Identification of Modulators of Autophagy

Abstract

Methods for identifying compounds that inhibit or stimulate the autophagy pathway are described. Devices for detecting the expression of autophagy-related genes and kits for assaying the expression of autophagy-related genes are also described. Also described are methods for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect.

Description

BACKGROUND OF THE INVENTION

Autophagy is a catabolic, cellular self-digestion process that is activated by starvation and stress whereby double membrane vesicles called autophagosomes form that engulf proteins and organelles. Autophagosomes then fuse with lysosomes where their cargo is degraded. The function of autophagy is to recycle intracellular nutrients to sustain metabolism during nutrient and growth factor deprivation, and to clear damaged proteins and organelles that accumulate during stress. Although elimination of individual proteins occurs by the ubiquitin-mediated proteasome degradation pathway, only the autophagy pathway can eliminate protein aggregates and organelles. Thus, autophagy complements and overlaps with proteasome function to prevent the accumulation of damaged cellular components during starvation and stress. Through these functions, autophagy is an important cellular stress response that functions to maintain protein and organelle quality control, protect the genome from damage, and sustain cell and mammalian viability.

Autophagy is controlled by ATG proteins that were initially identified in yeast for which there are mammalian homologues. ATG proteins are comprised of kinases, proteases, and two ubiquitin-like conjugation systems that likely function in concert with a host of unknown cellular proteins to control autophagosome formation, cargo recognition, engulfment, and trafficking to lysosomes. The ATG6/Beclin1-VPS34-ATG8/LC3 complex regulates autophagosome formation and LC3 cleavage, lipidation, and membrane translocation are frequently utilized to monitor autophagy induction and inhibition of flux through the autophagy pathway.

Targeting of cargo, including proteins and organelles, to autophagosomes for degradation is accomplished by tagging proteins with polyubiquitin. The ubiquitin-binding domain (UBA) on the adaptor protein p62 recognizes and binds these polyubiquitinated proteins. p62 oligomerizes by self-association of its PB1 domain and binds ATG8/LC3 on autophagosome membranes. p62 thereby identifies, collects and delivers cargo to autophagosomes for degradation. p62 itself is an autophagy substrate and is degraded by autophagy along with the cargo. As such, p62 accumulation in aggregates is indicative of autophagy inhibition and clearance of p62 following stress is indicative of functional autophagy. These properties of p62 have been demonstrated in vivo in autophagy-defective mutant mice and are mimicked by expression of EGFP-p62 in cell lines in vitro and in vivo (Mathew, R et al., (2009) Cell 137, 1062-1075).

The activation of autophagy by starvation and stress is controlled in part through the PI-3 kinase pathway via the protein kinase mTOR. Growth factor and nutrient availability promote mTOR activation that suppresses autophagy, whereas starvation and mTOR inactivation stimulate autophagy. While there are other mechanisms to regulate autophagy, and those that activate autophagy in response to stress are particularly poorly understood, mTOR provides a link between nutrient and growth factor availability, growth control, autophagy, and metabolism.

Autophagy dysfunction is believed to be a major contributor to human diseases including neurodegeneration, liver disease, and cancer. Many human neurodegenerative diseases are associated with aberrant protein accumulation and excessive neuronal cell death, and neurons of mice with targeted autophagy defects accumulate polyubiquitinated- and p62-containing protein aggregates that result in neurodegeneration. The human liver disease steatohepatitis and a major subset of hepatocellular carcinomas (HCCs) are associated with the formation of p62-containing protein aggregates (Mallory bodies), and livers of mice with autophagy defects have p62-containing protein aggregates, excessive cell death, and HCC.

Evidence from model organism disease models indicates that promoting autophagy with mTOR inhibitors such as rapamycin or CCI-779, and enhancing the clearance of misfolded, damaged or mutated proteins and protein aggregates prevents neurodegeneration, but that there also are mTOR-independent means to increase autophagy. Similarly, genetically eliminating the expression of p62 in hepatocytes and preventing p62 accumulation in autophagy-defective atg7^−/− hepatocytes dramatically suppresses the phenotype of steatohepatitis. In contrast, neurodegeneration due to expression and accumulation of polyglutamate expansion mutant proteins is greatly exacerbated by allelic loss of beclin1 and defective autophagy. Thus, while not intending to be bound by any theory of operation, autophagy is believed to be involved in limiting the buildup of misfolded, mutated proteins in p62-containing protein aggregates, which leads to cellular deterioration and disease.

Analogous to a wound-healing response, chronic tumor cell death in response to stress and induction of inflammation and cytokine production may provide a non-cell-autonomous mechanism by which tumorigenesis is promoted in autophagy-defective cells. Autophagy-defective tumor cells also display an elevated DNA damage response, gene amplification and chromosome instability in response to stress, suggesting that autophagy limits genome damage as a cell-autonomous mechanism of tumor suppression.

Therefore, while not intending to be bound by any theory of operation, stimulating autophagy may be involved in limiting disease progression, particularly neurodegeneration, liver disease, and also cancer, by facilitating the elimination of protein aggregates, damaged organelles, and the toxic consequences of their accumulation.

Autophagy has been identified also as a survival pathway in epithelial tumor cells that enables long-term survival to metabolic stress. Tumor cells with defined defects in autophagy accumulate p62-containing protein aggregates, DNA damage and die in response to stress, whereas those with intact autophagy can survive for weeks utilizing the autophagy survival pathway. Thus, autophagy appears to be required to prevent tumor cell damage and to maintain metabolism. Tumor cells can exploit this survival function to remain dormant only to reemerge under more favorable conditions. Interestingly, roughly half of human cancers may have impaired autophagy, either due to constitutive activation of the PI-3 kinase pathway or allelic loss of the essential autophagy gene beclin1, rendering them particularly susceptible to metabolic stress and autophagy inhibition.

Therefore, identification of the therapeutic means to inhibit the autophagy survival pathway in tumor cells would be advantageous. While not intending to be bound by any theory of operation, this may be of value as many therapeutics currently in use, such as kinase and angiogenesis inhibitors, inflict metabolic stress, which increases the dependency on autophagy for survival. Furthermore, tumor cells with impaired autophagy are particularly vulnerable to metabolic stress and further therapeutic suppression of autophagy may be able to exploit this vulnerability by promoting cell death by metabolic catastrophe or the failure to mitigate cell damage accumulation. Preclinical studies have been conducted using hydroxychloroquine to inhibit lysosome acidification and thereby autophagy in combination therapy. Specific inhibitors of the autophagy survival pathway in tumor cells are may be of great value in combination with agents such as angiogenesis and kinase inhibitors that promote metabolic stress.

Thus, the autophagy pathway represents fertile ground for novel therapeutic target identification for drug discovery for many diseases for both acute treatment and also disease prevention.

Accordingly, a need exists to identify nucleic acid sequences and their encoded proteins which are involved in modulation of the autophagy pathway.

BRIEF SUMMARY OF THE INVENTION

In certain aspects, the present invention relates to methods for identifying compounds that inhibit or stimulate the autophagy pathway.

Further aspects relate to methods for identifying individuals susceptible to or afflicted with a disease state associate with an autophagy pathway defect.

Additional aspects relate to devices for detecting the expression of autophagy-related genes.

Further aspects relate to kits for assaying expression of autophagy-related genes.

Other aspects are readily apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a cell-based shRNA screen for rescue of autophagy deficiency and p62 protein aggregate accumulation in metabolic stress: Screen for autophagy stimulators.

FIG. 2 illustrates representative images of shRNAs that modulate p62 aggregate elimination.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided to facilitate an understanding of the present invention:

A “polynucleotide,” “polynucleotide molecule” or “polynucleotide sequence” refers to a chain of nucleotides. It may refer to a DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there may be up to about a 30% mismatch in the sequences.

The term “oligonucleotide,” as used herein refers to sequences, primers and probes, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

The term “probe” as used herein refers to either a probe for a nucleic acid or a probe for a protein. When used in connection with nucleic acids, a “probe” refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single stranded or double stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. The probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. When used in connection with a polypeptide, a “probe” is a protein- or polypeptide-binding substance or agent, capable of specifically binding a particular protein or protein fragment to the substantial exclusion of other proteins or protein fragments. Such binding agents may be any molecule to which the protein or peptide specifically binds, including DNA (for DNA binding proteins), antibodies (as described in greater detail herein), cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organellar membranes.

“Array” refers to an ordered arrangement of at least two probes on a substrate. At least one of the probes represents a control or standard, and the other, a probe of diagnostic or screening interest.

“Specific binding” refers to a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups; for example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.

The term “specifically hybridize” refers to the association between two single stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”). For example, the term may refer to hybridization of a nucleic acid probe with a substantially complementary sequence contained within a single stranded DNA or RNA molecule according to an aspect of the invention, to the substantial exclusion of hybridization of the nucleic acid probe with single stranded nucleic acids of non-complementary sequence. When used in connection with the association between single stranded nucleic acid molecules, the term “specifically bind” may be used to indicate that the molecules “specifically hybridize” as described herein.

An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term includes polyclonal, monoclonal, chimeric, and bispecific antibodies. As used herein, antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule such as those portions known in the art as Fab, Fab′, F(ab′)2 and F(v).

“Sample” is used in its broadest sense as containing nucleic acids, proteins, antibodies, and the like. A sample may comprise, for example, a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue or a tissue biopsy; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like. Bodily fluids include, without limitation, whole blood, blood plasma, blood serum, sputum, urine, sweat, and lymph.

As used herein, the term “subject” or “patient” refers to both humans and animals, unless specified that the “subject” or “patient” is an animal or a human. An “individual” also refers to both humans and animals, unless specified that the “individual” is an animal or a human. Animal subjects are preferably vertebrates, and more preferably, mammals.

“Autophagy-associated” or “autophagy-related” as used herein with respect to a disease, condition or disorder refers to that which results from an increase or decrease in normal autophagy function and/or that which may be treated and/or prevented by modulation of the autophagy pathway. As used herein with respect to a biological molecule, such as, for example, a polynucleotide or polypeptide, “autophagy-associated” or “autophagy-related” refers to a molecule for which alteration of the expression, abundance and/or activity thereof leads to modulation of the autophagy pathway.

In certain embodiments, the present invention relates to the identification of genes whose expression modulates autophagy. These genes and their gene products may represent targets for therapeutic intervention in the autophagy pathway.

In accordance with aspects of the present invention, a number of polynucleotides comprising at least a fragment of a gene have been identified as representing molecules whose knockdown of expression modulates the function of the autophagy pathway. In certain aspects, knockdown of gene expression stimulates autophagy. In other aspects, knockdown of gene expression inhibits autophagy.

Embodiments of the invention include validation of the candidate genes and gene fragments described herein using known techniques for in vitro and in vivo analysis.

In accordance with various aspects of the present invention, combinations, compositions, devices and kits are provided that may be used in the practice of methods provided according to certain embodiments of the invention.

Certain embodiments relate to methods of detection of alterations in the autophagy pathway. Certain of these methods may be used to detect conditions in which autophagy is reduced. Certain of these methods may be used to detect conditions in which autophagy is increased.

In accordance with certain aspects of the invention, a combination is provided comprising a plurality of polynucleotide molecules wherein the polynucleotide molecules encode gene products associated with modulation of the autophagy pathway. In certain embodiments, the combination comprises a plurality of polynucleotides whose knockdown stimulates autophagy. In certain embodiments, the plurality of polynucleotide molecules comprise two or more molecules identified in Table 3 or fragments thereof.

In certain embodiments, the combination comprises a plurality of polynucleotides whose knockdown inhibits autophagy. In certain embodiments, the plurality of polynucleotide molecules comprise two or more molecules identified in Table 4 or fragments thereof.

An embodiment of the invention provides a method for identifying compounds that modulate autophagy-associated gene expression comprising: a) measuring standard expression by measuring transcription or translation products of one or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof, in a standard sample in the absence of a test compound; b) measuring test expression by measuring the transcription or translation products of one or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof, in a test sample in the presence of the test compound; and c) comparing the standard expression to the test expression, wherein a change in the test expression compared to the standard expression is indicative of an effect of the test compound on the expression of genes whose expression modulates the autophagy pathway. In certain embodiments, a plurality of two or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof are used.

One embodiment of the invention provides a method for identifying compounds that inhibit or stimulate the autophagy pathway for treatment of a disease state associated with an autophagy pathway defect, comprising measuring the effect of one or more test compounds on the inhibition or stimulation of a product of one or more of the genes or gene fragments identified in Table 3 or Table 4.

An embodiment provides a method for the detection of differential expression of autophagy-associated polypeptides in a sample, comprising the steps of: a) reacting protein binding molecules with polypeptides of the sample, thereby allowing specific binding to occur, wherein the polypeptides bound by the protein-binding molecules comprise one or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4 or fragments thereof; b) detecting specific binding; and c) comparing the specific binding in the sample with that of a standard, wherein differences between the standard and sample specific binding indicate differential expression of polypeptides in the sample. In certain embodiments, the protein-binding molecules are directed to polypeptides comprising a plurality of two or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4 or fragments thereof.

Another embodiment provides a method for the detection of differential expression of autophagy-associated nucleic acids in a sample, comprising the steps of: a) hybridizing polynucleotides comprising one or more molecules identified in Table 3 and/or Table 4 or fragments thereof with nucleic acids of the sample, thereby forming one or more hybridization complexes; b) detecting the hybridization complexes; and c) comparing the hybridization complexes with those of a standard, wherein differences between the standard and sample hybridization complexes indicate differential expression of nucleic acids in the sample. In certain embodiments, the polynucleotides comprise a plurality of two or more molecules identified in Table 3 and/or Table 4 or fragments thereof.

Another embodiment comprises a composition of matter comprising one or more probes for detecting expression of autophagy-associated genes, wherein the probes comprise one or more of: a) nucleic acid molecules that specifically hybridize to one or more of the genes or gene fragments identified in Table 3 and/or Table 4, or fragments thereof; or b) polypeptide binding agents that specifically bind to polypeptides produced by expression of one or more nucleic acid molecules comprising sequences selected from one or more of genes or gene fragments identified in Table 3 and/or Table 4, or fragments thereof. In certain embodiments, the composition of matter comprises a collection of two or more probes.

Another embodiment provides a device for detecting expression of a plurality of autophagy-related genes, comprising a substrate to which is affixed, at known locations, a plurality of probes, wherein the probes comprise: a) a plurality of oligonucleotides or polynucleotides, each of which specifically hybridizes to a different sequence selected from any of the sequences identified in Table 3 and/or Table 4 or fragments thereof; or b) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof produced by expression of a nucleic acid molecule comprising a sequence selected from the genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4 or fragments thereof.

In certain embodiments, a device is provided for detecting the expression of a plurality of autophagy-related genes associated with an autophagy pathway defect, said device comprising a substrate to which is affixed at known locations a plurality of probes, wherein the probes comprise:

• • a) a plurality of oligonucleotides or polynucleotides, each of which specifically binds to a different sequence selected from any of the sequences identified in Table 3 or Table 4 or fragments thereof; or
  • b) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof produced by expression of a gene or gene fragment comprising any of the sequences identified in Table 3 or Table 4 or fragments thereof.

Another embodiment provides a method for measuring the effect of a test compound on expression of an autophagy-associated gene, wherein the gene is selected from the group consisting of the genes or gene fragments identified in Table 3 and/or 4, the method comprising measuring production of transcription or translation products produced by expression of the gene or gene fragment in the presence or absence of the test compound, wherein a change in the production of transcription or translation products in the presence of the test compound is indicative of an effect of the test compound on expression of the gene or gene fragment.

In an embodiment, the gene expression is measured by providing a DNA construct comprising a reporter gene coding sequence operably linked to transcription regulatory sequences of the autophagy-associated gene, and measuring formation of a reporter gene product in the presence or absence of the test compound.

Another embodiment provides a kit for assaying the expression of autophagy-related genes, comprising at least one container comprising a collection of two or more probes, wherein the probes comprise: a) oligonucleotides or polynucleotides that specifically hybridize to two or more genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof; or b) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof. The kit preferably comprises instructions for performing an assay of gene expression.

In certain embodiments, the invention provides a kit for assaying the expression of autophagy-related genes associated with an autophagy pathway defect, comprising at least one container and a collection of two or more probes, wherein the probes comprise:

• • a) oligonucleotides or polynucleotides that specifically bind to two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof; or
  • b) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof. The kit preferably comprises instructions for performing an assay of gene expression.

The invention provides, in certain embodiments, methods for identifying compounds that are useful in modulating the autophagy pathway. Preferably, the methods include contacting at least one polypeptide encoded by the genes and/or gene fragments identified in Table 3 and/or Table 4 with a test substance and determining whether the test substance binds to the polypeptide. Further, in certain embodiments of the invention, a test substance may be determined to stimulate or inhibit the biological activity of the relevant gene product comprising at least one polypeptide encoded by the genes and/or gene fragments identified in Table 3 and/or Table 4 and thereby be identified as a compound useful for the modulation of the autophagy pathway. Such assays may, in certain embodiments, be performed in vitro and may, in certain embodiments, be performed in a cell-based assay. In some embodiments, substances identified as modulating expression or biological activity in vitro may be further tested in vivo to confirm relevant and effective activity.

Test substances or compounds contemplated by aspects of the invention include compounds from chemical libraries, including natural products and/or synthetic products from combinatorial chemical synthesis. Such substances may include, without limitation, polypeptides, oligonucleotides, polynucleotides, or organic molecules.

In a further embodiment is provided a method of modulating autophagy-associated gene expression in a cell by administering an effective amount of a composition under appropriate conditions to affect the expression of at least one gene associated with autophagy having a sequence selected from the sequences identified in Table 3 and/or Table 4, or fragments thereof.

In preferred embodiments, the composition comprises an inhibitor of gene expression. The inhibitor of gene expression may be selected from molecules including, but not limited to, an antisense RNA, a morpholino polynucleotide, and an interfering RNA (RNAi).

According to a still further aspect of the invention, there is provided a genetically-modified non-human animal that has been transformed to express higher, lower or absent levels of a protein according to any one of the aspects of the invention described herein. Preferably, said genetically-modified animal is a transgenic or knockout animal. Preferably, the genetically-modified animal is a rodent, most preferably a mouse.

An embodiment of the invention also provides a method for screening for a substance effective to treat an autophagy-associated disease condition, by contacting a non-human genetically-modified animal as described above with a candidate substance and determining the effect of the substance on the physiological state of the animal.

Certain embodiments of the invention provide methods and kits for diagnosis of, determining susceptibility to and/or developing a prognosis for an autophagy-associated disease state in a subject. In certain aspects, these may involve tests on subject samples. In certain embodiments, these may be nucleic acid based tests or polypeptide-based tests. In some embodiments, the method or kit may include probes that bind to at least one polynucleotide encoding an autophagy-associated polypeptode. In some embodiments, the a plurality of two or more probes may be used. In some embodiments, the method or kit may include polypeptide binding agents that bind to at least one autophagy-associated polypeptide. In some embodiments, a plurality of two or more polypeptide binding agents may be used. In certain embodiments, the polypeptide-binding agent comprises antibodies and/or antigen-binding portions of an antibody that specifically binds to one or more autophagy-associated polypeptides. Preferably, the autophagy-associated polypeptides are encoded by the gene or gene fragments identified in Table 3 and/or Table 4.

One embodiment provides a method for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect, comprising testing a biological sample from an individual for a characteristic of one or more polypeptides produced by expression of one or more of the genes or gene fragments identified in Table 3 or Table 4 that is indicative of said disease state, wherein said characteristic is selected from the presence of at least one of said polypeptides, the absence of at least one of said polypeptides, an elevated level of at least one of said polypeptides, a reduced level of at least one of said polypeptides and, for two or more of said polypeptides, combinations thereof.

An embodiment provides a method to diagnose or develop a prognosis for an autophagy-related disease in a subject, the method comprising: a) obtaining a sample from the subject; b) measuring in the sample the production of transcription or translation products produced by the expression of one or more autophagy-associated genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof; c) comparing the transcription or translation products of the sample with that of a standard, wherein a difference in the expression of any of the autophagy-associated genes or gene fragments is indicative of autophagy-related disease.

In one embodiment is provided a kit for the diagnosis of an autophagy-associated disease in a subject comprising polynucleotide probes that specifically bind to one or more autophagy-associated polynucleotides or a fragment thereof. Preferably the autophagy-associated polynucleotides or fragments thereof are selected from the polynucleotide sequences identified in Table 3 and/or Table 4 or fragments thereof. In certain embodiments, the kit comprises a plurality of two or more polynucleotide probes that specifically bind to polynucleotide sequences identified in Table 3 and/or Table 4 or fragments thereof. Preferably, the kit comprises also instructions for use.

The invention also provides kits for diagnosis of autophagy-associated conditions from patient samples that may be nucleic acid based tests or polypeptide-based tests. In some embodiments, the kit contains at least one polynucleotide that binds to a polynucleotide encoding an autophagy-related gene product. In some embodiments, the kit contains, preferably in separate containers, a plurality of probes to detect two or more polynucleotides encoding one or more autophagy-associated gene products. In preferred embodiments, the gene products are encoded by one or more of the genes or gene fragments identified in Table 3 and/or Table 4. In other embodiments, the kit contains at least one polypeptide binding agent that specifically binds to at least one autophagy-associated polypeptide. In some embodiments, the kit contains, preferably in separate containers, a plurality of polypeptide binding agents (or mixtures thereof) to detect one or more autophagy-associated polypeptide. In certain embodiments, the polypeptide binding agent may be an antibody or antigen-binding portion of an antibody. In certain embodiments, the autophagy-associated polypeptides include at least one polypeptide encoded by the genes or gene fragments identified in Table 3 and/or Table 4. In certain embodiments, the autophagy-associated polypeptides identified by the kit include a plurality of two or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4. In certain embodiments, the kits may also include instructions for use.

In certain embodiments, methods according to the invention may be used for high-throughput screening assays.

In certain embodiments, methods and kits useful in the methods of the invention may utilize nucleic acid, antibody and/or polypeptide arrays.

Using a cell-based loss-of function screen, the present inventors have identified candidate genes whose expression is involved in the autophagy pathway. In particular, the screen has been used to identify genes whose knockdown stimulates autophagy. Results from this screen are shown in Table 1. The screen has also been used to identify genes whose knockdown inhibits autophagy. Results from this screen are shown in Table 2.

A high-efficiency delivery method that enables stable long-term gene suppression in a broad range of cell types is virus-mediated integration of an RNAi expression cassette. After integration, the cassette produces a short dsRNA molecule, usually in the form of a hairpin structure, a short or small hairpin RNA (shRNA), which is processed into active small interfering RNA (siRNA). Although many types of viruses are suitable for this purpose, lentiviral vectors generate viruses of both high titer and broad tropism, permitting the infection of both dividing and nondividing cells. Lentiviral shRNA libraries for mouse gene clones were utilized that allow gene silencing in most dividing and nondividing cell types.

An image based, arrayed shRNA screen was employed. Lentiviral shRNA libraries developed by the RNA Consortium (TRC) at the Broad Institute were used in a cell-based screen. The screens utilized the publicly available kinase and vesicle trafficking lentiviral library subsets at the Broad Institute, as well as a custom library containing shRNAs targeting mouse GTPases. Lentiviruses are high-titer, individual clones with representation of at least five independent hairpins for each target gene supplied in a high-throughput format (Root, D. E., et al. (2006), Nature Methods 3, 715-719.) Fluorescence image analysis was used to capture the data. Gene were identified that were shown to promote or suppress autophagy (bimodal analysis).

The high content arrayed shRNA screen used to identify autophagy modulators utilized autophagy defective beclin1^+/− iBMK cells stably expressing the autophagy substrate EGFP-p62 (Mathew, R., Karantza-Wadsworth, V., and White, E. (2009) Methods Enzymol 453, 53-81; Mathew, R., et al. (2009) Cell 137, 1062-1075; and Mathew, R., et al. (2007) Genes Dev 21, 1367-1381). p62 accumulates and aggregates in response to metabolic stress and requires autophagy for degradation. p62 also accumulates in degenerative neuronal and liver diseases and in autophagy-defective mouse tissues, beclin1^+/− and atg5^−/− iBMK cells, and tumors. Genes were identified whose inactivation compensates for defective autophagy and restores p62 protein turnover. Since the image analysis captured every hairpin's p62 aggregation score (EGFP-p62 intensity divided by the nuclei in the field) it was also possible to identify genes whose inactivation lead to further accumulation of p62 aggregates, predicted to be autophagy inhibitors (FIG. 2). This was possible because the cell line employed in this screen is autophagy impaired rather than fully autophagy defective. Therefore, the disposition of p62 in the test cells serves as readout for both autophagy promotion (p62 degradation, low p62) and inhibition (autophagy inhibition, high p62) and is the basis for the identification of autophagy modulators in cell-based screens.

The shRNA libraries were screened using autophagy-impaired test cells expressing a marker of protein aggregation, subjecting the test cell to metabolic stress, and performing analysis on the test cell to determine the level of the marker. The marker of protein aggregation is a p62 protein linked to enhanced green fluorescent protein (EGFP) label. Image analysis is performed to determine the level of p62 aggregates in cells. The level of the marker found in p62 aggregates in the test cell is compared with that of a control cell. A lower level of p62 aggregates comprising the marker in the test cell compared to that demonstrated by the control cell demonstrates the rescue of the impairment in p62 clearance, indicating the lowered expression of a gene whose knockdown stimulates autophagy. A greater level of p62 aggregates in a test cell compared to that of a control cell demonstrates suppression of p62 clearance, indicating the knockdown of a gene whose lowered level of expression leads to inhibition of autophagy.

