally, active agents of the present invention can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. Further suitable formulations are found in Remington's Pharmaceutical Sciences, 17th ed., (Mack Publishing Company, Philadelphia, Pa.: 1985), and methods of drug delivery are reviewed in, for example, Langer, Science, 249, 1527-1533 (1990). Further provided by the present invention is a hybridoma cell line that produces a monoclonal antibody that is specific for an above-described isolated or purified polypeptide molecule and does not cross-react with an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a nuclear topoisomerase I. Methods of making hybridomas are known in the art (see, e.g., the references cited herein under "Examples."). Thus, the present invention also provides the monoclonal antibody produced by the hybridoma cell line. Similarly, the present invention provides a polyclonal antiserum raised against an above-described isolated or purified polypeptide molecule that does not cross-react with an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a nuclear topoisomerase I. Methods of raising polyclonal antiserum against a polypeptide molecule are also known in the art (see, e.g., the references cited herein under "Examples.").

A method of altering the level of mt topo I in a cell is also provided by the present invention. The method comprises contacting a cell with (i) an above-described isolated or purified nucleic acid, (ii) a vector comprising or encoding an antisense molecule that is specific for mt topo I, (iii) a vector comprising or encoding a ribozyme that is specific for mt topo I,

(iv) an above-described isolated or purified polypeptide or functional fragment thereof, or (v) a conjugate of an above-described isolated or purified polypeptide (or functional fragment thereof) and an antibody of an antigenically reactive fragment thereof, whereupon the level of mt topo I in the cell is altered. Preferably, the isolated or purified polypeptide is contained within a liposome comprising a cell-surface targeting moiety that binds to the cell being contacted. Herein, "contacting" is intended to mean that the cell, whether an individual cell or a collection of cells in the form of a tissue, organ or organism, is brought into contact with the isolated or purified nucleic acid molecule (or functional fragment thereof), vector or polypeptide molecule (or functional fragment thereof or conjugate thereof) in such a manner that the nucleic acid molecule (or functional fragment thereof), such as in the form of a vector, enters the cell and is expressed therein or the polypeptide molecule (or functional fragment thereof or conjugate thereof) is taken up by the cell. The cell can be contacted with mt topo I by any suitable manner, including by in vivo, in vitro and ex vivo methods. By "functional fragment thereof is meant that the fragment has activity characteristic of mt topo I. In other words, it relaxes DNA. The mt topo I fragment, however, can be more or less active than the complete mt topo I as desired in accordance with the present invention. Desirably, the functional fragment comprises at least the amino- terminal end of mt topo I or a variant thereof.

The nucleic acid sequence introduced in antisense suppression generally is substantially identical to at least a portion, preferably at least about 20 contiguous nucleotides, of the mt topo I gene, but need not be identical. The vectors can, thus be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantially homology to the target gene. The introduced sequence also need not be full-length relative to either of the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective. As an alternative to antisense suppression, interfering RNA can be used to achieve the same effect by a different mechanism of action. Ribozymes can be designed such that they specifically pair with virtually any target

RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered and is, thus, capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences with antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334: 585-591 (1988). Preferably, the ribozyme comprises at least about 20 contiguous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.

The above-described isolated or purified nucleic acid molecule or functional fragment thereof preferably is operably linked to a promoter. Preferably, the isolated or purified nucleic acid molecule or functional fragment thereof is in the form of a vector. Whether or not the nucleic acid molecule or functional fragment thereof is necessarily in the form of a vector depends, in part, on the particular method (e.g., transformation, transduction, electroporation, microinjection; etc.) used to contact the cell as is known in the art. The isolated or purified polypeptide molecule or functional fragment thereof can be contained within a liposome comprising a cell-surface targeting moiety that binds to the cell being contacted.

Preferably, the cell that is contacted is a cancerous cell and the method results in a decrease in the viability and/or metastatic potential of the cancerous cell, such as through the use of a vector comprising or encoding either of an antisense molecule or a ribozyme. Desirably, when the cell is a cancerous cell, the method further comprises the simultaneous or sequential administration, whether by the same or different route, of an anti-cancer agent, such as a chemotherapeutic agent.

