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Title:  cDNAs and proteins involved in hypoxia, circadian and orphan signal transduction pathways, and methods of use
United States Patent: 
September 12, 2006

Bradfield; Christopher A. (Madison, WI), Gu; Yi Zhong (Sunnyvale, CA), Hogenesch; John B. (San Diego, CA)
Wisconsin Alumni Research Foundation (Madison, WI)
Appl. No.:  09/555,362
November 27, 1998
PCT Filed: 
November 27, 1998
PCT No.: 
371(c)(1),(2),(4) Date: 
July 24, 2000
PCT Pub. No.: 
PCT Pub. Date: 
June 10, 1999


Woodbury College's Master of Science in Law


The present invention provides isolated nucleic acids and proteins that are new and distinct members of the bHLH-PAS superfamily of transcription regulators. These "MOPs" (members of PAS) are useful in a variety of research, diagnostic and therapeutic applications. Several of the MOPs of the present invention are .alpha.-class hypoxia-inducible factors. Several other of the MOPs of the invention are involved in circadian signal transduction.


Characterization of MOPS 1 9

Our hypothesis in accordance with the present invention was that additional bHLH-PAS proteins are encoded in the mammalian genome and that some of these proteins are involved in mediating the pleiotropic response to potent AHR agonists like TCDD. It has been observed that other bHLH superfamilies employ multiple dimeric partnerships to control complex biological processes, such as myogenesis (MyoD/myogenin), cellular proliferation (Myc, Max, Mad) and neurogenesis (achaete-scute/daughterless). The observation that bHLH proteins often restrict their dimerization to within members of the same gene family (i.e., "homotypic interactions") and that this restriction may occur as the result of constraints imposed by both primary (e.g., bHLH) and secondary dimerization surfaces (e.g., leucine zippers and PAS), prompted us to screen for additional bHLH-PAS proteins and test each protein for its capacity to interact with either the AHR or ARNT. The ultimate objective was to identify MOPs that were physiologically relevant partners of either the AHR or ARNT in vivo. Our prediction was that such proteins might respond to or modulate the AHR signaling pathway or other signaling pathways involving ARNT.

To rapidly identify expressed genes, the "expressed sequence tag" (EST) approach was developed, whereby a cDNA library is constructed and randomly selected clones are sequenced from both vector arms (Adams et al., Science 252: 1651 1656, 1991). These partial sequences, generally 200 400 bp, are deposited in a number of computer databases that can be readily analyzed using a variety of search algorithms. As of 1996, the I.M.A.G.E. Consortium has deposited over 300,000 human ESTs, generated from different tissues and developmental time periods into publicly accessible databases, identifying approximately 40,000 unique cDNA clones (Lennon et al., Genomics 33: 151 152, 1996). The availability of these sequences and plasmids harboring their corresponding cDNA clones provided a means by which to identify novel members of the bHLH-PAS family by nucleotide homology screening of available EST databases.

At the time this invention was initiated, the human AHR and ARNT and the drosophila SIM and PER were the only PAS protein that had been described. Therefore, we used the nucleotide sequences encoding their PAS domains as query sequences in BLASTN searches of the available EST databases. Using this strategy in an iterative fashion and confirming each hit with a reverse BLASTX search, we have identified eight cDNAs referred to herein as members of the PAS superfamily, or "MOPs". Using PCR, we were able to obtain the complete ORFs of MOPs 1 4, and extensive but incomplete ORFs of MOP5. We have also identified four more MOPs, MOPs 6, 7, 8 and 9, and obtained their complete ORFs.

While MOPs 1 5 were being characterized, Wang and colleagues identified two factors involved in cellular response to hypoxia, HIF1.alpha. and HIF1.beta.. These proteins are identical to MOP1 and ARNT, respectively (Wang et al., Proc. Natl. Acad. Sci. USA 92: 5510 5514, 1995). Thus, of the nine MOPs we have cloned, seven have not been previously characterized. For consistency herein, we describe MOP1 extensively, and describe heretofore undisclosed methods of using MOP1.

The experimental approach taken in accordance with the present invention has significantly expanded the number of known members of the emerging bHLH-PAS superfamily of transcriptional regulators. Along with the MOPs described herein, five additional mammalian bHLH-PAS proteins have been identified, HIF1.alpha. (MOP1, as described above), SIM1, SIM2, ARNT2, and SRC-1 (Wang et al., 1995, supra; Hirose et al., Mol. Cell. Biol. 16: 1706 1713, 1996; Fan et al., Mol. Cell. Neurosci. 7: 1 16, 1996; Ema et al., Mol. Cell. Biol. 16: 5865 5875, 1996; Chen et al., Nat. Genet. 10: 9 10, 1995; and Kamei et al., Cell 85: 403 414, 1996). To compare amino acid sequences of these proteins, we performed a CLUSTAL alignment with the bHLH-PAS domains of MOPs 1 5 and all the known family members using a PAM250 residue weight table (Higgins & Sharp, Gene (Amst.) 73: 237 244, 1988). The two most related members were MOP1/HIF1.alpha. and MOP2, which shared 66% identity in the PAS domain. A comparison of these two proteins reveals only a single amino acid difference in the basic region and 83% identity in the HLH region. This sequence similarity is in agreement with our contention (discussed in Example 1) that MOP1/HIF1.alpha. and MOP2 function analogously, interacting with the same heterodimeric partners and binding similar enhancer sequences in vivo. A comparison of MOP3 and ARNT and a comparison of MOP5 and SIM reveal 40% and 38% identity in the PAS domain, respectively. The basic regions of MOP3 and ARNT have only three substitutions, while the HLH domains share 66% identity, again suggesting that the two proteins may regulate similar or identical enhancer sequences (half sites).

A CLUSTAL alignment of the C-termini of MOPs 1 5 and the previously identified PAS members demonstrated that these regions are not well conserved (data not shown) (Burbach et al., Proc. Natl. Acad. Sci. USA 89: 8185 8189, 1992). This lack of conservation may indicate that the C-termini of these genes have divergent functions, or that the functions harbored in the C-termini can be accomplished by a variety of different sequences. For example, the C-termini of the AHR, ARNT, and SIM all harbor potent transactivation domains, yet display little sequence homology.

To characterize the evolutionary and functional relationships of these proteins, we performed a parsimony analysis to identify functionally related subsets. A dendrogram representing the primary amino acid relationship between the PAS domains of these proteins is illustrated in FIG. 4 (see Original Patent). This schematic suggests that major groups exist for eukaryotic PAS family members. The AHR, drSIMILAR, MOP1/HIF1.alpha., MOP2, drTRACHEALESS, MOP5, and SIM exist in one group, ARNT, muARNT2, MOP3, and MOP4 in another and PER and huSRC-1 exist in their own groups. Interestingly, this pattern reflects what is known functionally about the existing PAS members. The AHR, SIM, MOP1/HIF1.alpha. and MOP2 have all been shown to heterodimerize with the ARNT molecule and bind DNA. Additionally, the AHR and SIM are known to interact with HSP90, a chaperonin protein necessary for the signaling of the AHR and a number of steroid receptor family members in response to ligand. Based on these groupings, MOP5 may also be an ARNT-interacting protein and a candidate for interacting with Hsp90 and being activated by small molecule ligands. The observation that ARNT has been shown to be capable of forming DNA binding homodimers and as heterodimers with a number of previously identified members of the bHLH-PAS family (at least in vitro), suggests that it plays a role in a number of biological processes. Based on their similarity with ARNT, MOP3 and MOP4 may be candidates for binding DNA as homodimers, or for interacting with multiple bHLH-PAS members, possibly from the AHR group.

In addition to the relevance of the above data to TCDD signaling, they also reveal additional factors important to cellular responses to hypoxic stress. HIF1.alpha./MOP1 and MOP2 appear to share a common dimeric partner--ARNT, and are capable of regulating a common battery of genes. This notion is supported by three lines of evidence: (1) both MOP1 and MOP2 interact with ARNT as defined by coimmunoprecipitation or two-hybrid assay; (2) they have similar DNA half-site specificities when complexed with ARNT; and (3) they are both transcriptionally active from TACGTG enhancers in vivo. The observation that HIF1.alpha./MOP1 and MOP2 have markedly different tissue distributions suggests that these two proteins may be regulating similar batteries of genes in response to different environmental stimuli. Alternatively, these proteins may be involved in restricting expression of certain groups of genes regulated by TACGTG-dependent enhancers. Finally, it is now known that MOP2 and MOP7 are subunits of a "HIF1-like" complex (i.e. a "HIF2.alpha." and a "HIF3.alpha., respectively) that regulates hypoxia responsive genes in distinct sets of tissues.

From the foregoing discussion, it can be seen that, while the MOPs share certain common features among themselves and with other new members of the bHLH-PAS superfamily, each of MOPs 2 9 is a distinctive and unique member of that family. cDNA and deduced amino acid sequences for each of MOPs 1 9 is set forth at the end of this specification. General features of each MOP are summarized below. In addition, MOPs 1 5 are described in great detail in Example 1, MOP3 is specifically described in Example 2 and MOP 7 is described in Example 3.

MOP1: The nucleotide and deduced amino acid sequences of a cDNA encoding MOP1 are set forth herein as SEQ ID NOS: 1 and 10, respectively. The cDNA includes a complete coding sequence for MOP1. As discussed above, MOP1 is known more commonly in the literature as HIF (Hypoxia-Inducible Factor)-1.alpha. (Wang et al., 1995, supra). The factor is induced by low oxygen. It interacts with HSP90 and with ARNT (AHR's binding partner). The ARNT-dimerized factor regulates expression of erythropoietin, among other genes.

MOP2: The nucleotide and deduced amino acid sequences of a cDNA encoding MOP2 are set forth herein as SEQ ID NOS: 2 and 11, respectively. The cDNA includes a complete coding sequence for MOP2. MOP2 appears to be related structurally and functionally to MOP1. Similar to MOP1, MOP2 interacts with ARNT, but not AHR, and drives transcription in its ARNT-dimerized form. Unlike MOP1, MOP2 does not appear to interact significantly with HSP90. MOP2 is induced by low oxygen and may be involved in hypoxia responses in different cells and tissues than is MOP1. MOP2 is sometimes referred to herein as HIF2.alpha..

MOP3: The nucleotide and deduced amino acid sequences of a cDNA encoding MOP3 are set forth herein as SEQ ID NOS: 3 and 12, respectively. The cDNA includes a complete coding sequence for MOP3. MOP 3 and MOP 4 are related to each other as binding partners, analogous to ARNT and AHR, respectively. As described in greater detail in Example 2, in addition to being a specific partner for MOP4, MOP3 is a general dimerization partner for a subset of the bHLH/PAS superfamily of transcriptional regulators. MOP3 interacts with MOP4, CLOCK, HIF1.alpha. and HIF2.alpha.. The MOP3-MOP4 heterodimer binds a CACGTGA-containing DNA element. Moreover, MOP3-MOP4 and MOP3-CLOCK complexes bind this element in COS-1 cells and drive transcription from a linked luciferase reporter gene. A high-affinity DNA binding site has also been deduced for a MOP3-HIF1.alpha. complex (TACGTGA). MOP3-HIF1.alpha. and MOP3-HIF2.alpha. heterodimers bind this element, drive transcription, and respond to cellular hypoxia.

MOP3 also binds HSP90, and may be conditionally activated (like AHR) depending on whether it is bound to HSP90 (see Example 1) (of the MOP3/MOP4 dimerization pair, one appears to be conditionally activated, but as yet it is unclear which one). Evidence from Drosophila and rat suggest that MOP3 (cycle/bMAL1b) is regulated in a circadian manner.

MOP3 expression appears to be controlled by alternate 5' promoter regions. MOP3 mRNA expression overlaps in a number of tissues with each of its four potential partner molecules in vivo.

MOP4: The nucleotide and deduced amino acid sequences of a cDNA encoding MOP4 are set forth herein as SEQ ID NOS: 4 and 13, respectively. The cDNA includes an apparently complete coding sequence for MOP4. MOP4 appears to be a human ortholog of a recently identified murine gene called "Clock", for its involvement in circadian rhythms (King et al., Cell 89: 641 653). MOP4 also interacts with HSP90 and, as discussed above, is the dimerization partner of MOP3, and may be conditionally activated. MOP4 appears to be localized in the cytoplasm.

MOP5: The nucleotide and deduced amino acid sequences of a cDNA encoding MOP5 are set forth herein as SEQ ID NOS: 5 and 14, respectively. The cDNA includes a partial coding sequence for MOP5; however a complete coding sequence for MOP5 has become publicly available subsequent to the making of the present invention (GenBank Accession No. U77968, submitted Nov. 11, 1996, published Jan. 21, 1997 by Zhou et al., Proc. Natl. Acad. Sci. USA 94: 713 718).

MOP6: The nucleotide and deduced amino acid sequences of a cDNA encoding MOP6 (of human origin) are set forth herein as SEQ ID NOS: 6 and 15, respectively. The cDNA includes a complete coding sequence for MOP6. The nucleotide sequence of MOP6 is fairly unique. It is most similar in the 5' region to the bHLH-PAS member trachealess, which suggests that MOP6 may be a regulator (developmental or otherwise) of hypoxia. Functional data shows that MOP6 forms a partnership with ARNT and drives a hypoxia responsive element.

MOP7: The nucleotide and deduced amino acid sequences of a cDNA encoding MOP7 are set forth herein as SEQ ID NOS: 7 and 16, respectively. The cDNA includes a complete coding sequence for MOP7. In accordance with this invention, MOP7 has been characterized as a new hypoxia-inducible factor, and therefore is sometimes referred to herein as HIF3.alpha.. The expression profile of MOP7 is as follows: testis, thymus>[lung, brain, heart, liver, skeletal muscle]>[skin, stomach, small intestine, kidney]. This expression profile is distinct from any of MOP1, MOP2, MOP3, AHR and ARNT, suggesting a different functional role for MOP7. MOP7 is most closely related to MOP1/HIF1.alpha. and MOP2 (HIF2.alpha.), as described in greater detail in Example 3. Accordingly, MOP7 is likely to regulate the same genes as does HIF1.alpha. and HIF2.alpha., as evidenced by its dimerization with the same partners (ARNT, MOP3) and recognition of the same core response element. This, combined with the unique tissue-specific expression of MOP7 suggests that it may have a functional role associated with response to low oxygen in the tissues in which it is expressed.

MOP8: The nucleotide and deduced amino acid sequences of a cDNA encoding MOP8 are set forth herein as SEQ ID NOS: 8 and 17, respectively. The cDNA includes a complete coding sequence for MOP8. Like MOP4 and MOP3, MOP8 may be involved in regulation of circadian rhythm. MOP8 shows sequence similarity to other genes involved in the circadian pathway (human PER, Drosophila PER, human RIGUI).

MOP9: The nucleotide and deduced amino acid sequence of a cDNA encoding MOP9 are set forth herein as SEQ ID NOS: 9 and 18, respectively. Two ESTs (GenBank AA577389, AA576971) corresponding to a novel bHLH-PAS protein homologous to MOP3/bMAL1 were identified by TBLASTN searches of the Drosophila homolog of MOP3. Upon characterization, these clones were revealed to be truncated, and one of which appeared to be a splice variant. The cDNA was cloned from human brain mRNA, and alternative 5' splicing was found probably reflecting multiple promoters. A BLASTX search of the MOP 9 sequence reveals that it displays extended homology to MOP3 (E-154). These data suggest that MOP9 also pairs with CLOCK and MOP4 and binds an E-box element with flanking region specificity.

Although specific MOP clones are described and exemplified herein, this invention is intended to encompass nucleic acid sequences and proteins from humans and other species that are sufficiently similar to be used interchangeably with the exemplified MOP nucleic acids and proteins for the purposes described below. It will be appreciated by those skilled in the art that MOP-encoding nucleic acids from diverse species, and particularly mammalian species, should possess a sufficient degree of homology with human MOPs so as to be interchangeably useful in various applications. The present invention, therefore, is drawn to MOP-encoding nucleic acids and encoded proteins from any species in which they are found, preferably to MOPs of mammalian origin, and most preferably to MOPs of human origin. Additionally, in the same manner that structural homologs of human MOPs are considered to be within the scope of this invention, functional homologs are also considered to be within the scope of this invention.

Allelic variants and natural mutants of SEQ ID NOS: 1 9 or 10 17 are likely to exist within the human genome and within the genomes of other species. Because such variants are expected to possess certain differences in nucleotide and amino acid sequence, this invention provides isolated MOP-encoding nucleic acid molecules having at least about 65% (and preferably over 75%) sequence homology in the coding region with the nucleotide sequences set forth as SEQ ID NOS: 1 9 (and, most preferably, specifically comprising the coding regions of any of SEQ ID NOS: 1 9). This invention also provides isolated MOPs having at least about 75% (preferably 85% or greater) sequence homology with the amino acid sequence of SEQ ID NOS: 10 18. Because of the natural sequence variation likely to exist among the MOPs and nucleic acids encoding them, one skilled in the art would expect to find up to about 25 35% nucleotide sequence variation, while still maintaining the unique properties of the MOPs of the present invention. Such an expectation is due in part to the degeneracy of the genetic code, as well as to the known evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the protein. Accordingly, such variants are considered substantially the same as one another and are included within the scope of the present invention.

For purposes of this invention, the term "substantially the same" refers to nucleic acid or amino acid sequences having sequence variation that do not materially affect the nature of the protein. With particular reference to nucleic acid sequences, the term "substantially the same" is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide. With reference to amino acid sequences, the term "substantially the same" refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function of the protein. The terms "percent identity" and "percent similarity" are also used herein in comparisons among amino acid sequences. These terms are intended to be defined as they are in the UWGCG sequence analysis program (Devereaux et al., Nucl. Acids Res. 12: 387 397, 1984), available from the University of Wisconsin.

The following description sets forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. Unless otherwise specified, general cloning procedures, such as those set forth in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter "Sambrook et al.") or Ausubel et al. (eds) Current Protocols in Molecular Biology, John Wiley & Sons (1998) (hereinafter "Ausubel et al.") are used.

III. Preparation of MOP Nucleic Acid Molecules, MOP Proteins and Anti-MOP Antibodies

A. Nucleic Acid Molecules

Nucleic acid molecules encoding the MOPs of the invention may be prepared by two general methods: (1) They may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.

The availability of nucleotide sequence information, such as a full length cDNA having any of SEQ ID NOS: 1 9, enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a several-kilobase double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.

Nucleic acid sequences encoding MOPs may be isolated from appropriate biological sources using methods known in the art. In a preferred embodiment, cDNA clones are isolated from libraries of human origin. In an alternative embodiment, genomic clones encoding MOPs may be isolated. Alternatively, cDNA or genomic clones encoding MOPs from other species, preferably mammalian species, may be obtained.

In accordance with the present invention, nucleic acids having the appropriate level sequence homology with the coding regions of any of Sequence I.D. Nos. 1 9 may be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations may be performed, according to the method of Sambrook et al., using a hybridization solution comprising: 5.times.SSC, 5.times. Denhardt's reagent, 1.0% SDS, 100 .mu.g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37 C. for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2.times.SSC and 1% SDS; (2) 15 minutes at room temperature in 2.times.SSC and 0.1% SDS; (3) 30 minutes 1 hour at C. in 1.times.SSC and 1% SDS; (4) 2 hours at 42 in 1.times.SSC and 1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al., 1989): C.+16.6Log [Na+]+0.41(% G+C)-0.63 (% formamide)-600/#bp in duplex As an illustration of the above formula, using [N+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T.sub.m is C. The T.sub.m, of a DNA duplex decreases by 1 C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of C.

Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in plasmid cloning/expression vector, such as pGEM-T (Promega Biotech, Madison, Wis.) or pBluescript (Stratagene, La Jolla, Calif.), either of which is propagated in a suitable E. coli host cell.

MOP nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, such as selected segments of the cDNA having any of SEQ ID NOS: 1 9. Such oligonucleotides are useful as probes for detecting MOP genes or mRNA in test samples of cells, tissue or other biological sources, e.g. by PCR amplification, or for the positive or negative regulation of expression of MOP genes at or before translation of the mRNA into proteins.

B. Proteins

MOP proteins of the present invention may be prepared in a variety of ways, according to known methods. The proteins may be purified from appropriate sources, e.g., cultured or intact cells or tissues.

Alternatively, the availability of nucleic acids molecules encoding MOPs enables production of the MOP proteins using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such a pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville, Md.

According to a preferred embodiment, larger quantities of MOP proteins may be produced by expression in a suitable procaryotic or eucaryotic system. For example, part or all of a DNA molecule, such as any of the cDNAs having SEQ ID NOS: 1 9, may be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.

The MOPs produced by gene expression in a recombinant procaryotic or eucyarotic system may be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein. Such methods are commonly used by skilled practitioners. The MOP proteins of the invention, prepared by the aforementioned methods, may be analyzed according to standard procedures.

The present invention also provides antibodies capable of immunospecifically binding to MOP proteins of the invention. Polyclonal or monoclonal antibodies directed toward any of MOPs 1 9 may be prepared according to standard methods. Monoclonal antibodies may be prepared according to general methods of Kohler and Milstein, following standard protocols. In a preferred embodiment, antibodies have been prepared, which react immunospecifically with various epitopes of the MOPs.

Polyclonal or monoclonal antibodies that immunospecifically interact with MOPs can be utilized for identifying and purifying such proteins. For example, antibodies may be utilized for affinity separation of proteins with which they immunospecifically interact. Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules. Other uses of anti-MOP antibodies are described below.

IV. Uses of MOP-Encoding Nucleic Acids, MOP Proteins and Anti-MOP Antibodies

A. MOP-Encoding Nucleic Acids

MOP-encoding nucleic acids may be used for a variety of purposes in accordance with the present invention. MOP-encoding DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of genes encoding MOPs. Methods in which MOP-encoding nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR). In addition, recombinant cellular assay systems to examine signal transduction pathways in which the MOPs are involved are described below.

The MOP-encoding nucleic acids of the invention may also be utilized as probes to identify related genes either from humans or from other species. As is well known in the art, hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology. Thus, MOP-encoding nucleic acids may be used to advantage to identify and characterize other genes of varying degrees of relation to the respective MOPs, thereby enabling further characterization the AHR or related signaling cascades. Additionally, they may be used to identify genes encoding proteins that interact with MOPs (e.g., by the "interaction trap" technique, or modifications thereof, as described in Example 1), which should further accelerate elucidation of these cellular signaling mechanisms.

Nucleic acid molecules, or fragments thereof, encoding MOPs may also be utilized to control the production of the various MOPs, thereby regulating the amount of protein available to participate in cellular signaling pathways. In one embodiment, the nucleic acid molecules of the invention may be used to decrease expression of certain MOPs in cells. In this embodiment, full-length antisense molecules are employed which are targeted to respective MOP genes or RNAs, or antisense oligonucleotides, targeted to specific regions of MOP-encoding genes that are critical for gene expression, are used. The use of antisense molecules to decrease expression levels of a pre-determined gene is known in the art. In a preferred embodiment, antisense oligonucleotides are modified in various ways to increase their stability and membrane permeability, so as to maximize their effective delivery to target cells in vitro and in vivo. Such modifications include the preparation of phosphorothioate or methylphosphonate derivatives, among many others, according to procedures known in the art.

In another embodiment, the transcription regulation activity of bHLH-PAS homodimers or heterodimers involving MOPs may be blocked by genetically engineering a target cell to express a defective MOP--specifically one that has been modified to be unable to bind DNA. When the defective MOP dimerizes, the dimer is also unable to bind DNA, and therefore is unable to carry out its transcriptional regulatory function.

In another embodiment, overexpression of various MOPs is induced, which can lead to overproduction of a selected MOP. Overproduction of MOPs may facilitate the isolation and characterization of other components involved in protein--protein complex formation occurring during the MOP-related signal transduction in cells.

As described above, MOP-encoding nucleic acids are also used to advantage to produce large quantities of substantially pure MOP proteins, or selected portions thereof.

B. MOP Proteins and Anti-MOP Antibodies

Purified MOPs, or fragments thereof, may be used to produce polyclonal or monoclonal antibodies which also may serve as sensitive detection reagents for the presence and accumulation of MOPs (or complexes containing the MOPS) in cultured cells or tissues or in intact organisms. Recombinant techniques enable expression of fusion proteins containing part or all of a selected MOP protein. The full length protein or fragments of the protein may be used to advantage to generate an array of monoclonal or polyclonal antibodies specific for various epitopes of the protein, thereby providing even greater sensitivity for detection of the protein in cells or tissue.

Polyclonal or monoclonal antibodies immunologically specific for a MOP may be used in a variety of assays designed to detect and quantitate the protein. Such assays include, but are not limited to: (1) flow cytometric analysis; (2) immunochemical localization of a MOP in cells or tissues; and (3) immunoblot analysis (e.g., dot blot, Western blot) of extracts from various cells and tissues. Additionally, as described above, anti-MOPs can be used for purification of MOPs (e.g., affinity column purification, immunoprecipitation).

C. Recombinant Cells and Assay Systems

Genetically engineered cells, such as yeast cells or mammalian cells, may be produced to express any one, or a combination, of MOPs described herein. Such cells can be used to evaluate the binding interactions between MOPs, or between a MOP and another member of the bHLH-PAS superfamily (e.g., AHR, ARNT), and the requirement for homodimerization or heterodimerization of the MOPs for initiation of transcriptional control of a reporter gene driven by appropriate enhancer elements. In addition, such recombinant cells can be used to study the effect of external stimuli, such as hypoxia or TCDD, on activation of a selected MOP, or they can be used to screen panels of drugs for control of MOP-involved signal transduction pathways. U.S. Pat. No. 5,650,283 to Bradfield et al., the disclosure of which is incorporated herein by reference, describes recombinant cellular systems and assays for detecting agonists to the AHR. These materials and methods may be used similarly to design recombinant systems for evaluating any of MOP1-MOP8, in the presence or absence of an external stimulant.

Appropriate yeast cells for production of such recombinant systems include Saccharomyces cerevisiae and Saccharomyces pombe. Yeast strains carrying endogenous functional HSPs may be utilized (e.g., A303 obtained from Rick Gaber, Northwestern University, or commercially available equivalents). Yeast strains in which the genes encoding HSPs have been disrupted may also be utilized (e.g., GRS4, obtained from Susan Lindquist, University of Chicago), affording an opportunity to examine the relationship of various MOPs to HSPs.

Appropriate mammalian cells for production of such recombinant systems include COS, Hep3b, HepGr and Hepalclc7 cells, among others.

In one type of assay where the MOP signal transduction pathway is affected by an external stimulus (i.e. an agonist such as TCDD in the AHR-ARNT system, or cobalt chloride in the MOP1/HIF1.alpha.-ARNT system), an appropriate cell can be transformed with an expression plasmid expressing a full length agonist receptor MOP, along with its dimerization partner (if the MOP forms heterodimers) and a reporter plasmid expressing a reporter gene, such as LacZ or luciferin, which is driven by an appropriate enhancer element. The presence or potency of a selected agonist may be determined by its ability to activate transcription of the reporter gene in the recombinant system.

In another embodiment, a recombinant system that does not rely on heterodimerization can be constructed. In this case, a cell is transformed with an expression plasmid expressing a chimeric agonist-sensitive MOP, along with a reporter plasmid expressing a reporter gene driven by a suitable promoter. The chimeric MOP is modified to replace the heterodimerization domains (i.e. the bHLH-PAS domain) with a DNA binding domain, such as LexA or Gal4. Such chimeras will homodimerize and activate transcription of genes positioned downstream of LexA or Gal4 binding sites engineered into the reporter plasmid.

In a preferred embodiment, described in detail in Example 1 (see Original Patent), a modified yeast "two hybrid" system is used to assess binding interactions between MOPs (and other bHLH-PAS proteins) and the subsequent initiation of transcriptional control. For instance, as described in Example 1, fusion proteins were constructed in which the DNA binding domain of the bacterial repressor, LexA, was fused to the bHLH-PAS domains of the MOP proteins. Interactions were tested by cotransformation of each LexAMOP construct with either the full length AHR or ARNT into the L40 yeast strain, which harbors an integrated lacZ reporter gene driven by multiple LexA operator sites. In this system, LexAMOP fusions which interact with AHR or ARNT drive expression of the lacZ reporter gene. The effect of various agonists on reporter gene expression can also be evaluated using this system.

Any one or more of the aforementioned recombinant cell systems and assays can be used to screen panels of drugs for their effect on specific signal transduction pathways. For instance, recombinant systems employing any or MOPs 1, 2, 6 or 7 may be used to screen for drugs that stimulate red blood cell synthesis, angiogenesis or glucose metabolism.

Recombinant systems employing any of MOPs 3, 4, 8 or 9 may be used to screen for drugs that modify circadian rhythms. In connection with this embodiment, as described in greater detail in Example 2 (see Original Patent), we have determined the binding sequence for the MOP3/MOP4 heterodimer, and have constructed the following recombinant plasmids: PL833, a MOP3 expression vector for mammalian cells; PL834, a MOP4 expression vector for mammalian cells; and PL880, a reporter plasmid (expressing luciferase) driven by the MOP3/MOP4 consensus enhancer sequence GCA_CACGTG_ACC (SEQ ID NO: 124). When the three plasmids are introduced into a mammalian cell, the reporter gene responds to the presence of the MOP3/MOP4 dimer. This system is used in a high throughput microwell assay to screen for compounds that are specific activators or inhibitors of these transcription factors. A similar system has been established for MOP7 (HIF3.alpha.), as set forth in Example 3 (see Original Patent).


Claim 1 of 10 Claims

1. An isolated nucleic acid molecule having a sequence selected from the group consisting of: a) SEQ ID NO:3; b) a sequence encoding a polypeptide having amino acid SEQ ID NO:12.

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