[go: up one dir, main page]

US20020106733A1 - Fatty acid transport proteins - Google Patents

Fatty acid transport proteins Download PDF

Info

Publication number
US20020106733A1
US20020106733A1 US09/943,671 US94367101A US2002106733A1 US 20020106733 A1 US20020106733 A1 US 20020106733A1 US 94367101 A US94367101 A US 94367101A US 2002106733 A1 US2002106733 A1 US 2002106733A1
Authority
US
United States
Prior art keywords
leu
gly
val
ala
arg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US09/943,671
Inventor
Andreas Stahl
David Hirsch
Harvey Lodish
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Whitehead Institute for Biomedical Research
Original Assignee
Whitehead Institute for Biomedical Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Whitehead Institute for Biomedical Research filed Critical Whitehead Institute for Biomedical Research
Priority to US09/943,671 priority Critical patent/US20020106733A1/en
Publication of US20020106733A1 publication Critical patent/US20020106733A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • LCFAs Long chain fatty acids
  • LCFAs are an important source of energy for most organisms. They also function as blood hormones, regulating key metabolic functions such as hepatic glucose production.
  • LCFAs can diffuse through the hydrophobic core of the plasma membrane into cells, this nonspecific transport cannot account for the high affinity and specific transport of LCFAs exhibited by cells such as cardiac muscle, hepatocytes, enterocytes, and adipocytes.
  • the molecular mechanisms of LCFA transport remains largely unknown. Identifying these mechanisms can lead to pharmaceuticals that modulate fatty acid uptake by various organs, thereby alleviating certain medical conditions (e.g. obesity).
  • FATPs fatty acid transport proteins
  • FATPs fatty acid transport proteins
  • LCFAs fatty acid transport proteins
  • Members of the FATP family described herein are present in a wide variety of organisms, from mycobacteria to humans, and exhibit very different expression patterns in tissues. FATP family members are expressed in prokaryotic and eukaryotic organisms and comprise characteristic amino acid domains or sequences which are highly conserved across family members.
  • mmFATP2 mmFATP2, mmFATP3, mmFATP4 and mmFATP5
  • human FATPs referred to as, hsFATP2, hsFATP3, hsFATP4, hsFATP5 and hsFATP6, have been identified.
  • Human FATPs 2-5 have orthologs in mice; the sixth human FATP (hsFATP 6 ) does not as yet have a mouse ortholog. The expression patterns of these FATPs vary, as described below.
  • the present invention relates to FATP family members from prokaryotes and eukaryotes, nucleic acids (DNA, RNA) encoding FATPs, and nucleic acids which are useful as probes or primers (e.g., for use in hybridization methods, amplification methods) for example, in methods of detecting FATP-encoding genes, producing FATPs, and purifying or isolating FATP-encoding DNA or RNA.
  • antibodies polyclonal or monoclonal which bind an FATP or FATPs; methods of identifying additional FATP family members (for example, orthologs of those FATPs described herein by amino acid sequence) and variant alleles of known FATP genes; methods of identifying compounds which bind to an FATP or to a polypeptide comprising a portion of a FATP, or modulate or alter (enhance or inhibit) FATP function; compounds which modulate or alter FATP function; methods of modulating or altering (enhancing or inhibiting) FATP function and, thus, LCFA uptake into tissues of a mammal (e.g., human) by administering a compound or molecule (a drug or agent) which increases or reduces FATP activity; and methods of targeting compounds to tissues by administering a complex of the compound to be targeted to tissues and a component which is bound by an FATP present on cells of the tissues to which the compound is to be targeted.
  • additional FATP family members for example, orthologs of those FATPs described herein by amino acid sequence
  • a complex of a drug to be delivered into the liver and a component which is bound by an FATP present on liver cells can be administered.
  • LCFA uptake by the liver is modulated or altered (enhanced or reduced), in an individual.
  • a drug which inhibits the function of an FATP present in liver e.g., FATP 5
  • the present invention provides methods which are useful to alter, particularly reduce, LCFA uptake in individuals and, as a result, to alter (particularly reduce), availability of the LCFAs for further metabolism.
  • the present invention provides methods useful to reduce LCFA uptake and, thus, fatty acid metabolism in individuals, with the result that caloric availability from fats is reduced, and circulating fatty acid levels are lower than they otherwise would be.
  • These methods are useful, for example, as a means of weight control in individuals, (e.g., humans) and as a means of preventing elevated serum lipid levels or reducing serum lipid levels in humans.
  • FIG. 1 shows the amino acid sequences of mmFATP1 (SEQ ID NO:1), mmFATP5 (SEQ ID NO:2), ceFATPA (SEQ ID NO:3), scFATP (SEQ ID NO:4), and mtFATP (SEQ ID NO:5).
  • FIG. 2 is a phylogenetic tree showing the relationships among the FATP family members and VLACs, based on the 360 amino acid signature sequence of FATP.
  • FIGS. 3 A- 3 E are photographs of the results of northern analysis of issue distribution of the murine FATP genes.
  • FIGS. 4 A- 4 D shows results of FACs analysis of uptake of a BODIPY-labeled analog of a long chain fatty acid by COS cells transiently transfected with mmFATP1, mmFTP2, or mmFATP 5 (FIGS. 4B, 4C, and 4 D, respectively) and by control (untransfected COS cells; FIG. 4A).
  • FIGS. 5A and 5B are the mmFATP3 DNA sequence (SEQ ID NO:6).
  • FIG. 6 is the mmFATP3 protein sequence (SEQ ID NO:7).
  • FIGS. 7A and 7B are the mmFATP4 DNA sequence (SEQ ID NO:8).
  • FIG. 8 is the mmFATP4 protein sequence (SEQ ID NO:9).
  • FIGS. 9A and 9B are the rmFATP5 DNA sequence (SEQ ID NO:10).
  • FIG. 10 is the mmFATP5 protein sequence (SEQ ID NO:11).
  • FIGS. 11A and 11B are the hsFATP2 DNA sequence (SEQ ID NO:12).
  • FIG. 12 is the hsFATP2 protein sequence (SEQ ID NO:13).
  • FIGS. 13A and 13B are the hsFATP3 DNA sequence (SEQ ID NO:14).
  • FIG. 14 is the hsFATP3 protein sequence (SEQ ID NO:15).
  • FIG. 15A and 15B are the hsFATP4 DNA sequence (SEQ ID NO:16).
  • FIG. 16 is the hsFATP4 protein sequence (SEQ ID NO:17).
  • FIG. 17A and 17B are the hsFATP5 DNA sequence (SEQ ID NO:18).
  • FIG. 18 is the hsFATP5 protein sequence (SEQ ID NO:19).
  • FIGS. 19A and 19B are the hsFATP6 DNA sequence (SEQ ID NO:20).
  • FIG. 20 is the hsFATP6 protein sequence (SEQ ID NO:21).
  • FIGS. 21A and 21B are the mtFATP DNA sequence (SEQ ID NO:22).
  • FIG. 22 is the mtFATP protein sequence (SEQ ID NO:23).
  • FIG. 23A and 23B is a partial DNA sequence encoding a FATP of Drosophila melanogaster (SEQ ID NO:24).
  • FIG. 24 is a partial amino acid sequence of a Drosophila melanogaster FATP (SEQ ID NO:25).
  • FIG. 25 is a partial DNA sequence encoding a FATP of Danio rerio (SEQ ID NO:26).
  • FIG. 26 is a partial amino acid sequence of a Danio rerio (zebrafish) FATP (SEQ ID NO:27).
  • FIG. 27A and 27B is a DNA sequence encoding FATPa of Caenorhabditis elegans (SEQ ID NO:28).
  • FIG. 28 is an amino acid sequence of Caenorhabditis elegans FATPa (SEQ ID NO:29).
  • FIG. 29A and 29B is a DNA sequence encoding a FATPb of Caenorhabditis elegans (SEQ ID NO:30).
  • FIG. 30 is a amino acid sequence of Caenorhabditis elegans FATPb (SEQ ID NO:31).
  • FIG. 31A and 31B is a DNA sequence encoding a FATP of Cochliobolu heterostrophus (SEQ ID NO:32).
  • FIG. 32 is an amino acid sequence of a Cochliobolu heterostrophus FATP (SEQ ID NO:33).
  • FIG. 33 is a partial DNA sequence encoding a FATP of Magnaporthe grisea (SEQ ID NO:34).
  • FIG. 34 is a partial amino acid sequence of a Magnaporthe grisea FATP (SEQ ID NO:35).
  • FIG. 35A and 35B is a DNA sequence of a Mycobacterium tuberculosis FATP (SEQ ID NO:36).
  • FIG. 36 is an amino acid sequence of a Mycobacterium tuberculosis FATP (SEQ ID NO:37).
  • FATPs are a large evolutionary conserved family of proteins that mediate the transport of LCFAs into cells.
  • the family includes proteins which are conserved from mycobacteria to humans and exhibit very different expression patterns in tissues. Specific embodiments described include FATPs from mice, humans, nematodes, fungi, and mycobacteria.
  • FATPs fatty acid transport proteins
  • FATPs fatty acid transport proteins
  • FATPs refers to the proteins described herein as FATP1, FATP2, FATP3, FATP4, FATP5 and FATP6, which have been described in one or more species of mammals, as well as mtFATP, ceFATPa, ceFATPb, dmFATP, drFATP, mgFATP, and chFATP and other proteins sharing at least about 50% amino acid sequence similarity, preferably at least about 60% sequence similarity, more preferably at least about 70% sequence similarity, and still more preferably, at least about 80% sequence similarity, and most preferably, at least about 90% sequence similarity in the approximately 360 amino acid signature sequence.
  • the approximately 360 amino acid FATP signature sequence is shown in FIG. 1.
  • FATPs includes a species-specific prefix (e.g., mm, Mus musculus; hs or h, Homo sapiens or human; mt M. tuberculosis; ce, C. elegans; sc, Saccharomyces cerevisiae ) and a number such that mammalian homologues in different species share the same number.
  • hsFATP4 and mmFATP4 are the human and mouse orthologs.
  • LCFAs Long chain fatty acids
  • LCFAs are an important energy source for pro- and eukaryotes and are involved in diverse cellular processes, such as membrane synthesis, intracellular signaling, protein modification, and transcriptional regulation.
  • human dietary lipids are mainly di- and triglycerides and account for approximately 40% of caloric intake (Weisburger, J. H. (1997) J. Am. Diet. Assoc. 97:S16-S23). These lipids are broken down into fatty acids and glycerol by pancreatic lipases in the small intestine (Chapus, C., Rovery, M., Sarda, L & Verger, R.
  • the levels of fatty acids in the blood can be regulated by insulin and are dysregulated in diseases such as noninsulin-dependent diabetes and obesity (Boden, G. (1997) Diabetes 46:3-10).
  • the underlying mechanisms for the regulation of free fatty acid concentrations in the blood are not understood, but could be explained by hormonal modulation of FATPs.
  • Insulin-resistance is thought to be the major defect in non insulin-dependent diabetes mellitus (NIDDM) and is one of the earliest manifestations of NIDDM (McGarry (1992) Science 258:766-770).
  • Free fatty acids (FFAs) may provide an explanation for why obesity is a risk factor for NIDDM.
  • Plasma levels of FFAs are elevated in diabetic patients (Reaven et al. (1988) Diabetes 37:1020). Elevated plasma free fatty acids (FFAs) have been demonstrated to induce insulin-resistance in whole animals and humans (Boden (1998) Front. Biosci. 3:D169-D175).
  • FFAs added to adipocytes in vitro induce insulin resistance in this cell type as evidenced by inhibition of insulin-induced glucose transport (Van Epps-Fung et al. (1997) Endocrinology 138:4338-4345).
  • Rats fed a high fat diet developed skeletal muscle insulin resistance as evidenced by a decrease in insulin-induced glucose uptake by skeletal muscle (Han et al., (1997) Diabetes 46:1761-1767).
  • elevated plasma FFAs increase insulin-suppressed endogenous glucose production in the liver (Boden (1998) Front. Biosci. 3:D169-D175), thus increasing hepatic glucose output.
  • fatty acid induced insulin-resistance may be prevented by blocking uptake of FFAs into select tissues, in particular liver (by blocking FATP 2 and/or FATP 5 ), adipocyte (by blocking FATP 1 ), and skeletal muscle (by blocking FATP 1 ). Blocking intestinal fat absorption (by blocking FATP 4 ) is also expected to reduce plasma FFA levels and thus improve insulin resistance.
  • beta cell function decreases and overt diabetes results (McGarry (1992) Science 258: 766-770).
  • Manipulating beta cell function is a second point where fatty acid transporter blockers may be beneficial for diabetes. While no FATP homolog has been identified so far that is expressed in the beta cell of the pancreas, the data described below suggest the existence of such a transporter and the sequence information included herein provides the means to identify such a transporter by degenerate PCR, using primers to regions conserved in all FATP family members or by low stringency hybridization.
  • FATPs expressed in mycobacteria can be targeted to reduce or prevent replication of mycobacteria (e.g., to reduce or prevent replication of M. tuberculosis ) and, thus, reduce or prevent their adverse effects.
  • a FATP or FATPs expressed by M. tuberculosis can be targeted and inhibited, thus reducing or preventing growth of this pathogen (and tuberculosis in humans and other mammals).
  • An inhibitor of an M. tuberculosis FATP can be identified, using methods described herein (e.g., expressing the FATP in an appropriate host cell, such as E.
  • a drug or agent identified in this manner can be further tested for its ability to inhibit a M. tuberculosis FATP and M. tuberculosis infection in an appropriate animal model or in humans.
  • a method of inhibiting mycobacterial growth, particularly growth of M. tuberculosis, and compounds useful as drugs for doing so are also the subject of this invention.
  • An isolated polynucleotide encoding mtFATP can be incorporated into vectors, nucleic acids of viruses, and other nucleic acid constructs that can be used in various types of host cells to produce mtFATP.
  • This mtFATP can be used, as it appears on the surface of cells, or in various artificial membrane systems, to assess fatty acid transport function, to identify ligands and molecules that are modulators of fatty acid transport activity.
  • Molecules found to be inhibitors of mtFATP function can be incorporated into pharmaceutical compositions to administer to a human for the treatment of tuberculosis.
  • Particular embodiments of the invention are polynucleotides encoding a FATP of Cochliobolus (Helminthosporium) heterostrophus or portions or variants thereof, the isolated or recombinantly produced FATP, methods for assessing whether an agent binds to the chFATP, and further methods for assessing the effect of an agent being tested for its ability to modulate fatty acid transport activity.
  • Cochliobolus heterostrophus is an ascomycete that is the cause of southern corn leaf blight, an economically important threat to the corn crop in the U.S.
  • the related species C sativus causes crown rot and common root rot in wheat and barley.
  • One or more FATPs of C are polynucleotides encoding a FATP of Cochliobolus (Helminthosporium) heterostrophus or portions or variants thereof, the isolated or recombinantly produced FATP, methods for assessing whether an agent binds to the
  • heterostrophus can be targeted for the identification of an inhibitor of chFATP function, which can be then be used as an agent effective against infection of plants by C. heterostrophus and related organisms.
  • Methods described herein that were applied in studying the expression of a FATP gene and the function of the FATP in its natural site of expression or in a host cell, can be used in the study of the chFATP gene and protein.
  • Magnaporthe grisea (rice blast) is an economically important fungal pathogen of rice.
  • Further embodiments of the invention are nucleic acid molecules encoding a FATP of Magnaporthe grisea, portions thereof, or variants thereof, isolated mgFATP, nucleic acid constructs, and engineered cells expressing mgFATP.
  • Other aspects of the invention are assays to identify an agent which binds to mgFATP and assays to identify an agent which modulates the function of mgFATP in cells in which mgFATP is expressed or in artificial membrane systems. Agents identified as inhibiting mgFATP activity can be developed into anti-fungal agents to be used to treat rice infected with rice blast.
  • Caenorhabditis elegans is a nematode related to plant pathogens and human parasites.
  • An isolated polynucleotide which encodes ceFATP can be incorporated into nucleic acid vectors and other constructs that can be used in various types of cells to produce ceFATP.
  • ceFATP as it occurs in cells or as it can be isolated or incorporated into various artificial or reconstructed membrane systems can be used to assess fatty acid transport, and to identify ligands and agents that modulate fatty acid transport activity.
  • Agents found by such assays to be inhibitors of ceFATP activity can be incorporated into compositions for the treatment of diseases caused by genetically related organisms with a FATP of similar sensitivity to the agents.
  • One aspect of the invention relates to isolated nucleic acids that encode a FATP as described herein, such as those FATPs having an amino acid sequence shown in the figures, and nucleic acids closely related thereto as described herein.
  • FIGS. 5 A- 5 B SEQ ID NO: 6
  • FIGS. 7A and 7B SEQ ID NO: 8
  • FIGS. 9 A- 9 B SEQ ID NO:10
  • FIGS. 11 A- 11 B SEQ ID NO: 12
  • FIGS. 13A and 13B SEQ ID NO:14
  • FIGS. 15 A- 15 B SEQ ID NO:16
  • FIGS. 17A and 17B SEQ ID NO:18, FIGS. 19A and 19B (SEQ ID NO:20)
  • a nucleic acid of the invention encoding a FATP polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing cDNA library fragments, followed by obtaining a full length clone.
  • a library of clones of cDNA of human or other mammalian DNA can be probed with a labeled oligonucleotide, such as a radiolabeled oligonucleotide, preferably about 17 nucleotides or longer, derived from a partial sequence.
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent (also, “high stringency”) hybridization conditions.
  • stringent also, “high stringency”
  • sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full length sequence. Suitable techniques are described, for example, in Current Protocols in Molecular Biology (F. M. Ausubel et al, eds), containing supplements through Supplement 42, 1998, John Wiley and Sons, Inc., especially chapters 5, 6, and 7.
  • Embodiments of the invention include isolated nucleic acid molecules comprising any of the following nucleotide sequences: 1.) a nucleotide sequence which encodes a protein comprising the amino acid sequence of ceFATPa (SEQ ID NO:29), mgFATP (SEQ ID NO: 35), ceFATP6 (SEQ ID NO:31), mmFATP3 (SEQ ID NO:7), mmFATP4 (SEQ ID NO: 9), mmFATP5 (SEQ ID NO: 1), hsFATP2 (SEQ ID NO: 13), hsFATP3 (SEQ ID NO:15), hsFATP4 (SEQ ID NO:17), hsFATP5 (SEQ If) NO:19), hsFATP6 (SEQ ID NO:21), chFATP (SEQ ID NO:33), mtFATP (SEQ ID NO:23); 2.) nucleotide sequences described herein, such as those encoding hsFATP2, hsFATP3, h
  • the invention further relates to nucleic acids (nucleic acid molecules or polynucleotides) having nucleotide sequences identical over their entire length to those nucleotide sequences shown in the figures. It further relates to DNA, which due to the degeneracy of the genetic code, encodes a FATP encoded by one of the FATP-encoding DNAs, whose amino acid sequence is provided herein. Also provided by the invention are nucleic acids having the coding sequences for the mature polypeptides or fragments in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence.
  • the nucleic acids of the invention encompass nucleic acids that include a single continuous region or discontinuous regions encoding the polypeptide, together with additional regions, that may also contain coding or non-coding sequences.
  • the nucleic acids may also contain non-coding sequences, including, for example, but not limited to, non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequences which encode additional amino acids.
  • a marker sequence that facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence can be a hexa- histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), or an HA tag (Wilson et al, Cell 37: 767 (1984)), or a sequence encoding glutathione S-transferase of Schistosoma japonicum (vectors available from Pharmacia; see Smith, D. B. and Johnson K. S., Gene 67:31 (1988) and Kaelin, W. G. et al., Cell 70:351 (1992)).
  • Nucleic acids of the invention also include, but are not limited to, nucleic acids comprising a structural gene and its naturally associated sequences that control gene expression.
  • the invention further relates to variants, including naturally-occurring allelic variants, of those nucleic acids described specifically herein by DNA sequence, that encode variants of such polypeptides as those having the amino acid sequences shown in FIG. 28 (SEQ ID NO:29), FIG. 30 ( SEQ ID NO:31), FIG. 32 (SEQ ID NO:33), FIG. 34 (SEQ ID NO:35), FIG. 6 (SEQ ID NO:7), FIG. 8 (SEQ ID NO:9), FIG. 10 (SEQ ID NO:11), FIG. 12 (SEQ ID NO: 13) FIG. 14 (SEQ ID NO:15), FIG. 16 (SEQ ID NO:17), FIG. 18 (SEQ ID NO:19), FIG.
  • variants including naturally-occurring allelic variants, of those nucleic acids described specifically herein by DNA sequence, that encode variants of such polypeptides as those having the amino acid sequences shown in FIG. 28 (SEQ ID NO:29), FIG. 30 ( SEQ ID NO:31), FIG. 32 (SEQ
  • variants include nucleic acids encoding variants of the above-listed amino acid sequences, wherein those variants have several, such as 5 to 10, 1 to 5, or 3, 2 or 1 amino acids substituted, deleted, or added, in any combination.
  • Variants include polynucleotides encoding polypeptides with at least 95% but less than 100% amino acid sequence identity to the polypeptides described herein by amino acid sequence. Variant polynucleotides hybridize, under low to high stringency conditions, to the alleles described herein by DNA sequence. In one embodiment, nucleic acid variants have silent substitutions, additions and deletions that do not alter the properties and activities of the encoded FATP. 10 Orthologous genes are gene loci in different species that are sufficiently similar to each other in their nucleotide sequences to suggest that they originated from a common ancestral gene. Orthologous genes arise when a lineage splits into two species, rather than when a gene is duplicated within a genome.
  • Proteins that are orthologs are encoded by genes of two different species, wherein the genes are said to be orthologous. 15
  • the invention further relates to polynucleotides encoding polypeptides which are orthologous to those polypeptides having a specific amino acid sequence described herein.
  • These polynucleotides which can be called ortholog polynucleotides, encode orthologous polypeptides that can range in amino acid sequence identity to a reference amino acid sequence described herein, from about 65% to less than 100%, but preferably 70% to 80%, more preferably 80% to 90%, and still more preferably 90% to less than 100%.
  • Orthologous polypeptides can also be those polypeptides that range in amino acid sequence similarity to a reference amino acid sequence described herein from about 75% to 100%, within the signature sequence.
  • the amino acid sequence similarity between the signature sequences of orthologous polypeptides is preferably 80%, more preferably 90%, and still more preferably, 95%.
  • the ortholog polynucleotides encode polypeptides that have similar functional characteristics (e.g., fatty acid transport activity) and similar tissue distribution, as appropriate to the organism from which the ortholog polynucleotides can be isolated.
  • Ortholog polynucleotides can be isolated from (e.g., by cloning or nucleic acid amplification methods) a great number of species, as shown by the sample of FATPs from evolutionary divergent species described herein.
  • Ortholog polynucleotides corresponding to those having the nucleotide sequences shown in the figures are those which can be isolated from mammals such as rat, dog, chimpanzee, monkey, baboon, pig, rabbit and guinea pig, for example.
  • primer refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • the appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5′ (upstream) primer that hybridizes with the 5′ end of the DNA sequence to be amplified and a 3′ (downstream) primer that hybridizes with the complement of the 3′ end of the sequence to be amplified.
  • nucleic acids that are at least 80% identical over their entire length to a nucleic acid described herein. Additional embodiments are nucleic acids, and the complements of such nucleic acids, having at least 90% nucleotide sequence identity to the above-described sequences, and nucleic acids having at least 95% nucleotide sequence identity.
  • DNA of the present invention has 97% nucleotide sequence identity, 98% nucleotide sequence identity, or at least 99% nucleotide sequence identity with the DNA whose sequences are presented herein.
  • nucleic acids that are at least 80% identical in nucleotide sequence to a nucleic acid encoding a polypeptide having an amino acid sequence as set forth in herein, and nucleic acids that are complementary to such nucleic acids.
  • Specific embodiments are nucleic acids having at least 90% nucleotide sequence identity to a nucleic acid encoding a polypeptide having an amino acid sequence as described in the list above, nucleic acids having at least 95% sequence identity, and nucleic acids having at least 97% sequence identity.
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single-stranded molecules may be “partial” in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single-stranded molecules (that is, when A-T and G-C base pairing is 100% complete). The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend on binding between nucleic acid strands.
  • the invention further includes nucleic acids that hybridize to the above-described nucleic acids, especially those nucleic acids that hybridize under stringent hybridization conditions.
  • “Stringent hybridization conditions” or “high stringency conditions” generally occur within a range from about T m minus 5° C (5° C below the strand dissociation temperature or melting temperature (T m ) of the probe nucleic acid molecule) to about 20° C. to 25° C. below T m .
  • T m melting temperature
  • the stringency of hybridization may be altered in order to identify or detect molecules having identical or related polynucleotide sequences.
  • An example of high stringency hybridization follows.
  • Hybridization solution is (6x SSC/10 mM EDTAI 0 .5% SDS/5 ⁇ Denhardt's solution/100 ⁇ g/ml sheared and denatured salmon sperm DNA). Hybridization is at 64-65° C. for 16 hours. The hybridized blot is washed two times with 2 ⁇ SSC/0.5% SDS solution at room temperature for 15 minutes each, and two times with 0.2 ⁇ SSC/0.5% SDS at 65° C., for one hour each. Further examples of high stringency conditions can be found on pages 2.10.1-2.10.16 (see particularly 2 . 10 . 8 -11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., containing supplements up through Supplement 42, 1998). Examples of high, medium, and low stringency conditions can be found on pages 36 and 37 of WO 98/40404, which are incorporated herein by reference.
  • the invention further relates to nucleic acids obtainable by screening an appropriate library with a probe having a nucleotide sequence such as one set forth herein, or a probe which is a sufficiently long portion of these sequences; and isolating the nucleic acid.
  • probes generally can comprise at least 15 nucleotides.
  • Nucleic acids obtainable by such screenings may include RNAs, cDNAs and genomic DNA, for example, encoding FATPs of the FATP family described herein.
  • nucleic acid molecules of the invention whether encoding a full-length FATP or whether comprising a contiguous portion of a nucleic acid molecule described herein by sequence include use as markers for tissues in which the corresponding protein is preferentially expressed (to identify constitutively expressed proteins or proteins produced at a particular stage of tissue differentiation or stage of development of a disease state); as molecular weight markers on southern gels; as chromosome markers or tags (when labeled, for example with biotin, a radioactive label or a fluorescent label) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in a mammal to identify potential genetic disorders; as probes to hybridize and thus identify, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel nucleic acid molecules; for selecting and making oligomers for attachment to a “gene chip”
  • nucleic acids encoding FATPs of the FATP family include PCR and variations thereof (e.g., “RACE” PCR and semi-specific PCR methods). Portions of the nucleic acids having a nucleotide sequence set forth herein, (especially “flanking sequences” on either side of a coding region) can be used as primers in methods using the polymerase chain reaction, to produce DNA from an appropriate template nucleic acid.
  • a fragment of the FATP gene can be sequenced, and the sequence of the product can be compared to other DNA sequences, for example, by using the BLAST Network Service at the National Center for Biotechnology Information. The boundaries of the open reading frame can then be identified using semi-specific PCR or other suitable methods such as library screening. Once the 5′ initiator methionine codon and the 3′ stop codon have been identified, a PCR product encoding the full-length gene can be generated using genomic DNA as a template, with primers complementary to the extreme 5′ and 3′ ends of the gene or to their flanking sequences. The full-length genes can then be cloned into expression vectors for the production of functional proteins.
  • the invention also relates to isolated proteins or polypeptides such as those encoded by nucleic acids of the present invention.
  • Isolated proteins can be purified from a natural source or can be made recombinantly.
  • Proteins or polypeptides referred to herein as “isolated” are proteins or polypeptides that exist in a state different from the state in which they exist in cells in which they are normally expressed in an organism, and include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated.
  • isolated indicates that the polypeptide in question exists in a physical milieu distinct from that in which it occurs in nature.
  • isolated includes existing in membrane fragments and vesicles membrane fractions, liposomes, lipid bilayers and other artificial membrane systems.
  • An isolated FATP may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, and may even be purified essentially to homogeneity, for example as determined by PAGE or column chromatography (for example, HPLC), but may also have further cofactors or molecular stabilizers, such as detergents, added to the purified protein to enhance activity.
  • proteins or polypeptides are isolated to a state at least about 75% pure; more preferably at least about 85% pure, and still more preferably at least about 95% pure, as determined by Coomassie blue staining of proteins on SDS-polyacrylamide gels.
  • Proteins or polypeptides referred to herein as “recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids.
  • an isolated polypeptide comprising a FATP, a functional portion thereof, or a functional equivalent of the FATP has at least one function characteristic of a FATP, for example, transport activity, binding function (e.g., a domain which binds to AMP), or antigenic function (e.g., binding of antibodies that also bind to a naturally-occurring FATP, as that function is found in an antigenic determinant).
  • Functional equivalents can have activities that are quantitatively similar to, greater than, or less than, the reference protein.
  • proteins include, for example, naturally occurring FATPs that can be purified from tissues in which they are produced (including polymorphic or allelic variants), variants (e.g., mutants) of those proteins and/or portions thereof.
  • variants include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides in which one or more residues are modified, and mutants comprising one or more modified residues.
  • Portions or fragments of a FATP can range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
  • the isolated proteins of the invention preferably include mammalian fatty acid transport proteins of the FATP family of homologous proteins.
  • the extent of amino acid sequence similarity between a polypeptide having one of the amino acid sequences shown, for example, in FIGS. 1, 6, 8 , 10 , 12 , 14 , 16 , 18 , 20 , or 22 and the respective functional equivalents of these polypeptides is at least about 88%.
  • the degree of amino acid sequence similarity between a FATP and its respective functional equivalent is at least about 91%, at least about 94%, or at least about 97%.
  • polypeptides of the invention also include those FATPs encoded by polynucleotides which are orthologous to those polynucleotides, the sequences of which are described herein in whole or in part.
  • FATPs which are orthologs to those described herein by amino acid sequence, in whole or in part are, for example fatty acid transport proteins 1-6 of dog, rat chimpanzee, monkey, rabbit, guinea pig, baboon and pig, and are also embodiments of the invention.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment, and non-homologous (dissimilar) sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “similarity”.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptides described herein by amino acid sequence. Similarity for a polypeptide is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and lIle; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., Science 247:1306-1310 (1990).
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2 . 0 ) of Altschul, et al. ( J. Mol. Biol. 215:403-10 (1990)).
  • Gapped BLAST can be utilized as described in Altschul et al., ( Nucleic Acids Res. 25(17):3389-3402 (1997)).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See http://www.ncbi.nlm.nih.gov.
  • the invention further relates to fusion proteins, comprising a FATP or functional portion thereof (as described above) as a first moiety, linked to second moiety not occurring in the FATP as found in nature.
  • the second moiety can be, for example, an amino acid, peptide or polypeptide.
  • the first moiety can be in an N-terminal location, C-terminal location or internal to the fusion protein.
  • the fusion protein comprises a FATP as the first moiety, and a second moiety comprising a linker sequence and an affinity ligand. Fusion proteins can be produced by a variety of methods.
  • a fusion protein can be produced by the insertion of a FATP gene or portion thereof into a suitable expression vector, such as Bluescript SK +/ ⁇ (Stratagene), pGEX-4T-2 (Pharmacia), pET-24(+) (Novagen), or vectors of similar construction.
  • the resulting construct can be introduced into a suitable host cell for expression.
  • fusion protein can be purified from cells by means of a suitable affinity matrix (See e.g., Current Protocols in Molecular Biology, Ausubel, F. M. et al., eds., Vol. 2, pp. 16.4.1-16.7.8, containing supplements up through Supplement 42, 1998).
  • the invention also relates to enzymatically produced, synthetically produced, or recombinantly produced portions of a fatty acid transport protein.
  • Portions of a FATP can be made which have full or partial function on their own, or which when mixed together (though fully, partially, or nonfunctional alone), spontaneously assemble with one or more other polypeptides to reconstitute a functional protein having at least one function characteristic of a FATP.
  • Fragments of a FATP can be produced by direct peptide synthesis, for example those using solid-phase techniques (Roberge, J. Y. et al., Science 269:202-204 ( 1995 ); Merrifield, J., J. Am. Chem. Soc. 85:2149-2154 (1963)). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be carried out using, for instance, an Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of a FATP can be synthesized separately and combined using chemical methods.
  • One aspect of the invention is a peptide or polypeptide having the amino acid sequence of a portion of a fatty acid transport protein which is hydrophilic rather than hydrophobic, and ordinarily can be detected as facing the outside of the cell membrane.
  • a peptide or polypeptide can be thought of as being an extracellular domain of the FATP, or a mimetic of said extracellular domain. It is known, for example, that a portion of human FATP 4 that includes a highly conserved motif is involved in AMP-CoA binding function (Stuhlsatz-Krouper, S. M. et al., J. Biol Chem. 44:28642-28650 ( 1998 )).
  • the term “mimetic” as used herein, refers to a molecule, the structure of which is developed from knowledge of the structure of the FATP of interest, or one or more portions thereof, and, as such, is able to effect some or all of the functions of a FATP.
  • Portions of an FATP can be prepared by enzymatic cleavage of the isolated protein, or can be made by chemical synthesis methods. Portions of a FATP can also be made by recombinant DNA methods in which restriction fragments, or fragments that may have undergone further enzymatic processing, or synthetically made DNAs are joined together to construct an altered FATP gene.
  • the gene can be made such that it encodes one or more desired portions of a FATP. These portions of FATP can be entirely homologous to a known FATP, or can be altered in amino acid sequence relative to naturally occurring FATPs to enhance or introduce desired properties such as solubility, stability, or affinity to a ligand.
  • a further feature of the gene can be a sequence encoding an N-terninal signal peptide directed to the plasma membrane.
  • a polypeptide or peptide comprising all or a portion of a FATP extracellular domain can be used in a pharmaceutical composition.
  • the polypeptide or peptide When administered to a mammal by an appropriate route, the polypeptide or peptide can bind to fatty acids and compete with the native FATPs in the membrane of cells, thereby making fewer fatty acid molecules available as substrates for transport into cells, and reducing fatty acid uptake.
  • Another aspect of the invention relates to a method of producing a fatty acid transport protein, variants or portions thereof, and to expression systems and host cells containing a vector appropriate for expression of a fatty acid transport protein.
  • Cells that express a FATP, a variant or a portion thereof, or an ortholog of a FATP described herein by amino acid sequence can be made and maintained in culture, under conditions suitable for expression, to produce protein in the cells for cell-based assays, or to produce protein for isolation.
  • These cells can be procaryotic or eucaryotic. Examples of procaryotic cells that can be used for expression include Escherichia coli, Bacillus subtilis and other bacteria.
  • eucaryotic cells examples include yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and other lower eucaryotic cells, and cells of higher eucaryotes such as those from insects and mammals, such as primary cells and cell lines such as CHO, HeLa, 3T3 and BHK cells, COS cells, 293 cells, and Jurkat cells.
  • yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and other lower eucaryotic cells
  • cells of higher eucaryotes such as those from insects and mammals, such as primary cells and cell lines such as CHO, HeLa, 3T3 and BHK cells, COS cells, 293 cells, and Jurkat cells.
  • primary cells and cell lines such as CHO, HeLa, 3T3 and BHK cells, COS cells, 293 cells, and Jurkat cells.
  • host cells that produce a recombinant FATP, or a portion thereof, a variant, or an ortholog of a FATP described herein by amino acid sequence can be made as follows.
  • a gene encoding a FATP, variant or a portion thereof can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, phage, cosmid, phagemid, virus, virus-derived vector (e.g., SV40, vaccinia, adenovirus, fowl pox virus, pseudorabies viruses, retroviruses) or other suitable replicon, which can be present in a single copy or multiple copies, or the gene can be integrated in a host cell chromosome.
  • a DNA vector such as a plasmid, phage, cosmid, phagemid, virus, virus-derived vector (e.g., SV40, vaccinia, adenovirus, fowl pox virus, pseudo
  • a suitable replicon or integrated gene can contain all or part of the coding sequence for a FATP or variant, operably linked to one or more expression control regions whereby the coding sequence is under the control of transcription signals and linked to appropriate translation signals to permit translation.
  • the vector can be introduced into cells by a method appropriate to the type of host cells (e.g., transfection, electroporation, infection).
  • the host cells can be maintained under appropriate conditions (e.g., in the presence of inducer, normal growth conditions, etc.).
  • Proteins or polypeptides thus produced can be recovered (e.g., from the cells, as in a membrane fraction, from the periplasmic space of bacteria, from culture medium) using suitable techniques.
  • Appropriate membrane targeting signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Polypeptides of the invention can be recovered and purified from cell cultures (or from their primary cell source) by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and high performance liquid chromatography.
  • Known methods for refolding protein can be used to regenerate active conformation if the polypeptide is denatured during isolation or purification.
  • a further aspect of the invention are methods for assessing the transport function of any of the fatty acid transport proteins or polypeptides described herein, including orthologs, and in variations of these, methods for identifying an inhibitor (or an enhancer) of such function and methods for assessing the transport function in the presence of a candidate inhibitor or a known inhibitor.
  • a variety of systems comprising living cells can be used for these methods.
  • Cells for use in cell-based assays described herein can be drawn from a variety of sources, such as isolated primary cells of various organs and tissues wherein one or more FATPs are naturally expressed. In some cases, the cells can be from adult organs, and in some cases, from embryonic or fetal organs, such as heart, lung, liver, skeletal muscle, kidney and the like.
  • Cells for this purpose can also include cells cultured as fragments of organs or in conditions simulating the cell type and/or tissue organization of organs, in which artificial materials may be used as substrates for cell growth.
  • Other types of cells suitable for this purpose include cells of a cell strain or cell line (ordinarily comprising cells considered to be “transformed”) transfected to express one or more FATPs.
  • a further embodiment of the invention is a method for detecting, in a sample of cells, a fatty acid transport protein, a portion or fragment thereof, a fusion protein comprising a FATP or a portion thereof, or an ortholog as described herein, wherein the cells can be, for instance, cells of a tissue, primary culture cells, or cells of a cell line, including cells into which nucleic acid has been introduced.
  • the method comprises adding to the sample an agent that specifically binds to the protein, and detecting the agent specifically bound to the protein. Appropriate washing steps can be added to reduce nonspecific binding to the agent.
  • the agent can be, for example, an antibody, a ligand or a substrate mimic.
  • the agent can have incorporated into it, or have bound to it, covalently or by high affinity non-covalent interactions, for instance, a label that facilitates detection of the agent to which it is bound, wherein the label can be, but is not limited to, a phosphorescent label, a fluorescent label, a biotin or avidin label, or a radioactive label.
  • a label that facilitates detection of the agent to which it is bound can be, but is not limited to, a phosphorescent label, a fluorescent label, a biotin or avidin label, or a radioactive label.
  • the means of detection of a fatty acid transport protein can vary, as 10 appropriate to the agent and label used.
  • the means of detection may call for binding a second antibody, which has been conjugated to an enzyme, to the antibody which binds the fatty acid transport protein, and detecting the presence of the second antibody by means of the enzymatic activity of the conjugated enzyme.
  • Similar principles can also be applied to a cell lysate or a more purified preparation of proteins from cells that may comprise a fatty acid transport protein of interest, for example in the methods of immunoprecipitation, immunoblotting, immunoaffinity methods, that in addition to detection of the particular FATP, can also be used in purification steps, and qualitative and quantitative immunoassays. See, for instance, chapters 11 through 14 in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, eds., Cold Spring Harbor Laboratory, 1988.
  • Isolated fatty acid transport protein or, an antigenically similar portion thereof, especially a portion that is soluble can be used in a method to select and identify molecules which bind specifically to the FATP.
  • Fusion proteins comprising all of, or a portion of, the fatty acid transport protein linked to a second moiety not occurring in the FATP as found in nature, can be prepared for use in another embodiment of the method. Suitable fusion proteins for this purpose include those in which the second moiety comprises an affinity ligand (e.g., an enzyme, antigen, epitope).
  • FATP fusion proteins can be produced by the insertion of a gene encoding the FATP or a variant thereof, or a suitable portion of such gene into a suitable expression vector, which encodes an affinity ligand (e.g., pGEX-4T-2 and pET-1 Sb, encoding glutathione S-transferase and His-Tag affinity ligands, respectively).
  • the expression vector can be introduced into a suitable host cell for expression. Host cells are lysed and the lysate, containing fusion protein, can be bound to a suitable affinity matrix by contacting the lysate with an affinity matrix.
  • the fusion protein can be immobilized on a suitable affinity matrix under conditions sufficient to bind the affinity ligand portion of the fusion protein to the matrix, and is contacted with one or more candidate binding agents (e.g., a mixture of peptides) to be tested, under conditions suitable for binding of the binding agents to the FATP portion of the bound fusion protein.
  • candidate binding agents e.g., a mixture of peptides
  • the affinity matrix with bound fusion protein can be washed with a suitable wash buffer to remove unbound candidate binding agents and non-specifically bound candidate binding agents. Those agents which remain bound can be released by contacting the affinity matrix with fusion protein bound thereto with a suitable elution buffer.
  • Wash buffer can be formulated to permit binding of the fusion protein to the affinity matrix, without significantly disrupting binding of specifically bound binding agents.
  • elution buffer can be formulated to permit retention of the fusion protein by the affinity matrix, but can be formulated to interfere with binding of the candidate binding agents to the target portion of the fusion protein.
  • a change in the ionic strength or pH of the elution buffer can lead to release of specifically bound agent, or the elution buffer can comprise a release component or components designed to disrupt binding of specifically bound agent to the target portion of the fusion protein.
  • Immobilization can be performed prior to, simultaneous with, or after, contacting the fusion protein with candidate binding agent, as appropriate.
  • Various permutations of the method are possible, depending upon factors such as the candidate molecules tested, the affinity matrix-ligand pair selected, and elution buffer formulation.
  • a suitable elution buffer a matrix elution buffer, such as glutathione for a GST fusion.
  • the fusion protein comprises a cleavable linker, such as a thrombin cleavage site
  • cleavage from the affinity ligand can release a portion of the fusion with the candidate agent bound thereto.
  • Bound agent molecules can then be released from the fusion protein or its cleavage product by an appropriate method, such as extraction.
  • One or more candidate binding agents can be tested simultaneously. Where a mixture of candidate binding agents is tested, those found to bind by the foregoing processes can be separated (as appropriate) and identified by suitable methods (e.g., PCR, sequencing, chromatography). Large libraries of candidate binding agents (e.g., peptides, RNA oligonucleotides) produced by combinatorial chemical synthesis or by other methods can be tested (see e.g., Ohlmeyer, M. H. J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 ( 1993 ) and DeWitt, S. H. et al., Proc. Natl. Acad. Sci.
  • RNA molecules 17:89-93 can also be screened according to the present method to select RNA molecules which bind to a target FATP or FATP fusion protein.
  • binding agents selected from a combinatorial library by the present method carry unique tags
  • identification of individual biomolecules by chromatographic methods is possible.
  • chromatographic separation, followed by mass spectrometry to ascertain structure can be used to identify binding agents selected by the method, for example.
  • the invention also comprises a method for identifying an agent which inhibits interaction between a fatty acid transport protein and a ligand of said protein.
  • the FATP can be one described by amino acid sequence herein, a portion or fragment thereof, a variant thereof, or an ortholog thereof, or a FATP fusion protein.
  • a ligand can be, for instance, a substrate, or a substrate mimic, an antibody, or a compound, such as a peptide, that binds with specificity to a site on the protein.
  • the method comprises combining, not limited to a particular order, the fatty acid protein, the ligand of the protein, and a candidate agent to be assessed for its ability to inhibit interaction between the protein and the ligand, under conditions appropriate for interaction between the protein and the ligand (e.g., pH, salt, temperature conditions conducive to appropriate conformation and molecular interactions); determining the extent to which the protein and ligand interact; and comparing (1) the extent of protein- ligand interaction in the presence of candidate agent with (2) the extent of protein-ligand interaction in the absence of candidate agent, wherein if (1) is less than (2), then the candidate agent is one which inhibits interaction between the protein and the ligand.
  • conditions appropriate for interaction between the protein and the ligand e.g., pH, salt, temperature conditions conducive to appropriate conformation and molecular interactions
  • the method can be facilitated, for example, by using an experimental system which employs a solid support (column chromatography matrix, wall of a plate, microtiter wells, column pore glass, pins to be submerged in a solution, beads, etc.) to which the protein can be attached.
  • the protein can be fixed to a solid phase directly or indirectly, by a linker.
  • the candidate agent to be tested is added under conditions conducive for interaction and binding to the protein.
  • the ligand is added to the solid phase system under conditions appropriate for binding. Excess ligand is removed, as by a series of washes done under conditions that do not disrupt protein-ligand interactions.
  • Detection of bound ligand can be facilitated by using a ligand that carries a label (e.g., fluorescent, chemiluminescent, radioactive).
  • a label e.g., fluorescent, chemiluminescent, radioactive.
  • protein and ligand are allowed to interact in the absence of any candidate agent, under conditions otherwise identical to those used for the “test” conditions where candidate inhibiting agent is present, and any washes used in the test conditions are also used in the control.
  • the extent to which ligand binds to the protein in the presence of candidate agent is compared to the extent to which ligand binds to the protein in the absence of the candidate agent. If the extent to which interaction of the protein and the ligand occurs is less in the presence of the candidate agent than in the absence of the candidate agent, the candidate agent is an agent which inhibits interaction between the protein and the ligand of the protein.
  • an inhibitor (or an enhancer) of a fatty acid transport protein can be identified.
  • the method comprises steps which are, or are variations of the following: contacting the cells with fatty acid, wherein the fatty acid can be labeled for convenience of detection; contacting a first aliquot of the cells with an agent being tested as an inhibitor (or enhancer) of fatty acid uptake while maintaining a second aliquot of cells under the same conditions but without contact with the agent; and measuring (e.g., quantitating) fatty acid in the first and second aliquots of cells; wherein a lesser quantity of fatty acid in the first aliquot compared to that in the second aliquot is indicative that the agent is an inhibitor of fatty acid uptake by a fatty acid transport protein. A greater quantity of fatty acid in the first aliquot compared to that in the second aliquot is indicative that the agent is an enhancer of fatty acid uptake by a fatty acid transport protein.
  • a particular embodiment of identifying an inhibitor or enhancer of fatty acid transport function employs the above steps, but also employs additional steps preceding those given above: introducing into cells of a cell strain or cell line (“host cells” for the intended introduction of, or after the introduction of, a vector) a vector comprising a fatty acid transport protein gene, wherein expression of the gene can be regulatable or constitutive, and providing conditions to the host cells under which expression of the gene can occur.
  • host cells for the intended introduction of, or after the introduction of, a vector
  • a vector comprising a fatty acid transport protein gene, wherein expression of the gene can be regulatable or constitutive
  • contacting and “combining” as used herein in the context of bringing molecules into close proximity to each other, can be accomplished by conventional means. For example, when referring to molecules that are soluble, contacting is achieved by adding the molecules together in a solution. “Contacting” can also be adding an agent to a test system, such as a vessel containing cells in tissue culture.
  • inhibitor refers to an agent which blocks, diminishes, inhibits, hinders, limits, decreases, reduces, restricts or interferes with fatty acid transport into the cytoplasm of a cell, or alternatively and additionally, prevents or impedes the cellular effects associated with fatty acid transport.
  • antagonist refers to an agent which augments, enhances, or increases fatty acid transport into the cytoplasm of a cell. An antagonist will decrease fatty acid concentration, fatty acid metabolism and by-product levels in the cell, leading to phenotypic and molecular changes.
  • a nucleic acid vector can be constructed to comprise a gene encoding a fatty acid transport protein, for example, human FATP2, FATP3, FATP4, FATP5, FATP6, a mutant or variant thereof, an ortholog of the human proteins, such as mouse orthologs or orthologs found in other mammals, or a FATP family protein of origin in an organism other than a mammal.
  • a fatty acid transport protein for example, human FATP2, FATP3, FATP4, FATP5, FATP6, a mutant or variant thereof
  • an ortholog of the human proteins such as mouse orthologs or orthologs found in other mammals, or a FATP family protein of origin in an organism other than a mammal.
  • the gene of the vector can be regulatable, such as by the placement of the gene under the control of an inducible or repressible promoter in the vector (e.g., inducible or repressible by a change in growth conditions of the host cell harboring the vector, such as addition of inducer, binding or functional removal of repressor from the cell millieu, or change in temperature) such that expression of the FATP gene can be turned on or initiated by causing a change in growth conditions, thereby causing the protein encoded by the gene to be produced, in host cells comprising the vector, as a plasma membrane protein.
  • the FATP gene can be constitutively expressed.
  • a vector comprising an FATP gene can be introduced into host cells by a means appropriate to the vector and to the host cell type.
  • commonly used methods such as electroporation, transfection, for instance, transfection using CaCl 2, and transduction (as for a virus or bacteriophage) can be used.
  • Host cells can be, for example, mammalian cells such as primary culture cells or cells of cell lines such as COS cells, 293 cells or Jurkat cells.
  • Host cells can also be, in some cases, cells derived from insects, cells of insect cell lines, bacterial cells, such as E. coli , or yeast cells, such as S. cerevisiae .
  • the fatty acid transport protein whose function is to be assessed, with or without a candidate inhibitor or enhancer be produced in host cells whose ancestor cells originated in a species related to the species of origin of the FATP gene encoding the fatty acid transport protein.
  • tests of function or of inhibition or enhancement of a mammalian FATP be carried out in host mammalian cells producing the FATP, rather than bacterial cells or yeast cells.
  • Host cells comprising a vector comprising a regulatable FATP gene can be treated so as to allow expression of the FATP gene and production of the encoded protein (e.g., by contacting the cells with an inducer compound that effects transcription from an inducible promoter operably linked to the FATP gene).
  • the test agent e.g., an agonist or antagonist
  • the test agent is added to the cells to be used in a fatty acid transport assay, in the presence or absence of test agent, under conditions suitable for production and/or maintenance of the expressed FATP in a conformation appropriate for association of the FATP with test agent and substrate.
  • conditions under which an agent is assessed such as media and temperature requirements, can, initially, be similar to those necessary for transport of typical fatty acid substrates across the plasma membrane.
  • the test agent can be added to the cells in the presence of fatty acid, or in the absence of fatty acid substrate, with the fatty acid substrate being added following the addition of the test agent.
  • the concentration at which the test agent can be evaluated can be varied, as appropriate, to test for an increased effect with increasing concentrations.
  • Test agents to be assessed for their effects on fatty acid transport can be any chemical (element, molecule, compound), made synthetically, made by recombinant techniques or isolated from a natural source.
  • test agents can be peptides, polypeptides, peptoids, sugars, hormones, or nucleic acid molecules.
  • test agents can be small molecules or molecules of greater complexity made by combinatorial chemistry, for example, and compiled into libraries. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds.
  • Test agents can also be natural or genetically engineered products isolated from lysates of cells, bacterial, animal or plant, or can be the cell lysates themselves. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps.
  • the invention relates to a method for identifying agents which alter fatty acid transport, the method comprising providing the test agent to the cell (wherein “cell” includes the plural, and can include cells of a cell strain, cell line or culture of primary cells or organ culture, for example), under conditions suitable for binding to its target, whether to the FATP itself or to another target on or in the cell, wherein the transformed cell comprises a FATP.
  • the agent(s) can be contacted with the cells.
  • the cells can be contacted with a labeled fatty acid.
  • the fatty acid can be, for example, a known substrate of the fatty acid transport protein such as oleate or palmitate.
  • the fatty acid can itself be labeled with a radioactive isotope, (e.g., 3 H or 14 C) or can have a radioactively labeled adduct attached.
  • the fatty acid can have chemically attached to it a fluorescent label, or a substrate for an enzyme occurring within the cells, wherein the substrate yields a detectable product, such as a highly colored or fluorescent product.
  • Addition of candidate inhibitors and labeled substrate to the cells comprising fatty acid transport protein can be in either order or can be simultaneous.
  • a second aliquot of cells which can be called “control” cells (a “first” aliquot of cells can be called “test” cells), is treated, if necessary (as in the case of transformed “host” cells), so as to allow expression of the FATP gene, and is contacted with the labeled substrate of the fatty acid transport protein.
  • the second aliquot of cells is not contacted with one or more agents to be tested for inhibition of the transport function of the protein produced in the cells, but is otherwise kept under the same culture conditions as the first aliquot of cells.
  • the labeled fatty acid is measured in the first and second aliquots of cells.
  • a preliminary step of this measurement process can be to separate the external medium from the cells so as to be able to distinguish the labeled fatty acid external to the cells from that which has been transported inside the cells. This can be accomplished, for instance, by removing the cells from their growth container, centrifuging the cell suspension, removing the supernatant and performing one or more wash steps to extensively dilute the remaining medium which may contain labeled fatty acid.
  • Detection of the labeled fatty acid can be by a means appropriate to the label used. For example, for a radioactive label, detection can be by scintillation counting of appropriately prepared samples of cells (e.g., lysates or protein extracts); for a fluorescent label, by measuring fluorescence in the cells by appropriate instrumentation.
  • a compound tested as a candidate inhibitor of transport function causes the test cells to have less labeled fatty acid detected in the cells than that detected in the control cells, then the compound is an inhibitor of the fatty acid transport protein.
  • Procedures analogous to those above can be devised for identifying enhancers (agonists of FATPs) of fatty acid transport function wherein if the test cells contain more labeled fatty acid than that detected in the control cells, or if the fatty acid is taken up at a higher rate, then the compound being tested can be concluded to be an enhancer of the fatty acid transport protein.
  • Another assay to determine whether an agent is an inhibitor (or enhancer) of fatty acid transport employs animals, one or more of which are administered the agent, and one or more of which are maintained under similar conditions, but are not administered the agent. Both groups of animals are given fatty acids (e.g., orally, intravenously, by tube inserted into stomach or intestine), and the fatty acids taken up into a bodily fluid (e.g., serum) or into an organ or tissue of interest are measured from comparable samples taken from each group of animals.
  • the fatty acids may carry a label (e.g., radioactive) to facilitate detection and quantitation of fatty acids taken up into the fluid or tissue being sampled.
  • This type of assay can be used alone or can be used in addition to in vitro assays of a candidate inhibitor or enhancer.
  • An agent determined to be an inhibitor (or enhancer) of FATP function can be administered to cells in culture, or in vivo, to a mammal (e.g. human) to inhibit (or enhance) FATP function.
  • a mammal e.g. human
  • Such an agent may be one that acts directly on the FATP (for example, by binding) or can act on an intermediate in a biosynthetic pathway to produce FATP, such as transcription of the FATP gene, processing of the MRNA, or translation of the mRNA.
  • An example of such an agent is antisense oligonucleotide.
  • Cell-free assays can also be used to measure the transport of fatty acids across a membrane, and therefor also to assess a test treatment or test agent for its effect on the rate or extent of fatty acid transport.
  • An isolated FATP for example in the presence of a detergent that preserves the native 3-dimensional structure of the FATP, or partially purified FATP, can be used in an artificial membrane system typically used to preserve the native conformation and activity of membrane proteins.
  • Such systems include liposomes, artificial bilayers of phospholipids, isolated plasma membrane such as cell membrane fragrnents, cell membrane fractions, or cell membrane vesicles, and other systems in which the FATP can be properly oriented within the membrane to have transport activity.
  • Assays for transport activity can be performed using methods analogous to those that can be used in cells engineered to predominantly express one FATP whose function is to be measured.
  • a labeled (e.g., radioactively labeled) fatty acid substrate can be incubated with one side of a bilayer or in a suspension of liposomes constructed to integrate a properly oriented FATP.
  • the accumulation of fatty acids with time can be measured, using appropriate means to detect the label (e.g., scintillation counting of medium on each side of the bilayer, or of the contents of liposomes isolated from the surrounding medium).
  • Assays such as these can be adapted to use for the testing of agents which might interact with the FATP to produce an inhibitory or an enhancing effect on the rate or extent of fatty acid transport. That is, the above-described assay can be done in the presence or absence of the agent to be tested, and the results compared.
  • Another embodiment of the invention is a method for inhibiting fatty acid uptake in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of an inhibitor of the transport function of one or more of the fatty acid transport proteins, thereby decreasing fatty acid uptake by cells comprising the fatty acid protein(s).
  • a mammal e.g., a human
  • administering to the mammal a therapeutically effective amount of an inhibitor of the transport function of one or more of the fatty acid transport proteins, thereby decreasing fatty acid uptake by cells comprising the fatty acid protein(s).
  • one or more inhibitors of one or more of the fatty acid transport proteins can be administered in an effective dose, and by an effective route, for example, orally, or by an indwelling device.
  • the inhibitor can be one identified by methods described herein, or can be one that is, for instance, structurally related to an inhibitor identified by methods described herein (e.g., having chemical adducts to better stabilize or solubilize the inhibitor).
  • the invention further relates to compositions comprising inhibitors of fatty acid uptake in a mammal, which may further comprise pharmaceutical carriers suitable for administration to a subject mammal, such as sterile solubilizing or emulsifying agents.
  • a further embodiment of the present invention is a method of enhancing or increasing fatty acid uptake, such as enhancing or increasing LCFA uptake in the liver (e.g., by an enhancer of FATP 5 transport activity to treat acute liver failure) or in the kidney (e.g., by an enhancer of FATP 2 transport activity to treat kidney failure).
  • a therapeutically effective amount of an enhancer of the transport function of one or more of the fatty acid transport proteins can be administered to a mammalian subject, with the result that fatty acid uptake is enhanced.
  • one or more enhancers of one or more of fatty acid transport proteins is administered in an effective dose and by a route (e.g., orally or by a device, such as an indwelling catheter or other device) which can deliver doses to the gut.
  • a route e.g., orally or by a device, such as an indwelling catheter or other device
  • the enhancer of FATP function e.g., an enhancer of FATP 4 function
  • the invention further relates to antibodies that bind to an isolated or recombinant fatty acid transport protein of the FATP family, including portions of antibodies, which can specifically recognize and bind to one or more FATPs.
  • the antibodies and portions thereof of the invention include those which bind to one or more FATPs of mouse or other mammalian species.
  • the antibodies specifically bind to a naturally occurring FATP of humans.
  • the antibodies can be used in methods to detect or to purify a protein of the present invention or a portion thereof by various methods of immunoaffinity chromatography, to inhibit the function of a protein in a method of therapy, or to selectively inactivate an active site, or to study other aspects of the structure of these proteins, for example.
  • the antibodies of the present invention can be polyclonal or monoclonal.
  • the term antibody is intended to encompass both polyclonal and monoclonal antibodies.
  • Antibodies of the present invention can be raised against an appropriate immunogen, including proteins or polypeptides of the present invention, such as an isolated or recombinant FATP1, FATP2, FATP3, FATP4, FATP5, FATP6, mtFATP, ceFATPa, mgFATP, chFATP, dmFATP, drFATP or portions of any of the foregoing, or synthetic molecules, such as synthetic peptides (e.g., conjugated to a suitable carrier).
  • Preferred embodiments are antibodies that bind to any of the following:, hsFATP2, hsFATP3, hsFATP4, hsFATP5 or hsFATP6 polypeptides.
  • the immunogen can be a polypeptide comprising a portion of a FATP and having at least one function of a fatty acid transport protein, as described herein.
  • the term antibody is also intended to encompass single chain antibodies, chimeric, humanized or primatized (CDR-grafted) antibodies and the like, as well as chimeric or CDR-grafted single chain antibodies, comprising portions from more than one species.
  • the chimeric antibodies can comprise portions of proteins derived from two different species, joined together chemically by conventional techniques or prepared as a single contiguous protein using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous protein chain. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S.
  • Biologically functional antibody fragments which can be used include those fragments sufficient for binding of the antibody fragment to a FATP to occur, such as Fv, Fab, Fab′ and F(ab′) 2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab′) 2 fragments, respectively.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab′) 2 heavy chain portion can be designed to include DNA sequences encoding the CH 1 domain and hinge region of the heavy chain.
  • Preparation of immunizing antigen for example, whole cells comprising FATP on the cell surface or purified FATP
  • polyclonal and monoclonal antibody production can be performed using any suitable technique.
  • a variety of methods have been described (See e.g., Kohler et al, Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976); Milstein et al., Nature 266:550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D.
  • a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody producing cells.
  • a suitable immortal cell line e.g., a myeloma cell line such as SP2/0
  • the antibody producing cells preferably those obtained from the spleen or lymph nodes, can be obtained from animals immunized with the antigen of interest.
  • Immunization of animals can be by introduction of whole cells comprising fatty acid transport protein on the cell surface.
  • the fused cells can be isolated using selective culture conditions, and cloned by limiting dilution.
  • Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • Jakobovits et al. Proc. Natl. Acad. Sci. USA, 90:2551-2555 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Lonberg et al., U.S. Pat. No. 5,569,825; Lonberg et al., U.S. Pat. No. 5,545,806; Surani et al., U.S. Pat. No. 5,545,807; and Kucherlapati, R. et al., European Patent No. EP 0 463 151 B1).
  • An agent can be directed to the liver of a mammal, as FATP 5 is expressed in liver but not in other tissue types.
  • a targeting vehicle which specifically binds to FATP 5 can be conjugated to a drug for delivery of the drug to the liver, such as a drug to treat hepatitis, Wilson's disease, lipid storage diseases and liver cancer.
  • Targeting vehicles specific to FATP 5 can be used in studying tissue samples in vitro.
  • the invention also relates to compositions comprising a modulator of FATP function.
  • modulate refers to the ability of a molecule to alter the function of another molecule. Thus, modulate could mean, for example, inhibit, antagonize, agonize, upregulate, downregulate, induce, or suppress.
  • a modulator has the capability of altering function of its target. Such alteration can be accomplished at any stage of the transcription, translation, expression or function of the protein, so that, for example, modulation of a target gene can be accomplished by modulation of the DNA or RNA encoding the protein, and the protein itself.
  • Antagonists or agonists (inhibitors or enhancers) of the FATPs of the invention antibodies that bind a FATP, or mimetics of a FATP can be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a mammalian subject.
  • a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a mammalian subject.
  • Such compositions comprise, for instance, a media additive or a therapeutically effective amount of an inhibitor or enhancer compound to be identified by an assay of the invention and a pharmaceutically acceptable camrer or excipient.
  • Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, ethanol, surfactants, such as glycerol, excipients such as lactose and combinations thereof.
  • the formulation can be chosen by one of ordinary skill in the art to suit the mode of administration.
  • the chosen route of administration will be influenced by the predominant tissue or organ location of the FATP whose function is to be inhibited or enhanced.
  • a preferred administration can be oral or through a tube inserted into the stomach (e.g., direct stomach tube or nasopharyngeal tube), or through other means to accomplish delivery to the small intestine.
  • the invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Compounds of the invention which are FATPs, FATP fussion proteins, FATP mimetics, FATP gene-specific antisense poly- or oligonucleotides, inhibitors or enhancers of a FATP may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • the pharmaceutical compositions may be administered in any effective, convenient manner, including administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, transdermal or intradermal routes, among others.
  • the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
  • the composition may be formulated for topical application, for example, in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams.
  • Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
  • the amount of the compound will vary depending on the size, age, body weight, general health, sex, and diet of the host, and the time of administration, the biological half-life of the compound, and the particular characteristics and symptoms of the disorder to be treated. Adjustment and manipulation of established dose ranges are well within the ability of those of skill in the art.
  • a further aspect of the invention is a method to identify a polymnorphism, or the presence of an alternative or variant allele of a gene in the genome of an organism (of interest here, genes encoding FATPs).
  • polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.
  • a polymorphic locus may be as small as a base pair.
  • Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu.
  • the first identified alleleic form, or the most frequently occurring form can be arbitrarily designated as the reference (usually, “wildtype”) form, and other allelic forms are designated as alternative (sometimes, “mutant” or “variant”).
  • Dipolid organisms may be homozygous or heterozygous for allelic forms.
  • an “allele” or “allelic sequence” is an alternative form of a gene which may result from at least one mutation in the nucleotide sequence. Alleles may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms (polymorphism). Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • a restriction fragment length polymorphism is a variation in DNA sequence that alters the length of a restriction fragment (Botstein et al., Am. J Hum. Genet. 32:314-331 (1980)). The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment.
  • RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; WO 90/11369; Donis-Keller, Cell 51:319-337 (1987); Lander et al., Genetics 121:85-99 (1989)). When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the individual will also exhibit the trait.
  • VNTR variable number tandem repeat
  • polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPs, STRs (short tandem repeats) and VNTRs (variable number tandem repeats). Some single nucleotide polymorphisms occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • Another aspect of the invention is a method for detecting a variant allele of a human FATP gene, comprising preparing amplified, purified FATP DNA from a reference human and amplified, purified, FATP DNA from a “test” human to be compared to the reference as having a variant allele, using the same or comparable amplification procedures, and determining whether the reference DNA and test DNA differ in DNA sequence in the FATP gene, whether in a coding or a noncoding region, wherein, if the test DNA differs in sequence from the reference DNA, the test DNA comprises a variant allele of a human FATP gene.
  • the following is a discussion of some of the methods by which it can be determined whether the reference FATP DNA and test FATP DNA differ in sequence.
  • Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel eletrophoresis. Different alleles can be identified based on the different sequence-dependent strand dissociation properties and electrophoretic migration of DNA in solution (chapter 7 in Erlich, ed. PCR Technology, Principles and Applications for DNA Amplification, W. H. Freeman and Co., New York, 1992).
  • Single-strand Conformation Polymorphism Analysis Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al, Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single-stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.
  • Amplified DNA comprising the FATP gene or portion of the gene of interest from genomic DNA, for example, of a normal individual, is prepared, using primers designed on the basis of the DNA sequences provided herein. Amplified DNA is also prepared, in a similar manner, from genomic DNA of an individual to be tested for bearing a distinguishable allele.
  • the primers used in PCR carry different labels, for example, primer 1 with biotin, and primer 2 with 32 p. Unused primers are separated form the PCR products, and the products are quantitated.
  • the heteroduplexes are used in a mismatch detection assay using immobilized mismatch binding protein (MutS) bound to nitrocellulose.
  • MotS immobilized mismatch binding protein
  • MutS protein has also been used in the detection of point mutations in a gel-mobility-shift assay (Lishanski, A. et al., Proc. Natl. Acad. Sci. USA 91:2674-2678 (1994)).
  • Allele-specific probes The design and use of allele-specific probes for analyzing polymorphims is described by e.g., Saiki et al., Nature 324:163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed so that they hybridize to a segment of a target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.
  • Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms.
  • Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence.
  • Allele-specific Primers An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism, and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17:2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site.
  • the single-base mismatch prevents amplification and no detectable product is formed.
  • the method works best when the mismatch is included in the 3′ -most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • Gene Chips can also be identified by hybridization to nucleic acids immobilized on solid supports (gene chips), as described, for example, in WO 95/11995 and U.S. Pat. No. 5,143,854, both of which are incorporated herein by reference.
  • WO 95/11995 describes subarrays that are optimized for detection of a characterized variant allele. Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence.
  • FATP genes be given a species specific prefix (mm, mus musculus; hs, homo sapiens; mt, mycobacterium tuberculosis; ce caenorbiditis elegans) and numbered such that mammalian homologues in different species share the same number but differ in their prefix.
  • mmFATPI the gene cloned by Schaeffer and Lodish
  • mmFATP 2 the mouse homologue of the rat VLACS
  • the remaining genes would be numbered successively, starting with mmFATP 2 .
  • the mouse genes are approximately 70% identical to the consensus, the yeast and C. elegans genes 60% identical and the mycobacterial gene 55% identical.
  • the database When compared to the database, only one region shows homology to other proteins. This small stretch of amino acids (underlined in FIG. 1) is found in AMP binding proteins. The other regions in this sequence including stretches of amino acids over 90% identical from mycobacteria to mice are not found in any other class of proteins.
  • This FATP “signature sequence” of 360 amino acids was used to construct a phylogenetic tree (FIG. 2 ). As expected, mFATP2 is closer to mmFATP2 than hsFATP2. The fugu gene seems to be most homologous to mmFATP1 and the C. elegans genes are most closely related to each other. Very surprisingly, the mycobacterial gene seems to be more similar to mice genes than to the putative FATPs of other lower organisms.
  • mmFATP1 The tissue distribution of the murine genes was studied by northern analysis. Results are shown in FIG. 3. Probes from the 3′ untranslated region of these genes which shared no appreciable homology among each other were used to avoid cross-hybridization of these genes.
  • the expression pattern of mmFATP1 agrees with that previously found by Schaeffer and Lodish.
  • mmFATP2 is expressed exclusively in liver and kidney, which corresponds with the reported tissue distribution of the rat homologue (VLACS), as assessed by western blot.
  • mmFATP3 is expressed in lung, liver and testis, rmmFATP4 in heart brain lung liver and kidney, and mmFATP5 is exclusively expressed in liver.
  • the human homologue of mmFATP5, hsFATPs is liver specific in humans and cannot be detected in a wide array of other tissues, including fetal liver.
  • Cos cells were transiently transfected with the genes and the uptake of a Bodipy-labeled analog of a long chain fatty acid by FACS was measured.
  • mmFATP1, mmFATP2 and mmFATP5 increase uptake of the Bodipy-labeled lauric acid (FIG. 4).
  • FACS Bodipy-labeled lauric acid

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A family of fatty acid transport proteins (FATPs) mediate transport of long chain fatty acids (LCFAs) across cell membranes into cells. These proteins exhibit different expression patterns among the organs of mammals. Nucleic acids encoding FATPs of this family, are described. Also described are methods to test FATPs for fatty acid transport function, and methods to identify inhibitors or enhancers of transport function. The altering of LCFA uptake by administering to the mammal an inhibitor or enhancer of FATP transport function of a FATP can decrease or increase calories available as fats, and can decrease or increase circulating fatty acids. The organ specificity of FATP distribution can be exploited in methods to direct drugs, diagnostic indicators and so forth to an organ.