Preferred routes of administration in the treatment of cancer include intratumoral and peritumoral. Also preferred is administration that is targeted to a cancer cell. In this regard, examples of cancer-specific, cell-surface molecules include placental alkaline phosphatase (testicular and ovarian cancer), pan carcinoma (small cell lung cancer), polymorphic epithelial mucin (ovarian cancer), prostate-specific membrane antigen, α- fetoprotein, B-lymphocyte surface antigen (B-cell lymphoma), truncated EGFR (gliomas), idiotypes (B-cell lymphoma), gp95/gp97 (melanoma), N-CAM (small cell lung carcinoma), cluster w4 (small cell lung carcinoma), cluster 5A (small cell carcinoma), cluster 6 (small cell lung carcinoma), PLAP (seminomas, ovarian cancer, and non-small cell lung cancer), CA-125 (lung and ovarian cancers), ESA (carcinoma), CD 19, 22 or 37 (B-cell lymphoma), 250 kD proteoglycan (melanoma), P55 (breast cancer), TCR-IgH fusion (childhood T-cell leukemia), blood group A antigen in B or O type individual (gastric and colon tumors), and the like. Examples of cancer-specific, cell-surface receptors include erbB-2, erbB-3, erbB-4,

IL-2 (lymphoma and leukemia), IL-4 (lymphoma and leukemia), IL-6 (lymphoma and leukemia), MSH (melanoma), transferrin (gliomas), tumor vasculature integrins, and the like. Preferred cancer-specific, cell-surface receptors include erbB-2 and tumor vasculature integrins, such as CD1 la, CD1 lb, CD1 lc, CD 18, CD29, CD51, CD61, CD66d, CD66e, CD 106, and CDwl45.

There are a number of antibodies to cancer-specific, cell-surface molecules and receptors that are known. C46 Ab (Amersham) and 85A12 Ab (Unipath) to carcino- embryonic antigen, H17E2 Ab (ICRF) to placental alkaline phosphatase, NR-LU-10 Ab (NeoRx Corp.) to pan carcinoma, HMFC1 Ab (ICRF) to polymorphic epithelial mucin, W14 Ab to B-human chorionic gonadotropin, RFB4 Ab (Royal Free Hospital) to B- lymphocyte surface antigen, A33 Ab (Genex) to human colon carcinoma, TA-99 Ab (Genex) to human melanoma, antibodies to c-erbB2 (JP 7309780, JP 8176200 and JP 7059588), and the like. ScAbs can be developed, based on such antibodies, using techniques known in the art (see for example, Bind et al., Science 242: 423-426 (1988), and Whitlow et al., Methods 2(2): 97-105 (1991)).

In general, there are a number of databases for ligands, binding domains and cell- surface molecules. See, for example, ftp://kegg.genome.ad.jp, http://broweb.pasteur.fr/docs/versions, http://ampere.doe-mbi.ucla.edu:8801/dat/dip.dat or http://bones.biochem.ualberta.ca/pedro/rt- 1.html 1.

In addition to the treatment of cancer, there are a number of diseases and conditions involving lesions in the mitochondrion. Such diseases and conditions include, for example, neuromuscular diseases (such as myopathies, neuropathies and Alzheimer's disease), cardiac diseases (such as cardiomyopathy), diabetes, anemia, pancytopenia, pancreatitis, hepatic failure, isovaleric academia, citrullinemia, porphyria, Brunner's syndrome, Leigh syndrome, Leber's hereditary optic neuropathy, and Mohr-Tranebjaerg syndrome, among others. Cytoplasmic male sterility in some plants is believed to involve a lesion in the mitochondrion. Those diseases and conditions that involve a lesion directly or indirectly affecting the mt topo I could be treated in accordance with the above-described method of altering the level of mt topo I in a cell.

The dose administered to an animal, particularly a human, or a plant in the context of the present invention will vary with the nucleic acid molecule or polypeptide molecule administered, the composition employed, the route of administration, whether individual cells, a tissue, an organ or an organism is being contacted, and the particular site being treated.