The cell-based screen utilized autophagy-deficient beclin1^+/− immortalized baby mouse kidney (iBMK) cells stably expressing EGFP-p62. p62 accumulates and aggregates in response to metabolic stress and requires autophagy for degradation. FIG. 1 illustrates a cell-based shRNA screen for rescue of autophagy deficiency and p62 protein aggregate accumulation in metabolic stress and therefore represents a screen for autophagy stimulators. The autophagy-deficient beclin1^+/− iBMK cell line stably expressing EGFP-p62 accumulates p62-containing protein aggregates under stress, which fail to be cleared following recovery. Those shRNAs that facilitate p62 aggregate clearance, compensating for defective autophagy, are identified. The autophagy wild type beclin1^+/+ iBMK cell line stably expressing EGFP-p62 that effectively clears p62 aggregates following stress is used as a positive control.

FIG. 2 illustrates representative images of cells contacted with shRNAs that modulate p62 aggregate elimination. The screen is designed to identify genes whose loss results in restoration of autophagy (autophagy stimulators), manifested by successful clearance of p62 aggregates following a time course of stress and recovery. mTOR, a master negative regulator of autophagy, is shown here as an example of a gene whose loss restores autophagy and clearance of p62. Alternatively, loss of some genes is predicted to further inhibit autophagy (autophagy inhibitors). Loss of Ikbkb, a known autophagy promoter (Criollo, A, et al. EMBO J 29, 619-631), results in marked accumulation of p62. This accumulation is greater than observed in cells infected with an shRNA targeting luciferase.

The shRNAs shown to promote p62 elimination (autophagy stimulators) identify potential targets for drug discovery efforts for development of modulators of autophagy, including autophagy inhibitors. While not intending to be bound by any theory of operation, autophagy inhibitors are potentially useful as anti-cancer therapeutics by promoting cancer cell death.

The shRNAs shown to enhance p62 accumulation (autophagy inhibitors) identify potential targets for drug discovery efforts for development of modulators of autophagy, including autophagy stimulators. While not intending to be bound by any theory of operation, autophagy stimulators are potentially useful in preventing or delaying disease manifestation in the setting of cancer, neurodegenerative conditions, Crohn's disease, liver disease, aging and inflammatory diseases and in combating infections.

EXAMPLES

The following examples serve to more fully describe the manner of using the above-described invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

Materials and Methods

Cell Line

Murine kidney epithelial cells were isolated from beclin1^+/− mice and immortalized with dominant negative p53 and EIA as described previously (Degenhardt, K., and White, E. (2006). Clin Cancer Res 12, 5298-5304; Mathew, R., Degenhardt, K., Haramaty, L., Karp, C. M., and White, E. (2008) Methods Enzymol 446, 77-106.). The cells were subsequently engineered to overexpress Bcl-2 and eGFP-p62. Thus, these cells, known as 3BC2 EGFP-P62, contain an autophagy defect, are apoptotically impaired, and stably express EGFP-P62. (Mathew, R., et al. (2009). Cell 137, 1062-1075.).

Screening Protocol for Beclin^+/− eGFP-P62

Cells were plated into black barcoded 384 well plates (Corning 8793BC) at a density of 700 cells/well by the Biotek microfill and allowed to attach overnight. Infection and media changes for plates were achieved by use of two robotic liquid handlers at the Broad Institute, the Perkin Elmer Janus and EP3. Each viral plate was used to infect four target plates. Each virus plate contained 20 control hairpins (targeting either RFP, luciferase, or EGFP) in addition to wells containing no virus. Two hairpins targeting p62 were spiked into each plate at the time of infection to ensure that positive and negative controls were present on all plates. Immediately prior to infection, media was changed with the Janus robot (Perkin Elmer) to DMEM containing 8 ug/mL polybrene. The Perkin Elmer EP3 robot was used to add 6 ul of virus to each well. Cells were spin infected (2250 rpm 30 mins, 30° C.) in the presence of 6 ul of virus and Bug/ml of polybrene before returning to the 37 C incubator. Virus and polybrene containing media was removed 4 hours post infection and cells were incubated in normal growth media overnight (DMEM high glucose, 10% FBS, 1% penicillin streptomycin (PS)). Twenty four hours post infection, media was changed with the Janus to DMEM containing 3 ug/mL puromycin (for the three puro plus plates) or DMEM alone (for the puro minus replicate). Selection was allowed to continue for 72 hrs.

The assay employed in this screen is predicated on the ability of autophagy competent cells to successfully eliminate p62 aggregates that accumulate during metabolic stress during a recovery phase during which time oxygen and glucose are restored. Optimization experiments were conducted comparing the ability of autophagy competent beclin1^+/+-EGFP-p62 cells (WB3-EGFP-p62) and autophagy deficient beclin1^+/−-EGFP-p62 cells (3Bc2-EGFP-p62) to eliminate p62 aggregates during various time courses of metabolic stress (1% oxygen, glucose deprivation) and recovery within the setting of 384 well plates post infection and selection with puromycin. 7.5 hours of metabolic stress followed by 18 hours of recovery in high glucose DMEM 10% FBS was optimal, and these conditions were chosen for the large-scale screen.

Following puromycin selection, media containing DMEM high glucose was removed, and cells were washed twice in ischemia media (DMEM containing no glucose, 10% FBS, 1% PS) to remove residual glucose in wells prior to transfer into a hypoxia incubator set to 1% oxygen for 7.5 hours. They were then transferred to an incubator which could lower ambient oxygen levels to 1% by virtue of its attachment to a nitrogen tank. Cells stayed in this 1% oxygen, no glucose conditions, referred to as metabolic stress, for 7.5 h.

At the conclusion of the metabolic stress, normal growth media (DMEM high glucose 10% FBS, 1% PS) was added to the plates and the cells were allowed to recover overnight at 37° C. 18 hours post recovery, media was removed from plates, and cells were fixed by addition of 4% paraformaldehyde/PBS for 10 mins at RT.

Nuclei were visualized by inclusion of Hoechst 33342 at a dilution of 1:10,000 in the fixation solution. Plates were washed 3× with the ELx405 automated plate washer (Biotek). 80 ul of filtered PBS was left in each well at the end of washing to allow for evaporation during imaging.

Plates were imaged on the Arrayscan VTI (Thermo Scientific) housed within the Genome technology Core of the Whitehead Institute using a modified version of the Cellomics compartmental analysis bioapplication. Nuclei were visualized in channel 1. EGFP-p62 aggregates were visualized in channel 2. Nine images per channel were captured for each field, with an autofocus field interval of 3. MEAN_valid object count channel 1 represents the mean nuclear count within the field. MEAN_ring spot average integrated intensity channel 2 represents the mean intensity of the p62 aggregates in the field. To properly identify the p62 aggregates the following settings were employed: Spot kernel radius: 10, ring distance from nucleus: 0, ring width: 10 pixels. Data was exported to Excel for further analysis. Data quality (batch-to-batch variation, similarity of replicates) was examined with Spotfire decision software and RNAeyes, in house software developed by The RNAi Consortium (TRC) of the Broad Institute.

A p62 aggregate score equal to Mean Ring Spot total intensity/Mean nuclei was calculated for each well. Viral infections were done in quadruplicate, with three plates receiving puromycin, one not. A comparison of the nuclei counts from the puro+/puro− plates allowed calculation of the infection efficiency of each hairpin. Hairpins with less than 1500 nuclei per well or those that had an infection efficiency less than 25% were omitted from subsequent analysis. A robust Z-score, a standard metric for high throughput assays (Birmingham A., et al. (2009) Nat Methods 6, 569-575), was calculated for each well. The three puromycin selected replicates were averaged, and this value was used for further analysis.

The in-house Gene-E software ranked genes at both a hairpin and a gene level. Attached to this application are candidate results (‘hits’) from either end of our analysis: those that resulted in profound elimination of p62, predicted to be autophagy inducers (Table 1), and those that resulted in profound accumulation of p62, predicted to be autophagy inhibitors (Table 2). These tables represent the weighted sum ranking of the data. In this metric, 75% of the score is based on the robust z-score of the second best hairpin for a given gene, while the other 25% of the score is based on the rank of the robust z-score of the best hairpin. Similar data was obtained when three other analysis measures were employed: cut-off based on a given standard deviation from controls, second best ranking, or RNA Interference Gene Enrichment Rank (RIGER) analysis based on the KS statistic as described previously (Luo B., et al. (2008) Proc Natl Acad Sci USA 105, 20380-20385).

A subset of viral plates were re-screened to ensure reproducibility of the assay and analyses.

Example 1

Table 1 shows results of a screen that led to elimination of p62.

	TABLE 1
	Symbol	GeneID	Gene Rank
	Mtor	56717	1
	Tssk3	58864	2
	Pik3c3	225326	3
	Cask	12361	4
	Lrguk	74354	5
	GeneID: 218456	218456	6
	Rab9	56382	7
	GeneID: 381390	381390	8
	Gm4922	237300	9
	Cdk8	264064	10
	Ephb1	270190	11
	Prkaca	18747	12
	Kpna2	16647	13
	Pldn	18457	14
	Scfd1	76983	15
	Ripk3	56532	16
	Trib3	228775	17
	Vapa	30960	18
	Trrap	100683	19
	Mpp3	13384	20
	GeneID: 381082	381082	21
	Mapk14	26416	22
	Adk	11534	23
	Ern1	78943	24
	Hip1r	29816	25
	Nek5	330721	26
	Alpk3	116904	27
	4932415M13Rik	211496	28
	Vps33b	233405	29
	1810024B03Rik	329509	30
	Chmp1a	234852	31
	Atp6v0a1	11975	32
	Ddr2	18214	33
	Cdk6	12571	34
	Stxbp3a	20912	35
	Map4k3	225028	36
	Egfr	13649	37
	Tpr	108989	38
	Tlk2	24086	39
	Rhoh	74734	40
	Sar1a	20224	41
	Vta1	66201	42
	Rab34	19376	43
	Brdt	114642	44
	GFP	−10	45
	GeneID: 384481	384481	46
	Dgka	13139	47
	Rabl2a	68708	48
	Snx10	71982	49
	Rhoa	11848	50
	Map3k11	26403	51
	Gm5374	385049	52
	D1g4	13385	53
	Rab7l1	226422	54
	Vamp8	22320	55
	N4bp2	333789	56
	Arf3	11842	57
	GeneID: 381309	381309	58
	Plk2	20620	59
	Cpne3	70568	60
	Hip1	215114	61
	Musk	18198	62
	Rab39b	67790	63
	Akt1	11651	64
	Arhgap24	231532	65
	Eif2ak1	15467	66
	Pfkfb4	270198	67
	Txndc3	73412	68
	Pim1	18712	69
	5730410E15Rik	319613	70
	1190002A17Rik	68870	71
	Gvin1	74558	72
	Pank4	269614	73
	Bmpr1a	12166	74
	Grk4	14772	75
	Pip5k1a	18720	76
	Prkcz	18762	77
	Nek3	23954	78
	Pgk1	18655	79
	Kras	16653	80
	Tssk6	83984	81
	Rab2a	59021	82
	Mertk	17289	83
	Ap1m2	11768	84
	Snx2	67804	85
	Ilk	16202	86
	Dgkk	331374	87
	Csnk1d	104318	88
	Rps6kb2	58988	89
	Map3k4	26407	90
	Ca1m1	12313	91
	Trim24	21848	92
	Fyn	14360	93
	Sh3bp5	24056	94
	Fn3k	63828	95
	Ippk	75678	96
	Tspan1	66805	97
	Cd81	12520	98
	Met	17295	99
	Pdgfra	18595	100
	Vrk2	69922	101
	Gem	14579	102
	Camkk1	55984	103
	Pfkl	18641	104
	Stx8	55943	105
	Tspan9	109246	106
	Stx17	67727	107
	Tm4sf1	17112	108
	Chmp4c	66371	109
	Tbk1	56480	110
	Dgkh	380921	111
	Ephb4	13846	112
	Rhof	23912	113
	Pik3cd	18707	114
	Mark1	226778	115
	Tspan2	70747	116
	Dync1li1	235661	117
	Trim27	19720	118
	Aurkc	20871	119
	Itpkb	320404	120
	Cav1	12389	121
	Bub1	12235	122
	Rap1b	215449	123
	Mapk10	26414	124
	Mapk8	26419	125
	Rab24	19336	126
	Kdr	16542	127
	Rab2b	76338	128
	Irak3	73914	129
	Map3k8	26410	130
	Csnk1a1	93687	131
	Rhobtb1	69288	132
	Mast2	17776	133
	Raf1	110157	134
	Arl11	219144	135
	Dgkq	110524	136
	Arfrp1	76688	137
	Mknk2	17347	138
	Erbb3	13867	139
	Rheb	19744	140
	Clk4	12750	141
	Map2k1	26395	142
	Sbk1	104175	143
	Clk3	102414	144
	Irak1	16179	145
	Pik3cb	74769	146
	Map3k12	26404	147
	Tk1	21877	148
	Aatk	11302	149
	Fastkd2	75619	150