Description

    RELATED APPLICATIONS
  • This application is a divisional of U.S. application Ser. No. 09/232,191 filed Jan. 14, 1999 which claims the benefit of U.S. Provisional Application No.60/071,374, filed Jan. 15, 1998, U.S. Provisional Application No. 60/093,491 filed Jul. 20, 1998 and U.S. Provisional Application No. 60/110,941 filed Dec. 4, 1998. The teachings of each of these applications are incorporated herein by reference in their entirety.[0001]
    • GOVERNMENT SUPPORT
  • [0002] The invention was supported, in whole or in part, by National Institutes of Health Grant DK 47618 and National Institutes of Health Grant 5 T32 CA 09541. The U.S. Government has certain rights in the invention.
    • BACKGROUND OF THE INVENTION
  • Long chain fatty acids (LCFAs) are an important source of energy for most organisms. They also function as blood hormones, regulating key metabolic functions such as hepatic glucose production. Although LCFAs can diffuse through the hydrophobic core of the plasma membrane into cells, this nonspecific transport cannot account for the high affinity and specific transport of LCFAs exhibited by cells such as cardiac muscle, hepatocytes, enterocytes, and adipocytes. The molecular mechanisms of LCFA transport remains largely unknown. Identifying these mechanisms can lead to pharmaceuticals that modulate fatty acid uptake by various organs, thereby alleviating certain medical conditions (e.g. obesity). [0003]
    • SUMMARY OF THE INVENTION
  • Described herein are members of a diverse family of fatty acid transport proteins (FATPs) which are evolutionary conserved; these FATPs are plasma membrane proteins which mediate transport of LCFAs across the membranes and into cells. Members of the FATP family described herein are present in a wide variety of organisms, from mycobacteria to humans, and exhibit very different expression patterns in tissues. FATP family members are expressed in prokaryotic and eukaryotic organisms and comprise characteristic amino acid domains or sequences which are highly conserved across family members. [0004]
  • As described herein, four novel mouse FATPs, referred to as mmFATP2, mmFATP3, mmFATP4 and mmFATP5, and five human FATPs, referred to as, hsFATP2, hsFATP3, hsFATP4, hsFATP5 and hsFATP6, have been identified. Human FATPs 2-5 have orthologs in mice; the sixth human FATP (hsFATP[0005] 6) does not as yet have a mouse ortholog. The expression patterns of these FATPs vary, as described below.
  • The present invention relates to FATP family members from prokaryotes and eukaryotes, nucleic acids (DNA, RNA) encoding FATPs, and nucleic acids which are useful as probes or primers (e.g., for use in hybridization methods, amplification methods) for example, in methods of detecting FATP-encoding genes, producing FATPs, and purifying or isolating FATP-encoding DNA or RNA. Also the subject of this invention are antibodies (polyclonal or monoclonal) which bind an FATP or FATPs; methods of identifying additional FATP family members (for example, orthologs of those FATPs described herein by amino acid sequence) and variant alleles of known FATP genes; methods of identifying compounds which bind to an FATP or to a polypeptide comprising a portion of a FATP, or modulate or alter (enhance or inhibit) FATP function; compounds which modulate or alter FATP function; methods of modulating or altering (enhancing or inhibiting) FATP function and, thus, LCFA uptake into tissues of a mammal (e.g., human) by administering a compound or molecule (a drug or agent) which increases or reduces FATP activity; and methods of targeting compounds to tissues by administering a complex of the compound to be targeted to tissues and a component which is bound by an FATP present on cells of the tissues to which the compound is to be targeted. For example, a complex of a drug to be delivered into the liver and a component which is bound by an FATP present on liver cells (e.g., FATP[0006] 5) can be administered. In a further embodiment, LCFA uptake by the liver is modulated or altered (enhanced or reduced), in an individual. For example, a drug which inhibits the function of an FATP present in liver (e.g., FATP5) is administered to an individual who is diabetic, in order to reduce LCFA uptake by liver cells and, thus reduce insulin resistance.
  • The present invention, thus, provides methods which are useful to alter, particularly reduce, LCFA uptake in individuals and, as a result, to alter (particularly reduce), availability of the LCFAs for further metabolism. In a specific embodiment, the present invention provides methods useful to reduce LCFA uptake and, thus, fatty acid metabolism in individuals, with the result that caloric availability from fats is reduced, and circulating fatty acid levels are lower than they otherwise would be. These methods are useful, for example, as a means of weight control in individuals, (e.g., humans) and as a means of preventing elevated serum lipid levels or reducing serum lipid levels in humans. [0007]
  • The identification of this evolutionary conserved fatty acid transporter family will allow a better understanding of the mechanisms whereby LCFAs traverse the lipid bilayer as well as yield insight into the control of energy homeostasis and its dysregulation in diseases such as diabetes and obesity. [0008]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the amino acid sequences of mmFATP1 (SEQ ID NO:1), mmFATP5 (SEQ ID NO:2), ceFATPA (SEQ ID NO:3), scFATP (SEQ ID NO:4), and mtFATP (SEQ ID NO:5). [0009]
  • FIG. 2 is a phylogenetic tree showing the relationships among the FATP family members and VLACs, based on the 360 amino acid signature sequence of FATP. [0010]
  • FIGS. [0011] 3A-3E are photographs of the results of northern analysis of issue distribution of the murine FATP genes.
  • FIGS. [0012] 4A-4D shows results of FACs analysis of uptake of a BODIPY-labeled analog of a long chain fatty acid by COS cells transiently transfected with mmFATP1, mmFTP2, or mmFATP5 (FIGS. 4B, 4C, and 4D, respectively) and by control (untransfected COS cells; FIG. 4A).
  • FIGS. 5A and 5B are the mmFATP3 DNA sequence (SEQ ID NO:6). [0013]
  • FIG. 6 is the mmFATP3 protein sequence (SEQ ID NO:7). [0014]
  • FIGS. 7A and 7B are the mmFATP4 DNA sequence (SEQ ID NO:8). [0015]
  • FIG. 8 is the mmFATP4 protein sequence (SEQ ID NO:9). [0016]
  • FIGS. 9A and 9B are the rmFATP5 DNA sequence (SEQ ID NO:10). [0017]
  • FIG. 10 is the mmFATP5 protein sequence (SEQ ID NO:11). [0018]
  • FIGS. 11A and 11B are the hsFATP2 DNA sequence (SEQ ID NO:12). [0019]
  • FIG. 12 is the hsFATP2 protein sequence (SEQ ID NO:13). [0020]
  • FIGS. 13A and 13B are the hsFATP3 DNA sequence (SEQ ID NO:14). [0021]
  • FIG. 14 is the hsFATP3 protein sequence (SEQ ID NO:15). [0022]
  • FIG. 15A and 15B are the hsFATP4 DNA sequence (SEQ ID NO:16). [0023]
  • FIG. 16 is the hsFATP4 protein sequence (SEQ ID NO:17). [0024]
  • FIG. 17A and 17B are the hsFATP5 DNA sequence (SEQ ID NO:18). [0025]
  • FIG. 18 is the hsFATP5 protein sequence (SEQ ID NO:19). [0026]
  • FIGS. 19A and 19B are the hsFATP6 DNA sequence (SEQ ID NO:20). [0027]
  • FIG. 20 is the hsFATP6 protein sequence (SEQ ID NO:21). [0028]
  • FIGS. 21A and 21B are the mtFATP DNA sequence (SEQ ID NO:22). [0029]
  • FIG. 22 is the mtFATP protein sequence (SEQ ID NO:23). [0030]
  • FIG. 23A and 23B is a partial DNA sequence encoding a FATP of [0031] Drosophila melanogaster (SEQ ID NO:24).
  • FIG. 24 is a partial amino acid sequence of a [0032] Drosophila melanogaster FATP (SEQ ID NO:25).
  • FIG. 25 is a partial DNA sequence encoding a FATP of [0033] Danio rerio (SEQ ID NO:26).
  • FIG. 26 is a partial amino acid sequence of a [0034] Danio rerio (zebrafish) FATP (SEQ ID NO:27).
  • FIG. 27A and 27B is a DNA sequence encoding FATPa of [0035] Caenorhabditis elegans (SEQ ID NO:28).
  • FIG. 28 is an amino acid sequence of [0036] Caenorhabditis elegans FATPa (SEQ ID NO:29).
  • FIG. 29A and 29B is a DNA sequence encoding a FATPb of [0037] Caenorhabditis elegans (SEQ ID NO:30).
  • FIG. 30 is a amino acid sequence of [0038] Caenorhabditis elegans FATPb (SEQ ID NO:31).
  • FIG. 31A and 31B is a DNA sequence encoding a FATP of [0039] Cochliobolu heterostrophus (SEQ ID NO:32).
  • FIG. 32 is an amino acid sequence of a [0040] Cochliobolu heterostrophus FATP (SEQ ID NO:33).
  • FIG. 33 is a partial DNA sequence encoding a FATP of [0041] Magnaporthe grisea (SEQ ID NO:34).
  • FIG. 34 is a partial amino acid sequence of a [0042] Magnaporthe grisea FATP (SEQ ID NO:35).
  • FIG. 35A and 35B is a DNA sequence of a [0043] Mycobacterium tuberculosis FATP (SEQ ID NO:36).
  • FIG. 36 is an amino acid sequence of a [0044] Mycobacterium tuberculosis FATP (SEQ ID NO:37).
    • DETAILED DESCRIPTION OF THE INVENTION
  • As described herein, FATPs are a large evolutionary conserved family of proteins that mediate the transport of LCFAs into cells. The family includes proteins which are conserved from mycobacteria to humans and exhibit very different expression patterns in tissues. Specific embodiments described include FATPs from mice, humans, nematodes, fungi, and mycobacteria. The term “fatty acid transport proteins” (“FATPs”) as used herein, refers to the proteins described herein as FATP1, FATP2, FATP3, FATP4, FATP5 and FATP6, which have been described in one or more species of mammals, as well as mtFATP, ceFATPa, ceFATPb, dmFATP, drFATP, mgFATP, and chFATP and other proteins sharing at least about 50% amino acid sequence similarity, preferably at least about 60% sequence similarity, more preferably at least about 70% sequence similarity, and still more preferably, at least about 80% sequence similarity, and most preferably, at least about 90% sequence similarity in the approximately 360 amino acid signature sequence. The approximately 360 amino acid FATP signature sequence is shown in FIG. 1. The nomenclature used herein to refer to FATPs includes a species-specific prefix (e.g., mm, [0045] Mus musculus; hs or h, Homo sapiens or human; mt M. tuberculosis; ce, C. elegans; sc, Saccharomyces cerevisiae) and a number such that mammalian homologues in different species share the same number. For example, hsFATP4 and mmFATP4 are the human and mouse orthologs.
  • Expression patterns of human and mouse FATPs have been assessed and are described below. Briefly, results of these assessments show that FATP5 is a liver-specific gene. FATP2 is highly expressed in liver and kidney. [0046]
  • Long chain fatty acids (LCFAs) are an important energy source for pro- and eukaryotes and are involved in diverse cellular processes, such as membrane synthesis, intracellular signaling, protein modification, and transcriptional regulation. In developed Western countries, human dietary lipids are mainly di- and triglycerides and account for approximately 40% of caloric intake (Weisburger, J. H. (1997) [0047] J. Am. Diet. Assoc. 97:S16-S23). These lipids are broken down into fatty acids and glycerol by pancreatic lipases in the small intestine (Chapus, C., Rovery, M., Sarda, L & Verger, R. (1988) Biochimie 70:1223-34); LCFAs are then transported into brush border cells, where the majority is re-esterified and secreted into the lymphatic system as chylomicrons (Green, P. H. & Riley, J. W. (1981) Aust. NZ. J Med. 11:84-90). Fatty acids are liberated from lipoproteins by the enzyme lipoprotein lipase, which is bound to the luminal side of endothelial cells (Scow, R. O. & Blachette-Mackie, E. J. (1992) Mol. Cell. Biochem 116:181-191). “Free” fatty acids in the circulation are bound to serum albumin (Spector, A. A. (1984) Clin. Physiol. Biochem 2:123-134) and are rapidly incorporated by adipocytes, hepatocytes, and cardiac muscle cells. The latter derive 60-90% of their energy through the oxidation of LCFAs (Neely, J. F. Rovetto, M. J. & Oram, J. F. (1972) Prog. Cardiovasc. Dis: 15:289-329). Although saturable and specific uptake of LCFAs has been demonstrated for intestinal cells, hepatocytes, cardiac myocytes, and adipocytes, the molecular mechanisms of LCFA transport across the plasma membrane have remained controversial (Hui, T. Y. & Bemlohr, D. A. (1997) Front. Biosci. 15:d222-31-d231; Schaffer, J. E. & Lodish, H. F, (1995) Trends Cardiovasc. Med. 5:218-224). Described herein is a large family of highly homologous mammalian LCFA transporters which show wide expression. Further described are novel members of this family in other species, including mycobacterial, fungal and nematode FATPs.
  • The discovery of a diverse but highly homologous family of FATPs is reminiscent of the glucose transporter family. In a manner similar to the FATPs, the glucose transporters have very divergent patterns of tissue expression (McGowan, K. M., Long, S.D. & Pekala, P. H. (1995) [0048] Pharmacol. Ther. 66:465-505). The FATPs, like glucose transporters, may also differ in their substrate specificities, uptake kinetics, and hormonal regulation (Thorens, B. (1996) Am. J Physiol. 270:G541-G553). Indeed, the levels of fatty acids in the blood, like those of glucose, can be regulated by insulin and are dysregulated in diseases such as noninsulin-dependent diabetes and obesity (Boden, G. (1997) Diabetes 46:3-10). The underlying mechanisms for the regulation of free fatty acid concentrations in the blood are not understood, but could be explained by hormonal modulation of FATPs.
  • Insulin-resistance is thought to be the major defect in non insulin-dependent diabetes mellitus (NIDDM) and is one of the earliest manifestations of NIDDM (McGarry (1992) [0049] Science 258:766-770). Free fatty acids (FFAs) may provide an explanation for why obesity is a risk factor for NIDDM. Plasma levels of FFAs are elevated in diabetic patients (Reaven et al. (1988) Diabetes 37:1020). Elevated plasma free fatty acids (FFAs) have been demonstrated to induce insulin-resistance in whole animals and humans (Boden (1998) Front. Biosci. 3:D169-D175). This insulin-resistance is likely mediated by effects of FFAs on a variety of issues. FFAs added to adipocytes in vitro induce insulin resistance in this cell type as evidenced by inhibition of insulin-induced glucose transport (Van Epps-Fung et al. (1997) Endocrinology 138:4338-4345). Rats fed a high fat diet developed skeletal muscle insulin resistance as evidenced by a decrease in insulin-induced glucose uptake by skeletal muscle (Han et al., (1997) Diabetes 46:1761-1767). In addition, elevated plasma FFAs increase insulin-suppressed endogenous glucose production in the liver (Boden (1998) Front. Biosci. 3:D169-D175), thus increasing hepatic glucose output. It has been postulated that the adverse effects of plasma free fatty acids are due to the FFAs being taken up into the cell, leading to an increase in intracellular long chain fatty acyl CoA; intracellular long chain acyl CoAs are thought to mediate the effects of FFAs inside the cell. Thus, fatty acid induced insulin-resistance may be prevented by blocking uptake of FFAs into select tissues, in particular liver (by blocking FATP2 and/or FATP5), adipocyte (by blocking FATP1), and skeletal muscle (by blocking FATP1). Blocking intestinal fat absorption (by blocking FATP4) is also expected to reduce plasma FFA levels and thus improve insulin resistance.
  • During the pathogenesis of NIDDM insulin-resistance can initially be counteracted by increasing insulin output by the pancreatic beta cell. Ultimately, this compensation fails, beta cell function decreases and overt diabetes results (McGarry (1992) [0050] Science 258: 766-770). Manipulating beta cell function is a second point where fatty acid transporter blockers may be beneficial for diabetes. While no FATP homolog has been identified so far that is expressed in the beta cell of the pancreas, the data described below suggest the existence of such a transporter and the sequence information included herein provides the means to identify such a transporter by degenerate PCR, using primers to regions conserved in all FATP family members or by low stringency hybridization. It has been demonstrated that exposure of pancreatic beta- cells to FFAs increases the basal rate of insulin secretion; this in turn leads to a decrease in the intracellular stores of insulin, resulting in decreased capacity for insulin secretion after chronic exposure (Bollheimer et al., (1998) J. Clin. Invest. 101:1094-1101). The effects of FFAs are again likely to be mediated by intracellular long chain fatty acyl CoA molecules (Liu et al., (1998) J. Clin. Invest. 101:1870-1875). FFAs have also been demonstrated to increase beta cell apoptosis (Shimabukuro et al., (1998) Proc. Nat. Acad. Sci. USA 95:2498-2502), possibly contributing to the decrease in beta cell numbers in late stage NIDDM.
  • Another finding with potentially broad implications is the identification of a FATP homologue in [0051] M. tuberculosis. Tuberculosis causes more deaths worldwide than any other infectious agent and drug-resistant tuberculosis is re-emerging as a problem in industrialized nations (Bloom, B. R. & Small, P. M. (1998) N. Engl. J. Med. 338:677-678). Mycobacterium tuberculosis has about 250 enzymes involved in fatty acid metabolism, compared with only about 50 in E. coli. It has been suggested that, living as a pathogen, the mycobacteria are largely lipolytic, rather than lipogenic, relying on the lipds within mammalian cells and the tubercle (Cole, S. T. et al., Nature 393:537-544 (1998)). The de novo synthesis of fatty acids in Mycobacterium leprae is 2 5 insufficient to maintain growth (Wheeler, P. R., Bulmer, K & Ratledge, C. (1990) J. Gene. Microbiol. 136:211-217). Thus, it is reasonable to expect that inhibitors of mtFATP will serve as therapeutics for tuberculosis. FATPs expressed in mycobacteria can be targeted to reduce or prevent replication of mycobacteria (e.g., to reduce or prevent replication of M. tuberculosis) and, thus, reduce or prevent their adverse effects. For example, a FATP or FATPs expressed by M. tuberculosis can be targeted and inhibited, thus reducing or preventing growth of this pathogen (and tuberculosis in humans and other mammals). An inhibitor of an M. tuberculosis FATP can be identified, using methods described herein (e.g., expressing the FATP in an appropriate host cell, such as E. coli or COS cells; contacting the cells with an agent or drug to be assessed for its ability to inhibit the FATP and, as a result, mycobacterial growth, and assessing its effects on growth). A drug or agent identified in this manner can be further tested for its ability to inhibit a M. tuberculosis FATP and M. tuberculosis infection in an appropriate animal model or in humans. A method of inhibiting mycobacterial growth, particularly growth of M. tuberculosis, and compounds useful as drugs for doing so are also the subject of this invention.
  • An isolated polynucleotide encoding mtFATP, like other polynucleotides encoding FATPs of the FATP family, can be incorporated into vectors, nucleic acids of viruses, and other nucleic acid constructs that can be used in various types of host cells to produce mtFATP. This mtFATP can be used, as it appears on the surface of cells, or in various artificial membrane systems, to assess fatty acid transport function, to identify ligands and molecules that are modulators of fatty acid transport activity. Molecules found to be inhibitors of mtFATP function can be incorporated into pharmaceutical compositions to administer to a human for the treatment of tuberculosis. [0052]
  • Particular embodiments of the invention are polynucleotides encoding a FATP of [0053] Cochliobolus (Helminthosporium) heterostrophus or portions or variants thereof, the isolated or recombinantly produced FATP, methods for assessing whether an agent binds to the chFATP, and further methods for assessing the effect of an agent being tested for its ability to modulate fatty acid transport activity. Cochliobolus heterostrophus is an ascomycete that is the cause of southern corn leaf blight, an economically important threat to the corn crop in the U.S. The related species C sativus causes crown rot and common root rot in wheat and barley. One or more FATPs of C. heterostrophus can be targeted for the identification of an inhibitor of chFATP function, which can be then be used as an agent effective against infection of plants by C. heterostrophus and related organisms. Methods described herein that were applied in studying the expression of a FATP gene and the function of the FATP in its natural site of expression or in a host cell, can be used in the study of the chFATP gene and protein.
  • [0054] Magnaporthe grisea (rice blast) is an economically important fungal pathogen of rice. Further embodiments of the invention are nucleic acid molecules encoding a FATP of Magnaporthe grisea, portions thereof, or variants thereof, isolated mgFATP, nucleic acid constructs, and engineered cells expressing mgFATP. Other aspects of the invention are assays to identify an agent which binds to mgFATP and assays to identify an agent which modulates the function of mgFATP in cells in which mgFATP is expressed or in artificial membrane systems. Agents identified as inhibiting mgFATP activity can be developed into anti-fungal agents to be used to treat rice infected with rice blast.
  • [0055] Caenorhabditis elegans is a nematode related to plant pathogens and human parasites. An isolated polynucleotide which encodes ceFATP, like other polynucleotides encoding FATPs of the FATP family described herein, can be incorporated into nucleic acid vectors and other constructs that can be used in various types of cells to produce ceFATP. ceFATP as it occurs in cells or as it can be isolated or incorporated into various artificial or reconstructed membrane systems, can be used to assess fatty acid transport, and to identify ligands and agents that modulate fatty acid transport activity. Agents found by such assays to be inhibitors of ceFATP activity can be incorporated into compositions for the treatment of diseases caused by genetically related organisms with a FATP of similar sensitivity to the agents.
  • One aspect of the invention relates to isolated nucleic acids that encode a FATP as described herein, such as those FATPs having an amino acid sequence shown in the figures, and nucleic acids closely related thereto as described herein. [0056]
  • Using the information provided herein, such as a nucleic acid sequence set forth in FIGS. [0057] 5A-5B (SEQ ID NO:6), FIGS. 7A and 7B (SEQ ID NO:8), FIGS. 9A-9B (SEQ ID NO:10), FIGS. 11A-11B (SEQ ID NO:12), FIGS. 13A and 13B (SEQ ID NO:14), and FIGS. 15A-15B (SEQ ID NO:16), FIGS. 17A and 17B (SEQ ID NO:18, FIGS. 19A and 19B (SEQ ID NO:20), and FIGS. 21A and 21B (SEQ ID NO:22), a nucleic acid of the invention encoding a FATP polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing cDNA library fragments, followed by obtaining a full length clone. For example, to obtain a nucleic acid of the invention, a library of clones of cDNA of human or other mammalian DNA can be probed with a labeled oligonucleotide, such as a radiolabeled oligonucleotide, preferably about 17 nucleotides or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent (also, “high stringency”) hybridization conditions. By sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full length sequence. Suitable techniques are described, for example, in Current Protocols in Molecular Biology (F. M. Ausubel et al, eds), containing supplements through Supplement 42, 1998, John Wiley and Sons, Inc., especially chapters 5, 6, and 7. Embodiments of the invention include isolated nucleic acid molecules comprising any of the following nucleotide sequences: 1.) a nucleotide sequence which encodes a protein comprising the amino acid sequence of ceFATPa (SEQ ID NO:29), mgFATP (SEQ ID NO: 35), ceFATP6 (SEQ ID NO:31), mmFATP3 (SEQ ID NO:7), mmFATP4 (SEQ ID NO: 9), mmFATP5 (SEQ ID NO: 1), hsFATP2 (SEQ ID NO: 13), hsFATP3 (SEQ ID NO:15), hsFATP4 (SEQ ID NO:17), hsFATP5 (SEQ If) NO:19), hsFATP6 (SEQ ID NO:21), chFATP (SEQ ID NO:33), mtFATP (SEQ ID NO:23); 2.) nucleotide sequences described herein, such as those encoding hsFATP2, hsFATP3, hsFATP4, hsFATP5, hsFATP6, mmFATP3, mmFATP4, mmFATP5, or mtFATP, respectively); 3.) a nucleotide sequence which is complementary to the nucleotide sequences appearing herein; 4.) a nucleotide sequence which consists of the coding region for a FATP as described herein.
  • The invention further relates to nucleic acids (nucleic acid molecules or polynucleotides) having nucleotide sequences identical over their entire length to those nucleotide sequences shown in the figures. It further relates to DNA, which due to the degeneracy of the genetic code, encodes a FATP encoded by one of the FATP-encoding DNAs, whose amino acid sequence is provided herein. Also provided by the invention are nucleic acids having the coding sequences for the mature polypeptides or fragments in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence. The nucleic acids of the invention encompass nucleic acids that include a single continuous region or discontinuous regions encoding the polypeptide, together with additional regions, that may also contain coding or non-coding sequences. The nucleic acids may also contain non-coding sequences, including, for example, but not limited to, non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequences which encode additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence can be a hexa- histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., [0058] Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), or an HA tag (Wilson et al, Cell 37: 767 (1984)), or a sequence encoding glutathione S-transferase of Schistosoma japonicum (vectors available from Pharmacia; see Smith, D. B. and Johnson K. S., Gene 67:31 (1988) and Kaelin, W. G. et al., Cell 70:351 (1992)). Nucleic acids of the invention also include, but are not limited to, nucleic acids comprising a structural gene and its naturally associated sequences that control gene expression.
  • The invention further relates to variants, including naturally-occurring allelic variants, of those nucleic acids described specifically herein by DNA sequence, that encode variants of such polypeptides as those having the amino acid sequences shown in FIG. 28 (SEQ ID NO:29), FIG. 30 ( SEQ ID NO:31), FIG. 32 (SEQ ID NO:33), FIG. 34 (SEQ ID NO:35), FIG. 6 (SEQ ID NO:7), FIG. 8 (SEQ ID NO:9), FIG. 10 (SEQ ID NO:11), FIG. 12 (SEQ ID NO: 13) FIG. 14 (SEQ ID NO:15), FIG. 16 (SEQ ID NO:17), FIG. 18 (SEQ ID NO:19), FIG. 20 (SEQ ID NO:21), and FIG. [0059] 22 (SEQ ID NO:23). Such variants include nucleic acids encoding variants of the above-listed amino acid sequences, wherein those variants have several, such as 5 to 10, 1 to 5, or 3, 2 or 1 amino acids substituted, deleted, or added, in any combination.
  • Variants include polynucleotides encoding polypeptides with at least 95% but less than 100% amino acid sequence identity to the polypeptides described herein by amino acid sequence. Variant polynucleotides hybridize, under low to high stringency conditions, to the alleles described herein by DNA sequence. In one embodiment, nucleic acid variants have silent substitutions, additions and deletions that do not alter the properties and activities of the encoded FATP. [0060] 10 Orthologous genes are gene loci in different species that are sufficiently similar to each other in their nucleotide sequences to suggest that they originated from a common ancestral gene. Orthologous genes arise when a lineage splits into two species, rather than when a gene is duplicated within a genome. Proteins that are orthologs are encoded by genes of two different species, wherein the genes are said to be orthologous. 15 The invention further relates to polynucleotides encoding polypeptides which are orthologous to those polypeptides having a specific amino acid sequence described herein. These polynucleotides, which can be called ortholog polynucleotides, encode orthologous polypeptides that can range in amino acid sequence identity to a reference amino acid sequence described herein, from about 65% to less than 100%, but preferably 70% to 80%, more preferably 80% to 90%, and still more preferably 90% to less than 100%. Orthologous polypeptides can also be those polypeptides that range in amino acid sequence similarity to a reference amino acid sequence described herein from about 75% to 100%, within the signature sequence. The amino acid sequence similarity between the signature sequences of orthologous polypeptides is preferably 80%, more preferably 90%, and still more preferably, 95%. The ortholog polynucleotides encode polypeptides that have similar functional characteristics (e.g., fatty acid transport activity) and similar tissue distribution, as appropriate to the organism from which the ortholog polynucleotides can be isolated.
  • Ortholog polynucleotides can be isolated from (e.g., by cloning or nucleic acid amplification methods) a great number of species, as shown by the sample of FATPs from evolutionary divergent species described herein. Ortholog polynucleotides corresponding to those having the nucleotide sequences shown in the figures are those which can be isolated from mammals such as rat, dog, chimpanzee, monkey, baboon, pig, rabbit and guinea pig, for example. [0061]
  • Further variants that are fragments of the nucleic acids of the invention may be used to synthesize full-length nucleic acids of the invention, such as by use as primers in a polymerase chain reaction. As used herein, the term primer refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template. The term primer site refers to the area of the target DNA to which a primer hybridizes. The term primer pair refers to a set of primers including a 5′ (upstream) primer that hybridizes with the 5′ end of the DNA sequence to be amplified and a 3′ (downstream) primer that hybridizes with the complement of the 3′ end of the sequence to be amplified. [0062]
  • Further embodiments of the invention are nucleic acids that are at least 80% identical over their entire length to a nucleic acid described herein. Additional embodiments are nucleic acids, and the complements of such nucleic acids, having at least 90% nucleotide sequence identity to the above-described sequences, and nucleic acids having at least 95% nucleotide sequence identity. In preferred embodiments, DNA of the present invention has 97% nucleotide sequence identity, 98% nucleotide sequence identity, or at least 99% nucleotide sequence identity with the DNA whose sequences are presented herein. [0063]
  • Other embodiments of the invention are nucleic acids that are at least 80% identical in nucleotide sequence to a nucleic acid encoding a polypeptide having an amino acid sequence as set forth in herein, and nucleic acids that are complementary to such nucleic acids. Specific embodiments are nucleic acids having at least 90% nucleotide sequence identity to a nucleic acid encoding a polypeptide having an amino acid sequence as described in the list above, nucleic acids having at least 95% sequence identity, and nucleic acids having at least 97% sequence identity. [0064]
  • The terms “complementary” or “complementarity” as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single-stranded molecules may be “partial” in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single-stranded molecules (that is, when A-T and G-C base pairing is 100% complete). The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend on binding between nucleic acid strands. [0065]
  • The invention further includes nucleic acids that hybridize to the above-described nucleic acids, especially those nucleic acids that hybridize under stringent hybridization conditions. “Stringent hybridization conditions” or “high stringency conditions” generally occur within a range from about T[0066] m minus 5° C (5° C below the strand dissociation temperature or melting temperature (Tm ) of the probe nucleic acid molecule) to about 20° C. to 25° C. below Tm. As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect molecules having identical or related polynucleotide sequences. An example of high stringency hybridization follows. Hybridization solution is (6x SSC/10 mM EDTAI0.5% SDS/5×Denhardt's solution/100 μg/ml sheared and denatured salmon sperm DNA). Hybridization is at 64-65° C. for 16 hours. The hybridized blot is washed two times with 2×SSC/0.5% SDS solution at room temperature for 15 minutes each, and two times with 0.2×SSC/0.5% SDS at 65° C., for one hour each. Further examples of high stringency conditions can be found on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., containing supplements up through Supplement 42, 1998). Examples of high, medium, and low stringency conditions can be found on pages 36 and 37 of WO 98/40404, which are incorporated herein by reference.
  • The invention further relates to nucleic acids obtainable by screening an appropriate library with a probe having a nucleotide sequence such as one set forth herein, or a probe which is a sufficiently long portion of these sequences; and isolating the nucleic acid. Such probes generally can comprise at least 15 nucleotides. Nucleic acids obtainable by such screenings may include RNAs, cDNAs and genomic DNA, for example, encoding FATPs of the FATP family described herein. [0067]