Generally, when an above-described polypeptide is administered to an animal, such as a mammal, in particular a human, it is preferable that the polypeptide is administered in a dose of from about 1 to about 1,000 micrograms of the polypeptide per kg of the body weight of the host per day when given parenterally. However, this dosage range is merely preferred, and higher or lower doses may be chosen in appropriate circumstances. For instance, the actual dose and schedule can vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. One skilled in the art easily can make any necessary adjustments in accordance with the necessities of the particular situation.

If desired, the half-life of the polypeptide can be increased by conjugation to soluble macromolecules, such as polysaccharides, or synthetic polymers, such as polyethylene glycol, as described, for instance, in U.S. Patent Nos. 5,116,964, 5,336,603, and 5,428,130. Alternately, the polypeptides can be "protected" in vesicles composed of substances such as proteins, lipids (for example, liposomes), carbohydrates, or synthetic polymers. If liposomes are employed, liposome delivery can be carried out as described in U.S. Patent No. 5,468,481 , or using liposomes having increased transfer capacity and/or reduced toxicity in vivo (see, e.g., international patent application WO 95/21259 and the references cited therein). Furthermore, polypeptides can be administered in conjunction with adenovirus (preferably replication- deficient adenovirus) to allow the intracellular uptake of the polypeptides by adenoviral- mediated uptake of bystander molecules (e.g., as described in international patent application WO 95/21259). Similarly, a fusion of a conjugate of an above-described polypeptide and an antibody (or an antigenically reactive fragment thereof) that recognizes a cell surface antigen; etc. as described below with respect to nucleic acids can be employed to deliver the resultant fusion protein to a specific target cell or tissue (e.g., as described in U.S. Patent No. 5,314,995).

Those of ordinary skill in the art can easily make a determination of the amount of an above-described isolated and purified nucleic acid molecule to be administered to an animal, such as a mammal, in particular a human. The dosage will depend upon the particular method of administration, including any vector or promoter utilized. For purposes of considering the dose in terms of particle units (pu), also referred to as viral particles, it can be assumed that there are 100 particles/pfu (e.g., 1x10 pfu is equivalent to lxlO¹⁴ pu). An amount of recombinant virus, recombinant DNA vector or RNA genome sufficient to achieve a tissue concentration of about 10²to about 10¹² particles per ml is preferred, especially of about 10⁶to about 10¹⁰ particles per ml. In certain applications, multiple daily doses are preferred. Moreover, the number of doses will vary depending on the means of delivery and the particular recombinant virus, recombinant DNA vector or RNA genome administered.

A targeting moiety also can be used in the contact of a cell with an above-described isolated or purified nucleic acid molecule. In this regard, any molecule that can be linked with the therapeutic nucleic acid directly or indirectly, such as through a suitable delivery vehicle, such that the targeting moiety binds to a cell-surface receptor, can be used. The targeting moiety can bind to a cell through a receptor, a substrate, an antigenic determinant or another binding site on the surface of the cell. Examples of a targeting moiety include an antibody (i.e., a polyclonal or a monoclonal antibody), an immunologically reactive fragment of an antibody, an engineered immunoprotein and the like, a protein (target is receptor, as substrate, or regulatory site on DNA or RNA), a polypeptide (target is receptor), a peptide (target is receptor), a nucleic acid, which is DNA or RNA (i.e., single-stranded or double-stranded, synthetic or isolated and purified from nature; target is complementary nucleic acid), a steroid (target is steroid receptor), and the like. In general, there are a number of computer databases for targeting moieties (see, e.g., ftp://kegg.genome.ad.jp, http://broweb.pasteur.fr/docs/versions, http://ampere.doe-mbi.ucla.edu:8801/dat/dip.dat, or http://bones.biochem.ualberta.ca/pedro/rt-l.htmll). Analogs of targeting moieties that retain the ability to bind to a defined target also can be used. In addition, synthetic targeting moieties can be designed, such as to fit a particular epitope. Alternatively, the therapeutic nucleic acid can be encapsulated in a liposome comprising on its surface the targeting moiety. The targeting moiety includes any linking group that can be used to join a targeting moiety to, in the context of the present invention, an above-described nucleic acid molecule. It will be evident to one skilled in the art that a variety of linking groups, including bifunctional reagents, can be used. The targeting moiety can be linked to the therapeutic nucleic acid by covalent or non-covalent bonding. If bonding is non-covalent, the conjugation can be through hydrogen bonding, ionic bonding, hydrophobic or van der Waals interactions, or any other appropriate type of binding.

