JP2002523112A - Toxicological markers - Google Patents

Abstract

  (57) [Summary] The invention relates to a composition comprising a plurality of nucleic acid molecules. This composition can be used for hybridizable array elements of a microarray. The invention also relates to methods of screening for compounds and methods of treating metabolic reactions indicative of toxic compounds.

Description

DETAILED DESCRIPTION OF THE INVENTION

RELATED APPLICATIONS The present invention is a Patent Cooperation Treaty (PCT) application, claiming the benefit of the following United States application: US Provisional Application No. 09 / 141,825, filed August 28, 1998, which is not a provisional application
No. 09-172,711, non-provisional application filed on Oct. 13, 1998, attorney reference number PA-0011US, Jan. 1998
US Provisional Application Ser. No. 09 / 172,108, non-provisional application filed on Jan. 13, attorney docket number PA-0012US.

TECHNICAL FIELD The present invention relates to compositions and methods for detecting metabolic and toxicological reactions. BACKGROUND ART Toxicity testing is one of the time-consuming and essential pharmaceutical processes. For high-speed screening to determine metabolic effects and detect the toxicity of lead drug candidates,
Gene expression microarrays can be used. For example, various microarrays can be made using full-length genes or gene fragments. These arrays can then be used to examine samples treated with drug candidates to elucidate gene expression patterns associated with drug treatment. This genetic pattern can be compared to gene expression patterns associated with compounds with known toxicological and metabolic responses.

[0003] Benzopyrene is a known rodent carcinogen and is likely to be a human carcinogen. Benzopyrene is a prototype of a class of compounds that are polycyclic aromatic hydrocarbons (PAH). Benzopyrene is also available in some cytochrome P45
It is metabolized by type 0 (P450 isozyme) and related enzymes that form active and detoxified metabolites. The final metabolite is the groove domain diol epoxide (ba
y-region diol epoxide, benzopyrene-7,8-diol-9,10-epoxide (BPDE), K-region diol epoxide
And 9-hydroxybenzopyrene-4,5-oxide which induces the formation of DNA adducts. DNA adducts are given at a single dose of 10 mg / kg (per body weight) three times a week.
It has been shown that when benzopyrene is administered twice a week to rats, it remains in the liver of the rats for 56 days thereafter (Qu and Stacey (1996) Carcinogenesis 17: 53-5).
9).

[0004] Acetaminophen is widely used as an analgesic. Acetaminophen is metabolized by certain cytochrome P450 isozymes, and most drugs are detoxified by the glucuronic acid and sulfate, glutathione conjugation pathways. However, above a given drug capacity, acetaminophen is converted to N-acetyl-P-benzoquinone imine (N
APQI). In turn, NAPQI binds to the sulfhydryl groups of proteins that cause inactivation leading to cell death (Kroger et al. (1997) Gen. Pharmacol.
. 28: 257-263).

[0005] Clofibrate is a therapeutic agent for hyperlipidemia that reduces elevated serum triglyceride levels. In rodents, prolonged treatment develops hepatoma and increases liver peroxisomes. Clofibrate is cytochrome P450 4
It has been shown to increase the level of A and decrease the level of P450 4F. Clofibrate is also involved in the induction of peroxisome proliferator (PP) -activated receptors and in the transcription of β-oxidation genes (Kawashima et al. (1997) Arch. Biochem.
Biophys. 347: 148-154). Peroxisome proliferation induced by clofibrate and the chemically related compound fenofibrate,
It is mediated by a general inhibitory effect on mitochondrial membrane depolarization (Zhou and Wa
llace (1999) Toxicol. Sci. 48: 82-89).

[0006] The present invention provides compositions and methods for screening metabolic and toxicological reactions of compounds.

DISCLOSURE OF THE INVENTION The present invention provides nucleic acid molecules of a sample whose transcription levels are modulated upon a metabolic reaction to a toxic compound. The invention also provides nucleic acid molecules of a sample in which the level of transcription is up-regulated upon a metabolic reaction to a toxic compound. The present invention also provides nucleic acid molecules in which the level of transcription of the sample is down-regulated upon a metabolic reaction to a toxic compound. Up-regulation or down-regulation is at least 2 times or more, preferably 2.5 times or more, and more preferably 3 times or more. The metabolic reaction to a toxic compound can be a toxicological reaction.

[0008] In another embodiment, the invention provides a method of screening a compound to determine the metabolic response to the compound or molecule under test. The method includes treating the tissue with a known toxic compound, determining the level of multiple nucleic acid molecules, and treating the tissue with a known toxic compound, as compared to a sample that has not been treated with the known toxic compound. Selecting from a plurality of nucleic acid molecules of the sample whose level has been modulated. One nucleic acid molecule is upregulated in transcription level by a toxic compound, another nucleic acid molecule is downregulated by a toxic compound, and
A nucleic acid molecule may be up-regulated with one known toxic compound and down-regulated with another known toxic compound. Selected nucleic acid molecules that are up-regulated and down-regulated by known toxic compounds are placed on a substrate. The method further includes determining the level of the nucleic acid molecule in the sample after being treated with the compound. The level of nucleic acid molecules in the sample thus treated is compared to a plurality of arrayed nucleic acid molecules disposed on a substrate to identify which sample nucleic acid molecules have been up- or down-regulated by the compound.

[0009] Preferably, the comparison includes contacting the sample nucleic acid molecule with the array nucleic acid molecule under conditions that effectively form a hybridization complex between the array nucleic acid molecule and the sample nucleic acid molecule. Determining the presence or absence of the complex. As used herein, similarity is defined as an increase of at least once over a longer period of time than a probe from a sample that has not been treated with a test compound or a known toxic compound forms a hybridization complex with an array nucleic acid molecule. At least one, preferably at least five, more preferably at least ten of the regulated array nucleic acid molecules form a hybridization complex with the sample nucleic acid molecule. Similarity can also be measured at least once in a shorter period of time than when a sample nucleic acid molecule of a sample not treated with a test compound or a known toxic compound forms a hybridization complex with a down-regulated array nucleic acid molecule. At least one of the regulated array nucleic acid molecules
At least three, preferably at least three, form a hybridization complex with the sample nucleic acid molecule.

[0010] Suitable tissues are selected from the group consisting of liver and kidney, brain, spleen, pancreas, lung. Suitable toxic compounds are selected from a group. This group includes antilipidemics, n-alkyl carboxylic acids, n-alkyl carboxylic acid precursors, azole antifungal compounds, leukotriene D4 antagonists, herbicides, insecticides, phthalates, phenyl acetate, dehydroepiandrates. Sterone sulfate, oleic acid, methanol and their corresponding metabolites, acetaminophen and its corresponding metabolite, benzopyrene, 3 methylcholanthrene, benzanthracene,
7,12-ditylbenzanthracene and their corresponding metabolites and the like. The array nucleic acid molecules have been treated with a known toxic compound and have been up-regulated or down-regulated at least 2 times, preferably 2.5 times or more, more preferably 3 times or more compared to the untreated sample. Contains fragments of the messenger RNA transcript of the sample gene. Suitable array nucleic acid molecules include those whose transcription levels are regulated or those whose transcription levels are down-regulated.
ID NO: 1-117 or a fragment thereof. More preferably, the up-regulated SEQ ID NOs: 3, 9, 10, 1
3, 19, 26, 31, 33, 35, 36, 37, 39, 42, 57, 67, 7
8, 81, 82, 94, and 98, and down-regulated SEQ ID
NO: 43, 49, 50, 52, 53, 54, 55, 56, 59, 61, 63
, 68, 71, 74, 85, 87, 90, 95, 102, 103, 105, and 115. Most preferred are those with SEQ ID NOs: 31, 33, 35, 36, 3
9, 52, 53, 54, 55, 63, 74, 81, 90, 94, and 95. In some embodiments, the target elements are hybridizable microarray array elements.

[0011] Alternatively, the invention provides a method of screening a test compound or molecule for a metabolic reaction, or a method of screening a sample for a metabolic response to a test compound or molecule.

[0012] Alternatively, the invention provides a method of screening a test compound or molecule for a previously unknown metabolic response, or a method of screening a sample for a metabolic response to a previously unknown test compound or molecule. A method for screening is provided.

In another embodiment, the present invention administers a nucleic acid molecule that is complementary to one or more selected up-regulated nucleic acid molecules, or a ribozyme that specifically cleaves such molecules. Providing a method for inhibiting toxicological reactions. Alternatively, a toxicological response can be prevented by administering a sense nucleotide molecule to one or more selected down-regulated nucleic acid molecules.

In another embodiment, the invention provides a method of blocking an toxic response by administering an agonist that initiates transcription of a gene comprising a down-regulated nucleic acid molecule of the invention. Alternatively, the toxicological response can be blocked by administering an antagonist that blocks the transcription of a gene comprising an up-regulated nucleic acid molecule of the invention.

[0015] The invention also provides a substantially purified mammalian protein or a portion thereof. The invention further provides an isolated and purified protein encoded by the nucleic acid molecule of SEQ ID NO: 1-117. Further, the invention provides a pharmaceutical composition comprising a substantially purified mammalian protein or a portion thereof, together with a pharmaceutical carrier.

[0016] The invention further provides a method of making an antibody using at least a portion of the protein encoded by SEQ ID NO: 1-117. The present invention also provides
Methods for screening a library of molecules using a protein or a portion thereof to identify at least one ligand that specifically binds to the protein are provided. This method involves binding the protein to a library molecule under conditions that allow specific binding, and detecting the specific binding to identify a ligand that specifically binds to the protein. included. Such libraries include DN
A and RNA molecules, peptides, agonists, antagonists, antibodies, immunoglobulins, drug compounds, pharmaceutical agents, and other ligands are included. In one embodiment, the ligand identified in this way modulates the activity of a mammalian protein. In a similar manner, the ligand is purified using the protein or a portion of the protein.
This method involves binding the protein or a part thereof to a sample under conditions that allow specific binding, detecting specific binding between the protein and a ligand, and recovering the bound protein. And a step of separating the ligand from the protein to obtain a purified ligand.

[0017] The invention further provides a method of introducing a marker gene into the genomic DNA of an animal to disrupt expression of a naturally occurring nucleic acid molecule. The present invention also provides a method for producing an animal model system using a nucleic acid molecule. The method includes the steps of producing a vector containing the nucleic acid molecule, introducing the vector into totipotent embryonic stem cells,
Selecting embryonic stem cells containing the vector transferred into the genomic DNA; microinjecting the selected cells into blastocysts to form chimeric blastocysts; Transferring the blastocyst, wherein the female gives birth to a chimeric animal comprising an additional copy of at least one nucleic acid molecule of its germline;
Mating the chimeric animal to create a homozygous animal model system.

Definition of Implementation Method of the Present Invention "Sample" as used herein is used in the broadest sense. Samples containing nucleic acid molecules include body fluids, cells or chromosomes, organelles, extracts from membranes isolated from cells, and genomic DNA and RNA, c in solution or bound to a substrate.
It includes DNA, cells, biological tissues such as biopsy needles or isolated fragments thereof, fingerprints and tissue prints, and natural or synthetic fibers, which are solutions or suspensions of solutions. , In gaseous suspensions, in aerosols and the like.

As used herein, “plurality” refers to a group of one or more members,
Preferably, it is a group consisting of at least about 10 or more, more preferably a group consisting of about 100 or more members, and most preferably a group consisting of 10,000 or more members.

As used herein, “substrate” refers to a solid or semi-solid support to which nucleic acid molecules or proteins bind. Among them are membranes and filters, chips, slides,
Includes wafers, fibers, magnetic or non-magnetic beads, gels, capillaries or other tubes, plates, polymers, microparticles, the surfaces of which form wells, grooves, pins, channels, perforations, and the like.

As used herein, “modulate” refers to altering the activity (biological or chemical, immunological) or life span of a molecule by binding specifically to a nucleic acid molecule or a molecule to a protein. .

As used herein, a “microarray” is a regularly arrayable hybridizable array element on a substrate. The array elements are arranged such that there are at least 10 or more, more preferably 100 or more, more preferably 1000 or more, and most preferably 10,000 or more array elements. Further, the hybridization signals from each array element can be individually identified. In a preferred embodiment, the array elements include nucleic acid molecules.

As used herein, a “nucleic acid molecule” is a nucleic acid, oligonucleotide, nucleotide, polynucleotide, or any fragment thereof. Also, they are of genomic or synthetic origin, are double-stranded or single-stranded, have specific activities such as sugars or lipids, proteins, or transformations, or form useful compositions such as peptide nucleic acids (PNA). DNA or RNA that binds to other substances is included. "Oligonucleotide" refers to amplimers and primers, oligomers, elements, targets,
Single-stranded, substantially equivalent to the probe, are preferred.

As used herein, “protein” is a naturally occurring or synthetic amino acid sequence or an oligopeptide, peptide, polypeptide or part thereof.

“Up-regulated” according to the present invention means that the level of nucleic acid molecules in the treated sample is increased compared to the nucleic acid molecules in the untreated sample.

As used herein, “down-regulated” means that the level of nucleic acid molecules in the treated sample has been reduced compared to the nucleic acid molecules in the untreated sample.

The “toxic compound” or “toxic drug” of the present invention refers to an individual or an animal, for example,
any compound or molecule or agent that elicits a biochemical and metabolic, physiological response such as i) DNA damage, ii) cell damage, iii) organ damage or cell death, iv) disease morbidity or mortality. That is.

The “toxicological response” of the present invention refers to a biochemical, metabolic, or biochemical response in an individual or animal or a test system exposed to a toxic compound or a toxic drug.

As used herein, a “fragment” is any part of an Incyte clone or nucleic acid molecule that retains usable functional characteristics. Useful fragments include oligonucleotides and polynucleotides useful for hybridization or propagation techniques or for controlling replication, transcription, and translation. Specific examples of fragments include SEQ ID NO: 1-11
There are 7 first consecutive 20 nucleotides.

As used herein, “hybridization complex” refers to a complex of two nucleic acid molecules by forming a hydrogen bond between a purine and a pyrimidine.

As used herein, “ligand” refers to any molecule, agent or compound that specifically binds to a complementary site on a nucleic acid molecule or protein. Such a ligand stabilizes or modulates the activity of the protein or nucleic acid molecule of the present invention. Further, the ligand may include at least one of an inorganic substance and an organic substance including nucleic acids and proteins, sugars, fats, and lipids.

As used herein, “substantially purified” is a nucleic acid molecule or protein that has been removed from its natural environment and contains at least 60% of other compounds that are isolated or separated and naturally associated. No, preferably no about 75%, most preferably about 90%
% Nucleic acid molecule or protein.

[0033] INVENTION The present invention, in order to examine the toxicological reactions, provides a method of screening for test compounds and molecules using compositions and compositions thereof. The invention further provides a method for characterizing the toxicological response of a sample to a test compound or molecule. In particular, the invention provides compositions comprising a plurality of nucleic acid molecules from a library. This library includes a human cDNA library for test system and monkey cDN.
A library, mouse cDNA library, normal rat liver cDNA library, normalized rat liver cDNA library, pre-hybridized rat liver cDNA library, rat kidney cDNA library. When rats are exposed to known hepatotoxins including peroxisome proliferator (PP), acetaminophen or its corresponding metabolite, and polycyclic aromatic hydrocarbons (PAH), it is down-regulated in rat liver Alternatively, the nucleic acid molecule is selected to show up-regulated gene expression.

PP includes clofibrate and fenofibrate, clofenica
cid, nafenopin, gemfibrozil, ciprofibra
te, bezafibrate, halofenate, cinfibrate, b
antilipidemic agents such as enzofibrate, etofibrate, WY-14,643, n-alkyl carboxylic acids such as trichloronucleic acid and palproic acid and hexanoic acid; n-alkyl carboxylic acid precursors such as trichlorethylene and trachloroethylene; and bifenazole Azole antifungal compounds, leukotriene D4 antagonists, herbicides, insecticides, di- [2-ethylhexyl] phthalic acid, mono- [2-ethylhexyl] phthala
Phthalates such as te and natural chemicals such as phenyl acetate and dehydroepiandrosterone sulfate, oleic acid, and methanol. In a preferred embodiment, the toxic compound is clofibrate or one of its corresponding metabolites. In another preferred embodiment, the toxic compound is fenofibrate or one of its corresponding metabolites.

PAHs include compounds such as benzopiene and 3-methylcholanthrene, benzanthracene, 7,12-dimethylbenzanthracene, and their corresponding metabolites. In a preferred embodiment, the toxic compound is benzopyrene or one of its corresponding metabolites.

[0036] SEQ ID NOs: 1-117 were identified by their pattern being at least 2-fold up- or down-regulated after hybridization with sample nucleic acid molecules from treated rat liver tissue. . These nucleic acid molecules and other nucleic acid molecules can be immobilized on a substrate as a microarray-type hybridizable array element. Characterize gene expression patterns associated with novel compounds to monitor the effects of treatment and elucidate any metabolic reactions in clinical treatments where metabolic reactions to toxic compounds are expected using microarrays It is possible to add.

When the nucleic acid molecule is used as a hybridizable array element of a microarray, the array element is arranged to be aligned at a specified position on the substrate. Since the array elements are at designated locations on the substrate, the hybridization pattern and its intensity (they create unique expression profiles for each other) are blocked for the expression levels of specific genes and the toxicology associated with the test compound or molecule. Response can be correlated.

The present invention further provides methods of screening test compounds and / or molecules for potential toxicological reactions, and screening samples for toxicological responses to specific test compounds or molecules. Provide a way. Briefly, these methods include treating a sample with a test compound or molecule to induce a change in a gene expression pattern that includes the expression of a plurality of sample nucleic acid molecules. When treated with a known toxic compound, it is at least twice, preferably 2.
Nucleic acid molecules are selected by identifying rat liver or kidney nucleic acid molecules that are expressed at up-regulated or down-regulated levels at least 5-fold, more preferably at least 3-fold. The nucleic acid molecules are arranged on a substrate. The array nucleic acid molecules and the sample nucleic acid molecules are then combined under conditions sufficient to form a hybridization complex detectable by methods well known in the art. Is the level of such a hybridization complex higher than that of a sample treated with a compound that does not elicit a toxicological response that correlates with a toxicological response to the test compound or molecule? Detect if low.

Complementary DNA Library Molecules that reflect most or all of the genes expressed in rat tissues have been identified.
The molecule is a normal rat cDNA library and a normalized rat cDN.
Clones from various types of rat cDNA libraries, including the A library, a prehybridized rat cDNA library, can be isolated and identified.
Clone inserts from these clones can be partially sequenced to create an expression sequence tab (EST).

In one example, two collections of ESTs were identified and sequenced. The first collection of ESTs (starting molecules) is derived from rat liver and kidney, and the cDNA libraries shown in the Examples section. The second collection of ESTs is derived from other rat cDNA libraries in the ZOOSEQ database (Incyte Pharmaceuticals, Inc., Palo Alto CA).

The two collections of the EST are electrically clustered to form the main cluster of the EST. The main cluster is formed by the identification of overlapping EST molecules and the construction of these ESTs. For this purpose, the Phrap tool (Phil Gr
een, University of Washington) and GELVIEW fragment construction system (GCG, Madison
WI) can be used. The minimum number of clones that can form a cluster is two. In another example, using a collection of human genes known to be expressed in response to toxic drugs, 113 rat cdD
Select a representative EST from the NA library. The process of main cluster formation is the repetition of these molecules.

After constructing the clustered consensus nucleic acid sequence, a representative 5 ′ clone is nominated from each main cluster. Most 5 'clones are preferred because they are likely to contain the complete gene. For a description of this nomination process, see USSN 09 / 034,807, "Relational Database," filed March 4, 1998.
and System for Storing Information Relating to Biomolecular Sequences an
d Reagents, "which is incorporated herein by reference in its entirety. This EST molecule is used as an array element of a microarray.

A selected sample of array nucleic acid molecules is treated with one or more known toxic compounds for a predetermined time. At this time, a subchronic dose is preferable. This drug is a peroxisome proliferator (PP)
), Acetaminophen or one of its corresponding metabolites, and polycyclic aromatic hydrocarbons (PAH) are preferred.

Comparing the gene expression pattern from the biological sample thus treated to the gene expression pattern from an untreated biological sample, the expression of the nucleic acid molecule is up-regulated by the response to the toxic compound Is identified or down-regulated. These molecules can then be used alone as array elements or with other array element molecules. Such microarrays are particularly useful in detecting and characterizing gene expression patterns associated with known toxic compounds. Such gene expression patterns can then be used in comparisons to identify other compounds that elicit a metabolic response to the toxic compound.

[0045] The array nucleic acid molecules can be manipulated to increase efficiency in hybridization. To optimize hybridization, the array nucleic acid molecules are examined by a computer program to identify portions of the gene that do not contain potential secondary structure. Such computer programs are known in the art and OLIGO4
.06 Primer analysis software (National Biosciences, Plymouth MN) or LA
Part of the SERGENE software (DNASTAR, Madison WI). These programs search within nucleic acid molecule sequences to identify stem-loop structures and tandem repeats,
Analyze the total G and C content of the sequence (molecules with a total G and C content greater than 60% are excluded). Alternatively, the array nucleic acid molecules can be optimized by trial and error. Under experimental conditions, experiments can be performed to determine whether an array nucleic acid molecule complementary to a sample nucleic acid molecule optimally hybridizes.

Array nucleic acid molecules include mRNA and cDNA, genomic DNA, peptide nucleic acids,
Any RNA-like or DNA-like peptide, such as branched DNA, can be used. The array nucleic acid molecules can be in either sense or antisense orientation.

In one embodiment, the array nucleic acid molecules are cDNA. Suitable DNA sequences preferably have a length of 50 to 10,000 nucleotides, more preferably 150 to 3,
It is 500 nucleotides long. In a second embodiment, the nucleic acid molecule is a vector DNA. In this case, the suitable length of the DNA sequence, ie, the length of the inserted sequence, varies, and is about 50 to 10,000 nucleotides, preferably 150 to 3,500 nucleotides.

The nucleic acid molecule sequences in the Sequence Listing have been generated in an automated fashion of the current state of the art, but sometimes contain sequence errors or undefined nucleotides. Nucleotide analogs can be incorporated into nucleic acid molecules by methods well known in the art. However,
The incorporated nucleotides themselves must form base pairs with the sample nucleic acid molecule. For example, certain guanine nucleotides can be replaced with hypoxanthine, which base pairs with cytosine residues. However, these base pairs are not as stable as guanine and cytosine base pairs. Alternatively, adenine nucleotides can form 2,6-diamin, which can form stronger base pairs than adenine and thymidine base pairs.
Can be replaced with opurine. In addition, nucleic acid molecules can include nucleotides that are chemically or enzymatically derivatized. Typical modifications include derivatization with an acyl, alkyl, aryl, or amino group.

[0049] Nucleic acid molecules can be immobilized on a substrate by chemical bonding. In addition, rather than attaching the molecule directly to the substrate, it can be attached to the substrate via a linker group. Usually, it is about 6 to 50 atoms long. The linker strand exposes the bound nucleic acid molecule. Suitable linker chains include ethylene glycol oligomers and diamines, diacids, and the like. One end of the linker reacts with a reactive group on the substrate surface to bind the linker to the substrate. The other end of the linker binds to the nucleic acid molecule. A suitable substrate is any suitable solid or semi-solid support. This support includes membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles and capillaries. The substrate surface can take a variety of forms, such as wells, grooves, pins, channels and perforations to which the array nucleic acid molecules bind.

The sample can be any body fluid (blood, urine, saliva, sputum, gastric juice, etc.) or any sample obtained from cultured cells, biological tissues, other specimen tissues, and sample nucleic acid molecules. . Samples can be taken from any species, but are preferably eukaryotes, more preferably mammals such as rats and humans.

[0051] DNA or RNA can be isolated from a sample by any of a variety of methods known in the art. For example, purification of nucleic acid molecules can be performed using Laboratory Techniques in Bio
chemistry and Molecular Biology: Hybridization With Nucleic Acid Probes,
Part I, Theory and Nucleic Acid Preparation , P. Tijssen, ed.Elsevier (
1993). In one preferred embodiment, total RNA is isolated using TRIZOL total RNA isolation reagent (Life Technologies, Gaithersburg MD) and mRNA is isolated using oligo d (T) column chromatography or glass beads. It is. If the sample nucleic acid molecule is amplified, it is preferred that the sample nucleic acid molecule be amplified to maintain the relative abundance of the original sample, including the low transcript abundance. RNA can be amplified in vitro, in situ , or in vivo (Eberwine US Patent No. 5
, 514,545).

It is also preferable to include in-sample adjustments that allow amplification and labeling without altering the actual distribution of the nucleic acid molecules in the sample. To this end, the sample is spiked with a fixed amount of a regulatory nucleic acid molecule that is detectable upon hybridization with a complementary array nucleic acid molecule, and the composition of nucleic acid molecules specifically hybridizes to the array regulatory nucleic acid molecule. Reference nucleic acid molecule. After hybridization and processing, the resulting hybridization signal should accurately reflect the amount of array regulatory nucleic acid molecules added to the sample.

Prior to hybridization, it may be desirable to fragment the sample nucleic acid molecule. This fragmentation minimizes cross-hybridization and secondary structure with other sample nucleic acid molecules or non-complementary nucleic acid molecules in the sample and improves hybridization. This fragmentation can be performed by a mechanical or chemical method.

The sample nucleic acid molecule can be labeled with one or more labeling components so that a complex of the labeled hybridized array nucleic acid molecule and the sample nucleic acid molecule can be detected. The labeling component can include a composition that can be detected by spectroscopic or photochemical, biochemical, bioelectronic, immunochemical, electrical, optical, or chemical methods. The labeling component includes a radioisotope such as ³² P or ³³ P, ³⁵ S,
Includes chemical fermentation compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, magnetic labels, binding enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors. Suitable fluorescent markers include Cy3
And Cy5 fluorophore (Amersham Pharmacia Biotech, Piscataw)
ay NJ).

Hybridization The nucleic acid molecule sequences of SEQ ID NOs: 1-117 and fragments thereof can be used in testing for various purposes and for various hybridization techniques. Hybridization probes can be derived or designed from SEQ ID NO: 1-117. Such probes can be made from highly specific regions, such as the 5 'regulatory region, or conserved motifs, and can be used in protocols to identify naturally occurring sequences that encode mammalian proteins or allelic variants and related sequences. It must be used and preferably has at least 50% sequence identity with any protein sequence. The hybridization probe of the present invention can be DNA or RNA, and has SEQ ID NO:
1-117 or a genomic sequence containing promoters, enhancers and introns of mammalian genes. Hybridization or PCR probes can be made by oligolabeling or nick translation, end labeling, PCR amplification in the presence of labeled nucleotides.
Vectors containing nucleic acid sequences can be used for the production of mRNA probes in vitro , with the addition of RNA polymerase and labeled nucleic acid molecules. These methods can be performed using kits such as those sold by Amersham Pharmacia Biotech.

The stringency of hybridization is determined by the total content of G and C of the probe, salt concentration, and temperature. In particular, stringency can be increased by lowering the salt concentration or raising the hybridization temperature. By adding an organic solvent such as formamide to a region used for some membrane-based hybridizations, a reaction at a low temperature becomes possible. For example, a buffer such as 5 × SSC at 60 ° C. containing 1% sodium dodecyl sulfate (SDS) enables low-stringency hybridization, and forms a hybridization complex between nucleotide sequences containing mismatches. Become. Subsequent washing was performed with 0.1% SD
0.2xSS at 45 ° C (medium stringency) or 68 ° C (high stringency) containing S
A higher stringency can be achieved with a buffer such as C. With high stringency, the hybridization complex remains stable only if the nucleic acid sequences are perfectly complementary. For some membrane-based hybridizations, the hybridization solution is preferably added to the hybridization solution to reduce the temperature at which hybridization is possible.
%, Most preferably 50% formamide can be added.
Background signals can be reduced using detergents such as yl or Triton X-100 and blocking agents such as salmon sperm DNA. The selection of hybridization components and conditions is well known to those skilled in the art and is described in Ausubel (supra) and Sambrook et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,
Plainview NY). The specificity of the hybridization can be assessed by comparing the hybridization of the specificity-regulating nucleic acid molecule to a specificity-regulating sample nucleic acid molecule added to a defined amount of sample. An array specificity regulating nucleic acid molecule can have one or more sequence mismatches when compared to the corresponding array nucleic acid molecule. In this method, it can be determined whether the sequenced complementary nucleic acid molecule has hybridized to the sample nucleic acid molecule or whether a hybrid duplex having a mismatched portion has been formed.

The hybridization reaction can be performed in an absolute hybridization type or a differential hybridization type. In absolute hybridization, nucleic acid molecules in a sample hybridize to molecules in a microarray, and the signal detected after the formation of the hybridization complex correlates to the level of nucleic acid molecules in the sample. In a differential hybridization format, two biological samples are analyzed for differential expression of a group of genes. For differential hybridization, both biological samples prepare nucleic acid molecules and label them with different labeling components. The two labeled nucleic acid molecules are mixed and added to the microarray. The microarray is then tested under conditions where luminescence from two different labels can be individually detected. The molecules of the microarray that hybridize substantially the same number of nucleic acid molecules from both biological samples emit a unique, bound fluorescence (Shalon et al., PCT publicatio
n WO95 / 35505). In a preferred embodiment, Cy3 with distinct excitation and emission ranges
And fluorescent markers such as Cy5 fluorophores are labels.

After hybridization, the microarray is washed to remove unhybridized nucleic acid molecules, and a complex formed between the hybridizable array element and the nucleic acid molecule is detected. Methods for detecting formed complexes are well known in the art. In a preferred embodiment, the nucleic acid molecules are labeled with a fluorescent label, and the measurement of the fluorescence pattern and its level indicating the complex formed is performed with a fluorescence microscope, preferably a confocal fluorescence microscope.

In a differential hybridization experiment, nucleic acid molecules from two or more different biological samples are labeled with two or more different fluorescent labels having different excitation and emission wavelengths. The labeled sample is excited at a specific excitation wavelength. The fluorescent signals are each detected with a different photomultiplier tube set to detect a particular emission wavelength. The relative abundance / expression level of two or more sample nucleic acid molecules is obtained.

In general, when one or more microarrays are used under similar test conditions, the emission intensity of a microarray can be normalized in consideration of the variation in hybridization intensity. In a preferred embodiment, the hybridization intensity of each array sample nucleic acid molecule complex is normalized by the intensity from the internal normalization control of each microarray.

The labeled sample emits a particular wavelength and is detected with a plurality of photomultipliers. The relative abundance / expression level of the array nucleic acid molecules can be used as a hybridizable element of a microarray. Using such a microarray, it is possible to identify expression profiles associated with a particular toxicological response. Next, a particular subset of these photomultipliers are set to detect a particular wavelength. The relative expression level of an array nucleic acid molecule can be identified as the expression of the array nucleic acid molecule is modulated in response to a particular toxic agent. These photomultiplier tubes are set so that a specific wavelength can be detected.
The relative expression level of a nucleic acid molecule can be used in identifying other compounds that have a similar toxicological response.

Alternatively, the therapy is fine-tuned using the microarray and expression patterns from the microarray for certain therapies with known side effects. Dosages are determined to minimize the pattern of expression associated with undesired side effects. This method can be more acute and more sensitive than waiting for the patient to show toxic side effects before changing therapy.

In general, methods for screening a library of test compounds or molecules to identify those with a toxicological response result in modulated expression levels in tissues treated with a known toxic compound compared to untreated tissues. Selecting the plurality of arrayed nucleic acid molecules. The sample is then treated with a test compound or molecule to induce a gene expression pattern that includes the expression of the plurality of nucleic acid molecules. Test compounds are screened at several doses to determine which doses are likely and which are not.

Next, the compound that induces the expression level of a sample nucleic acid molecule similar to the array nucleic acid molecule is identified by comparing the expression level of the array nucleic acid molecule with the expression level of the sample nucleic acid molecule. In a preferred embodiment, gene expression levels are compared by contacting the array nucleic acid molecule with the sample nucleic acid molecule under conditions that effectively form a hybridization complex between the array nucleic acid molecule and the sample nucleic acid molecule. , To determine the presence or absence of the hybridization complex.

Similarity is defined as an array nucleic acid molecule that has been up-regulated at least once for a longer time than a nucleic acid molecule of a sample that has not been treated with the test compound forms a hybridization complex with the array nucleic acid molecule. At least one, preferably at least five, more preferably at least ten form a hybridization complex with the sample nucleic acid molecule. Similarity can also be attributed to the at least one down-regulated array nucleic acid in a shorter time than the nucleic acid molecules of the sample not treated with the test compound form a hybridization complex with the down-regulated array nucleic acid molecules. At least one, preferably three or more of the molecules form a hybridization complex with the sample nucleic acid molecule.

Such similarity in expression patterns is that the toxicological response is associated with the test compound or the molecule being tested. Toxic compounds belonging to the class of peroxisome proliferators (PP) include clofibrate and fenofibrate, clo
fenic acid, nafenopin, gemfibrozil, chip
rofibrate, bezafibrate, halofenate, symfibrate, benzofibrate, etofibrate, WY-14
An antilipidemic agent such as 643, trichloronucleic acid and n-alkyl carboxylic acids such as palproic acid and hexanoic acid, and trichlorethylene and trachloroethyl
an n-alkyl carboxylic acid precursor such as ne, an azole antifungal compound such as bifenazole, a leukotriene D4 antagonist, a herbicide, an insecticide,
Di- [2-ethylhexyl] phthalic acid, mono- [2-ethylhexyl] p
phthalates such as phthalate and natural chemicals such as phenyl acetate and dehydroepiandrosterone sulfate, oleic acid, and methanol. In another embodiment, the toxic compound is acetaminophen or a corresponding metabolite thereof. In another embodiment, the toxic compound is a polycyclic aromatic hydrocarbon (PA
H), which includes compounds such as benzopiene and 3-methylcholanthrene, benzanthracene, 7,12-dimethylbenzanthracene, and their corresponding metabolites. Useful for studying the metabolic reactions of these compounds in the liver and kidney, brain, treasured, pancreas, and lung.

Altering Gene Expression Using Nucleic Acids Gene expression can be altered by designing complementary or antisense molecules to the 5 'or 3' regulatory regions of mammalian genes, or other regulatory regions. It is. Oligonucleotides designed for the transcription start site are preferred. Similarly, gene expression can be blocked by triple helix base pairing, which blocks the binding of polymerases or transcription factors, regulatory molecules (Gee et al. In: Huber and Carr (1994) Molecul ) .
ar and Immunologic Approaches , Futura Publishing, Mt.Kisco NY, pp. 163-
177). It is also possible to design a complementary molecule so as to block translation by preventing the binding of ribosome to mRNA. In one alternative, a library of nucleic acid molecules or fragments thereof can be screened to identify those that specifically bind to untranslated regulatory sequences.

It is possible to catalyze the specific cleavage of RNA using ribozymes, which are RNA molecules with enzymatic activity. The mechanism of ribozyme action is based on sequence-specific hybridization of the ribozyme molecule to a complementary target RNA followed by GUA.
And cleavage of nucleotide chains at sites such as GUU and GUC. Once such sites have been identified, oligonucleotides having the same sequence can be evaluated for secondary structural properties that render the oligonucleotide dysfunctional. In addition, RNase protection assays can be used to test hybridization with complementary oligonucleotides to assess the suitability of candidate targets.

The complementary nucleic acids and ribozymes of the invention can be produced by in vitro or in vivo recombinant expression using solid phase phosphoramidite chemical synthesis. In addition, RNA molecules can be stabilized in cells using phosphorothionate or 2'O-methyl rather than lateral sequences at the 5 'and / or 3' end of the molecule, or phosphodiesterase linkages in the backbone of the molecule. Can be modified to increase gender and half-life. This modification is unique to the production of PNAs, but can be extended to other nucleic acid molecules. For example, inosine, queosine, wybutosine and other non-traditional bases, or acetyl, methyl, uridine having a thio group and modification of adenine, cytidine, guanine, thymine, the effectiveness as a substrate for endogenous endonuclease. Lower.

Screening Assays Nucleic acid molecules encoding mammalian proteins can be used to screen libraries of molecules for specific binding affinities. This library can also be DNA and RNA molecules, PNAs, peptides, ligands and transcription factors that regulate translation or activity, replication and transcription of biological nucleic acid molecules, proteins such as enhancers, repressors, and the like. This assay involves binding a mammalian nucleic acid molecule or fragment thereof to a library of molecules under conditions that permit specific binding, and specific binding to identify at least one molecule that specifically binds to the nucleic acid molecule. The step of detecting

Similarly, mammalian proteins, or portions thereof, can be used to screen libraries of molecules in any of a variety of screening assays.
Some of the proteins used in such screening may be free in solution, immobilized on an abiotic or biological substrate, or present at an intracellular site. It is possible to measure the specific binding between a protein and a molecule. Depending on the type of library to be screened, DNA, RNA, DNA molecules, agonists, antagonists, antibodies,
Identify immunoglobulins, inhibitors, peptides, proteins, drugs, and any other ligands. One high-throughput screening method using very low assay volumes and very low amounts of test compound is disclosed in USPN 5,876,94.
No. 6 and incorporated herein by reference. The method also screens a large number of molecules for enzymatic inhibition or receptor binding.

Production of Ligands The nucleic acids or fragments thereof can be used to purify ligands from samples. A method for purifying a ligand using a mammalian nucleic acid molecule or a fragment thereof includes a step of binding the nucleic acid molecule or a fragment thereof to a sample under conditions capable of specific binding,
The steps may include detecting specific binding, recovering the bound protein, and separating the nucleic acid molecule from the purified ligand using a suitable agent.

Similarly, it is possible to purify a ligand from a sample using a protein or a portion thereof. The method of purifying a ligand using a mammalian protein or a part thereof includes a process of binding a sample to a protein or a part thereof under conditions capable of specific binding, and a method of specifically binding a protein to a ligand. , Recovering the bound protein, and separating the protein from the purified ligand using a suitable chaotropic agent.

Pharmacopharmaceutical compositions are substances which contain an effective amount of the active ingredient to achieve the desired intended purpose. Determining the effective dosage depends in large part on the capabilities of those skilled in the art. For any given compound, the therapeutically effective dose is initially estimated either by cell culture assays or animal models. In addition, the route of administration and suitable concentration range are determined using animal models. Such information is then used to determine the effective route of administration and dosage for humans.

[0075] A therapeutically effective dose is that amount of the protein or inhibitor that ameliorates the symptoms or condition. Medicinal efficacy and toxicity can be measured, for example, by cell culture or experiment, such as ED50 (50% of the population has a medicinal effect on taking) and LD50 (50% of the population is lethal on taking). It can be determined by standard drug procedures in animals. The ratio between toxic and therapeutic effects is the therapeutic index and the LD
50 / ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. The data obtained from the cell culture assays and animal studies is used in formulating a range of dosage for human application.

Model System The animal model can be used as a bioassay in which the animal model exhibits a toxic reaction similar to humans under exposure conditions corresponding to humans. Mammals are the most common model, and most toxicology studies are primarily performed on rodents such as rats or mice because of their low cost and availability of reference toxins. Syngeneic rodents are a convenient model for investigating the physiological causes of over- or under-expression of a gene of interest, and are also useful in developing methods for diagnosing and treating diseases. Because of overexpression of a particular gene, syngeneic animals that secrete the protein into milk can be a convenient source of the protein expressed by the gene.

Toxicology Toxology is a test compound or molecule in a biological system for identifying drug side effects.
A study that studies the effects of poisons. Most toxicology studies use rats or mice to estimate whether adverse effects of the drug will occur in humans. Observe physiological and qualitative and quantitative changes and use behavioral, homeostatic, developmental, reproductive, and mortality profiles to create a safety or toxic response profile that can be used after exposure to the drug. Assess human health.

Genetic toxicology identifies and analyzes the ability of an agent to cause damage at the cellular or subcellular level. Typically, such genotoxic agents have a common chemical or physical toxicity that promotes interaction with the nucleic acid and are often harmful if the mutated chromosome is passed on to progeny. Toxicological studies identify drugs that increase the frequency of structural or functional abnormalities in offspring when administered to either pre-conceptional parents or pregnant mothers, or any developing organism I do. Mice and rats are often used in these tests because the reproductive cycle of mice and rats is short and yields as many organisms as necessary to meet statistical needs.

[0079] Acute toxicology testing determines the symptoms or mortality from the drug in a single dose of the drug. These experiments include 1) an initial dose range determination experiment, 2) an experiment that defines the range of effective doses, and 3) a final experiment to determine a dose response curve.

A long-term toxin test is performed by repeatedly administering a drug. Such studies generally use rats and dogs to collect data from different taxonomic species. Except for carcinogens, there are many instances where intensive and routine administration of drugs at high doses for three to four months has revealed most forms of toxicity in adult animals. Long-term toxicity tests, of one year or more, are used to demonstrate the absence of drug toxicity or the potential for carcinogens. If the study is performed in rats, at least one test group of animals and one control group of animals are used.
Animals are quarantined, health checked, and monitored throughout the experiment.

Genetically Modified Animal Models Genetically modified rodents which over- or under-express a gene of interest are inbred and used as human disease models, or compounds for examining therapeutic or toxicological side effects. And molecular testing (USPN 4,736,866; USPN 5,175,3
83; and USPN 5,767,337, which are incorporated herein by reference). In some cases, the introduced gene may be activated at a particular developmental stage or after birth, in a particular tissue type, for a particular period of time. Expression of the recombinant gene
Monitored by analysis of phenotypic or tissue-specific mRNA expression in transgenic animals before and during drug treatment experiments.

Embryonic stem cells (ES) isolated from rodent embryos maintain the potential to form embryos. When ES cells are introduced into the carrier fetus, they resume normal development and contribute to all tissues of the live born animal. ES cells are suitable for use in certain experimental knock-out and knock-in rodent systems. Mouse 1
Mouse ES cells, such as the 29 / SvJ cell line, are obtained from early mouse fetuses and grown under culture conditions well known in the art. Knockout vectors include disease gene candidates that have been modified to include marker genes that disrupt transcription and / or translation in vivo . The vector to be introduced into the ES cell is obtained by a transformation method such as electroporation and ribosome transport and microinjection which are well known in the art. The rodent endogenous gene is replaced by a disease gene that has been disrupted by recombination during cell differentiation and homologous recombination. Preferably, the transformed ES cells are selected under predetermined conditions and microinjected into mouse cell blastocysts such as the C57 / BL / 6 mouse line. The blastocyst is surgically transferred to a pseudopregnant female and the genotype is passed on to offspring chimeras, creating a heterozygous or homozygous line.

In addition, ES cells are used to study differences in various cell types and tissues in vitro , such as nerve cells, hematopoietic lineages, and fungal cells (Bain et al. (1995) Dev. Biol. 168: 34).
2-357; Wiles and Keller (1991) Development 111: 259-267; and Klug et al. (199
6) J. Clin. Invest. 98: 216-224). Recent studies have shown that human blastocyst-derived ES
Cells can also be manipulated in vitro to differentiate into eight distinct cell lineages, including endoderm and mesoderm, ectodermal cell types (Thomson (1998) Science 282: 1145-1147).
.

Knockout Analysis In gene knockout analysis, regions of human disease gene candidates are enzymatically modified to include non-mammalian genes such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244: 1288-1292). . The inserted coding sequence disrupts the transcription and translation of the target gene, preventing physiological synthesis of the disease candidate protein. The altered gene is transformed into cultured embryonic stem cells (described above), the transformed cells are injected into rodent germ spores, and the germ spores are introduced into pseudopregnant females. The transgenic progeny are cross-bred to create homozygous inbred lines.

Knock-in analysis It is possible to create knock-in humanized animals (pigs) or transgenic animal models of human disease (mouse or rat) using totipotent ES cells that appear early in fetal development. . In the knock-in technique, a region of the human gene is injected into animal ES cells, and the human sequence is transferred into the animal cell genome by genetic recombination. Totipotent ES cells containing the transferred human gene are manipulated as described above. The syngeneic animal is treated and studied to gather information similar to human conditions. These methods are used to model some human diseases (eg, Lee et al. (1998) Proc. Natl. Acad. Sci. 95: 11371-11376; Baudoin
Et al. (1998) Genes Dev. 12: 1202-1216; and Zhuang et al. (1998) Mol. Cell Biol.
18: 3340-3349).

Non-Human Primate Models This field of animal experiments deals with basic chemistry methodologies and data such as physiology and genetics, chemistry, pharmacology, statistics, etc. These data are important in assessing the effects of a non-human primate test compound or molecule. Because they are relevant to human health. Monkeys were used as surrogates for humans in the evaluation of vaccines and drugs, and their responses corresponded to human exposure under similar conditions. Cynomolgus and rhesus monkeys (Macaca fascicularis and Macaca mulatta, respectively) and common marmosets (Callithrix jacchus) are the most common non-human primates (NHPs) used in these studies. Initial and toxicological studies are usually performed in rodent models because of the high cost of building and maintaining NHPs. In studies examining behavior such as drug addiction, NHP is the best experimental animal.
In addition, NHPs and individual humans show different sensitivities to many drugs and toxins, and can be classified as high metabolizers and poor metabolizers for these drugs.

In a further embodiment, where the new technology relies on currently known properties of the nucleic acid molecule, including but not limited to the triplet genetic code and properties such as specific base-forming interactions, Nucleic acid molecules encoding mammalian proteins may be used in any molecular biology technique still under development.

[0088]

【Example】

It is to be understood that this invention is not limited to particular methodology, protocols, and reagents described herein, which may vary. Further, the terminology used in the present specification is used for the purpose of describing specific examples only, and is intended to limit the scope of the present invention which is limited only by the description of the claims. It should be understood that it is not. The following examples have been presented as the best description of the invention and its representative configurations.

[0089]1. Preparation of cDNA library RALINOT01 cDNA library was transferred to 50 10- to 11-week SD strain Mesra
(Pharmacon, Waverly PA) from liver tissue collected from a pool
. Animals are housed in standard laboratory cages and awarded PMI-approved Rodent Diet # 5002
I got it. The animals were in good health at the time of tissue collection. Animals are CO ₂ After anesthesia by inhalation, cardiac puncture was performed.

[0091] Frozen tissue was prepared using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instrume
nts, Westbury, NY) using TRIZOL reagent (1 g tissue / 10 ml TRIZOL; L
homogenized and lysed in a solution of monoplastic phenol and guanidine isothiocyanate. After brief incubation with ice cooling, chloroform (1: 5 v / v) was mixed with the reagent and centrifuged at 1000 rpm. The upper liquid phase was removed and transferred to a new tube, the RNA was precipitated by adding isopropanol, resuspended in DEPC-treated water and treated with DNase I at 37 ° C. for 25 minutes. RNA was re-extracted once with phenol chloroform pH 4.7 and precipitated with 0.3 M sodium acetate and 2.5 volumes of ethanol. Next, the mRNA was transferred to the OLIGOTEX kit (QIAGEN, Inc., Ch.
atsworth, CA) and used to generate a cDNA library.

This mRNA was handled according to the recommended protocol of the SUPERSCRIPT Plasmid System (Life Technologies). The cDNA was transferred to a SEPHAROSE CL4B column (Amersham P
Harmacia Biotech), and cDNA having a size exceeding 400 bp was
Ligation to the INCY1 plasmid (Incyte Pharmaceuticals). The recombinant plasmid is then transferred to DH5α or DH10B competent cells (Life Technologies
).

The RAKINOT01 library was prepared using mRNA isolated from kidney cells collected from a pool of 50 7-8 week old male SD rats as described above.

[0093] The RAKINOT02 library contains 50 female SD-11 weeks as described above.
It was prepared using mRNA isolated from kidney tissue collected from a pool of strain rats.

2 Normalization of cDNA Library In some cases, Soares et al. (1994, Proc. NatI. Acad. Sci. 91: 9228-9
232) Normalize the cDNA library once (normalize)
did. However, the following changes were made in the procedure of Soares. The ratio of primer to template in the primer extension reaction was increased from 2: 1 to 10: 1. By reducing the concentration of each dNTP in the reaction to 150 μm, longer (400-
It enabled the generation of a 1000 nucleotide (nt) primer extension. Rear annealing hybridization was extended to 13-19 hours. The single-stranded circular DNA of the normalized library is purified by hydroxyapatite chromatography, partially converted to double-stranded by random priming, and electroporated to DH10B competent bacteria (Life Technologies).
).

The Soares normalization procedure is designed to reduce variability in the frequency of initial cDNA occurrences and to achieve abundance within an order of magnitude while maintaining the complexity of the entire sequence of the library. Have been. In the normalization process, the abundance of high abundance cDNA clones is significantly reduced, medium abundance clones are relatively unaffected, and rare transcript clone abundance is increased. In this modified Soares normalization procedure, gene discovery is performed by using very long hybridization times to bias the normalized library so that low-abundance cDNAs are well represented in standard transcript images. Increasing rates.

Normalized rat liver cd of RALINON03, RALINON04, and RALINON07
The NA library was 2.0 × 1 each from the RALINOT01 cDNA library.
0 ⁶ was produced as having ^{4.6 × 10 5, 2.0 × 10} 6 independent clones. The RALINOT01 library was normalized once under conditions adapted to that of Soares (supra) except that very long (48 hours) reannealing hybridization was used.

[0097]3 Prehybridization of cDNA library RALINOH01 cDNA library is a clone from RALINOT01 library.
Prepared. After preparing the RALINOT01 cDNA library,
Spot on a nylon filter, dissolve, and bind plasmid DNA to the filter.
I combined. Incubate the filter with the preheated hybridization buffer.
And then 0.75M NaCl, 0.1M Na₂HPO₄/ NaH ₂ PO₄0.15M tris-HCl (pH 7.5), 5x Denhardt's solution
2% SDS, 100 μg / ml sheared salmon sperm DNA, 50%
Formamide and reverse transcripts of untreated rat liver mRNA [³² P] Among labeled oligonucleotide molecules, at 42 ° C. for 14-16 hours
Hybridized. Filter the filter at ambient temperature for 5 minutes in 2 × SSC (citric acid
Rinsing with sodium salt) followed by a preheated wash solution (2 × SSC, 1% SDS)
) At 68 ° C. for 30 minutes. Using a new washing solution at 68 ° C for 30 minutes
The washing process was repeated. The filter was then preheated at 68 ° C. for 30 minutes (0.
6 × SSC, 1% SDS). Approximately 4224 clones
It has a low hybridization signal and about 20% of the clones
No, two groups were isolated and sequenced.

4 Isolation and Sequencing of cDNA Clones DNA was isolated using the following protocol. One bacterial clone using a toothpick in a 384-well plate (Genetix Ltd, Christchurch, United Kingdom)
Was transferred to each well. Wells were sterilized with 25 mg / l carbonicillin and 0.4% glycerol (v / v) in sterile Terrific Broth (Life Technolog).
ies) was included. Cover the plate and incubate at 37 ° C (The
rmodyne, Newtown Square PA) for 8-10 hours. Plasmid DNA is released from the cells and is subjected to direct link PCR (Rao, VB (1994) Ana
l. Biochem. 216: 1-14). The direct link PCR solution is 3
0 ml of NUCLEIX PLUS PCR nucleotide mix (Amersham Pharmacia Biosech
, Piscataway NJ) and 300 μl of Taq DNA polymerase (Amersham Phar
macia Biotech). 5 μl of the PCR solution was added to each of 384 wells using a MICROLAB 2200 system (Hamilton, Reno NV). Plates were centrifuged at 1000 rpm for 20 seconds and refrigerated until use. 38
Bacterial cells were transferred from the incubation plate to the plate containing the PCR solution using a 4-pin pin tool (V & P Scientific mc, San Diego CA). In the PCR solution, 0.1% Tween 20 lyses the cells and releases the plasmid DNA. After lysis, the plates are centrifuged at up to 500 rpm, covered with a cycle sealer and programmed dPCR
384-well DNA ENGINE thermal cycler (MJ Resea
rch, Watertown MA), the following parameters: Step (1) 95 ° C
Step (2) 94 ° C. for 30 seconds; Step (3) 55 ° C. for 30 seconds; Step (4) 72 ° C. for 2 minutes; Step (5) Steps 2, 3, and 4
Repeat 9 times; step (6) at 72 ° C. for 10 seconds; and step (7) store at 4 ° C.,
It was subjected to a thermal cycle with the following parameters.

The determination of the concentration of DNA in each well was performed by adding 100 μl of P dissolved in 1 × TE.
ICO GREEN quantitative reagent (0.25% (v / v), Molecular Probes, Euge
ne OR) and 0.5 μl of the undiluted PCR product were transferred to an opaque fluorometric plate (Comin
g Costar, Acton MA) was dispensed into each well to allow DNA to bind to the quantification reagent. Plates are loaded with Fluoroscan II (Labsystems Oy, Hels
inki, Finland), and the fluorescence of the sample was measured to quantify the DNA concentration. Typical concentrations for each DNA sample ranged from 100 to 500 ng / ml.

HYDRA microdispenser (Robbins Scientific, Sunnyvale CA) or MICRO
Use one of the LAB 2200 systems (Hamilton) with DNA ENGINE thermal cycler (MJ Res
cDNA was prepared for sequencing by using in combination with earch). The cDNA was obtained from Sanger, F. and AR. Coulson (J. Mol. Biol. (1975) 94: 441-448.
) And the ABI 377 sequencing system (PE Biosystems). Most isolates are prepared using standard ABI kits (PE Biosystems) using standard AB
Sequenced according to the I protocol. Solutions were used at concentrations of 0.25x to 1.0x. Typically, 500-700 base pairs were sequenced in 3.5-4 hours. Alternatively, cDNA may be sequenced using solutions and dyes from Amersham Pharmacia Biotech.

5 Selection of Rat Liver and Kidney Genes As a first step, the originator molecules from the high-throughput sequencing experiments were RALINOT01, RAKINOT01, RAKINOT02, RALINOH01, R
It was derived from clone inserts from ALINON03, RALINON04, and RALINON07. A cDNA library clone was obtained. There were 18140 rat liver molecules and 5779 rat kidney molecules.

Furthermore, 1500 rat molecules derived from any of the clone inserts of 113 rat cDNA libraries were used to convert peroxisomal-related genes, lysosomal-related genes, apoptosis-related genes, P450 thyrochrome, sulfotransferase, glutathione S-transferase and cysteine. Selections were based on their homology to genes encoding polypeptides involved in toxicological reactions, including detoxification genes such as proteases.

Next, all remaining molecules from all rat cDNA library clones were clustered based on the source molecules described above. In the clustering process, BLAST was used to identify overlapping molecules with a quality of match as indicated by the product score 50. 6581 master clusters were identified.

After forming a clone cluster, Phrap (Phil Green, University of Washi
(ngton) was used to derive a consensus sequence based on the composition of the cloned molecule. Next, the assembled molecules are annotated by screening the assembled molecules against GenBank using BLASTn and then screening the assembled molecules against GenPept using FASTX. Was. About two thirds of the assembled molecules were annotated, and about one third were not.

6 Preparation of Substrate and Array Element / Probe Array elements were purified using the clones specified in the process described in Example 5. Each array element was amplified from bacterial cells. In the amplification by the PCR method, primers complementary to the sequence of the vector adjacent to the cDNA insert were used. The array elements were amplified by 30 cycles of PCR,
Amplification was performed from 2 ng to a final amount of 5 μg or more. The amplified array elements were then purified using SEPHACRYL-400 (Amershans Pharmacia Biotech).

The purified array elements were immobilized on polymer-coated glass slides. Microscope slide glass (Coming, Corning NY) is 0.1% SDS
And ultrasonically in acetone and with and after a large amount of distilled water during and after treatment. Slide glass with 4% hydrofluoric acid (VWR, West Chester
Etch in PA), rinse thoroughly with distilled water, and dry in 95% ethanol.
Coated with 05% aminopropyl salt (Sigma-Aldrich, St. Louis MO). The coated slides were stored in a 110 ° C. oven.

The array elements were deposited on a coated glass substrate using the procedure described in US Pat. No. 5,807,522. The US patent is hereby incorporated by reference. Briefly, 1 μl of array element DNA at an average concentration of 0.5 μg / ml in 3 × SSC was loaded onto an open capillary printing element by a high speed robotic device. The device was then immersed in approximately 5 ml of the array element sample per slide. Array elements representing a total of 7404 rat liver and kidney genes and various regulatory elements (
Fourteen synthetic control molecules, including human genomic DNA and yeast genomic DNA) were arranged in homologous quadrants within a 1.8 cm ² area of the glass substrate.

Microarrays were UV-coated using STRATALINKER UV-crosslinker (Stratagene)
Crosslinked. The microarray was washed once with 0.2% SDS at room temperature and three times with distilled water. Blocking of non-specific binding sites was performed by microarraying at 60 ° C. in 0.2% casein in phosphate buffered saline (PBS) (Tropix Inc., Bedford MA).
For 30 minutes and then washed as before with 0.2% SDS and distilled water.

7 Preparation of Target A male SD rat (6 to 8 weeks) was given 250 m / kg body weight (bw).
g of clofibrate (CLO; Acros, Geel, Belgium), 10 per kg bw
00 mg of acetaminophen (APAP; Acros), 10 mg of benzoprene / kg of bw (B (a) P; Acros), or less than 2 ml of dimethyl sulfoxide carrier (DMSO; Acros) per kg of bw were injected intraperitoneally, after which the animals were injected. Euthanized by CO ₂ anesthesia. Animals were monitored daily for physical condition and weight. Three animals per group were sacrificed on days 1, 3, 7, 14, and 28 after a single injection for approximately 12 hours. Prior to sacrifice, a blood sample from each animal was removed and serum alanine transferase (ALT) and aspartate aminotransferase (AST) were used using a diagnostic kit (Sigma-Aldrich).
A) assayed for concentration; At necropsy, grossly observed lesions and liver weights were recorded. Liver, kidney, brain, spleen, and pancreas from each rat were harvested, flash frozen in liquid nitrogen, and stored at -80C.

For the preparation of each probe, a modified TRIZOL reagent (Li
Liver livers were homogenized and lysed in fe Technologies, Gaithersburg MD). Messenger RNA was isolated using the OLIGOTEX kit (QIAGEN) and
The cells were labeled with Cy3 or Cy5 labeled primers (Operon Technologies, Alameda CA) using an MBRIGHT labeling kit (Incyte Pharmaceuticals). Messenger RNA isolated from tissues of rats treated with clofibrate, acetaminophen, or benzopyrene was labeled with Cy5, and mRNA isolated from tissues of rats treated with DMSO was labeled with Cy3. CDNAs controlling different expression patterns for quantification were added to each labeling reaction. 85 labeled cDNA
The RNA was degraded by treatment with 0.5 M sodium bicarbonate (pH 9.2) at 20 ° C. for 20 minutes and two successive CHROMA SPIN 30 gel filtration spin columns (Clontech, Palo A).
lto CA). Cy3-labeled control samples and Cy5-labeled experimental samples were combined and precipitated with glycogen, sodium acetate, and ethanol.

Probes are also prepared from tissue needle biopsy samples. The sample is used to identify, for example, toxic compounds, potentially toxic compounds, compounds that cause an unknown metabolic reaction, or changes in tissue following exposure to pharmacological compounds.

8. Hybridization Hybridization was performed using the method described by Shalon (supra).

9 The microscope used to detect the hybridization complexes labeled with the detection reporter molecule is capable of generating a 488 nm spectral line for exciting Cy3 and a 432 nm spectral line for exciting Cy5.
a 70 mixed gas 10 W laser (Coherent Lasers, Santa Clara CA). A 20 × microscope objective lens (Nikon, Melville)
NY). The slide containing the array was placed on a computer controlled XY stage on a microscope and raster scanned through an objective. The 1.8 cm × 1.8 cm array used in this example was scanned at a resolution of 20 μm.

In two separate scans, the mixed gas multiline laser excited the two phosphors sequentially. The emitted light is divided into two photomultiplier detectors (PMT R1477, Hamamatsu Photonics, San Jose C) corresponding to two kinds of fluorescent substances based on the wavelength.
A) was split. The signal was filtered using a suitable filter located between the array and the photomultiplier. The maximum emission of the used fluorescent substance is Cy3
Was 565 nm and Cy5 was 650 nm. Generally, each array is scanned twice and once for each type of phosphor using a suitable filter in the laser light source, but some devices can record spectra from both phosphors simultaneously.

[0115] The sensitivity of the scan is usually the cDN added to the probe mixture at a known concentration.
Calibrated using the signal intensity purified by the A control species. Certain locations on the array contained complementary DNA sequences such that the intensity of the signal at that location could be correlated with a weight ratio of hybridizing species of 1: 100,000. When two probes from different sources (eg, test and control cells) are labeled with different fluorophores and hybridized to an array to identify differentially expressed genes, the calibration is: A sample of the calibration cDNA was labeled with the two fluorescent substances, and each was added by adding the same amount of the hybridization mixture.

The output of the photomultiplier tube is a 12-bit RTI-835H analog-to-digital (A / D) converter board (Analog Device) installed on an IBM compatible PC computer.
es, Norwood MA). The digitized data is displayed as an image in which the signal intensity is mapped to a pseudo-color scale from blue (weak signal) to red (strong signal) using a linear 20-color transform. The data were also analyzed quantitatively. When two different fluorescent substances are excited and measured at the same time, the data is due to the optical crosstalk between the fluorescent substances using the emission spectrum of each fluorescent substance (due to overlapping emission spectra). Was corrected first.

The grid was overlaid on the fluorescent signal image such that the signal from each spot was centered on each element of the grid. The fluorescent signals within each element were then integrated to obtain a value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte Pharmaceuti
cals). In one analysis, when two different samples were prepared from similarly treated cell media, the expression pattern of the cDNA, which varied between 1.6-1.7 fold, showed a 95% accuracy of Poisson distribution. (I. Therlault
, pert. communication).

Experimental Results Eight tyrochrome P4s known to be induced in toxicological reactions
The expression pattern of the 50 isozymes was monitored over 28 days. Tables 1, 2, and 3 show the results when clofibrate, acetaminophen, and benzopyrene were used, respectively. Each of the known genes was up-regulated at least once during that period, more than twice.

[0119]

[Table 1]

[0120]

[Table 2]

[0121]

[Table 3]

We have discovered new nucleotide molecules that are up-regulated at least twice, or down-regulated by less than half, at least once during that period. These molecules are shown in SEQ ID NOs: 1 to 117 in the sequence listing. These polynucleotide molecules can be used to screen compounds or molecules for toxicological effects.

Table 4 shows the gene expression patterns of selected molecules that were up-regulated at least once and at least 2-fold over time after treatment with clofibrate (CLO). Shows the gene expression pattern of the selected molecule down-regulated at least once, at least less than half, over the time course following treatment with clofibrate.

[0124]

[Table 4]

[0125]

[Table 5]

Table 6 shows the gene expression patterns of selected molecules that were up-regulated at least once, at least 2-fold over the course of time after treatment with acetaminophen (APAP), Table 7 Was down-regulated at least once in the time course following treatment with acetaminophen, at least one-half or less,
Figure 2 shows the gene expression pattern of the selected molecule.

[0127]

[Table 6]

[0128]

[Table 7]

Table 8 shows the gene expression patterns of selected molecules that were up-regulated at least once and at least 2-fold over the time course following treatment with benzopyrene (B (a) P), Table 9 shows the gene expression patterns of selected molecules that were down-regulated at least once in the time course following treatment with benzopyrene to at least one-half or less.

[0130]

[Table 8]

[0131]

[Table 9]

BRIEF DESCRIPTION OF SEQUENCE LISTING A portion of the disclosure of this patent specification contains material whose copyright must be protected.
The copyright owner has no objection to facsimile copying any part of the specification relating to the present disclosure in patent office files or records, but otherwise reserves the copyright.

The Sequence Listing contains the nucleic acid sequence of SEQ ID NO: 1-117, which is a specific example of a nucleic acid molecule of the present invention.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 G01N 37/00 102 37/00 102 103 103 103 C12N 15/00 ZNAA (31) Priority claim number 09 / 172,108 (32) Priority Date October 13, 1998 (Oct. 13, 1998) (33) Priority Claimed States United States (US) (81) Designated States 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, UG, ZW), EA (AM, AZ, BY, KG, Z, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, 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, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US , UZ, VN, YU, ZW (72) Inventor Zeger, Gary B. 95023, California, United States Mountain View # 124 Delmedio Court 2700 (72) Inventor Panther, Scott Earl 94087, Sunny, California, United States Vail # 621 East El Camino 965 (72) Inventor Silehammer, Jeffrey J. 94022, Los Angeles, CA, USA United States 12555 F-term (reference) 2G045 AA37 AA40 BB20 CA25 CB01 CB03 CB07 CB08 CB21 CB26 DA12 DA13 DA14 DA36 FA12 FB FB03 FB04 FB06 FB12 FB15 FB16 4B024 AA11 CA01 CA04 CA09 CA11 HA12 4B063 QA01 QA05 QQ02 QQ42 QQ61 QQ91 QQ98 QQ99 QR32 QR42 QR56 QR66 QR84 QS34 QS36 QX02

Claims (8)

[Claims] 1. A method for detecting or diagnosing the effect of a test compound or molecule associated with an increase or decrease in the concentration of a nucleic acid molecule in a mammalian subject, comprising the steps of: Treating an animal subject; (b) obtaining a sample containing nucleic acid from the mammalian subject treated with the toxic compound or molecule; and (c) obtaining one or more hybridization complexes. Contacting said sample with a microarray comprising a plurality of nucleic acid molecules comprising SEQ ID NO: 1 to SEQ ID NO: 117 or fragments thereof under conditions for formation; (d) said hybridization complex Detecting the hybridization activity when compared to a hybridization complex formed from nucleic acid molecules from an untreated mammalian subject. A process wherein the presence, absence, or change in the amount of the complex indicates a metabolic reaction to the toxic compound or molecule, and the method of (e), (c), (d) Measuring the concentration of the nucleic acid molecule in a sample from a mammalian subject treated with the molecule; (f) detecting the concentration detected in the process of (e); Comparing the amount of nucleic acid molecule present with the amount of nucleic acid molecule present, wherein the increase or decrease of the concentration of the nucleic acid molecule as compared to a standard concentration indicates a toxicological reaction. 2. The method of claim 1, wherein the toxic compound or molecule is an antilipidemic, n-alkyl carboxylic acid, n-alkyl carboxylic acid precursor, azole antifungal compound, leukotriene D4 antagonist, herbicide, insecticide, phthalate ester. , Phenyl acetate,
Dehydroepiandrosterone sulfate, methanol and its corresponding metabolite, anthracene and its corresponding metabolite, benzopyrene, 3-methylcholanthrene, benzanthracene, 7,12-dimitylbenzanthracene, and the corresponding metabolite The method of claim 1, wherein the method is selected from: 3. The method of claim 1, wherein said sample is a tissue selected from the group consisting of liver, kidney, brain, spleen, spleen, pancreas, and lung. 4. The method according to claim 1, wherein the test compound that induces a metabolic reaction is a compound that causes an unknown metabolic reaction. 5. The test compound or molecule that induces a metabolic reaction causes at least a two-fold change in the amount of the hybridization complex formed from at least one of the nucleic acid molecules of the sample. The method according to claim 1, wherein 6. An isolated and purified nucleic acid molecule selected from SEQ ID NO: 1 to SEQ ID NO: 117 or a fragment thereof. 7. A method for screening a library of molecules or compounds using the nucleic acid molecule of claim 6 to identify at least one molecule or compound that specifically binds to the nucleic acid molecule, (A) binding the nucleic acid molecule of claim 6 to a molecule or compound library under conditions that allow specific binding; and (b) detecting specific binding to the nucleic acid molecule. Identifying a molecule or compound that specifically binds. 8. The method of claim 7, wherein said library is selected from a DNA molecule, an RNA molecule, a peptide nucleic acid, an artificial chromosome construct, a peptide, and a protein.