  • Further uses for the nucleic acid molecules of the invention, whether encoding a full-length FATP or whether comprising a contiguous portion of a nucleic acid molecule described herein by sequence include use as markers for tissues in which the corresponding protein is preferentially expressed (to identify constitutively expressed proteins or proteins produced at a particular stage of tissue differentiation or stage of development of a disease state); as molecular weight markers on southern gels; as chromosome markers or tags (when labeled, for example with biotin, a radioactive label or a fluorescent label) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in a mammal to identify potential genetic disorders; as probes to hybridize and thus identify, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel nucleic acid molecules; for selecting and making oligomers for attachment to a “gene chip” or other support, to be used, for example, for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or to elicit another immune response. [0068]
  • Further methods to obtain nucleic acids encoding FATPs of the FATP family include PCR and variations thereof (e.g., “RACE” PCR and semi-specific PCR methods). Portions of the nucleic acids having a nucleotide sequence set forth herein, (especially “flanking sequences” on either side of a coding region) can be used as primers in methods using the polymerase chain reaction, to produce DNA from an appropriate template nucleic acid. [0069]
  • Once a fragment of the FATP gene is generated by PCR, it can be sequenced, and the sequence of the product can be compared to other DNA sequences, for example, by using the BLAST Network Service at the National Center for Biotechnology Information. The boundaries of the open reading frame can then be identified using semi-specific PCR or other suitable methods such as library screening. Once the 5′ initiator methionine codon and the 3′ stop codon have been identified, a PCR product encoding the full-length gene can be generated using genomic DNA as a template, with primers complementary to the extreme 5′ and 3′ ends of the gene or to their flanking sequences. The full-length genes can then be cloned into expression vectors for the production of functional proteins. [0070]
  • The invention also relates to isolated proteins or polypeptides such as those encoded by nucleic acids of the present invention. Isolated proteins can be purified from a natural source or can be made recombinantly. Proteins or polypeptides referred to herein as “isolated” are proteins or polypeptides that exist in a state different from the state in which they exist in cells in which they are normally expressed in an organism, and include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. Thus, the term “isolated” as used herein, indicates that the polypeptide in question exists in a physical milieu distinct from that in which it occurs in nature. Thus, “isolated” includes existing in membrane fragments and vesicles membrane fractions, liposomes, lipid bilayers and other artificial membrane systems. An isolated FATP may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, and may even be purified essentially to homogeneity, for example as determined by PAGE or column chromatography (for example, HPLC), but may also have further cofactors or molecular stabilizers, such as detergents, added to the purified protein to enhance activity. In one embodiment, proteins or polypeptides are isolated to a state at least about 75% pure; more preferably at least about 85% pure, and still more preferably at least about 95% pure, as determined by Coomassie blue staining of proteins on SDS-polyacrylamide gels. Proteins or polypeptides referred to herein as “recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids. [0071]
  • In a preferred embodiment, an isolated polypeptide comprising a FATP, a functional portion thereof, or a functional equivalent of the FATP, has at least one function characteristic of a FATP, for example, transport activity, binding function (e.g., a domain which binds to AMP), or antigenic function (e.g., binding of antibodies that also bind to a naturally-occurring FATP, as that function is found in an antigenic determinant). Functional equivalents can have activities that are quantitatively similar to, greater than, or less than, the reference protein. These proteins include, for example, naturally occurring FATPs that can be purified from tissues in which they are produced (including polymorphic or allelic variants), variants (e.g., mutants) of those proteins and/or portions thereof. Such variants include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides in which one or more residues are modified, and mutants comprising one or more modified residues. Portions or fragments of a FATP can range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. [0072]
  • The isolated proteins of the invention preferably include mammalian fatty acid transport proteins of the FATP family of homologous proteins. In one embodiment, the extent of amino acid sequence similarity between a polypeptide having one of the amino acid sequences shown, for example, in FIGS. 1, 6, [0073] 8, 10, 12, 14, 16, 18, 20, or 22 and the respective functional equivalents of these polypeptides is at least about 88%. In other embodiments, the degree of amino acid sequence similarity between a FATP and its respective functional equivalent is at least about 91%, at least about 94%, or at least about 97%.
  • The polypeptides of the invention also include those FATPs encoded by polynucleotides which are orthologous to those polynucleotides, the sequences of which are described herein in whole or in part. FATPs which are orthologs to those described herein by amino acid sequence, in whole or in part, are, for example fatty acid transport proteins 1-6 of dog, rat chimpanzee, monkey, rabbit, guinea pig, baboon and pig, and are also embodiments of the invention. [0074]
  • To determine the percent identity or similarity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment, and non-homologous (dissimilar) sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “similarity”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. [0075]
  • The invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptides described herein by amino acid sequence. Similarity for a polypeptide is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and lIle; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., [0076] Science 247:1306-1310 (1990).
  • The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. ([0077] Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereaux, J., eds., M. Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version [0078] 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, word length=12 to obtain nucleotide sequences homologous to (with calculatably significant similarity to) the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
  • Similarity for nucleotide and amino acid sequences can be defined in terms of the parameters set by the Advanced Blast search available from NCBI (the National Center for Biotechnology Information; see, for Advanced BLAST page, www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast?Jform=1). These default parameters, recommended for a query molecule of length greater than 85 amino acid residues or nucleotides have been set as follows: gap existence cost, [0079] 11, per residue gap cost, 1; lambda ratio, 0.85. Further explanation of version 2.0 of BLAST can be found on related website pages and in Altschul, S. F. et al., Nucleic Acids Res. 25:3389-3402 (1997).
  • The invention further relates to fusion proteins, comprising a FATP or functional portion thereof (as described above) as a first moiety, linked to second moiety not occurring in the FATP as found in nature. Thus, the second moiety can be, for example, an amino acid, peptide or polypeptide. The first moiety can be in an N-terminal location, C-terminal location or internal to the fusion protein. In one embodiment, the fusion protein comprises a FATP as the first moiety, and a second moiety comprising a linker sequence and an affinity ligand. Fusion proteins can be produced by a variety of methods. For example, a fusion protein can be produced by the insertion of a FATP gene or portion thereof into a suitable expression vector, such as Bluescript SK +/− (Stratagene), pGEX-4T-2 (Pharmacia), pET-24(+) (Novagen), or vectors of similar construction. The resulting construct can be introduced into a suitable host cell for expression. Upon expression, fusion protein can be purified from cells by means of a suitable affinity matrix (See e.g., [0080] Current Protocols in Molecular Biology, Ausubel, F. M. et al., eds., Vol. 2, pp. 16.4.1-16.7.8, containing supplements up through Supplement 42, 1998).
  • The invention also relates to enzymatically produced, synthetically produced, or recombinantly produced portions of a fatty acid transport protein. Portions of a FATP can be made which have full or partial function on their own, or which when mixed together (though fully, partially, or nonfunctional alone), spontaneously assemble with one or more other polypeptides to reconstitute a functional protein having at least one function characteristic of a FATP. [0081]
  • Fragments of a FATP can be produced by direct peptide synthesis, for example those using solid-phase techniques (Roberge, J. Y. et al., [0082] Science 269:202-204 (1995); Merrifield, J., J. Am. Chem. Soc. 85:2149-2154 (1963)). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be carried out using, for instance, an Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of a FATP can be synthesized separately and combined using chemical methods.
  • One aspect of the invention is a peptide or polypeptide having the amino acid sequence of a portion of a fatty acid transport protein which is hydrophilic rather than hydrophobic, and ordinarily can be detected as facing the outside of the cell membrane. Such a peptide or polypeptide can be thought of as being an extracellular domain of the FATP, or a mimetic of said extracellular domain. It is known, for example, that a portion of human FATP[0083] 4 that includes a highly conserved motif is involved in AMP-CoA binding function (Stuhlsatz-Krouper, S. M. et al., J. Biol Chem. 44:28642-28650 (1998)).
  • The term “mimetic” as used herein, refers to a molecule, the structure of which is developed from knowledge of the structure of the FATP of interest, or one or more portions thereof, and, as such, is able to effect some or all of the functions of a FATP. [0084]
  • Portions of an FATP can be prepared by enzymatic cleavage of the isolated protein, or can be made by chemical synthesis methods. Portions of a FATP can also be made by recombinant DNA methods in which restriction fragments, or fragments that may have undergone further enzymatic processing, or synthetically made DNAs are joined together to construct an altered FATP gene. The gene can be made such that it encodes one or more desired portions of a FATP. These portions of FATP can be entirely homologous to a known FATP, or can be altered in amino acid sequence relative to naturally occurring FATPs to enhance or introduce desired properties such as solubility, stability, or affinity to a ligand. A further feature of the gene can be a sequence encoding an N-terninal signal peptide directed to the plasma membrane. [0085]
  • A polypeptide or peptide comprising all or a portion of a FATP extracellular domain can be used in a pharmaceutical composition. When administered to a mammal by an appropriate route, the polypeptide or peptide can bind to fatty acids and compete with the native FATPs in the membrane of cells, thereby making fewer fatty acid molecules available as substrates for transport into cells, and reducing fatty acid uptake. [0086]
  • Another aspect of the invention relates to a method of producing a fatty acid transport protein, variants or portions thereof, and to expression systems and host cells containing a vector appropriate for expression of a fatty acid transport protein. [0087]
  • Cells that express a FATP, a variant or a portion thereof, or an ortholog of a FATP described herein by amino acid sequence, can be made and maintained in culture, under conditions suitable for expression, to produce protein in the cells for cell-based assays, or to produce protein for isolation. These cells can be procaryotic or eucaryotic. Examples of procaryotic cells that can be used for expression include [0088] Escherichia coli, Bacillus subtilis and other bacteria. Examples of eucaryotic cells that can be used for expression include yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and other lower eucaryotic cells, and cells of higher eucaryotes such as those from insects and mammals, such as primary cells and cell lines such as CHO, HeLa, 3T3 and BHK cells, COS cells, 293 cells, and Jurkat cells. (See, e.g., Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, Inc., containing Supplements up through Supplement 42, 1998)).
  • In one embodiment, host cells that produce a recombinant FATP, or a portion thereof, a variant, or an ortholog of a FATP described herein by amino acid sequence, can be made as follows. A gene encoding a FATP, variant or a portion thereof can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, phage, cosmid, phagemid, virus, virus-derived vector (e.g., SV40, vaccinia, adenovirus, fowl pox virus, pseudorabies viruses, retroviruses) or other suitable replicon, which can be present in a single copy or multiple copies, or the gene can be integrated in a host cell chromosome. A suitable replicon or integrated gene can contain all or part of the coding sequence for a FATP or variant, operably linked to one or more expression control regions whereby the coding sequence is under the control of transcription signals and linked to appropriate translation signals to permit translation. The vector can be introduced into cells by a method appropriate to the type of host cells (e.g., transfection, electroporation, infection). For expression from the FATP gene, the host cells can be maintained under appropriate conditions (e.g., in the presence of inducer, normal growth conditions, etc.). Proteins or polypeptides thus produced can be recovered (e.g., from the cells, as in a membrane fraction, from the periplasmic space of bacteria, from culture medium) using suitable techniques. Appropriate membrane targeting signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals. [0089]
  • Polypeptides of the invention can be recovered and purified from cell cultures (or from their primary cell source) by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and high performance liquid chromatography. Known methods for refolding protein can be used to regenerate active conformation if the polypeptide is denatured during isolation or purification. [0090]
  • In a further aspect of the invention are methods for assessing the transport function of any of the fatty acid transport proteins or polypeptides described herein, including orthologs, and in variations of these, methods for identifying an inhibitor (or an enhancer) of such function and methods for assessing the transport function in the presence of a candidate inhibitor or a known inhibitor. [0091]
  • A variety of systems comprising living cells can be used for these methods. Cells to be used in fatty acid transport assays, and further in methods for identifying an inhibitor or enhancer of this function, express one or more FATPs. Cells for use in cell-based assays described herein can be drawn from a variety of sources, such as isolated primary cells of various organs and tissues wherein one or more FATPs are naturally expressed. In some cases, the cells can be from adult organs, and in some cases, from embryonic or fetal organs, such as heart, lung, liver, skeletal muscle, kidney and the like. Cells for this purpose can also include cells cultured as fragments of organs or in conditions simulating the cell type and/or tissue organization of organs, in which artificial materials may be used as substrates for cell growth. Other types of cells suitable for this purpose include cells of a cell strain or cell line (ordinarily comprising cells considered to be “transformed”) transfected to express one or more FATPs. [0092]
  • A further embodiment of the invention is a method for detecting, in a sample of cells, a fatty acid transport protein, a portion or fragment thereof, a fusion protein comprising a FATP or a portion thereof, or an ortholog as described herein, wherein the cells can be, for instance, cells of a tissue, primary culture cells, or cells of a cell line, including cells into which nucleic acid has been introduced. The method comprises adding to the sample an agent that specifically binds to the protein, and detecting the agent specifically bound to the protein. Appropriate washing steps can be added to reduce nonspecific binding to the agent. The agent can be, for example, an antibody, a ligand or a substrate mimic. The agent can have incorporated into it, or have bound to it, covalently or by high affinity non-covalent interactions, for instance, a label that facilitates detection of the agent to which it is bound, wherein the label can be, but is not limited to, a phosphorescent label, a fluorescent label, a biotin or avidin label, or a radioactive label. The means of detection of a fatty acid transport protein can vary, as [0093] 10 appropriate to the agent and label used. For example, for an antibody that binds to the fatty acid transport protein, the means of detection may call for binding a second antibody, which has been conjugated to an enzyme, to the antibody which binds the fatty acid transport protein, and detecting the presence of the second antibody by means of the enzymatic activity of the conjugated enzyme.
  • Similar principles can also be applied to a cell lysate or a more purified preparation of proteins from cells that may comprise a fatty acid transport protein of interest, for example in the methods of immunoprecipitation, immunoblotting, immunoaffinity methods, that in addition to detection of the particular FATP, can also be used in purification steps, and qualitative and quantitative immunoassays. See, for instance, [0094] chapters 11 through 14 in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, eds., Cold Spring Harbor Laboratory, 1988.
  • Isolated fatty acid transport protein or, an antigenically similar portion thereof, especially a portion that is soluble, can be used in a method to select and identify molecules which bind specifically to the FATP. Fusion proteins comprising all of, or a portion of, the fatty acid transport protein linked to a second moiety not occurring in the FATP as found in nature, can be prepared for use in another embodiment of the method. Suitable fusion proteins for this purpose include those in which the second moiety comprises an affinity ligand (e.g., an enzyme, antigen, epitope). FATP fusion proteins can be produced by the insertion of a gene encoding the FATP or a variant thereof, or a suitable portion of such gene into a suitable expression vector, which encodes an affinity ligand (e.g., pGEX-4T-2 and pET-1 Sb, encoding glutathione S-transferase and His-Tag affinity ligands, respectively). The expression vector can be introduced into a suitable host cell for expression. Host cells are lysed and the lysate, containing fusion protein, can be bound to a suitable affinity matrix by contacting the lysate with an affinity matrix. [0095]
  • In one embodiment, the fusion protein can be immobilized on a suitable affinity matrix under conditions sufficient to bind the affinity ligand portion of the fusion protein to the matrix, and is contacted with one or more candidate binding agents (e.g., a mixture of peptides) to be tested, under conditions suitable for binding of the binding agents to the FATP portion of the bound fusion protein. Next, the affinity matrix with bound fusion protein can be washed with a suitable wash buffer to remove unbound candidate binding agents and non-specifically bound candidate binding agents. Those agents which remain bound can be released by contacting the affinity matrix with fusion protein bound thereto with a suitable elution buffer. Wash buffer can be formulated to permit binding of the fusion protein to the affinity matrix, without significantly disrupting binding of specifically bound binding agents. In this aspect, elution buffer can be formulated to permit retention of the fusion protein by the affinity matrix, but can be formulated to interfere with binding of the candidate binding agents to the target portion of the fusion protein. For example, a change in the ionic strength or pH of the elution buffer can lead to release of specifically bound agent, or the elution buffer can comprise a release component or components designed to disrupt binding of specifically bound agent to the target portion of the fusion protein. [0096]
  • Immobilization can be performed prior to, simultaneous with, or after, contacting the fusion protein with candidate binding agent, as appropriate. Various permutations of the method are possible, depending upon factors such as the candidate molecules tested, the affinity matrix-ligand pair selected, and elution buffer formulation. For example, after the wash step, fusion protein with binding agent molecules bound thereto can be eluted from the affinity matrix with a suitable elution buffer (a matrix elution buffer, such as glutathione for a GST fusion). Where the fusion protein comprises a cleavable linker, such as a thrombin cleavage site, cleavage from the affinity ligand can release a portion of the fusion with the candidate agent bound thereto. Bound agent molecules can then be released from the fusion protein or its cleavage product by an appropriate method, such as extraction. [0097]
  • One or more candidate binding agents can be tested simultaneously. Where a mixture of candidate binding agents is tested, those found to bind by the foregoing processes can be separated (as appropriate) and identified by suitable methods (e.g., PCR, sequencing, chromatography). Large libraries of candidate binding agents (e.g., peptides, RNA oligonucleotides) produced by combinatorial chemical synthesis or by other methods can be tested (see e.g., Ohlmeyer, M. H. J. et al., [0098] Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to tagged compounds; see also Rutter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D. et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No. 4,833,092). Random sequence RNA libraries (see Ellington, A. D. et al., Nature 346:818-822 (1990); Bock, L. C. et al., Nature 355:584-566 (1992); and Szostak, J. W., Trends in Biochem. Sci. 17:89-93 (March, 1992)) can also be screened according to the present method to select RNA molecules which bind to a target FATP or FATP fusion protein. Where binding agents selected from a combinatorial library by the present method carry unique tags, identification of individual biomolecules by chromatographic methods is possible. Where binding agents do not carry tags, chromatographic separation, followed by mass spectrometry to ascertain structure, can be used to identify binding agents selected by the method, for example.
  • The invention also comprises a method for identifying an agent which inhibits interaction between a fatty acid transport protein and a ligand of said protein. The FATP can be one described by amino acid sequence herein, a portion or fragment thereof, a variant thereof, or an ortholog thereof, or a FATP fusion protein. Here, a ligand can be, for instance, a substrate, or a substrate mimic, an antibody, or a compound, such as a peptide, that binds with specificity to a site on the protein. The method comprises combining, not limited to a particular order, the fatty acid protein, the ligand of the protein, and a candidate agent to be assessed for its ability to inhibit interaction between the protein and the ligand, under conditions appropriate for interaction between the protein and the ligand (e.g., pH, salt, temperature conditions conducive to appropriate conformation and molecular interactions); determining the extent to which the protein and ligand interact; and comparing (1) the extent of protein- ligand interaction in the presence of candidate agent with (2) the extent of protein-ligand interaction in the absence of candidate agent, wherein if (1) is less than (2), then the candidate agent is one which inhibits interaction between the protein and the ligand. [0099]
  • The method can be facilitated, for example, by using an experimental system which employs a solid support (column chromatography matrix, wall of a plate, microtiter wells, column pore glass, pins to be submerged in a solution, beads, etc.) to which the protein can be attached. Accordingly, in one embodiment, the protein can be fixed to a solid phase directly or indirectly, by a linker. The candidate agent to be tested is added under conditions conducive for interaction and binding to the protein. The ligand is added to the solid phase system under conditions appropriate for binding. Excess ligand is removed, as by a series of washes done under conditions that do not disrupt protein-ligand interactions. Detection of bound ligand can be facilitated by using a ligand that carries a label (e.g., fluorescent, chemiluminescent, radioactive). In a control experiment, protein and ligand are allowed to interact in the absence of any candidate agent, under conditions otherwise identical to those used for the “test” conditions where candidate inhibiting agent is present, and any washes used in the test conditions are also used in the control. The extent to which ligand binds to the protein in the presence of candidate agent is compared to the extent to which ligand binds to the protein in the absence of the candidate agent. If the extent to which interaction of the protein and the ligand occurs is less in the presence of the candidate agent than in the absence of the candidate agent, the candidate agent is an agent which inhibits interaction between the protein and the ligand of the protein. [0100]
  • In a further embodiment, an inhibitor (or an enhancer) of a fatty acid transport protein can be identified. The method comprises steps which are, or are variations of the following: contacting the cells with fatty acid, wherein the fatty acid can be labeled for convenience of detection; contacting a first aliquot of the cells with an agent being tested as an inhibitor (or enhancer) of fatty acid uptake while maintaining a second aliquot of cells under the same conditions but without contact with the agent; and measuring (e.g., quantitating) fatty acid in the first and second aliquots of cells; wherein a lesser quantity of fatty acid in the first aliquot compared to that in the second aliquot is indicative that the agent is an inhibitor of fatty acid uptake by a fatty acid transport protein. A greater quantity of fatty acid in the first aliquot compared to that in the second aliquot is indicative that the agent is an enhancer of fatty acid uptake by a fatty acid transport protein. [0101]
  • A particular embodiment of identifying an inhibitor or enhancer of fatty acid transport function employs the above steps, but also employs additional steps preceding those given above: introducing into cells of a cell strain or cell line (“host cells” for the intended introduction of, or after the introduction of, a vector) a vector comprising a fatty acid transport protein gene, wherein expression of the gene can be regulatable or constitutive, and providing conditions to the host cells under which expression of the gene can occur. [0102]
  • The terms “contacting” and “combining” as used herein in the context of bringing molecules into close proximity to each other, can be accomplished by conventional means. For example, when referring to molecules that are soluble, contacting is achieved by adding the molecules together in a solution. “Contacting” can also be adding an agent to a test system, such as a vessel containing cells in tissue culture. [0103]
  • The term “inhibitor” or “antagonist”, as used herein, refers to an agent which blocks, diminishes, inhibits, hinders, limits, decreases, reduces, restricts or interferes with fatty acid transport into the cytoplasm of a cell, or alternatively and additionally, prevents or impedes the cellular effects associated with fatty acid transport. The term “enhancer” or “agonist”, as used herein, refers to an agent which augments, enhances, or increases fatty acid transport into the cytoplasm of a cell. An antagonist will decrease fatty acid concentration, fatty acid metabolism and by-product levels in the cell, leading to phenotypic and molecular changes. [0104]
  • In order to produce a “host cell” type suitable for fatty acid uptake assays and for assays derived therefrom for identifying inhibitors or enhancers thereof, a nucleic acid vector can be constructed to comprise a gene encoding a fatty acid transport protein, for example, human FATP2, FATP3, FATP4, FATP5, FATP6, a mutant or variant thereof, an ortholog of the human proteins, such as mouse orthologs or orthologs found in other mammals, or a FATP family protein of origin in an organism other than a mammal. The gene of the vector can be regulatable, such as by the placement of the gene under the control of an inducible or repressible promoter in the vector (e.g., inducible or repressible by a change in growth conditions of the host cell harboring the vector, such as addition of inducer, binding or functional removal of repressor from the cell millieu, or change in temperature) such that expression of the FATP gene can be turned on or initiated by causing a change in growth conditions, thereby causing the protein encoded by the gene to be produced, in host cells comprising the vector, as a plasma membrane protein. Alternatively, the FATP gene can be constitutively expressed. [0105]
  • A vector comprising an FATP gene, such as a vector described herein, can be introduced into host cells by a means appropriate to the vector and to the host cell type. For example, commonly used methods such as electroporation, transfection, for instance, transfection using CaCl[0106] 2, and transduction (as for a virus or bacteriophage) can be used. Host cells can be, for example, mammalian cells such as primary culture cells or cells of cell lines such as COS cells, 293 cells or Jurkat cells. Host cells can also be, in some cases, cells derived from insects, cells of insect cell lines, bacterial cells, such as E. coli, or yeast cells, such as S. cerevisiae. It is preferred that the fatty acid transport protein whose function is to be assessed, with or without a candidate inhibitor or enhancer, be produced in host cells whose ancestor cells originated in a species related to the species of origin of the FATP gene encoding the fatty acid transport protein. For example, it is preferable that tests of function or of inhibition or enhancement of a mammalian FATP be carried out in host mammalian cells producing the FATP, rather than bacterial cells or yeast cells.
  • Host cells comprising a vector comprising a regulatable FATP gene can be treated so as to allow expression of the FATP gene and production of the encoded protein (e.g., by contacting the cells with an inducer compound that effects transcription from an inducible promoter operably linked to the FATP gene). [0107]
  • The test agent (e.g., an agonist or antagonist) is added to the cells to be used in a fatty acid transport assay, in the presence or absence of test agent, under conditions suitable for production and/or maintenance of the expressed FATP in a conformation appropriate for association of the FATP with test agent and substrate. For example, conditions under which an agent is assessed, such as media and temperature requirements, can, initially, be similar to those necessary for transport of typical fatty acid substrates across the plasma membrane. One of ordinary skill in the art will know how to vary experimental conditions depending upon the biochemical nature of the test agent. The test agent can be added to the cells in the presence of fatty acid, or in the absence of fatty acid substrate, with the fatty acid substrate being added following the addition of the test agent. The concentration at which the test agent can be evaluated can be varied, as appropriate, to test for an increased effect with increasing concentrations. [0108]
  • Test agents to be assessed for their effects on fatty acid transport can be any chemical (element, molecule, compound), made synthetically, made by recombinant techniques or isolated from a natural source. For example, test agents can be peptides, polypeptides, peptoids, sugars, hormones, or nucleic acid molecules. hi addition, test agents can be small molecules or molecules of greater complexity made by combinatorial chemistry, for example, and compiled into libraries. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Test agents can also be natural or genetically engineered products isolated from lysates of cells, bacterial, animal or plant, or can be the cell lysates themselves. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps. [0109]
  • Thus, the invention relates to a method for identifying agents which alter fatty acid transport, the method comprising providing the test agent to the cell (wherein “cell” includes the plural, and can include cells of a cell strain, cell line or culture of primary cells or organ culture, for example), under conditions suitable for binding to its target, whether to the FATP itself or to another target on or in the cell, wherein the transformed cell comprises a FATP. [0110]
  • In greater detail, to test one or more agents or compounds (e.g., a mixture of compounds can conveniently be screened initially) for inhibition of the transport function of a fatty acid transport protein, the agent(s) can be contacted with the cells. The cells can be contacted with a labeled fatty acid. The fatty acid can be, for example, a known substrate of the fatty acid transport protein such as oleate or palmitate. The fatty acid can itself be labeled with a radioactive isotope, (e.g., [0111] 3H or 14C) or can have a radioactively labeled adduct attached. In other variations, the fatty acid can have chemically attached to it a fluorescent label, or a substrate for an enzyme occurring within the cells, wherein the substrate yields a detectable product, such as a highly colored or fluorescent product. Addition of candidate inhibitors and labeled substrate to the cells comprising fatty acid transport protein can be in either order or can be simultaneous.
  • A second aliquot of cells, which can be called “control” cells (a “first” aliquot of cells can be called “test” cells), is treated, if necessary (as in the case of transformed “host” cells), so as to allow expression of the FATP gene, and is contacted with the labeled substrate of the fatty acid transport protein. The second aliquot of cells is not contacted with one or more agents to be tested for inhibition of the transport function of the protein produced in the cells, but is otherwise kept under the same culture conditions as the first aliquot of cells. [0112]
  • In a further step of a method to identify inhibitors of a fatty acid transport protein, the labeled fatty acid is measured in the first and second aliquots of cells. A preliminary step of this measurement process can be to separate the external medium from the cells so as to be able to distinguish the labeled fatty acid external to the cells from that which has been transported inside the cells. This can be accomplished, for instance, by removing the cells from their growth container, centrifuging the cell suspension, removing the supernatant and performing one or more wash steps to extensively dilute the remaining medium which may contain labeled fatty acid. Detection of the labeled fatty acid can be by a means appropriate to the label used. For example, for a radioactive label, detection can be by scintillation counting of appropriately prepared samples of cells (e.g., lysates or protein extracts); for a fluorescent label, by measuring fluorescence in the cells by appropriate instrumentation. [0113]
  • If a compound tested as a candidate inhibitor of transport function causes the test cells to have less labeled fatty acid detected in the cells than that detected in the control cells, then the compound is an inhibitor of the fatty acid transport protein. Procedures analogous to those above can be devised for identifying enhancers (agonists of FATPs) of fatty acid transport function wherein if the test cells contain more labeled fatty acid than that detected in the control cells, or if the fatty acid is taken up at a higher rate, then the compound being tested can be concluded to be an enhancer of the fatty acid transport protein. [0114]
  • Another assay to determine whether an agent is an inhibitor (or enhancer) of fatty acid transport employs animals, one or more of which are administered the agent, and one or more of which are maintained under similar conditions, but are not administered the agent. Both groups of animals are given fatty acids (e.g., orally, intravenously, by tube inserted into stomach or intestine), and the fatty acids taken up into a bodily fluid (e.g., serum) or into an organ or tissue of interest are measured from comparable samples taken from each group of animals. The fatty acids may carry a label (e.g., radioactive) to facilitate detection and quantitation of fatty acids taken up into the fluid or tissue being sampled. This type of assay can be used alone or can be used in addition to in vitro assays of a candidate inhibitor or enhancer. [0115]
  • An agent determined to be an inhibitor (or enhancer) of FATP function, such as fatty acid binding and/or fatty acid uptake, can be administered to cells in culture, or in vivo, to a mammal (e.g. human) to inhibit (or enhance) FATP function. Such an agent may be one that acts directly on the FATP (for example, by binding) or can act on an intermediate in a biosynthetic pathway to produce FATP, such as transcription of the FATP gene, processing of the MRNA, or translation of the mRNA. An example of such an agent is antisense oligonucleotide. [0116]
  • Cell-free assays can also be used to measure the transport of fatty acids across a membrane, and therefor also to assess a test treatment or test agent for its effect on the rate or extent of fatty acid transport. An isolated FATP, for example in the presence of a detergent that preserves the native 3-dimensional structure of the FATP, or partially purified FATP, can be used in an artificial membrane system typically used to preserve the native conformation and activity of membrane proteins. Such systems include liposomes, artificial bilayers of phospholipids, isolated plasma membrane such as cell membrane fragrnents, cell membrane fractions, or cell membrane vesicles, and other systems in which the FATP can be properly oriented within the membrane to have transport activity. Assays for transport activity can be performed using methods analogous to those that can be used in cells engineered to predominantly express one FATP whose function is to be measured. A labeled (e.g., radioactively labeled) fatty acid substrate can be incubated with one side of a bilayer or in a suspension of liposomes constructed to integrate a properly oriented FATP. The accumulation of fatty acids with time can be measured, using appropriate means to detect the label (e.g., scintillation counting of medium on each side of the bilayer, or of the contents of liposomes isolated from the surrounding medium). Assays such as these can be adapted to use for the testing of agents which might interact with the FATP to produce an inhibitory or an enhancing effect on the rate or extent of fatty acid transport. That is, the above-described assay can be done in the presence or absence of the agent to be tested, and the results compared. [0117]
  • Another embodiment of the invention is a method for inhibiting fatty acid uptake in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of an inhibitor of the transport function of one or more of the fatty acid transport proteins, thereby decreasing fatty acid uptake by cells comprising the fatty acid protein(s). Where it is desirable to reduce the uptake of fatty acids, for example, in the treatment of chronic obesity or as a part of a program of weight control or hyperlipidemia control in a human, one or more inhibitors of one or more of the fatty acid transport proteins can be administered in an effective dose, and by an effective route, for example, orally, or by an indwelling device. The inhibitor can be one identified by methods described herein, or can be one that is, for instance, structurally related to an inhibitor identified by methods described herein (e.g., having chemical adducts to better stabilize or solubilize the inhibitor). The invention further relates to compositions comprising inhibitors of fatty acid uptake in a mammal, which may further comprise pharmaceutical carriers suitable for administration to a subject mammal, such as sterile solubilizing or emulsifying agents. [0118]
  • A further embodiment of the present invention is a method of enhancing or increasing fatty acid uptake, such as enhancing or increasing LCFA uptake in the liver (e.g., by an enhancer of FATP[0119] 5 transport activity to treat acute liver failure) or in the kidney (e.g., by an enhancer of FATP2 transport activity to treat kidney failure). In this embodiment, a therapeutically effective amount of an enhancer of the transport function of one or more of the fatty acid transport proteins can be administered to a mammalian subject, with the result that fatty acid uptake is enhanced. In this embodiment, one or more enhancers of one or more of fatty acid transport proteins is administered in an effective dose and by a route (e.g., orally or by a device, such as an indwelling catheter or other device) which can deliver doses to the gut. The enhancer of FATP function (e.g., an enhancer of FATP4 function) can be identified by methods described herein or can be one that is structurally similar to an enhancer identified by methods described herein.
  • The invention further relates to antibodies that bind to an isolated or recombinant fatty acid transport protein of the FATP family, including portions of antibodies, which can specifically recognize and bind to one or more FATPs. The antibodies and portions thereof of the invention include those which bind to one or more FATPs of mouse or other mammalian species. In a preferred embodiment, the antibodies specifically bind to a naturally occurring FATP of humans. The antibodies can be used in methods to detect or to purify a protein of the present invention or a portion thereof by various methods of immunoaffinity chromatography, to inhibit the function of a protein in a method of therapy, or to selectively inactivate an active site, or to study other aspects of the structure of these proteins, for example. [0120]
  • The antibodies of the present invention can be polyclonal or monoclonal. The term antibody is intended to encompass both polyclonal and monoclonal antibodies. Antibodies of the present invention can be raised against an appropriate immunogen, including proteins or polypeptides of the present invention, such as an isolated or recombinant FATP1, FATP2, FATP3, FATP4, FATP5, FATP6, mtFATP, ceFATPa, mgFATP, chFATP, dmFATP, drFATP or portions of any of the foregoing, or synthetic molecules, such as synthetic peptides (e.g., conjugated to a suitable carrier). Preferred embodiments are antibodies that bind to any of the following:, hsFATP2, hsFATP3, hsFATP4, hsFATP5 or hsFATP6 polypeptides. The immunogen can be a polypeptide comprising a portion of a FATP and having at least one function of a fatty acid transport protein, as described herein. [0121]
  • The term antibody is also intended to encompass single chain antibodies, chimeric, humanized or primatized (CDR-grafted) antibodies and the like, as well as chimeric or CDR-grafted single chain antibodies, comprising portions from more than one species. For example, the chimeric antibodies can comprise portions of proteins derived from two different species, joined together chemically by conventional techniques or prepared as a single contiguous protein using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous protein chain. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; [0122] Boss et al., U.S.
  • Pat. No. 4,816,397; [0123] Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al., U.S. Pat. No. 5,585,089; and Queen et al., European Patent No. EP 0 451 216 B1. See also, Newman, R. et al., Biotechnology, 10:1455-1460 (1992), regarding primatized antibody, and Ladner et al, U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242:423-426 (1988) regarding single chain antibodies.)
  • Whole antibodies and biologically functional fragments thereof are also encompassed by the term antibody. Biologically functional antibody fragments which can be used include those fragments sufficient for binding of the antibody fragment to a FATP to occur, such as Fv, Fab, Fab′ and F(ab′)[0124] 2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab′)2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab′)2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain.
  • Preparation of immunizing antigen (for example, whole cells comprising FATP on the cell surface or purified FATP), and polyclonal and monoclonal antibody production can be performed using any suitable technique. A variety of methods have been described (See e.g., Kohler et al, [0125] Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976); Milstein et al., Nature 266:550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.); Chapter 11 In Current Protocols In Molecular Biology, Vol. 2 (containing supplements up through Supplement 42, 1998), Ausubel, F. M. et al., eds., (John Wiley & Sons: New York, N.Y.)). Generally, a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody producing cells. The antibody producing cells, preferably those obtained from the spleen or lymph nodes, can be obtained from animals immunized with the antigen of interest. Immunization of animals can be by introduction of whole cells comprising fatty acid transport protein on the cell surface. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • Other suitable methods of producing or isolating antibodies (including human antibodies) of the requisite specificity can used, including, for example, methods which select recombinant antibody from a library (e.g., Hoogenboom et al, WO 93/06213; Hoogenboom et al., U.S. Pat. No. 5,565,332; WO 94/13804, published Jun. 23, 1994; and Dower, W. J. et al., U.S. Pat. No. 5,427,908), or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies (see e.g., Jakobovits et al., [0126] Proc. Natl. Acad. Sci. USA, 90:2551-2555 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Lonberg et al., U.S. Pat. No. 5,569,825; Lonberg et al., U.S. Pat. No. 5,545,806; Surani et al., U.S. Pat. No. 5,545,807; and Kucherlapati, R. et al., European Patent No. EP 0 463 151 B1).
  • An agent can be directed to the liver of a mammal, as FATP[0127] 5 is expressed in liver but not in other tissue types. A targeting vehicle which specifically binds to FATP5 can be conjugated to a drug for delivery of the drug to the liver, such as a drug to treat hepatitis, Wilson's disease, lipid storage diseases and liver cancer. Targeting vehicles specific to FATP5 can be used in studying tissue samples in vitro.
  • The invention also relates to compositions comprising a modulator of FATP function. The term “modulate” as used herein refers to the ability of a molecule to alter the function of another molecule. Thus, modulate could mean, for example, inhibit, antagonize, agonize, upregulate, downregulate, induce, or suppress. A modulator has the capability of altering function of its target. Such alteration can be accomplished at any stage of the transcription, translation, expression or function of the protein, so that, for example, modulation of a target gene can be accomplished by modulation of the DNA or RNA encoding the protein, and the protein itself. [0128]
  • Antagonists or agonists (inhibitors or enhancers) of the FATPs of the invention, antibodies that bind a FATP, or mimetics of a FATP can be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a mammalian subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of an inhibitor or enhancer compound to be identified by an assay of the invention and a pharmaceutically acceptable camrer or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, ethanol, surfactants, such as glycerol, excipients such as lactose and combinations thereof. The formulation can be chosen by one of ordinary skill in the art to suit the mode of administration. The chosen route of administration will be influenced by the predominant tissue or organ location of the FATP whose function is to be inhibited or enhanced. For example, for affecting the function of FATP4, a preferred administration can be oral or through a tube inserted into the stomach (e.g., direct stomach tube or nasopharyngeal tube), or through other means to accomplish delivery to the small intestine. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. [0129]
  • Compounds of the invention which are FATPs, FATP fussion proteins, FATP mimetics, FATP gene-specific antisense poly- or oligonucleotides, inhibitors or enhancers of a FATP may be employed alone or in conjunction with other compounds, such as therapeutic compounds. The pharmaceutical compositions may be administered in any effective, convenient manner, including administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, transdermal or intradermal routes, among others. In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. [0130]
  • Alternatively, the composition may be formulated for topical application, for example, in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. [0131]
  • In addition, the amount of the compound will vary depending on the size, age, body weight, general health, sex, and diet of the host, and the time of administration, the biological half-life of the compound, and the particular characteristics and symptoms of the disorder to be treated. Adjustment and manipulation of established dose ranges are well within the ability of those of skill in the art. [0132]
  • A further aspect of the invention is a method to identify a polymnorphism, or the presence of an alternative or variant allele of a gene in the genome of an organism (of interest here, genes encoding FATPs). As used herein, polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic locus may be as small as a base pair. Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu. The first identified alleleic form, or the most frequently occurring form can be arbitrarily designated as the reference (usually, “wildtype”) form, and other allelic forms are designated as alternative (sometimes, “mutant” or “variant”). Dipolid organisms may be homozygous or heterozygous for allelic forms. [0133]
  • An “allele” or “allelic sequence” is an alternative form of a gene which may result from at least one mutation in the nucleotide sequence. Alleles may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms (polymorphism). Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. [0134]
  • Several different types of polymorphisms have been reported. A restriction fragment length polymorphism (RFLP) is a variation in DNA sequence that alters the length of a restriction fragment (Botstein et al., [0135] Am. J Hum. Genet. 32:314-331 (1980)). The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; WO 90/11369; Donis-Keller, Cell 51:319-337 (1987); Lander et al., Genetics 121:85-99 (1989)). When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the individual will also exhibit the trait.
  • Other polymorphisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetra-nucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analysis (U.S. Pat. No. 5,075,217; Armour et al., FEBS Lett. 307:113-115 (1992); Horn et al., WO 91/14003; Jeffreys, EP 370,719), and in a large number of genetic mapping studies. [0136]
  • Other polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPs, STRs (short tandem repeats) and VNTRs (variable number tandem repeats). Some single nucleotide polymorphisms occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects. [0137]
  • Many of the methods described below require amplification of DNA from target samples and purification of the amplified products. This can be accomplished by PCR, for instance. See generally, PCR Technology, Principles and Applications for DNA [0138] Amplification (ed. H. A. Erlich), Freeman Press, New York, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al.), Academic Press, San Diego, CA, 1990; Mattila et al, Nucleic Acids Res. 19:4967 (1991); Eckert et al., PCR Methods and Applications 1:17 (1991); PCR (eds. McPherson et al., IRS Press, Oxford); and U.S. Pat. No. 4,683,202.
  • Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989); Landegren et al.; Science 241:1077 (1988)), transcription amplification (Kwoh et al., [0139] Proc. Natl. Acad. Sci. USA 86:1173 (1989), self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874 (1990), and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • Another aspect of the invention is a method for detecting a variant allele of a human FATP gene, comprising preparing amplified, purified FATP DNA from a reference human and amplified, purified, FATP DNA from a “test” human to be compared to the reference as having a variant allele, using the same or comparable amplification procedures, and determining whether the reference DNA and test DNA differ in DNA sequence in the FATP gene, whether in a coding or a noncoding region, wherein, if the test DNA differs in sequence from the reference DNA, the test DNA comprises a variant allele of a human FATP gene. The following is a discussion of some of the methods by which it can be determined whether the reference FATP DNA and test FATP DNA differ in sequence. [0140]
  • Direct Sequencing. The direct analysis of the sequence of variant alleles of the present invention can be accomplished using either the dideoxy chain termination method or the Maxam and Gilbert method (see Sambrook et al., [0141] Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, New York 1989; Zyskind et al., Recombinant DNA Laboratory Manual, Acad. Press, 1988)).
  • Denaturing Gradient Gel Electrophoresis. Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel eletrophoresis. Different alleles can be identified based on the different sequence-dependent strand dissociation properties and electrophoretic migration of DNA in solution (chapter 7 in Erlich, ed. PCR [0142] Technology, Principles and Applications for DNA Amplification, W. H. Freeman and Co., New York, 1992).
  • Single-strand Conformation Polymorphism Analysis. Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al, [0143] Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single-stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.
  • Detection of Binding by Protein That Binds to Mismatches. Amplified DNA comprising the FATP gene or portion of the gene of interest from genomic DNA, for example, of a normal individual, is prepared, using primers designed on the basis of the DNA sequences provided herein. Amplified DNA is also prepared, in a similar manner, from genomic DNA of an individual to be tested for bearing a distinguishable allele. The primers used in PCR carry different labels, for example, [0144] primer 1 with biotin, and primer 2 with 32p. Unused primers are separated form the PCR products, and the products are quantitated. The heteroduplexes are used in a mismatch detection assay using immobilized mismatch binding protein (MutS) bound to nitrocellulose. The presence of biotin-labeled DNA wherein mismatched regions are bound to the nitrocellulose via MutS protein, is detected by visualizing the binding of streptavidin to biotin. See WO 95/12689. MutS protein has also been used in the detection of point mutations in a gel-mobility-shift assay (Lishanski, A. et al., Proc. Natl. Acad. Sci. USA 91:2674-2678 (1994)).
  • Other methods, such as those described below, can be used to distinguish a FATP allele from a reference allele, once a particular allele has been characterized as to DNA sequence. [0145]
  • Allele-specific probes. The design and use of allele-specific probes for analyzing polymorphims is described by e.g., Saiki et al., [0146] Nature 324:163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed so that they hybridize to a segment of a target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms.
  • Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence. [0147]
  • Allele-specific Primers. An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism, and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, [0148] Nucleic Acid Res. 17:2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3′ -most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • Gene Chips. Allelic variants can also be identified by hybridization to nucleic acids immobilized on solid supports (gene chips), as described, for example, in WO 95/11995 and U.S. Pat. No. 5,143,854, both of which are incorporated herein by reference. WO 95/11995 describes subarrays that are optimized for detection of a characterized variant allele. Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence. [0149]
  • The present method is illustrated by the following examples, which are not intended to be limiting in any way. [0150]
  • EXEMPLIFICATION [0151]
  • To identify novel FATPs we searched the NCBI expressed sequence tag (EST) database using the FATP protein sequence. This strategy led to the identification of more than 50 murine EST sequences which could be assembled into five distinct contiguous DNA sequences (contigs). One of these contigs represented the previously cloned FATP (FATP1). Another was identified as the mouse homologue of the previously cloned rat very long chain acyl Co-A synthase (FATP2). The other three contigs represented new genes (FATP3 to 5). Screening of mouse fetal 10.5 day embryo and adult liver cDNA libraries resulted in full length clones for FATP2 and FATP5 and nearly complete sequences for FATP3 and FATP4. Human homologues for each of the murine genes were also identified. Additionally, a sixth human gene was present in the EST database. It is not clear if this gene does not occur in the mouse or is merely not present in the mouse database. Sequences conserved among the 5 murine FATP genes were used to carry out database searches to include other organisms. This resulted in identification of the previously described FATP homologue in [0152] S. cerevisiae and of novel genes in fugu, C. elegans, Mycobacterium tuberculosis, Deinoccoccus radiodurans, and Archaebacterium fulgidus.
  • In order to compare FATPs from different species we propose that the FATP genes be given a species specific prefix (mm, mus musculus; hs, homo sapiens; mt, mycobacterium tuberculosis; ce caenorbiditis elegans) and numbered such that mammalian homologues in different species share the same number but differ in their prefix. Thus, the gene cloned by Schaeffer and Lodish would be designated mmFATPI, the mouse homologue of the rat VLACS would be designated mmFATP[0153] 2, and the remaining genes would be numbered successively, starting with mmFATP2. For multiple fatty acid transporters in a single non-mammalian species for which the corresponding mammalian FATP counterpart cannot be identified we suggest a small letter suffix, e.g. ceFATPa and ceFATPb for the two C. elegans genes. Faergeman et al. have described three regions of very strong sequence conservation between the yeast FATP gene and the mouse FATP gene. The sequences of mmFATP1, mmFATP5, ceFATPa, scFATP, and mtFATP were compared over a 360 amino acid stretch which includes these regions. The DNAstar program was used to determine a consensus sequence for this region and align these genes with that sequence (FIG. 1). Over this interval, the mouse genes are approximately 70% identical to the consensus, the yeast and C. elegans genes 60% identical and the mycobacterial gene 55% identical. When compared to the database, only one region shows homology to other proteins. This small stretch of amino acids (underlined in FIG. 1) is found in AMP binding proteins. The other regions in this sequence including stretches of amino acids over 90% identical from mycobacteria to mice are not found in any other class of proteins. This FATP “signature sequence” of 360 amino acids was used to construct a phylogenetic tree (FIG. 2). As expected, mFATP2 is closer to mmFATP2 than hsFATP2. The fugu gene seems to be most homologous to mmFATP1 and the C. elegans genes are most closely related to each other. Very surprisingly, the mycobacterial gene seems to be more similar to mice genes than to the putative FATPs of other lower organisms.
  • The tissue distribution of the murine genes was studied by northern analysis. Results are shown in FIG. 3. Probes from the 3′ untranslated region of these genes which shared no appreciable homology among each other were used to avoid cross-hybridization of these genes. The expression pattern of mmFATP1 agrees with that previously found by Schaeffer and Lodish. mmFATP2 is expressed exclusively in liver and kidney, which corresponds with the reported tissue distribution of the rat homologue (VLACS), as assessed by western blot. mmFATP3 is expressed in lung, liver and testis, rmmFATP4 in heart brain lung liver and kidney, and mmFATP5 is exclusively expressed in liver. The human homologue of mmFATP5, hsFATPs, is liver specific in humans and cannot be detected in a wide array of other tissues, including fetal liver. To assess whether the newly identified mouse genes are functional fatty acid transporters, Cos cells were transiently transfected with the genes and the uptake of a Bodipy-labeled analog of a long chain fatty acid by FACS was measured. When overexpressed in Cos cells, mmFATP1, mmFATP2 and mmFATP5 increase uptake of the Bodipy-labeled lauric acid (FIG. 4). Interestingly, when others et al. transfected the rat homologue of mmFATP[0154] 2 into Cos cells, they observed an increase in very long chain acyl-CoA synthase activity, leading to the assumption that the protein was a VLACS. It appears that the increase in VLACS activity may be explained by previous data demonstrating that exogenously applied long chain fatty acids directly activate transcription of the long-chain acyl-CoA synthase gene. Thus, overexpression of mmFATP2 (or the rat homologue) may increase fatty acid uptake of cells from the media and subsequently lead to activation of VLACS gene expression.
  • All references cited herein are incorporated by reference in their entirety. [0155]
  • While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. [0156]
  • 1 37 1 340 PRT Mus musculus 1 Phe Ile Phe Thr Ser Gly Thr Thr Gly Leu Pro Lys Pro Ala Ile Leu 1 5 10 15 Ser His Glu Arg Val Ile Gln Val Ser Asn Val Leu Ser Phe Cys Gly 20 25 30 Cys Arg Ala Asp Asp Val Val Tyr Asp Val Leu Pro Leu Tyr His Thr 35 40 45 Ile Gly Leu Val Leu Gly Phe Leu Gly Cys Leu Gln Val Gly Ala Thr 50 55 60 Cys Val Leu Ala Pro Lys Phe Ser Ala Ser Arg Phe Trp Ala Glu Cys 65 70 75 80 Arg Gln His Gly Val Thr Val Ile Gln Tyr Ile Gly Glu Ile Cys Arg 85 90 95 Tyr Leu Leu Arg Gln Pro Val Arg Asp Val Glu Gln Arg His Arg Val 100 105 110 Arg Leu Ala Val Gly Asn Gly Leu Arg Pro Ala Ile Trp Glu Glu Phe 115 120 125 Thr Gln Arg Phe Gly Val Pro Gln Ile Gly Glu Phe Tyr Gly Ala Thr 130 135 140 Glu Cys Asn Cys Ser Ile Ala Asn Met Asp Gly Lys Val Gly Ser Cys 145 150 155 160 Gly Phe Asn Ser Arg Ile Leu Thr His Val Tyr Pro Ile Arg Leu Val 165 170 175 Lys Val Asn Glu Asp Thr Met Glu Pro Leu Arg Asp Ser Glu Gly Leu 180 185 190 Cys Ile Pro Cys Gln Pro Gly Glu Pro Gly Leu Leu Val Gly Gln Ile 195 200 205 Asn Gln Gln Asp Pro Leu Arg Arg Phe Asp Gly Tyr Val Ser Asp Ser 210 215 220 Ala Thr Asn Lys Lys Ile Ala His Ser Val Phe Arg Lys Gly Asp Ser 225 230 235 240 Ala Tyr Leu Ser Gly Asp Val Leu Val Met Asp Glu Leu Gly Tyr Met 245 250 255 Tyr Phe Arg Asp Arg Ser Gly Asp Thr Phe Arg Trp Arg Gly Glu Asn 260 265 270 Val Ser Thr Thr Glu Val Glu Ala Val Leu Ser Arg Leu Leu Gly Gln 275 280 285 Thr Asp Val Ala Val Tyr Gly Val Ala Val Pro Gly Val Glu Gly Lys 290 295 300 Ala Gly Met Ala Ala Ile Ala Asp Pro His Ser Gln Leu Asp Pro Asn 305 310 315 320 Ser Met Tyr Gln Glu Leu Gln Lys Val Leu Ala Ser Tyr Ala Arg Pro 325 330 335 Ile Phe Leu Arg 340 2 339 PRT Mus musculus 2 Tyr Ile Tyr Thr Ser Gly Thr Thr Gly Asn Pro Lys Pro Ala Val Ile 1 5 10 15 Lys His Phe Arg Tyr Phe Trp Ile Ala Met Gly Ala Gly Lys Ala Phe 20 25 30 Gly Ile Asn Lys Ser Asp Val Val Tyr Ile Thr Met Pro Met Tyr His 35 40 45 Ser Ala Ala Gly Ile Met Gly Ile Gly Ser Leu Ile Ala Phe Gly Ser 50 55 60 Thr Ala Val Ile Arg Lys Lys Phe Ser Ala Ser Asn Phe Trp Lys Asp 65 70 75 80 Cys Val Lys Tyr Asn Val Thr Ala Thr Leu Tyr Val Gly Glu Ile Leu 85 90 95 Arg Tyr Leu Cys Asn Val Pro Glu Gln Pro Glu Asp Lys Ile His Thr 100 105 110 Val Arg Leu Ala Met Gly Thr Gly Leu Arg Ala Asn Val Trp Lys Asn 115 120 125 Phe Gln Gln Arg Phe Gly Pro Ile Arg Ile Trp Glu Phe Tyr Gly Ser 130 135 140 Thr Glu Gly Asn Val Gly Leu Met Asn Tyr Val Gly His Cys Gly Ala 145 150 155 160 Val Gly Arg Thr Ser Cys Ile Leu Arg Met Leu Thr Pro Phe Glu Leu 165 170 175 Val Gln Phe Asp Ile Glu Thr Ala Glu Pro Leu Arg Asp Lys Gln Gly 180 185 190 Phe Cys Ile Pro Val Glu Pro Gly Lys Pro Gly Leu Leu Leu Thr Lys 195 200 205 Val Arg Lys Asn Gln Pro Phe Leu Gly Tyr Arg Gly Ser Gln Ala Glu 210 215 220 Ser Asn Arg Lys Leu Val Ala Asn Val Arg Arg Val Gly Asp Leu Tyr 225 230 235 240 Phe Asn Thr Gly Asp Val Leu Thr Leu Asp Gln Glu Gly Phe Phe Tyr 245 250 255 Phe Gln Asp Arg Leu Gly Asp Thr Phe Arg Trp Lys Gly Glu Asn Val 260 265 270 Ser Thr Gly Glu Val Glu Cys Val Leu Ser Ser Leu Asp Phe Leu Glu 275 280 285 Glu Val Asn Val Tyr Gly Val Pro Val Pro Gly Cys Glu Gly Lys Val 290 295 300 Gly Met Ala Ala Val Lys Leu Ala Pro Gly Lys Thr Phe Asp Gly Lys 305 310 315 320 Lys Tyr Gln His Val Arg Ser Trp Leu Pro Ala Tyr Ala Thr Pro His 325 330 335 Phe Ile Arg 3 345 PRT Caenorhabditis elegans 3 Ile Tyr Thr Ser Gly Thr Thr Gly Leu Pro Lys Ser Ala Ile Met Ser 1 5 10 15 Trp Arg Lys Ser Ser Val Gly Cys Gln Val Phe Gly His Val Leu His 20 25 30 Met Thr Asn Glu Ser Thr Val Phe Thr Ala Met Pro Leu Phe His Ser 35 40 45 Thr Ala Ala Leu Leu Gly Ala Cys Ala Ile Leu Ser His Gly Gly Cys 50 55 60 Leu Ala Leu Ser His Lys Phe Ser Ala Ser Thr Phe Trp Lys Gln Val 65 70 75 80 Tyr Leu Thr Gly Ala Thr His Ile Gln Tyr Ile Gly Glu Ile Cys Arg 85 90 95 Tyr Leu Leu Ala Ala Asn Pro Cys Pro Glu Glu Lys Gln His Asn Val 100 105 110 Arg Leu Met Trp Gly Asn Gly Leu Arg Gly Gln Ile Trp Lys Glu Phe 115 120 125 Val Gly Arg Phe Gly Ile Lys Lys Ile Gly Glu Leu Tyr Gly Ser Thr 130 135 140 Glu Gly Asn Ser Asn Ile Val Asn Val Asp Asn His Val Gly Ala Cys 145 150 155 160 Gly Phe Met Pro Ile Tyr Pro His Ile Gly Ser Leu Tyr Pro Val Arg 165 170 175 Leu Ile Lys Val Asp Arg Ala Thr Gly Glu Leu Glu Arg Asp Lys Asn 180 185 190 Gly Leu Cys Val Pro Cys Val Pro Gly Glu Thr Gly Glu Met Val Gly 195 200 205 Val Ile Lys Glu Lys Asp Ile Leu Leu Lys Phe Glu Gly Tyr Val Ser 210 215 220 Glu Gly Asp Thr Ala Lys Lys Ile Tyr Arg Asp Val Phe Lys His Gly 225 230 235 240 Asp Lys Val Phe Ala Ser Gly Asp Ile Leu His Trp Asp Asp Leu Gly 245 250 255 Tyr Leu Tyr Phe Val Asp Arg Cys Gly Asp Thr Phe Arg Trp Lys Gly 260 265 270 Glu Asn Val Ser Thr Thr Glu Val Glu Gly Ile Leu Gln Pro Val Met 275 280 285 Asp Val Glu Asp Ala Thr Val Tyr Gly Val Thr Val Gly Lys Met Glu 290 295 300 Gly Arg Ala Gly Met Ala Gly Ile Val Val Lys Asp Gly Thr Asp Val 305 310 315 320 Glu Lys Phe Ile Ala Asp Ile Thr Ser Arg Leu Thr Glu Asn Leu Ala 325 330 335 Ser Tyr Ala Ile Pro Val Phe Ile Arg 340 345 4 356 PRT Saccharomyces cerevisiae 4 Tyr Ile Tyr Thr Ser Gly Thr Thr Gly Leu Pro Lys Ala Ala Ile Val 1 5 10 15 Val His Ser Arg Tyr Tyr Arg Ile Ala Ala Phe Gly His His Ser Tyr 20 25 30 Ser Met Arg Ala Ala Asp Val Leu Tyr Asp Cys Leu Pro Leu Tyr His 35 40 45 Ser Ala Gly Asn Ile Met Gly Val Gly Gln Cys Val Ile Tyr Gly Leu 50 55 60 Thr Val Val Leu Arg Lys Lys Phe Ser Ala Ser Arg Phe Trp Asp Asp 65 70 75 80 Cys Val Lys Tyr Asn Cys Thr Val Val Gln Tyr Val Gly Glu Val Cys 85 90 95 Arg Tyr Leu Leu His Thr Pro Ile Ser Lys Tyr Glu Lys Met His Lys 100 105 110 Val Lys Val Ala Tyr Gly Asn Gly Leu Arg Pro Asp Ile Trp Gln Asp 115 120 125 Phe Arg Lys Arg Phe Asn Ile Glu Val Ile Gly Glu Phe Tyr Ala Ala 130 135 140 Thr Glu Ala Pro Phe Ala Thr Thr Thr Phe Gln Lys Gly Asp Phe Gly 145 150 155 160 Ile Gly Ala Cys Arg Asn Tyr Gly Thr Ile Ile Gln Trp Phe Leu Ser 165 170 175 Phe Gln Gln Thr Leu Val Arg Met Asp Pro Asn Asp Asp Ser Val Ile 180 185 190 Tyr Arg Asn Ser Lys Gly Phe Cys Glu Val Ala Pro Val Gly Glu Pro 195 200 205 Gly Glu Met Leu Met Arg Ile Phe Phe Pro Lys Lys Pro Glu Thr Ser 210 215 220 Phe Gln Gly Tyr Leu Gly Asn Ala Lys Glu Thr Lys Ser Lys Val Val 225 230 235 240 Arg Asp Val Phe Arg Arg Gly Asp Ala Trp Tyr Arg Cys Gly Asp Leu 245 250 255 Leu Lys Ala Asp Glu Tyr Gly Leu Trp Tyr Phe Leu Asp Arg Met Gly 260 265 270 Asp Thr Phe Arg Trp Lys Ser Glu Asn Val Ser Thr Thr Glu Val Glu 275 280 285 Asp Gln Leu Thr Ala Ser Asn Lys Glu Gln Tyr Ala Gln Val Leu Val 290 295 300 Val Gly Ile Lys Val Pro Lys Tyr Glu Gly Arg Ala Gly Phe Ala Val 305 310 315 320 Ile Lys Leu Thr Asp Asn Ser Leu Asp Ile Thr Ala Lys Thr Lys Leu 325 330 335 Leu Asn Asp Ser Leu Ser Arg Leu Asn Leu Pro Ser Tyr Ala Met Pro 340 345 350 Leu Phe Val Lys 355 5 334 PRT Mycobacterium tuberculosis 5 Tyr Ile Phe Thr Ser Gly Thr Thr Gly Phe Pro Lys Ala Ser Val Met 1 5 10 15 Thr His His Arg Trp Leu Arg Ala Leu Ala Val Phe Gly Gly Met Gly 20 25 30 Leu Arg Leu Lys Gly Ser Asp Thr Leu Tyr Ser Cys Leu Pro Leu Tyr 35 40 45 His Asn Asn Ala Leu Thr Val Ala Val Ser Ser Val Ile Asn Ser Gly 50 55 60 Ala Thr Leu Ala Leu Gly Lys Ser Phe Ser Ala Ser Arg Phe Trp Asp 65 70 75 80 Glu Val Ile Ala Asn Arg Ala Thr Ala Phe Val Tyr Ile Gly Glu Ile 85 90 95 Cys Arg Tyr Leu Leu Asn Gln Pro Ala Lys Pro Thr Asp Arg Ala His 100 105 110 Gln Val Arg Val Ile Cys Gly Asn Gly Leu Arg Pro Glu Ile Trp Asp 115 120 125 Glu Phe Thr Thr Arg Phe Gly Val Ala Arg Val Cys Glu Phe Tyr Ala 130 135 140 Ala Ser Glu Gly Asn Ser Ala Phe Ile Asn Ile Phe Asn Val Pro Arg 145 150 155 160 Thr Ala Gly Val Ser Pro Met Pro Leu Ala Phe Val Glu Tyr Asp Leu 165 170 175 Asp Thr Gly Asp Pro Leu Arg Asp Ala Ser Gly Arg Val Arg Arg Val 180 185 190 Pro Asp Gly Glu Pro Gly Leu Leu Leu Ser Arg Val Asn Arg Leu Gln 195 200 205 Pro Phe Asp Gly Tyr Thr Asp Pro Val Ala Ser Glu Lys Lys Leu Val 210 215 220 Arg Asn Ala Phe Arg Asp Gly Asp Cys Trp Phe Asn Thr Gly Asp Val 225 230 235 240 Met Ser Pro Gln Gly Met Gly His Ala Ala Phe Val Asp Arg Leu Gly 245 250 255 Asp Thr Phe Arg Trp Lys Gly Glu Asn Val Ala Thr Thr Gln Val Glu 260 265 270 Ala Ala Leu Ala Ser Asp Gln Thr Val Glu Glu Cys Thr Val Tyr Gly 275 280 285 Val Gln Ile Pro Arg Thr Gly Gly Arg Ala Gly Met Ala Ala Ile Thr 290 295 300 Leu Arg Ala Gly Ala Glu Phe Asp Gly Gln Ala Leu Ala Arg Thr Val 305 310 315 320 Tyr Gly His Leu Pro Gly Tyr Ala Leu Pro Leu Phe Val Arg 325 330 6 2087 DNA Mus musculus 6 acgactcact atagggagag agctatgacg tcgcatgcac gcgtaagctt gggcccctcg 60 agggatcctc tagagcggcc gccgaccccg aaagctctga gagcgggtgc agtctggcct 120 ggcgtctcgc gtacctggcc cgggagcagc cgacacacac cttcctcatc cacggcgcgc 180 agcgctttag ctacgcggag gctgagcgcg agagcaaccg gattgctcgc gcctttctgc 240 gcgcacgggg ctggaccggg ggccgccgag gctcgggcag gggcagcact gaggaaggcg 300 cacgcgtggc gcctccggct ggagatgcgg ctgctagagg gacgaccgcg ccccctctgg 360 cacccggggc gaccgtggcg ctgctcctcc cagcgggccc ggatttcctt tggatttggt 420 tcggactggc caaagctggc ctgcgcacgg cctttgtgcc caccgcttta cgccgaggac 480 ccctgctgca ctgcctccgc agctgcggtg cgagtgcgct cgtgctggcc acagagttcc 540 tggagtccct ggagccggac ctgccggcct tgagagccat ggggctccac ctatgggcga 600 cgggccctga aactaatgta gctggaatca gcaatttgct atcggaagca gcagaccaag 660 tggatgagcc agtgccgggg tacctctctg ccccccagaa cataatggac acctgcctgt 720 acatcttcac ctctggcact actggcctgc ccaaggctgc tcgaatcagt catctgaagg 780 ttctacagtg ccagggattc taccatctgt gtggagtcca ccaggaggac gtgatctacc 840 tcgcactccc actgtaccac atgtctggct cccttctggg cattgtgggc tgcttgggca 900 ttggggccac cgtggtgctg aaacccaagt tctcagctag ccagttctgg gacgattgcc 960 agaaacacag ggtgacagtg ttccagtaca ttggggagtt gtgccgatac ctcgtcaacc 1020 agcccccgag caaggcagag tttgaccata aggtgcgctt ggcagtgggc agtgggttgc 1080 gcccagacac ctgggagcgt ttcctgcggc gatttggacc tctgcagata ctggagacgt 1140 atggcatgac agagggcaac gtagctacgt tcaattacac aggacggcag ggtgcagtgg 1200 ggcgagcttc ctggctttac aagcacatct tccccttctc cttgattcga tacgatgtca 1260 tgacagggga gcctattcgg aatgcccagg ggcactgcat gaccacatct ccaggtgagc 1320 caggcctact ggtggcccca gtgagccagc agtccccctt cctgggctat gctggggctc 1380 cggagctggc caaggacaag ctgctgaagg atgtcttctg gtctggggac gttttcttca 1440 atactgggga cctcttggtc tgtgatgagc aaggctttct tcacttccac gatcgtactg 1500 gagacaccat caggtggaag ggagagaatg tggccacaac tgaagtggct gaggtcttgg 1560 agaccctgga cttccttcag gaggtgaaca tctatggagt cacggtgcca gggcacgaag 1620 gcagggcagg catggcggcc ttggctctgc ggcccccgca ggctctgaac ctggtgcagc 1680 tctacagcca tgtttctgag aacttgccac cgtatgcccg acctcggttt ctcaggctcc 1740 aggaatcttt ggccactact gagaccttca aacagcagaa ggttaggatg gccaatgagg 1800 gctttgaccc cagtgtactg tctgacccac tctatgttct ggaccaagat ataggggcct 1860 acctgcccct cacacctgcc cggtacagtg ccctcctgtc tggagacctt cgaatctgaa 1920 accttccact tgagggaggg gctcggaggg tacaggccac catggctgca ccagggaggg 1980 ttttcgggta tcttttgtat atggagtcat tattttgtaa taaacagctg gagcttaaaa 2040 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 2087 7 613 PRT Mus musculus 7 Ala Ala Asp Pro Glu Ser Ser Glu Ser Gly Cys Ser Leu Ala Trp Arg 1 5 10 15 Leu Ala Tyr Leu Ala Arg Glu Gln Pro Thr His Thr Phe Leu Ile His 20 25 30 Gly Ala Gln Arg Phe Ser Tyr Ala Glu Ala Glu Arg Glu Ser Asn Arg 35 40 45 Ile Ala Arg Ala Phe Leu Arg Ala Arg Gly Trp Thr Gly Gly Arg Arg 50 55 60 Gly Ser Gly Arg Gly Ser Thr Glu Glu Gly Ala Arg Val Ala Pro Pro 65 70 75 80 Ala Gly Asp Ala Ala Ala Arg Gly Thr Thr Ala Pro Pro Leu Ala Pro 85 90 95 Gly Ala Thr Val Ala Leu Leu Leu Pro Ala Gly Pro Asp Phe Leu Trp 100 105 110 Ile Trp Phe Gly Leu Ala Lys Ala Gly Leu Arg Thr Ala Phe Val Pro 115 120 125 Thr Ala Leu Arg Arg Gly Pro Leu Leu His Cys Leu Arg Ser Cys Gly 130 135 140 Ala Ser Ala Leu Val Leu Ala Thr Glu Phe Leu Glu Ser Leu Glu Pro 145 150 155 160 Asp Leu Pro Ala Leu Arg Ala Met Gly Leu His Leu Trp Ala Thr Gly 165 170 175 Pro Glu Thr Asn Val Ala Gly Ile Ser Asn Leu Leu Ser Glu Ala Ala 180 185 190 Asp Gln Val Asp Glu Pro Val Pro Gly Tyr Leu Ser Ala Pro Gln Asn 195 200 205 Ile Met Asp Thr Cys Leu Tyr Ile Phe Thr Ser Gly Thr Thr Gly Leu 210 215 220 Pro Lys Ala Ala Arg Ile Ser His Leu Lys Val Leu Gln Cys Gln Gly 225 230 235 240 Phe Tyr His Leu Cys Gly Val His Gln Glu Asp Val Ile Tyr Leu Ala 245 250 255 Leu Pro Leu Tyr His Met Ser Gly Ser Leu Leu Gly Ile Val Gly Cys 260 265 270 Leu Gly Ile Gly Ala Thr Val Val Leu Lys Pro Lys Phe Ser Ala Ser 275 280 285 Gln Phe Trp Asp Asp Cys Gln Lys His Arg Val Thr Val Phe Gln Tyr 290 295 300 Ile Gly Glu Leu Cys Arg Tyr Leu Val Asn Gln Pro Pro Ser Lys Ala 305 310 315 320 Glu Phe Asp His Lys Val Arg Leu Ala Val Gly Ser Gly Leu Arg Pro 325 330 335 Asp Thr Trp Glu Arg Phe Leu Arg Arg Phe Gly Pro Leu Gln Ile Leu 340 345 350 Glu Thr Tyr Gly Met Thr Glu Gly Asn Val Ala Thr Phe Asn Tyr Thr 355 360 365 Gly Arg Gln Gly Ala Val Gly Arg Ala Ser Trp Leu Tyr Lys His Ile 370 375 380 Phe Pro Phe Ser Leu Ile Arg Tyr Asp Val Met Thr Gly Glu Pro Ile 385 390 395 400 Arg Asn Ala Gln Gly His Cys Met Thr Thr Ser Pro Gly Glu Pro Gly 405 410 415 Leu Leu Val Ala Pro Val Ser Gln Gln Ser Pro Phe Leu Gly Tyr Ala 420 425 430 Gly Ala Pro Glu Leu Ala Lys Asp Lys Leu Leu Lys Asp Val Phe Trp 435 440 445 Ser Gly Asp Val Phe Phe Asn Thr Gly Asp Leu Leu Val Cys Asp Glu 450 455 460 Gln Gly Phe Leu His Phe His Asp Arg Thr Gly Asp Thr Ile Arg Trp 465 470 475 480 Lys Gly Glu Asn Val Ala Thr Thr Glu Val Ala Glu Val Leu Glu Thr 485 490 495 Leu Asp Phe Leu Gln Glu Val Asn Ile Tyr Gly Val Thr Val Pro Gly 500 505 510 His Glu Gly Arg Ala Gly Met Ala Ala Leu Ala Leu Arg Pro Pro Gln 515 520 525 Ala Leu Asn Leu Val Gln Leu Tyr Ser His Val Ser Glu Asn Leu Pro 530 535 540 Pro Tyr Ala Arg Pro Arg Phe Leu Arg Leu Gln Glu Ser Leu Ala Thr 545 550 555 560 Thr Glu Thr Phe Lys Gln Gln Lys Val Arg Met Ala Asn Glu Gly Phe 565 570 575 Asp Pro Ser Val Leu Ser Asp Pro Leu Tyr Val Leu Asp Gln Asp Ile 580 585 590 Gly Ala Tyr Leu Pro Leu Thr Pro Ala Arg Tyr Ser Ala Leu Leu Ser 595 600 605 Gly Asp Leu Arg Ile 610 8 2301 DNA Mus musculus 8 cccacgcgtc cgcccacgcg tccggcatgg ccaagctggg cgtggaggcg gctctcatca 60 acaccaacct taggcgggat gccctgcgcc actgtcttga cacctcaaag gcacgagctc 120 tcatctttgg cagtgagatg gcctcagcta tctgtgagat ccatgctagc ctggagccca 180 cactcagcct cttctgctct ggatcctggg agcccagcac agtgcccgtc agcacagagc 240 atctggaccc tcttctggaa gatgccccga agcacctgcc cagtcaccca gacaagggtt 300 ttacagataa gctcttctac atctacacat cgggcaccac ggggctaccc aaagctgcca 360 ttgtggtgca cagcaggtat tatcgtatgg cttccctggt gtactatgga ttccgcatgc 420 ggcctgatga cattgtctat gactgcctcc ccctctacca ctcaagcagg aaacatcgtg 480 gggattggca gtgcttactc cacggcatga ctgtggtgat ccggaagaag ttctcagcct 540 cccggttctg ggatgattgt atcaagtaca actgcacagt ggtacagtac attggcgagc 600 tctgccgcta cctcctgaac cagccacccc gtgaggctga gtctcggcac aaggtgcgca 660 tggcactggg caacggtctc cggcagtcca tctggaccga cttctccagc cgtttccaca 720 tcccccaggt ggctgagttc tatggggcca ctgaatgcaa ctgtagcctg ggcaactttg 780 acagccgggt gggggcctgt ggcttcaata gccgcatcct gtcctttgtg taccctatcc 840 gtttggtacg tgtcaatgag gataccatgg aactgatccg gggacccgat ggagtctgca 900 ttccctgtca accaggtcag ccaggccagc tggtgggtcg catcatccag caggaccctc 960 tgcgccgttt cgacgggtac ctcaaccagg gtgccaacaa caagaagatt gctaatgatg 1020 tcttcaagaa gggggaccaa gcctacctca ctggtgacgt cctggtgatg gatgagctgg 1080 gttacctgta cttccgagat cgcactgggg acacgttccg ctggaaaggg gagaatgtat 1140 ctaccactga ggtggagggc acactcagcc gcctgcttca tatggcagat gtggcagttt 1200 atggtgttga ggtgccagga actgaaggcc gagcaggaat ggctgccgtt gcaagtccca 1260 tcagcaactg tgacctggag agctttgcac agaccttgaa aaaggagctg cctctgtatg 1320 cccgccccat cttcctgcgc ttcttgcctg agctgcacaa gacagggacc ttcaagttcc 1380 agaagacaga gttgcggaag gagggctttg acccatctgt tgtgaaagac ccgctgttct 1440 atctggatgc tcggaagggc tgctacgttg cactggacca ggaggcctat acccgcatcc 1500 aggcaggcga ggagaagctg tgatttcccc ctacatccct ctgagggcca gaagatgctg 1560 gattcagagc cctagcgtcc accccagagg gtcctgggca atgccagacc aaagctagca 1620 gggcccgcac ctccgcccct aggtgctgat ctcccctctc ccaaactgcc aagtgactca 1680 ctgccgcttc cccgaccctc cagaggcttt ctgtgaaagt ctcatccaag ctgtgtcttc 1740 tggtccaggc gtggcccctg gccccagggt ttctgatagg ctcctttagg atggtatctt 1800 gggtccagcg ggccagggtg tgggagagga gtcactaaga tccctccaat cagaagggag 1860 cttacaaagg aaccaaggca aagcctgtag actcaggaag ctaagtggcc agagactata 1920 gtggccagtc atcccatgtc cacagaggat cttggtccag agctgccaaa gtgtcacctc 1980 tccctgcctg cacctctggg gaaaagagga cagcatgtgg ccactgggca cctgtctcaa 2040 gaagtcagga tcacacactc agtccttgtt tctccaggtt cccttgttct tgtctcgggg 2100 agggagggac gagtgtcctg tctgtccttc ctgcctgtct gtgagtctgt gttgcttctc 2160 catctgtcct agcctgagtg tgggtggaac aggcatgagg agagtgtggc tcaggggcca 2220 ataaactctg ccttgactcc tcttaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2280 aaaaaaaaaa aaaaaaaaaa a 2301 9 506 PRT Mus musculus 9 His Ala Ser Ala His Ala Ser Gly Met Ala Lys Leu Gly Val Glu Ala 1 5 10 15 Ala Leu Ile Asn Thr Asn Leu Arg Arg Asp Ala Leu Arg His Cys Leu 20 25 30 Asp Thr Ser Lys Ala Arg Ala Leu Ile Phe Gly Ser Glu Met Ala Ser 35 40 45 Ala Ile Cys Glu Ile His Ala Ser Leu Glu Pro Thr Leu Ser Leu Phe 50 55 60 Cys Ser Gly Ser Trp Glu Pro Ser Thr Val Pro Val Ser Thr Glu His 65 70 75 80 Leu Asp Pro Leu Leu Glu Asp Ala Pro Lys His Leu Pro Ser His Pro 85 90 95 Asp Lys Gly Phe Thr Asp Lys Leu Phe Tyr Ile Tyr Thr Ser Gly Thr 100 105 110 Thr Gly Leu Pro Lys Ala Ala Ile Val Val His Ser Arg Tyr Tyr Arg 115 120 125 Met Ala Ser Leu Val Tyr Tyr Gly Phe Arg Met Arg Pro Asp Asp Ile 130 135 140 Val Tyr Asp Cys Leu Pro Leu Tyr His Ser Ser Arg Lys His Arg Gly 145 150 155 160 Asp Trp Gln Cys Leu Leu His Gly Met Thr Val Val Ile Arg Lys Lys 165 170 175 Phe Ser Ala Ser Arg Phe Trp Asp Asp Cys Ile Lys Tyr Asn Cys Thr 180 185 190 Val Val Gln Tyr Ile Gly Glu Leu Cys Arg Tyr Leu Leu Asn Gln Pro 195 200 205 Pro Arg Glu Ala Glu Ser Arg His Lys Val Arg Met Ala Leu Gly Asn 210 215 220 Gly Leu Arg Gln Ser Ile Trp Thr Asp Phe Ser Ser Arg Phe His Ile 225 230 235 240 Pro Gln Val Ala Glu Phe Tyr Gly Ala Thr Glu Cys Asn Cys Ser Leu 245 250 255 Gly Asn Phe Asp Ser Arg Val Gly Ala Cys Gly Phe Asn Ser Arg Ile 260 265 270 Leu Ser Phe Val Tyr Pro Ile Arg Leu Val Arg Val Asn Glu Asp Thr 275 280 285 Met Glu Leu Ile Arg Gly Pro Asp Gly Val Cys Ile Pro Cys Gln Pro 290 295 300 Gly Gln Pro Gly Gln Leu Val Gly Arg Ile Ile Gln Gln Asp Pro Leu 305 310 315 320 Arg Arg Phe Asp Gly Tyr Leu Asn Gln Gly Ala Asn Asn Lys Lys Ile 325 330 335 Ala Asn Asp Val Phe Lys Lys Gly Asp Gln Ala Tyr Leu Thr Gly Asp 340 345 350 Val Leu Val Met Asp Glu Leu Gly Tyr Leu Tyr Phe Arg Asp Arg Thr 355 360 365 Gly Asp Thr Phe Arg Trp Lys Gly Glu Asn Val Ser Thr Thr Glu Val 370 375 380 Glu Gly Thr Leu Ser Arg Leu Leu His Met Ala Asp Val Ala Val Tyr 385 390 395 400 Gly Val Glu Val Pro Gly Thr Glu Gly Arg Ala Gly Met Ala Ala Val 405 410 415 Ala Ser Pro Ile Ser Asn Cys Asp Leu Glu Ser Phe Ala Gln Thr Leu 420 425 430 Lys Lys Glu Leu Pro Leu Tyr Ala Arg Pro Ile Phe Leu Arg Phe Leu 435 440 445 Pro Glu Leu His Lys Thr Gly Thr Phe Lys Phe Gln Lys Thr Glu Leu 450 455 460 Arg Lys Glu Gly Phe Asp Pro Ser Val Val Lys Asp Pro Leu Phe Tyr 465 470 475 480 Leu Asp Ala Arg Lys Gly Cys Tyr Val Ala Leu Asp Gln Glu Ala Tyr 485 490 495 Thr Arg Ile Gln Ala Gly Glu Glu Lys Leu 500 505 10 2277 DNA Mus musculus 10 cactcatcag agctaagaga gactacacgc tctcatctac ttcagaaaga gccaatgcca 60 tgggtatttg gaagaaacta accttactgc tgttgctgct tctgctggtt ggcctggggc 120 agcccccatg gccagcagct atggctctgg ccctgcgttg gttcctggga gaccccacat 180 gccttgtgct gcttggcttg gcattgctgg gcagaccctg gatcagctcc tggatgcccc 240 actggctgag cctggtagga gcagctctta ccttattcct attgcctcta cagccacccc 300 cagggctacg ctggctgcat aaagatgtgg ctttcacctt caagatgctt ttctatggcc 360 taaagttcag gcgacgcctt aacaaacatc ctccagagac ctttgtggat gctttagagc 420 ggcaagcact ggcatggcct gaccgggtgg ccttggtgtg tactgggtct gagggctcct 480 caatcacaaa tagccagctg gatgccaggt cctgtcaggc agcatgggtc ctgaaagcaa 540 agctgaagga tgccgtaatc cagaacacaa gagatgctgc tgctatctta gttctcccgt 600 ccaagaccat ttctgctttg agtgtgtttc tggggttggc caagttgggc tgccctgtgg 660 cctggatcaa tccacacagc cgagggatgc ccttgctaca ctctgtacgg agctctgggg 720 ccagtgtgct gattgtggat ccagacctcc aggagaacct ggaagaagtc cttcccaagc 780 tgctagctga gaacattcac tgcttctacc ttggccacag ctcacccacc ccgggagtag 840 aggctctggg agcttccctg gatgctgcac cttctgaccc agtacctgcc agccttcgag 900 ctacgattaa gtggaaatct cctgccatat tcatctttac ttcagggacc actggactcc 960 caaagccagc catcttatca catgagcggg tcatacaagt gagcaacgtg ctgtccttct 1020 gtggatgcag agctgatgat gtggtctatg acgtcctacc tctgtaccat acgatagggc 1080 ttgtccttgg attccttggc tgcttacaag ttggagccac ctgtgtcctg gcccccaagt 1140 tctctgcctc ccgattctgg gctgagtgcc ggcagcatgg cgtaacagtg atcttgtatg 1200 tgggtgaaat cctgcggtac ttgtgtaacg tccctgagca accagaagac aagatacata 1260 cagtgcgctt ggccatggga actggacttc gggcaaatgt gtggaaaaac ttccagcaac 1320 gctttggtcc cattcggatc tgggaattct acggatccac agagggcaat gtgggcttaa 1380 tgaactatgt gggccactgc ggggctgtgg gaaggaccag ctgcatcctt cgaatgctga 1440 ctccctttga gcttgtacag ttcgacatag agacagcaga gcctctgagg gacaaacagg 1500 gtttttgcat tcctgtggag ccaggaaagc caggacttct tttgaccaag gttcgaaaga 1560 accaaccctt cctgggctac cgtggttccc aggccgagtc caatcggaaa cttgttgcga 1620 atgtacgacg cgtaggagac ctgtacttca acactgggga cgtgctgacc ttggaccagg 1680 aaggcttctt ctactttcaa gaccgccttg gtgacacctt ccggtggaag ggcgaaaacg 1740 tatctactgg agaggtggag tgtgttttgt ctagcctaga cttcctagag gaagtcaatg 1800 tctatggtgt gcctgtgcca gggtgtgagg gtaaggttgg catggctgct gtgaaactgg 1860 ctcctgggaa gacttttgat gggcagaagc tataccagca tgtccgctcc tggctccctg 1920 cctatgccac acctcatttc atccgtatcc aggattccct ggagatcaca aacacctaca 1980 agctggtaaa gtcacggctg gtgcgtgagg gttttgatgt ggggatcatt gctgaccccc 2040 tctacatact ggacaacaag gcccagacct tccggagtct gatgccagat gtgtaccagg 2100 ctgtgtgtga aggaacctgg aatctctgac cacctagcca actggaaggc aatccaaaag 2160 tgtagagatt gacactagtc agcttcacaa agttgtccgg gttccagatg cccatggccc 2220 agtagtactt agagaataaa cttgaatgtg tatacaaaaa aaaaaaaaaa aaaaaaa 2277 11 662 PRT Mus musculus 11 Met Ala Leu Ala Leu Arg Trp Phe Leu Gly Asp Pro Thr Cys Leu Val 1 5 10 15 Leu Leu Gly Leu Ala Leu Leu Gly Arg Pro Trp Ile Ser Ser Trp Met 20 25 30 Pro His Trp Leu Ser Leu Val Gly Ala Ala Leu Thr Leu Phe Leu Leu 35 40 45 Pro Leu Gln Pro Pro Pro Gly Leu Arg Trp Leu His Lys Asp Val Ala 50 55 60 Phe Thr Phe Lys Met Leu Phe Tyr Gly Leu Lys Phe Arg Arg Arg Leu 65 70 75 80 Asn Lys His Pro Pro Glu Thr Phe Val Asp Ala Leu Glu Arg Gln Ala 85 90 95 Leu Ala Trp Pro Asp Arg Val Ala Leu Val Cys Thr Gly Ser Glu Gly 100 105 110 Ser Ser Ile Thr Asn Ser Gln Leu Asp Ala Arg Ser Cys Gln Ala Ala 115 120 125 Trp Val Leu Lys Ala Lys Leu Lys Asp Ala Val Ile Gln Asn Thr Arg 130 135 140 Asp Ala Ala Ala Ile Leu Val Leu Pro Ser Lys Thr Ile Ser Ala Leu 145 150 155 160 Ser Val Phe Leu Gly Leu Ala Lys Leu Gly Cys Pro Val Ala Trp Ile 165 170 175 Asn Pro His Ser Arg Gly Met Pro Leu Leu His Ser Val Arg Ser Ser 180 185 190 Gly Ala Ser Val Leu Ile Val Asp Pro Asp Leu Gln Glu Asn Leu Glu 195 200 205 Glu Val Leu Pro Lys Leu Leu Ala Glu Asn Ile His Cys Phe Tyr Leu 210 215 220 Gly His Ser Ser Pro Thr Pro Gly Val Glu Ala Leu Gly Ala Ser Leu 225 230 235 240 Asp Ala Ala Pro Ser Asp Pro Val Pro Ala Ser Leu Arg Ala Thr Ile 245 250 255 Lys Trp Lys Ser Pro Ala Ile Phe Ile Phe Thr Ser Gly Thr Thr Gly 260 265 270 Leu Pro Lys Pro Ala Ile Leu Ser His Glu Arg Val Ile Gln Val Ser 275 280 285 Asn Val Leu Ser Phe Cys Gly Cys Arg Ala Asp Asp Val Val Tyr Asp 290 295 300 Val Leu Pro Leu Tyr His Thr Ile Gly Leu Val Leu Gly Phe Leu Gly 305 310 315 320 Cys Leu Gln Val Gly Ala Thr Cys Val Leu Ala Pro Lys Phe Ser Ala 325 330 335 Ser Arg Phe Trp Ala Glu Cys Arg Gln His Gly Val Thr Val Ile Leu 340 345 350 Tyr Val Gly Glu Ile Leu Arg Tyr Leu Cys Asn Val Pro Glu Gln Pro 355 360 365 Glu Asp Lys Ile His Thr Val Arg Leu Ala Met Gly Thr Gly Leu Arg 370 375 380 Ala Asn Val Trp Lys Asn Phe Gln Gln Arg Phe Gly Pro Ile Arg Ile 385 390 395 400 Trp Glu Phe Tyr Gly Ser Thr Glu Gly Asn Val Gly Leu Met Asn Tyr 405 410 415 Val Gly His Cys Gly Ala Val Gly Arg Thr Ser Cys Ile Leu Arg Met 420 425 430 Leu Thr Pro Phe Glu Leu Val Gln Phe Asp Ile Glu Thr Ala Glu Pro 435 440 445 Leu Arg Asp Lys Gln Gly Phe Cys Ile Pro Val Glu Pro Gly Lys Pro 450 455 460 Gly Leu Leu Leu Thr Lys Val Arg Lys Asn Gln Pro Phe Leu Gly Tyr 465 470 475 480 Arg Gly Ser Gln Ala Glu Ser Asn Arg Lys Leu Val Ala Asn Val Arg 485 490 495 Arg Val Gly Asp Leu Tyr Phe Asn Thr Gly Asp Val Leu Thr Leu Asp 500 505 510 Gln Glu Gly Phe Phe Tyr Phe Gln Asp Arg Leu Gly Asp Thr Phe Arg 515 520 525 Trp Lys Gly Glu Asn Val Ser Thr Gly Glu Val Glu Cys Val Leu Ser 530 535 540 Ser Leu Asp Phe Leu Glu Glu Val Asn Val Tyr Gly Val Pro Val Pro 545 550 555 560 Gly Cys Glu Gly Lys Val Gly Met Ala Ala Val Lys Leu Ala Pro Gly 565 570 575 Lys Thr Phe Asp Gly Gln Lys Leu Tyr Gln His Val Arg Ser Trp Leu 580 585 590 Pro Ala Tyr Ala Thr Pro His Phe Ile Arg Ile Gln Asp Ser Leu Glu 595 600 605 Ile Thr Asn Thr Tyr Lys Leu Val Lys Ser Arg Leu Val Arg Glu Gly 610 615 620 Phe Asp Val Gly Ile Ile Ala Asp Pro Leu Tyr Ile Leu Asp Asn Lys 625 630 635 640 Ala Gln Thr Phe Arg Ser Leu Met Pro Asp Val Tyr Gln Ala Val Cys 645 650 655 Glu Gly Thr Trp Asn Leu 660 12 1622 DNA Homo sapiens misc_feature (1)...(1621) n = A,T,C or G 12 atgggattga ctctttcctg gacaaagtgg atgaagtatc aactgaacct atcccagagt 60 catggaggtc tgaagtcact ttttccactc ctgccttata catttatact tctggaacca 120 caggtcttcc aaaagcagcc atgatcactc atcagcgcat atggtatgga actggcctca 180 cttttgtaag cggattgaag gcagatgatg tcatctatat cactctgccc ttttaccaca 240 gtgctgcact actgattggc attcacggat gtattgtggc tggtgctact cttgccttgc 300 ggactaaatt ttcagccagc cagttttggg atgactgcag aaaatacaac gtcactgtca 360 ttcagtatat cggtgaactg cttcggtatt tatgcaactc accacagaaa ccaaatgacc 420 gtgatcataa agtgagactg gcactgggaa atggcttacg aggagatgtg tggagacaat 480 ttgtcaagag atttggggac atatgcatct atgagttcta tgctgccact gaaggcaata 540 ttggatttat gaattatgcg agaaaagttg gtgctgttgg aagagtaaac tacctacaga 600 aaaaaatcat aacttatgac ctgattaaat atgatgtgga gaaagatgaa cctgtccgtg 660 atgaaaatgg atattgcgtc agagttccca aaggtgaagt tggacttctg gtttgcaaaa 720 tcacacaact tacaccattt aatggctatg ctggagcaaa ggctcagaca gagaagaaaa 780 aactgagaga tgtctttaag aaaggagacc tctatttcaa cagtggagat ctcttaatgg 840 ttgaccatga aaatttcatc tatttccacg acagagttgg agatacattc cggtggaaag 900 gggaaaatgt ggccaccact gaagttgctg atatagttgg actggttgat ttttttccaa 960 ggaagtaaaa tgtttatggg agtgcatggg ccaagatnat ggaggttcga attggcatgg 1020 cnttccnttc aaaatggaaa gaaaaccatg gaatttgatg gaaagaaatt ttttcagnac 1080 attgctgata accnacctag ttatgcaagg ccccggtttt ntaagaanac aggacaccat 1140 tgagatcact ggaattttta aacaccgcaa aatgaccttt ggtggaggag ggctttaacc 1200 cngctgtcat caaagatgcc ttgtattttc ttggatgaca cagcaaaaat gtatgtgcct 1260 atgactgagg acatntataa tgccataagt gntaaaaccc tgaaattntg aatattccca 1320 ggaggataat tcaacatttc cagaaagaaa ctgaatggac agccacttga tataatccaa 1380 ctttaatttg attgaagatt gtgaggaaat tttgtaggaa atttgcatac ccgtaaaggg 1440 agactttttt aaataacagt tgagtctttg caagtaaaaa gatttagaga ttattatttt 1500 tcagtgtgca cctactgttt gtatttgcaa actgagcttg ttggagggaa ggcattattt 1560 tttaaaatac ttagtaaatt aaagaacacc aacatgtgaa aaaaaaaaaa aaaaaaaaaa 1620 aa 1622 13 286 PRT Homo sapiens 13 Tyr Ile Tyr Thr Ser Gly Thr Thr Gly Leu Pro Lys Ala Ala Met Ile 1 5 10 15 Thr His Gln Arg Ile Trp Tyr Gly Thr Gly Leu Thr Phe Val Ser Gly 20 25 30 Leu Lys Ala Asp Asp Val Ile Tyr Ile Thr Leu Pro Phe Tyr His Ser 35 40 45 Ala Ala Leu Leu Ile Gly Ile His Gly Cys Ile Val Ala Gly Ala Thr 50 55 60 Leu Ala Leu Arg Thr Lys Phe Ser Ala Ser Gln Phe Trp Asp Asp Cys 65 70 75 80 Arg Lys Tyr Asn Val Thr Val Ile Gln Tyr Ile Gly Glu Leu Leu Arg 85 90 95 Tyr Leu Cys Asn Ser Pro Gln Lys Pro Asn Asp Arg Asp His Lys Val 100 105 110 Arg Leu Ala Leu Gly Asn Gly Leu Arg Gly Asp Val Trp Arg Gln Phe 115 120 125 Val Lys Arg Phe Gly Asp Ile Cys Ile Tyr Glu Phe Tyr Ala Ala Thr 130 135 140 Glu Gly Asn Ile Gly Phe Met Asn Tyr Ala Arg Lys Val Gly Ala Val 145 150 155 160 Gly Arg Val Asn Tyr Leu Gln Lys Lys Ile Ile Thr Tyr Asp Leu Ile 165 170 175 Lys Tyr Asp Val Glu Lys Asp Glu Pro Val Arg Asp Glu Asn Gly Tyr 180 185 190 Cys Val Arg Val Pro Lys Gly Glu Val Gly Leu Leu Val Cys Lys Ile 195 200 205 Thr Gln Leu Thr Pro Phe Asn Gly Tyr Ala Gly Ala Lys Ala Gln Thr 210 215 220 Glu Lys Lys Lys Leu Arg Asp Val Phe Lys Lys Gly Asp Leu Tyr Phe 225 230 235 240 Asn Ser Gly Asp Leu Leu Met Val Asp His Glu Asn Phe Ile Tyr Phe 245 250 255 His Asp Arg Val Gly Asp Thr Phe Arg Trp Lys Gly Glu Asn Val Ala 260 265 270 Thr Thr Glu Val Ala Asp Ile Val Gly Leu Val Asp Phe Phe 275 280 285 14 753 DNA Homo sapiens 14 caattcggga cccccagggg cactgtatgg ccacatctcc aggtgagcca ggggaagttg 60 ctaaaggatg tcttccggcc tggggatgtt ttcttcaaca ctggggacct gctggtctgc 120 gatgaccaag gttttctccg cttccatgat cgtactggag acaccttcag gtggaaaggg 180 gagaatgtgg ccacaaccga ggtggcagag gtcttcgagg ccctagattt tcttcaggag 240 gtgaacgtct atggagtcac tgtgccaggg catgaaggca gggctggaat ggcagcccta 300 gttctgcgtc ccccccacgc tttggacctt atgcagctct acacccacgt gtctgagaac 360 ttgccacctt atgcccggcc ccgattcctc aggctccagg agtctttggc caccacagag 420 accttcaaac agcagaaagt tcggatggca aatgagggct tcgaccccag caccctgtct 480 gacccactgt acgttctgga ccaggctgta ggtgcctacc tgcccctcac aactgcccgg 540 tacagcgccc tcctggcagg aaaccttcga atctgagaac ttccacacct gaggcacctg 600 agagaggaac tctgtggggt gggggccgtt gcaggtgtac tgggctgtca gggatctttt 660 ctataccaga actgcggtca ctattttgta ataaatgtgg ctggagctga tccagctgtc 720 tctgacctac aaaaaaaaaa aaaaaaaaaa aaa 753 15 191 PRT Homo sapiens 15 Gln Phe Gly Thr Pro Arg Gly Thr Val Trp Pro His Leu Gln Val Ser 1 5 10 15 Gln Gly Lys Leu Leu Lys Asp Val Phe Arg Pro Gly Asp Val Phe Phe 20 25 30 Asn Thr Gly Asp Leu Leu Val Cys Asp Asp Gln Gly Phe Leu Arg Phe 35 40 45 His Asp Arg Thr Gly Asp Thr Phe Arg Trp Lys Gly Glu Asn Val Ala 50 55 60 Thr Thr Glu Val Ala Glu Val Phe Glu Ala Leu Asp Phe Leu Gln Glu 65 70 75 80 Val Asn Val Tyr Gly Val Thr Val Pro Gly His Glu Gly Arg Ala Gly 85 90 95 Met Ala Ala Leu Val Leu Arg Pro Pro His Ala Leu Asp Leu Met Gln 100 105 110 Leu Tyr Thr His Val Ser Glu Asn Leu Pro Pro Tyr Ala Arg Pro Arg 115 120 125 Phe Leu Arg Leu Gln Glu Ser Leu Ala Thr Thr Glu Thr Phe Lys Gln 130 135 140 Gln Lys Val Arg Met Ala Asn Glu Gly Phe Asp Pro Ser Thr Leu Ser 145 150 155 160 Asp Pro Leu Tyr Val Leu Asp Gln Ala Val Gly Ala Tyr Leu Pro Leu 165 170 175 Thr Thr Ala Arg Tyr Ser Ala Leu Leu Ala Gly Asn Leu Arg Ile 180 185 190 16 734 DNA Homo sapiens misc_feature (1)...(733) n = A,T,C or G 16 tcaagtacaa ctgcacgatt gtcatancat tggtgaactg tgccgntacc tcctgaacca 60 gccaccgcgg gaggcagaaa accagcacca ggttcgcatg gcactaggca atggcctccg 120 gcagtccatc tggaccaact tttccagccg cttccacata ccccaggtgg ctgagttyta 180 cggggccaca gagtgcaact gtagcctggg caacttcgac agccaggtgg gggcctgtgg 240 tttcaatagc cgcatcctgt ccttcgtgta ccccatccgg ttggtacgtg tcaacgagga 300 caccatggag ctgatccggg ggcccgacgg cgtctgcatt ccctgccagc caggtgagcc 360 gggccagctg gtgggccgca tcatccagaa agaccccctg cgccgcttcg atggctacct 420 caaccagggc gccaacaaca agaagattgc caaggatgtc ttcaagaagg gggaccaggc 480 ctaccttact ggtgatgtgc tggtgatgga cgagctgggc tacctgtact tccgagaccg 540 cactggggac acgttccgct ggaaaggtga gaacgtgtcc accaccgagg tggaaggcac 600 actcagccgc ctgctggaca tggctgacgt ggccgtgtat ggtgtcgagg tgccaggaac 660 cgagggccgg gccggaatgg ctgctgtggc cagccccact ggcaactgtg acctgggagc 720 gctttgctca ggtc 734 17 213 PRT Homo sapiens 17 Ile Gly Glu Leu Cys Arg Tyr Leu Leu Asn Gln Pro Pro Arg Glu Ala 1 5 10 15 Glu Asn Gln His Gln Val Arg Met Ala Leu Gly Asn Gly Leu Arg Gln 20 25 30 Ser Ile Trp Thr Asn Phe Ser Ser Arg Phe His Ile Pro Gln Val Ala 35 40 45 Glu Phe Tyr Gly Ala Thr Glu Cys Asn Cys Ser Leu Gly Asn Phe Asp 50 55 60 Ser Gln Val Gly Ala Cys Gly Phe Asn Ser Arg Ile Leu Ser Phe Val 65 70 75 80 Tyr Pro Ile Arg Leu Val Arg Val Asn Glu Asp Thr Met Glu Leu Ile 85 90 95 Arg Gly Pro Asp Gly Val Cys Ile Pro Cys Gln Pro Gly Glu Pro Gly 100 105 110 Gln Leu Val Gly Arg Ile Ile Gln Lys Asp Pro Leu Arg Arg Phe Asp 115 120 125 Gly Tyr Leu Asn Gln Gly Ala Asn Asn Lys Lys Ile Ala Lys Asp Val 130 135 140 Phe Lys Lys Gly Asp Gln Ala Tyr Leu Thr Gly Asp Val Leu Val Met 145 150 155 160 Asp Glu Leu Gly Tyr Leu Tyr Phe Arg Asp Arg Thr Gly Asp Thr Phe 165 170 175 Arg Trp Lys Gly Glu Asn Val Ser Thr Thr Glu Val Glu Gly Thr Leu 180 185 190 Ser Arg Leu Leu Asp Met Ala Asp Val Ala Val Tyr Gly Val Glu Val 195 200 205 Pro Gly Thr Glu Gly 210 18 1278 DNA Homo sapiens misc_feature (1)...(1278) n = A,T,C or G 18 cntgcctctt gtaccacgtg atgggacttt gtcgttggga tcctcggctg cttagatctc 60 ggagccacct gtgttctggc ccccaagttc tctacttcct gcttctggga tgactgtcgg 120 cagcatggcg tgacagtgat cctgtatgtg ggcgagctcc tgcgntactt gtgtaacatt 180 ccccagcaac cagaggaccg gacacataca gtccgcctgg caatgggcaa tggactacgg 240 gctgatgtgt ggggagacct tccagcagcg tttcggtcct atttcggatc tngggaagtc 300 ttacgggcty ccacagaagg gcaacatggg gctttagttc aactattgtt gggggcgctg 360 cggggscctg grggcaaaga tggagcttgc ctcctccgaa tgctgtcccc ctttgagctg 420 gtgcagttcg acatggaggc ggcggagcct gtgagggaca atcagggctt ctgcatccct 480 gtagggctag gggagccggg gctgctgttg accaaggtgg taagccagca acccttcgtg 540 ggctaccgcg gcccccgaga gctgtcggaa cggaagctgg tgcgcaacgt gcggcaatcg 600 ggcgacgttt actacaacac cggggacgta ctggccatgg accgcgaagg cttcctctac 660 ttccgcgacc gactcgggga caccttccga tggaagggcg agaacgtgtc cacgcacgag 720 gtggagggcg tgttgtcgca ggtggacttc ttgcaacagg ttaacgtgta tggcgtgtgc 780 gtgccaggtt gtgagggtaa ggtgggcatg gctgctgtgg cattagcccc cggccagact 840 ttcgacgggg agaagttgta ccagcacgtt cgcgcttggc tccctgccta cgctaccccc 900 catttcatcc gcatccagga cgccatggag gtcaccagca cgttcaaact gatgaagacc 960 cggttggtgc gtgagggctt caatgtgggg atcgtggttg accctctgtt tgtactggac 1020 aaccgggccc agtccttccg gcccctgacg gcagaaatgt accaggctgt gtgtgaggga 1080 acctggaggc tctgatcacc tggccaaccc actggggtag ggatcaaagc cagccacccc 1140 caccccaaca cactcggtgt ccctttcatc ctgggcctgt gtgaatccca gcctggccat 1200 accctcaacc tcagtgggct ggaaatgaca gtgggccctg tagcagtggc agaataaact 1260 cagmtgygtt cacagaaa 1278 19 199 RT Homo sapiens 19 Glu Gly Gln His Gly Ala Leu Val Gln Leu Leu Leu Gly Ala Leu Arg 1 5 10 15 Gly Pro Gly Gly Lys Asp Gly Ala Cys Leu Leu Arg Met Leu Ser Pro 20 25 30 Phe Glu Leu Val Gln Phe Asp Met Glu Ala Ala Glu Pro Val Arg Asp 35 40 45 Asn Gln Gly Phe Cys Ile Pro Val Gly Leu Gly Glu Pro Gly Leu Leu 50 55 60 Leu Thr Lys Val Val Ser Gln Gln Pro Phe Val Gly Tyr Arg Gly Pro 65 70 75 80 Arg Glu Leu Ser Glu Arg Lys Leu Val Arg Asn Val Arg Gln Ser Gly 85 90 95 Asp Val Tyr Tyr Asn Thr Gly Asp Val Leu Ala Met Asp Arg Glu Gly 100 105 110 Phe Leu Tyr Phe Arg Asp Arg Leu Gly Asp Thr Phe Arg Trp Lys Gly 115 120 125 Glu Asn Val Ser Thr His Glu Val Glu Gly Val Leu Ser Gln Val Asp 130 135 140 Phe Leu Gln Gln Val Asn Val Tyr Gly Val Cys Val Pro Gly Cys Glu 145 150 155 160 Gly Lys Val Gly Met Ala Ala Val Ala Leu Ala Pro Gly Gln Thr Phe 165 170 175 Asp Gly Glu Lys Leu Tyr Gln His Val Arg Ala Trp Leu Pro Ala Tyr 180 185 190 Ala Thr Pro His Phe Ile Arg 195 20 1361 DNA Homo sapiens 20 cgcttgtgtg ttaaagaaga aattttcagc aagccagttt tggagtgact gcaagaagta 60 tgatgtgact gtgtttcagt atattggaga actttgtcgc tacctttgca aacaatctaa 120 gagagaagga gaaaaggatc ataaggtgcg tttggcaatt ggaaatggca tacggagtga 180 tgtatggaga gaatttttag acagatttgg aaatataaag gtgtgtgaac tttatgcagc 240 taccgaatca agcatatctt tcatgaacta cactgggaga attggagcaa ttgggagaac 300 aaatttgttt tacaaacttc tttccacttt tgacttaata aagtatgact ttcagaaaga 360 tgaacccatg agaaatgagc agggttgggt attcatgaga aaaaggagac ctggacttct 420 catttctcga gtgaatgcaa aaaatccctt ctttggctat gctgggcctt ataagcacac 480 aaaagacaaa ttgctttgtg atgtttttaa gaagggagat gtttacctta atactggaga 540 cttaatagtc caggatcagg acaatttcct ttatttttgg gaccgtactg gagacacttt 600 cagatggaaa ggagaaaatg tcgcaaccac tgaggttgct gatgttattg gaatgttgga 660 tttcatacag gaagcaaacg tctatggtgt ggctatatca ggttatgaag gaagagcagg 720 aatggcttct attattttaa aaccaaatac atctttagat ttggaaaaag tttatgaaca 780 agttgtaaca tttctaccag cttatgcttg tccacgattt ttaagaattc aggaaaaaat 840 ggaagcaaca ggaacattca aactattgaa gcatcagttg gtggaagatg gatttaatcc 900 actgaaaatt tctgaaccac tttacttcat ggataacttg aaaaagtctt atgttctact 960 gaccagggaa ctttatgatc aaataatgtt aggggaaata aaactttaag atttttatat 1020 ctagaacttt catatgcttt cttaggaaga gtgagagggg ggtatatgat tctttatgaa 1080 atggggaaag ggagctaaca ttaattatgc atgtactata tttccttaat atgagagata 1140 attttttaat tgcataagaa ttttaatttc ttttaattga tataaacaga gttgattatt 1200 ctttttatct atttggagat tcagtgcata actaagtatt ttccttaata ctaaagattt 1260 taaataataa atagtggcta gcggtttgga caatcactaa aaatgtactt tctaataagt 1320 aaaatttcta attttgaata aaagattaaa ttttactgaa a 1361 21 335 PRT Homo sapiens 21 Ala Cys Val Leu Lys Lys Lys Phe Ser Ala Ser Gln Phe Trp Ser Asp 1 5 10 15 Cys Lys Lys Tyr Asp Val Thr Val Phe Gln Tyr Ile Gly Glu Leu Cys 20 25 30 Arg Tyr Leu Cys Lys Gln Ser Lys Arg Glu Gly Glu Lys Asp His Lys 35 40 45 Val Arg Leu Ala Ile Gly Asn Gly Ile Arg Ser Asp Val Trp Arg Glu 50 55 60 Phe Leu Asp Arg Phe Gly Asn Ile Lys Val Cys Glu Leu Tyr Ala Ala 65 70 75 80 Thr Glu Ser Ser Ile Ser Phe Met Asn Tyr Thr Gly Arg Ile Gly Ala 85 90 95 Ile Gly Arg Thr Asn Leu Phe Tyr Lys Leu Leu Ser Thr Phe Asp Leu 100 105 110 Ile Lys Tyr Asp Phe Gln Lys Asp Glu Pro Met Arg Asn Glu Gln Gly 115 120 125 Trp Val Phe Met Arg Lys Arg Arg Pro Gly Leu Leu Ile Ser Arg Val 130 135 140 Asn Ala Lys Asn Pro Phe Phe Gly Tyr Ala Gly Pro Tyr Lys His Thr 145 150 155 160 Lys Asp Lys Leu Leu Cys Asp Val Phe Lys Lys Gly Asp Val Tyr Leu 165 170 175 Asn Thr Gly Asp Leu Ile Val Gln Asp Gln Asp Asn Phe Leu Tyr Phe 180 185 190 Trp Asp Arg Thr Gly Asp Thr Phe Arg Trp Lys Gly Glu Asn Val Ala 195 200 205 Thr Thr Glu Val Ala Asp Val Ile Gly Met Leu Asp Phe Ile Gln Glu 210 215 220 Ala Asn Val Tyr Gly Val Ala Ile Ser Gly Tyr Glu Gly Arg Ala Gly 225 230 235 240 Met Ala Ser Ile Ile Leu Lys Pro Asn Thr Ser Leu Asp Leu Glu Lys 245 250 255 Val Tyr Glu Gln Val Val Thr Phe Leu Pro Ala Tyr Ala Cys Pro Arg 260 265 270 Phe Leu Arg Ile Gln Glu Lys Met Glu Ala Thr Gly Thr Phe Lys Leu 275 280 285 Leu Lys His Gln Leu Val Glu Asp Gly Phe Asn Pro Leu Lys Ile Ser 290 295 300 Glu Pro Leu Tyr Phe Met Asp Asn Leu Lys Lys Ser Tyr Val Leu Leu 305 310 315 320 Thr Arg Glu Leu Tyr Asp Gln Ile Met Leu Gly Glu Ile Lys Leu 325 330 335 22 2007 DNA Mycobacterium tuberculosis 22 tagtcgataa cgtcaaggac gctctgcggg cctgcgcacc ttcctgaggt tggtcgacaa 60 ccaattcgac atttcgcaaa cgaatcgagg gcttacgtgt ccgattacta cggcggcgca 120 cacacaacgg tcaggctgat cgacctggca actcggatgc cgcgagtgtt ggcggacacg 180 ccggtgattg tgcgtggggc aatgaccggg ctgctggccc ggccgaattc caaggcgtcg 240 atcggcacgg tgttccagga ccgggccgct cgctacggtg accgagtctt cctgaaattc 300 ggcgatcagc agctgaccta ccgcgacgct aacgccaccg ccaaccggta cgccgcggtg 360 ttggccgccc gcggcgtcgg ccccggcgac gtcgttggca tcatgttgcg taactcaccc 420 agcacagtct tggcgatgct ggccacggtc aagtgcggcg ctatcgccgg catgctcaac 480 taccaccagc gcggcgaggt gttggcgcac agcctgggtc tgctggacgc gaaggtactg 540 atcgcagagt ccgacttggt cagcgccgtc gccgaatgcg gcgcctcgcg cggccgggta 600 gcgggcgacg tgctgaccgt cgaggacgtg gagcgattcg ccacaacggc gcccgccacc 660 aacccggcgt cggcgtcggc ggtgcaagcc aaagacaccg cgttctacat cttcacctcg 720 ggcaccaccg gatttcccaa ggccagtgtc atgacgcatc atcggtggct gcgggcgctg 780 gccgtcttcg gagggatggg gctgcggctg aagggttccg acacgctcta cagctgcctg 840 ccgctgtacc acaacaacgc gttaacggtc gcggtgtcgt cggtgatcaa ttctggggcg 900 accctggcgc tgggtaagtc gttttcggcg tcgcggttct gggatgaggt gattgccaac 960 cgggcgacgg cgttcgtcta catcggcgaa atctgccgtt atctgctcaa ccagccggcc 1020 aagccgaccg accgtgccca ccaggtgcgg gtgatctgcg gtaacgggct gcggccggag 1080 atctgggatg agttcaccac ccgcttcggg gtcgcgcggg tgtgcgagtt ctacgccgcc 1140 agcgaaggca actcggcctt tatcaacatc ttcaacgtgc ccaggaccgc cggggtatcg 1200 ccgatgccgc ttgcctttgt ggaatacgac ctggacaccg gcgatccgct gcgggatgcg 1260 agcgggcgag tgcgtcgggt acccgacggt gaacccggcc tgttgcttag ccgggtcaac 1320 cggctgcagc cgttcgacgg ctacaccgac ccggttgcca gcgaaaagaa gttggtgcgc 1380 aacgcttttc gagatggcga ctgttggttc aacaccggtg acgtgatgag cccgcagggc 1440 atgggccatg ccgccttcgt cgatcggctg ggcgacacct tccgctggaa gggcgagaat 1500 gtcgccacca ctcaggtcga agcggcactg gcctccgacc agaccgtcga ggagtgcacg 1560 gtctacggcg tccagattcc gcgcaccggc gggcgcgccg gaatggccgc gatcacactg 1620 cgcgctggcg ccgaattcga cggccaggcg ctggcccgaa cggtttacgg tcacttgccc 1680 ggctatgcac ttccgctctt tgttcgggta gtggggtcgc tggcgcacac cacgacgttc 1740 aagagtcgca aggtggagtt gcgcaaccag gcctatggcg ccgacatcga ggatccgctg 1800 tacgtactgg ccggcccgga cgaaggatat gtgccgtact acgccgaata ccctgaggag 1860 gtttcgctcg gaaggcgacc gcagggctag cggattccgg gcgcagtctc gatacccgca 1920 ctggacgctc gacggtaacc aggcactatg gatgcgtgcg ttcaacaccg ccggcctcag 1980 ccggtcgttc aacaccgccg gcgttag 2007 23 597 PRT Mycobacterium tuberculosis 23 Met Ser Asp Tyr Tyr Gly Gly Ala His Thr Thr Val Arg Leu Ile Asp 1 5 10 15 Leu Ala Thr Arg Met Pro Arg Val Leu Ala Asp Thr Pro Val Ile Val 20 25 30 Arg Gly Ala Met Thr Gly Leu Leu Ala Arg Pro Asn Ser Lys Ala Ser 35 40 45 Ile Gly Thr Val Phe Gln Asp Arg Ala Ala Arg Tyr Gly Asp Arg Val 50 55 60 Phe Leu Lys Phe Gly Asp Gln Gln Leu Thr Tyr Arg Asp Ala Asn Ala 65 70 75 80 Thr Ala Asn Arg Tyr Ala Ala Val Leu Ala Ala Arg Gly Val Gly Pro 85 90 95 Gly Asp Val Val Gly Ile Met Leu Arg Asn Ser Pro Ser Thr Val Leu 100 105 110 Ala Met Leu Ala Thr Val Lys Cys Gly Ala Ile Ala Gly Met Leu Asn 115 120 125 Tyr His Gln Arg Gly Glu Val Leu Ala His Ser Leu Gly Leu Leu Asp 130 135 140 Ala Lys Val Leu Ile Ala Glu Ser Asp Leu Val Ser Ala Val Ala Glu 145 150 155 160 Cys Gly Ala Ser Arg Gly Arg Val Ala Gly Asp Val Leu Thr Val Glu 165 170 175 Asp Val Glu Arg Phe Ala Thr Thr Ala Pro Ala Thr Asn Pro Ala Ser 180 185 190 Ala Ser Ala Val Gln Ala Lys Asp Thr Ala Phe Tyr Ile Phe Thr Ser 195 200 205 Gly Thr Thr Gly Phe Pro Lys Ala Ser Val Met Thr His His Arg Trp 210 215 220 Leu Arg Ala Leu Ala Val Phe Gly Gly Met Gly Leu Arg Leu Lys Gly 225 230 235 240 Ser Asp Thr Leu Tyr Ser Cys Leu Pro Leu Tyr His Asn Asn Ala Leu 245 250 255 Thr Val Ala Val Ser Ser Val Ile Asn Ser Gly Ala Thr Leu Ala Leu 260 265 270 Gly Lys Ser Phe Ser Ala Ser Arg Phe Trp Asp Glu Val Ile Ala Asn 275 280 285 Arg Ala Thr Ala Phe Val Tyr Ile Gly Glu Ile Cys Arg Tyr Leu Leu 290 295 300 Asn Gln Pro Ala Lys Pro Thr Asp Arg Ala His Gln Val Arg Val Ile 305 310 315 320 Cys Gly Asn Gly Leu Arg Pro Glu Ile Trp Asp Glu Phe Thr Thr Arg 325 330 335 Phe Gly Val Ala Arg Val Cys Glu Phe Tyr Ala Ala Ser Glu Gly Asn 340 345 350 Ser Ala Phe Ile Asn Ile Phe Asn Val Pro Arg Thr Ala Gly Val Ser 355 360 365 Pro Met Pro Leu Ala Phe Val Glu Tyr Asp Leu Asp Thr Gly Asp Pro 370 375 380 Leu Arg Asp Ala Ser Gly Arg Val Arg Arg Val Pro Asp Gly Glu Pro 385 390 395 400 Gly Leu Leu Leu Ser Arg Val Asn Arg Leu Gln Pro Phe Asp Gly Tyr 405 410 415 Thr Asp Pro Val Ala Ser Glu Lys Lys Leu Val Arg Asn Ala Phe Arg 420 425 430 Asp Gly Asp Cys Trp Phe Asn Thr Gly Asp Val Met Ser Pro Gln Gly 435 440 445 Met Gly His Ala Ala Phe Val Asp Arg Leu Gly Asp Thr Phe Arg Trp 450 455 460 Lys Gly Glu Asn Val Ala Thr Thr Gln Val Glu Ala Ala Leu Ala Ser 465 470 475 480 Asp Gln Thr Val Glu Glu Cys Thr Val Tyr Gly Val Gln Ile Pro Arg 485 490 495 Thr Gly Gly Arg Ala Gly Met Ala Ala Ile Thr Leu Arg Ala Gly Ala 500 505 510 Glu Phe Asp Gly Gln Ala Leu Ala Arg Thr Val Tyr Gly His Leu Pro 515 520 525 Gly Tyr Ala Leu Pro Leu Phe Val Arg Val Val Gly Ser Leu Ala His 530 535 540 Thr Thr Thr Phe Lys Ser Arg Lys Val Glu Leu Arg Asn Gln Ala Tyr 545 550 555 560 Gly Ala Asp Ile Glu Asp Pro Leu Tyr Val Leu Ala Gly Pro Asp Glu 565 570 575 Gly Tyr Val Pro Tyr Tyr Ala Glu Tyr Pro Glu Glu Val Ser Leu Gly 580 585 590 Arg Arg Pro Gln Gly 595 24 2221 DNA Drosophila melanogaster 24 gctctctggg cctatatcaa gctgctgagg tacacgaagc gccatgagcg gctcaactac 60 acggtggcgg acgtcttcga acgaaatgtt caggcccatc cggacaaggt ggctgtggtc 120 agtgagacgc aacgctggac cttccgtcag gtgaacgagc atgcgaacaa ggtggccaat 180 gtgctgcagg ctcagggcta caaaaagggc gatgtggtgg ccctgttgct ggagaaccgc 240 gccgagtacg tggccacctg gctgggtctc tccaagatcg gtgtgatcac accgctgatc 300 aacacgaatc tgcgcggtcc ctccctgctg cacagcatca cggtggccca ttgctcggct 360 ctcatttacg gcgaggactt cctggaagct gtcaccgacg tggccaagga tctgccagcg 420 aacctcacac tcttccagtt caacaacgag aacaacaaca gcgagacgga aaagaacata 480 ccgcaggcca agaatctgaa cgcgctgctg accacggcca gctatgagaa gcctaacaag 540 acgcaggtta accaccacga caagctggtc tacatctaca cctccggcac cacaggattg 600 ccaaaggctg cggttatctc tcactcccgt tatctgttta tcgctgctgg catccactac 660 accatgggtt tccaggagga ggacatcttc tacacgccct tgcctttgta ccacaccgct 720 ggtggcatta tgtgcatggg tcagtcggtg ctctttggct ccacggtctc cattcgcaag 780 aagttctcgg catccaacta tttcgccgac tgcgccaagt ataatgcaac tattggtcag 840 tatatcggtg agatggctcg ctacattcta gctacgaaac cctcggaata cgaccagaaa 900 caccgagtgc gtctggtctt tggaaacgga ctgcgaccgc agatttggcc acagtttgtg 960 cagcgcttca acattgccaa ggttggcgag ttctacggcg ccaccgaggg taatgcgaac 1020 atcatgaatc atgacaacac ggtgggcgcc atcggctttg tgtcgcgcat cctgcccaag 1080 atctacccaa tctcgatcat tcgcgccgat ccggacaccg gagagcccat tagagatagg 1140 aatggcctat gccaactgtg cgctcccaac gagccaggcg tattcatcgg caagatcgtc 1200 aaaggaaatc cttctcgcga attcctcgga tacgtcgatg aaaaggcctc cgcgaagaag 1260 attgttaagg atgtgttcaa gcatggcgat atggctttca tctccggaga tctgctggtt 1320 gccgacgaga agggttatct gtacttcaag gatcgcaccg gtgacacctt ccgctggaag 1380 ggcgagaatg tttccaccag cgaggtggag gcgcaagtca gcaatgtggc cggttacaag 1440 gataccgtcg tttacggcgt aaccattccg cacaccgagg gaagggccgg catggccgcc 1500 atctatgatc cggagcgaga attggacctc gacgtcttcg ccgctagctt ggccaaggtg 1560 ctgcccgcgt acgctcgtcc ccagatcatt cgattgctca ccaaggtgga cctgactgga 1620 acctttaagc tgcgcaaggt agacctgcag aaggagggct acgatccgaa cgcgatcaag 1680 gacgcgctgt actaccagac ttccaagggt cggtacgagc tgctcacgcc ccaggtttac 1740 gaccaggtgc agcgcaacga aatccgcttc taagagctgc aatagagttg tgtctgaacc 1800 ttgccttttg cccaatatgc tgttaattag tttgtaaggc taagtgtagt agaggaaaat 1860 cgggggaaat cggcagcaaa gatcattcag cctaggagag atgcatccga agcacatttc 1920 catgtcaaca atgcactttt gtatatcgta agcatatata tatcgtatat cgtaaacgta 1980 gttgtatctg catttgtgta gatgatagcc tcctatacgc atttcaattg tttttagcgt 2040 gctaaagaac cttgttaaat gcaatttcag ctattgttta gtcagtttta gtggcattta 2100 cacttccatt ctcgttgcgt ttcgtttttg cctgtacata tgagaagctc tgatgttttt 2160 gtatcaaata aagttttttc cttcaccacg gaccacgtga aaaaaaaaaa aaaaaaaaaa 2220 a 2221 25 590 PRT Drosophila melanogaster 25 Ala Leu Trp Ala Tyr Ile Lys Leu Leu Arg Tyr Thr Lys Arg His Glu 1 5 10 15 Arg Leu Asn Tyr Thr Val Ala Asp Val Phe Glu Arg Asn Val Gln Ala 20 25 30 His Pro Asp Lys Val Ala Val Val Ser Glu Thr Gln Arg Trp Thr Phe 35 40 45 Arg Gln Val Asn Glu His Ala Asn Lys Val Ala Asn Val Leu Gln Ala 50 55 60 Gln Gly Tyr Lys Lys Gly Asp Val Val Ala Leu Leu Leu Glu Asn Arg 65 70 75 80 Ala Glu Tyr Val Ala Thr Trp Leu Gly Leu Ser Lys Ile Gly Val Ile 85 90 95 Thr Pro Leu Ile Asn Thr Asn Leu Arg Gly Pro Ser Leu Leu His Ser 100 105 110 Ile Thr Val Ala His Cys Ser Ala Leu Ile Tyr Gly Glu Asp Phe Leu 115 120 125 Glu Ala Val Thr Asp Val Ala Lys Asp Leu Pro Ala Asn Leu Thr Leu 130 135 140 Phe Gln Phe Asn Asn Glu Asn Asn Asn Ser Glu Thr Glu Lys Asn Ile 145 150 155 160 Pro Gln Ala Lys Asn Leu Asn Ala Leu Leu Thr Thr Ala Ser Tyr Glu 165 170 175 Lys Pro Asn Lys Thr Gln Val Asn His His Asp Lys Leu Val Tyr Ile 180 185 190 Tyr Thr Ser Gly Thr Thr Gly Leu Pro Lys Ala Ala Val Ile Ser His 195 200 205 Ser Arg Tyr Leu Phe Ile Ala Ala Gly Ile His Tyr Thr Met Gly Phe 210 215 220 Gln Glu Glu Asp Ile Phe Tyr Thr Pro Leu Pro Leu Tyr His Thr Ala 225 230 235 240 Gly Gly Ile Met Cys Met Gly Gln Ser Val Leu Phe Gly Ser Thr Val 245 250 255 Ser Ile Arg Lys Lys Phe Ser Ala Ser Asn Tyr Phe Ala Asp Cys Ala 260 265 270 Lys Tyr Asn Ala Thr Ile Gly Gln Tyr Ile Gly Glu Met Ala Arg Tyr 275 280 285 Ile Leu Ala Thr Lys Pro Ser Glu Tyr Asp Gln Lys His Arg Val Arg 290 295 300 Leu Val Phe Gly Asn Gly Leu Arg Pro Gln Ile Trp Pro Gln Phe Val 305 310 315 320 Gln Arg Phe Asn Ile Ala Lys Val Gly Glu Phe Tyr Gly Ala Thr Glu 325 330 335 Gly Asn Ala Asn Ile Met Asn His Asp Asn Thr Val Gly Ala Ile Gly 340 345 350 Phe Val Ser Arg Ile Leu Pro Lys Ile Tyr Pro Ile Ser Ile Ile Arg 355 360 365 Ala Asp Pro Asp Thr Gly Glu Pro Ile Arg Asp Arg Asn Gly Leu Cys 370 375 380 Gln Leu Cys Ala Pro Asn Glu Pro Gly Val Phe Ile Gly Lys Ile Val 385 390 395 400 Lys Gly Asn Pro Ser Arg Glu Phe Leu Gly Tyr Val Asp Glu Lys Ala 405 410 415 Ser Ala Lys Lys Ile Val Lys Asp Val Phe Lys His Gly Asp Met Ala 420 425 430 Phe Ile Ser Gly Asp Leu Leu Val Ala Asp Glu Lys Gly Tyr Leu Tyr 435 440 445 Phe Lys Asp Arg Thr Gly Asp Thr Phe Arg Trp Lys Gly Glu Asn Val 450 455 460 Ser Thr Ser Glu Val Glu Ala Gln Val Ser Asn Val Ala Gly Tyr Lys 465 470 475 480 Asp Thr Val Val Tyr Gly Val Thr Ile Pro His Thr Glu Gly Arg Ala 485 490 495 Gly Met Ala Ala Ile Tyr Asp Pro Glu Arg Glu Leu Asp Leu Asp Val 500 505 510 Phe Ala Ala Ser Leu Ala Lys Val Leu Pro Ala Tyr Ala Arg Pro Gln 515 520 525 Ile Ile Arg Leu Leu Thr Lys Val Asp Leu Thr Gly Thr Phe Lys Leu 530 535 540 Arg Lys Val Asp Leu Gln Lys Glu Gly Tyr Asp Pro Asn Ala Ile Lys 545 550 555 560 Asp Ala Leu Tyr Tyr Gln Thr Ser Lys Gly Arg Tyr Glu Leu Leu Thr 565 570 575 Pro Gln Val Tyr Asp Gln Val Gln Arg Asn Glu Ile Arg Phe 580 585 590 26 173 DNA Danio rerio 26 agtgtagata ccacaggaac gtttaaaatc cagaagacca gactgcaaag ggaaggatac 60 gatccacggc tcacaactga ccagatctac ttcctaaact ccagagcagg gcgttacgag 120 cttgtcaacg aggagctgta caatgcattt gaacaagggc aggatttccc ttt 173 27 57 PRT Danio rerio 27 Ser Val Asp Thr Thr Gly Thr Phe Lys Ile Gln Lys Thr Arg Leu Gln 1 5 10 15 Arg Glu Gly Tyr Asp Pro Arg Leu Thr Thr Asp Gln Ile Tyr Phe Leu 20 25 30 Asn Ser Arg Ala Gly Arg Tyr Glu Leu Val Asn Glu Glu Leu Tyr Asn 35 40 45 Ala Phe Glu Gln Gly Gln Asp Phe Pro 50 55 28 1953 DNA Caenorhabditis elegans 28 atgaagctgg aggagcttgt gacagttatg cttctcacag tggctgtcat tgctcagaat 60 cttccgattg gagtaatatt ggctggagtt cttattttat acatcacagt ggttcatgga 120 gatttcattt atagaagtta tcttacgttg aatagggatt taacaggatt ggctctaatt 180 attgaagtca aaatcgacct atggtggagg ttgcatcaga ataaaggaat ccatgaactg 240 tttttggata ttgtgaaaaa gaatccaaat aagccggcga tgattgacat cgagacgaat 300 acaacagaaa catacgcaga gttcaatgca cattgtaata gatatgccaa ttatttccag 360 ggtcttggct atcgatccgg agacgttgtc gccttgtaca tggagaactc ggtcgagttt 420 gtggccgcgt ggatgggact cgcaaaaatc ggagttgtaa cggcttggat caactcgaat 480 ttgaaaagag agcaacttgt tcattgtatc actgcgagca agacaaaggc gattatcaca 540 agtgtaacac ttcagaatat tatgcttgat gctatcgatc agaagctgtt tgatgttgag 600 ggaattgagg tttactctgt cggagagccc aagaagaatt ctggattcaa gaatctcaag 660 aagaagttgg atgctcaaat tactacggaa ccaaagaccc ttgacatagt agattttaaa 720 agtattcttt gcttcatcta tacaagtggt actactggaa tgccaaaagc cgctgtcatg 780 aagcacttca gatattactc gattgccgtt ggagccgcaa aatcattcgg aatccgccct 840 tctgatcgta tgtacgtctc gatgccaatt tatcacactg cagctggaat tcttggagtt 900 gggcaagctc tgttgggtgg atcatcgtgt gtcattagaa aaaaattctc ggctagcaac 960 ttttggaggg attgtgtaaa gtatgattgt acagtttcac aatacattgg agagatttgt 1020 cggtacttgt tggctcagcc agttgtggaa gaggaatcca ggcatagaat gagattgttg 1080 gttggaaacg gactccgtgc tgaaatctgg caaccatttg tagatcgatt ccgtgtcaga 1140 attggagaac tttatggttc aactgaagga acttcatctc tcgtgaacat tgacggacat 1200 gtcggagctt gcggattctt gccaatatcc ccattaacaa agaaaatgca tccggttcga 1260 ttaattaagg ttgatgatgt cactggagaa gcaatccgaa cttccgatgg actttgcatt 1320 gcatgtaatc caggagagtc tggagcaatg gtgtcgacga tcagaaaaaa taatccatta 1380 ttgcaattcg agggatatct gaataagaag gaaacgaata aaaagattat cagagatgtc 1440 ttcgcaaagg gagatagttg ctttttgact ggagatcttc ttcattggga tcgtcttggt 1500 tatgtatatt tcaaggatcg tactggagat actttccgtt ggaagggaga gaatgtgtcg 1560 actactgaag tcgaggcaat tcttcatcca attactggat tgtctgatgc aactgtttat 1620 ggtgtagagg ttcctcaaag agagggaaga gttggaatgg cgtcagttgt tcgagttgta 1680 tcgcatgagg aagatgaaac tcaatttgtt catagagttg gagcaagact tgcctcttcg 1740 cttaccagct acgcgattcc tcagtttatg cgaatttgtc aggatgttga gaaaacaggt 1800 acattcaaac ttgtgaagac gaatctacaa cgattaggta tcatggatgc tccttcagat 1860 tcaatttaca tctacaattc tgaaaatcgc aattttgtgc cgttcgacaa tgatttgagg 1920 tgcaaggtct cactgggaag ttatccattt taa 1953 29 650 PRT Caenorhabditis elegans 29 Met Lys Leu Glu Glu Leu Val Thr Val Met Leu Leu Thr Val Ala Val 1 5 10 15 Ile Ala Gln Asn Leu Pro Ile Gly Val Ile Leu Ala Gly Val Leu Ile 20 25 30 Leu Tyr Ile Thr Val Val His Gly Asp Phe Ile Tyr Arg Ser Tyr Leu 35 40 45 Thr Leu Asn Arg Asp Leu Thr Gly Leu Ala Leu Ile Ile Glu Val Lys 50 55 60 Ile Asp Leu Trp Trp Arg Leu His Gln Asn Lys Gly Ile His Glu Leu 65 70 75 80 Phe Leu Asp Ile Val Lys Lys Asn Pro Asn Lys Pro Ala Met Ile Asp 85 90 95 Ile Glu Thr Asn Thr Thr Glu Thr Tyr Ala Glu Phe Asn Ala His Cys 100 105 110 Asn Arg Tyr Ala Asn Tyr Phe Gln Gly Leu Gly Tyr Arg Ser Gly Asp 115 120 125 Val Val Ala Leu Tyr Met Glu Asn Ser Val Glu Phe Val Ala Ala Trp 130 135 140 Met Gly Leu Ala Lys Ile Gly Val Val Thr Ala Trp Ile Asn Ser Asn 145 150 155 160 Leu Lys Arg Glu Gln Leu Val His Cys Ile Thr Ala Ser Lys Thr Lys 165 170 175 Ala Ile Ile Thr Ser Val Thr Leu Gln Asn Ile Met Leu Asp Ala Ile 180 185 190 Asp Gln Lys Leu Phe Asp Val Glu Gly Ile Glu Val Tyr Ser Val Gly 195 200 205 Glu Pro Lys Lys Asn Ser Gly Phe Lys Asn Leu Lys Lys Lys Leu Asp 210 215 220 Ala Gln Ile Thr Thr Glu Pro Lys Thr Leu Asp Ile Val Asp Phe Lys 225 230 235 240 Ser Ile Leu Cys Phe Ile Tyr Thr Ser Gly Thr Thr Gly Met Pro Lys 245 250 255 Ala Ala Val Met Lys His Phe Arg Tyr Tyr Ser Ile Ala Val Gly Ala 260 265 270 Ala Lys Ser Phe Gly Ile Arg Pro Ser Asp Arg Met Tyr Val Ser Met 275 280 285 Pro Ile Tyr His Thr Ala Ala Gly Ile Leu Gly Val Gly Gln Ala Leu 290 295 300 Leu Gly Gly Ser Ser Cys Val Ile Arg Lys Lys Phe Ser Ala Ser Asn 305 310 315 320 Phe Trp Arg Asp Cys Val Lys Tyr Asp Cys Thr Val Ser Gln Tyr Ile 325 330 335 Gly Glu Ile Cys Arg Tyr Leu Leu Ala Gln Pro Val Val Glu Glu Glu 340 345 350 Ser Arg His Arg Met Arg Leu Leu Val Gly Asn Gly Leu Arg Ala Glu 355 360 365 Ile Trp Gln Pro Phe Val Asp Arg Phe Arg Val Arg Ile Gly Glu Leu 370 375 380 Tyr Gly Ser Thr Glu Gly Thr Ser Ser Leu Val Asn Ile Asp Gly His 385 390 395 400 Val Gly Ala Cys Gly Phe Leu Pro Ile Ser Pro Leu Thr Lys Lys Met 405 410 415 His Pro Val Arg Leu Ile Lys Val Asp Asp Val Thr Gly Glu Ala Ile 420 425 430 Arg Thr Ser Asp Gly Leu Cys Ile Ala Cys Asn Pro Gly Glu Ser Gly 435 440 445 Ala Met Val Ser Thr Ile Arg Lys Asn Asn Pro Leu Leu Gln Phe Glu 450 455 460 Gly Tyr Leu Asn Lys Lys Glu Thr Asn Lys Lys Ile Ile Arg Asp Val 465 470 475 480 Phe Ala Lys Gly Asp Ser Cys Phe Leu Thr Gly Asp Leu Leu His Trp 485 490 495 Asp Arg Leu Gly Tyr Val Tyr Phe Lys Asp Arg Thr Gly Asp Thr Phe 500 505 510 Arg Trp Lys Gly Glu Asn Val Ser Thr Thr Glu Val Glu Ala Ile Leu 515 520 525 His Pro Ile Thr Gly Leu Ser Asp Ala Thr Val Tyr Gly Val Glu Val 530 535 540 Pro Gln Arg Glu Gly Arg Val Gly Met Ala Ser Val Val Arg Val Val 545 550 555 560 Ser His Glu Glu Asp Glu Thr Gln Phe Val His Arg Val Gly Ala Arg 565 570 575 Leu Ala Ser Ser Leu Thr Ser Tyr Ala Ile Pro Gln Phe Met Arg Ile 580 585 590 Cys Gln Asp Val Glu Lys Thr Gly Thr Phe Lys Leu Val Lys Thr Asn 595 600 605 Leu Gln Arg Leu Gly Ile Met Asp Ala Pro Ser Asp Ser Ile Tyr Ile 610 615 620 Tyr Asn Ser Glu Asn Arg Asn Phe Val Pro Phe Asp Asn Asp Leu Arg 625 630 635 640 Cys Lys Val Ser Leu Gly Ser Tyr Pro Phe 645 650 30 1968 DNA Caenorhabditis elegans 30 atgagggaaa tgccggacag tcccaagttt gcgttagtca cgtttgttgt gtatgcagtg 60 gttttgtaca atgtcaacag cgttttctgg aaatttgtat tcatcggata tgttgtattt 120 aggctgcttc gcactgattt tggaagaaga gcacttgcca cgttacctag agattttgcg 180 ggactgaagc tcttaatatc ggttaagtcg acaattcgtg gcttgttcaa gaaagatcgc 240 ccaattcatg aaatcttttt gaatcaggtg aaacagcatc caaacaaagt ggcgattatt 300 gaaattgaaa gtggtaggca gttgacgtat caagaattga atgcgttagc taatcagtat 360 gctaaccttt acgtgagtga aggttacaaa atgggcgacg ttgtcgcttt gtttatggaa 420 aatagcatcg acttctttgc aatttggctg ggactttcca agattggagt cgtgtcggcg 480 ttcatcaact caaacttgaa gttggagcca ttggcacatt cgattaatgt ttcgaagtgc 540 aaatcatgca ttaccaatat caatctgttg ccgatgttca aagccgctcg tgaaaagaat 600 ctgatcagtg acgagatcca cgtgtttctg gctggaactc aggttgatgg acgtcataga 660 agtcttcagc aagatctcca tcttttctct gaggatgaac ctccagttat agacggactc 720 aattttagaa gcgttctgtg ttatatttac acttccggta ctaccggaaa tccaaagcca 780 gccgtcatta aacacttccg ttacttctgg attgcgatgg gagcaggaaa agcatttgga 840 attaataagt cagacgttgt gtacattacg atgccaatgt atcactctgc cgccggtatc 900 atgggtattg gatcattaat tgcattcggg tcgaccgctg ttattaggaa aaagttttcg 960 gcaagcaact tctggaaaga ttgcgtcaag tacaacgtca cagcgacaca gtacattgga 1020 gaaatctgca ggtatcttct ggcagcgaat ccatgtcctg aagagaaaca acacaacgtg 1080 cgattgatgt ggggaaatgg tttgagagga caaatttgga aagagtttgt aggaagattt 1140 ggaattaaga aaattggaga gttgtacggc tcaacagaag gaaactccaa tattgttaac 1200 gtggataacc atgttggagc ttgtggattc atgccaattt atccccatat tggatccctc 1260 tacccagttc gacttattaa ggttgataga gccactggag agcttgaacg tgataagaac 1320 ggactctgtg tgccgtgtgt gcctggtgaa actggggaaa tggttggcgt tatcaaggag 1380 aaagatattc ttctaaagtt cgaaggatat gtcagcgaag gggatactgc aaagaaaatc 1440 tacagagatg tgttcaagca tggagataag gtgtttgcaa gtggagatat tcttcattgg 1500 gatgatcttg gatacttgta ctttgtggac cgttgtggag acactttccg ttggaaaggg 1560 gagaacgtgt caactactga agttgaggga attcttcagc ctgtgatgga tgtggaagat 1620 gcaactgttt atggagtcac tgtcggtaaa atggaggggc gtgccggaat ggctggtatt 1680 gtcgtcaagg atggaacgga tgttgagaaa ttcatcgccg atattacttc tcgactgacc 1740 gaaaatctgg cgtcttacgc aatccctgtt ttcattcggc tgtgcaagga agttgatcga 1800 accggaacct tcaaactcaa gaagactgat cttcaaaaac aaggttacga cctggttgct 1860 tgtaaaggag acccaattta ctactggtca gctgcagaaa aatcctacaa accactgact 1920 gacaaaatgc aacaggatat tgacactggt gtttatgatc gcatttaa 1968 31 655 PRT Caenorhabditis elegans 31 Met Arg Glu Met Pro Asp Ser Pro Lys Phe Ala Leu Val Thr Phe Val 1 5 10 15 Val Tyr Ala Val Val Leu Tyr Asn Val Asn Ser Val Phe Trp Lys Phe 20 25 30 Val Phe Ile Gly Tyr Val Val Phe Arg Leu Leu Arg Thr Asp Phe Gly 35 40 45 Arg Arg Ala Leu Ala Thr Leu Pro Arg Asp Phe Ala Gly Leu Lys Leu 50 55 60 Leu Ile Ser Val Lys Ser Thr Ile Arg Gly Leu Phe Lys Lys Asp Arg 65 70 75 80 Pro Ile His Glu Ile Phe Leu Asn Gln Val Lys Gln His Pro Asn Lys 85 90 95 Val Ala Ile Ile Glu Ile Glu Ser Gly Arg Gln Leu Thr Tyr Gln Glu 100 105 110 Leu Asn Ala Leu Ala Asn Gln Tyr Ala Asn Leu Tyr Val Ser Glu Gly 115 120 125 Tyr Lys Met Gly Asp Val Val Ala Leu Phe Met Glu Asn Ser Ile Asp 130 135 140 Phe Phe Ala Ile Trp Leu Gly Leu Ser Lys Ile Gly Val Val Ser Ala 145 150 155 160 Phe Ile Asn Ser Asn Leu Lys Leu Glu Pro Leu Ala His Ser Ile Asn 165 170 175 Val Ser Lys Cys Lys Ser Cys Ile Thr Asn Ile Asn Leu Leu Pro Met 180 185 190 Phe Lys Ala Ala Arg Glu Lys Asn Leu Ile Ser Asp Glu Ile His Val 195 200 205 Phe Leu Ala Gly Thr Gln Val Asp Gly Arg His Arg Ser Leu Gln Gln 210 215 220 Asp Leu His Leu Phe Ser Glu Asp Glu Pro Pro Val Ile Asp Gly Leu 225 230 235 240 Asn Phe Arg Ser Val Leu Cys Tyr Ile Tyr Thr Ser Gly Thr Thr Gly 245 250 255 Asn Pro Lys Pro Ala Val Ile Lys His Phe Arg Tyr Phe Trp Ile Ala 260 265 270 Met Gly Ala Gly Lys Ala Phe Gly Ile Asn Lys Ser Asp Val Val Tyr 275 280 285 Ile Thr Met Pro Met Tyr His Ser Ala Ala Gly Ile Met Gly Ile Gly 290 295 300 Ser Leu Ile Ala Phe Gly Ser Thr Ala Val Ile Arg Lys Lys Phe Ser 305 310 315 320 Ala Ser Asn Phe Trp Lys Asp Cys Val Lys Tyr Asn Val Thr Ala Thr 325 330 335 Gln Tyr Ile Gly Glu Ile Cys Arg Tyr Leu Leu Ala Ala Asn Pro Cys 340 345 350 Pro Glu Glu Lys Gln His Asn Val Arg Leu Met Trp Gly Asn Gly Leu 355 360 365 Arg Gly Gln Ile Trp Lys Glu Phe Val Gly Arg Phe Gly Ile Lys Lys 370 375 380 Ile Gly Glu Leu Tyr Gly Ser Thr Glu Gly Asn Ser Asn Ile Val Asn 385 390 395 400 Val Asp Asn His Val Gly Ala Cys Gly Phe Met Pro Ile Tyr Pro His 405 410 415 Ile Gly Ser Leu Tyr Pro Val Arg Leu Ile Lys Val Asp Arg Ala Thr 420 425 430 Gly Glu Leu Glu Arg Asp Lys Asn Gly Leu Cys Val Pro Cys Val Pro 435 440 445 Gly Glu Thr Gly Glu Met Val Gly Val Ile Lys Glu Lys Asp Ile Leu 450 455 460 Leu Lys Phe Glu Gly Tyr Val Ser Glu Gly Asp Thr Ala Lys Lys Ile 465 470 475 480 Tyr Arg Asp Val Phe Lys His Gly Asp Lys Val Phe Ala Ser Gly Asp 485 490 495 Ile Leu His Trp Asp Asp Leu Gly Tyr Leu Tyr Phe Val Asp Arg Cys 500 505 510 Gly Asp Thr Phe Arg Trp Lys Gly Glu Asn Val Ser Thr Thr Glu Val 515 520 525 Glu Gly Ile Leu Gln Pro Val Met Asp Val Glu Asp Ala Thr Val Tyr 530 535 540 Gly Val Thr Val Gly Lys Met Glu Gly Arg Ala Gly Met Ala Gly Ile 545 550 555 560 Val Val Lys Asp Gly Thr Asp Val Glu Lys Phe Ile Ala Asp Ile Thr 565 570 575 Ser Arg Leu Thr Glu Asn Leu Ala Ser Tyr Ala Ile Pro Val Phe Ile 580 585 590 Arg Leu Cys Lys Glu Val Asp Arg Thr Gly Thr Phe Lys Leu Lys Lys 595 600 605 Thr Asp Leu Gln Lys Gln Gly Tyr Asp Leu Val Ala Cys Lys Gly Asp 610 615 620 Pro Ile Tyr Tyr Trp Ser Ala Ala Glu Lys Ser Tyr Lys Pro Leu Thr 625 630 635 640 Asp Lys Met Gln Gln Asp Ile Asp Thr Gly Val Tyr Asp Arg Ile 645 650 655 32 1932 DNA Cochliobolu heterostrophus 32 atggcgtgta tgcatcaggc tcagctatac aatgatctag aggaattgct aactggtcca 60 tcagtaccca tcgttgctgg agctgctgga gctgcagctc tcactgccta cattaacgcc 120 aaataccaca tagcccatga tctcaagacc ctcggtggtg gattgacaca atcgtccgaa 180 gcgattgatt tcataaaccg ccgcgtcgca caaaagcgcg tcctcacgca ccacatcttc 240 caggagcagg tccaaaaaca atcaaatcat ccctttctta tctttgaggg caagacatgg 300 tcttacaagg agttctctga ggcatacacg agggtcgcga actggctgat tgatgagctg 360 gacgtacaag taggggagat ggtcgcaatt gatggcggaa atagtgcaga gcacctgatg 420 ctttggcttg cacttgatgc aatcggtgcg gctacgagtt ttttgaactg gaacctgaca 480 ggggcagggt taattcattg cataaagcta tgcgaatgtc gattcgttat cgcagacatc 540 gatattaaag cgaacattga accgtgccgt ggcgaactgg aggagacggg catcaacatt 600 cactactatg acccatcctt catctcatcg ctaccgaata acacgccaat tcccgacagc 660 cgcactgaga acattgaatt agattcagta cgaggactga tatacacatc tggaaccact 720 ggtctaccta aaggcgtgtt tataagcact ggccgcgagc ttaggactga ctggtcgatt 780 tcaaagtatc taaatctcaa gcccacggat cgaatgtata catgtatgcc gctctaccat 840 gccgctgcac acagcctctg tacagcatca gttattcatg gtggaggtac cgtggtattg 900 agcaggaaat tctcacacaa gaagttctgg cctgaagttg tggcttcgga agcaaatatc 960 attcagtacg ttggtgaatt aggtcgatat ctcctgaatg gtccaaagag tccttacgac 1020 agggcccata aagtccagat ggcgtggggc aatggcatgc gtccagacgt gtgggaagcg 1080 tttcgtgaac gcttcaacat accaattatt catgagctct atgccgcaac cgatgggctc 1140 gggtcaatga ccaatcgtaa cgcgggccct tttacagcaa actgtattgc gctgcgaggg 1200 ctgatctggc actggaaatt tcgaaatcag gaagtgctgg tcaagatgga tctcgatact 1260 gatgagatca tgagagatcg caatgggttt gcgatacgat gcgctgtcaa tgaacctgga 1320 cagatgcttt ttcggctgac acccgaaact ctggctggtg caccaagcta ctacaacaac 1380 gaaacggcca cacagagcag gcggattaca gatgtgtttc aaaagggtga cctgtggttc 1440 aagtccggtg acatgctacg gcaagacgcc gaaggccgcg tctactttgt cgatcgacta 1500 ggcgatacgt tccgctggaa atccgaaaac gtttctacca atgaagtcgc ggacgtgatg 1560 ggcacatttc ctcagattgc tgaaacgaat gtatacggtg tccttgtgcc gggtaacgat 1620 ggtcgagtgc gcagcctcaa ttgtcatggc agacggcgtg acagagtcga cattcgcttc 1680 gctgcccttg caaagcacgc ccgagatcgg ttaccgggtt atgctgtacc actgtttctg 1740 agggtaactc cagcacttga atatacgggc acattaaaga ttcagaaagg acgcctcaag 1800 caggaaggta tagacccaga taagatttcc ggcgaagata agttatactg gctgccgcct 1860 ggtagcgata tatatttacc atttggaaag atggagtggc agggaattgt agataagcgt 1920 atacggctgt ga 1932 33 643 PRT Cochliobolu heterostrophus 33 Met Ala Cys Met His Gln Ala Gln Leu Tyr Asn Asp Leu Glu Glu Leu 1 5 10 15 Leu Thr Gly Pro Ser Val Pro Ile Val Ala Gly Ala Ala Gly Ala Ala 20 25 30 Ala Leu Thr Ala Tyr Ile Asn Ala Lys Tyr His Ile Ala His Asp Leu 35 40 45 Lys Thr Leu Gly Gly Gly Leu Thr Gln Ser Ser Glu Ala Ile Asp Phe 50 55 60 Ile Asn Arg Arg Val Ala Gln Lys Arg Val Leu Thr His His Ile Phe 65 70 75 80 Gln Glu Gln Val Gln Lys Gln Ser Asn His Pro Phe Leu Ile Phe Glu 85 90 95 Gly Lys Thr Trp Ser Tyr Lys Glu Phe Ser Glu Ala Tyr Thr Arg Val 100 105 110 Ala Asn Trp Leu Ile Asp Glu Leu Asp Val Gln Val Gly Glu Met Val 115 120 125 Ala Ile Asp Gly Gly Asn Ser Ala Glu His Leu Met Leu Trp Leu Ala 130 135 140 Leu Asp Ala Ile Gly Ala Ala Thr Ser Phe Leu Asn Trp Asn Leu Thr 145 150 155 160 Gly Ala Gly Leu Ile His Cys Ile Lys Leu Cys Glu Cys Arg Phe Val 165 170 175 Ile Ala Asp Ile Asp Ile Lys Ala Asn Ile Glu Pro Cys Arg Gly Glu 180 185 190 Leu Glu Glu Thr Gly Ile Asn Ile His Tyr Tyr Asp Pro Ser Phe Ile 195 200 205 Ser Ser Leu Pro Asn Asn Thr Pro Ile Pro Asp Ser Arg Thr Glu Asn 210 215 220 Ile Glu Leu Asp Ser Val Arg Gly Leu Ile Tyr Thr Ser Gly Thr Thr 225 230 235 240 Gly Leu Pro Lys Gly Val Phe Ile Ser Thr Gly Arg Glu Leu Arg Thr 245 250 255 Asp Trp Ser Ile Ser Lys Tyr Leu Asn Leu Lys Pro Thr Asp Arg Met 260 265 270 Tyr Thr Cys Met Pro Leu Tyr His Ala Ala Ala His Ser Leu Cys Thr 275 280 285 Ala Ser Val Ile His Gly Gly Gly Thr Val Val Leu Ser Arg Lys Phe 290 295 300 Ser His Lys Lys Phe Trp Pro Glu Val Val Ala Ser Glu Ala Asn Ile 305 310 315 320 Ile Gln Tyr Val Gly Glu Leu Gly Arg Tyr Leu Leu Asn Gly Pro Lys 325 330 335 Ser Pro Tyr Asp Arg Ala His Lys Val Gln Met Ala Trp Gly Asn Gly 340 345 350 Met Arg Pro Asp Val Trp Glu Ala Phe Arg Glu Arg Phe Asn Ile Pro 355 360 365 Ile Ile His Glu Leu Tyr Ala Ala Thr Asp Gly Leu Gly Ser Met Thr 370 375 380 Asn Arg Asn Ala Gly Pro Phe Thr Ala Asn Cys Ile Ala Leu Arg Gly 385 390 395 400 Leu Ile Trp His Trp Lys Phe Arg Asn Gln Glu Val Leu Val Lys Met 405 410 415 Asp Leu Asp Thr Asp Glu Ile Met Arg Asp Arg Asn Gly Phe Ala Ile 420 425 430 Arg Cys Ala Val Asn Glu Pro Gly Gln Met Leu Phe Arg Leu Thr Pro 435 440 445 Glu Thr Leu Ala Gly Ala Pro Ser Tyr Tyr Asn Asn Glu Thr Ala Thr 450 455 460 Gln Ser Arg Arg Ile Thr Asp Val Phe Gln Lys Gly Asp Leu Trp Phe 465 470 475 480 Lys Ser Gly Asp Met Leu Arg Gln Asp Ala Glu Gly Arg Val Tyr Phe 485 490 495 Val Asp Arg Leu Gly Asp Thr Phe Arg Trp Lys Ser Glu Asn Val Ser 500 505 510 Thr Asn Glu Val Ala Asp Val Met Gly Thr Phe Pro Gln Ile Ala Glu 515 520 525 Thr Asn Val Tyr Gly Val Leu Val Pro Gly Asn Asp Gly Arg Val Arg 530 535 540 Ser Leu Asn Cys His Gly Arg Arg Arg Asp Arg Val Asp Ile Arg Phe 545 550 555 560 Ala Ala Leu Ala Lys His Ala Arg Asp Arg Leu Pro Gly Tyr Ala Val 565 570 575 Pro Leu Phe Leu Arg Val Thr Pro Ala Leu Glu Tyr Thr Gly Thr Leu 580 585 590 Lys Ile Gln Lys Gly Arg Leu Lys Gln Glu Gly Ile Asp Pro Asp Lys 595 600 605 Ile Ser Gly Glu Asp Lys Leu Tyr Trp Leu Pro Pro Gly Ser Asp Ile 610 615 620 Tyr Leu Pro Phe Gly Lys Met Glu Trp Gln Gly Ile Val Asp Lys Arg 625 630 635 640 Ile Arg Leu 34 522 DNA Magnaporthe grisea misc_feature (1)...(522) n = A,T,C or G 34 gcaaaggccg acgcgtggct gcggacgggt aacgtgatca gggcggacaa cgaagggcga 60 ctcttcttcc acgaccggat cggagacacg ttccgatgga agggagagac ngtcagcaca 120 caagaggtca gtttggtgct cggacgacac gactcaatca aggaggccaa cgtgtacggc 180 gtgacggtgc cgaaccacga cgggcgggcc ggctgcgctg cgctcacgct atcagacgct 240 ctggcgactg aaaagaagct gggcgatgag ctgctaaagg gattggctac tcactcgtcg 300 acttcgcttc ccaagtttgc ggtgccgcag ttcctacggg tggtgcgcgg cgagatgcag 360 tcaacgggca ccaacaagca acagaagcac gacctgaggg tgcagggtgt agagccgggc 420 aaggtgggcg tagacgaggt gtactggttg cggggaggga catatgtacc attcggaaca 480 gaggattggg atgggttgaa gaagggtctt gtgaagttgt ga 522 35 173 PRT Magnaporthe grisea 35 Ala Lys Ala Asp Ala Trp Leu Arg Thr Gly Asn Val Ile Arg Ala Asp 1 5 10 15 Asn Glu Gly Arg Leu Phe Phe His Asp Arg Ile Gly Asp Thr Phe Arg 20 25 30 Trp Lys Gly Glu Thr Val Ser Thr Gln Glu Val Ser Leu Val Leu Gly 35 40 45 Arg His Asp Ser Ile Lys Glu Ala Asn Val Tyr Gly Val Thr Val Pro 50 55 60 Asn His Asp Gly Arg Ala Gly Cys Ala Ala Leu Thr Leu Ser Asp Ala 65 70 75 80 Leu Ala Thr Glu Lys Lys Leu Gly Asp Glu Leu Leu Lys Gly Leu Ala 85 90 95 Thr His Ser Ser Thr Ser Leu Pro Lys Phe Ala Val Pro Gln Phe Leu 100 105 110 Arg Val Val Arg Gly Glu Met Gln Ser Thr Gly Thr Asn Lys Gln Gln 115 120 125 Lys His Asp Leu Arg Val Gln Gly Val Glu Pro Gly Lys Val Gly Val 130 135 140 Asp Glu Val Tyr Trp Leu Arg Gly Gly Thr Tyr Val Pro Phe Gly Thr 145 150 155 160 Glu Asp Trp Asp Gly Leu Lys Lys Gly Leu Val Lys Leu 165 170 36 1794 DNA Mycobacterium tuberculosis 36 gtgtccgatt actacggcgg cgcacacaca acggtcaggc tgatcgacct ggcaactcgg 60 atgccgcgag tgttggcgga cacgccggtg attgtgcgtg gggcaatgac cgggctgctg 120 gcccggccga attccaaggc gtcgatcggc acggtgttcc aggaccgggc cgctcgctac 180 ggtgaccgag tcttcctgaa attcggcgat cagcagctga cctaccgcga cgctaacgcc 240 accgccaacc ggtacgccgc ggtgttggcc gcccgcggcg tcggccccgg cgacgtcgtt 300 ggcatcatgt tgcgtaactc acccagcaca gtcttggcga tgctggccac ggtcaagtgc 360 ggcgctatcg ccggcatgct caactaccac cagcgcggcg aggtgttggc gcacagcctg 420 ggtctgctgg acgcgaaggt actgatcgca gagtccgact tggtcagcgc cgtcgccgaa 480 tgcggcgcct cgcgcggccg ggtagcgggc gacgtgctga ccgtcgagga cgtggagcga 540 ttcgccacaa cggcgcccgc caccaacccg gcgtcggcgt cggcggtgca agccaaagac 600 accgcgttct acatcttcac ctcgggcacc accggatttc ccaaggccag tgtcatgacg 660 catcatcggt ggctgcgggc gctggccgtc ttcggaggga tggggctgcg gctgaagggt 720 tccgacacgc tctacagctg cctgccgctg taccacaaca acgcgttaac ggtcgcggtg 780 tcgtcggtga tcaattctgg ggcgaccctg gcgctgggta agtcgttttc ggcgtcgcgg 840 ttctgggatg aggtgattgc caaccgggcg acggcgttcg tctacatcgg cgaaatctgc 900 cgttatctgc tcaaccagcc ggccaagccg accgaccgtg cccaccaggt gcgggtgatc 960 tgcggtaacg ggctgcggcc ggagatctgg gatgagttca ccacccgctt cggggtcgcg 1020 cgggtgtgcg agttctacgc cgccagcgaa ggcaactcgg cctttatcaa catcttcaac 1080 gtgcccagga ccgccggggt atcgccgatg ccgcttgcct ttgtggaata cgacctggac 1140 accggcgatc cgctgcggga tgcgagcggg cgagtgcgtc gggtacccga cggtgaaccc 1200 ggcctgttgc ttagccgggt caaccggctg cagccgttcg acggctacac cgacccggtt 1260 gccagcgaaa agaagttggt gcgcaacgct tttcgagatg gcgactgttg gttcaacacc 1320 ggtgacgtga tgagcccgca gggcatgggc catgccgcct tcgtcgatcg gctgggcgac 1380 accttccgct ggaagggcga gaatgtcgcc accactcagg tcgaagcggc actggcctcc 1440 gaccagaccg tcgaggagtg cacggtctac ggcgtccaga ttccgcgcac cggcgggcgc 1500 gccggaatgg ccgcgatcac actgcgcgct ggcgccgaat tcgacggcca ggcgctggcc 1560 cgaacggttt acggtcactt gcccggctat gcacttccgc tctttgttcg ggtagtgggg 1620 tcgctggcgc acaccacgac gttcaagagt cgcaaggtgg agttgcgcaa ccaggcctat 1680 ggcgccgaca tcgaggatcc gctgtacgta ctggccggcc cggacgaagg atatgtgccg 1740 tactacgccg aataccctga ggaggtttcg ctcggaaggc gaccgcaggg ctag 1794 37 597 PRT Mycobacterium tuberculosis 37 Met Ser Asp Tyr Tyr Gly Gly Ala His Thr Thr Val Arg Leu Ile Asp 1 5 10 15 Leu Ala Thr Arg Met Pro Arg Val Leu Ala Asp Thr Pro Val Ile Val 20 25 30 Arg Gly Ala Met Thr Gly Leu Leu Ala Arg Pro Asn Ser Lys Ala Ser 35 40 45 Ile Gly Thr Val Phe Gln Asp Arg Ala Ala Arg Tyr Gly Asp Arg Val 50 55 60 Phe Leu Lys Phe Gly Asp Gln Gln Leu Thr Tyr Arg Asp Ala Asn Ala 65 70 75 80 Thr Ala Asn Arg Tyr Ala Ala Val Leu Ala Ala Arg Gly Val Gly Pro 85 90 95 Gly Asp Val Val Gly Ile Met Leu Arg Asn Ser Pro Ser Thr Val Leu 100 105 110 Ala Met Leu Ala Thr Val Lys Cys Gly Ala Ile Ala Gly Met Leu Asn 115 120 125 Tyr His Gln Arg Gly Glu Val Leu Ala His Ser Leu Gly Leu Leu Asp 130 135 140 Ala Lys Val Leu Ile Ala Glu Ser Asp Leu Val Ser Ala Val Ala Glu 145 150 155 160 Cys Gly Ala Ser Arg Gly Arg Val Ala Gly Asp Val Leu Thr Val Glu 165 170 175 Asp Val Glu Arg Phe Ala Thr Thr Ala Pro Ala Thr Asn Pro Ala Ser 180 185 190 Ala Ser Ala Val Gln Ala Lys Asp Thr Ala Phe Tyr Ile Phe Thr Ser 195 200 205 Gly Thr Thr Gly Phe Pro Lys Ala Ser Val Met Thr His His Arg Trp 210 215 220 Leu Arg Ala Leu Ala Val Phe Gly Gly Met Gly Leu Arg Leu Lys Gly 225 230 235 240 Ser Asp Thr Leu Tyr Ser Cys Leu Pro Leu Tyr His Asn Asn Ala Leu 245 250 255 Thr Val Ala Val Ser Ser Val Ile Asn Ser Gly Ala Thr Leu Ala Leu 260 265 270 Gly Lys Ser Phe Ser Ala Ser Arg Phe Trp Asp Glu Val Ile Ala Asn 275 280 285 Arg Ala Thr Ala Phe Val Tyr Ile Gly Glu Ile Cys Arg Tyr Leu Leu 290 295 300 Asn Gln Pro Ala Lys Pro Thr Asp Arg Ala His Gln Val Arg Val Ile 305 310 315 320 Cys Gly Asn Gly Leu Arg Pro Glu Ile Trp Asp Glu Phe Thr Thr Arg 325 330 335 Phe Gly Val Ala Arg Val Cys Glu Phe Tyr Ala Ala Ser Glu Gly Asn 340 345 350 Ser Ala Phe Ile Asn Ile Phe Asn Val Pro Arg Thr Ala Gly Val Ser 355 360 365 Pro Met Pro Leu Ala Phe Val Glu Tyr Asp Leu Asp Thr Gly Asp Pro 370 375 380 Leu Arg Asp Ala Ser Gly Arg Val Arg Arg Val Pro Asp Gly Glu Pro 385 390 395 400 Gly Leu Leu Leu Ser Arg Val Asn Arg Leu Gln Pro Phe Asp Gly Tyr 405 410 415 Thr Asp Pro Val Ala Ser Glu Lys Lys Leu Val Arg Asn Ala Phe Arg 420 425 430 Asp Gly Asp Cys Trp Phe Asn Thr Gly Asp Val Met Ser Pro Gln Gly 435 440 445 Met Gly His Ala Ala Phe Val Asp Arg Leu Gly Asp Thr Phe Arg Trp 450 455 460 Lys Gly Glu Asn Val Ala Thr Thr Gln Val Glu Ala Ala Leu Ala Ser 465 470 475 480 Asp Gln Thr Val Glu Glu Cys Thr Val Tyr Gly Val Gln Ile Pro Arg 485 490 495 Thr Gly Gly Arg Ala Gly Met Ala Ala Ile Thr Leu Arg Ala Gly Ala 500 505 510 Glu Phe Asp Gly Gln Ala Leu Ala Arg Thr Val Tyr Gly His Leu Pro 515 520 525 Gly Tyr Ala Leu Pro Leu Phe Val Arg Val Val Gly Ser Leu Ala His 530 535 540 Thr Thr Thr Phe Lys Ser Arg Lys Val Glu Leu Arg Asn Gln Ala Tyr 545 550 555 560 Gly Ala Asp Ile Glu Asp Pro Leu Tyr Val Leu Ala Gly Pro Asp Glu 565 570 575 Gly Tyr Val Pro Tyr Tyr Ala Glu Tyr Pro Glu Glu Val Ser Leu Gly 580 585 590 Arg Arg Pro Gln Gly 595