A method of identifying an inhibitor or an activator of mt topo I also is provided by the present invention. The method comprises contacting an above-described polypeptide with an agent in vitro and assaying for topoisomerase activity. A decrease in topoisomerase activity after contact of the polypeptide with the agent indicates that the agent is an inhibitor of mt topo I. An increase in topoisomerase activity after contact of the polypeptide with the agent indicates that the agent is an activator of mt topo I. Methods of assessing topoisomerase activity are known in the art.

EXAMPLES

The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

Birren et al., Genome Analysis: A Laboratory Manual Series, Volume 1, Analyzing DNA, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1997),

Birren et al., Genome Analysis: A Laboratory Manual Series, Volume 2, Detecting Genes, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998),

Birren et al., Genome Analysis: A Laboratory Manual Series, Volume 3, Cloning Systems, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999),

Birren et al., Genome Analysis: A Laboratory Manual Series, Volume 4, Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY (1988), Harlow et al., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999),

Hoffman, Cancer and the Search for Selective Biochemical Inhibitors, CRC Press (1999),

Pratt, The Anticancer Drugs, 2nd edition, Oxford University Press, NY (1994),

QIAexpress Detection and Assay Handbook, 2nd edition, QIAGEN Inc., 28159 Avenue Stanford, Valencia, CA 91355 (April 1999), and

Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

Example 1

This example describes the cloning and sequence of human mt topo I cDNA. Sequences from the nuclear topoisomerase I were used to search for homologs. A clone, Genbank accession number AI872335, was identified and ordered from GenomeSystem Inc. The clone, which comprised an insert in the vector pCMV-SPORT6 was sequenced.

Based on the sequence, two PCR primer pairs were designed. A 1074 bp product was obtained from the middle of the gene. A 136 bp product was obtained from the 5' end and GeneRacer (Invitrogen, Carlsbad, CA) was used to amplify the 5' end. A 262 bp fragment was spliced with a 1661 bp fragment from AI872335 to generate the 1923 bp mt topo I cDNA. The cDNA was cloned into a pET- 15b vector.

The nucleotide sequence (SEQ ID NO: 1) is shown in Figure 1, in which the sequence is set forth from 5' to 3' from left to right, starting at the upper left and ending at the bottom right. The start codon is underlined. The sequence differs significantly from the nuclear mt topo I. The longest stretch of contiguous nucleotides in the mt topo I that is identical to a contiguous stretch of nucleotides in the nuclear topo I is 15 nucleotides.

The deduced amino acid sequence indicates that the mt topo I is highly homologous to the nuclear topo I, except for the N-terminal domain, which is much shorter in the mt topo I. See Figure 6, which is a ClustalW formatted alignment of the mt topo I sequence (top line in each row; SEQ ID NO: 2) with the nuclear topo I sequence (bottom line in each row).

Example 2

This example describes the use of the cDNA sequence of Example 1 to isolate the corresponding genomic sequence encoding mt topo I. A genomic library was probed with two pairs of PCR primers derived from the cDNA.

A clone was obtained that contained the genomic sequence encoding mt topo I. Exon structure was determined by direct sequencing of the genomic DNA probe (PI) using primers derived from the cDNA sequence and determining the boundaries between exons and introns. The gene has 14 exons as shown in Figure 4 A. Figure 4 A is a schematic representation of the exons for the human mt topo I gene (designated mTopl in the figure).

Figure 4B is a schematic representation of the exons for the human nuclear topoisomerase I (designated topi in the figure). The nuclear topoisomerase I gene contains 21 exons. The first 8 exons of the nuclear topoisomerase I are different from the first exon of the mt topo I. Nuclear localization signals for nuclear topoisomerase I are located in the first 8 exons, whereas the mitochondrial localization signal for mt topo I is located in the first exon. Catalytic tyrosine residues are indicated with arrows. Alternative splicing in the first intron of the mt topo I gene yields three possible transcripts as shown in Figure 5. Figure 5 is a diagram of the three possible alternative splices in the first intron of the human mt topo I gene. The most common transcript is spliced between residues 41 and 42 and yields the full-length mt topo I. The two alternative transcripts (la and lb) result in a stop codon and, therefore, do not yield functional topoisomerase. Note that the alternative transcript lb has two alternative 5' splice sites, namely at nucleotide 2224 and nucleotide 2280.

Example 3

This example describes the use of the complete genomic sequence as a probe to identify the chromosomal location of the gene.

The genomic probe (PI) carrying the whole mt topo I gene was used as the probe to map the chromosomal position of mtTopl by fluorescence in situ hybridization (FISH). This genomic clone was screened using two pairs of PCR primers from the mtTopl cDNA, one pair from the 5' end of the cDNA and another from the 3' primer end. The FISH experiments were according to previously published procedures (Zimonjic et al., Cancer Genet Cvtogenet 80:100-102 (1995)). The FISH probe was labeled with biotin (Random- Primer DNA labeling kit; Boehringer Mannheim Corp., Indianapolis, IN) and used for in situ hybridization of chromosomes derived from normal mouse spleen cultures. The hybridization conditions, processing, analysis, and direct fluorescent signal localization on banded chromosomes were performed as previously described in detail (Zimonjic et al.

(1995), supra). The mt topo I gene was localized to chromosome 8q24.3 as shown in Figure 3, which is a map of chromosome 8.

Example 4 This example demonstrates that the cDNA-expressed polypeptide exhibits topoisomerase I activity. The plasmid shown in Figure 2 was generated and used to express the mt topo I cDNA in E. coli. The expressed polypeptide exhibited topoisomerase I activity. The mt topo I belongs to the group of type IB topoisomerases. .

Mt topo I-mediated DNA relaxation assays were also conducted. Reaction mixtures (10 μl) contained 0.3 μg native supercoiled SV40 DNA per reaction. Reactions were performed in the presence and absence of Mg, Ca and ΕDTA at room temperature in reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 0.1 mM Na2ΕDTA, 15 μg/ml bovine serum albumin, 0.2 mM dithiothreitol [DTT]) for 30 minutes, using no topoisomerase and nuclear topoisomerase I as controls, and were terminated by the addition of sodium dodecylsulfate (SDS) (0.5% final concentration). An amount of 1.1 μl of 1 OX loading buffer (20%) Ficoll 400, 0.1 M Na₂EDTA, pH 8, 1.0% SDS, 0.25% bromophenol blue) was then added, and reactions were loaded onto 1% agarose gels run in TBE (Tris.borate.EDTA) buffer. After electrophoresis, the gels were stained with IX buffer solution containing 10 μg/ml of ethidium bromide, and were visualized by transillumination with UV light (300 nm). The results showed that mt topo I relaxed DNA; i.e., has topoisomerase activity.

Example 5

This example demonstrates that the topoisomerase I is concentrated in mitochondria and the mitochondrial localization signal resides in the N-terminal domain. The mt topo I cDNA from Example 1 was cloned into the Eco RI site of pEGFP-N2

(Clontech, Palo Alto, CA), which contains the green fluorescent protein (GFP) gene. The encoded polypeptide comprises the full-length mt topo I fused at its C-terminus to GFP. The resulting plasmid was transfected (using 1 μg of plasmid DNA in 100 μl solution containing 5 μl of FuGENE 6 (Boehringer Mannheim Corp.) and 95 μl of the cell culture medium RPMI 1640) into human glioblastoma M059J cells in culture. Other cultured human cells was transfected with a control marker. After 24 hours, the tissue culture plates were examined under a fluorescence microscope. The results showed that the mt topo I localized in the mitochondria.

A plasmid comprising the GFP gene as a marker at the 3' end of a segment of the cDNA of Example 1 encoding the first 41 amino acids was constructed and transfected into human glioblastoma M059J cells. Another plasmid comprising the GFP gene as a marker at the 3' end of a segment of the cDNA of Example 1 encoding all but the first 41 amino acids was constructed and transfected into another sample of human glioblastoma M059J cells. The results showed that the N-terminal segment of the mt topo I polypeptide is sufficient and necessary to confer mitochondrial localization. Example 6

This example demonstrates that the expression of mt topo I in various tissues of adult and fetal humans is consistent with the mitochondrial content of various organs.

Membranes from Clontech with RNA samples from various tissues from adult and fetal humans were hybridized with radiolabeled mt topo I cDNA. The blots were prehybridized with 15 ml of Ultrahyb hybridization buffer (Ambion, Austin, TX) for 15 min at 42 °C and hybridized for 12 hrs at the same temperature. The blots were washed with the following solutions: 5 min, 42 °C, 2X with 2XSSC + 0.1 %SDS, 15 min, 42 °C, 3X with 0.1XSSC + 0.1 %SDS, and 15 min, 45 °C, 3X with 0.1XSSC + 0.1 %SDS. The blots were exposed to phosphorlmager screens (Molecular Dynamics) overnight and scanned. Northern blots of RNA samples from heart, brain, kidney, liver, lung, pancreas, spleen and skeletal muscle tissues of adult humans and heart, liver skeletal muscle, and skin tissues of fetal humans probed with mt topo I cDNA. The results showed that the expression of mt topo I is consistent with the mitochondrial content of the various organs.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incoφorated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a mitochondrial topoisomerase I (mt topo I) or a 5' fragment thereof.

2. The isolated or purified nucleic acid molecule of claim 1 , which (i) encodes the amino acid sequence of SEQ ID NO: 2 or (ii) consists essentially of the nucleotide sequence of SEQ ID NO: 1 or a 5' fragment thereof comprising at least the first 15 contiguous nucleotides.

3. The isolated or purified nucleic acid molecule of claim 1 , which (i) encodes the amino acid sequence of SEQ ID NO: 4 or (ii) consists essentially of the nucleotide sequence of SEQ ID NO: 3 or a fragment thereof.

4. An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a variant mt topo I, which comprises one or more insertions, deletions and/or substitutions and, when the variant mt topo I is a variant human mt topo I, the one or more insertions, deletions and/or substitutions are in a region other than that which encodes alanine at amino acid positions 88 and 93, wherein the amino acid positions are given with respect to SEQ ID NO: 2 and wherein the variant mt topo I encoded by said isolated or purified nucleic acid molecule does not differ functionally from the corresponding unmodified mt topo I.

5. The isolated or purified nucleic acid molecule of claim 4, wherein the variant mt topo I relaxes DNA at least about 90% as well as the unmodified mt topo I comprising SEQ ID NO: 2 as determined by in vitro assay.

6. An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding mt topo I or a 5' fragment thereof.

7. The isolated or purified nucleic acid molecule of claim 6, which (i) is complementary to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or (ii) is complementary to the nucleotide sequence of SEQ ID NO: 1 or a 5' fragment thereof comprising at least the first 15 contiguous nucleotides.

8. The isolated or purified nucleic acid molecule of claim 6, which (i) is complementary to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4 or (ii) is complementary to the nucleotide sequence of SEQ ID NO: 3 or a fragment thereof.

9. An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding a variant mt topo I, which comprises one or more insertions, deletions and/or substitutions and, when the variant mt topo I is a variant human mt topo I, the one or more insertions, deletions and/or substitutions are in a region other than that which encodes alanine at amino acid positions 88 and 93, wherein the amino acid positions are given with respect to SEQ ID NO: 2.

10. A vector comprising the isolated or purified nucleic acid molecule of any of claims 1-3.

11. A vector comprising the isolated or purified nucleic acid molecule of claim 4 or 5.

12. A vector comprising the isolated or purified nucleic acid molecule of any of claims 6-8.

13. A vector comprising the isolated or purified nucleic acid molecule of claim 9.

14. A composition comprising the isolated or purified nucleic acid molecule of any of claims 1-3 and a pharmaceutically acceptable carrier.

15. A composition comprising the isolated or purified nucleic acid molecule of claim 4 or 5 and a pharmaceutically acceptable carrier.

16. A composition comprising the isolated or purified nucleic acid molecule of any of claims 6-8 and a pharmaceutically acceptable carrier.

17. A composition comprising the isolated or purified nucleic acid molecule of claim 9 and a pharmaceutically acceptable carrier.

18. A cell comprising the vector of claim 10.

19. A cell comprising the vector of claim 11.

20. A cell comprising the vector of claim 12.

21. A cell comprising the vector of claim 13.

22 An isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding mt topo I, which is optionally glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated or converted into an acid addition salt.

23 The isolated or purified polypeptide molecule of claim 22, which consists essentially of the amino acid sequence of SEQ ID NO: 2.

24. The isolated or purified polypeptide molecule of claim 22, which consists essentially of the amino acid sequence of SEQ ID NO: 4

25. An isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a variant mt topo I, which, when the variant mt topo I is a human mt topo I, comprises an alanine at amino acid position 88 and an alanine at amino acid position 93, and which is optionally glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated or converted into an acid addition salt.

26. A composition comprising the isolated or purified polypeptide molecule of any of claims 22-24 and a pharmaceutically acceptable carrier.

27. A composition comprising the isolated or purified polypeptide molecule of claim 25 and a pharmaceutically acceptable carrier.

28. A conjugate comprising the isolated or purified polypeptide molecule of any of claims 22-24 and a cell-surface targeting moiety.

29. The conjugate of claim 28, wherein said targeting moiety is an antibody or an antigenically reactive fragment thereof.

30. A conjugate comprising the isolated or purified polypeptide molecule of claim 25 and a cell-surface targeting moiety.

31. The conjugate of claim 30, wherein said targeting moiety is an antibody or an antigenically reactive fragment thereof.

32. A composition comprising the conjugate of claim 28 or 29 and a pharmaceutically acceptable carrier.

33. A composition comprising the conjugate of claim 30 and a pharmaceutically acceptable carrier.

34. A hybridoma cell line that produces a monoclonal antibody that is specific for the isolated or purified polypeptide molecule of any of claims 22-24 and does not cross- react with an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a nuclear topoisomerase I.

35. The monoclonal antibody produced by the hybridoma cell line of claim 34.

36. A hybridoma cell line that produces a monclonal antibody that is specific for the isolated or purified polypeptide molecule of claim 25 and does not cross-react with an isolated and purified polypeptide molecule consisting essentially of an amino acid sequence encoding a nuclear topoisomerase I.

37. The monoclonal antibody produced by the hybridoma cell line of claim 36.

38. A polyclonal antiserum raised against the isolated or purified polypeptide molecule of any of claims 22-24 that does not cross-react with an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a nuclear topoisomerase I.

39. A polyclonal antiserum raised against the isolated or purified polypeptide molecule of claim 25 that does not cross-react with an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a nuclear topoisomerase I.

40. A method of altering the level of mitochondrial topoisomerase I (mt topo I) in a cell, which method comprises contacting said cell with (i) an isolated or purified nucleic acid of any of claims 1-9, (ii) a vector comprising or encoding an antisense molecule that is specific for mt topo I, (iii) a vector comprising or encoding a ribozyme that is specific for mt topo I, (iv) an isolated or purified polypeptide of any of claims 22-25, or (v) a conjugate of claim 28 or 30, whereupon the level of mt topo I in said cell is altered.

41. The method of claim 40, wherein the isolated or purified polypeptide of (iv) is contained within a liposome comprising a cell-surface targeting moiety that binds to the cell being contacted.

42. A method of identifying an inhibitor or an activator of mt topo I, which method comprises contacting a polypeptide of any of claims 22-25 with an agent in vitro and assaying for topoisomerase activity, wherein a decrease in topoisomerase activity after contact of the polypeptide with the agent indicates that the agent is an inhibitor of mt topo I and wherein an increase in topoisomerase activity after contact of the polypeptide with the agent indicates that the agent is an activator of mt topo I.

43. An isolated or purified DNA molecule consisting essentially of the genomic sequence of mt topo I or a fragment thereof, wherein said fragment comprises at least the first 15 contiguous nucleotides from exon I of mt topo I.