JP2003513613A - Methods for identifying virulence factors using differential gene expression - Google Patents

Abstract

  (57) [Summary] Methods for identifying virulence factors (eg, hepatotoxicity factors) using differential gene expression are disclosed. Also disclosed is a novel nucleic acid sequence, the expression of which is differentially regulated by troglitazone. The present invention also relates to a method of screening for a test agent for hepatotoxicity, the method comprising procuring a test cell population comprising cells capable of expressing one or more nucleic acid sequences selected from the group consisting of HEPATOX: 1-168 and 169. Contacting the test cell population with a test agent, measuring the expression of one or more nucleic acid sequences in the test cell population, and comparing the nucleic acid sequence expression in the test cell population with the nucleic acid expression in the reference cell population (reference The cell population comprises at least one cell whose exposure to the hepatotoxic agent is known), and, if present, identifies differences in the expression levels of the HEPATOX sequence in the test and reference cell populations, thereby Screen test factors for toxicity.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates generally to nucleic acids and polypeptides. In particular, it relates to the identification of hepatotoxic agents in liver tissue using differential gene expression.

BACKGROUND OF THE INVENTION An inadequate drawback associated with another potential drug is that they induce unwanted side effects and their intended therapeutic effect. Often, these side effects are not apparent until the drug enters clinical trials, or even when it is completed. For example, troglitazone has recently been used to treat non-insulin dependent type II diabetes. Despite its demonstrated efficacy in alleviating the symptoms associated with diabetes, significant side effects are associated with this drug. In particular, it has been reported to result in liver damage in the subset of patients to which it is administered.

Hepatotoxicity associated with administration of troglitazone can range from mild idiosyncratic reactions to severe hepatocyte necrosis, subsequent liver injury and death. Clinical symptoms of troglitazone-related toxicity may include, for example: jaundice, nausea, abdominal pain, low-grade fever, malaise, vomiting, hematuria, edema of lower extremities and fatigue. Histological changes can include, for example: cholestasis, hepatocyte necrosis, fibrosis, bile duct proliferation, and acute and chronic inflammation.
Other changes in toxicity associated with troglitazone can include, for example: significant increase in levels of bilirubin, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase, and increase in prothrombin time.

SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery that certain nucleic acids are differentially expressed in liver tissue of animals treated with troglitazone. These differentially expressed nucleic acids include novel sequences and nucleic acid sequences, which have been previously described but have not previously been identified as troglitazone responsive.

In various aspects, the invention includes methods of screening test compounds for toxicity (eg, hepatotoxicity). For example, in one aspect, the invention provides a test cell population comprising cells capable of expressing one or more nucleic acid sequences responsive to troglitazone, contacting the test cell population with a test agent, and A method of identifying a hepatotoxic agent is provided by comparing the expression of a nucleic acid sequence in a test cell population with the expression of a nucleic acid sequence in a reference cell population. An alteration in the expression of the nucleic acid sequence in the test cell population compared to the expression of the nucleic acid sequence in the reference cell population indicates that the test agent is hepatotoxic.

In a further aspect, the invention provides a method of assessing hepatotoxicity of a test agent in a subject. This method comprises the steps of preparing from a subject a cell population comprising cells capable of expressing one or more troglitazone responsive genes, and expressing the nucleic acid sequence from a subject known to be exposed to hepatotoxic agents. Comparing to the expression of the nucleic acid sequence in a reference cell population comprising the cells of An alteration in the expression of the nucleic acid sequence in the test cell population compared to the expression of the gene in the reference cell population is indicative of hepatotoxicity of the test agent in the subject.

[0007] Novel nucleic acids, and their encoded polypeptides, whose expression is responsive to the effects of troglitazone, are also provided.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the accompanying detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based in part on the discovery of alterations in expression patterns of multiple nucleic acid sequences in rodent hepatocytes following exposure to troglitazone, a hepatotoxic compound. This compound is described by Spencer et al., Drugs 54 (1): 89-102 (1997).
), Which is incorporated herein by reference in its entirety.

The differentially expressed nucleic acid was administered to male 12-week-old Sprague Dawley rats or Wistar Kyoto rats at OD of 617 mg / kg / day p. o
． Was identified by dosing for 3 days. Control animals received dimethyl sulfoxide. The animals were sacrificed 24 hours after dosing. Liver tissue was excised from the animal and total RNA was recovered from the excised tissue. prepare the cDNA,
The resulting sample is then used in US Pat. No. 5,871,697 and Shimk.
ets et al., Nature Biotechnology 17: 798-803.
Processed through the use of GENECALLING ^™ differential expression analysis as described (1990). The contents of these patents and publications are incorporated herein by reference in their entirety.

The 819 gene fragment was first found to be differentially expressed in rat liver tissue in response to troglitazone in both Sprague Dawley and Wistar Kyoto rats. Gene fragments whose expression levels were regulated more than 3-fold were selected for further analysis.

A summary of the sequences analyzed is shown in Table 1. Differential expression of the 169 gene fragment was determined by Shimkets et al., Nature Biotechnology 17
: 198-803 to confirm using an unlabeled oligonucleotide competition assay. The 169 signal nucleic acid sequences identified herein are HEP
Referred to herein as ATOX1-169. The sixth column of Table 1 (titled "Function") lists the classification types assigned to proteins based on their function.

The 21 sequences (HEPATOX: 1-21) represent a novel rat gene, which has a high degree of sequence identity to sequences found in public databases (ie> 90% for 5 fragments). Observed), moderate (ie, about 70% to about 90%, observed for 15 genes), or low (ie, less than 70%, observed for two genes) (these are , Suggests a putative homology).

Other sequences of 148 identified have been previously described. For some of the new sequences (ie HEPATOX: 1-21), the cloned sequences are
Provided with one or more additional sequence fragments (eg EST or contig). It contains a sequence that is substantially identical to the cloned sequence. Consensus sequences are also provided, including composite sequences constructed from cloned fragments and additional fragments. Predetermined HEPATOX
For sequences, their expression can be measured using any of the relevant nucleic acid sequences that can be used in the methods described herein. The previously described sequence (H
Database registration numbers are provided for EPATO X: 22-169).
This information allows one of skill in the art to derive the information necessary to detect and measure the expression of HEPATOX nucleic acid sequences.

Troglitazone-a representative nucleic acid discussed herein, including:

[0017]

[Table 1] Following is further disclosure of nucleic acid sequences whose expression is differentially regulated in the presence of troglitazone.

(HEPATOX1) HEPATOX1 is a novel 185 bp gene fragment. The nucleic acid has the following sequence:

[0019]

[Chemical 1] (HEPATOX2) HEPATOX2 is a novel 56 bp gene fragment. This nucleic acid has the following sequence:

[0020]

[Chemical 2] (HEPATOX3) HEPATOX3 is a novel 572 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0021]

[Chemical 3] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0022]

[Chemical 4] (HEPATOX4) HEPATOX4 is a novel 1770 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0023]

[Chemical 5] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0024]

[Chemical 6] (HEPATO5) HEPATOX5 is a novel 755 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0025]

[Chemical 7] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0026]

[Chemical 8] (HEPATOX6) HEPATOX6 is a novel 675 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0027]

[Chemical 9] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0028]

[Chemical 10] (HEPATOX7) HEPATOX7 is a novel 874 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0029]

[Chemical 11] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0030]

[Chemical 12] (HEPATOX8) HEPATOX8 is a novel 546 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0031]

[Chemical 13] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0032]

[Chemical 14] (HEPATOX9) HEPATOX9 is a novel 407 bp gene fragment. This nucleic acid has the following sequence:

[0033]

[Chemical 15] (HEPATOX10) HEPATOX10 is a novel 1149 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0034]

[Chemical 16] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0035]

[Chemical 17] (HEPATOX11) HEPATOX11 is a novel 572 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0036]

[Chemical 18] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0037]

[Chemical 19] (HEPATOX12) HEPATOX12 is a novel 667 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0038]

[Chemical 20] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0039]

[Chemical 21] (HEPATOX13) HEPATOX13 is a novel 580 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0040]

[Chemical formula 22] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0041]

[Chemical formula 23] (HEPATOX14) HEPATOX14 is a novel 327 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0042]

[Chemical formula 24] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0043]

[Chemical 25] (HEPATOX15) HEPATOX15 is a novel 650 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0044]

[Chemical formula 26] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0045]

[Chemical 27] (HEPATOX16) HEPATOX16 is a novel 499 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0046]

[Chemical 28] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0047]

[Chemical 29] (HEPATOX17) HEPATOX17 is a novel 315 bp gene fragment. This nucleic acid has the following sequence:

[0048]

[Chemical 30] (HEPATOX18) HEPATOX18 is a novel 760 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0049]

[Chemical 31] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0050]

[Chemical 32] (HEPATOX19) HEPATOX19 is a novel 499 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0051]

[Chemical 33] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0052]

[Chemical 34] (HEPATOX20) HEPATOX20 is a novel 1083 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0053]

[Chemical 35] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0054]

[Chemical 36] (HEPATOX21) HEPATOX21 is a novel 580 bp gene fragment. This nucleic acid was first identified in a cloned fragment with the following sequence:

[0055]

[Chemical 37] This cloned sequence was constructed in a contig that yielded the following consensus sequence:

[0056]

[Chemical 38] HEPATOX nucleic acids and encoded polypeptides can be identified using the information provided above. In some embodiments, this HEPATOX
Nucleic acids and peptides correspond to nucleic acids or polypeptides that include the various sequences published for each HEPATOX polypeptide (referenced by SEQ ID NO :).

In this various aspects and embodiments, the invention relates to the sequence HEPATOX1-
Providing a test cell population comprising at least one cell capable of expressing one or more of 169. By "expressible" is meant that the gene exists and can be expressed intracellularly in intact form. One, some, or all HE
Expression of the PATOX sequence is detected and, if present, preferably measured. Using the sequence information provided by database entries for known sequences, or for newly described sequences, expression of the HEPATOX sequence is detected (present) using techniques well known to those of skill in the art. Case) and can be measured. For example, a sequence within a sequence database entry that corresponds to a HEPATOX sequence, or a sequence within a sequence disclosed herein may be used, for example, in a Northern blot hybridization analysis or, particularly and preferably, to quantify a particular nucleic acid sequence. Can be used to construct probes for detecting HEPATOX RNA sequences. As another example, this sequence may be used in HE-based detection methods, such as in reverse transcription-based polymerase chain reaction.
It can be used to construct primers for specific amplification of PATOX sequences. Where changes in gene expression are associated with gene amplification or deletion, sequence comparisons in the test and reference populations can be made by comparison of the relative amounts of the tested DNA sequences in the test and reference cell populations.

Expression can also be measured at the protein level (ie, by measuring the level of the polypeptide encoded by the gene product described herein). Such methods are well known in the art and include, for example, antibody-based immunoassays against proteins encoded by genes.

The expression level of one or more HEPATOX sequences in the test cell population is then compared to the expression level of the sequences in one or more cells from the reference cell population.
Expression of sequences in test and control populations of cells can be compared using any art-recognized method for comparing expression of nucleic acid sequences. For example, expression is described in US Pat.
at. Biotechnol. 17: 798-803 as described in GEN
They can be compared using the ECALLING® method.

In various embodiments, the expression of one or more sequences encoding genes with related functions as listed in Table 1 is compared. These features include
For example, "Molecular Toxicology Genes" (for example, HEPATOX22 to 24) and "PPAR Ligands" (HEPATO).
X25-34), "Ligand Activated Genes", "Ac
ute Phase Protein Genes / Immune Respo
"Nse Genes" (HEPATOX35-53), "Stress Res"
"Ponse Genes" (HEPATOX 54-56), "Serum Pr"
"Otein Related Genes" (HEPATOX 57 and 58)
, And "Drug Metabolism and Free Radic
al Scavenger Genes "(HEPATOX 59-73). In some embodiments, the expression of two or more functional family members is compared.

In various embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 35, 40, 5 of the sequences represented by HEPATOX 1-169.
Expression of 0, 100 or all is measured. If desired, expression of these sequences can be measured, along with other sequences whose expression is known to be altered according to one of the parameters or conditions described herein.

The reference cell population comprises one or more cells with known compared parameters. The parameter to be compared can be, for example, the expression status of the hepatotoxic factor. By "hepatotoxic factor expression status" is meant that it is known whether the reference cell has contact with the hepatotoxic factor. Examples of hepatotoxic factors are eg troglitazone (tro
a thiazolidinedione such as glitazone). Comparison of the gene expression profile in the test cell population to the reference cell population relies on comparison of the reference cell population without revealing the presence or extent of the measured parameter.
For example, if the reference cell population consists of cells that have not been treated with a known hepatotoxic factor, expression levels of similar genes in the test and reference cell populations indicate that the test factor is not a hepatotoxic factor. Conversely, if the reference cell population consists of cells treated with a hepatotoxic factor, the expression profile of similar genes between the test cell population and the reference cell population indicates that the test factor is a hepatotoxic factor.

[0063] In various embodiments, the HEPATOX sequences in the test cell population have an expression level that is relative to the level of the HEPATOX transcript in the reference cell population.
An expression level is considered comparable to that of a HEPATOX sequence if it varies within 0, 1.5 or 1.0 fold. In various embodiments, the HEPATOX sequence in the test cell population has an expression level 1.0, 1.
Changes in expression levels can be considered to be altered by a factor of 5, 2.0 or more from the reference cell population.

If desired, a comparison of the differential expression of sequences between a test cell population and a reference cell population can be made to a control nucleic acid independent of the parameter or condition for which expression is measured. The expression level of the control nucleic acid in the test and reference nucleic acids can be used to normalize signal levels in the compared populations.

In some embodiments, the test cell population is compared to multiple reference cell populations. Each of the multiple reference populations may differ in known parameters. Therefore, the test cell population is the first reference cell population known to be exposed to hepatotoxic factors,
And a second reference cell population known not to be exposed to hepatotoxic factors.

The test cell population exposed to (ie, contacted with) the test hepatotoxic factor can be any number of cells (ie, one or more cells) and provided in vitro, in vivo, or ex vivo. obtain.

In other embodiments, the test cell population may be divided into two or more subpopulations. The subpopulation can be created by dividing the first cell population to make the subpopulation as identical as possible. This is suitable, for example, in in vitro or ex vivo screening methods. In some embodiments, different subpopulations are administered as control agents, and / or one or several test agents, ie, different dosages of one or more test agents that are administered together or in different combinations. Can be exposed to.

Preferably, the cells in the reference cell population are from a tissue type that is as similar as possible to the test cells (eg liver tissue). In some embodiments, the control cell is from the same subject as the test cell (eg, from a region adjacent to the region of origin of the test cell). In other embodiments, the reference cell population is from multiple cells. For example, the reference cell population is a database of expression patterns derived from cells that have been previously tested and for which one of the parameters or conditions (hepatotoxic drug expression pattern) described herein is known. possible.

The test agent can be a compound not previously described, or it can be a compound known in advance, but not known to be a hepatotoxic agent.

The subject is preferably a mammal. The mammal can be, for example, a human, non-human primate, mouse, rat, dog, cat, horse or cow.

Screening for Toxic Drugs In one aspect, the invention provides methods for identifying toxic drugs (eg, hepatotoxic drugs). Hepatotoxic agents can be identified by providing a cell population comprising cells capable of expressing one or more nucleic acid sequences homologous to the nucleic acid sequences listed in Table 1 as HEPATOX 1-169. This sequence will be labeled as long as HEPATOX 1-, as long as it is sufficiently similar that specific hybridization can be detected.
It need not be identical to the sequence containing 169. Preferably, the cells contain sequences that are identical or nearly identical to the sequences identifying the HEPATOX nucleic acids shown in Table 1.

Expression of the nucleic acid sequence in the test cell population is then determined by measuring the reference cell population (which may be a cell population that has not been exposed to the test agent or, in some embodiments, cells that have been exposed to the test agent). The population of nucleic acid sequences is compared. The comparison can be performed on test and reference samples measured at the same time or at different time points. An example of the latter is the use of compiled expression information (eg, sequence databases) that collects information about the expression levels of known sequences after administration of various agents. For example, a change in expression level after administration of a test drug is
For example, it can be compared to the changes in expression observed in the nucleic acid sequences after administration of troglitazone.

A change in the sequence of the nucleic acid sequence in the test cell population compared to the expression of the nucleic acid sequence in the reference cell population that has not been exposed to the test agent indicates that the test agent is a hepatotoxic agent.

The present invention also includes hepatotoxic agents identified by this screening method.

Evaluation of Toxicity of Drugs in a Subject The differentially expressed HEPATOX sequences identified herein also allow for measuring or monitoring the hepatotoxicity of hepatotoxic drugs. In this method, a test cell population from a subject is exposed to a test agent (ie, a hepatotoxic agent). If desired, the test cell population may be exposed to the test agent prior to exposure to the test agent,
Subjects may be harvested during exposure to the test agent or at various times after exposure to the test agent. Then, one or more HEPATOX sequences (eg, HE
The expression of PATOX: 1-169) is measured and compared to a reference cell population that includes cells with known expression status of the hepatotoxic drug. Preferably, the reference cells have not been exposed to the test agent.

HE in the test cell population if the reference cell population does not include cells exposed to treatment
The similarity in expression between the PATOX sequence and the HEPATOX sequence in the reference cell population indicates that this treatment is non-hepatotoxic. However, HE in the test cell population
Differences in expression between the PATOX sequence and the HEPATOX sequence in this reference cell population indicate that this treatment is hepatotoxic.

“Hepatoxicity” refers to an agent that, when administered to a subject, damages the liver or is destructive to the liver, leading to liver damage.

(HEPATOX Nucleic Acid) The present invention also provides a novel nucleic acid containing a nucleic acid sequence selected from the group consisting of HEPATOX: 1 to 21 or a complement thereof, and vectors and cells containing these nucleic acids.

Accordingly, one aspect of the invention relates to an isolated HEPATOX nucleic acid molecule encoding a HEPATOX protein or biologically active portion thereof. HE
A nucleic acid fragment sufficient for use as a hybridization probe to identify a PATOX-encoding nucleic acid (eg, HEPATOX mRNA)
, And fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of HEPATOX nucleic acid molecules. As used herein, the term "nucleic acid molecule" refers to a DNA molecule (eg, cDNA
A or genomic DNA), RNA molecules (eg, mRNA), analogs of DNA or RNA produced using nucleotide analogs, and their derivatives, fragments, and homologs. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

“Probes” may be of various lengths (preferably at least about 1), depending on the application.
A nucleic acid sequence of 0 nucleotides (nt) and a length as large as about 6,000 nt, for example. Probes are used for the detection of identical nucleic acid sequences, similar nucleic acid sequences, or complementary nucleic acid sequences. Longer length probes are usually
It is obtained from natural or recombinant sources and is more specific than oligomers and hybridizes more slowly. Probes can be single-stranded or double-stranded and designed to have specificity in PCR, membrane-based hybridization techniques or ELISA-like techniques.

An “isolated” nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are present within the natural source of the nucleic acid. Examples of isolated nucleic acid molecules include recombinant DNA molecules contained in vectors, recombinant DNA molecules maintained in heterologous host cells, partially or substantially purified nucleic acid molecules, and synthetic DNA molecules or Examples include, but are not limited to, synthetic RNA molecules. Preferably, an "isolated" nucleic acid has sequences that naturally flank the nucleic acid within the genomic DNA of the organism in which the nucleic acid is derivatized (ie, sequences located at the 5'and 3'ends of the nucleic acid). do not do. For example, in various embodiments, an isolated HEPATOX nucleic acid molecule is less than about 50 kb, less than about 25 kb, less than about 5 kb, about 5 kb naturally adjacent to the nucleic acid molecule within the genomic DNA of the cell in which the nucleic acid is derivatized. It may comprise less than 4 kb, less than about 3 kb, less than about 2 kb, less than about 1 kb, less than about 0.5 kb or less than about 0.1 kb of nucleotide sequence. Furthermore, an "isolated" nucleic acid molecule (eg, a cDNA molecule) may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or when chemically synthesized. May be substantially free of chemical precursors or other chemicals.

Nucleic acid molecules of the invention (eg, nucleic acid molecules having a nucleotide sequence of any of HEPATOX: 1-21, or the complement of any of these nucleotide sequences) may be prepared using standard molecular biology techniques and the methods described herein. It can be isolated using the sequence information provided in the text. Using all or part of these nucleic acid sequences as hybridization probes, HEPATOX nucleic acid sequences can be isolated using standard hybridization and cloning techniques (eg Samb).
Look et al., Molecular Cloning: A Laborator
y Manual 2nd edition, Cold Spring Harbor Labo
rally Press, Cold Spring Harbor, NY, 1
989; and edited by Ausubel et al., Current Protocols i.
n Molecular Biology, John Wiley & Sons,
New York, NY, 1993).

The nucleic acids of the invention can be used as templates and suitable oligonucleotide primers according to standard PCR amplification techniques as cDNA, mRNA or genomic DNA.
Can be amplified using The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, HEPAT
Oligonucleotides corresponding to OX nucleotide sequences can be prepared by standard synthetic techniques, eg, using an automated DNA synthesizer.

The term “oligonucleotide” as used herein refers to a series of linked nucleotide residues, where the oligonucleotide has a sufficient number of nucleotide bases to be used in the PCR reaction. Short oligonucleotide sequences can be based on, or designed from, genomic or cDNA sequences, and the presence of identical, similar, or complementary DNA or RNA within a particular cell or tissue. Is used to amplify, confirm, or reveal. The oligonucleotide comprises a portion of the nucleic acid sequence having at least about 10 nt, and at most 50 nt, preferably about 15 nt to 30 nt. They can be chemically synthesized and used as probes.

In another embodiment, the isolated nucleic acid molecule of the invention is HEPATOX: 1.
A nucleic acid molecule that is the complement of the nucleotide sequence set forth in -21. In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is the complement of a nucleotide sequence set forth in any of these sequences, or a portion of any of these nucleotide sequences. A nucleic acid molecule that is complementary to a nucleotide sequence shown in HEPATOX: 1-21 is a nucleic acid molecule that is sufficiently complementary to the nucleotide sequence shown so that it has little or no nucleotide sequence shown. It can hydrogen bond without any mismatch, thereby forming a stable duplex.

As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairs between nucleotide units of nucleic acid molecules,
The term "binding" refers to a physical or chemical interaction between two polypeptides or compounds or a subsidiary polypeptide or compound or a combination thereof. As a bond, ionic interaction, nonionic interaction, Von d
er Waals interaction, hydrophobic interaction and the like. Physical interactions can be either direct or indirect. Indirect binding can be through or due to the influence of another polypeptide or compound. Direct binding is an interaction that does not occur through the effect of another polypeptide or compound, or due to the effect of another polypeptide or compound, but instead of other substantial chemical mediators. Does not have.

Furthermore, the nucleic acid molecule of the present invention comprises only a part of the nucleic acid sequence of HEPATOX: 1 to 21 (for example, a fragment that can be used as a probe or a primer, or a fragment encoding a biologically active portion of HEPATOX). ) May be included. The fragments provided herein are of sufficient length to allow specific hybridization in the case of nucleic acids, or allow specific recognition of epitopes in the case of amino acids. Is defined as a sequence of at least 6 (consecutive) nucleic acids or at least 4 (consecutive) amino acids that is of sufficient length and at most some portion less than the full length sequence. Fragments can be derived from any contiguous portion of the selected nucleic acid or amino acid sequences. Derivatives are nucleic acid or amino acid sequences formed from a native compound, either directly or by modification or partial substitution. An analog is a nucleic acid or amino acid sequence that has a structure that is similar, but not identical, to the native compound, but that differs with respect to the particular compound or side chain from the native compound. Analogs can be of synthetic or different evolutionary origin, and have similar or opposite metabolic activity as compared to wild type.

Derivatives and analogs may or may not be full length if the derivative or analog contains a modified nucleic acid or amino acid, as described below. As the derivative or analog of the nucleic acid or protein of the present invention,
Containing regions of substantial homology to the nucleic acids or proteins of the invention (in various embodiments, alignments made over sequences of nucleic acids or amino acids of the same length, or alignments made by computer homology programs known in the art). At least about 45%, 50%, 70%, 80%, 95% when compared to the sequence
, 98%, or even 99% identical (preferably 80-99% identity), or the encoding nucleic acid is stringent, moderately stringent, or under low stringent conditions. And a molecule capable of hybridizing with the complement of the sequence encoding the above protein in (1) to (3). For example, Ausubel et al., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons,
See New York, NY, 1993 and below. An exemplary program is a Gap program (Wisconsin Se) that uses default settings.
sequence Analysis Package, Version 8 fo
r UNIX, Genetics Computer Group, Universal
rsity Research Park, Madison, WI), which is based on the algorithm of Smith and Waterman (Adv. Appl.
Math. , 1981, 2: 482-489, which is hereby incorporated by reference in its entirety).

A “homologous nucleic acid sequence” or “homologous amino acid sequence” or a variant thereof means
Refers to sequences characterized by homology at the nucleotide or amino acid level as discussed above. The homologous nucleotide sequence encodes a sequence that encodes an isoform of HEPATOX polypeptide. The isoform is
It can be expressed in different tissues of the same organ, eg as a result of alternative splicing of RNA. Alternatively, the isoforms can be encoded by different genes. In the present invention, homologous nucleotide sequences include species other than human (including but not limited to mammals, therefore, for example, mouse, rat, rabbit,
Can include dogs, cats, cows, horses, and other organisms) HEPATOX polypeptides. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variants and variants of the nucleotide sequences described herein. However, the homologous nucleotide sequence does not include the nucleotide sequence encoding the human HEPATOX protein. Homologous nucleic acid sequences include conserved amino acid sequences in HEPATOX polypeptides (see below), as well as nucleic acid sequences that encode polypeptides having HEPATOX activity. A homologous amino acid sequence does not encode the amino acid sequence of human HEPATOX polypeptide.

Nucleotide sequences determined from cloning of the human HEPATOX gene are used in the identification and / or cloning of HEPATOX homologs in other cell types (eg, from other tissues), as well as from other mammals. Allows the generation of probes and primers designed for. The probe / primer typically comprises a substantially purified oligonucleotide. The oligonucleotide is typically at least about 12, 25, 50 of the nucleic acid containing the HEPATOX sequence, under stringent conditions.
100, 150, 200, 250, 300, 350 or 400 contiguous sense strand nucleotide sequences, or antisense strand nucleotide sequences of nucleic acids containing HEPATOX sequences, or sequences of naturally occurring variants of these sequences. Includes a region of nucleotide sequence that is soybean.

Probes based on human HEPATOX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a labeling group attached to the probe. For example, the labeling group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Cells or tissues that misexpress the HEPATOX protein,
For example, determining the level of HEPATOX-encoding nucleic acid in a sample of cells from a subject (eg, detection of HEPATOX mRNA levels or genomic HE
Determination of whether the PATOX gene is mutated or deleted).
Such probes can be used as part of a diagnostic test kit.

A “polypeptide having a biologically active portion of HEPATOX” refers to a polypeptide of the invention (including the mature form) as measured in a particular biological assay with or without dose dependence. A polypeptide showing an activity similar to that of the above-mentioned but not necessarily the same. A nucleic acid fragment encoding a “biologically active portion of HEPATOX” is a part of HEPATOX: 1 to 21 (HEPAT).
Encoding a polypeptide having the biological activity of OX), HE
It can be prepared by expressing the encoded portion of the PATOX protein (eg, by in vitro recombinant expression) and assessing the activity of the encoded portion of HEPATOX. For example, a nucleic acid fragment encoding a biologically active portion of a HEPATOX polypeptide can optionally include an ATP binding domain. In another embodiment, the nucleic acid fragment encoding the biologically active portion of HEPATOX comprises one or more regions.

Variants of HEPATOX The invention further encompasses nucleic acid molecules that differ from the disclosed or referenced HEPATOX nucleotide sequences due to the degeneracy of the genetic code. Thus, these nucleic acids can be labeled, for example, in HE
Encodes a HEPATOX protein encoded by a nucleotide sequence that includes a PATOX nucleic acid.

In addition to the rat HEPATOX nucleotide sequence shown in HEPATOX: 1-21, DN leading to changes in the amino acid sequence of the HEPATOX polypeptide.
It will be appreciated by those of skill in the art that A sequence polymorphisms can exist within a population (eg, the human population). Such genetic polymorphisms in the HEPATOX gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to HEPATOX.
A nucleic acid molecule containing an open reading frame encoding a protein, preferably a mammalian HEPATOX protein. Such natural allelic variations typically result in 1-5% variability in the nucleotide sequence of the HEPATOX gene. Any and all such nucleotide variations within HEPATOX and resulting amino acid polymorphisms that are the result of natural allelic variation and do not alter the functional activity of HEPATOX are intended to be within the scope of the present invention. To be done.

In addition, it encodes HEPATOX proteins from other species, and thus ADI
Nucleic acid molecules having a nucleotide sequence that differs from the human sequence of PO1-21 are intended to be within the scope of the present invention. Nucleic acid molecules directed to naturally occurring allelic variants and homologues of HEPATOX DNA of the present invention, based on their homology to the human HEPATOX nucleic acids disclosed herein, under standard stringent hybridization conditions, Can be isolated using human cDNA or a portion thereof as a hybridization probe according to various hybridization techniques. For example, soluble human HEPATOX DNA is a product of human membrane-bound HE.
It can be isolated based on its homology to PATOX. Similarly, membrane-bound human H
EPATO X DNA can be isolated based on its homology to soluble human HEPATOX.

Thus, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and, under stringent conditions, HEPATOX: 1-2.
Hybridizes to a nucleic acid molecule containing one nucleotide sequence. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250 or 500 nucleotides in length. In another embodiment, the isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridize under stringent conditions” typically leaves hybridized nucleotide sequences that are at least 60% homologous to each other due to hybridization and washing. It is intended to describe the conditions.

Homologs (ie, nucleic acids encoding HEPATOX proteins from species other than human) or other related sequences (eg, paralogs) are
All or part of a particular human sequence may be probed and obtained by low, moderate or high stringency hybridization using methods well known in the art for nucleic acid hybridization and cloning.

As used herein, the phrase “stringent hybridization conditions” refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but not hybridize to other sequences. . Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected at a defined ionic strength and pH of about 5 ° C. below the temperature melting point (Tm) for the specific sequence. The Tm is the temperature (defined ionic strength, pH and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes to the target sequence at equilibrium. Since the target sequence is generally over-represented in the Tm, 50% of the probes are occupied in equilibrium. Typically, stringent conditions are less than about 1.0 M sodium ion in salt concentration, typically about 0.01-1.0 M sodium ion (or other salt),
pH 7.0-8.3 and the temperature is at least about 30 ° C. for short probes, primers or oligonucleotides (eg 10 nucleotides to 50 nucleotides), and longer probes, primers and oligonucleotides. For at least about 60 ° C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Stringent conditions are known to those of skill in the art, and may be CURRENT PR
OTOCOLS IN MOLECULAR BIOLOGY, John Wi
ley & Sons, N.M. Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences that are at least about 65%, about 70%, about 75%, about 85%, about 90%, about 95%, about 98% or about 99% homologous to one another, typically to each other. Conditions are such that they will remain hybridized. A non-limiting example of stringent hybridization conditions is 6 × SSC, 50 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,
Hybridization at 65 ° C. in high salt buffer containing 0.02% Ficoll, 0.02% BSA, and 500 mg / ml denatured salmon sperm DNA. This hybridization is followed by 0.2 x SSC, 0.01% BS.
One or more washes in A at 50 ° C. HEPAT under stringent conditions
An isolated nucleic acid molecule of the invention which hybridizes to the sequence of OX: 1-21 is:
Corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule that has a naturally-occurring (eg, encoding a native protein) nucleotide sequence.

In the second embodiment, HEPATOX: 1-21 or a fragment thereof,
Nucleic acid molecules that include the nucleotide sequences of analogs or derivatives are provided with nucleic acid sequences that can hybridize under moderate stringency conditions. A non-limiting example of moderate stringency hybridization conditions is 6 × at 55 ° C.
Hybridization in SSC, 5 × Denhardt's solution, 0.5% SDS and 100 mg / ml denatured salmon sperm DNA, followed by 1 × SSC, 0.1% S.
One or more washes at 37 ° C in DS. Other conditions of moderate stringency that can be used are well known in the art. For example, Ausubel et al. (Eds.), 1993, CURRENT PROTOCOLS IN MOLECULA.
R BIOLOGY, John Wiley & Sons, NY and Kri
egler, 1990, GENE TRANSFER AND EXPRESS
ION, A LABORATORY MANUAL, Stockton Pre
See ss, NY.

In a third embodiment, there is provided a nucleic acid capable of hybridizing to a nucleic acid molecule comprising a nucleotide sequence of HEPATOX: 1-21 or a fragment, analog or derivative thereof under low stringency conditions. A non-limiting example of low stringency hybridization conditions is 35% formamide, 5X.
SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.
02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg /
Hybridization in ml of denatured salmon sperm DNA, 10% (weight / volume) dextran sulphate at 40 ° C., followed by 2 × SSC, 25 mM Tri.
5 in s-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS
One or more washes at 0 ° C. Other conditions of low stringency that can be used are well known in the art (eg, as used for cross-species hybridization). For example, Ausubel et al. (Eds.), 1993, CUREN.
T PROTOCOLS IN MOLECULAR BIOLOGY, Joh
n Wiley & Sons, NY and Kriegler, 1990, G
ENE TRANSFER AND EXPRESSION, A LABORA
TORY MANUAL, Stockton Press, NY; Shiro et al., 1981, Proc Natl Acad Sci USA 78: 6789.
See -6792.

Conservative Mutations In addition to allelic variants of naturally occurring HEPATOX sequences that may be present in the population, those of skill in the art will further appreciate that alterations do not alter the functional capacity of the HEPATOX protein. Can be introduced into HEPATOX nucleic acid or HAPPATOX
It is understood that it can be introduced directly into the polypeptide sequence. In some embodiments, the nucleotide sequence of HEPATOX: 1-21 is altered, resulting in a change in the amino acid sequence of the encoded HEPATOX protein. For example, nucleotide substitutions resulting in amino acid substitutions at various "non-essential" amino acid residues can be made in the sequence of HEPATOX: 1-21. "Not required"
Amino acid residues are those that may be altered from the wild-type sequence of HEPATOX whose biological activity has not been altered, whereas the "essential" amino acid residues are necessary for biological activity. For example, amino acid residues conserved among the HEPATOX proteins of the invention are expected to be particularly awkward to alter.

Furthermore, amino acid residues conserved among the family members of the HEPATOX proteins of the invention are also expected to be particularly awkward to alter.
As such, these conserved domains are probably awkward to mutation. However, other amino acid residues (eg, residues that are not conserved or only semi-conserved among members of the HEPATOX protein) may not be essential for activity and are therefore likely to be awkward to alter. .

Another aspect of the invention is HEPAT that includes changes in amino acid residues that are not essential for activity.
A nucleic acid molecule encoding an OX protein. Such a HEPATOX protein differs in amino acid sequence from the amino acid sequence of a polypeptide encoded by a nucleic acid containing HEPATOX: 1-21, but still retains biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein, where the protein is HEPATO.
At least about 45% homology, more preferably 60% homology, and even more preferably at least about 70%, 80%, 90 to the amino acid sequence of a polypeptide encoded by a nucleic acid comprising X: 1-21. %, 95%, 98%, and most preferably at least about 99% homologous amino acid sequences.

An isolated nucleic acid molecule encoding a homologous HEPATOX protein is HEP
ATOX: 1-21 can be produced by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the nucleic acid comprising, resulting in one or more amino acid substitutions, additions, or deletions, It can be introduced into the encoded protein.

Mutations can be introduced into nucleic acids containing HEPATOX: 1-21 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made with one or more putative nonessential amino acid residues. "
A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acids with similar side chains has been defined in the art. These families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, Tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg, threonine, valine, isoleucine),
And aromatic side chains (eg, tyrosine, phenylalanine, tryptophan,
Amino acid with (histidine) is included. Therefore, a putative nonessential amino acid residue in HEPATOX is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, the mutations may be randomly introduced along all or part of the HEPATOX coding sequence (eg, by saturation mutagenesis), and the resulting mutations screened for HEPATOX biological activity. Can be identified to identify mutations that retain activity. After nucleic acid mutagenesis, the encoded protein can be expressed by any recombinant technique known in the art,
And its activity can be determined.

In one embodiment, the mutant HEPATOX protein comprises (1) other HE
(2) Mutant H for its ability to form protein: protein interactions with PATOX protein, other cell surface proteins, or biologically active portions thereof.
For complex formation between EPATO X protein and HEPATOX ligand; (3) For the ability of mutant HEPATOX protein to bind to intracellular target protein or biologically active portion thereof (eg, avidin protein);
4) ATP binding capacity; or (5) HEPATOX protein antibody specific binding capacity can be assayed.

In another embodiment, a fragment of the complementary polynucleotide sequence is set forth in claim 1, wherein the fragment of the complementary polynucleotide sequence hybridizes to the first sequence.

In another particular embodiment, the nucleic acid is RNA or DNA. A fragment of the complementary polynucleotide sequence is set forth in claim 38, wherein the fragment is between about 10 and about 100 nucleotides in length, eg, about 10 to about 90 nucleotides, or about 10 to about 75. It is between nucleotide length, about 10 to about 50 bases, about 10 to about 40 bases, or about 15 to about 30 bases.

Antisense Another aspect of the invention is the isolation of a hybridizable or complementary nucleic acid molecule comprising a nucleotide sequence of a HEPATOX sequence, or a fragment, analog, or derivative thereof. Antisense nucleic acid molecule. "
An "antisense" nucleic acid is complementary to a "sense" nucleic acid encoding a protein, which is, for example, complementary to the coding strand of a double-stranded cDNA branch or complementary to an mRNA sequence. Containing a unique nucleotide sequence. In certain aspects, there is provided an antisense nucleic acid molecule comprising at least about 10, 25, 50, 100, 250, or 500 nucleotides or a sequence complementary to the complete HEPATOX coding strand, or to only a portion thereof. To be done. Further provided are nucleic acid molecules that encode fragments, homologues, derivatives, and analogs of HEPATOX proteins, or nucleic acid molecules that encode antisense nucleic acids that are complementary to nucleic acids that include HEPATOX nucleic acid sequences.

[0111] In one embodiment, the antisense nucleic acid molecule is antisense to the "coding region" of the code strand of the nucleotide sequence encoding HEPATOX. The term "coding region" refers to the region of nucleotide sequence that contains codons translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is HEP.
It is antisense to the "non-coding sequence" of the coding strand of the nucleotide sequence encoding ATOX. The term "noncoding sequence" refers to 5'and 3'sequences which flank the coding region that are not translated into amino acids (ie, also referred to as 5'and 3'untranslated regions).

Given the coding strand sequences encoding HEPATOX described herein, the antisense nucleic acids of the invention may be modified according to Watson and Crick's law or H.
It can be designed according to Oogsteen base pairing. Antisense nucleic acid molecule
Although it may be complementary to the entire coding region of HEPATOX mRNA, more preferably, it is an oligonucleotide that is antisense to only a part of the coding region or non-coding region of HEPATOX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of HEPATOX mRNA. Antisense nucleotides include, for example, about 5, 10, 15,
It can be 20, 25, 30, 35, 40, 45 or 50 nucleotides long. Antisense nucleic acids of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) increase the biological stability of natural nucleotides or molecules, or the physical properties of duplexes formed between antisense and sense nucleic acids. It may be chemically synthesized using variously modified nucleotides designed to increase biological stability, such as phosphorothioate derivatives and acridine substituted nucleotides may be used.

Examples of modified nucleotides that can be used to generate antisense nucleic acids include:
The following may be mentioned: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-. 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- Methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-
Mannosyl cuocosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5
-Oxyacetic acid (v), wybutoxosine, pseudouracil, cuocosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-
Oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2
-Thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid is subcloned such that the nucleic acid is subcloned in an antisense orientation (ie, the RNA transcribed from the inserted nucleic acid is in the antisense orientation relative to the target nucleic acid of interest, and is further described in the subsection below). Can be produced biologically using the expression vector.

Antisense nucleic acid molecules of the invention are typically administered to a patient or produced in situ so that they are expressed in cellular mRNA and / or genomic DNA (which encodes the HEPATOX protein). Hybridize or
Alternatively, it binds and thereby inhibits the expression of the protein, for example by inhibiting transcription and / or translation. Hybridization may be by conventional nucleotide complementarity to form a stable duplex, or, for example, D
In the case of antisense nucleic acid molecules that bind to NA duplexes, they may be through specific interactions in the major groove of the double helix. Examples of routes of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules are expressed on the surface of cells of which they are selected, eg, by linking the antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. It can be modified to specifically bind to a receptor or antigen. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. In order to achieve a sufficient intracellular concentration of the antisense molecule, the antisense nucleic acid molecule has strong pol II
Vector constructs that are placed under the control of a promoter or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. Alpha-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA, where the strands are parallel to each other, as opposed to the normal β-unit (Gaultier et al., (1987) Nucleic Acids. Res
15: 6625-6641). Antisense nucleic acid molecules also include 2'-o-methyl ribonucleotides (Inoue et al., (1987) Nucleic Acids.
Res 15: 6131-6148) or chimeric RNA-DNA analog (I
Noue et al. (1987) FEBS Lett 215: 327-330).

Ribozymes and PNA Sites In yet another embodiment, the antisense nucleic acids of the invention are ribozymes. Ribozymes are catalytic RNA molecules that have ribonuclease activity and are capable of cleaving single-stranded nucleic acids (eg, mRNA) (ribozymes have regions complementary to them). Thus, ribozymes (eg, hammerhead ribozyme (Haselhoff and Gerlach (1988) Nature 334).
: 585-591)) can be used to catalytically cleave HEPATOX mRNA transcripts, thereby inhibiting translation of HEPATOX mRNA. Ribozymes with specificity for HEPATOX-encoding nucleic acids can be designed based on the nucleotide sequences of HEPATOX DNA disclosed herein. For example, if the nucleotide sequence of the active site is HEPATOX coded mRN
Tetrahymena L-19, which is complementary to the nucleotide sequence cleaved at A.
Derivatives of IVS RNA can be constructed. For example, Cech et al., US Pat.
, 987,071; and Cech et al., U.S. Patent No. 5,116,742. Alternatively, HEPATOX mRNA may be used to select for catalytic RNA having a particular ribonuclease activity from a pool of RNA molecules. See, for example, Bartel et al., (1993) Science 261: 1411-141.
See 8.

Alternatively, HEPATOX gene expression can be achieved by using HEPATOX nucleic acid (eg HE
HEPATO in target cells, which is inhibited by targeting a nucleotide sequence complementary to the regulatory region of the PATOX promoter and / or enhancer).
It may form a triple helix structure that prevents transcription of the X gene. Generally, Helene
(1991) Anticancer Drug Des. 6: 569-84; H
elene et al., (1992) Ann. N. Y. Acad. Sci. 660: 27
-36; and Maher (1992) Bioassays 14: 807-15.
checking ...

In various embodiments, HEPATOX nucleic acids may be modified with base moieties, sugar moieties or phosphate backbones to improve, for example, molecular stability, hybridization, or solubility. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to produce peptide nucleic acids (Hyru).
p., et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the term "peptide nucleic acid" or "PNA" refers to a nucleic acid mimetic (eg, a DNA mimic), where the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone. , And 4
One neutral nucleobase is maintained. The neutral backbone of PNA has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers is described by Hyrup et al.
996); Perry-O'Keefe et al. (1996) PNAS 93: 1467.
It can be performed using standard solid phase peptide synthesis protocols as described in 0-675.

HEPATOX PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigenic agents for sequence-specific regulation of gene expression, eg, by inducing transcription or translation arrest or inhibiting replication. HEPATOX PNAs can also be labeled with other enzymes (eg, S1 nuclease (Hyr as described above), eg, by PNA-directed PCR clamps.
up B. (1996)) as an artificial restriction enzyme; or a probe or primer for DNA sequences and hybridization (Hyrup et al. (1996) supra; Perry-O'Keefe (1 supra).
996)) can be used in the analysis of single base pair mutations in a gene.

In another embodiment, the PEPA of HEPATOX may be prepared by, for example, attaching a lipophilic group or other helper group to PNA, forming a PNA-DNA chimera, or liposomes or other drug delivery known in the art. Can be modified to enhance their stability or cellular uptake. For example, PNA and DN
A PEPA-DNA chimera of HEPATOX can be generated that can bind the preferred peptide of A. Such chimeras have DNA recognition enzymes (eg, RNase).
H and DNA polymerase) to interact with the DNA moiety, while the PNA moiety provides high binding affinity and specificity. PNA-DNA chimeras can be ligated using a suitable length of bond selected by the conditions of base stacking, the number of bonds between nucleobases, and orientation (Hyrup (1996) supra).
. The synthesis of PNA-DNA chimeras was performed according to Hyrup (1996) and Fin described above.
n et al. (1996) Nucl Acids Res 24: 3357-63. For example, DNA strands can be synthesized on solid supports using standard phosphoramidite coupling chemistry and modified nucleoside analogs such as 5 '-(4-methoxytrityl) amino-5'-deoxy-. Thymidine phosphoramidites) can be used between the PNA and the 5'end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88).
. The PNA monomers are then combined in a stepwise fashion to produce a chimeric molecule having a 5'PNA segment and a 3'DNA segment (Finn et al. (199
6) Above). Alternatively, the chimeric molecule comprises a 5'DNA segment and a 3'PNA
It can be synthesized using segments. Petersen et al. (1975) Bioor
g Med Chem Lett 5: 1119-11124.

In other embodiments, the oligonucleotides are labeled with other addition groups (eg, peptides (eg, for in vivo targeting of host receptors)) or agents that facilitate transport across cell membranes (eg, Letsinger). , 1989, Proc.
Natl. Acad. Sci. U. S. A. 86: 6553-6556; Lem.
aitre et al., 1987, Proc. Natl. Acad. Sci. 84:64
8-652; PCT Publication No. WO88 / 09810) or blood-brain barrier (see, eg, PCT Publication No. WO89 / 10134). In addition, the oligonucleotide may be a hybridization-triggered cleavage agent (see, eg, Krol et al., 1988, BioTechniques 6: 958-976) or an intercalating agent (eg, Zon, 1988, Pharm. Res. 5).
: 539-549)). At this end, the oligonucleotide can be conjugated to another molecule (eg, a peptide, hybridization-triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.).

HEPATOX Polypeptides One aspect of the invention pertains to isolated HEPATOX proteins and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Polypeptide fragments suitable for use as an immunogen for raising anti-HEPATOX antibodies are also provided. In one embodiment, native HEPATOX protein can be isolated from a cell or tissue source by a suitable purification scheme using standard protein purification techniques. In another embodiment, the HEPATOX protein is produced by recombinant DNA technology. As an alternative to recombinant expression, HEPATOX proteins or polypeptides can be chemically synthesized using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or substantially from the cell or cell source from which the HEPATOX protein is derived. Are other contaminating proteins that are not present, or are substantially absent chemical precursors or substantially absent other chemicals when chemically synthesized. The term "substantially free of cellular material" includes a preparation of HEPATOX protein, where the cellular component of the cell is either isolated or recombinantly produced from this cellular component. Be separated. In one embodiment, the term "substantially free of cellular material" comprises less than about 30% of non-HEPATOX proteins (also referred to herein as "contaminant proteins") (
Dry weight), more preferably less than about 20% of non-HEPATOX protein, even more preferably less than 10% of non-HEPATOX protein, and most preferably less than about 5% of non-HEPATOX protein. If the HEPATOX protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, ie, less than about 20%, more preferably of the volume of the protein preparation. About 10
%, And most preferably less than about 5%.

The term “substantially free of chemical precursors or other chemicals” refers to HEP
ATOX proteins include preparations of this protein that have been separated from the chemical precursors or other chemicals involved in the synthesis of the protein. In one embodiment, the term "substantially free of chemical precursors or other chemicals" means less than about 30% (by dry weight) of chemical precursors or non-HEPATOX chemicals, more preferably. Less than about 20% chemical precursors or non-HEPATOX chemicals, even more preferably less than about 10% chemical precursors or non-HEPATOX chemicals, and most preferably chemical precursors or non-HEPATOX chemicals.
It includes a preparation of HEPATOX protein having less than about 5% chemicals.

The biologically active portion of the HEPATOX protein is the full-length HEPATOX protein.
Is sufficiently homologous to an amino acid sequence of a HEPATOX protein (eg, an amino acid sequence encoded by a nucleic acid containing HEPATOX1-20) that contains less amino acid sequence than the X protein and exhibits at least one activity of the HEPATOX protein, Or a peptide containing the amino acid sequence derived therefrom. Typically, the biologically active portion comprises a domain or motif with at least one activity of HEPATOX protein. The biologically active portion of the HEPATOX protein can be, for example, a polypeptide that is 10, 25, 50, 100 or more amino acids in length.

The biologically active portion of the HEPATOX protein of the invention may be a HEPATOX protein.
It may comprise at least one of the structural domains identified above, conserved in the protein tract. The alternative biologically active portion of the HEPATOX protein may include at least two of the domains identified above. Another biologically active portion of the HEPATOX protein may include at least three of the domains identified above. Yet another biologically active portion of the HEPATOX proteins of the invention may include at least four of the above-identified domains.

In addition, other biologically active portions in which other regions of the protein have been deleted can be prepared by recombinant techniques and evaluated for one or more functional activities of the native HEPATOX protein.

In some embodiments, the HEPATOX proteins are labeled with these HEP
It is substantially homologous to one of the ATOX proteins and, as described in detail below, retains its functional activity even though the amino acid sequences differ due to natural allelic variation or mutagenesis. .

In certain embodiments, the invention comprises an isolated polypeptide comprising an amino acid sequence that is 80% or more identical to the sequence of the polypeptide, the expression of which polypeptide is administered with a hepatotoxic agent. Are regulated in mammals.

Determination of Homology between Two or More Sequences To determine the percent homology of two amino acid sequences or two nucleic acids, these sequences are aligned for the purpose of optimal comparison ( For example, a gap may be introduced in the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides are then compared at corresponding amino acid positions or nucleotide positions. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is homologous at that position (ie, as used herein, the amino acid or Nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").

Nucleic acid sequence homology can be determined as the degree of identity between two sequences. This homology can be determined using computer programs known in the art such as GAP software provided in the GCG program package. Needl
eman and Wunsch 1970 J Mol Biol 48: 443.
See -453. The GCG GAP software uses the following settings for nucleic acid sequence comparisons: a GAP creation penalty of 5.0 and a GAP extension penalty of 0.3, the coding region for similar nucleic acid sequences referred to above is preferably HE.
A CDS (ie, coding) portion of the DNA sequence containing PATHOX 1-21, and
It exhibits a degree of identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

The term “sequence identity” refers to two polynucleotide or polypeptide sequences
It refers to the degree to which each residue is identical over the particular region of comparison. The term "percentage sequence identity" refers to the comparison of two optimally aligned sequences over the region of the comparison, the nucleobase being identical in both sequences (eg, in the case of nucleic acids,
A, T, C, G, U, or I) determines the number of positions that are present and obtains the number of matched positions, the total number of positions in the comparison region (ie, the undone size) Calculated by dividing by the number and multiplying this result by 100 to get the percent sequence identity. The term "substantially identical" as used herein refers to a characteristic of a polynucleotide sequence, where the polynucleotide has at least 80% sequence identity when compared to a reference sequence over a comparison region, It preferably comprises sequences having at least 85% sequence identity and often 90-95% sequence identity, more usually at least 99% sequence identity.

Chimeric and Fusion Proteins The present invention provides HEPATOX chimeric or fusion proteins. As used herein, HEPATOX "chimeric protein" or "
A "fusion protein" is an H-operably linked to a non-HEPATOX polypeptide.
Includes EPATOX polypeptide. "HEPATOX polypeptide" refers to HEP
It refers to a polypeptide having an amino acid sequence corresponding to ATOX. On the other hand, "non-HE
A "PATOX polypeptide" refers to a protein that is not substantially homologous to a HEPATOX protein (eg, a polypeptide that differs from the HEPATOX protein and has an amino acid sequence that matches the protein from the same or a different organism. HEPATOX fusions. Within the protein, the HEPATOX polypeptide corresponds to all or a portion of the HEPATOX protein, hi one embodiment, the HEPATOX fusion protein comprises at least one biologically active portion of the HEPATOX protein. In, the HEPATOX fusion protein comprises at least two biologically active portions of the HEPATOX protein, hi yet another embodiment, the HEPATOX fusion protein comprises H
It comprises at least three biologically active portions of the EPATO X protein. Within this fusion protein, the term "operably linked" is intended to allow the HEPATOX and non-HEPATOX polypeptides to be fused in-frame to each other. The non-HEPATOX polypeptide can be fused to the N-terminus and C-terminus of the HEPATOX polypeptide.

For example, in one embodiment, the HEPATOX fusion protein comprises a HEPATOX domain operably linked to the extracellular domain of a second protein.
Such fusion proteins can further be used in screening assays for compounds that modulate HEPATOX activity, such assays are described in detail below.

In yet another embodiment, the fusion protein has a HEPATOX sequence of GST (
That is, the C fused to the C-terminus of the glutathione S-transferase) sequence.
ST-HEPATOX fusion protein. Such a fusion protein may facilitate the production of recombinant HEPATOX.

In another embodiment, the fusion protein is a HEPATOX protein containing a heterologous signal sequence at its N-terminus. For example, native HEPATOX
The signal sequence is removed and replaced with a signal sequence from another protein. In certain host cells, such as mammalian host cells, HEPATOX expression and / or secretion can be increased through the use of heterologous signal sequences.

[0137] In yet another embodiment, the fusion protein is HE comprising one or more domains.
The PATOX sequence is a HEPATOX-immunoglobulin fusion protein fused to sequences from members of the immunoglobulin protein family. The HEPATOX-immunoglobulin fusion proteins of the present invention can be incorporated into pharmaceutical compositions and administered to a subject, with HEPATOX ligand and HE on the surface of cells.
It may inhibit the interaction with the PATOX protein, which may suppress HEPATOX-mediated signaling in vivo. This HEPATOX-immunoglobulin fusion protein can be used to influence the bioavailability of HEPATOX cognate ligands. Inhibition of HEPATOX ligand / HEPATOX interactions regulates treatment of both proliferative and differentiation disorders as well as cell survival (
Therapeutically useful (eg, to promote or inhibit). Further, the HEPATOX-immunoglobulin fusion protein of the present invention can be used in
It can be used as an immunogen for producing OX antibodies, as an immunogen for purifying HEPATOX ligand, and in screening assays to identify molecules that inhibit the interaction of HEPATOX and HEPATOX ligand.

The HEPATOX chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragment coding for different polypeptide sequences has been described using conventional techniques (eg, using blunt or staggered ends for ligation, using restriction enzyme digests to provide suitable ends, appropriately protruding). Filling the ends, using alkaline phosphatase treatment to avoid unwanted conjugation, and by enzymatic ligation)
In-frame, connected together. In another embodiment, the fusion gene is an automated DN.
It can be synthesized by conventional techniques including A synthesizer. Alternatively, PCR amplification of the gene fragment can be performed using an anchor primer that complementarily overhangs between two consecutive gene fragments that can be subsequently annealed or reamplified to produce a chimeric gene sequence. (For example, Ausubel et al. (Ed.)
CURRENT PROTOCOLS IN MOLECULAR BIOLO
GY, Jhon Wiley & Sons, 1992). In addition, many expression vectors are commercially available and these are already fusion moieties (eg GST
Polypeptide). The HEPATOX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the HEPATOX protein.

HEPATOX Agonists and HEPATOX Antagonists The present invention also relates to various HEPATOX proteins that function as either HEPATOX agonists (mimetics) or HEPATOX antagonists. Variants of HEPATOX protein can be generated by mutagenesis (eg, individual point mutations or cleavage of HEPATOX protein). An agonist of a HEPATOX protein retains substantially the same biological activity as the naturally occurring form of the HEPATOX protein, or retains a biologically active subset. An antagonist of HEPATOX protein is, for example, HEP
One of the naturally occurring forms of the HEPATOX protein by competitively binding downstream or upstream of the cell signaling cascade containing the ATOX protein.
It may inhibit more than one activity. Thus, a particular biological effect can be elicited by treatment with a variant of limited function. In one embodiment, treatment of the subject with a variant having a subset of biological activities of the naturally occurring form of the protein is compared to treatment of the subject with a naturally occurring form of the HEPATOX protein. Have less side effects in.

HEPATOX protein variants, which function as either HEPATOX agonists (mimetics) or HEPATOX antagonists, generate a combinatorial library of HEPATOX protein variants (eg truncation variants).
It can be identified by screening for EPATO X protein agonist or antagonist activity. In one embodiment, HEPAT
A variegated library of OX variants was generated by nucleic acid level combinatorial mutagenesis and is encoded by a variegated gene library. A variegated library of HEPATOX variants is, for example, a mixture of synthetic oligonucleotides that is enzymatically linked into a gene sequence such that the degenerate set of possible HEPATOX sequences is either as individual polypeptides or in the set of HEPATOX sequences. To be expressible (eg, for phage display) as a larger set of fusion proteins containing There are a variety of methods that can be used to generate a library of possible HEPATOX variants from degenerate oligonucleotide sequences. Chemical synthesis of the degenerate gene sequence can be carried out in an automated DNA synthesizer, which synthetic gene can then be ligated into an appropriate vector. The use of a degenerate set of genes makes it possible, in one mixture, to provide all of the sequences encoding the desired set of possible HEPATOX sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (eg, Narang (1983) Tetrahedron 39).
: 3; Itakura et al. (1984) Annu Rev Biochem 53.
: 323; Itakura et al. (1984) Science 198: 1056;
See Ike et al. (1983) Nucl Acid Res 11: 477).

Polypeptide Library In addition, a library of fragments of the HEPATOX protein coding sequence can be used to generate a variegated population of HEPATOX fragments for screening and subsequent selection of variants of the HEPATOX protein. In one embodiment, the library of coding sequence fragments comprises treating the double-stranded PCR fragment of the HEPATOX coding sequence with a nuclease under conditions where nicking occurs only about once per molecule. Denaturing double-stranded DNA, regenerating it to form double-stranded DNA that may contain sense / antisense pairs from different nicking products, double-strands reformed by treatment with S1 nuclease Can be produced by removing the single-stranded portion from the product and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes various sized N-terminal and internal fragments of the HEPATOX protein.

Several techniques are known in the art for screening the gene products of combinatorial libraries produced by point mutations or truncations, and for screening cDNA libraries for gene products with selected properties. Is. Such techniques are amenable to rapid screening of gene libraries generated by combinatorial mutagenesis of HEPATOX proteins. The most widely used technique for screening large gene libraries, which follows high throughput analysis, typically involves cloning the gene library into a replicable expression vector. Transforming suitable cells with a library of different vectors and expressing the combinatorial gene under conditions which facilitate isolation of the vector encoding the gene whose product was detected by detection of the desired activity. , Can be mentioned.
Repetitive Ensemble Mutagenesis (REM), a novel technique that increases the frequency of functional variants in libraries, can be combined with screening assays to identify HEPATOX variants (Arkin and Yourvan (1992).
PNAS 89: 7811-7815; Delgrave et al. (1993) Pro.
tain Engineering 6: 327-331).

Anti-HEPATOX Antibodies For the production of antibodies that the isolated HEPATOX protein, or a portion or fragment thereof, binds HEPATOX using standard techniques for the preparation of polyclonal and monoclonal antibodies. It can be used as an immunogen. The full length HEPATOX protein may be used, or the invention may be
Provided are antigenic peptide fragments of HEPATOX for use as immunogens. The antigenic peptide of HEPATOX is at least 8 of the amino acid sequences encoded by the nucleic acids including the nucleic acid sequences shown in HEPATOX: 1-21.
An antibody containing an amino acid residue and raised against the peptide is HEPAT
It contains an epitope of HEPATOX so as to form a specific immune complex with OX. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. A preferred epitope comprised by the antigenic peptide is a region of HEPATOX (eg, a hydrophobic region) located on the surface of the protein. As a means to target antibody production, hydropathic plots showing hydrophilic and hydrophobic regions can be obtained by any method known in the art (eg, Kyte Doolittle method or Hopp Woods method) with Fourier transformation. It can be generated either with or without. For example, Hoop and Woods, 198.
1, Proc. Natl. Acad. Sci. USA 78: 3824-382.
8; Kyte and Doolittle 1982, J. Am. Mol. Biol. 1
57: 105-142, each of which is incorporated herein by reference in its entirety.

HEPATOX polypeptides, or derivatives, fragments, analogs or homologs thereof, can be utilized as immunogens in the production of antibodies that immunospecifically bind these protein components. The term "antibody," as used herein, refers to immunoglobulin molecules, and immunologically active portions of immunoglobulin molecules (ie, that specifically bind to (immunoreact with) an antigen. Molecule containing the antigen-binding site). Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, _Fab and F _{(ab) 2} fragments, and _Fab expression libraries.
Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies against the HEPATOX protein sequence, or a derivative, fragment, analog or homologue thereof. Some of these proteins are discussed below.

For the production of polyclonal antibodies, a variety of suitable host animals (eg, rabbits, goats, mice or other animals) can be injected with the native protein, or synthetic variants thereof, or derivatives thereof as described above. Can be immunized. A suitable immunogenic preparation can include, for example, recombinantly expressed HEPATOX protein or a chemically synthesized HEPATOX polypeptide. The preparation may further include an adjuvant. Various adjuvants used to increase immunological response include Freund's (complete and incomplete) adjuvants, mineral gels (eg aluminum hydroxide), surface-active substances (eg lysolecithin, polyfunctional polyols, polyanions). , Peptides, oil emulsions, dinitrophenol, etc.), human adjuvants (eg, Bacillus Calmette-Guerin and Corynebacte).
rum parvum, or similar immune stimulators), but is not limited thereto. If desired, antibody molecules to HEPATOX can be isolated from mammals (eg, from blood) and further purified by well-known techniques (eg, Protein A chromatography) to yield IgG fractions. .

As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to a population of antibody molecules that contains only one species of antigen binding site capable of immunoreacting with a particular epitope of HEPATOX. Say. Therefore, a monoclonal antibody composition will typically be used to specifically detect the specific HEP with which it immunoreacts.
It exhibits a single binding affinity for the ATOX protein. Any technique which provides for the production of antibody molecules by continuous cell line cultures may be utilized, particularly for the preparation of monoclonal antibodies against the HEPATOX protein, or derivatives, fragments, analogs or homologs thereof. Such techniques include, but are not limited to: hybridoma technology (Kohler and Mil.
Stein, 1975 Nature 256: 495-497).
Trioma technology; human B cell hybridoma technology (Kozbor et al., 1983).
Immunol Today 4:72) and EBV hybridoma technology for producing human monoclonal antibodies (Cole et al., 1985).
MONOCLONAL ANTIBODIES AND CANCER THE
RAPY, Alan R .; Liss, Inc. , 77-96).
Human monoclonal antibodies can be utilized in the practice of the present invention, and by using human hybridomas (Cote et al., 1983. Proc Natl.
Acad Sci USA 80: 2026-2030), or transformation of human B cells in vitro with Epstein-Barr virus (C
ole et al., 1985: MONOCLONAL ANTIBODIES AND
CANCER THERAPY, Alan R.M. Liss, Inc. , 77-9
See page 6).

[0147]   For the production of single chain antibodies specific for HEPATOX protein according to the invention
For example, the technique may be adapted (see, eg, US Pat. No. 4,946,778).
When). Further, the method is F_abAdapted to the construction of expression libraries (eg Hu
See et al., 1989 Science 246: 1275-1281.
), HEPATOX protein, or a derivative, fragment, analog thereof,
Alternatively, a monoclonal F having desired properties for homologues_abFragment
Enables rapid and effective identification of Non-human antibodies are prepared by techniques well known in the art.
Can be “humanized”. See, for example, US Pat. No. 5,225,539.
. An antibody fragment containing the idiotype for the HEPATOX protein is
, Can be produced by techniques known in the art including, but not limited to:
(I) F produced by antibody molecule pepsin production_{(ab ') 2}Fragment; (i
i) F_{(ab ') 2}F generated by reducing the disulfide bridge of the fragment _ab A fragment; (iii) by treatment of the antibody molecule with papain and a reducing agent
Arises F_abFragment, as well as (iv) F_vFragment.

In addition, recombinant anti-HEPATOX antibodies (eg, chimeric and humanized monoclonal antibodies), such as chimeric and humanized monoclonal antibodies that include both human and non-human portions, are available from standard recombinant DNA techniques. Can be made using) is within the scope of the present invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. This technique uses, for example, the following method: PCT International Application No. PCT / US8.
6/02269; European Patent Application No. 184,187; European Patent Application No. 171
, 496; European Patent Application No. 173,494; PCT International Publication No. WO 86 /.
01533; U.S. Pat. No. 4,816,567; European Patent Application No. 125,02.
No. 3; Better et al. (1988) Science 240: 1041-104.
3; Liu et al. (1987) PNAS 84: 3439-3443; Liu et al. (1).
987) J Immunol. 139: 3521-3526; Sun et al. (198
7) PNAS 84: 214-218; Nishimura et al. (1987) Ca.
ncer Res 47: 999-1005; Wood et al. (1985) Natu.
Re 314: 446-449; Shaw et al. (1988) J Natl Can.
cer Inst 80: 1553-1559); Morrison (1985).
) Science 229: 1202-1207; Oi et al. (1986) BioT.
techniques 4: 214; US Pat. No. 5,225,539; Jones.
s et al. (1986) Nature 321: 552-525; Verhoeyan.
(1988) Science 239: 1534; and Beidler et al. (1988) J Immunol 141: 4053-4060.

In one embodiment, methods for screening antibodies with the desired specificity include enzyme-linked immunosorbent assay (ELISA) and other immunologically mediated techniques known in the art. However, it is not limited to this. In certain embodiments, selection of antibodies that are specific for particular domains of the HEPATOX protein is facilitated by the production of hybridomas that bind fragments of the HEPATOX protein that have such domains. Antibodies specific for one or more domains within the HEPATOX protein (eg, the conserved regions identified above of HEPATOX family proteins, or domains spanning derivatives, fragments, analogs or homologs thereof) are also provided herein. Provided to.

Anti-HEPATOX antibodies can be used in methods known in the art for the localization and / or quantification of HEPATOX protein (eg, use in determining levels of HEPATOX protein in a suitable physiological sample). For use in diagnostic methods, for use in protein imaging, etc.). In certain embodiments, an antibody to the HEPATOX protein, or derivative, fragment, analog or homolog thereof, which comprises the antibody-derived binding domain, is a pharmacologically active compound [hereinbelow " Therapeutics ”].

Anti-HEPATOX antibodies (eg, monoclonal antibodies) can be isolated by standard techniques (eg, affinity chromatography or immunoprecipitation) from HEPATOX.
Can be used to isolate Anti-HEPATOX antibody is a natural HE from cells.
Purification of PATOX and recombinantly produced HEP expressed in host cells
It may facilitate the purification of ATOX. Furthermore, using anti-HEPATOX antibody, H
To assess the amount and pattern of EPATOX protein expression, HEPA
TOX protein can be detected (eg in cell lysate or cell supernatant)
. Anti-HEPATOX antibodies can be used, for example, to diagnostically monitor protein levels in tissues as part of a clinical trial procedure to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of such fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine and dichlorotriazinamine.
ylamine) fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; examples of bioluminescent substances include luciferase, luciferin and aequorin; and examples of suitable radioactive substances ^{Includes 125} I, ¹³¹ I, ³⁵ S or ³ H.

(HEPATOX Recombinant Expression Vector and Host Cell) Another aspect of the present invention relates to a vector, preferably an expression vector, containing a nucleic acid encoding a HEPATOX protein, or a derivative, fragment, analog or homologue thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a "plasmid." This refers to a linear or circular double stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors)
Is integrated into the host cell's genome upon introduction into the host cell and is thereby replicated with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." In general, useful expression vectors in recombinant DNA technology are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors that serve equivalent functions (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses).

The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for expressing the nucleic acid in a host cell. This means that the recombinant expression vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected on the basis of the host cell to be used for expression. . In the recombinant expression vector,
"Operably linked" means (for example, in an in vitro transcription / translation system,
Alternatively, when the vector is introduced into a host cell, it is intended to mean that the nucleotide sequence of interest (in the host cell) is linked to regulatory sequences in a manner that allows expression of the nucleotide sequence. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, by Goeddel; GENE.
EXPRESSION TECHNOLOGY: METHODS IN EN
ZYMOLOGY 185, Academic Press, San Diego
o, Calif. (1990). Regulatory sequences include sequences that direct constitutive expression of nucleotide sequences in many types of host cells, and sequences that direct expression of nucleotide sequences only in particular host cells (eg, tissue-specific regulatory sequences). The selection of the host cell to be transformed by the design of the expression vector,
It will be appreciated by those skilled in the art that it may depend on factors such as the level of expression of the desired protein and the like. The expression vector of the present invention may be introduced into a host cell, whereby a protein or peptide encoded by a nucleic acid as described herein ((
Fusion proteins or peptides) (eg, HEPATOX proteins, or mutant forms of HEPATOX, fusion proteins, etc.) can be produced.

Recombinant expression vectors of the invention can be used in prokaryotic or eukaryotic cells,
It can be designed for the expression of HEPATOX. For example, HEPATOX can be expressed in bacterial cells (eg, E. coli), insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are Goeddel, GENE EXPRESSION TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic P
less, San Diego, Calif. (1990) for further discussion. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. executed in E. coli. Fusion vectors add multiple amino acids to the protein encoded therein (usually at the amino terminus of recombinant proteins). Such fusion vectors typically serve three purposes: (1) increasing the expression of the recombinant protein; (2) increasing the solubility of the recombinant protein; and ( 3) To assist in the purification of recombinant proteins by acting as a ligand in affinity purification. Often, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein, allowing the recombinant protein to be separated from the fusion moiety after purification of the fusion protein. . Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin,
And enterokinase. As a typical fusion expression vector, glutathione S-transferase (GST), maltose E binding protein, or protein A is fused to a target recombinant protein, respectively, pGEX (Ph
armia Biotech Inc; Smith and Johnso
n (1988) Gene 67: 31-40), pMAL (New Engla).
nd Biolabs, Beverly, Mass. ) And pRIT5 (Ph
armacia, Piscataway, N .; J. ) Is mentioned.

Suitable inducible unfused E. An example of an E. coli expression vector is pTrc (Am
runn et al. (1988) Gene 69: 301-315) and pET 11
d (Studier et al., GENE EXPRESSION TECHNOLOG
Y: METHODS IN ENZYMOLOGY 185, Academic
Press, San Diego, Calif. (1990) 60-89).

E. One strategy for maximizing recombinant protein expression in E. coli is to express the protein in a host bacterium with impaired ability to proteolytically cleave the recombinant protein. Gottesman, GENE
EXPRESSION TECHNOLOGY: METHODS IN EN
ZYMOLOGY 185, Academic Press, San Diego
o, Calif. (1990) 119-128. Another strategy is that the individual codons for each amino acid are to change the nucleic acid sequence of the nucleic acid inserted into the expression vector so that it is the preferentially utilized codon in E. coli (Wada et al. (1992) Nucleic Acids Res.
0: 2111-2118). Such alterations of the nucleic acid sequence of the present invention are standard D
It can be performed by NA synthesis technology.

[0158] In another embodiment, the HEPATOX expression vector is a yeast expression vector. Yeast S. Examples of vectors for expression in C. cerevisiae include pYepSec1 (Baldari et al., (1987) EMBO J 6: 2.
29-234), pMFa (Kurjan and Herskowitz, (19
82) Cell 30: 933-943), pJRY88 (Schultz et al.,
(1987) Gene 54: 113-123, pYES2 (Invitro.
gen Corporation, San Diego, Calif. ), And picZ (InVitrogen Corp., San Diego, Cal.
if. ) Is mentioned.

Alternatively, HEPATOX can be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (eg, SF9 cells) include pAc series (S
Smith et al. (1983) Mol Cell Biol 3: 2156-2165.
) And pVL series (Lucklow and Summers (1989) V
irology 170: 31-39).

In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. An example of a mammalian expression vector is pCDM8
(Seed (1987) Nature 329: 840) and pMT2PC (
Kaufman et al. (1987) EMBO J 6: 187-195). When used in mammalian cells, the expression vector's control functions are often
Provided by the regulatory elements of the virus. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, eg, Sambrook et al., MOLECUL.
AR CLONING: A LABORATORY MANUAL. Second edition, C
old Spring Harbor Laboratory, Cold Sp
ring Harbor Laboratory Press, Cold Sp
ring Harbor, N.M. Y. , 1989, chapters 16 and 17.

In another embodiment, the recombinant mammalian expression vector may direct the expression of the nucleic acid preferentially in a particular cell type (eg, use the tissue-specific regulatory elements to express the nucleic acid). . Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev 1: 268-27.
7), lymphoid-specific promoter (Calame and Eaton (1988)
Adv Immunol 43: 235-275), at a specific promoter of the T cell receptor (Winoto and Baltimore (1989) E.
MBO J 8: 729-733) and immunoglobulins (Banerji et al.
1983) Cell 33: 729-740; Queen and Baltimo.
re (1983) Cell 33: 741-748), neuron-specific promoters (eg, neurofilament promoter; Byrne and Rud).
dle (1989) PNAS 86: 5473-5477), pancreas-specific promoter (Edlund et al. (1985) Science 230: 912-916).
), And mammary gland-specific promoters (eg, milk whey promoter; US Pat. No. 4,873,316 and EP-A-264,166). Developmentally regulated promoters are also included (eg, murine hox promoter (Kessel and Gruss (19
90) Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes.
Dev 3: 537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in the antisense orientation. That is, the DNA molecule is operably linked to regulatory sequences in a manner that allows expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to HEPATOX mRNA. A regulatory sequence operably linked to the cloned nucleic acid in the antisense orientation can be chosen to direct the continuous expression of the antisense RNA molecule in a variety of cell types. For example, viral promoters and / or enhancers, or regulatory sequences that direct constitutive, tissue-specific, or cell-specific expression of antisense RNA can be selected. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus, where the antisense nucleic acid is produced under the control of a high efficiency regulatory region, the activity of which is introduced into the vector. Cell type. For a discussion of the regulation of gene expression using antisense genes, see Weintraub et al., “An.
tissue RNA as a molecular tool for
genetic analysis ", Reviews--Trends in
See Genetics, Volume 1 (1) 1986.

Another aspect of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Such progeny may, in fact, not be identical to the parental cell, as the particular modification may be present in the next generation, either due to mutations or environmental influences, but is still referred to herein. Within the scope of the term as used in.

The host cell can be any prokaryotic or eukaryotic cell. For example, H
The EPATO X protein can be expressed in bacterial cells (eg, E. coli), insect cells, yeast or mammalian cells (eg, Chinese Hamster Ovary cells (CHO) or COS cells). Other suitable host cells are known to those of skill in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to various art-recognized techniques for introducing exogenous nucleic acid (eg, DNA) into a host cell. As intended, these include calcium phosphate or calcium chloride co-precipitation, DEAE dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are described by Sambrook et al. (MOLECULAR C
LONING: A LABORATORY MANUAL. Second Edition, Cold
Spring Harbor Laboratory, Cold Spring
Harbor Laboratory Press, Cold Spring
Harbor, N.M. Y. , 1989) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that only a fraction of the cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select for these elements, a gene encoding a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cell along with the gene of interest.
Various selectable markers confer resistance to a drug (eg, G
418, hygromycin, and methotrexate). The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the vector encoding HEPATOX, or can be introduced on a separate vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated the selectable marker gene will survive while other cells die).

Host cells of the invention (eg, prokaryotic or eukaryotic host cells in culture) can be used to produce (ie, express) HEPATOX protein. Accordingly, the present invention further uses the host cells of the invention to provide HEPATO
Methods for producing an X protein are provided. In one embodiment, the method comprises the step of culturing a host cell of the invention, into which a recombinant expression vector encoding HEPATOX has been introduced, in a suitable medium in which the HEPATOX protein is produced. Includes. In another embodiment, the method further comprises
Isolating HEPATOX from the medium or host cells.

Pharmaceutical Compositions HEPATOX nucleic acid molecules, HEPATOX proteins, and anti-HE of the present invention.
PATOX antibody (also referred to herein as "active compound")
, And their derivatives, fragments, analogs, and homologs can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include a nucleic acid molecule, a protein or antibody, and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents compatible with pharmaceutical administration. And absorption delaying agents and the like. A suitable carrier is Remi, a standard reference book in the field.
Latest version of ngton's Pharmaceutical Sciences (
Which is incorporated herein by reference). Preferred examples of such carriers or diluents include water, saline, finger solution (
finger's solution), dextrose solution and 5% human serum albumin, but are not limited thereto. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except to the extent that any conventional vehicle or agent is incompatible with the active compound, their use in the composition is considered. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (eg, intravenous, intradermal, subcutaneous) administration, oral (
For example, inhalation), transdermal (topical) administration, transmucosal
) Administration, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may contain the following components: water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic materials. Sterile diluents such as solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;
Chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or aqueous dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water,
Cremophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases,
The composition should be sterile and should be easy to inject.
It should be fluid to the extent that litty) is present. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example: water, ethanol, polyols such as glycerol, propylene glycol, and liquid polyethylene glycols, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

If necessary, incorporating the active compound (eg, HEPATOX protein, or anti-HEPATOX antibody) in the required amount of a suitable solvent together with one or a combination of the above enumerated components, followed by filter sterilization. By doing so, sterile water for injection can be prepared. In general, the active compounds are incorporated into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above,
Prepare the dispersant. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and lyophilization, whereby a powder of the active ingredient and its prefilter-sterilized solution are used to remove any further desired ingredients. Produces a powder.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared with a liquid carrier for use as a mouthwash. Here the compound in a liquid carrier is applied orally and shaken off and exhaled or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, etc. may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose. , Disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or peppermint, methyl salicylate. Or a flavoring like orange flavor.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a compressed container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or from a nebulizer. It

Systemic administration can also be administered by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compound is an ointment, as is commonly known in the art.
nt), salves, gels, or creams.

The compounds can also be prepared in the form of suppositories (eg, with conventional suppositories such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound is prepared with a carrier that protects the compound against rapid elimination from the body (eg, a sustained release formulation (contro)).
Led release formulation)) This includes implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods of preparing such formulations will be apparent to those of skill in the art. This material is also Alza
Corporation and Nova Pharmaceuticals, I
nc. Can be purchased from. Liposomal suspensions, including liposomes targeted to cells infected with monoclonal antibodies against viral antigens, can also be used as pharmaceutically acceptable carriers. These are, for example, US Pat. No. 4,522,
It can be prepared according to methods known to those skilled in the art, as described in No. 811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically defined unit adapted as a unit dosage for the subject to be treated. Here each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are set forth by the unique characteristics of the active compounds, and the particular therapeutic effect to be achieved, as well as limitations inherent in the field of compounds such as active compounds for treatment of individuals. , And depends on this.

The nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors can be administered, for example, by intravenous injection, topical administration (see US Pat. No. 5,328,470), or stereotactic injection (eg, Ch.
en et al. (1994) PNAS 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent or can include a sustained release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells (eg, a retroviral vector), the pharmaceutical preparation can include one or more cells that give rise to the gene delivery system.

The pharmaceutical composition may be included in a container, pack or dispenser together with instructions for administration.

Kits and Nucleic Acid Harvests for Identifying HEPATOX Nucleic Acids In another aspect, the invention provides kits useful for testing the hepatotoxicity of drugs. The kit can include nucleic acids that detect more than one HEPATOX sequence. In a preferred embodiment, the kit comprises 3, of HEPATOX nucleic acid sequences.
It includes reagents that detect 4, 5, 6, 8, 10, 12, 15, 20, 25, 50, 100 or all.

The present invention also includes a number of isolated sequences that can identify one or more HEPATOX-responsive nucleic acid sequences.

The kit or multiplicities may include, for example, sequences homologous to the HEPATOX nucleic acid sequence,
Alternatively, one or more HEPATOX nucleic acid sequences can be specifically identified.

(Other Embodiments) Although the present invention has been described in combination with the detailed description thereof,
It is understood that the scope of the invention defined by the claims set forth above is intended to be illustrative, but not limiting. Other aspects, advantages and modifications are within the scope of the above claims.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12Q 1/02 5/10 1/68 A C12Q 1/02 C12N 15/00 ZNAA 1/68 5/00 A (31) Priority claim number 09 / 548,589 (32) Priority date April 13, 2000 (April 13, 2000) (33) Country of priority claim United States (US) (81) Designation Country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG) , CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, K , KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE , DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F term (reference) 4B024 AA11 BA80 CA04 DA02 DA06 EA02 GA11 HA12 4B063 QA19 QQ08 QQ43 QR32 QR55 QR62 QS25 QS34 4B065 AA90X AA99Y AB01 AC14 BA02 CA24 CA46 4H045 AA10 AA11 AA30 BA10 CA40 DA76 DA86 EA28 FA74

Claims (33)

[Claims] 1. A method for screening a test factor for hepatotoxicity, which comprises: (a) a cell capable of expressing one or more nucleic acid sequences selected from the group consisting of HEPATOX: 1-168 and 169. Providing a test cell population; (b) contacting the test cell population with a test factor; (c) measuring the expression of one or more of the nucleic acid sequences in the test cell population; (d) Comparing the expression of the nucleic acid sequence in the test cell population to the expression of the nucleic acid in a reference cell population, the reference cell population comprising at least one cell known to be exposed to a hepatotoxic factor. And (e) HEPATO in the test cell population and reference cell population, if present.
Identifying differences in the expression levels of the X sequences, thereby screening the test agent for liver toxicity. 2. The method comprises HEPATOX 22-24 and HEPATO.
The method of claim 1, comprising comparing the expression of one or more genes selected from the group consisting of X59-73. 3. The method of claim 1, wherein the method comprises comparing the expression of one or more genes selected from the group consisting of HEPATOX 25-34. 4. The method comprises HEPATOX 22-24 and HEPATO.
The method of claim 2, comprising comparing the expression of one or more genes selected from the group consisting of X59-73. 5. The method of claim 1, wherein the method comprises comparing the expression of 40 or more nucleic acid sequences. 6. The method of claim 1, wherein expression of said nucleic acid sequence in said test cell population is reduced compared to said reference cell population. 7. The method of claim 1, wherein expression of the nucleic acid sequence in the test cell population is increased compared to the reference cell population. 8. The method of claim 1, wherein the test cell population is provided in vitro. 9. The method of claim 1, wherein the test cell population is provided ex vivo from a mammalian subject. 10. The method of claim 1, wherein the test cell population is provided in vivo in a mammalian subject. 11. The method of claim 1, wherein the test cell population is from a human or rodent subject. 12. The method of claim 1, wherein the test cell population comprises hepatocytes. 13. The method of claim 1, wherein the test factor is a ligand for the PPARγ receptor. 14. The hepatotoxic factor is a thiazolidinedione.
The method described in. 15. The method of claim 14, wherein the thiazolidinedione is troglitazone. 16. A method for evaluating hepatotoxicity of a test factor in a subject, which method comprises: (a) one or more nucleic acid sequences selected from the group consisting of HEPATOX: 1-168 and 169 from the subject. Procuring a test cell population that includes cells capable of expressing: (b) contacting the test cell population with a test factor; (c) measuring the expression of one or more of the nucleic acid sequences in the test cell population. And (d) comparing the expression of the nucleic acid sequence in the test cell population with the expression of the nucleic acid sequence in a reference cell population, wherein the reference cell population is known for exposure to hepatotoxic factors. Including at least one cell; (e) identifying a difference in the expression level of the nucleic acid sequence in the test cell population and the reference cell population, if present, Evaluating liver toxicity of the test factor in the subject by Les method. 17. The method according to claim 16, wherein the method comprises the step of comparing the expression of one or more genes selected from the group consisting of HEPATOX 59-73. 18. The method according to claim 16, wherein the method comprises the step of comparing the expression of one or more genes selected from the group consisting of HEPATOX 25-34. 19. The method of claim 18, wherein the method comprises comparing the expression of one or more genes selected from the group consisting of HEPATOX 59-73. 20. The method of claim 16, wherein expression of said nucleic acid expression in said test cell population is reduced compared to said reference cell population. 21. The method of claim 16, wherein expression of the nucleic acid sequence in the test cell population is increased compared to the reference cell population. 22. The method of claim 16, wherein the subject is a human or a rodent. 23. The method of claim 16, wherein the test cell population is sourced ex vivo from the subject. 24. The method of claim 16, wherein the test cell population is sourced from the subject in vivo. 25. An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of HEPATOX 1-21 nucleic acids, or the complement thereof. 26. A vector comprising the nucleic acid of claim 25. 27. A cell comprising the vector of claim 26. 28. A pharmaceutical composition comprising the nucleic acid of claim 25. 29. A polypeptide encoded by the nucleic acid of claim 25. 30. An antibody that specifically binds to the polypeptide of claim 29. 31. A kit for detecting two or more nucleic acid sequences selected from the group consisting of HEPATOX: 1-168 and 169. 32. An array that detects one or more nucleic acids selected from the group consisting of HEPATOX: 1-168 and 169. 33. A plurality of nucleic acids comprising one or more nucleic acids selected from the group consisting of HEPATOX: 1-168 and 169.