Claims (54)

What is claimed is:
1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO.: 7.
2. The isolated polypeptide encoded by the nucleic acid comprising the nucleotide sequence of SEQ ID NO.6.
3. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO.:7.
4. An isolated polypeptide having fatty acid transport activity and comprising an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:7.
5. A method of producing a fatty acid transport protein 3, FATP3, polypeptide comprising the steps of
transfecting a suitable host cell with the nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 6; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP3 polypeptide.
6. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO.: 9.
7. The isolated polypeptide encoded by the nucleic acid comprising the nucleotide sequence of SEQ ID NO.8.
8. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO.:9.
9. An isolated polypeptide having fatty acid transport activity and comprising an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:9.
10. A method of producing a fatty acid transport protein 4, FATP4, polypeptide comprising the steps of
transfecting a suitable host cell with the nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 8; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP4 polypeptide.
11. An isolated polypeptide comprising the amino acid sequence of SEQ ID) NO.:11.
12. The isolated polypeptide encoded by the nucleic acid comprising the nucleotide sequence of SEQ ID) NO.100.
13. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO.:11.
14. An isolated polypeptide having fatty acid transport activity and comprising an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:11.
15. A method of producing a fatty acid transport protein 5, FATP5, polypeptide comprising the steps of
transfecting a suitable host cell with the nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 10; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP5 polypeptide.
16. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO.:13.
17. The isolated polypeptide encoded by the nucleic acid comprising the nucleotide sequence of SEQ ID NO. 12.
18. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO.:13.
19. An isolated polypeptide having fatty acid transport activity and comprising an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:13.
20. A method of producing a fatty acid transport protein 2, FATP2, polypeptide comprising the steps of
transfecting a suitable host cell with the nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 12; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP2 polypeptide.
21. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO.:15.
22. The isolated polypeptide encoded by the nucleic acid comprising the nucleotide sequence of SEQ ID NO. 14.
23. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO.:15.
24. An isolated polypeptide having fatty acid transport activity and comprising an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:15.
25. A method of producing a FATP3 polypeptide comprising the steps of
transfecting a suitable host cell with the nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 14; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP3 polypeptide.
26. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO.:17.
27. The isolated polypeptide encoded by the nucleic acid comprising the nucleotide sequence of SEQ ID NO. 16.
28. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO.:17.
29. An isolated polypeptide having fatty acid transport activity and comprising an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:17.
30. A method of producing a FATP4 polypeptide comprising the steps of
transfecting a suitable host cell with the nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 16; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP4 polypeptide.
31. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO.:19.
32. The isolated polypeptide encoded by the nucleic acid comprising the nucleotide sequence of SEQ ID NO. 18.
33. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO.:19.
34. An isolated polypeptide having fatty acid transport activity and comprising an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:19.
35. A method of producing a FATP5 polypeptide comprising the steps of
transfecting a suitable host cell with the nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 18; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP5 polypeptide.
36. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO.:21.
37. The isolated polypeptide encoded by the nucleic acid comprising the nucleotide sequence of SEQ ID NO.20.
38. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO.:21.
39. An isolated polypeptide having fatty acid transport activity and comprising an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:21.
40. A method of producing a fatty acid transport protein, FATP6, polypeptide comprising the steps of transfecting a suitable host cell with the nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 20; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP6 polypeptide.
41. A method of producing a fatty acid transport protein, FATP, polypeptide comprising the steps of
transfecting a suitable host cell with a nucleic acid encoding a FATP polypeptide; and
culturing the host cell under conditions such that the nucleic acid is expressed, thereby producing a FATP polypeptide.
42. The method of claim 41, wherein the FATP is fatty acid transport protein 1, FATP1.
43. The method of claim 41, wherein the FATP is FATP2.
44. The method of claim 41, wherein the FATP is FATP3.
45. The method of claim 41, wherein the FATP is FATP4.
46. The method of claim 41, wherein the FATP is FATP5.
47. The method of claim 41, wherein the FATP is FATP6.
48. A method of producing a FATP polypeptide comprising the steps of
transfecting a suitable host cell with a nucleic acid encoding a FATP polypeptide; and
culturing the host cell under conditions such that the nucleic acid is overexpressed, thereby producing a FATP polypeptide.
49. The method of claim 48, wherein the FATP is fatty acid transport protein 1, FATP1.
50. The method of claim 48, wherein the FATP is FATP2.
51. The method of claim 48, wherein the FATP is FATP3.
52. The method of claim 48, wherein the FATP is FATP4.
53. The method of claim 48, wherein the FATP is FATP5.
54. The method of claim 48, wherein the FATP is FATP6.
US09/943,671 1998-01-15 2001-08-31 Fatty acid transport proteins Abandoned US20020106733A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US09/943,671 US20020106733A1 (en) 1998-01-15 2001-08-31 Fatty acid transport proteins

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US7137498P 1998-01-15 1998-01-15
US9349198P 1998-07-20 1998-07-20
US11094198P 1998-12-04 1998-12-04
US09/232,191 US6284487B1 (en) 1998-01-15 1999-01-14 Polynucleotides encoding fatty acid transport proteins
US09/943,671 US20020106733A1 (en) 1998-01-15 2001-08-31 Fatty acid transport proteins

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/232,191 Division US6284487B1 (en) 1998-01-15 1999-01-14 Polynucleotides encoding fatty acid transport proteins

Publications (1)

Publication Number Publication Date
US20020106733A1 true US20020106733A1 (en) 2002-08-08

Family

ID=27490889

Family Applications (2)

Application Number Title Priority Date Filing Date
US09/232,191 Expired - Fee Related US6284487B1 (en) 1998-01-15 1999-01-14 Polynucleotides encoding fatty acid transport proteins
US09/943,671 Abandoned US20020106733A1 (en) 1998-01-15 2001-08-31 Fatty acid transport proteins

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US09/232,191 Expired - Fee Related US6284487B1 (en) 1998-01-15 1999-01-14 Polynucleotides encoding fatty acid transport proteins

Country Status (1)

Country Link
US (2) US6284487B1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7070944B2 (en) 2001-03-14 2006-07-04 Clf Medical Technology Acceleration Program, Inc. System for screening fatty acid transport inhibitors, methods of use and modulators identified thereby

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070012896A1 (en) * 1997-09-30 2007-01-18 Richard Sapienza Environmentally benign anti-icing or deicing fluids
US20030232363A1 (en) * 1998-01-15 2003-12-18 Andreas Stahl Fatty acid transport proteins
US7033772B1 (en) 1998-01-15 2006-04-25 Millennium Pharmaceuticals, Inc. Methods of identifying inhibitors of fatty acid transport proteins (FATP)
US7888477B2 (en) * 1998-12-17 2011-02-15 Corixa Corporation Ovarian cancer-associated antibodies and kits
WO2000077201A1 (en) * 1999-06-15 2000-12-21 Astrazeneca Ab Livin; inhibitor-of-apoptosis protein-3 (iap-3)
US6656703B1 (en) * 1999-12-29 2003-12-02 Millennium Pharamaceuticals, Inc. High throughput screen for inhibitors of fatty acid biosynthesis in bacteria
US20110177082A1 (en) * 2000-07-17 2011-07-21 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US20030165430A1 (en) * 2001-12-13 2003-09-04 Whitehead Institute For Biomedical Research Transgenic fatty acid transport one (FATP1) non-human knockout mammals uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4935450A (en) 1982-09-17 1990-06-19 Therapeutical Systems Corporation Cancer therapy system for effecting oncolysis of malignant neoplasms

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7070944B2 (en) 2001-03-14 2006-07-04 Clf Medical Technology Acceleration Program, Inc. System for screening fatty acid transport inhibitors, methods of use and modulators identified thereby

Also Published As

Publication number Publication date
US6284487B1 (en) 2001-09-04

Similar Documents

Publication Publication Date Title
Hoops et al. Isolation of the cDNA encoding glycoprotein-2 (GP-2), the major zymogen granule membrane protein: homology to uromodulin/Tamm-Horsfall protein
Otera et al. Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants
Ghaedi et al. The peroxin Pex3p initiates membrane assembly in peroxisome biogenesis
Flood et al. Identification of the proteoglycan binding site in apolipoprotein B48
JP4726302B2 (en) Methods and reagents for regulating ABC1 polypeptide and cholesterol levels
Hui et al. Fatty acid transporters in animal cells
AU2003261280B2 (en) Identification of a novel bitter taste receptor, T2R76
US6284487B1 (en) Polynucleotides encoding fatty acid transport proteins
EP1045904B1 (en) Fatty acid transport proteins
JP2000511062A (en) G-beta-gamma regulated phosphatidylinositol-3 'kinase
US6348321B1 (en) Methods of identifying agents inhibiting fatty acid transport proteins
US6300096B1 (en) Polynucleotides encoding fatty acid transport proteins
Kumar et al. Cloning and Expression of a Major Rat Lens Membrane Protein, M P20
US6288213B1 (en) Fatty acid transport proteins
AU742098B2 (en) Vascular adhesion protein-1 having amine oxidase activity
WO2000006087A2 (en) Uncoupling protein-2(hcp2): compositions and methods of use
US6657049B1 (en) Fatty acid transport proteins
WO2001021795A9 (en) Fatty acid transport proteins
US20030232363A1 (en) Fatty acid transport proteins
US7033772B1 (en) Methods of identifying inhibitors of fatty acid transport proteins (FATP)
Hill et al. Lecithin: cholesterol acyltransferase deficiency: identification of a causative gene mutation and a co-inherited protein polymorphism
AU1272197A (en) Nucleic acid molecule encoding a 11-cis retinol dehydrogenase
US7056719B2 (en) Polypeptide-phosphatidic acid phosphatase 29.81 and the polynucleotide encoding said polypeptide
CA2289945A1 (en) Human uncoupling protein 3
US20040126858A1 (en) Novel polypeptide-nadp dependent leukotriene b412-hydroxydehydrogenase-36 and the polynucleotide encoding said polypeptide

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION