US 5,811,231 AGranted

Methods and kits for eukaryotic gene profiling

AssigneesPres. & Fellows of Harvard College, Xenometrix, Inc.

InventorsSpencer B. Farr, Marque D. Todd

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Abstract

This invention provides methods and diagnostic kits for identifying and characterizing toxic compounds. These methods and diagnostic kits measure transcription or translation levels from genes linked to native eukaryotic stress promoters, especially those of mammals. The kits and methods of this invention utilize at least one stress promoter from each of the following groups: redox stress, DNA stress, protein stress and energy/ionic stress. The invention also provides methods and diagnostic kits for identifying and characterizing compounds that are toxic to specific organs, such as skin and the eye, as well as for each of the individual stresses indicated above. The methods and diagnostic kits of this invention yield information concerning the action of a compound on a subcellular level. This information may be utilized to design antitoxins to compounds found to be toxic and in active drug design.

Claims40

Dependent claims

A diagnostic kit for identifying disturbances of the biochemical and biophysical homeostasis of a cell caused by a compound comprising:

(a) an eukaryotic cell comprising:
(i) at least one promoter or response element which responds to disturbances of the homeostatic redox state in the cell, wherein said disturbances include conditions which vary the normal reduction or oxidation potential state of a cell and conditions which cause proliferation of peroxisomes;
(ii) at least one promoter or response element which responds to disturbances of the homeostatic state of DNA in the cell, wherein said disturbances include alterations of deoxyribonucleic acids, alterations of precursor nucleotides, inhibition of DNA synthesis, inhibition of DNA replication, inhibition of mitosis or meiosis;
(iii) at least one promoter or response element which responds to disturbances of the homeostatic state of proteins in the cell, wherein said disturbances include alterations of proteins or individual amino acids, inhibition of enzyme functions, and perturbations of intracellular transport of proteins;
and
(iv) at least one promoter or response element which responds to disturbances of the homeostatic energy and/or ionic state of the cell, wherein said disturbances include conditions which affect ATP levels, or ionic gradients across a cell membrane,
each of said promoters or response elements being operatively linked to a separate gene which encodes a detectable product;
(b) at least four different nucleic acid sequences, each of said nucleic acid sequences being capable of hybridizing under conditions of high stringency to an mRNA transcript of a different one of the genes that is operatively linked to said promoters or response elements or to a single stranded cDNA prepared from said mRNA transcript;
and
(c) means for quantifying the nucleic acid sequences that hybridize to each of said mRNA transcripts or to single stranded cDNA prepared from each of said mRNA transcripts.

7 more elements

A method for identifying disturbances of the biochemical and biophysical homeostasis of a cell caused by a compound comprising the steps of:

(a) exposing to said compound:
(i) a eukaryotic cell comprising at least one promoter or response element which responds to disturbances of the homeostatic redox state in the cell, wherein said disturbances include conditions which vary the normal reduction or oxidation potential state of a cell and conditions which cause proliferation of peroxisomes;
(ii) a eukaryotic cell comprising at least one promoter or response element which responds to disturbances of the homeostatic state of DNA in the cell, wherein said disturbances include alterations of deoxyribonucleic acids, alterations of precursor nucleotides, inhibition of DNA synthesis, inhibition of DNA replication, inhibition of mitosis or meiosis;
(iii) a eukaryotic cell comprising at least one promoter or response element which responds to disturbances to the homeostatic state of proteins in the cell, including alterations to proteins or individual amino acids, inhibition of enzyme functions, and perturbations of intracellular transport of proteins;
and
(iv) a eukaryotic cell comprising at least one promoter or response element which responds to disturbances to the homeostatic energy or ionic state of the cell, including conditions which affect ATP levels, or ionic gradients across a cell membrane;
wherein each of said promoters or response elements is operatively linked to a gene, either the same or different, encoding a detectable product;
(b) quantifying the product of each of said genes;
and
(c) creating a promoter induction profile for said compound.

7 more elements

A diagnostic kit for identifying disturbances of the biochemical and biophysical homeostasis of a cell caused by a compound comprising:

(a) an eukaryotic cell which comprises at least one promoter or response element that responds to disturbances of the homeostatic redox state in the cell, including conditions which vary the normal reduction or oxidation potential state of a cell and conditions which cause proliferation of peroxisomes;
(b) an eukaryotic cell which comprises at least one promoter or response element that responds to disturbances of the homeostatic state of DNA in the cell, wherein said disturbances include alterations of deoxyribonucleic acids, alterations of precursor nucleotides, inhibition of DNA synthesis, inhibition of DNA replication, inhibition of mitosis or meiosis;
(c) an eukaryotic cell which comprises at least one promoter or response element that responds to disturbances of the homeostatic state of proteins in the cell, wherein said disturbances include alterations of proteins or individual amino acids, inhibition of enzyme functions, and perturbations of intracellular transport of proteins;
and
(d) an eukaryotic cell which harbors at least one promoter or response element that responds to disturbances of the homeostatic energy and/or ionic state of the cell, wherein said disturbances include conditions which affect ATP levels, or ionic gradients across a cell membrane;
wherein, each of said promoters or response elements is operatively linked to a heterologous gene encoding a detectable product;
and
(e) means for quantifying the amount of said detectable product from each of said eukaryotic cells.

5 more elements

A method for identifying the disturbances of the biochemical and biophysical homeostasis of a cell caused by a compound comprising the steps of:

(a) culturing:
(i) a eukaryotic cell comprising at least one promoter or response element which responds to disturbances of the homeostatic redox state in the cell, wherein said disturbances include conditions which vary the normal reduction or oxidation potential state of a cell and conditions which cause proliferation of peroxisomes;
(ii) a eukaryotic cell comprising at least one promoter or response element which responds to disturbances to the homeostatic state of DNA in the cell, wherein said disturbances include alterations of deoxyribonucleic acids, alterations of precursor nucleotides, inhibition of DNA synthesis, inhibition of DNA replication, inhibition of mitosis or meiosis;
(iii) a eukaryotic cell comprising at least one promoter or response element which responds to disturbances of the homeostatic state of proteins in the cell, including alterations of proteins or individual amino acids, inhibition of enzyme functions, and perturbations of intracellular transport of proteins;
and
(iv) a eukaryotic cell comprising at least one promoter or response element which responds to disturbances to the homeostatic energy or ionic state of the cell, including conditions which affect ATP levels, or ionic gradients across a cell membrane;
wherein each of said promoters or response elements is operatively linked to a gene, either the same or different, encoding a detectable product;
(b) exposing said cells to said compound;
(c) quantifying the product of each of said genes;
and
(d) creating a promoter induction profile for said compound.

8 more elements

Dependencies are inferred from the claim text.