TABLE 3
GeneID	RefSeq	Species	Description
270198	NM_001039217	MUS	6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-
		MUSCULUS	2,6-BIPHOSPHATASE 4
270198	NM_001039215	MUS	6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-
		MUSCULUS	2,6-BIPHOSPHATASE 4
270198	NM_001039216	MUS	6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-
		MUSCULUS	2,6-BIPHOSPHATASE 4
270198	NM_173019	MUS	6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-
		MUSCULUS	2,6-BIPHOSPHATASE 4
11768	NM_009678	MUS	ADAPTOR PROTEIN COMPLEX AP-1, MU 2
		MUSCULUS	SUBUNIT
11534	NM_134079	MUS	ADENOSINE KINASE
		MUSCULUS
11842	NM_007478	MUS	ADP-RIBOSYLATION FACTOR 3
		MUSCULUS
76688	NM_029702	MUS	ADP-RIBOSYLATION FACTOR RELATED
		MUSCULUS	PROTEIN 1
219144	NM_177337	MUS	ADP-RIBOSYLATION FACTOR-LIKE 11
		MUSCULUS
116904	NM_054085	MUS	ALPHA-KINASE 3
		MUSCULUS
11302	NM_007377	MUS	APOPTOSIS-ASSOCIATED TYROSINE
		MUSCULUS	KINASE
11975	NM_016920	MUS	ATPASE, H+ TRANSPORTING, LYSOSOMAL
		MUSCULUS	V0 SUBUNIT A1
20871	NM_020572	MUS	AURORA KINASE C
		MUSCULUS
333789	NM_001024917	MUS	BCL3 BINDING PROTEIN
		MUSCULUS
12166	NM_009758	MUS	BONE MORPHOGENETIC PROTEIN
		MUSCULUS	RECEPTOR, TYPE 1A
114642	NM_054054	MUS	BROMODOMAIN, TESTIS-SPECIFIC
		MUSCULUS
12235	NM_009772	MUS	BUDDING UNINHIBITED BY
		MUSCULUS	BENZIMIDAZOLES 1 HOMOLOG (S. CEREVISIAE)
17289	NM_008587	MUS	C-MER PROTO-ONCOGENE TYROSINE
		MUSCULUS	KINASE
55984	NM_018883	MUS	CALCIUM/CALMODULIN-DEPENDENT
		MUSCULUS	PROTEIN KINASE KINASE 1, ALPHA
12361	NM_009806	MUS	CALCIUM/CALMODULIN-DEPENDENT
		MUSCULUS	SERINE PROTEIN KINASE (MAGUK
			FAMILY)
12313	NM_007589	MUS	CALMODULIN 1
		MUSCULUS
12313	NM_007590	MUS	CALMODULIN 1
		MUSCULUS
12313	NM_009790	MUS	CALMODULIN 1
		MUSCULUS
104318	NM_027874	MUS	CASEIN KINASE 1, DELTA
		MUSCULUS
104318	NM_139059	MUS	CASEIN KINASE 1, DELTA
		MUSCULUS
93687	NM_146087	MUS	CASEIN KINASE I-ALPHA
		MUSCULUS
12389	NM_007616	MUS	CAVEOLIN, CAVEOLAE PROTEIN 1
		MUSCULUS
12520	NM_133655	MUS SP.	CD 81 ANTIGEN
12520	NM_133655	MUS	CD 81 ANTIGEN
		MUSCULUS
12750	NM_007714	MUS	CDC LIKE KINASE 4
		MUSCULUS
102414	NM_007713	MUS	CDC-LIKE KINASE 3
		MUSCULUS
70568	NM_027769	MUS	COPINE III
		MUSCULUS
12571	NM_009873	MUS	CYCLIN-DEPENDENT KINASE 6
		MUSCULUS
264064	NM_181570	MUS	CYCLIN-DEPENDENT KINASE 8
		MUSCULUS
264064	NM_153599	MUS	CYCLIN-DEPENDENT KINASE 8
		MUSCULUS
331374	NM_177914	MUS	DIACYLGLYCEROL KINASE KAPPA
		MUSCULUS
13139	NM_016811	MUS	DIACYLGLYCEROL KINASE, ALPHA
		MUSCULUS
380921	NM_001081336	MUS	DIACYLGLYCEROL KINASE, ETA
	XM_895030	MUSCULUS
380921	XM_902438	MUS	DIACYLGLYCEROL KINASE, ETA
		MUSCULUS
380921	XM_484397	MUS	DIACYLGLYCEROL KINASE, ETA
		MUSCULUS
380921	XM_916777	MUS	DIACYLGLYCEROL KINASE, ETA
		MUSCULUS
380921	XM_924467	MUS	DIACYLGLYCEROL KINASE, ETA
		MUSCULUS
110524	NM_199011	MUS	DIACYLGLYCEROL KINASE, THETA
		MUSCULUS
18214	NM_022563	MUS	DISCOIDIN DOMAIN RECEPTOR FAMILY,
		MUSCULUS	MEMBER 2
13385	NM_007864	MUS	DISCS, LARGE HOMOLOG 4 (DROSOPHILA)
		MUSCULUS
235661	NM_146229	MUS	DYNEIN CYTOPLASMIC 1 LIGHT
		MUSCULUS	INTERMEDIATE CHAIN 1
78943	NM_023913	MUS	ENDOPLASMIC RETICULUM (ER) TO
		MUSCULUS	NUCLEUS SIGNALLING 1
270190	NM_173447	MUS	EPH RECEPTOR B1
		MUSCULUS
13846	NM_010144	MUS	EPH RECEPTOR B4
		MUSCULUS
13649	NM_007912	MUS	EPIDERMAL GROWTH FACTOR RECEPTOR
		MUSCULUS
13649	NM_207655	MUS	EPIDERMAL GROWTH FACTOR RECEPTOR
		MUSCULUS
15467	NM_013557	MUS	EUKARYOTIC TRANSLATION INITIATION
		MUSCULUS	FACTOR 2 ALPHA KINASE 1
56717	NM_020009	MUS	FK506 BINDING PROTEIN 12-RAPAMYCIN
		MUSCULUS	ASSOCIATED PROTEIN 1
56717	NM_001039554	MUS	FK506 BINDING PROTEIN 12-RAPAMYCIN
		MUSCULUS	ASSOCIATED PROTEIN 1
63828	NM_001038699	MUS	FRUCTOSAMINE 3 KINASE
		MUSCULUS
63828	NM_022014	MUS	FRUCTOSAMINE 3 KINASE
		MUSCULUS
14360	NM_008054	MUS	FYN PROTO-ONCOGENE
		MUSCULUS
14772	NM_019497	MUS	G PROTEIN-COUPLED RECEPTOR KINASE
		MUSCULUS	2, GROUCHO GENE RELATED
			(DROSOPHILA)
14579	NM_010276	MUS	GTP BINDING PROTEIN (GENE
		MUSCULUS	OVEREXPRESSED IN SKELETAL MUSCLE)
74558	NM_001039160	MUS	GTPASE, VERY LARGE INTERFERON
		MUSCULUS	INDUCIBLE 1
74558	NM_029000	MUS	GTPASE, VERY LARGE INTERFERON
		MUSCULUS	INDUCIBLE 1
29816	NM_145070	MUS	HUNTINGTIN INTERACTING PROTEIN 1
		MUSCULUS	RELATED
237300	NM_177706	MUS	HYPOTHETICAL PROTEIN 4933423E17
		MUSCULUS
228775	NM_175093	MUS	INDUCED IN FATTY LIVER DYSTROPHY 2
		MUSCULUS
228775	NM_144554	MUS	INDUCED IN FATTY LIVER DYSTROPHY 2
		MUSCULUS
75678	NM_199056	MUS	INOSITOL 1,3,4,5,6-PENTAKISPHOSPHATE
		MUSCULUS	2-KINASE
320404	NM_001081175	MUS	INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE B
	XM_205854	MUSCULUS
320404	XM_923874	MUS	INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE B
		MUSCULUS
320404	XM_915655	MUS	INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE B
		MUSCULUS
320404	XM_900404	MUS	INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE B
		MUSCULUS
16202	NM_010562	MUS	INTEGRIN LINKED KINASE
		MUSCULUS
16179	NM_008363	MUS	INTERLEUKIN-1 RECEPTOR-ASSOCIATED
		MUSCULUS	KINASE 1
73914	NM_028679	MUS	INTERLEUKIN-1 RECEPTOR-ASSOCIATED
		MUSCULUS	KINASE 3
16647	NM_010655	MUS	KARYOPHERIN (IMPORTIN) ALPHA 2
		MUSCULUS
16542	NM_010612	MUS SP.	KINASE INSERT DOMAIN PROTEIN
		RECEPTOR
16542	NM_010612	MUS	KINASE INSERT DOMAIN PROTEIN
		MUSCULUS	RECEPTOR
17347	NM_021462	MUS	MAP KINASE-INTERACTING
		MUSCULUS	SERINE/THREONINE KINASE 2
226778	NM_145515	MUS	MAP/MICROTUBULE AFFINITY-
		MUSCULUS	REGULATING KINASE 1
13384	NM_007863	MUS	MEMBRANE PROTEIN, PALMITOYLATED 3
		MUSCULUS	(MAGUK P55 SUBFAMILY MEMBER 3)
17295	NM_008591	MUS	MET PROTO-ONCOGENE
		MUSCULUS
17776	NM_008641	MUS	MICROTUBULE ASSOCIATED
		MUSCULUS	SERINE/THREONINE KINASE 2
26414	NM_009158	MUS	MITOGEN ACTIVATED PROTEIN KINASE
		MUSCULUS	10
26416	NM_011951	MUS	MITOGEN ACTIVATED PROTEIN KINASE
		MUSCULUS	14
26419	NM_016700	MUS	MITOGEN ACTIVATED PROTEIN KINASE 8
		MUSCULUS
26395	NM_008927	MUS	MITOGEN ACTIVATED PROTEIN KINASE
		MUSCULUS	KINASE 1
26403	NM_022012	MUS	MITOGEN ACTIVATED PROTEIN KINASE
		MUSCULUS	KINASE KINASE 11
26404	NM_009582	MUS	MITOGEN ACTIVATED PROTEIN KINASE
		MUSCULUS	KINASE KINASE 12
26407	NM_011948	MUS	MITOGEN ACTIVATED PROTEIN KINASE
		MUSCULUS	KINASE KINASE 4
26410	NM_007746	MUS	MITOGEN ACTIVATED PROTEIN KINASE
		MUSCULUS	KINASE KINASE 8
18198	NM_001037128	MUS	MUSCLE, SKELETAL, RECEPTOR
		MUSCULUS	TYROSINE KINASE
18198	NM_010944	MUS	MUSCLE, SKELETAL, RECEPTOR
		MUSCULUS	TYROSINE KINASE
18198	NM_001037127	MUS	MUSCLE, SKELETAL, RECEPTOR
		MUSCULUS	TYROSINE KINASE
18198	NM_001037129	MUS	MUSCLE, SKELETAL, RECEPTOR
		MUSCULUS	TYROSINE KINASE
18198	NM_001037130	MUS	MUSCLE, SKELETAL, RECEPTOR
		MUSCULUS	TYROSINE KINASE
23954	NM_011848	MUS	NIMA (NEVER IN MITOSIS GENE A)-
		MUSCULUS	RELATED EXPRESSED KINASE 3
330721	NM_177898	MUS	NIMA (NEVER IN MITOSIS GENE A)-
		MUSCULUS	RELATED EXPRESSED KINASE 5
18457	NM_019788	MUS	PALLIDIN
		MUSCULUS
269614	NM_172990	MUS	PANTOTHENATE KINASE 4
		MUSCULUS
18707	NM_001029837	MUS	PHOSPHATIDYLINOSITOL 3-KINASE
		MUSCULUS	CATALYTIC DELTA POLYPEPTIDE
18707	NM_008840	MUS	PHOSPHATIDYLINOSITOL 3-KINASE
		MUSCULUS	CATALYTIC DELTA POLYPEPTIDE
74769	NM_029094	MUS	PHOSPHATIDYLINOSITOL 3-KINASE,
		MUSCULUS	CATALYTIC, BETA POLYPEPTIDE
18720	NM_008847	MUS	PHOSPHATIDYLINOSITOL-4-PHOSPHATE 5-
		MUSCULUS	KINASE, TYPE 1 BETA
18641	NM_008826	MUS	PHOSPHOFRUCTOKINASE, LIVER, B-TYPE
		MUSCULUS
18655	NM_008828	MUS	PHOSPHOGLYCERATE KINASE 1
		MUSCULUS
18655	XM_484116	MUS	PHOSPHOGLYCERATE KINASE 1
		MUSCULUS
18655	XM_485239	MUS	PHOSPHOGLYCERATE KINASE 1
		MUSCULUS
225326	NM_181414	MUS	PHOSPHOINOSITIDE-3-KINASE, CLASS 3
		MUSCULUS
18595	NM_011058	MUS	PLATELET DERIVED GROWTH FACTOR
		MUSCULUS	RECEPTOR, ALPHA POLYPEPTIDE
20620	NM_152804	MUS	POLO-LIKE KINASE 2 (DROSOPHILA)
		MUSCULUS
234852	NM_145606	MUS	PROCOLLAGEN (TYPE III) N-
		MUSCULUS	ENDOPEPTIDASE
18762	NM_001039079	MUS	PROTEIN KINASE C, ZETA
		MUSCULUS
18762	NM_008860	MUS	PROTEIN KINASE C, ZETA
		MUSCULUS
18747	NM_008854	MUS	PROTEIN KINASE, CAMP DEPENDENT,
		MUSCULUS	CATALYTIC, ALPHA
18712	NM_008842	MUS	PROVIRAL INTEGRATION SITE 1
		MUSCULUS
68708	NM_026817	MUS	RAB, MEMBER OF RAS ONCOGENE
		MUSCULUS	FAMILY-LIKE 2A
59021	NM_021518	MUS	RAB2, MEMBER RAS ONCOGENE FAMILY
		MUSCULUS
19336	NM_009000	MUS	RAB24, MEMBER RAS ONCOGENE FAMILY
		MUSCULUS
76338	NM_172601	MUS	RAB2B, MEMBER RAS ONCOGENE FAMILY
		MUSCULUS
19376	NM_033475	MUS SP.	RAB34, MEMBER OF RAS ONCOGENE
			FAMILY
19376	NM_033475	MUS	RAB34, MEMBER OF RAS ONCOGENE
		MUSCULUS	FAMILY
67790	NM_175122	MUS	RAB39B, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
226422	NM_144875	MUS	RAB7, MEMBER RAS ONCOGENE FAMILY-
		MUSCULUS	LIKE 1
56382	NM_019773	MUS	RAB9, MEMBER RAS ONCOGENE FAMILY
		MUSCULUS
11848	NM_016802	MUS	RAS HOMOLOG GENE FAMILY, MEMBER A
		MUSCULUS
23912	NM_175092	MUS	RAS HOMOLOG GENE FAMILY, MEMBER F
		MUSCULUS
74734	NM_001081105	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
	XM_132051	MUSCULUS
74734	XM_903893	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
		MUSCULUS
74734	XM_903680	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
		MUSCULUS
74734	XM_924029	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
		MUSCULUS
74734	XM_622908	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
		MUSCULUS
74734	XM_924031	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
		MUSCULUS
74734	XM_915950	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
		MUSCULUS
74734	XM_900704	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
		MUSCULUS
74734	XM_132051	MUS	RAS HOMOLOG GENE FAMILY, MEMBER H
		MUSCULUS
215449	NM_024457	MUS	RAS RELATED PROTEIN 1B
		MUSCULUS
19744	NM_053075	MUS	RAS-HOMOLOG ENRICHED IN BRAIN
		MUSCULUS
56532	NM_019955	MUS	RECEPTOR-INTERACTING SERINE-
		MUSCULUS	THREONINE KINASE 3
69288	NM_001081347	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
	XM_897555	MUSCULUS
69288	XM_897555	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_125637	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_920631	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_920652	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_897548	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_907869	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_897523	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_920622	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_897577	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_920637	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_920646	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_897586	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_920664	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_887557	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
69288	XM_920656	MUS	RHO-RELATED BTB DOMAIN CONTAINING 1
		MUSCULUS
58988	NM_021485	MUS	RIBOSOMAL PROTEIN S6 KINASE,
		MUSCULUS	POLYPEPTIDE 2
231532	NM_146161	MUS	RIKEN CDNA 0610025G21 GENE
		MUSCULUS
231532	NM_029270	MUS	RIKEN CDNA 0610025G21 GENE
		MUSCULUS
66201	NM_025418	MUS	RIKEN CDNA 1110059P08 GENE
		MUSCULUS
68870	NM_001033874	MUS	RIKEN CDNA 1190002A17 GENE
		MUSCULUS
329509	NM_198630	MUS	RIKEN CDNA 1810024B03 GENE
		MUSCULUS
66371	NM_025519	MUS	RIKEN CDNA 2310010I16 GENE
		MUSCULUS
75619	NM_172422	MUS	RIKEN CDNA 2810421I24 GENE
		MUSCULUS
225028	NM_001081357	MUS	RIKEN CDNA 4833416M01 GENE
	XM_898848	MUSCULUS
225028	XM_898825	MUS	RIKEN CDNA 4833416M01 GENE
		MUSCULUS
225028	XM_898819	MUS	RIKEN CDNA 4833416M01 GENE
		MUSCULUS
225028	XM_898848	MUS	RIKEN CDNA 4833416M01 GENE
		MUSCULUS
225028	XM_898843	MUS	RIKEN CDNA 4833416M01 GENE
		MUSCULUS
225028	XM_898830	MUS	RIKEN CDNA 4833416M01 GENE
		MUSCULUS
225028	XM_898852	MUS	RIKEN CDNA 4833416M01 GENE
		MUSCULUS
225028	XM_898838	MUS	RIKEN CDNA 4833416M01 GENE
		MUSCULUS
74354	XM_910825	MUS	RIKEN CDNA 4921528H16 GENE
		MUSCULUS
74354	XM_895665	MUS	RIKEN CDNA 4921528H16 GENE
		MUSCULUS
74354	NM_028886	MUS	LEUCINE-RICH REPEATS AND
	XM_133060	MUSCULUS	GUANYLATE KINASE DOMAIN
	XM_910825		CONTAINING (LRGUK)
74354	XM_921792	MUS	RIKEN CDNA 4921528H16 GENE
		MUSCULUS
211496	NM_177599	MUS	RIKEN CDNA 4932415M13 GENE
		MUSCULUS
211496	NM_001037718	MUS	RIKEN CDNA 4932415M13 GENE
		MUSCULUS
319613	NM_176998	MUS	RIKEN CDNA 5730410E15 GENE
		MUSCULUS
319613	NM_001032727	MUS	RIKEN CDNA 5730410E15 GENE
		MUSCULUS
319613	NM_178765	MUS	RIKEN CDNA 5730410E15 GENE
		MUSCULUS
110157	NM_029780	MUS	RIKEN CDNA 6430402F14 GENE
		MUSCULUS
215114	NM_146001	MUS	RIKEN CDNA A930014B11 GENE
		MUSCULUS
20224	NM_009120	MUS	SAR1 GENE HOMOLOG A (S. CEREVISIAE)
		MUSCULUS
76983	NM_029825	MUS	SEC1 FAMILY DOMAIN CONTAINING 1
		MUSCULUS
104175	NM_145587	MUS	SH3-BINDING KINASE 1
		MUSCULUS
24056	NM_011894	MUS	SH3-DOMAIN BINDING PROTEIN 5 (BTK-
		MUSCULUS	ASSOCIATED)
385049	XM_001473528	MUS	SIMILAR TO RHO-ASSOCIATED COILED-
	XM_904204	MUSCULUS	COIL FORMING KINASE 1
385049	XM_358017	MUS	SIMILAR TO RHO-ASSOCIATED COILED-
		MUSCULUS	COIL FORMING KINASE 1
381390	XM_485079	MUS	SIMILAR TO SERINE/THREONINE KINASE
		MUSCULUS
381390	XM_355352	MUS	GM14147 PREDICTED GENE 14147
		MUSCULUS
71982	NM_028035	MUS	SORTING NEXIN 10
		MUSCULUS
67804	NM_026386	MUS	SORTING NEXIN 2
		MUSCULUS
67727	NM_026343	MUS	SYNTAXIN 17
		MUSCULUS
55943	NM_018768	MUS	SYNTAXIN 8
		MUSCULUS
20912	NM_011504	MUS	SYNTAXIN BINDING PROTEIN 3A
		MUSCULUS
20912	NM_198326	MUS	SYNTAXIN BINDING PROTEIN 3A
		MUSCULUS
56480	NM_019786	MUS	TANK-BINDING KINASE 1
		MUSCULUS
58864	NM_080442	MUS	TESTIS-SPECIFIC SERINE KINASE 3
		MUSCULUS
83984	NM_032004	MUS	TESTIS-SPECIFIC SERINE KINASE 6
		MUSCULUS
66805	NM_133681	MUS	TETRASPANIN 1
		MUSCULUS
70747	NM_027533	MUS	TETRASPANIN 2
		MUSCULUS
109246	NM_175414	MUS	TETRASPANIN 9
		MUSCULUS
73412	NM_181591	MUS	THIOREDOXIN DOMAIN CONTAINING 3
		MUSCULUS	(SPERMATOZOA)
21877	NM_009387	MUS SP.	THYMIDINE KINASE 1
21877	NM_009387	MUS	THYMIDINE KINASE 1
		MUSCULUS
11651	NM_009652	MUS	THYMOMA VIRAL PROTO-ONCOGENE 1
		MUSCULUS
24086	NM_011903	MUS	TOUSLED-LIKE KINASE 2 (ARABIDOPSIS)
		MUSCULUS
100683	XM_891798	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_899747	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_918276	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	NM_001081362	MUS	TRANSFORMATION/TRANSCRIPTION
	XM_899741	MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_899763	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_917315	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_886427	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_899754	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_925613	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_925614	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_899771	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_918275	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_917317	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_899733	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
100683	XM_925612	MUS	TRANSFORMATION/TRANSCRIPTION
		MUSCULUS	DOMAIN-ASSOCIATED PROTEIN
108989	NM_133780	MUS	TRANSLOCATED PROMOTER REGION
		MUSCULUS
17112	NM_008536	MUS	TRANSMEMBRANE 4 SUPERFAMILY
		MUSCULUS	MEMBER 1
21848	NM_145076	MUS SP.	TRIPARTITE MOTIF PROTEIN 24
21848	NM_145076	MUS	TRIPARTITE MOTIF PROTEIN 24
		MUSCULUS
19720	NM_009054	MUS	TRIPARTITE MOTIF PROTEIN 27
		MUSCULUS
13867	NM_010153	MUS	V-ERB-B2 ERYTHROBLASTIC LEUKEMIA
		MUSCULUS	VIRAL ONCOGENE HOMOLOG 3 (AVIAN)
16653	NM_010937	MUS	V-KI-RAS2 KIRSTEN RAT SARCOMA VIRAL
		MUSCULUS	ONCOGENE HOMOLOG
16653	NM_021284	MUS	V-KI-RAS2 KIRSTEN RAT SARCOMA VIRAL
		MUSCULUS	ONCOGENE HOMOLOG
69922	NM_027260	MUS	VACCINIA RELATED KINASE 2
		MUSCULUS
233405	NM_178070	MUS	VACUOLAR PROTEIN SORTING 33B
		MUSCULUS	(YEAST)
22320	NM_016794	MUS	VESICLE-ASSOCIATED MEMBRANE
		MUSCULUS	PROTEIN 8
30960	NM_013933	MUS	VESICLE-ASSOCIATED MEMBRANE
		MUSCULUS	PROTEIN, ASSOCIATED PROTEIN A
218456	N/A	MUS	SIMILAR TO NUCLEOSIDE DIPHOSPHATE
		MUSCULUS	KINASE B (NDK B) (NDP KINASE B) (P18
381082	N/A	MUS	SIMILAR TO MITOGEN-ACTIVATED
		MUSCULUS	PROTEIN KINASE 14 ISOFORM 1;
			CYTOKINE SUP
381309	N/A	MUS	SIMILAR TO CDC42-BINDING PROTEIN
		MUSCULUS	KINASE ALPHA; MYTONIC DYSTROPHY
			KINAS
384481	N/A	MUS	SIMILAR TO URIDINE MONOPHOSPHATE
		MUSCULUS	KINASE

Example 2

Table 2 shows the results of a screen that led to accumulation of p62. The results are presented in a positive to negative ranking according to p62.

	TABLE 2
	Symbol	Gene ID	Gene rank
	Chmp4b	75608	1
	Kpnb1	16211	2
	Irak1	16179	3
	Rhov	228543	4
	Gm5285	383956	5
	Eef1a2	13628	6
	Ulk1	22241	7
	Epha5	13839	8
	LUCIFERASE	−14	9
	Rhot1	59040	10
	Stk35	67333	11
	Gtpbp4	69237	12
	Prkaa2	108079	13
	Pfkfb4	270198	14
	Map3k14	53859	15
	Rhog	56212	16
	Mylk4	238564	17
	Rhoc	11853	18
	Cdc42bpg	240505	19
	GeneID: 245619	245619	20
	Tssk3	58864	21
	Ap1s1	11769	22
	Smok2a	27263	23
	Agap3	213990	24
	Shpk	74637	25
	Cdk3	69681	26
	N4bp2	333789	27
	Ap3m2	64933	28
	Rab20	19332	29
	Vps4b	20479	30
	Hgs	15239	31
	Pik3c2a	18704	32
	Etnk1	75320	33
	Sh3gl2	20404	34
	Prkar2b	19088	35
	Rac1	19353	36
	Gpn1	74254	37
	Nkiras2	71966	38
	Pik3cg	30955	39
	Prkag1	19082	40
	Phkg1	18682	41
	GFP	−10	42
	Vps13a	271564	43
	Txk	22165	44
	Dab2	13132	45
	Mast3	234385	46
	Pdgfrl	68797	47
	Vps36	70160	48
	GeneID: 233024	#N/A	49
	Prkaa1	105787	50
	Pdpk1	18607	51
	Doc2b	13447	52
	Gm5374	385049	53
	Nuak1	77976	54
	Acvr2a	11480	55
	Snx3	54198	56
	Mapk9	26420	57
	Camk4	12326	58
	Grk6	26385	59
	Nme2	18103	60
	Pak4	70584	61
	Stradb	227154	62
	Rab19	19331	63
	Pi4ka	224020	64
	Camk2g	12325	65
	Dlg4	13385	66
	Cdadc1	71891	67
	Mapkapk3	102626	68
	Tjp2	21873	69
	Gm318	240091	70
	Cdkl5	382253	71
	Snx16	74718	72
	Cdc2l1	12537	73
	Trpm6	225997	74
	Ap1g1	11765	75
	Rab14	68365	76
	RFP	−12	77
	Pmvk	68603	78
	Pank3	211347	79
	Pkmyt1	268930	80
	Crkl	12929	81
	Fastkd5	380601	82
	Etnk2	214253	83
	Ephb6	13848	84
	GeneID: 229309	#N/A	85
	Aldh18a1	56454	86
	Ak1	11636	87
	Eif2ak1	15467	88
	Kdr	16542	89
	Gsk3a	606496	90
	Gck	103988	91
	Araf	11836	92
	Arf5	11844	93
	Dgkb	217480	94
	Ltk	17005	95
	Rab8a	17274	96
	Ryk	20187	97
	Gm4862	229879	98
	Mknk2	17347	99
	GeneID: 243968	#N/A	100
	Arpc1a	56443	101
	2310079N02Rik	66566	102
	GeneID: 381981	#N/A	103
	Nme7	171567	104
	Ak2	11637	105
	Tesk2	230661	106
	Sik1	17691	107
	Synj2	20975	108
	Chek1	12649	109
	GeneID: 384257	#N/A	110
	Mapk3	26417	111
	Itpka	228550	112
	Gm1078	381835	113
	Ciita	12265	114
	GeneID: 381061	#N/A	115
	Uck1	22245	116
	Rps6ka2	20112	117
	Prps111	75456	118
	Rap1a	109905	119
	Rac3	170758	120
	Gm4776	212225	121
	Eif2s3y	26908	122
	Trpd5213	66745	123
	GeneID: 245068	#N/A	124
	lacZ	−15	125
	Sec23a	20334	126
	Arfrp1	76688	127
	Ras110b	276952	128
	Hspb8	80888	129
	Rab3d	19340	130
	Arl4d	80981	131
	GeneID: 384894	#N/A	132
	GeneID: 381446	#N/A	133
	GeneID: 268321	#N/A	134
	Anxa7	11750	135
	Hras1	15461	136
	Wnk4	69847	137
	Melk	17279	138
	Frk	14302	139
	Mapkapk2	17164	140
	Rragc	54170	141
	Phka1	18679	142
	Becn1	56208	143
	Pik3c2g	18705	144
	Acvr2b	11481	145
	Tie1	21846	146
	Camk1g	215303	147
	Phkg2	68961	148
	Csnk1g1	214897	149
	Khk	16548	150
	Fgfr4	14186	151
	Gucy2e	14919	152
	Gm1893	381599	153
	Rras2	66922	154
	Ikbkb	16150	155
	Gsg2	14841	156
	Tspan7	21912	157
	Keap1	50868	158
	Rab6b	270192	159
	Ern1	78943	160
	Golga5	27277	161
	Mlkl	74568	162
	Epha6	13840	163
	Ephb2	13844	164
	Camkk1	55984	165
	Psmc1	19179	166
	Ankk1	244859	167
	B2m	12010	168
	Nkiras1	69721	169
	Rab15	104886	170
	Smg1	233789	171
	Ckmt2	76722	172
	Gm9824	432447	173
	Nek4	23955	174
	Trp53rk	76367	175
	Mtor	56717	176
	Vps39	269338	177
	Rps6kl1	238323	178
	Drg2	13495	179
	Mark4	232944	180
	Syt15	319508	181
	Rerg	232441	182
	Hipk2	15258	183
	Cav3	12391	184
	Grk5	14773	185
	Map2k3	26397	186
	Smok4a	272667	187
	Rab7	19349	188
	2810408M09Rik	381406	189
	Chmp5	76959	190
	Prkcd	18753	191
	Snx2	67804	192
	Mst1r	19882	193
	Stk19	54402	194
	Gk2	14626	195
	Acvrl1	11482	196
	Syt8	55925	197
	Cyth3	19159	198
	Tspan6	56496	199
	Irak3	73914	200

TABLE 4
GeneID	RefSeq	Species	Description
18607	NM_011062	MUS	3-PHOSPHOINOSITIDE DEPENDENT
		MUSCULUS	PROTEIN KINASE-1
270198	NM_173019	MUS	6-PHOSPHOFRUCTO-2-
		MUSCULUS	KINASE/FRUCTOSE-2,6-
			BIPHOSPHATASE 4
270198	NM_001039217	MUS	6-PHOSPHOFRUCTO-2-
		MUSCULUS	KINASE/FRUCTOSE-2,6-
			BIPHOSPHATASE 4
270198	NM_001039216	MUS	6-PHOSPHOFRUCTO-2-
		MUSCULUS	KINASE/FRUCTOSE-2,6-
			BIPHOSPHATASE 4
270198	NM_001039215	MUS	6-PHOSPHOFRUCTO-2-
		MUSCULUS	KINASE/FRUCTOSE-2,6-
			BIPHOSPHATASE 4
56443	NM_019767	MUS	ACTIN RELATED PROTEIN ⅔
		MUSCULUS	COMPLEX, SUBUNIT 1A
11482	NM_009612	MUS	ACTIVIN A RECEPTOR, TYPE II-LIKE 1
		MUSCULUS
11480	NM_007396	MUS	ACTIVIN RECEPTOR IIA
		MUSCULUS
11481	NM_007397	MUS	ACTIVIN RECEPTOR IIB
		MUSCULUS
11765	NM_009677	MUS	ADAPTOR PROTEIN COMPLEX AP-1,
		MUSCULUS	GAMMA 1 SUBUNIT
11769	NM_007457	MUS	ADAPTOR PROTEIN COMPLEX AP-1,
		MUSCULUS	SIGMA 1
64933	NM_029505	MUS	ADAPTOR-RELATED PROTEIN
		MUSCULUS	COMPLEX 3, MU 2 SUBUNIT
11636	NM_021515	MUS	ADENYLATE KINASE 1
		MUSCULUS
11637	NM_001033966	MUS	ADENYLATE KINASE 2
		MUSCULUS
11637	NM_016895	MUS	ADENYLATE KINASE 2
		MUSCULUS
11844	NM_007480	MUS	ADP-RIBOSYLATION FACTOR 5
		MUSCULUS
76688	NM_029702	MUS	ADP-RIBOSYLATION FACTOR
		MUSCULUS	RELATED PROTEIN 1
80981	NM_031160	MUS	ADP-RIBOSYLATION FACTOR-LIKE 4D
		MUSCULUS
56454	NM_019698	MUS	ALDEHYDE DEHYDROGENASE 18
		MUSCULUS	FAMILY, MEMBER A1
56454	NM_153554	MUS	ALDEHYDE DEHYDROGENASE 18
		MUSCULUS	FAMILY, MEMBER A1
227154	NM_172656	MUS	AMYOTROPHIC LATERAL SCLEROSIS 2
		MUSCULUS	(JUVENILE) CHROMOSOME REGION,
			CANDIDAT . . .
244859	NM_172922	MUS	ANKYRIN REPEAT AND KINASE
		MUSCULUS	DOMAIN CONTAINING 1
11750	NM_009674	MUS	ANNEXIN A7
		MUSCULUS
333789	NM_001024917	MUS	BCL3 BINDING PROTEIN
		MUSCULUS
56208	NM_019584	MUS	BECLIN 1 (COILED-COIL, MYOSIN-LIKE
		MUSCULUS	BCL2-INTERACTING PROTEIN)
12010	NM_009735	MUS	BETA-2 MICROGLOBULIN
		SPRETUS
12010	NM_009735	MUS	BETA-2 MICROGLOBULIN
		MUSCULUS
215303	NM_144817	MUS	CALCIUM/CALMODULIN-DEPENDENT
		MUSCULUS	PROTEIN KINASE I GAMMA
12325	NM_178597	MUS	CALCIUM/CALMODULIN-DEPENDENT
		MUSCULUS	PROTEIN KINASE II GAMMA
12325	NM_001039139	MUS	CALCIUM/CALMODULIN-DEPENDENT
		MUSCULUS	PROTEIN KINASE II GAMMA
12325	NM_001039138	MUS	CALCIUM/CALMODULIN-DEPENDENT
		MUSCULUS	PROTEIN KINASE II GAMMA
12326	NM_009793	MUS	CALCIUM/CALMODULIN-DEPENDENT
		MUSCULUS	PROTEIN KINASE IV
55984	NM_018883	MUS	CALCIUM/CALMODULIN-DEPENDENT
		MUSCULUS	PROTEIN KINASE KINASE 1, ALPHA
74637	NM_029031	MUS	CARBOHYDRATE KINASE-LIKE
		MUSCULUS
214897	NM_173185	MUS	CASEIN KINASE 1, GAMMA 1
		MUSCULUS
12391	NM_007617	MUS	CAVEOLIN 3
		MUSCULUS
240505	NM_001033342	MUS	CDC42 BINDING PROTEIN KINASE
	XM_906449	MUSCULUS	GAMMA (DMPK-LIKE)
240505	NM_001033342	MUS	CDC42 BINDING PROTEIN KINASE
	XM_140553	MUSCULUS	GAMMA (DMPK-LIKE)
12537	NM_007661	MUS	CELL DIVISION CYCLE 2 HOMOLOG (S. POMBE)-
		MUSCULUS	LIKE 1
213990	NM_139153	MUS	CENTAURIN, GAMMA 3
		MUSCULUS
12649	NM_007691	MUS	CHECKPOINT KINASE 1 HOMOLOG (S. POMBE)
		MUSCULUS
13848	NM_007680	MUS	CHICKEN EPH/ELK RECEPTOR-LIKE
		MUSCULUS	PROTEIN
75608	NM_029362	MUS	CHROMATIN MODIFYING PROTEIN 4B
		MUSCULUS
76959	NM_029814	MUS	CHROMATIN MODIFYING PROTEIN 5
		MUSCULUS
12265	NM_007575	MUS	CLASS II TRANSACTIVATOR
		MUSCULUS
76722	NM_198415	MUS	CREATINE KINASE, MITOCHONDRIAL 2
		MUSCULUS
69681	NM_027165	MUS	CYCLIN-DEPENDENT KINASE 3
		MUSCULUS
382253	XM_912167	MUS	CYCLIN-DEPENDENT KINASE-LIKE 5
	NM_001024624	MUSCULUS
	NM_027986
	XM_914101
	XM_001000245
	XM_001000259
	XM_001000276
	XM_001000293
71891	XM_001000308	MUS	CYTIDINE AND DCMP DEAMINASE
	XM_001000321	MUSCULUS	DOMAIN CONTAINING 1
	XM_001000336
	XM_001002774
	XM_127813
	XM_914101
	XM_985192
71891	XM_901860	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_923095	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_894723	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_923089	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_901847	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_901853	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_923082	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_923072	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_923086	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_901857	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_127813	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_901866	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
71891	XM_923093	MUS	CYTIDINE AND DCMP DEAMINASE
		MUSCULUS	DOMAIN CONTAINING 1
13495	NM_021354	MUS	DEVELOPMENTALLY REGULATED GTP
		MUSCULUS	BINDING PROTEIN 2
217480	NM_178681	MUS	DIACYLGLYCEROL KINASE, BETA
		MUSCULUS
13132	NM_001037905	MUS	DISABLED HOMOLOG 2 (DROSOPHILA)
		MUSCULUS
13132	NM_001008702	MUS	DISABLED HOMOLOG 2 (DROSOPHILA)
		MUSCULUS
13132	NM_023118	MUS	DISABLED HOMOLOG 2 (DROSOPHILA)
		MUSCULUS
13385	NM_007864	MUS	DISCS, LARGE HOMOLOG 4
		MUSCULUS	(DROSOPHILA)
13447	NM_007873	MUS	DOUBLE C2, BETA
		MUSCULUS
13839	NM_007937	MUS	ECK-LIKE SEQUENCE 1
		MUSCULUS
78943	NM_023913	MUS	ENDOPLASMIC RETICULUM (ER) TO
		MUSCULUS	NUCLEUS SIGNALLING 1
13840	NM_007938	MUS	EPH RECEPTOR A6
		MUSCULUS
13844	NM_010142	MUS	EPH RECEPTOR B2
		MUSCULUS
75320	XM_284250	MUS	ETHANOLAMINE KINASE 1
		MUSCULUS
75320	NM_029250	MUS	ETHANOLAMINE KINASE 1
	XM_908334	MUSCULUS
	XM_979562
214253	NM_175443	MUS	ETHANOLAMINE KINASE 2
		MUSCULUS
13628	NM_007906	MUS	EUKARYOTIC TRANSLATION
		MUSCULUS	ELONGATION FACTOR 1 ALPHA 2
15467	NM_013557	MUS	EUKARYOTIC TRANSLATION
		MUSCULUS	INITIATION FACTOR 2 ALPHA KINASE 1
26908	NM_012011	MUS	EUKARYOTIC TRANSLATION
		MUSCULUS	INITIATION FACTOR 2, SUBUNIT 3,
			STRUCTURAL GENE . . .
18103	NM_008705	MUS	EXPRESSED IN NON-METASTATIC
		MUSCULUS	CELLS 2, PROTEIN
380601	NM_198176	MUS	EXPRESSED SEQUENCE C78212
		MUSCULUS
14186	NM_008011	MUS	FIBROBLAST GROWTH FACTOR
		MUSCULUS	RECEPTOR 4
56717	NM_020009	MUS	FK506 BINDING PROTEIN 12-
		MUSCULUS	RAPAMYCIN ASSOCIATED PROTEIN 1
56717	NM_001039554	MUS	FK506 BINDING PROTEIN 12-
		MUSCULUS	RAPAMYCIN ASSOCIATED PROTEIN 1
14302	NM_010237	MUS	FYN-RELATED KINASE
		MUSCULUS
14773	NM_018869	MUS	G PROTEIN-COUPLED RECEPTOR
		MUSCULUS	KINASE 5
26385	NM_011938	MUS	G PROTEIN-COUPLED RECEPTOR
		MUSCULUS	KINASE 6
26385	NM_001038018	MUS	G PROTEIN-COUPLED RECEPTOR
		MUSCULUS	KINASE 6
381835	XM_355840	MUS	GENE MODEL 1078, (NCBI)
		MUSCULUS
381835	XM_911944	MUS	GENE MODEL 1078, (NCBI)
		MUSCULUS
381599	XM_355556	MUS	GENE MODEL 1893, (NCBI)
		MUSCULUS
240091	XM_139919	MUS	GENE MODEL 318, (NCBI)
[replaced	XM_619462	MUSCULUS
with	XM_001481303
545204]	XM_622850
14841	NM_010353	MUS	GERM CELL-SPECIFIC GENE 2
		MUSCULUS
103988	NM_010292	MUS	GLUCOKINASE
		MUSCULUS
14626	NM_010294	MUS	GLYCEROL KINASE 2
		MUSCULUS
606496	NM_001031667	MUS	GLYCOGEN SYNTHASE KINASE 3
		MUSCULUS	ALPHA
27277	NM_013747	MUS	GOLGI AUTOANTIGEN, GOLGIN
		MUSCULUS	SUBFAMILY A, 5
69237	NM_027000	MUS	GTP BINDING PROTEIN 4
		MUSCULUS
14919	NM_008192	MUS	GUANYLATE CYCLASE 2E
		MUSCULUS
15461	NM_008284	MUS SP.	HARVEY RAT SARCOMA VIRUS
			ONCOGENE 1
15461	NM_008284	MUS	HARVEY RAT SARCOMA VIRUS
		MUSCULUS	ONCOGENE 1
80888	NM_030704	MUS	HEAT SHOCK PROTEIN 8
		MUSCULUS
15239	NM_008244	MUS	HGF-REGULATED TYROSINE KINASE
		MUSCULUS	SUBSTRATE
15258	NM_010433	MUS	HOMEODOMAIN INTERACTING
		MUSCULUS	PROTEIN KINASE 2
212225	NM_172504	MUS	HYPOTHETICAL PROTEIN 4930509O22
		MUSCULUS
16150	NM_010546	MUS	INHIBITOR OF KAPPAB KINASE BETA
		MUSCULUS
228550	NM_146125	MUS	INOSITOL 1,4,5-TRISPHOSPHATE 3-
		MUSCULUS	KINASE A
16179	NM_008363	MUS	INTERLEUKIN-1 RECEPTOR-
		MUSCULUS	ASSOCIATED KINASE 1
73914	NM_028679	MUS	INTERLEUKIN-1 RECEPTOR-
		MUSCULUS	ASSOCIATED KINASE 3
16211	NM_008379	MUS	KARYOPHERIN (IMPORTIN) BETA 1
		MUSCULUS
50868	NM_016679	MUS	KELCH-LIKE ECH-ASSOCIATED
		MUSCULUS	PROTEIN 1
16548	NM_008439	MUS	KETOHEXOKINASE
		MUSCULUS
16542	NM_010612	MUS	KINASE INSERT DOMAIN PROTEIN
		MUSCULUS	RECEPTOR
16542	NM_010612	MUS SP.	KINASE INSERT DOMAIN PROTEIN
			RECEPTOR
17005	NM_008523	MUS	LEUKOCYTE TYROSINE KINASE
		MUSCULUS
17005	NM_206942	MUS	LEUKOCYTE TYROSINE KINASE
		MUSCULUS
17005	NM_206941	MUS	LEUKOCYTE TYROSINE KINASE
		MUSCULUS
17005	NM_203345	MUS	LEUKOCYTE TYROSINE KINASE
		MUSCULUS
19882	NM_009074	MUS SP.	MACROPHAGE STIMULATING 1
			RECEPTOR (C-MET-RELATED
			TYROSINE KINASE)
19882	NM_009074	MUS	MACROPHAGE STIMULATING 1
		MUSCULUS	RECEPTOR (C-MET-RELATED
			TYROSINE KINASE)
17164	NM_008551	MUS	MAP KINASE-ACTIVATED PROTEIN
		MUSCULUS	KINASE 2
17347	NM_021462	MUS	MAP KINASE-INTERACTING
		MUSCULUS	SERINE/THREONINE KINASE 2
232944	NM_172279	MUS	MAP/MICROTUBULE AFFINITY-
		MUSCULUS	REGULATING KINASE 4
17279	NM_010790	MUS	MATERNAL EMBRYONIC LEUCINE
		MUSCULUS	ZIPPER KINASE
268930	NM_023058	MUS	MEMBRANE-ASSOCIATED TYROSINE-
		MUSCULUS	AND THREONINE-SPECIFIC CDC2-
			INHIBITORY KI . . .
234385	NM_199308	MUS	MICROTUBULE ASSOCIATED
[replaced	XM_134245	MUSCULUS	SERINE/THREONINE KINASE 3
with	XM_913385
546071]
234385	XM_888290	MUS	MICROTUBULE ASSOCIATED
		MUSCULUS	SERINE/THREONINE KINASE 3
234385	XM_922751	MUS	MICROTUBULE ASSOCIATED
		MUSCULUS	SERINE/THREONINE KINASE 3
234385	XM_897983	MUS	MICROTUBULE ASSOCIATED
		MUSCULUS	SERINE/THREONINE KINASE 3
234385	XM_897989	MUS	MICROTUBULE ASSOCIATED
		MUSCULUS	SERINE/THREONINE KINASE 3
234385	XM_620670	MUS	MICROTUBULE ASSOCIATED
		MUSCULUS	SERINE/THREONINE KINASE 3
234385	XM_897962	MUS	MICROTUBULE ASSOCIATED
		MUSCULUS	SERINE/THREONINE KINASE 3
234385	XM_897954	MUS	MICROTUBULE ASSOCIATED
		MUSCULUS	SERINE/THREONINE KINASE 3
26420	NM_016961	MUS	MITOGEN ACTIVATED PROTEIN
		MUSCULUS	KINASE 9
26420	NM_207692	MUS	MITOGEN ACTIVATED PROTEIN
		MUSCULUS	KINASE 9
26397	NM_008928	MUS	MITOGEN ACTIVATED PROTEIN
		MUSCULUS	KINASE KINASE 3
53859	NM_016896	MUS	MITOGEN-ACTIVATED PROTEIN
		MUSCULUS	KINASE KINASE KINASE 14
102626	NM_178907	MUS	MITOGEN-ACTIVATED PROTEIN
		MUSCULUS	KINASE-ACTIVATED PROTEIN KINASE 3
74568	NM_029005	MUS	MIXED LINEAGE KINASE DOMAIN-
	XM_001003995	MUSCULUS	LIKE
	XM_001003998
	XM_356104
	XM_895027
	XM_916960
	XM_924589
	XM_924589
74568	XM_895027	MUS	MIXED LINEAGE KINASE DOMAIN-
		MUSCULUS	LIKE
74568	XM_902435	MUS	MIXED LINEAGE KINASE DOMAIN-
		MUSCULUS	LIKE
74568	XM_916960	MUS	MIXED LINEAGE KINASE DOMAIN-
		MUSCULUS	LIKE
74568	XM_356104	MUS	MIXED LINEAGE KINASE DOMAIN-
		MUSCULUS	LIKE
74568	XM_924585	MUS	MIXED LINEAGE KINASE DOMAIN-
		MUSCULUS	LIKE
69721	NM_023526	MUS	NFKB INHIBITOR INTERACTING RAS-
		MUSCULUS	LIKE PROTEIN 1
71966	NM_028024	MUS	NFKB INHIBITOR INTERACTING RAS-
		MUSCULUS	LIKE PROTEIN 2
23955	NM_011849	MUS	NIMA (NEVER IN MITOSIS GENE A)-
		MUSCULUS	RELATED EXPRESSED KINASE 4
171567	NM_178071	MUS	NON-METASTATIC CELLS 7, PROTEIN
		MUSCULUS	EXPRESSED IN
171567	NM_138314	MUS	NON-METASTATIC CELLS 7, PROTEIN
		MUSCULUS	EXPRESSED IN
70584	NM_027470	MUS	P21 (CDKN1A)-ACTIVATED KINASE 4
		MUSCULUS
211347	NM_145962	MUS	PANTOTHENATE KINASE 3
		MUSCULUS
18704	NM_011083	MUS	PHOSPHATIDYLINOSITOL 3-KINASE,
		MUSCULUS	C2 DOMAIN CONTAINING, ALPHA
			POLYPEPTIDE
18705	NM_207683	MUS	PHOSPHATIDYLINOSITOL 3-KINASE,
		MUSCULUS	C2 DOMAIN CONTAINING, GAMMA
			POLYPEPTIDE
18705	NM_011084	MUS	PHOSPHATIDYLINOSITOL 3-KINASE,
		MUSCULUS	C2 DOMAIN CONTAINING, GAMMA
			POLYPEPTIDE
224020	NM_001001983	MUS	PHOSPHATIDYLINOSITOL 4-KINASE,
		MUSCULUS	CATALYTIC, ALPHA POLYPEPTIDE
30955	NM_020272	MUS	PHOSPHOINOSITIDE-3-KINASE,
		MUSCULUS	CATALYTIC, GAMMA POLYPEPTIDE
68603	NM_026784	MUS	PHOSPHOMEVALONATE KINASE
		MUSCULUS
18679	NM_008832	MUS	PHOSPHORYLASE KINASE ALPHA 1
		MUSCULUS
18679	NM_173021	MUS	PHOSPHORYLASE KINASE ALPHA 1
		MUSCULUS
18682	NM_011079	MUS	PHOSPHORYLASE KINASE GAMMA 1
		MUSCULUS
68961	NM_026888	MUS	PHOSPHORYLASE KINASE, GAMMA 2
		MUSCULUS	(TESTIS)
68797	NM_026840	MUS	PLATELET-DERIVED GROWTH FACTOR
		MUSCULUS	RECEPTOR-LIKE
19159	NM_011182	MUS	PLECKSTRIN HOMOLOGY, SEC7 AND
		MUSCULUS	COILED-COIL DOMAINS 3
19179	NM_008947	MUS	PROTEASE (PROSOME, MACROPAIN)
		MUSCULUS	26S SUBUNIT, ATPASE 1
18753	NM_011103	MUS	PROTEIN KINASE C, DELTA
		MUSCULUS
105787	NM_001013367	MUS	PROTEIN KINASE, AMP-ACTIVATED,
		MUSCULUS	ALPHA 1 CATALYTIC SUBUNIT
108079	NM_178143	MUS	PROTEIN KINASE, AMP-ACTIVATED,
		MUSCULUS	ALPHA 2 CATALYTIC SUBUNIT
19082	NM_016781	MUS	PROTEIN KINASE, AMP-ACTIVATED,
		MUSCULUS	GAMMA 1 NON-CATALYTIC SUBUNIT
19088	NM_011158	MUS	PROTEIN KINASE, CAMP DEPENDENT
		MUSCULUS	REGULATORY, TYPE II BETA
26417	NM_011952	MUS SP.	PROTEIN KINASE, MITOGEN
			ACTIVATED KINASE 3
26417	NM_011952	MUS	PROTEIN KINASE, MITOGEN
		MUSCULUS	ACTIVATED KINASE 3
68365	NM_026697	MUS	RAB14, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
104886	NM_134050	MUS	RAB15, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
19331	NM_011226	MUS	RAB19, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
19332	NM_011227	MUS	RAB20, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
19340	NM_031874	MUS	RAB3D, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
270192	NM_173781	MUS	RAB6B, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
19349	NM_009005	MUS	RAB7, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
17274	NM_023126	MUS SP.	RAB8A, MEMBER RAS ONCOGENE
			FAMILY
17274	NM_023126	MUS	RAB8A, MEMBER RAS ONCOGENE
		MUSCULUS	FAMILY
11853	NM_007484	MUS	RAS HOMOLOG GENE FAMILY,
		MUSCULUS	MEMBER C
56212	NM_019566	MUS	RAS HOMOLOG GENE FAMILY,
		MUSCULUS	MEMBER G
59040	NM_021536	MUS	RAS HOMOLOG GENE FAMILY,
		MUSCULUS	MEMBER T1
228543	NM_145530	MUS	RAS HOMOLOG GENE FAMILY,
		MUSCULUS	MEMBER V
232441	NM_181988	MUS	RAS-LIKE, ESTROGEN-REGULATED,
		MUSCULUS	GROWTH-INHIBITOR
276952	NM_001013386	MUS	RAS-LIKE, FAMILY 10, MEMBER B
		MUSCULUS
19353	NM_009007	MUS	RAS-RELATED C3 BOTULINUM
		MUSCULUS	SUBSTRATE 1
170758	NM_133223	MUS	RAS-RELATED C3 BOTULINUM
		MUSCULUS	SUBSTRATE 3
54170	NM_017475	MUS	RAS-RELATED GTP BINDING C
		MUSCULUS
109905	NM_145541	MUS	RAS-RELATED PROTEIN-1A
		MUSCULUS
20187	NM_013649	MUS SP.	RECEPTOR-LIKE TYROSINE KINASE
20187	NM_013649	MUS	RECEPTOR-LIKE TYROSINE KINASE
		MUSCULUS
66922	NM_025846	MUS	RELATED RAS VIRAL (R-RAS)
		MUSCULUS	ONCOGENE HOMOLOG 2
20112	NM_011299	MUS	RIBOSOMAL PROTEIN S6 KINASE,
		MUSCULUS	RELATED SEQUENCE 1
238323	NM_146244	MUS	RIBOSOMAL PROTEIN S6 KINASE-LIKE 1
		MUSCULUS
75456	NM_029294	MUS	RIKEN CDNA 1700011K15 GENE
		MUSCULUS
66566	NM_025636	MUS	RIKEN CDNA 2310079N02 GENE
		MUSCULUS
233789	NM_001031814	MUS	RIKEN CDNA 2610207I05 GENE
		MUSCULUS
272667	XM_895217	MUS	RIKEN CDNA 4930513D10 GENE
		MUSCULUS
272667	XM_142762	MUS	RIKEN CDNA 4930513D10 GENE
		MUSCULUS
272667	XM_912174	MUS	RIKEN CDNA 4930513D10 GENE
		MUSCULUS
271564	NM_173028	MUS	RIKEN CDNA 4930516E05 GENE
		MUSCULUS
20334	NM_009147	MUS	SEC23A (S. CEREVISIAE)
		MUSCULUS
54402	NM_019442	MUS	SERINE/THREONINE KINASE 19
		MUSCULUS
67333	NM_001038635	MUS	SERINE/THREONINE KINASE 35
		MUSCULUS
67333	NM_183262	MUS	SERINE/THREONINE KINASE 35
		MUSCULUS
20404	NM_019535	MUS	SH3-DOMAIN GRB2-LIKE 2
		MUSCULUS
383956	XM_916921	MUS	SIMILAR TO MAP/MICROTUBULE
		MUSCULUS	AFFINITY-REGULATING KINASE 3
383956	XM_357348	MUS	SIMILAR TO MAP/MICROTUBULE
		MUSCULUS	AFFINITY-REGULATING KINASE 3
245068	XM_918167	MUS	SIMILAR TO MAP/MICROTUBULE
		MUSCULUS	AFFINITY-REGULATING KINASE 4
			(MAP/MICROTUBU . . .
245068	XM_142402	MUS	SIMILAR TO MAP/MICROTUBULE
		MUSCULUS	AFFINITY-REGULATING KINASE 4
			(MAP/MICROTUBU . . .
238564	NM_001166030	MUS	SIMILAR TO MYOSIN LIGHT CHAIN
	XM_910560	MUSCULUS	KINASE
238564	XM_111421	MUS	SIMILAR TO MYOSIN LIGHT CHAIN
		MUSCULUS	KINASE
229879	XM_143595	MUS	GM4862 PREDICTED GENE 4862
	XM_001478899	MUSCULUS	SIMILAR TO NON-METASTATIC CELLS
			2, PROTEIN (NM23B) EXPRESSED IN
			ISOFOR . . .
			GM5374 PREDICTED GENE 5374
385049	XM_904204	MUS	SIMILAR TO RHO-ASSOCIATED
	XM_001473528	MUSCULUS	COILED-COIL FORMING KINASE 1
385049	XM_358017	MUS	SIMILAR TO RHO-ASSOCIATED
		MUSCULUS	COILED-COIL FORMING KINASE 1
17691	NM_010831	MUS	SNF1-LIKE KINASE
		MUSCULUS
74718	NM_029068	MUS	SORTING NEXIN 16
		MUSCULUS
67804	NM_026386	MUS	SORTING NEXIN 2
		MUSCULUS
54198	NM_017472	MUS	SORTING NEXIN 3
		MUSCULUS
27263	XM_889037	MUS	SPERM MOTILITY KINASE 2
		MUSCULUS
27263	XM_620264	MUS	SPERM MOTILITY KINASE 2
		MUSCULUS
27263	XM_135656	MUS	SPERM MOTILITY KINASE 2
		MUSCULUS
27263	XM_889060	MUS	SPERM MOTILITY KINASE 2
		MUSCULUS
27263	XM_620265	MUS	SPERM MOTILITY KINASE 2
		MUSCULUS
27263	XM_907097	MUS	SPERM MOTILITY KINASE 2
		MUSCULUS
20975	NM_011523	MUS	SYNAPTOJANIN 2
		MUSCULUS
55925	NM_018802	MUS	SYNAPTOTAGMIN VIII
		MUSCULUS
319508	NM_176931	MUS	SYNAPTOTAGMIN XV
		MUSCULUS
319508	NM_181529	MUS	SYNAPTOTAGMIN XV
		MUSCULUS
230661	NM_146151	MUS	TESTIS-SPECIFIC KINASE 2
		MUSCULUS
58864	NM_080442	MUS	TESTIS-SPECIFIC SERINE KINASE 3
		MUSCULUS
56496	NM_019656	MUS	TETRASPANIN 6
		MUSCULUS
21912	NM_019634	MUS	TETRASPANIN 7
		MUSCULUS
21873	NM_011597	MUS	TIGHT JUNCTION PROTEIN 2
		MUSCULUS
225997	NM_153417	MUS	TRANSIENT RECEPTOR POTENTIAL
		MUSCULUS	CATION CHANNEL, SUBFAMILY M,
			MEMBER 6
381406	NM_023815	MUS	TRP53 REGULATING KINASE
		MUSCULUS
76367	NM_023815	MUS	TRP53 REGULATING KINASE
		MUSCULUS
76367	NM_001007581	MUS	TRP53 REGULATING KINASE
		MUSCULUS
381406	NM_001007581	MUS	TRP53 REGULATING KINASE
		MUSCULUS
66745	NM_025741	MUS	TUMOR PROTEIN D52-LIKE 3
		MUSCULUS
22165	NM_013698	MUS	TXK TYROSINE KINASE
		MUSCULUS
21846	NM_011587	MUS	TYROSINE KINASE RECEPTOR 1
		MUSCULUS
21846	NM_011587	MUS SP.	TYROSINE KINASE RECEPTOR 1
22241	NM_009469	MUS	UNC-51 LIKE KINASE 1 (C. ELEGANS)
		MUSCULUS
22245	NM_011675	MUS	URIDINE-CYTIDINE KINASE 1
		MUSCULUS
12929	NM_007764	MUS	V-CRK SARCOMA VIRUS CT10
		MUSCULUS	ONCOGENE HOMOLOG (AVIAN)-LIKE
11836	NM_009703	MUS	V-RAF MURINE SARCOMA 3611 VIRAL
		MUSCULUS	ONCOGENE HOMOLOG
70160	NM_027338	MUS	VACUOLAR PROTEIN SORTING 36
		MUSCULUS	(YEAST)
20479	NM_009190	MUS	VACUOLAR PROTEIN SORTING 4B
		MUSCULUS	(YEAST)
269338	NM_178851	MUS	VPS39
		MUSCULUS
269338	NM_147153	MUS	VPS39
		MUSCULUS
69847	NM_175638	MUS	WNK LYSINE DEFICIENT PROTEIN
		MUSCULUS	KINASE 4
74254	NM_133756	MUS	XPA BINDING PROTEIN 1
		MUSCULUS
77976	NM_001004363	MUS	ZNUAK FAMILY, SNF1-LIKE KINASE, 1
		MUSCULUS
229309	N/A	MUS	SIMILAR TO PHOSPHOGLYCERATE
		MUSCULUS	KINASE (EC 2.7.2.3) - MOUSE
243968	N/A	MUS	SIMILAR TO KIAA1883 PROTEIN
		MUSCULUS
245619	NM_027067	MUS	GLYCOGEN SYNTHASE KINASE 3
[Replaced		MUSCULUS	ALPHA PROBABLE PSEUDOGENE
with
69389]
268321	N/A	MUS	SIMILAR TO NUCLEOSIDE
[Replaced		MUSCULUS	DIPHOSPHATE KINASE B
with
432482]
381061	NM_013741	MUS	SPERM MOTILITY KINASE 2A
[Replaced		MUSCULUS
with
27263]
381446	N/A	MUS	SIMILAR TO PHOSPHOGLYCERATE
		MUSCULUS	KINASE (EC 2.7.2.3) - MOUSE
381981	N/A	MUS	SIMILAR TO AARF DOMAIN
		MUSCULUS	CONTAINING KINASE 4
384257	N/A	MUS	SIMILAR TO NUCLEOSIDE
		MUSCULUS	DIPHOSPHATE KINASE B
384894	N/A	MUS	SIMILAR TO CYTOSOLIC THYMIDINE
		MUSCULUS	KINASE
432447	N/A	MUS	PHOSPHATIDYLETHANOLAMINE-
		MUSCULUS	BINDING PROTEIN PSEUDOGENE

In Tables 1 and 2, the numbers in the column labeled GeneID correspond with the accession numbers in the Entrez GeneID database made available by the National Center for Biotechnology Information (NCBI). The Entrez GeneID may be used to identify corresponding sequences such as, for example, genomic DNA, mRNA and protein sequences.

In Tables 3 and 4, each GeneID is presented along with its corresponding accession number(s) from the NCBI Reference Sequences (RefSeq) database for mRNA transcripts. Through the accession numbers, the sequences are readily available. Table 3 contains sequences from Table 1. Table 4 contains sequences from Table 2. The rank order of the GenelDs in Tables 1 and 2 are not maintained in Tables 3 and 4.

The terms and expressions which have been employed are used as terms of descriptions and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

In addition, where features or aspects of the invention are described in terms of Markush group or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

All references, including the disclosures of each patent, patent application, publication and accession number to database sequences, cited or described in this document are hereby incorporated herein by reference, in their entireties.

Claims

1. A method for identifying compounds that inhibit or stimulate the autophagy pathway for treatment of a disease state associated with an autophagy pathway defect, comprising measuring the effect of one or more test compounds on the inhibition or stimulation of a product of one or more of the genes or gene fragments identified in Table 3 or Table 4.

2. A method for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect, comprising testing a biological sample from an individual for a characteristic of one or more polypeptides produced by expression of one or more of the genes or gene fragments identified in Table 3 or Table 4 that is indicative of said disease state, wherein said characteristic is selected from the presence of at least one of said polypeptides, the absence of at least one of said polypeptides, an elevated level of at least one of said polypeptides, a reduced level of at least one of said polypeptides and, for two or more of said polypeptides, combinations thereof.

3. A device for detecting the expression of a plurality of autophagy-related genes associated with an autophagy pathway defect, said device comprising a substrate to which is affixed at known locations a plurality of probes, wherein the probes comprise: a) a plurality of oligonucleotides or polynucleotides, each of which specifically binds to a different sequence selected from any of the sequences identified in Table 3 or Table 4 or fragments thereof; orb) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof produced by expression of a gene or gene fragment comprising any of the sequences identified in Table 3 or Table 4 or fragments thereof.

4. A kit for assaying the expression of autophagy-related genes associated with an autophagy pathway defect, comprising at least one container and a collection of two or more probes, wherein the probes comprise: a) oligonucleotides or polynucleotides that specifically bind to two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof; orb) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof.