WO2001032873A1 - Insulin-responsive sequence dna binding protein-1' (irsdbp-1), gene encoding it and uses thereof - Google Patents

Insulin-responsive sequence dna binding protein-1' (irsdbp-1), gene encoding it and uses thereof Download PDF

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Publication number
WO2001032873A1
WO2001032873A1 PCT/US2000/030096 US0030096W WO0132873A1 WO 2001032873 A1 WO2001032873 A1 WO 2001032873A1 US 0030096 W US0030096 W US 0030096W WO 0132873 A1 WO0132873 A1 WO 0132873A1
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Prior art keywords
irsbp
nucleic acid
sequence
seq
protein
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PCT/US2000/030096
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French (fr)
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WO2001032873A8 (en
Inventor
Betty C. Villafuerte
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Emory University
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Priority to AT00976787T priority Critical patent/ATE312918T1/en
Priority to CA002389615A priority patent/CA2389615A1/en
Priority to EP00976787A priority patent/EP1228209B1/en
Priority to DE60024862T priority patent/DE60024862T2/en
Priority to AU14518/01A priority patent/AU1451801A/en
Publication of WO2001032873A1 publication Critical patent/WO2001032873A1/en
Publication of WO2001032873A8 publication Critical patent/WO2001032873A8/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to the identification of an Insulin-
  • IRSBP Responsive Sequence DNA Binding Protein
  • Insulin resistance is a key factor in the pathogenesis of Type II diabetes
  • insulin action involves all major target tissues, i.e., skeletal muscle, liver and fat
  • the action of insulin is initiated by binding to cell surface receptors.
  • IRS-1 insulin receptor substrate-1
  • IRS-2 insulin receptor substrate-1
  • IRS-1 interacts with several proteins that
  • SH2 Src homology 2 domains, including the p85 subunits of PI3' -kinase
  • This target backward approach has
  • IGFBP-3 insulin protein-3
  • NIDDM Type II diabetes mellitus
  • twins approaches 100% (Barnett et al., (1981); Newman et al, (1987)), while the risk
  • IRBPs responsive binding proteins
  • Insulin-like growth factors I and II are proteins that have
  • IGF production in cancer cells such as from breast and kidney
  • IGFs have also been implicated in diabetic retinopathy by stimulating endothelial and
  • IGFs The actions of IGFs are modulated by a family of six IGF-binding proteins
  • IGFBPs insulin receptor binders
  • IGFBP-1 One binding protein, has a molecular weight of
  • IGF-I and IGF-II are associated with IGFBP-3 and an acid-labile subunit
  • IGFBP-3 is critical for understanding the regulation of systemic IGFBP-3.
  • IGFBP-3 can: (a) mediate the growth inhibitory actions of
  • TGF- ⁇ transforming growth factor- ⁇
  • retinoic acid retinoic acid
  • anti-estrogens fibroblast growth factor- ⁇
  • IGFBPs IGFBPs, IGFBP-3 levels increase in the presence of anabolic hormones such as
  • IGFBP-3 may be regulated by the mechanisms by which IGFBP-3 is regulated.
  • IGFBP-3 may be regulated by the mechanisms by which IGFBP-3 is regulated.
  • dissociation of cell-associated IGFBP-3 is one mechanism by which IGF-1 promotes release of IGFBP-3 into conditioned medium
  • Insulin increases IGFBP-3 expression by stimulating the rate of gene
  • IRE regulatory insulin-responsive element
  • the IGFBP-3 IRE comprises the nucleotide dyad ACC(A/G)A which has a
  • the present invention relates to a novel protein called
  • IRSBP-1 Insulin-Responsive Sequence DNA Binding Protein- 1
  • IRSBP-1 is capable of binding to nucleic acid regions
  • IRSBP-1 regulates genes important in mediating the insulin response in
  • One aspect of the present invention relates to isolated or non-naturally
  • nucleic acid molecules that encode at least a portion of a human or animal
  • IRSBP-1 protein or a variant thereof.
  • the present invention provides isolated molecules that can hybridize to nucleic
  • the present invention further provides nucleic acids that are fragments or
  • derivatives of the cDNA molecules comprising at least in part a region of the IRSBP-1
  • IRSBP-1 encoding IRSBP-1, including expression vectors for the expression of IRSBP-1,
  • invention is the development of therapeutic and diagnostic agents that mimic, facilitate or inhibit the action of IRSBP-1, and/or are based on relationships to the
  • IRSBP-1 structure and action of IRSBP-1.
  • the invention further provides non-naturally occurring recombinant nucleic acid
  • IRSBP-1 amino acids that can be in a cell or an organism.
  • recombinant nucleic acid may comprise IRSBP-1 -related sequences, functional
  • the recombinant nucleic acid molecule can alternatively contain
  • transcription regulatory sequences functional in a particular cell, a sequence
  • transcriptional control sequences functional in that cell.
  • the present invention still further provides oligopeptides having amino acid
  • IRSBP-1 amino acid sequences derived from the amino acid sequence of a human or animal IRSBP-1
  • the antibodies may be used for, but are not limited to, the detection
  • the invention also provides a recombinant cell or tissue containing non-recombinant cell or tissue containing non-recombinant cell or tissue containing non-recombinant cell or tissue containing non-recombinant cell or tissue containing non-recombinant cell or tissue containing non-recombinant cell or tissue containing non-recombinant cell or tissue containing non-recombinant cell or tissue containing non-recombinant cell or tissue containing non-
  • IRSBP-1 naturally occurring recombinant nucleic acid molecules coding for an IRSBP-1
  • the IRSBP-1 coding sequence may be
  • present invention further provides for the production of animals that have modified
  • nucleic acids encoding at least a portion of the IRSBP-1 protein or have the IRSBP-1
  • the present invention further provides for methods of gene therapy
  • compositions including antisense and sense nucleic acids that are present in various organs and pharmaceutical compositions including antisense and sense nucleic acids that are present in various organs and pharmaceutical compositions including antisense and sense nucleic acids that are present in various organs and pharmaceutical compositions including antisense and sense nucleic acids that are present in various organs and pharmaceutical compositions including antisense and sense nucleic acids that are present in various organs and pharmaceutical compositions including antisense and sense nucleic acids that
  • the invention features methods for identifying mammalian cells containing an
  • IRSBP-1 polypeptide or a related sequence.
  • Such methods comprise identifying the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids, or amino acids, amino acids, amino acids, amino acids, amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids in the amino acids
  • IRSBP-1 polypeptide in mammalian cells using techniques that are routine and
  • the present invention also relates to methods of detecting proliferating cells
  • the present invention discloses and the cells of humans or animals having diabetic disorders.
  • the present invention further contemplates that modulation of the
  • Fig. 1 shows the nucleotide sequence of the rat clone 52 cDNA (SEQ ID NO:
  • Fig. 2 shows the protein sequence of the rat clone 52 (SEQ ID NO: 3)
  • Fig. 3 shows the nucleotide sequence of a nucleic acid probe (SEQ ID NO: 4)
  • Figs. 4A-4B show the nucleotide sequence of the rat cDNA (SEQ ID NO: 5)
  • Fig. 4A is the rat IRSBP-1 coding region (SEQ ID NO: 1
  • Fig. 4B is the untranslated region that is 3' of the
  • Fig. 5 A is a partial region of a rat IRSBP-1 coding region with the corresponding 3-letter amino acid designation listed below its respective nucleotide
  • Fig. 5B is the untranslated region that is 3' of the coding region in Fig. 5A.
  • Figs. 6A-6D show the nucleotide sequence of the human cDNA (SEQ ID NO:
  • Fig. 6B is a human IRSBP-1 coding region (SEQ ID NO: 9),
  • Fig. 6C is the untranslated region that is 3' of the coding region
  • Fig. 6B shows the exons (SEQ ID NOS: 16-41) that comprise
  • Fig. 7A is a partial region of a human variant IRSBP-1 coding region
  • Fig. 7B is the untranslated region that is 3' of the coding region
  • Fig. 8 shows the amino acid sequence (SEQ ID NO: 11) of the rat IRSBP-1
  • Fig. 9 shows the amino acid sequence (SEQ ID NO: 12) of the human IRSBP-
  • Fig. 10 shows the amino acid sequence (SEQ ID NO: 13) of the variant human
  • IRSBP-1 protein translated from the coding region of the human IRSBP-1 cDNA, the
  • Figs. 11A-11B show the nucleotide sequence of the truncated rat cDNA (SEQ ID NO: 1
  • Fig. 11A is a partial region of the rat IRSBP-1 coding region
  • Fig. 1 IB is the untranslated region that is 3' of the coding
  • Fig. 12 illustrates gel mobility shift of the -1150/-1117 bp IRE fragment of
  • IGFBP-3 (SEQ ID NO: 1) by polypeptides derived from cDNA clones isolated using
  • Fig. 13 illustrates gel mobility shift analysis of the -1150/-1117 IRE fragment
  • IGFBP-3 SEQ ID NO: 1
  • Fig. 14 illustrates a competition-binding assay of the polypeptide encoded by
  • Fig. 15 illustrates IREs derived from other genes competing with the IRE of
  • IGFBP-3 (SEQ ID NO: 1) for binding to the protein product of clone 52.
  • Fig. 16 illustrates IGFBP-1 IRE reporter activity in CHO cells transfected with
  • Fig. 18 A illustrates the effect of insulin on the expression of the IRSBP-1
  • Fig 18B illustrates the effect of an IRSBP-1 expressing clone (SEQ ID NO:
  • Fig. 19 illustrates the effect of IRSBP-1 (SEQ ID NO: 14) on the glycogen
  • Fig. 20 illustrates the tissue distribution of clone 52 mRNA.
  • Fig. 21 illustrates ribonuclease protection assays of the expression of IRSBP-1
  • Fig. 22 illustrates the detection of IRSBP-1 mRNA within the hypothalamic
  • Fig. 23 illustrates the detection of IRSBP-1 mRNA in the lateral hypothalamus
  • Fig. 24 shows a comparison of IRSBP-1 mRNA in the lateral hypothalamic
  • Fig. 25 illustrates IRSBP-1 expression in the solitary tract nuclei of lean
  • Fig. 26 illustrates an in-situ hybridization analysis of a rat brain section
  • Fig. 27 illustrates an in-situ hybridization analysis showing the expression
  • IRSBP-1 in the pyramidal area of the brain of a lean Zucker rat.
  • Fig. 28 illustrates IRSBP-1 expression in the olfactory bulb.
  • Fig. 29 illustrates IRSBP-1 expression in the amygdala.
  • Fig. 30 illustrates immunohistochemical staining with an anti-IRSBP-1
  • Fig. 32 illustrates immunohistochemical staining of the endothelial lining of
  • Fig. 33 illustrates immunohistochemical staining of the rat brain, using anti-
  • Fig. 34 illustrates a Western blot analysis using anti-rat IRSBP-1 peptide
  • Fig. 35 illustrates a Western blot analysis showing IRSBP-1 in COS 7 cell
  • Fig. 36 illustrates the position of clone 52 in the compartment of the cell
  • Fig. 37 illustrates differential phosphorylation of IRSBP-1 in normal and streptozotocin-induced diabetic rats, and in Zucker lean and obese rats.
  • Fig. 38 A illustrates the effect of administered IRSBP-1 antisense (AS) or
  • Fig. 39 illustrates phase contrast micrographs of L6 cells stably transfected
  • Fig. 40 illustrates analysis by flow cytometry of the cell cycle compartments
  • animal is used herein to include all vertebrate animals, including
  • transgenic animal is any animal containing one or
  • transgenic animal is not intended to encompass classical cross-breeding or in vitro
  • acid molecule may be specifically targeted to a defined genetic locus, may be
  • chromosome randomly integrated within a chromosome, or it may be extrachromosomally
  • the term "germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was
  • transgenic animals they are transgenic animals as well.
  • organism of the taxonomic class mammalia such as, but not limited to, such
  • mice as mice, rats, rabbits, sheep, cattle, and primates, including humans.
  • IRSBP-1 refers to an Insulin-Responsive Sequence
  • Binding Protein- 1 capable of binding to at least one insulin responsive element
  • insulin-responsive gene insulin-responsive gene.
  • IMSBP-1 insulin-responsive gene.
  • nucleic acid refers to any natural and synthetic nucleic acid
  • nucleotides and nucleosides for example cDNA
  • genomic DNA mRNA, RNA, oligonucleotides, oligonucleosides and derivatives
  • nucleic acids may be collectively referred to
  • nucleic acids of the present invention include bacterial plasmid vectors including
  • animal viral vectors such as, but not limited to, modified adenovirus,
  • nucleic acid further includes modified or derivatised
  • nucleotides and nucleosides such as, but not limited to, halogenated nucleotides
  • isolated nucleic acid refers to a nucleic acid with a
  • RNA spanning more than three separate genes, and includes DNA, RNA, or derivatives or
  • variants thereof covers, for example, (a) a DNA which has the sequence of
  • nucleic acid incorporated into a vector or into the
  • genomic nucleic acid of a prokaryote or eukaryote in a manner such that the resulting
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • hybrid gene i.e., a gene encoding a fusion protein
  • nucleic acid molecules of the present invention can include, for example, natural
  • allelic variants as well as nucleic acid molecules modified by nucleotide deletions, insertions, inversions, or substitutions such that the resulting nucleic acid molecule
  • IRSBP-1 protein still essentially encodes an IRSBP-1 protein or a variant thereof of the present
  • a yeast or bacterial genome may, for example, be derived from a yeast or bacterial genome, or a cloning vector
  • plasmid such as a plasmid or a viral vector.
  • viral vector such as a plasmid or a viral vector.
  • polypeptide includes proteins, protein fragments, protein
  • polypeptides as defined above that are encoded by nucleic acids, produced through
  • polypeptides are synthesized.
  • polypeptides further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids
  • nucleic acid refers to an isolated portion of the subject nucleic acid constructed artificially (e.g., by
  • fragment may also be referred to as well known to one of skill in the art.
  • fragment may also be referred to as well known to one of skill in the art.
  • protease or is a portion of the naturally occurring polypeptide synthesized by
  • modulates refers to the ability of a compound to
  • a modulator preferably increases the
  • a modulator can alternatively decrease
  • IRSBP-1 polypeptide or fragments thereof the binding or activating potential of IRSBP-1 polypeptide or fragments thereof.
  • RNA whole RNA
  • protein whole protein
  • gene product refers to RNAs or proteins that are encoded by the
  • endogenous gene products are RNA or proteins encoded by endogenous genes.
  • heterologous or exogenous genes which, therefore, are not naturally expressed in
  • locus refers to the site of a gene on a
  • chromosome Pairs of genes control hereditary traits, each in the same position on a
  • allelic variants with similar, but not identical, nucleotide sequences. Such allelic
  • variants typically encode proteins with similar activity to that of the protein encoded by the gene to which they are compared, because natural selection typically selects
  • Allelic variants can also comprise alterations in
  • genomic sequence refers to the total DNA in the genome of
  • an organism and includes non-coding regions like nitrons.
  • transcription regulatory sequences refers to transcription regulatory sequences
  • nucleotide sequences that are associated with a gene nucleic acid sequence and which
  • sequences may be isolated and incorporated into a vector nucleic acid to enable
  • transcription regulatory sequence may precede, but are not limited to, the region of
  • nucleic acid sequence that is in the region 5' of the end of a protein coding sequence
  • Transcriptional regulatory sequences may also be transcribed into mRNA.
  • Transcriptional regulatory sequences may also be transcribed into mRNA.
  • “intron” regions may be in regions of nucleic acid sequence that are in the region
  • coding region refers to a continuous linear
  • coding region is translated into a full length protein; that is, a complete protein as
  • a full length coding region may also include any leader protein sequence or any other
  • adenine base within one strand of a nucleic acid can form two hydrogen bonds with
  • nucleic acid can form three hydrogen bonds with cytosine within a second nucleic
  • Complementary nucleic acids as referred to herein, may further comprise modified
  • modified thymine, and a modified cytosine may form hydrogen bonds with a guanine
  • probe when referring to a nucleic acid, refers to a
  • nucleotide sequence that can be used to hybridize with and thereby identify the
  • the probe may be modified with labels
  • Stringent hybridization conditions typically permit the
  • nucleic acid molecule being used as a probe in the hybridization
  • Hybridization of the first and second nucleic acids may be any combination of the first and second nucleic acids.
  • hybridization of the first and second nucleic acid may be conducted under reduced
  • reagents such as, but not limited to, dimethyl sulfoxide (DMSO)
  • a suitable hybridization protocol may, for example, involve hybridization
  • IX SSC comprises 0.015 M sodium citrate and 0.15 M sodium
  • nucleic acid region refers to a sequence
  • nucleic acid that is not present in any other nucleic acid sequence.
  • nucleic acid region is a nucleotide sequence
  • nucleic acid sequences present in two or more nucleic acid sequences, to which a particular nucleic acid
  • sequence can hybridize under low, medium or high stringency conditions. The greater
  • Gapped BLAST is utilized as described in Attschul et al. (1997) Nuc. Acids Res. 25:
  • sense strand refers to a single stranded DNA
  • antisense strand is used herein
  • antisense DNA refers to a gene sequence DNA that
  • transcriptional or translational level For example, transcription of the subject nucleic acid
  • acids may produce antisense transcripts that are capable of inhibiting transcription by
  • the antisense transcripts may inhibit transport of the "target RNA", or, the
  • antisense transcripts may inhibit translation of "target RNA”.
  • antisense therapy refers to the administration or in
  • genomic DNA encoding a IRSBP-1 protein so as to inhibit expression of that protein
  • binding may be by
  • DNA duplexes through specific interactions in the major groove of the double helix.
  • antisense therapy refers to the range of techniques generally employed in
  • nucleic acid vector refers to a natural or synthetic
  • a circular double stranded plasmid can be linearized by treatment with
  • a nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together.
  • the nucleic acid molecule can be
  • expression vector refers to a nucleic acid vector
  • regulatory sequences includes promoters, enhancers, and other
  • nucleic acid sequences that are not naturally found in a eukaryotic or
  • nucleic acid sequences may include, but are not limited to
  • nucleic acid vectors genes expression regulatory elements, origins of replication,
  • recombinant polypeptide will be present in a cell in an amount different from that
  • regenerator cell refers to a cell that has a new combination of
  • combination of nucleic acid segments can be introduced into an organism using a
  • a recombinant cell can be a single eukaryotic cell, or a single prokaryotic cell, or a
  • the recombinant cell can harbor a vector that is extragenomic.
  • extragenomic nucleic acid vector does not insert into the cell's genome.
  • recombinant cell can further harbor a vector or a portion thereof that is intragenomic.
  • intragenomic defines a nucleic acid construct incorporated within the
  • antibody refers to polyclonal and monoclonal
  • IRSBP-1 -related polypeptides.
  • Such antibodies may include, but are not limited to polyclonal antibodies,
  • Such antibodies may be used, for example, in the detection of a
  • various host animals may be immunized by injection with a differentially expressed or pathway gene protein, or a portion thereof.
  • Such host animals may include but are not
  • polyanions polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and
  • BCG Bacille Calmette-Guerin
  • Polyclonal antibodies are heterogeneous populations of antibody molecules
  • Monoclonal antibodies which are homogeneous populations of antibodies to a
  • Hybridomas can be screened
  • antibodies may be analogous to those described in Cassone et al. (1988) J. Med.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM,
  • inventions may be cultivated in vitro or in vivo. Production of high titers of mAbs in
  • F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule
  • Forms of administration may include
  • intradermal, intramuscular or intravenous administration e.g., injectable
  • compositions can contain auxiliary substances such as wetting or emulsifying agents,
  • pH buffering agents for example, sodium bicarbonate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium tartrate, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite, sodium metabisulfite
  • preservatives flavoring agents, colors, and the like, depending upon the route of
  • transgenic animal is any animal, preferably a non-human
  • nucleic acid is introduced into the cell, directly or indirectly by
  • This molecule rather is directed to the introduction of a recombinant DNA molecule. This molecule
  • chromosome may be integrated within a chromosome, or it may be extrachromosomally replicating
  • the transgene causes cells to express a
  • IRSBP-1 protein e.g. either agonistic or antagonistic
  • transgenic animals in which the recombinant IRSBP-1 gene is silent are also provided.
  • non-human animals include vertebrates such as vertebrates
  • rodents non-human primates, sheep, dog, cow, birds, amphibians, reptiles, etc.
  • Preferred non-human animals are selected from the rodent family including rat and
  • mouse most preferably mouse.
  • chimeric animal is used herein to refer to
  • transgene means a nucleic acid sequence (encoding,
  • IRSBP-1 polypeptide e.g., a IRSBP-1 polypeptide that is partly or entirely heterologous, i.e., foreign, to the
  • transgenic animal or cell into which it is introduced or, is homologous to an
  • a transgene can include one or more transcriptional regulatory sequences
  • an "IRSBP-1 nucleic acid molecule” includes nucleic acid
  • IRSBP-1 protein or production of the IRSBP-1 protein encoded by the gene (such as,
  • an IRSBP-1 variant is an IRSBP-1 polypeptide that
  • IRSBP-1 variant retains its useful function, i.e., for
  • epitope refers to a part of the protein that can
  • IGFBP IGF-binding protein
  • IRE insulin response element
  • PEPCK phosphoenol pryuvate carboxykinase
  • GAPDH glyceraldehyde-3-phosphate
  • nt nucleotide(s)
  • SSC sodium chloride-sodium citrate
  • the present invention provides isolated nucleic acids, derivatives and variants
  • the present invention further provides an isolated nucleic acid encoding a fragment of
  • IRE Insulin Responsive Element
  • IGFBP-3 Insulin-like Growth Factor Binding Protein-3
  • nucleotide sequence 5'-AATTCAAGGGTATCCAGGAAAGTCTCC-3 * (SEQ ID NO:
  • IREs are regulatory nucleic acid sequences of insulin-regulated
  • nucleotide sequence of SEQ ID NO: 1 is localized between the -1150 and the -1124 bp positions
  • Example 1 The cDNA library screening provided a novel 952-bp cDNA
  • SEQ ID NO: 2 encodes a polypeptide having the amino acid sequence of SEQ ID NO:
  • IGFBP-3 (SEQ ID NO: 1), as well as to other insulin-responsive genes.
  • Trx thioredoxin
  • IRE nucleic acids but not by nucleic acids of sequences unrelated to the IRE.
  • IRSBP-1 -related polypeptide SEQ ID NO: 3
  • the IRSBP-1 polypeptide fragment (SEQ ID NO: 3) encoded by clone 52 also has a unique peptide sequence (SEQ ID NO: 3) and a unique peptide sequence (SEQ ID NO: 3) encoded by clone 52 also has a unique peptide sequence (SEQ ID NO: 3) and a unique peptide sequence (SEQ ID NO: 3) encoded by clone 52 also has
  • the polypeptide interacts with the IREs from insulin-responsive genes encoding IGF-
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • IGFBP-1 is a hepatic acute phase reactant protein that coordinates the level of
  • GAPDH catalyzes the
  • IRSBP-1 polypeptide S ⁇ Q ID NO: 3
  • RNA probe obtained by transcribing a Kpnl-Xhol fragment of clone 52 (SEQ ID NO:
  • liver, kidney, brain, small intestine, muscle, and fat pads is expressed in at least liver, kidney, brain, small intestine, muscle, and fat pads.
  • nucleic acids capable of hybridizing with clone 52 as discussed in Example 5.
  • the present invention further provides two rat cDNA clones hybridizing to the
  • a first rat IRSBP-1 cDNA clone (SEQ ID NO 5; shown in Fig 4 A and
  • 4B comprises about 4998 bp, and includes at least one open reading frame (ORF) as
  • SEQ ID NO: 5 as shown in Fig. 4.
  • SEQ ID NO: 6 A second rat cDNA clone (SEQ ID NO: 6, shown
  • Fig. 5A and 5B is a partial cDNA comprising a partial open-reading frame (ORF)
  • FIG. 5A having sequence similarity to a region of SEQ ID NO: 5 (Fig. 4A), and a
  • the present invention also provides for the human cDNA clones (SEQ ID NO:
  • Fig. 7A may be, but is not limited to, the sequence SEQ ID NO: 8 as shown in Fig. 7A and
  • nucleic acid molecules can include nucleotides in addition to those included
  • the human chromosome lp31.31.3-32.2 and at least one human gene encoding the
  • IRSBP-1 transcribed nucleic acid and protein derived therefrom is comprised of at
  • nucleic acids comprising at least one exon, or a
  • fragment, variant or derivative thereof capable of hybridizing with at least one region
  • hybridization is specific for an IRSBP-1 -encoding nucleic acid
  • the salt concentration in the wash step can be any suitable salt concentration in the wash step.
  • the salt concentration in the wash step can be any suitable salt concentration in the wash step.
  • nucleic acids can encode functionally
  • acids may be used to generate variant animals with inactive or functionally modified
  • Codons that specify the same amino acid or synonyms (for example, CAU and CAC
  • IRSBP-1 protein of the present invention will exist from one human or animal
  • Nucleic acid fragments within the scope of the invention also include those
  • IRSBP-1 clones in other eukaryotes can be used to detect IRSBP-1 homologs in other vertebrate species, such as, but
  • IRSBP-1 phenotype a natural IRSBP-1 gene, an IRSBP-1 cDNA molecule, a
  • nucleic acid sequence substantially similar to the cDNA sequence for a
  • rat IRSBP-1 SEQ ID NO: 5 as shown in Figs. 4A and 4B, or any variant thereof.
  • the nucleic acid molecules of the present invention can include an isolated deletion
  • IRSBP-1 cDNA molecule a degenerate variant thereof, a truncated variant thereof or
  • nucleic acid is provided
  • nucleic acid sequence substantially similar to the cDNA sequence for a
  • rat IRSBP-1 (SEQ ID NO: 6) as shown in Figs. 5A and 5B comprising a variant of
  • an isolated nucleic acid that comprises the nucleic acid sequence corresponding to a human IRSBP-1
  • an isolated nucleic acid is
  • IRSBP-1 SEQ ID NO: 8 as shown in Figs. 7A and 7B.
  • gene or nucleic acid molecule can be allelic variants of SEQ ID NOS: 5 - 10.
  • allelic variant is a gene that occurs essentially at the same locus or loci in the
  • an isolated nucleic acid molecule in one embodiment, an isolated nucleic acid molecule
  • nucleic acid that is at least about 75%, preferably at
  • IRSBP-1 -encoding nucleic acid molecule as depicted in SEQ ID NO: 5, and/or a
  • an isolated nucleic acid in another embodiment of the present invention, an isolated nucleic acid
  • molecule of the present invention includes a nucleic acid that is at least about 75%
  • nucleic acid molecule as depicted in SEQ ID NO: 7, and/or a variant thereof, such
  • SEQ ID NO: 8 The nucleic acid sequences of a IRSBP-1 nucleic acid molecules (SEQ ID NO: 8.
  • nucleic acid molecules including full-length genes, full-length
  • the probe and the target nucleic acid are the probe and the target nucleic acid.
  • nucleic acid homologs can be obtained in a variety of ways including by
  • nucleic acid molecules from which to amplify nucleic acid molecules, include but are not limited to
  • species homologous to IRSBP-1 include, but are not limited to, GenBank and the
  • Another aspect of the present invention is to provide protein sequences that

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Abstract

The present invention relates to a novel protein called Insulin-Responsive Sequence DNA Binding Protein-1 (IRSBP-1) and nucleotide sequences that encode it. IRSBP-1 is capable of binding to nucleic acid regions associated with genes that respond when cells are exposed to insulin or insulin-like factors. IRSBP-1 regulates genes important in mediating the insulin response in humans and animals and in regulating pathological conditions such as diabetes, obesity, insulin-resistant syndrome and cell proliferative disorders. The present invention provides nucleic acids that may be used as probes specific for hybridizing and detecting nucleic acid molecules that encode at least in part a region of the IRSBP-1 protein. Within the scope of the present invention are recombinant cells, tissues and animals containing non-naturally occurring recombinant nucleic acid molecules encoding IRSBP-1, including expression vectors for the expression of IRSBP-1, antibodies to the IRSBP-1 proteins, assays utilizing the IRSBP-1 polypeptide, and methods relating to all of the foregoing. Also within the scope of the present invention is the development of therapeutic and diagnostic agents that mimic, facilitate or inhibit the action of IRSBP-1, and/or are based on relationships to the structure and action of IRSBP-1.

Description

Title of the Invention
INSULIN-RESPONSIVE SEQUENCE DNA BINDING PROTEIN- 1 AND METHODS TO REGULATE INSULIN- RESPONSIVE GENES
Reference to Related Application
This application claims priority from U.S. Provisional Application Serial No.: 60/162,687 filed November 1, 1999.
Acknowledgment of Federal Research Support
This invention was made, at least in part, with funding from the National
Institutes of Health (Grants NO. K08 DK022115 and RO1 DK52965). Accordingly,
the United States Government has certain rights in this invention.
Field of the Invention
The present invention relates generally to the identification of an Insulin-
Responsive Sequence DNA Binding Protein (IRSBP) gene, specifically IRSBP-1, from
mammals, and to corresponding proteins and coding sequences thereof. More
specifically, the invention relates to a nucleotide sequence encoding the IRSBP-1
protein, to methods and compositions that employ this coding sequence, to the use of
therapeutic agents that mimic or facilitate the action of IRSBP-1, and to nucleotide
sequences useful in diagnostic methods and treatment of diabetes, obesity, insulin
resistance syndrome and other pathological ailments. Background
It is estimated that there are more than 6.5 million people in the U.S.
diagnosed as having diabetes mellitus (Harris (1993) Diabetes Care 16: 642-652). Of
those diagnosed, more than 90% have Type II diabetes mellitus. Although β-cell
dysfunction is detectable in all diabetic patients whose pancreas exhibits an inability
to produce sufficient insulin to maintain glucose levels in the normal range (Defronzo
et al. (1992) Diabetes Care 15: 318-368; Taylor et al. (1994) Diabetes 43: 735-774),
the rapid increase in the prevalence of diabetes over the past several decades is
apparently more likely to be due to insulin resistance (diminished insulin action on
target tissues) (Defronzo et al. (1992) Diabetes Care 15: 318-368; Taylor et al. (1994)
Diabetes 43: 735-774; Muller-Wieland et al. (1993) Exp. Clin. Endocrinol. 101: 17-
29; Defronzo & Ferrannini (1991) Diabetes Care 14: 173-194). Thus, the current
epidemic of Type II diabetes in the United States is usually attributed to the aging of
the population, the increased prevalence of obesity and sedentary activity, and the
enrichment of the population with ethnic groups that may have a genetically
predisposed inability of the pancreas to meet the challenge of increased insulin
resistance or pancreatic dysfunction (Colditz et al. (1995) Ann. Internal Med. 122:
481-486; Helmrich et al. (1991) N. Eng. J. Med. 325: 147-152). The high incidence
of diabetes represents a significant economic burden, such that approximately $92
billion in health care expenditures in 1992 were diverted to the treatment of diabetes
(Rubin et al. (1994) J. Clin. Endocrinol. 78: 809A-809F). Insulin resistance is a key factor in the pathogenesis of Type II diabetes, and
can precede abnormal insulin secretion and the onset of clinical diabetes by decades
(Kahn (1994) Diabetes 43: 1066-1084; Ericksson et al. (1989) N. Engl. J. Med. 321:
337-343; Lillioja et al. (1988) N. Engl. J. Med. 318: 1217-1225). Resistance to
insulin action involves all major target tissues, i.e., skeletal muscle, liver and fat
(Schanlin-Jantti et al. (1992) Diabetes 41: 598-604; Haring & Hehnert (1993)
Diabetol. 36: 176-182). Although insulin resistance appears to involve defects in
insulin signaling at the post-receptor level, the mechanism of insulin resistance
remains poorly understood.
The action of insulin is initiated by binding to cell surface receptors.
Autophosphorylation and activation of the intrinsic tyrosine kinase of the insulin
receptor β-subunit leads to phosphorylation of several proximal interacting proteins,
including insulin receptor substrate-1 (IRS-1), IRS-2, and She (Sun et al. (1992) J.
Biol. Chem. 267: 22662-22672; Sun et al. (1991) Nature 352: 73-77; Kerouz et al.
(1997) J. Clin. Invest. 100: 3164-3172; Zhou et al. (1997) J. Biol. Chem. 272: 29829-
29833; Pellicci et al. (1992) Cell 70: 93-104; Kovacina & Roth (1993) Biochem.
Biophys. Res. Comm. 192: 1303-1311). IRS-1 interacts with several proteins that
contain Src homology 2 (SH2) domains, including the p85 subunits of PI3' -kinase,
GRB-2, Syp and Nek (Myers et al. (1992) Proc. Natl. Acad. Sci. 89: 10350-10354;
Skolnik et al. (1991) Cell 65: 83-90; Backer et al. (1992); Lowenstein et al. (1992)
Cell 70: 431-442). Activation of these proteins and the subsequent cascade activation
of other intracellular signaling molecules, such as p21ras, raf-1, MAP kinases, and S6 kinase, account for many of insulin's pleiotropic effects (Medina et al. (1993) Mol.
Cell Biol. 13: 155-162; Medema & Box (1993) Crit. Reviews in Oncogenesis 4: 615-
661; Wood et al. (1992) Cell 68: 1041-1050; Kyriakis et al. (1992) Nature 358: 417-
421; Sturgill et al. (1988) Nature 33.4: 715-718). Each of these cytoplasmic
substrates and the activating regulatory loop involved represents a potential linkage to
the development of insulin resistance.
The substantial number of signaling circuits involved, including interacting,
bypassing and overlapping pathways, the involvement of numerous serine/threonine
kinases and phosphatases (Reik et al. (1994) Mol. Endocrinol. 8: 490-497; Reusch et
al. (1995) Endocrinol. 136: 2464-2469; McGuire et al. (1991) Diabetes 40: 939-942;
Kusari et al. (1992) Diabetes 41: 184 A), and still uncharacterized links, characterize
the complexity of the signaling from the insulin signal at the cell surface receptor to
targets within the cell. One approach to the study of insulin interactions with cells is
to select a physiological action of insulin and then trace back toward the receptor, an
approach known as the target backward approach. This target backward approach has
yielded information concerning the mechanism of insulin regulation by focusing on
the genetic regulation of the insulin-regulated gene insulin-like growth factor binding
protein-3 (IGFBP-3).
Genetic factors also contribute to the development of non-insulin dependent
Type II diabetes mellitus (NIDDM). The concordance rate for NIDDM in identical
twins approaches 100% (Barnett et al., (1981); Newman et al, (1987)), while the risk
to other siblings of a diabetic proband is between 30 and 40% (Kobberling & Tillil (1982) ). Despite considerable investigative efforts, the genetic heterogeneity of
diabetes and the contribution of environmental factors in the development of the
phenotype make the identification of specific diabetes-related genes difficult.
Methods used in the study of the genetics of NIDDM include association of case
control studies, positional searches, parametric linkage, and molecular screening using
single-strand conformation polymorphism analysis (Elbein et al. (1994); Cox & Bell
(1989); Cox et al. (1988); Cook et al. (1993); Orita et al. (1989)). In addition, cloned
genes, including genes important for both insulin secretion and insulin action, have
been examined for sequence abnormalities. Specific mutations associated with insulin
resistance and the development of diabetes have been identified for the α- and β-
subunits of the insulin receptor (Sestz et al. (1991); van der Vorm & Maassen (1994);
Benecke et al. (1992); Hansen et al. (1992)), the glucose transporter GLUT4 (Kusari
et al. (1991); Choi et al. (1991)), Rad (Ras-associated with diabetes) (Reynet & Kahn
(1993)), and the glucokinase gene implicated in MODY (maturity onset diabetes of
the young), as well as HNF-1 and HNF-4 (Vionnet et al. (1992); Lesage et al. (1995);
Bell et al. (1991); Kaisaki et al. (1997); Moller et al. (1997)). Such mutations,
however, appear to account for less than 5% of patients with Type II diabetes.
A series of adapter proteins or substrates link the receptor tyro sine kinases to
gene transcription, and determine the response to insulin in a given cell or tissue.
Each of the proteins in the signaling cascade is a potential candidate for an acquired or
genetic defect contributing to insulin resistance. Thus, characterization of the insulin-
responsive binding proteins (IRBPs) that may bind to gene transcriptional regulatory sequences essential for insulin-regulated expression of target genes, and delineation of
the pattern of signal transduction to the IRBPs constitutes an important strategy to
identify genes important in mediating insulin resistance.
Insulin-like growth factors I and II (IGF-I and -II) are proteins that have
insulin-like metabolic and trophic effects and mediate some of the peripheral actions
of growth hormone (Daughaday & Reeder (1966)). IGFs also have a role in wound
healing by stimulating fibroblasts to produce collagen and induce hematopoiesis
through an erythropoietin-like activity. Studies have also shown that certain cancer
cells, such as from breast and kidney, produce IGFs. IGF production in cancer cells
auto-regulates cell proliferation and the production of a vascular system required for
growth of the tumor mass (LeRoth et al. (1995) Ann. Intern. Med. 122: 154-159).
IGFs have also been implicated in diabetic retinopathy by stimulating endothelial and
fibroblast proliferation (Pfeiffer et al. (1997) Diabetes 46: S26-S30; Paques et al.
(1997) Diabetes and Metabolism 23: 125-130; Flyvbjerg (1997) Kidney Int. (Suppl.)
60: S12-S19).
The actions of IGFs are modulated by a family of six IGF-binding proteins
(IGFBPs) that have different tissue distribution and production sites (Albiston &
Herington (1992) Endocrinol. 130: 497-502; Shimasaki et al. (1991) J. Biol. Chem.
266: 10646-10653). One binding protein, IGFBP-1, has a molecular weight of
approximately 30-40 kd in the human and the rat. Most of the circulating plasma
IGF-I and IGF-II, however, are associated with IGFBP-3 and an acid-labile subunit
thereof that serve as reservoirs for IGFs (Martin & Baxter (1986) J. Biol. Chem. 261: 8754-8760). Diabetes mellitus in humans and animal models is associated with
decreased levels of serum IGFBP-3 (Zapfet al. (1986) J. Clin. Invest. 77: 1768-1775;
Graubert et al. (1991) Diabetes, 40: 959-965). Hepatic expression of IGFBP-3 is
correlated with circulating IGFBP-3 levels in streptozotocin-diabetic and BB/W rats
(Luo & Murphy (1992) J. Mol. Endocrinol. 8: 155-163; Binoux et al. (1984) J. Clin.
Endocrinol. Metab. 59: 453-462; Umezawa et al. (1991) Brit. J. Nutrition. 66: 105-
116). Thus, hepatic expression of IGFBP-3 appears to determine systemic IGFBP-3
levels; and the study of the mechanisms by which insulin stimulates hepatic synthesis
of IGFBP-3 is critical for understanding the regulation of systemic IGFBP-3.
Most evidence indicates that IGFBP-3 is inhibitory to IGF action (Blat et al.
(1989) J. Biol. Chem. 264: 12449-12454; Valentinis et al. (1995) Mol. Endocrinol.
9:361-367; DeMellow & Baxter (1988) Biochem. Biophys. Res. Commun. 156: 199-
204). Furthermore, IGFBP-3 can: (a) mediate the growth inhibitory actions of
transforming growth factor-β (TGF-β), retinoic acid, anti-estrogens and fibroblast
growth factor (Oh et al. (1995), J. Biol. Chem. 270: 13589-13592; Gucev et al. (1996)
Cancer Res. 56: 1545-1550; Huynh et al. (1996) J. Biol. Chem. 271: 1016-1021;
Zadeh & Binoux (1997) Endocrinol. 138: 3069-3072), (b) mediate the induction of
apoptosis by the tumor suppressor gene p53 (Buckbinder et al. (1995) Nature 377:
646-649; Rajah et al. (1997) J. Biol. Chem. 272: 12181-12188), and (c) travel to the
cell nucleus, potentially directly regulating the transcription of critical growth
inhibitory genes independent of IGF-I (Jaques et al. (1997) Endocrinol. 134: 1767-
1770, Li et al. (1997) Endocrinol. 138: 1763-1766). The levels of IGFBP-3 in serum and liver mRNA are highest during puberty
and adult life (Romanus et al. (1986) Endocrinol. 118: 1743-1758). Unlike other
IGFBPs, IGFBP-3 levels increase in the presence of anabolic hormones such as
insulin and growth hormone (Unterman et al. (1991) Endocrinol. 128: 2693-2701;
Binoux et al (1984) J. Clin. Endocrinol. Metab. 59: 453-462). Dependence on growth
hormone (GH) has been inferred from the deceased levels of IGFBP-3 in
hypopituitary subjects and GH-deficient children and increased levels in acromegalic
patients (Boxer & Martin (1986) J. Clin. Invest. 78: 1504-1512; Blum et al. (1990) J.
Clin. Endocrinol. Metab. 70: 1292-1298). Additionally, IGFBP-3 production is
inhibited at the level of gene expression by glucocorticoids (Villafuerte et al. (1995)
Endocrinol. 136: 1928-1933).
The mechanisms by which IGFBP-3 is regulated are complex. IGFBP-3 may
undergo post-translational processing to yield various proteolytically cleaved,
phosphorylated, and glycosylated products (Blum et al. (1989) Endocrinol. 125: 766-
772; Busby et al. (1989) Endocrinol. 125: 773-777; Hoeck & Mukku (1994) J. Cell
Biochem. 56: 262-273; Hossenlopp et al. (1990) J. Clin. Endocrinol. & Metab. 71 :
797-805; Grimes & Hammond (1994) Endocrinol. 134: 337-343; Lassaree et al.
(1994) Growth Reg. 4: 48-55; Young & Rechler (1995) Endocrinol. 136: 668-678).
These processes have been shown to alter the binding of IGFBP-3 to the acid-labile
subunit, cell surfaces and to affect the affinity of IGFBP-3 for IGFs (Coverly &
Baxter (1995) Endocrinol. 136: 5778-5781). IGFBP-3 can also associate with the cell
surface and extracellular matrix; dissociation of cell-associated IGFBP-3 is one mechanism by which IGF-1 promotes release of IGFBP-3 into conditioned medium
by fibroblasts and breast cancer cells (Martin et al. (1992) Endocrinol. 131: 1703-
1710; Oh et al (1992) Endocrinol. 131: 3123-3125).
Insulin increases IGFBP-3 expression by stimulating the rate of gene
transcription rather than by stabilization of mRNA transcripts (Villafuerte et al.
(1996) Mol. Endocrinol. 10: 622-630). This enhancement is mediated through a cis-
regulatory insulin-responsive element (IRE) localized to the -1150 to -1124 bp region
of the gene encoding IGFBP-3 (Villafuerte et al. (1997) J. Biol. Chem. 272: 5024-
5030). The IGFBP-3 IRE comprises the nucleotide dyad ACC(A/G)A which has a
strong resemblance to the recognition sequence of ETS-related transcription factors,
namely AGGAA, which is within the IRE of both the prolactin and somatostatin
genes (Jacob et al. (1995) J. Biol. Chem. 270: 27773-27779). The 10-bp core
sequence of the IGFBP-3 IRE that is most critical for insulin responses (base
positions- 1148 to -1139) had no significant consensus sequence similarity to
previously identified transcription factor binding sites. What was not known,
however, was any protein or other factor that would mediate a cellular response to
insulin and which directly binds to such insulin-response elements like the IRE of
IGFBP-3.
Summary of the Invention
Briefly described, the present invention relates to a novel protein called
Insulin-Responsive Sequence DNA Binding Protein- 1 (IRSBP-1) and nucleotide sequences that encode it. IRSBP-1 is capable of binding to nucleic acid regions
associated with genes that respond when cells are exposed to insulin or insulin-like
factors. IRSBP-1 regulates genes important in mediating the insulin response in
humans and animals and in regulating pathological conditions such as diabetes,
obesity, insulin-resistant syndrome and cell proliferative disorders.
One aspect of the present invention relates to isolated or non-naturally
occurring nucleic acid molecules that encode at least a portion of a human or animal
IRSBP-1 protein or a variant thereof.
The present invention provides isolated molecules that can hybridize to nucleic
acid sequences of the genome of a human or animal and which encode an IRSBP-1
protein or variants thereof.
The present invention further provides nucleic acids that are fragments or
derivatives of the cDNA molecules comprising at least in part a region of the IRSBP-1
coding region and or an untranslated region of the cDNA, wherein the fragments may
be used as probes specific for hybridizing and detecting nucleic acid molecules that
encode at least in part a region of the IRSBP-1 protein.
Also within the scope of the present invention are recombinant cells, tissues
and animals containing non-naturally occurring recombinant nucleic acid molecules
encoding IRSBP-1, including expression vectors for the expression of IRSBP-1,
antibodies to the IRSBP-1 proteins, assays utilizing the IRSBP-1 polypeptide, and
methods relating to all of the foregoing. Also within the scope of the present
invention is the development of therapeutic and diagnostic agents that mimic, facilitate or inhibit the action of IRSBP-1, and/or are based on relationships to the
structure and action of IRSBP-1.
The invention further provides non-naturally occurring recombinant nucleic
acid molecules encoding IRSBP-1 that can be in a cell or an organism. The
recombinant nucleic acid may comprise IRSBP-1 -related sequences, functional
derivatives thereof, and a vector or a promoter effective to initiate transcription in a
host cell. The recombinant nucleic acid molecule can alternatively contain
transcription regulatory sequences functional in a particular cell, a sequence
complementary to a mRNA sequence encoding an IRSBP-1 polypeptide and
transcriptional control sequences functional in that cell.
The present invention still further provides oligopeptides having amino acid
sequences derived from the amino acid sequence of a human or animal IRSBP-1
protein that may be used to induce the formation of polyclonal or monoclonal
antibodies that specifically bind to at least one region of the IRSBP-1 protein from
human or animal. The antibodies may be used for, but are not limited to, the detection
and assay of IRSBP-1 in biological samples, or the purification of the IRSBP-1
protein. Diagnostic kits for the detection of IRSBP-1 in biological samples are also
within the scope of the present invention.
The invention also provides a recombinant cell or tissue containing non-
naturally occurring recombinant nucleic acid molecules coding for an IRSBP-1
polypeptide or a portion thereof. In such cells, the IRSBP-1 coding sequence may be
expressed under the control of its genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. The
present invention further provides for the production of animals that have modified
nucleic acids encoding at least a portion of the IRSBP-1 protein, or have the IRSBP-1
gene inactivated. The present invention further provides for methods of gene therapy
and pharmaceutical compositions including antisense and sense nucleic acids that
will modulate, in the human or animal, the activity of the IRSBP-1 gene or the IRSBP-
1 protein encoded therein.
The invention features methods for identifying mammalian cells containing an
IRSBP-1 polypeptide, or a related sequence. Such methods comprise identifying the
IRSBP-1 polypeptide in mammalian cells using techniques that are routine and
standard in the art, for example, PCR amplification, and Northern, Western, Southern
and Southwestern blotting using oligonucleotides and derivatives thereof, or
antibodies specific to the IRSBP-1 protein.
The present invention also relates to methods of detecting proliferating cells,
and the cells of humans or animals having diabetic disorders. The present invention
further relates to methods of activating or inhibiting the expression of the gene in
humans or animals that encode an IRSBP-1 protein, wherein the proliferation of cells
may be modified. The present invention further contemplates that modulation of the
activity of the IRSBP-1 protein or the expression thereof may be used to relieve the
symptomatic effects of diabetes, particularly type II diabetes. Additional objects and aspects of the present invention will become more
apparent upon review of the detailed description set forth below when taken in
conjunction with the accompanying figures, which are briefly described as follows.
Brief Description of the Figures
Fig. 1 shows the nucleotide sequence of the rat clone 52 cDNA (SEQ ID NO:
2) isolated by the yeast one-hybrid screening procedure with the translated protein
sequence (SEQ ID NO: 3) therefrom depicted in Fig. 2.
Fig. 2 shows the protein sequence of the rat clone 52 (SEQ ID NO: 3)
translated from the cDNA nucleic acid sequence (SEQ ID NO: 2) depicted in Fig. 1.
Fig. 3 shows the nucleotide sequence of a nucleic acid probe (SEQ ID NO: 4)
derived from the rat clone 52 cDNA (SEQ ID NO: 2) depicted in Fig. 1 that was
subcloned into a transcription plasmid vector, transcribed and used as a riboprobe.
Figs. 4A-4B show the nucleotide sequence of the rat cDNA (SEQ ID NO: 5)
encoding a rat ERSBP-1 protein having the translated protein sequence (SEQ ID NO:
11 as depicted in Fig. 8), wherein Fig. 4A is the rat IRSBP-1 coding region (SEQ ID
NO: 9) with the corresponding 3 -letter amino acid designation listed below its
respective nucleotide triplet, and Fig. 4B is the untranslated region that is 3' of the
coding region in Fig. 4A.
Figs. 5 A-5B show the nucleotide sequence of a truncated rat cDNA (SEQ ID
NO: 6) wherein Fig. 5 A is a partial region of a rat IRSBP-1 coding region with the corresponding 3-letter amino acid designation listed below its respective nucleotide
triplet, and Fig. 5B is the untranslated region that is 3' of the coding region in Fig. 5A.
Figs. 6A-6D show the nucleotide sequence of the human cDNA (SEQ ID NO:
7) encoding a human IRSBP-1 protein having the translated protein sequence (SEQ
ID NO: 12) (ATCC as shown in Fig. 9) wherein Fig. 6A shows a region 5'
to the coding region, Fig. 6B is a human IRSBP-1 coding region (SEQ ID NO: 9),
with the corresponding 3-letter amino acid designation listed below each respective
nucleotide triplet, Fig. 6C is the untranslated region that is 3' of the coding region
shown in Fig. 6B, and Fig. 6D shows the exons (SEQ ID NOS: 16-41) that comprise
SEQ ID NO: 7.
Figs. 7A-7B show the nucleotide sequence of a variant human cDNA (SEQ ID
NO: 8), wherein Fig. 7A is a partial region of a human variant IRSBP-1 coding region
with the corresponding 3-letter amino acid designation listed below its respective
nucleotide triplet, and Fig. 7B is the untranslated region that is 3' of the coding region
in Fig. 7 A.
Fig. 8 shows the amino acid sequence (SEQ ID NO: 11) of the rat IRSBP-1
protein translated from the coding region of the rat IRSBP-1 cDNA, the sequence
(SEQ ID NO: 9) of which is depicted in Fig. 4A.
Fig. 9 shows the amino acid sequence (SEQ ID NO: 12) of the human IRSBP-
1 protein translated from the coding region of the human IRSBP-1 cDNA, the
sequence (SEQ ID NO: 10) of which is depicted in Fig. 6B. Fig. 10 shows the amino acid sequence (SEQ ID NO: 13) of the variant human
IRSBP-1 protein translated from the coding region of the human IRSBP-1 cDNA, the
sequence (SEQ ID NO: 8) of which is depicted in Fig. 7 A.
Figs. 11A-11B show the nucleotide sequence of the truncated rat cDNA (SEQ
ID NO: 14), wherein Fig. 11A is a partial region of the rat IRSBP-1 coding region
with the corresponding 3-letter amino acid designation listed below its respective
nucleotide triplet, and Fig. 1 IB is the untranslated region that is 3' of the coding
region in Fig. 11 A.
Fig. 12 illustrates gel mobility shift of the -1150/-1117 bp IRE fragment of
IGFBP-3 (SEQ ID NO: 1) by polypeptides derived from cDNA clones isolated using
the yeast one-hybrid system.
Fig. 13 illustrates gel mobility shift analysis of the -1150/-1117 IRE fragment
of IGFBP-3 (SEQ ID NO: 1) by cDNAs expressed as thioredoxin fusion proteins.
Fig. 14 illustrates a competition-binding assay of the polypeptide encoded by
clone 52 binding to the - 1150/- 1117 bp IRE fragment of IGFBP-3 (SEQ ID NO: 1 ).
Fig. 15 illustrates IREs derived from other genes competing with the IRE of
IGFBP-3 (SEQ ID NO: 1) for binding to the protein product of clone 52.
Fig. 16 illustrates IGFBP-1 IRE reporter activity in CHO cells transfected with
a vector containing HBP1 encoding sequences, a rat IRSBP-1 encoding sequence
(SEQ ID NO: 2), or both IRSBP-1 and HBPl . Fig. 17 illustrates the effect of insulin on IGFBP-3 IRE-regulated reporter gene
activity in response to the addition of insulin to cultured cells containing either
expression vector or expression vector with clone 52 (SEQ ID NO: 14).
Fig. 18 A illustrates the effect of insulin on the expression of the IRSBP-1
gene, and Fig 18B illustrates the effect of an IRSBP-1 expressing clone (SEQ ID NO:
14) on insulin regulation of glucose uptake in myoblasts.
Fig. 19 illustrates the effect of IRSBP-1 (SEQ ID NO: 14) on the glycogen
content of cultured cells.
Fig. 20 illustrates the tissue distribution of clone 52 mRNA.
Fig. 21 illustrates ribonuclease protection assays of the expression of IRSBP-1
in tissues that utilize glucose as a main energy source.
Fig. 22 illustrates the detection of IRSBP-1 mRNA within the hypothalamic
portion of the brain by in-situ hybridization.
Fig. 23 illustrates the detection of IRSBP-1 mRNA in the lateral hypothalamus
by in-situ hybridization.
Fig. 24 shows a comparison of IRSBP-1 mRNA in the lateral hypothalamic
area of obese and lean rats by in-situ hybridization.
Fig. 25 illustrates IRSBP-1 expression in the solitary tract nuclei of lean and
obese rats.
Fig. 26 illustrates an in-situ hybridization analysis of a rat brain section
showing that IRSBP-1 -specific mRNA is expressed in the pyramidal tract and
decussations of the pyramidal tract in obese Zucker rats. Fig. 27 illustrates an in-situ hybridization analysis showing the expression
level of IRSBP-1 in the pyramidal area of the brain of a lean Zucker rat.
Fig. 28 illustrates IRSBP-1 expression in the olfactory bulb.
Fig. 29 illustrates IRSBP-1 expression in the amygdala.
Fig. 30 illustrates immunohistochemical staining with an anti-IRSBP-1
antibody, or with pre-immune serum, of a section of rat pancreas.
Fig. 31 illustrates immunohistochemical staining of rat kidney mesangial cells,
using anti-IRSBP-1 antibody (right) or pre-immune serum (left).
Fig. 32 illustrates immunohistochemical staining of the endothelial lining of
blood vessels of the kidney, using anti-IRSBP-1 antibody (right) or pre-immune
serum (left).
Fig. 33 illustrates immunohistochemical staining of the rat brain, using anti-
IRSBP-1 antibody.
Fig. 34 illustrates a Western blot analysis using anti-rat IRSBP-1 peptide
polyclonal antibody and cell extracts from human vascular endothelial cells treated
with and without insulin for 6 hours.
Fig. 35 illustrates a Western blot analysis showing IRSBP-1 in COS 7 cell
extracts and co-immunoprecipitation of IRSBP-1 with both anti-Akt 1 (lanes 1, 2) and
anti-Erk2 (lanes 3, 4) that is increased with the addition of insulin.
Fig. 36 illustrates the position of clone 52 in the compartment of the cell and
the insulin signal transduction cascade.
Fig. 37 illustrates differential phosphorylation of IRSBP-1 in normal and streptozotocin-induced diabetic rats, and in Zucker lean and obese rats. Upper panel:
western blot; lower panel: Erk2 -phosphorylation of IRSBP-1.
Fig. 38 A illustrates the effect of administered IRSBP-1 antisense (AS) or
sense (S) oligonucleo tides on the body weight of rats. Fig. 38B illustrates the loss in
body weight of females rats receiving sense IRSBP-1 DNA.
Fig. 39 illustrates phase contrast micrographs of L6 cells stably transfected
with control vector alone (left) or with vector containing the IRSBP-1 nucleic acid
sequence (right) and grown in soft agar for 4 weeks.
Fig. 40 illustrates analysis by flow cytometry of the cell cycle compartments
of L6 cells with (right panel) or without (left panel) over-expression of the IRSBP-1
nucleic acid sequence.
Detailed Description of the Preferred Embodiments
Reference now will be made in detail to the presently preferred embodiments
of the invention, one or more examples of which are illustrated in the accompanying
drawings. Each example is provided by way of explanation of the invention, not
limitation of the invention. In fact, it will be apparent to those skilled in the art that
various modifications, combination, additions, deletions and variations can be made in
the present invention without departing from the scope or spirit of the invention. For
instance, features illustrated or described as part of one embodiment can be used in
another embodiment to yield a still further embodiment. It is intended that the present invention covers such modifications, combinations, additions, deletions and variations
as come within the scope of the appended claims and their equivalents.
This description uses gene nomenclature accepted by the Cucurbit Genetics
Cooperative as it appears in the Cucurbit Genetics Cooperative Report 18:85 (1995);
herein incorporated by reference in its entirety. Using this gene nomenclature, genes
are symbolized by italicized Roman letters. If a mutant gene is recessive to the
normal type, then the symbol and name of the mutant gene appear in italicized lower
case letters.
For convenience, certain terms employed in the specification, examples, and
appended claims are collected here.
The term "animal" is used herein to include all vertebrate animals, including
humans. It also includes an individual animal in all stages of development, including
embryonic and fetal stages. A "transgenic animal" is any animal containing one or
more cells bearing genetic information altered or received, directly or indirectly, by
deliberate genetic manipulation at a subcellular level, such as by targeted
recombination or microinjection or by infection with recombinant virus. The term
"transgenic animal" is not intended to encompass classical cross-breeding or in vitro
fertilization, but rather is meant to encompass animals in which one or more cells are
altered by, or receive, a recombinant nucleic acid molecule. This recombinant nucleic
acid molecule may be specifically targeted to a defined genetic locus, may be
randomly integrated within a chromosome, or it may be extrachromosomally
replicating nucleic acid. The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was
introduced into a germ line cell, thereby conferring the ability to transfer the genetic
information to offspring. If such offspring in fact possess some or all of that alteration
or genetic information, they are transgenic animals as well.
The term ''mammalian" as used herein refers to any species, subspecies or race
of organism of the taxonomic class mammalia, such as, but not limited to, such
organisms as mice, rats, rabbits, sheep, cattle, and primates, including humans.
As used herein, the term "IRSBP-1" refers to an Insulin-Responsive Sequence
Binding Protein- 1 capable of binding to at least one insulin responsive element
associated with a gene or genes, and by so doing may regulate the expression of an
insulin-responsive gene. The term "IRSBP-1" is also intended to apply to proteins,
peptides or polypeptides capable of binding to at least one insulin-responsive element
of any organism, including fungi or animals.
The term "nucleic acid" as used herein refers to any natural and synthetic
linear and sequential arrays of nucleotides and nucleosides, for example cDNA,
genomic DNA, mRNA, RNA, oligonucleotides, oligonucleosides and derivatives
thereof. For ease of discussion, such nucleic acids may be collectively referred to
herein as "constructs," "plasmids," or "vectors." Representative examples of the
nucleic acids of the present invention include bacterial plasmid vectors including
expression, cloning, cosmid and transformation vectors such as, but not limited to,
pBR322, animal viral vectors such as, but not limited to, modified adenovirus,
influenza virus, polio virus, pox virus, retrovirus, and the like, vectors derived from bacteriophage nucleic acid, and synthetic oligonucleotides like chemically synthesized
DNA or RNA. The term "nucleic acid" further includes modified or derivatised
nucleotides and nucleosides such as, but not limited to, halogenated nucleotides such
as, but not only, 5-bromouracil, and derivatised nucleotides such as biotin-labeled
nucleotides.
The term "isolated nucleic acid" as used herein refers to a nucleic acid with a
structure (a) not identical to that of any naturally occurring nucleic acid or (b) not
identical to that of any fragment of a naturally occurring genomic nucleic acid
spanning more than three separate genes, and includes DNA, RNA, or derivatives or
variants thereof. The term covers, for example, (a) a DNA which has the sequence of
part of a naturally occurring genomic molecule but is not flanked by at least one of the
coding sequences that flank that part of the molecule in the genome of the species in
which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the
genomic nucleic acid of a prokaryote or eukaryote in a manner such that the resulting
molecule is not identical to any vector or naturally occurring genomic DNA; (c) a
separate molecule such as a cDNA, a genomic fragment, a fragment produced by
polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis,
or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a
hybrid gene, i.e., a gene encoding a fusion protein, and (e) a recombinant nucleotide
sequence that is part of a hybrid sequence that is not naturally occurring. Isolated
nucleic acid molecules of the present invention can include, for example, natural
allelic variants as well as nucleic acid molecules modified by nucleotide deletions, insertions, inversions, or substitutions such that the resulting nucleic acid molecule
still essentially encodes an IRSBP-1 protein or a variant thereof of the present
invention.
By the use of the term "enriched" in reference to nucleic acid it is meant that
the specific DNA or RNA sequence constitutes a significantly higher fraction of the
total DNA or RNA present in the cells or solution of interest than in normal or
diseased cells or in the cells from which the sequence was taken. Enriched does not
imply that there are no other DNA or RNA sequences present, just that the relative
amount of the sequence of interest has been significantly increased. The other DNA
may, for example, be derived from a yeast or bacterial genome, or a cloning vector,
such as a plasmid or a viral vector. The term significant as used herein is used to
indicate that the level of increase is useful to the person making such an increase.
It is advantageous for some purposes that a nucleotide sequence is in purified
form. The term "purified" in reference to nucleic acid represents that the sequence
has increased purity relative to the natural environment.
As used herein the terms "polypeptide" and "protein" refer to a polymer of
amino acids of three or more amino acids in a serial array, linked through peptide
bonds. The term "polypeptide" includes proteins, protein fragments, protein
analogues, oligopeptides and the like. The term "polypeptides" contemplates
polypeptides as defined above that are encoded by nucleic acids, produced through
recombinant technology, isolated from an appropriate source such as a mammal, or
are synthesized. The term "polypeptides" further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids
covalently or noncovalently linked to labeling ligands.
The term "fragment" as used herein to refer to a nucleic acid (e.g., cDNA)
refers to an isolated portion of the subject nucleic acid constructed artificially (e.g., by
chemical synthesis) or by cleaving a natural product into multiple pieces, using
restriction endonucleases or mechanical shearing, or a portion of a nucleic acid
synthesized by PCR, DNA polymerase or any other polymerizing technique well
known in the art, or expressed in a host cell by recombinant nucleic acid technology
well known to one of skill in the art. The term "fragment" as used herein may also
refer to an isolated portion of a polypeptide, wherein the portion of the polypeptide is
cleaved from a naturally occurring polypeptide by proteolytic cleavage by at least one
protease, or is a portion of the naturally occurring polypeptide synthesized by
chemical methods well known to one of skill in the art.
The term "modulates" as used herein refers to the ability of a compound to
alter the function of an IRE binding protein. A modulator preferably increases the
binding or activating potential of an IRSBP-1. A modulator can alternatively decrease
the binding or activating potential of IRSBP-1 polypeptide or fragments thereof.
The term "gene" or "genes" as used herein refers to nucleic acid sequences
(including both RNA or DNA) that encode genetic information for the synthesis of a
whole RNA, a whole protein, or any portion of such whole RNA or whole protein.
Genes that are not naturally part of a particular organism's genome are referred to as
"foreign genes", "heterologous genes" or "exogenous genes" and genes that are naturally a part of a particular organism's genome are referred to as "endogenous
genes". The term "gene product" refers to RNAs or proteins that are encoded by the
gene. "Foreign gene products" are RNA or proteins encoded by "foreign genes" and
"endogenous gene products" are RNA or proteins encoded by endogenous genes.
"Heterologous gene products" are RNAs or proteins encoded by "foreign,
heterologous or exogenous genes" and which, therefore, are not naturally expressed in
the cell.
The term "expressed" or "expression" as used herein refers to the transcription
from a gene to give an RNA nucleic acid molecule at least complementary in part to a
region of one of the two nucleic acid strands of the gene. The term "expressed" or
"expression" as used herein also refers to the translation from said RNA nucleic acid
molecule to give a protein or polypeptide or a portion thereof.
As used herein, the term "locus" or "loci" refers to the site of a gene on a
chromosome. Pairs of genes control hereditary traits, each in the same position on a
pair of chromosomes. These gene pairs, or alleles, may both be dominant or both be
recessive in expression of that trait. In either case, the individual is said to be
homozygous for the trait controlled by that gene pair. If the gene pair (alleles)
consists of one dominant and one recessive trait, the individual is heterozygous for the
trait controlled by the gene pair. Natural variation in genes or nucleic acid molecules
caused by, for example, recombination events or resulting from mutation, gives rise to
allelic variants with similar, but not identical, nucleotide sequences. Such allelic
variants typically encode proteins with similar activity to that of the protein encoded by the gene to which they are compared, because natural selection typically selects
against variations that alter function. Allelic variants can also comprise alterations in
the untranslated regions of the gene as, for example, in the 3' or 5' untranslated
regions or can involve alternate splicing of a nascent transcript, resulting in alternative
exons being positioned adjacently.
As used herein "genomic sequence" refers to the total DNA in the genome of
an organism, and includes non-coding regions like nitrons.
The term "transcription regulatory sequences" as used herein refers to
nucleotide sequences that are associated with a gene nucleic acid sequence and which
regulate the transcriptional expression of the gene. The "transcription regulatory
sequences" may be isolated and incorporated into a vector nucleic acid to enable
regulated transcription in appropriate cells of portions of the vector DNA. The
"transcription regulatory sequence" may precede, but are not limited to, the region of
a nucleic acid sequence that is in the region 5' of the end of a protein coding sequence
that may be transcribed into mRNA. Transcriptional regulatory sequences may also
be located within a protein coding region, in regions of a gene that are identified as
"intron" regions, or may be in regions of nucleic acid sequence that are in the region
of nucleic acid.
The term "coding region" as used herein refers to a continuous linear
arrangement of nucleotides which may be translated into a protein. A full length
coding region is translated into a full length protein; that is, a complete protein as
would be translated in its natural state absent any post-translational modifications. A full length coding region may also include any leader protein sequence or any other
region of the protein that may be excised naturally from the translated protein.
The term "complementary" as used herein refers to two nucleic acid molecules
that can form specific interactions with one another. In the specific interactions, an
adenine base within one strand of a nucleic acid can form two hydrogen bonds with
thymine within a second nucleic acid strand when the two nucleic acid strands are in
opposing polarities. Also in the specific interactions, a guanine base within one strand
of a nucleic acid can form three hydrogen bonds with cytosine within a second nucleic
acid strand when the two nucleic acid strands are in opposing polarities.
Complementary nucleic acids as referred to herein, may further comprise modified
bases wherein a modified adenine may form hydrogen bonds with a thymine or
modified thymine, and a modified cytosine may form hydrogen bonds with a guanine
or a modified guanine.
The term "probe" as used herein, when referring to a nucleic acid, refers to a
nucleotide sequence that can be used to hybridize with and thereby identify the
presence of a complementary sequence, or a complementary sequence differing from
the probe sequence but not to a degree that prevents hybridization under the
hybridization stringency conditions used. The probe may be modified with labels
such as, but not only, radioactive groups, biotin, or any other label that is well known
in the art.
The term "capable of hybridizing under stringent conditions" as used herein
refers to annealing a first nucleic acid to a second nucleic acid under stringent conditions as defined below. Stringent hybridization conditions typically permit the
hybridization of nucleic acid molecules having at least 70% nucleic acid sequence
identity with the nucleic acid molecule being used as a probe in the hybridization
reaction. For example, the first nucleic acid may be a test sample or probe, and the
second nucleic acid may be the sense or antisense strand of a IRSBP-1 gene or a
fragment thereof. Hybridization of the first and second nucleic acids may be
conducted under stringent conditions, e.g., high temperature and/or low salt content
that tend to disfavor hybridization of dissimilar nucleotide sequences. Alternatively,
hybridization of the first and second nucleic acid may be conducted under reduced
stringency conditions, e.g. low temperature and/or high salt content that tend to favor
hybridization of dissimilar nucleotide sequences. Low stringency hybridization
conditions may be followed by high stringency conditions to increase the selectivity
of the binding of the first and second nucleic acids. The hybridization conditions
may further include reagents such as, but not limited to, dimethyl sulfoxide (DMSO)
or formamide to disfavor still further the hybridization of dissimilar nucleotide
sequences. A suitable hybridization protocol may, for example, involve hybridization
in 6X SSC (wherein IX SSC comprises 0.015 M sodium citrate and 0.15 M sodium
chloride), at 65° Celsius in aqueous solution, followed by washing with IX SSC at 65°
Celsius. Formulae to calculate appropriate hybridization and wash conditions to
achieve hybridization permitting 30% or less mismatch between two nucleic acid
molecules are disclosed, for example, in Meinkoth et al. (1984) Anal. Biochem. 138:
267-284; the content of which is herein incorporated by reference in its entirety. Protocols for hybridization techniques are well known to those of skill in the art and
standard molecular biology manuals may be consulted to select a suitable
hybridization protocol without undue experimentation. See, for example, Sambrook
et al (1989) Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor
Press, the contents of which are herein incorporated by reference in their entirety.
The term "unique" nucleic acid region as used herein refers to a sequence
present in a nucleic acid that is not present in any other nucleic acid sequence. The
term "conserved nucleic acid region" as referred to herein is a nucleotide sequence
present in two or more nucleic acid sequences, to which a particular nucleic acid
sequence can hybridize under low, medium or high stringency conditions. The greater
the degree of conservation between the conserved regions of two or more nucleic acid
sequences, the higher the hybridization stringency that will allow hybridization
between the conserved region and a particular nucleic acid sequence.
The terms "percent sequence identity" or "percent sequence similarity" as used
herein refer to the degree of sequence identity between two nucleic acid sequences or
two amino acid sequences as determined using the algorithm of Karlin & Attschul
(1990) Proc. Natl. Acad. Sci. 87: 2264-2268, modified as in Karlin & Attschul (1993)
Proc. Natl. Acad. Sci. 90: 5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs of Attschul et al. (1990) T. Mol. Biol. Q15: 403-
410. BLAST nucleotide searches are performed with the NBLAST program, score =
100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid
molecule of the invention. BLAST protein searches are performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a
reference polypeptide. To obtain gapped alignments for comparison purposes,
Gapped BLAST is utilized as described in Attschul et al. (1997) Nuc. Acids Res. 25:
3389-3402. When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g. XBLAST and NBLAST) are used. See
http://www.ncbi.nlm.nih.gov.
Other algorithms, programs and default settings may also be suitable such as,
but not only, the GCG-Sequence Analysis Package of the U.K. Human Genome
Mapping Project Resource Centre that includes programs for nucleotide or amino acid
sequence comparisons.
The term "sense strand" as used herein refers to a single stranded DNA
molecule from a genomic DNA that may be transcribed into RNA and translated into
the natural polypeptide product of the gene. The term "antisense strand" is used herein
to mean the single strand DNA molecule of a genomic DNA that is complementary
with the sense strand of the gene.
The term "antisense DNA" as used herein refers to a gene sequence DNA that
has a nucleotide sequence complementary to the "sense strand" of a gene when read in
reverse orientation, i.e., DNA read into RNA in a 3' to 5' direction rather than in the
5' to 3' direction. The term "antisense RNA" is used to mean an RNA nucleotide
sequence (for example that encoded by an antisense DNA or synthesized
complementary with the antisense DNA). Antisense RNA is capable of hybridizing
under stringent conditions with an antisense DNA. The antisense RNA of the invention is useful for regulating expression of a "target gene" either at the
transcriptional or translational level. For example, transcription of the subject nucleic
acids may produce antisense transcripts that are capable of inhibiting transcription by
inhibiting initiation of transcription or by competing for limiting transcription factors;
or, the antisense transcripts may inhibit transport of the "target RNA", or, the
antisense transcripts may inhibit translation of "target RNA".
The term "antisense therapy" as used herein refers to the administration or in
situ generation of ohgonucleotide probes or their derivatives that specifically
hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or
genomic DNA encoding a IRSBP-1 protein so as to inhibit expression of that protein,
e.g. by inhibiting transcription and/or translation. The binding may be by
conventional base pair complementation, or, for example, in the case of binding to
DNA duplexes, through specific interactions in the major groove of the double helix.
In general, "antisense" therapy refers to the range of techniques generally employed in
the art, and includes any therapy that relies on specific binding to ohgonucleotide
sequences.
The term "nucleic acid vector" as used herein refers to a natural or synthetic
single or double stranded plasmid or viral nucleic acid molecule that can be
transfected or transformed into cells and replicate independently of, or within, the host
cell genome. A circular double stranded plasmid can be linearized by treatment with
an appropriate restriction enzyme based on the nucleotide sequence of the plasmid
vector. A nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together. The nucleic acid molecule can be
RNA or DNA.
The term "expression vector" as used herein refers to a nucleic acid vector
that may further include at least one regulatory sequence operably linked to the
nucleotide sequence coding for the IRSBP-1 protein. Regulatory sequences are well
recognized in the art and may be selected to ensure good expression of the linked
nucleotide sequence without undue experimentation by those skilled in the art. As
used herein, the term "regulatory sequences" includes promoters, enhancers, and other
elements that may control expression. Standard molecular biology textbooks such as
Sambrook et al. eds "Molecular Cloning: A Laboratory Manual" 2nd ed. Cold Spring
Harbor Press (1989) may be consulted to design suitable expression vectors,
promoters, and other expression control elements. It should be recognized, however,
that the choice of a suitable expression vector depends upon multiple factors including
the choice of the host cell to be transformed and/or the type of protein to be expressed.
The terms "transformation" and "transfection" as used herein refer to the
process of inserting a nucleic acid into a host. Many techniques are well known to
those skilled in the art to facilitate transformation or transfection of a nucleic acid into
a prokaryotic or eukaryotic organism. These methods involve a variety of techniques,
such as treating the cells with high concentrations of salt such as, but not only a
calcium or magnesium salt, an electric field, detergent, or liposome mediated
transfection, to render the host cell competent for the uptake of the nucleic acid
molecules. The term "recombinant nucleic acid" as used herein refers to combinations of
at least two nucleic acid sequences that are not naturally found in a eukaryotic or
prokaryotic cell. The nucleic acid sequences may include, but are not limited to
nucleic acid vectors, gene expression regulatory elements, origins of replication,
sequences that when expressed confer antibiotic resistance, and protein-encoding
sequences. The term "recombinant polypeptide" it is meant to include a polypeptide
produced by recombinant DNA techniques such that it is distinct from a naturally
occurring polypeptide either in its location, purity or structure. Generally, such a
recombinant polypeptide will be present in a cell in an amount different from that
normally observed in nature.
The term "recombinant cell" refers to a cell that has a new combination of
nucleic acid segments that are not covalently linked to each other in nature. A new
combination of nucleic acid segments can be introduced into an organism using a
wide array of nucleic acid manipulation techniques available to those skilled in the art.
A recombinant cell can be a single eukaryotic cell, or a single prokaryotic cell, or a
mammalian cell. The recombinant cell can harbor a vector that is extragenomic. An
extragenomic nucleic acid vector does not insert into the cell's genome. A
recombinant cell can further harbor a vector or a portion thereof that is intragenomic.
The term intragenomic defines a nucleic acid construct incorporated within the
recombinant cell's genome.
The term "antibody" as used herein refers to polyclonal and monoclonal
antibodies and fragments thereof, and immunologic binding equivalents thereof that are capable of specifically binding to the IRSBP-1 polypeptides and fragments
thereof, including epitopes thereof, or to polynucleotide sequences from the IRSBP-1
region, particularly from the IRSBP-1 locus or a portion thereof. The term "antibody"
refers to a homogeneous molecular entity, or a mixture such as a serum product made
up of a plurality of different molecular entities, and may further comprise any
modified or derivatised variant thereof that retains the ability to specifically bind to
IRSBP-1 -related polypeptides.
Described herein are methods for the production of antibodies capable of
specifically recognizing one or more differentially expressed or pathway gene
epitopes. Such antibodies may include, but are not limited to polyclonal antibodies,
monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain
antibodies, Fab fragments, F(ab').sub.2 fragments, fragments produced by a FAb
expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments
of any of the above. Such antibodies may be used, for example, in the detection of a
fingerprint, target, or pathway gene in a biological sample, or, alternatively, as a
method for the inhibition of abnormal target gene activity. Thus, such antibodies may
be utilized as part of body weight disorder treatment methods, and/or may be used as
part of diagnostic techniques whereby patients may be tested for abnormal levels of
fingerprint, target, or pathway gene proteins, or for the presence of abnormal forms of
the such proteins.
For the production of antibodies to a differentially expressed or pathway gene,
various host animals may be immunized by injection with a differentially expressed or pathway gene protein, or a portion thereof. Such host animals may include but are not
limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used
to increase the immunologic response, depending on the host species, including but
not limited to Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
Corynebacterium parvum.
Polyclonal antibodies are heterogeneous populations of antibody molecules
derived from the sera of animals immunized with an antigen, such as a target gene
product, or an antigenic functional derivative thereof. For the production of
polyclonal antibodies, host animals such as those described above, may be immunized
by injection with differentially expressed or pathway gene product supplemented with
adjuvants as also described above.
Monoclonal antibodies, which are homogeneous populations of antibodies to a
particular antigen, may be obtained by any technique that provides for the production
of antibody molecules by continuous cell lines in culture. These include, but are not
limited to the hybridoma technique of Kohler & Milstein (1975) Nature 256: 495-497;
and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al.
(1983) Immunology Today 4: 72; Cole et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-
2030), and the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies
And Cancer Therapy Alan R. Liss, Inc. pp. 77-96). Briefly, spleen cells are harvested from an immunized mouse and fused with immortalizing cells (i.e., myeloma cells) to
yield antibody-producing hybridomas. Hybridomas can be screened
immunochemically for production of monoclonal antibodies specifically reactive with
the IRSBP-1 protein.
Protocols for producing, isolating and purifying conventional and monoclonal
antibodies may be analogous to those described in Cassone et al. (1988) J. Med.
Microbiol. 27: 233-238; Hancock & Evan Production and Characterization of
Antibodies against Synthetic Peptides pp23-33 in Immunochemical Protocols ed.
M.M. Manson, (1992) (Humana Press, Totowa, NJ); Goding, J. W., Monoclonal
Antibodies: Principles and Practice, 2d ed., (1986) (Academic Press Ltd., London)
and Lam & Mutharia, "Antigen- Antibody Reactions," pp 104- 132 in Methods for
General and Molecular Bacteriology, ed. P. Gerhardt, (1994) (ASM Press,
Washington, DC) the contents of which are incorporated herein by reference in their
entirety. Such antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this
invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in
vivo makes this the presently preferred method of production.
In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al. (1984) Proc. Natl. Acad. Sci. 81 : 6851-6855; Neuberger et al. (1984)
Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from
different animal species, such as those having a variable region derived from a murine
mAb and a human immunoglobulin constant region.
Alternatively, techniques described for the production of single chain
antibodies such as, but not only U.S. Patent No. 4,946,778; Bird (1988) Science 242:
423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. 85: 5879-5883; and Ward et al.
(1989) Nature 334: 544-546 can be adapted to produce differentially expressed or
pathway gene-single chain antibodies. Single chain antibodies are formed by linking
the heavy and light chain fragments of the Fv region via an amino acid bridge,
resulting in a single chain polypeptide.
Antibody fragments which recognize specific epitopes may be generated by
known techniques. For example, such fragments include but are not limited to: the
F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule
and the Fab fragments which can be generated by reducing the disulfide bridges of the
F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et
al. (1989) Science 246: 1275-1281) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity.
Pharmaceutical compositions comprising agents that will modulate the
physiological activity of the IRSBP-1 gene product or the regulation of the expression
of the IRSBP-1 gene can be administered in dosages and by techniques well known to
those skilled in the medical or veterinary arts, taking into consideration such factors as
the age, sex, weight, species and condition of the particular patient, and the route of administration. The route of administration can be percutaneous, via mucosal
administration (e.g., oral, nasal, anal, vaginal) or via a parenteral route (intradermal,
intramuscular, subcutaneous, intravenous, or mtraperitoneal). Pharmaceutical
compositions can be administered alone, or can be co-administered or sequentially
administered with other treatments or therapies. Forms of administration may include
suspensions, syrups or elixirs, and preparations for parenteral, subcutaneous,
intradermal, intramuscular or intravenous administration (e.g., injectable
administration) such as sterile suspensions or emulsions. Pharmaceutical
compositions may be administered in admixture with a suitable carrier, diluent, or
excipient such as sterile water, physiological saline, glucose, or the like. The
compositions can contain auxiliary substances such as wetting or emulsifying agents,
pH buffering agents, adjuvants, gelling or viscosity enhancing additives,
preservatives, flavoring agents, colors, and the like, depending upon the route of
administration and the preparation desired. Standard pharmaceutical texts, such as
"Remmington's Pharmaceutical Science," 17th edition, 1985 may be consulted to
prepare suitable preparations, without undue experimentation. The effective dosage
and route of administration are determined by the therapeutic range and nature of the
compound, and by known factors, such as the age, weight, and condition of the host,
as well as LD50 and other screening procedures that are known and do not require
undue experimentation. Dosages can generally range from a few hundred milligrams
to a few grams. As used herein, a "transgenic animal" is any animal, preferably a non-human
mammal, in which one or more of the cells of the animal contain heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques well
known in the art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell, by way of deliberate genetic manipulation,
such as by microinjection or by infection with a recombinant virus. The term genetic
manipulation does not include classical cross-breeding, or in vitro fertilization, but
rather is directed to the introduction of a recombinant DNA molecule. This molecule
may be integrated within a chromosome, or it may be extrachromosomally replicating
DNA. In the typical transgenic animals, the transgene causes cells to express a
recombinant form of the subject IRSBP-1 protein, e.g. either agonistic or antagonistic
forms, or in which the endogenous IRSBP-1 gene has been disrupted. However,
transgenic animals in which the recombinant IRSBP-1 gene is silent are also
contemplated, as for example, the FLP or CRE recombinase dependent constructs
described below. The "non-human animals" of the invention include vertebrates such
as rodents, non-human primates, sheep, dog, cow, birds, amphibians, reptiles, etc.
Preferred non-human animals are selected from the rodent family including rat and
mouse, most preferably mouse. The term "chimeric animal" is used herein to refer to
animals in which the recombinant gene is found, or in which the recombinant is
expressed in some but not all cells of the animal. The term "tissue-specific chimeric
animal" indicates that the recombinant IRSBP-1 gene is present and/or expressed in
some tissues but not others. As used herein, the term "transgene" means a nucleic acid sequence (encoding,
e.g., a IRSBP-1 polypeptide) that is partly or entirely heterologous, i.e., foreign, to the
transgenic animal or cell into which it is introduced, or, is homologous to an
endogenous gene of the transgenic animal or cell into which it is introduced, but
which is designed to be inserted, or is inserted, into the animal's genome in such a
way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a
location which differs from that of the natural gene or its insertion results in a
knockout). A transgene can include one or more transcriptional regulatory sequences
and any other nucleic acid, such as introns, that may be necessary for optimal
expression of a selected nucleic acid.
As used herein, an "IRSBP-1 nucleic acid molecule" includes nucleic acid
sequences related to a natural IRSBP-1 gene and includes all regions such as
regulatory regions that control production of an RNA nucleic acid encoding the
IRSBP-1 protein or production of the IRSBP-1 protein encoded by the gene (such as,
but not limited to, transcription, translation or post-translation regulatory sequences)
as well as the coding region itself, and any introns or non-translated coding regions.
In the present context, an IRSBP-1 variant is an IRSBP-1 polypeptide that
differs from an exemplified sequence in that one or more amino acids have been
changed, added or deleted. An IRSBP-1 variant retains its useful function, i.e., for
example, ability to bind IREs or activate or suppress insulin-regulating genes.
As used herein, the term "epitope" refers to a part of the protein that can
specifically bind to an antibody by fitting into the antigen-binding site of the antibody. The techniques used to isolate and characterize the nucleic acids and proteins
of the present invention are well known to those of skill in the art and standard
molecular biology and biochemical manuals may be consulted to select suitable
protocols without undue experimentation. See, for example, Sambrook et al (1989)
Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press, the
content of which is herein incorporated by reference in its entirety.
Abbreviations used in the present specification include the following: aa,
amino acid(s); bp, base ρair(s); cDNA, DNA complementary to RNA; IGF, insulin¬
like growth factor; IGFBP, IGF-binding protein; IRE, insulin response element;
PEPCK, phosphoenol pryuvate carboxykinase; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase enzyme; nt, nucleotide(s); SSC, sodium chloride-sodium citrate ;
DMSO, dimethyl sulfoxide.
The present invention provides isolated nucleic acids, derivatives and variants
thereof that encode human and rat IRSBP-1 proteins, derivatives or variants thereof.
The present invention further provides an isolated nucleic acid encoding a fragment of
a rat IRSBP-1 protein isolated based on the ability of the expressed protein product
thereof to bind to the nucleic acid Insulin Responsive Element (IRE), associated with
the rat Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) that has the
nucleotide sequence 5'-AATTCAAGGGTATCCAGGAAAGTCTCC-3* (SEQ ID NO:
1). As used herein, IREs are regulatory nucleic acid sequences of insulin-regulated
genes that are necessary to enable an insulin-dependent response. The nucleotide sequence of SEQ ID NO: 1 is localized between the -1150 and the -1124 bp positions
of the promoter region of the IGFBP-3 encoding gene of the rat.
A rat liver cDNA library using the yeast one-hybrid system was screened
using concatemerized IREs of rat IGFBP-3, as described by Wang & Reed (1993)
Nature 364: 121-126, incorporated herein by reference in its entirety, and discussed in
Example 1 below. The cDNA library screening provided a novel 952-bp cDNA
(clone 52) encoding a portion of the Insulin-Responsive Sequence DNA Binding
Protein- 1 (IRSBP-1) that was identified and sequenced (SEQ ID NO: 2)
(ATCC ), as illustrated in Fig. 1. The nucleic acid sequence of clone 52
(SEQ ID NO: 2) encodes a polypeptide having the amino acid sequence of SEQ ID
NO: 3 (ATCC ), as shown in Fig. 2, that binds to the IRE region of the rat
IGFBP-3 (SEQ ID NO: 1), as well as to other insulin-responsive genes. A clone 52-
thioredoxin (Trx) fusion protein also binds to the IRE of rat IGFBP-3 (SEQ ID NO:
1). The amino acid sequence (SEQ ID NO: 3) deduced from the nucleotide sequence
(SEQ ID NO: 2) of clone 52 contains a homeodomain motif typical of transcription
factors. Binding by the polypeptide (SEQ ID NO: 3) encoded by clone 52 (SEQ ID
NO: 2) to the IRE of IGFBP-3 (SEQ ID NO: 1) could be competed away by IGFBP-3
IRE nucleic acids but not by nucleic acids of sequences unrelated to the IRE. The
interaction between the IRSBP-1 -related polypeptide (SEQ ID NO: 3) and the IGFBP-
3 IRE nucleic acid (SEQ ID NO: 1) was specific.
The IRSBP-1 polypeptide fragment (SEQ ID NO: 3) encoded by clone 52 also
interacts with IREs associated with other insulin-responsive genes besides IGFBP-3. The polypeptide interacts with the IREs from insulin-responsive genes encoding IGF-
1, IGFBP-1, phosphoenol pyruvate carboxykinase (PEPCK), amylase, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
IGFBP-1 is a hepatic acute phase reactant protein that coordinates the level of
IGF-1 in response to changes in insulin levels (Eee et al. (1993)). Amylase is
important for intestinal hydrolysis of carbohydrates. GAPDH catalyzes the
conversion of glyceraldehyde-3 -phosphate to 1,3-diphosphoglycerate, a rate-limiting
step in adipose tissue glycolysis. While not being bound by any theory, the naturally
occurring IRSBP-1 protein is likely a transcription factor that coordinates the
responses of several genes to insulin. The IRSBP-1 polypeptide (SΕQ ID NO: 3) of
clone 52 can regulate critical genes in target tissues implicated in insulin resistance
and insulin secretion. It is believed to modulate the pleiotropic actions of insulin in
the normal metabolism and storage of ingested carbohydrate and other fuels, in the
modulation of intermediary metabolism, and in normal cellular growth and
differentiation.
Ribonuclease protection assays (discussed in Example 8) using an antisense
RNA probe obtained by transcribing a Kpnl-Xhol fragment of clone 52 (SEQ ID NO:
4, as shown in Fig. 3) showed that at least one gene, encoding at least one nucleic acid
with sequence similarity to a region of the clone 52 cDNA sequence (SEQ ID NO: 2)
is expressed in at least liver, kidney, brain, small intestine, muscle, and fat pads.
The abundance of a rat RNA transcript capable of hybridizing to a probe
having a nucleic acid sequence of the clone 52 (SEQ ID NO: 4) was increased with the addition of physiological concentrations of insulin (10~9 M) in cell culture. It was
also decreased in the livers of diabetic rats.
The present invention provides for the use of the isolated cDNA clone 52
(SEQ ID NO: 2) as a probe to screen rat and human cDNA libraries to obtain isolated
nucleic acids capable of hybridizing with clone 52, as discussed in Example 5.
Nucleic acid regions extending the cDNA sequences in the 5' direction from the
isolated human and rat partial cDNA clones were obtained by primer extension
reactions and then sequenced.
The present invention further provides two rat cDNA clones hybridizing to the
clone 52 probe identified and sequenced as SEQ ID NOS: 5 and 6 (ATCC
and ATCC respectively) and shown in Figs. 4A and 4B and 5A and 5B
respectively. A first rat IRSBP-1 cDNA clone (SEQ ID NO 5; shown in Fig 4 A and
4B) comprises about 4998 bp, and includes at least one open reading frame (ORF) as
in Fig. 4A and which encodes a rat ISRBP-1 protein. The nucleotides at positions 68-
349 of clone 52 (SEQ ID NO: 1) correspond to the nucleotide positions 2123-2404 of
SEQ ID NO: 5 as shown in Fig. 4. A second rat cDNA clone (SEQ ID NO: 6, shown
in Fig. 5A and 5B) is a partial cDNA comprising a partial open-reading frame (ORF)
(Fig. 5A) having sequence similarity to a region of SEQ ID NO: 5 (Fig. 4A), and a
3 'untranslated region (Fig. 5B) longer than that of SEQ ID NO: 5 (shown in Fig. 5B).
The present invention also provides for the human cDNA clones (SEQ ID NO:
7 as shown in Fig. 6A-6C and SEQ ID NO: 8 as shown in Fig. 7 A and 7B) also
identified by hybridization with the nucleic acid probe comprising the clone 52 nucleic acid sequence (SEQ ID NO: 2) during the screening of a human cDNA
library.
It is contemplated that any nucleic acid molecule of the present invention can
comprise one or more regulatory regions, full-length or partial coding regions such as,
but not limited to, fragments SEQ ID NOS: 16-41 as shown in Fig. 6D, or any
combinations thereof. The minimal size of a nucleic acid molecule of the present
invention is a size sufficient to allow the formation of a stable hybridization product
with the complementary sequence of another nucleic acid molecule under selected
stringency conditions.
Embodiments of the present invention may, therefore, include, but are not
limited to, nucleic acid molecules such as: a) an IRSBP-1 cDNA molecule derived
from the rat and comprising the protein coding region (SEQ ID NO: 9, shown in Fig.
4A) of SEQ ID NO: 5, and a 3' non-coding, or untranslated, region of SEQ ID NO: 5,
shown in Fig. 4 A and Fig. 4B respectively; b) an IRSBP-1 cDNA molecule derived
from the rat nucleic acid SEQ ID NO: 5 and comprising the isolated coding region
(SEQ ID NO: 9), the sequence of which is illustrated in Fig. 4A, or a substantial
region thereof; or nucleic acid molecules representing degenerate variants, derivatives,
modified sequences and truncated variants such as, but not limited to, SEQ ID NO: 6
(ATCC ) shown in Figs. 5 A and 5B, thereof; c) an IRSBP-1 cDNA molecule
derived from the human comprising 5' and 3' non-coding regions and the protein
coding region of the sequence SEQ ID NO: 7 (ATCC ) as shown in Fig. 6A-
C; d) a nucleic acid molecule derived from the human IRSBP-1 cDNA sequence SEQ ID NO: 7 and comprising the human IRSBP-1 coding region alone (SEQ ID NO: 10),
as depicted in Fig. 6B; and/or nucleic acid molecules representing degenerate variants,
derivatives, alternatively spliced variants and modified variants thereof. A variant
may be, but is not limited to, the sequence SEQ ID NO: 8 as shown in Fig. 7A and
7B. Such nucleic acid molecules can include nucleotides in addition to those included
in SEQ ID NOS: 2, 5-10 such as, but not limited to, a full-length gene, a full-length
coding region, or a nucleic acid molecule encoding a fusion protein. BLASTN
algorithm searching of the Genbank database using the human IRSBP-1 nucleic acid
sequence SEQ ID NO: 7 as the search target found that there was almost 100%
identity with regions of the human genomic DNA sequence ATCC AC005237 from
the human chromosome lp31.31.3-32.2 and at least one human gene encoding the
IRSBP-1 transcribed nucleic acid and protein derived therefrom is comprised of at
least 26 exons as shown in Table 1, Example 5. The present invention, therefore, is
intended also to provide isolated nucleic acids comprising at least one exon, or a
fragment, variant or derivative thereof, capable of hybridizing with at least one region
of the sequences SEQ ID NO:2, 5 - 10 under low, medium or high stringency
conditions, wherein the hybridization is specific for an IRSBP-1 -encoding nucleic
acid, or a fragment, variant or derivative thereof.
One aspect of the invention therefore also provides nucleic acids that hybridize
under high or low stringency conditions to a nucleic acid that encodes a peptide
having all, a derivative of, or a portion of an amino acid sequence derived from the
nucleic acid sequences SEQ ID NOS: 2, 5-10. Appropriate stringency conditions which promote DNA hybridization, for example, 6 x SSC at about 45°C, followed by
a wash of 2 x SSC at 50°C, are well known to those skilled in the art or can be found,
for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be
selected from a low stringency of about 2 x SSC at 50°C. to a high stringency of about
0.2 x SSC at 50°C. In addition, the temperature in the wash step can be increased
from low stringency conditions at room temperature, about 22°C, to high stringency
conditions at about 65°C.
Isolated nucleic acids that differ in sequence from the nucleotide sequences
represented in SEQ ID NOS: 2, 5-10 due to degeneracy in the genetic code are also
within the scope of the invention. Such nucleic acids can encode functionally
equivalent peptides (i.e., a polypeptide having a biological activity of a IRSBP-1
protein) but differ in sequence from the sequence shown in SEQ ID NO: 2, 5-10 due
to degeneracy in the genetic code. Isolated nucleic acid sequence variants may also
encode non-functional polypeptides, the sequences of which are substantially similar,
but not identical, those of functional variants of IRSBP-1. These isolated nucleic
acids may be used to generate variant animals with inactive or functionally modified
IRSBP-1 polypeptides or fragments, variants or derivatives thereof.
For example, a number of amino acids are designated by more than one triplet.
Codons that specify the same amino acid, or synonyms (for example, CAU and CAC
are synonyms for histidine) may result in "silent" mutations which do not affect the
amino acid sequence of the subject protein. However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the
present IRSBP-1 protein of the present invention will exist from one human or animal
subject to the next of the same species. One skilled in the art will appreciate that these
variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the
nucleic acids encoding peptides having an activity of, for example, an IRSBP-1
protein may exist among individuals due to natural allelic variation. Any and all such
nucleotide variations and resulting amino acid polymorphisms are within the scope of
this invention.
Fragments of a nucleic acid encoding an active portion of one of the subject
IRSBP-1 proteins are also within the scope of the invention. As used herein, a
fragment of the nucleic acid encoding an active portion of a IRSBP-1 protein refers to
a nucleotide sequence having fewer nucleotides than the nucleotide sequence
encoding the entire amino acid sequence of the protein but which encodes a peptide
that possesses agonistic or antagonistic activity relative to a naturally occurring form
of the protein.
Nucleic acid fragments within the scope of the invention also include those
capable of hybridizing under high or low stringency conditions with nucleic acids
from other species for use in screening protocols to detect IRSBP-1 homo logs.
Comparison of the nucleic acid sequences of rat and human IRSBP-1 show that
ohgonucleotide primers can be generated that are suitable for detecting and isolating
IRSBP-1 clones in other eukaryotes. For example, the cDNA clone 52 (SEQ ID NO: 1) can be used to detect IRSBP-1 homologs in other vertebrate species, such as, but
not only, human, mice, rats, chickens.
One embodiment of the present invention provides a nucleic acid comprising a
nucleic acid sequence substantially similar to the clone 52 cDNA sequence (SEQ ID
NO: 2) encoding at least a region of a rat IRSBP-1 protein (SEQ ID NO: 3) as shown
in Figs. 1 and 2 respectively, or any variants thereof. The nucleic acid molecules of
the present invention can include an isolated deletion mutation corresponding to the
IRSBP-1 phenotype, a natural IRSBP-1 gene, an IRSBP-1 cDNA molecule, a
degenerate variant, a truncated form thereof, a homolog thereof or any other modified
versions.
In another embodiment of the present invention, a nucleic acid is provided
comprising a nucleic acid sequence substantially similar to the cDNA sequence for a
rat IRSBP-1 (SEQ ID NO: 5) as shown in Figs. 4A and 4B, or any variant thereof.
The nucleic acid molecules of the present invention can include an isolated deletion
mutation corresponding to the IRSBP-1 phenotype, a natural IRSBP-1 gene, an
IRSBP-1 cDNA molecule, a degenerate variant thereof, a truncated variant thereof or
a homolog thereof or any other variant thereof
In yet another embodiment of the present invention, a nucleic acid is provided
comprising a nucleic acid sequence substantially similar to the cDNA sequence for a
rat IRSBP-1 (SEQ ID NO: 6) as shown in Figs. 5A and 5B comprising a variant of
SEQ ID NO: 5.
In yet another embodiment of the present invention, an isolated nucleic acid is provided that comprises the nucleic acid sequence corresponding to a human IRSBP-1
sequence (SEQ ID NO: 7) as shown in Figs. 6A - 6C.
In another embodiment of the present invention, an isolated nucleic acid is
provided that comprises the nucleic acid sequence corresponding to a variant human
IRSBP-1 (SEQ ID NO: 8) as shown in Figs. 7A and 7B.
In still another embodiment of the present invention, a mammalian IRSBP-1
gene or nucleic acid molecule can be allelic variants of SEQ ID NOS: 5 - 10. An
allelic variant is a gene that occurs essentially at the same locus or loci in the
mammalian genome as the genes comprising SEQ ID NOS: 5 - 10, but which has
similar, but not identical, sequences to that of SEQ ID NO: 5 - 10.
In one embodiment of the present invention, an isolated nucleic acid molecule
of the present invention includes a nucleic acid that is at least about 75%, preferably at
least about 80%, more preferably at least about 85%, even more preferably at least
about 90%, and even more preferably at least about 95% identical to a rat-derived
IRSBP-1 -encoding nucleic acid molecule as depicted in SEQ ID NO: 5, and/or a
variant thereof, such as, but not limited to, SEQ ID NO: 6.
In another embodiment of the present invention, an isolated nucleic acid
molecule of the present invention includes a nucleic acid that is at least about 75%,
preferably at least about 80%, more preferably at least about 85%, even more
preferably at least about 90%, and even more preferably at least about 95% identical
to a nucleic acid molecule as depicted in SEQ ID NO: 7, and/or a variant thereof, such
as, but not limited to, SEQ ID NO: 8. The nucleic acid sequences of a IRSBP-1 nucleic acid molecules (SEQ ID
NOS: 2, 5 - 10) of the present invention allow one skilled in the art to, for example,
(a) make copies of those nucleic acid molecules by procedures such as, but not limited
to, insertion into a cell for replication by the cell, by chemical synthesis or by
procedures such as PCR or LCR, (b) obtain nucleic acid molecules which include at
least a portion of such nucleic acid molecules, including full-length genes, full-length
coding regions, regulatory control sequences, truncated coding regions and the like,
(c) obtain IRSBP-1 nucleic acid homologs in other mammalian species such as the
dog, cat, cow, pig or primates other than human and, (d) to obtain isolated nucleic
acids capable of hybridizing to a mammalian IRSBP-1 nucleic acid and be used to
detect the presence of IRSBP-1 nucleic acid sequences by complementation between
the probe and the target nucleic acid.
Such nucleic acid homologs can be obtained in a variety of ways including by
screening appropriate expression libraries with antibodies of the present invention;
using traditional cloning techniques employing ohgonucleotide probes made
according to the present invention to screen appropriate libraries; amplifying
appropriate libraries or DNA using ohgonucleotide primers of the present invention in
a polymerase chain reaction or other amplification method; and screening public
and/or private databases containing genetic sequences using nucleic acid molecules of
the present invention to identify targets. Examples of preferred libraries to screen, or
from which to amplify nucleic acid molecules, include but are not limited to
mammalian BAC libraries, genomic DNA libraries, and cDNA libraries. Similarly, preferred sequence databases useful for screening to identify sequences in other
species homologous to IRSBP-1 include, but are not limited to, GenBank and the
mammalian Gene Index database of The Institute of Genomics Research (TIGR).
Another aspect of the present invention is to provide protein sequences that
comprise a mammalian IRSBP-1 protein, and derivatives and fragments thereof. One
embodiment of the present invention, therefore, comprises a protein sequence (SEQ
ID NO: 3, as in Fig 2) encoded by the rat cDNA clone 52 nucleic acid sequence (SEQ
ID NO: 2; as shown in Fig. 1).
In another embodiment of the present invention, a rat IRSBP-1 protein is
provided having an amino acid sequence (SEQ ID NO: 11, illustrated in Fig. 8)
derived from the coding region of the rat cDNA clone IRSBP-1 (SEQ ID NO: 9, as in
Fig. 4A).
In yet another embodiment of the present invention, a human IRSBP-1 protein
sequence is provided (SEQ ID NO: 12, illustrated in Fig 9) that is encoded by a
coding region of the human nucleic acid sequence SEQ ID NO: 10, shown in Fig. 6B.
In still other embodiments of the present invention, peptide fragments of a
human or animal IRSBP-1 protein are provided, wherein the fragments may be
immunogenic peptides, capable of inducing an immune response when administered
to an animal, and which will be recognized and bound by an antibody or not
immunogenic when administered to an animal.
In one embodiment of the present invention, the peptide fragment is an epitope
of the rat IRSBP-1 protein (SEQ ID NO: 3 as in Fig. 2) and has the amino acid sequence: Acetylated Cys-Thr-Ser-Gln-Asn-Thr-Lys-Ser-Arg-Ty-Iso-Pro-Asn-Gly-
Lys-Leu (SEQ ID NO: 15) (ATCC ) at amino acid positions 62-76 of the
rat IRSBP-1 amino acid sequence SEQ ID NO: 3 as shown in Fig. 2.
Further contemplated to be within the scope of the present invention are
proteins having substantial similarity to the rat or human protein amino acid
sequences SEQ ID NOS: 11 and 12 respectively. Isolated peptides and polypeptides
of the present invention may also include any protein fragments thereof, a protein
analogue, or any immunologic fragments thereof.
In another embodiment of the present invention, an IRSBP-1 nucleic acid
molecule of the present invention encodes a protein having an amino acid sequence
that is at least about 75%, preferably at least about 80%, more preferably at least about
85%), even more preferably at least about 90%, and more preferably still at least about
95%o identical to a rat IRSBP-1 protein whose amino acid sequence is disclosed in
SEQ ID NO: 11, as well as allelic variants of an IRSBP-1 nucleic acid molecule
encoding a protein having these sequences, including nucleic acid molecules that have
been modified to accommodate codon usage properties of the cells in which such
nucleic acid molecules are to be expressed.
In an embodiment of the present invention, an IRSBP-1 nucleic acid molecule
of the present invention encodes a protein having an amino acid sequence that is at
least about 75%, preferably at least about 80%, more preferably at least about 85%>,
even more preferably at least about 90%), and more preferably still at least about 95%
identical to a human IRSBP-1 protein whose amino acid sequence is disclosed in SEQ ID NO: 12, as well as allelic variants of an IRSBP-1 nucleic acid molecule encoding a
protein having these sequences, including nucleic acid molecules that have been
modified to accommodate codon usage properties of the cells in which such nucleic
acid molecules are to be expressed.
Isolated peptidyl portions of the subject IRSBP-1 proteins within the scope of
the present invention can be obtained by screening peptides recombinantly produced
from the coπesponding fragment of the nucleic acid encoding such peptides. In
addition, fragments can be chemically synthesized using techniques known in the art
such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example,
one of the subject IRSBP-1 proteins may be arbitrarily divided into fragments of
desired length with no overlap of the fragments, or preferably divided into
overlapping fragments of a desired length. The fragments can be produced
recombinantly or by chemical synthesis and tested to identify those peptidyl
fragments which can function as either agonists or antagonists of, for example,
IRSBP-1 binding to nucleic acids. In an illustrative embodiment, peptidyl portions of
IRSBP-1 can tested for nucleic acid-binding activity, as well as preventing inhibitory
ability, by expression as, for example, thioredoxin fusion proteins each of which
contains a discrete fragment of the IRSBP-1 protein (see, for example, U.S. Patent
Nos. 5,270,181 and 5,292,646; and PCT publication WO94/02502) incorporated
herein by reference in their entireties.
Furthermore, it is also possible to modify the structure of an IRSBP-1
polypeptide for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo).
Such modified peptides are considered functional equivalents of peptides having an
activity of, or which antagonize, a IRSBP-1 protein as defined herein. A modified
polypeptide can be produced in which the amino acid sequence has been altered, such
as by amino acid substitution, deletion, or addition.
For example, it is reasonable to expect that an isolated replacement of a
leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a
serine, or a similar replacement of an amino acid with a structurally related amino acid
(i.e. conservative mutations) will not have a major effect on the biological activity of
the resulting molecule. Conservative replacements are those that take place within a
family of amino acids that are related in their side chains. Genetically encoded amino
acids can be divided into four families: (1) acidic = aspartate, glutamate; (2) basic =
lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine,
glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and
tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion,
the amino acid repertoire can be grouped as (1) acidic = aspartate, glutamate; (2) basic
= lysine, arginine histidine, (3) aliphatic = glycine, alanine, valine, leucine, isoleucine,
serine, threonine, with serine and threonine optionally be grouped separately as
aliphatic-hydroxyl; (4) aromatic = phenylalanine, tyrosine, tryptophan; (5) amide =
asparagine, glutamine; and (6) sulfur-containing = cysteine and methionine. (see, for
example, Biochemistry, 2nd ed, Ed. by L. Stryer, WH Freeman and Co.: 1981). Whether a change in the amino acid sequence of a peptide results in a functional
IRSBP-1 homolog can be readily determined by assessing the ability of the variant
peptide to, for instance, mediate ubiquitination in a fashion similar to the wild-type
IRSBP-1. Peptides in which more than one replacement has taken place can readily
be tested in the same manner.
In one embodiment of the present invention, therefore, a host cell is
transformed with a nucleic acid comprising the sequences SEQ ID NOS: 5 - 8, or
variants thereof. The transformed cell may, but not necessarily, express the
transformed nucleic acid to yield rat or human IRSBP-1 polypeptides SEQ ID NOS:
11 or 12 respectively, or any fragment or derivative thereof. A recombinant
expression vector suitable for transformation of a host cell means that the recombinant
expression vector contains a nucleic acid molecule, or an ohgonucleotide fragment
thereof, of the present invention coupled to a regulatory sequence selected on the basis
of the host cell used for expression. For example, the nucleic acid sequence coding
for the IRSBP-1 protein of the present invention may be operatively linked to a
regulatory sequence selected to direct expression of the desired protein in an
appropriate host cell.
The protein of the present invention may be produced in purified form by any
known conventional techniques. For example, rat or human cells may be
homogenized and centrifuged. The supernatant is then subjected to sequential
ammonium sulfate precipitation and heat treatment. The fraction containing the
protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein
fraction may be further purified by HPLC.
The present invention provides novel compositions comprising nucleotide
sequences encoding IRSBP-1 fragments. Also provided are recombinant proteins
produced using the novel coding sequences, and methods of using the recombinant
proteins.
The DNA nucleic acid molecules of the present invention can be incorporated
into cells using conventional recombinant DNA technology. The DNA molecule may
be inserted into an expression system to which the DNA molecule is heterologous (i.e.
not normally present). Alternatively, as described more fully below, the DNA
molecule may be introduced into cells which normally contain the DNA molecule, as,
for example, to correct a deficiency or defect in IRSBP-1 expression, or where over-
expression of the IRSBP-1 protein is desired.
For expression in heterologous systems, the heterologous DNA molecule is
inserted into the expression system or vector in proper sense orientation and correct
reading frame. The vector contains the necessary elements for the transcription and
translation of the inserted protein-coding sequences.
U.S. Patent No. 4,237,224 to Cohen & Boyer, which is hereby incorporated by
reference, describes the production of expression systems in the form of recombinant
plasmids using restriction enzyme cleavage and ligation with DNA ligase. These
recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in
tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccinia
virus. Recombinant viruses can be generated by transfection of plasmids into cells
infected with virus. Suitable vectors include, but are not limited to, the following
viral vectors such as lambda vector system gtl l, gt WES.tB, Charon 4, and plasmid
vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18,
pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/-
(see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif,
which is hereby incorporated by reference), pQE, pIH821, PGEX, pET series (see
Studier, F. W. et. al. (1990) "Use of T7 RNA Polymerase to Direct Expression of
Cloned Genes" Gene Expression Technology, vol. 185, which is hereby incorporated
by reference) and any derivatives thereof. Recombinant molecules can be introduced
into cells via transformation, particularly transduction, conjugation, mobilization, or
electroporation. The DNA sequences are cloned into the vector using standard
cloning procedures in the art, as described by Maniatis et al. Molecular Cloning: A
Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982),
which is hereby incorporated by reference.
A variety of host-vector systems may be utilized to express the protein-
encoding sequence(s). Primarily, the vector system must be compatible with the host
cell used. Host- vector systems include but are not limited to the following: bacteria
transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems
infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected
with virus (e.g., baculovirus). The expression elements of these vectors vary in their
strength and specificities. Depending upon the host-vector system utilized, any one of
a number of suitable transcription and translation elements can be used.
Different genetic signals and processing events control many levels of gene
expression (e.g., DNA transcription and messenger RNA (mRNA) translation).
Transcription of DNA is dependent upon the presence of a promoter which is a DNA
sequence that directs the binding of RNA polymerase and thereby promotes mRNA
synthesis. The DNA sequences of eukaryotic promoters differ from those of
prokaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic
signals may not be recognized in or may not function in a prokaryotic system, and,
further, prokaryotic promoters are not recognized and do not function in eukaryotic
cells.
Similarly, translation of mRNA in prokaryotes depends upon the presence of
the proper prokaryotic signals that differ from those of eukaryotes. Efficient
translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-
Dalgarno (SD) sequence on the mRNA. This sequence is a short nucleotide sequence
of mRNA that is located before the start codon, usually AUG, which encodes the
amino-terminal methionine of the protein. The SD sequences are complementary to
the 3'-end of the 16S rRNA (ribosomal RNA) and probably promote binding of
mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts & Lauer (1979)
Methods in Enzymology 68: 473, which is hereby incorporated by reference in its
entirety.
Promoters vary in their "strength" (i.e. their ability to promote transcription).
For the purposes of expressing a cloned gene, it is desirable to use strong promoters in
order to obtain a high level of transcription and, hence, expression of the gene.
Depending upon the . host cell system utilized, any one of a number of suitable
promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or
plasmids, promoters such as the T7 phage promoter, lac promotor, trp promotor, recA
promotor, ribosomal RNA promotor, the PR and PL promoters of coliphage lambda
and others, including but not limited, to lacUN5, ompF, bla, lpp, and the like, may be
used to direct high levels of transcription of adjacent DΝA segments. Additionally, a
hybrid trp-lacUV5 (tac) promotor or other E. coli promoters produced by recombinant
DΝA or other synthetic DΝA techniques may be used to provide for transcription of
the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit
the action of the promotor unless specifically induced. In certain operons, the
addition of specific inducers is necessary for efficient transcription of the inserted
DΝA. For example, the lac operon is induced by the addition of lactose or IPTG
(isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc.,
are under different controls. Specific initiation signals are also required for efficient gene transcription and
translation in prokaryotic cells. These transcription and translation initiation signals
may vary in "strength" as measured by the quantity of gene specific messenger RNA
and protein synthesized, respectively. The DNA expression vector, which contains a
promoter, may also contain any combination of various "strong" transcription and/or
translation initiation signals. For instance, efficient translation in E. coli requires a
Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to
provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized
by host cell ribosomes may be employed. Such combinations include but are not
limited to the SD-ATG combination from the cro gene or the N gene of coliphage
lambda, or from the E. coli tryptophan Ε, D, C, B or A genes. Additionally, any SD-
ATG combination produced by recombinant DNA or other techniques involving
incorporation of synthetic nucleotides may be used.
Once the isolated DNA molecule of the present invention has been cloned into
an expression system, it is ready to be incorporated into a host cell. Such
incorporation can be carried out by the various forms of transformation noted above,
depending upon the vector/host cell system. Suitable host cells include, but are not
limited to, bacteria, virus, yeast, mammalian cells, and the like.
Recombinant expression vectors can be designed for the expression of the
encoded proteins in prokaryotic or eukaryotic cells. The prokaryotic expression
system may comprise the host bacterial species E. coli, B. subtilis or any other host
cell known to one of skill in the art. Useful vectors may comprise constitutive or inducible promoters to direct expression of either fusion or non- fusion proteins. With
fusion vectors, a number of amino acids are usually added to the expressed target gene
sequence such as, but not limited to, a protein sequence for thioredoxin. A proteolytic
cleavage site may further be introduced at a site between the target recombinant
protein and the fusion sequence. Additionally, a region of amino acids such as a
polymeric histidine region may be introduced to allow binding to the fusion protein by
metallic ions such as nickel bonded to a solid support, and thereby allow purification
of the fusion protein. Once the fusion protein has been purified, the cleavage site
allows the target recombinant protein to be separated from the fusion sequence.
Enzymes suitable for use in cleaving the proteolytic cleavage site includes, but are not
limited to, Factor Xa and thrombin. Fusion expression vectors that may be useful in
the present invention include pGex (Amrad Corp., Melbourne, Australia), pRIT5
(Pharmacia, Piscataway, NJ) and pMAL (New England Biolabs, Beverly, MA), that
fuse glutathione S-transferase, protein A, or maltose E binding protein, respectively,
to the target recombinant protein.
Expression of unfused foreign genes in E. coli may be accomplished with
recombinant vectors including, but not limited to, the E. coli expression vector
pUR278 as described in Ruther et al. (1983) E.M.B.O.J. 2: 1791, incorporated herein
by reference in its entirety. Using the pUR278 vector, the nucleotide sequence coding
for the IRSBP-1 gene product may be ligated in frame with the lacV coding region to
produce a fusion protein. Expression of a foreign gene can also be obtained using eukaryotic vectors
such as mammalian, yeast or insect cells. The use of eukaryotic vectors permits
partial or complete post-translational modification such as, but not only, glycosylation
and/or the formation of the relevant inter- or intra-chain disulfide bonds. Examples of
vectors useful for expression in the yeast Saccharomyces cerevisiae include pYepSecl
as in Baldari et al, (1987), E.M.B.O.J., 6: 229-234 and pYES2 (Invitrogen Corp., San
Diego, CA), incorporated herein by reference in their entirety.
Baculovirus vectors are also available for the expression of proteins in
cultured insect cells (F9 cells). The use of recombinant Baculovirus vectors can be, or
is, analogous to the methods disclosed in Richardson CD. ed., (1995), "Baculovirus
Expression Protocol" Humana Press Inc.; Smith et al. (1983) Mol. Cell. Biol. 3: 2156-
2165; Pennock et al. (1984) Mol. Cell. Biol. 4: 399-406 and incorporated herein by
reference in their entirety.
Other vectors useful for expressing the IRSBP-1 protein, or an epitope of a
IRSBP-1 protein, include viral vectors. Methods for making a viral recombinant
vector useful for expressing the IRSBP-1 protein are analogous to the methods
disclosed in U.S. Patent Nos. 4,603,112; 4,769,330; 5,174,993; 5,505,941; 5,338,683;
5,494,807; 4,722,848; Paoletti, E. (1996) Proc. Natl. Acad. Sci. 93: 11349-11353;
Moss (1996) Proc. Natl. Acad. Sci. 93: 11341-11348; Roizman (1996) Proc. Natl.
Acad. Sci. 93: 11307-11302; Frolov et al. (1996) Proc. Natl. Acad. Sci. 93: 11371-
11377; Grunhaus et al. (1993) Seminars in Virology 3: 237-252 and U.S. Patent Nos. 5,591,639; 5,589,466; and 5,580,859 relating to DNA expression vectors, mter alia;
the contents of which are incorporated herein by reference in their entireties.
Another aspect of the present invention pertains to the use of an isolated
nucleic acid molecule for constructing nucleotide probes and primers useful for a
variety of functions. For example, synthetic ohgonucleotide probes are useful for
detecting complementary nucleotide sequences in biological materials such as cells,
cell extracts or tissues (as well as in an in situ hybridization technique). For example,
isolated nucleic acids synthesized according to the present invention can determine
whether a cell expresses an mRNA transcript encoding the IRSBP-1 protein. The
present invention also contemplates the use of antisense nucleic acid molecules, which
are designed to be complementary to a coding strand of a nucleic acid (i.e.,
complementary to an mRNA sequence) or, alternatively, complimentary to a 5' or 3'
untranslated region of the mRNA. Another use of synthetic nucleotides is as primers
(DNA or RNA) for a polymerase chain reaction (PCR), ligase chain reaction (LCR),
or the like.
Synthesized nucleotides can be produced in variable lengths - the number of
bases synthesized will depend upon a variety of factors, including the desired use for
the probes or primers. Additionally, sense or anti-sense nucleic acids or
oligonucleotides can be chemically synthesized using modified nucleotides to increase
the biological stability of the molecule or of the binding complex formed between the
anti-sense and sense nucleic acids. For example, acridine substituted nucleotides can
be synthesized. Protocols for designing isolated nucleotides, nucleotide probes, and/or nucleotide primers are well-known to those of ordinary skill, and can be
purchased commercially from a variety of sources (e.g., Sigma Genosys, The
Woodlands, TX or The Great American Gene Co., Ramona, CA).
Nucleotides constructed in accordance with the present invention can be
labeled to provide a signal as a means of detection. For example, radioactive elements
such as 32P, 3H, and 35S or the like provide sufficient half-life to be useful as
radioactive labels. Other materials useful for labeling synthetic nucleotides include
fluorescent compounds, enzymes and chemiluminescent moieties. Methods useful in
selecting appropriate labels and binding protocols for binding the labels to the
synthetic nucleotides are well known to those of skill in the art. Standard
immunology manuals such as Promega: Protocol and Applications Guide, 2nd
Edition, 1991 (Promega Corp., Madison, WI; the content of which is incorporated
herein in its entirety) may be consulted to select an appropriate labeling protocol
without undue experimentation.
It is further contemplated to be within the scope of the present invention to
produce and use antibodies specifically reactive with an IRSBP-1 protein or a region
thereof. The antibody may be monoclonal or polyclonal and may be produced by
conventional methodology using the IRSBP-1 protein, or an immunologic fragment
thereof, as an immunogen. For example, a mammal (i.e., a mouse, rabbit, horse,
sheep, or goat) may be immunized with a IRSBP-1 protein of the present invention,
an immunogenic fragment thereof, or an IRSBP-1 fusion protein or fragment thereof,
using an immunization protocol conducive to producing antibodies reactive with the IRSBP-1 protein or a fragment thereof. Following completion of the immunization
steps, antiserum reactive with the jointed protein may be collected and, if desired,
polyclonal anti-IRSBP-1 antibodies isolated.
One embodiment of the present invention, therefore, is a fragment of an amino
acid sequence of the rat IRSBP-1 protein of SEQ ID NOS: 3 or 11 , or human IRSBP-
1 protein (SEQ ID NO: 12) may be synthesized and used as an immunogen to produce
an anti-IRSBP-1 polyclonal antibody. The sequence of the immunogenic peptide
synthesized was: Acetylated Cys-Thr-Ser-Gln-Asn-Thr-Lys-Ser-Arg-Tyr-Ile-Pro-Asn-
Gly-Lys-Leu (SEQ ID NO: 15) at amino acid positions 746-760 of the rat IRSBP-1
amino acid sequence SEQ ID NO: 11. The polyclonal raised against the peptide SEQ
ID NO: 13 was specific for the rat IRSBP-1 protein and cross-reacted with the human
IRSBP-1 protein.
Antibodies that specifically bind, for example, IRSBP-1 epitopes can also be
used in immunohistochemical staining of tissue samples in order to evaluate the
abundance and pattern of expression of IRSBP-1. Anti-IRSBP-1 antibodies can be
used diagnostically in immuno-precipitation and immuno-blotting to detect and
evaluate IRSBP-1 levels in tissue or bodily fluid as part of a clinical testing procedure.
For instance, such measurements can be useful in predictive valuations of the onset or
progression of diabetes or cell proliferation disorders. Likewise, the ability to monitor
IRSBP-1 levels in an individual can allow determination of the efficacy of a given
treatment regimen for an individual afflicted with such a disorder. The level of
IRSBP-1 can be measured in cells isolated from bodily fluid, such as in samples of cerebral spinal fluid or blood, or can be measured in tissue, such as produced by
biopsy. Diagnostic assays using anti-IRSBP-1 antibodies can include, for example,
immunoassays designed to aid in early diagnosis of a diabetic, neoplastic or
hyperplastic disorder, e.g. the presence of insulin-responsive negative cells in the
sample, e.g. to detect cells in which a lesion of the IRSBP-1 gene has occurred.
Another application of anti-IRSBP-1 antibodies is in the immuno logical
screening of cDNA libraries constructed in expression vectors, such as λgtll, λgtlδ-
23, λZAP, and λORF8. Messenger libraries of this type, having coding sequences
inserted in the correct reading frame and orientation, can produce fusion proteins. For
instance, λgtl l will produce fusion proteins whose amino termini consist of .beta.-
galactosidase amino acid sequences and whose carboxy termini consist of a foreign
polypeptide. Antigenic epitopes of IRSBP-1 can then be detected with antibodies, as,
for example, reacting nitrocellulose filters lifted from infected plates with anti-IRSBP-
1 antibodies. Phage, scored by this assay, can then be isolated from the infected plate.
Thus, the presence of IRSBP-1 homologs can be detected and cloned from other
human sources, i.e. to identified other closely homologous human isoforms, as well as
to identify IRSBP-1 homologs in other mammals.
It is further contemplated to be within the scope of the present invention for an
assay to detect natural serum antibodies specific for the IRSBP-1 protein. These
antibodies may be induced as a result of the release of IRSBP-1 or fragments thereof,
during the onset of deterioration and destruction of the cells of the islets of Langerhan. The detection of the antibodies will provide a diagnostic indication of the onset of
diabetes, cancer and the progressive loss of pancreatic activity.
Moreover, the nucleotide sequence determined from the cloning of subject
IRSBP-1 from a human or animal cell line will further allow for the generation of
probes designed for use in identifying IRSBP-1 homologs in other animal cell-types,
particularly cells associated with the onset and maintenance of diabetes and obesity,
cancer or other transformed or immortalized cells, as well as IRSBP-1 homologs from
other non-human mammals.
In addition the present invention contemplates nucleotide probes can be
generated from a cloned nucleic acid sequence of the IRSBP-1 protein, which allow
for histological screening of intact tissue and tissue samples for the presence of
IRSBP-1 mRNA. Similar to the diagnostic uses of anti-IRSBP-1 antibodies, the use
of probes directed to IRSBP-1 mRNA, or to genomic IRSBP-1 sequences, can be used
for both predictive and therapeutic evaluation of allelic mutations which might be
manifest in, for example, diabetes or other metabolic disorders directly or indirectly
attributed to a failure of the cells to respond or over-respond to insulin as well as
neoplastic or hyperplastic disorders such as, but not limited to, unwanted cell growth.
Used in conjunction with anti-IRSBP-1 antibody immunoassays, the nucleotide
probes can help facilitate the determination of the molecular basis for a disorder or
ailment that may involve some abnormality associated with expression (or lack
thereof) of an IRSBP-1 protein and perturbation of insulin regulation of a gene
expression or activity. For instance, nucleic acid molecules complementary to an IRSBP-1 coding sequence can be used to determine if cells contain IRSBP-1 coding
sequences using Southern hybridization analysis. Nucleic acid molecules can also be
used to determine the level of expression of IRSBP-1 mRNA in cells using Northern
analysis as discussed in Example 8.
For example, the present method provides a method for determining if a
subject is at risk for a disorder characterized by unwanted insulin non-responsiveness
or cell proliferation. In preferred embodiments, the subject method can be generally
characterized as comprising: detecting, in a tissue of a subject (e.g. a human patient),
the presence or absence of a genetic lesion characterized by at least one of (i) a
mutation of a gene encoding IRSBP-1 or (ii) the mis-expression of the IRSBP-1 gene.
To illustrate, such genetic lesions can be detected by ascertaining the existence of at
least one of (i) a deletion of one or more nucleotides from the IRSBP-1 gene, (ii) an
addition of one or more nucleotides to the IRSBP-1 gene, (iii) a substitution of one or
more nucleotides of the IRSBP-1 gene, (iv) a gross chromosomal rearrangement of the
IRSBP-1 gene, (v) a gross alteration in the level of a messenger RNA transcript of the
IRSBP-1 gene, (vi) the presence of a non-wild type splicing pattern of a messenger
RNA transcript of the IRSBP-1 gene, and (vii) a non- wild type level of the IRSBP-1
protein. In one aspect of the invention there is provided a probe/primer comprising an
ohgonucleotide containing a region of nucleotide sequence which is capable of
hybridizing to a sense or antisense sequence of the rat or human IRSBP-1 SEQ ID
NOS: 5 - 8, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or
intronic sequences naturally associated with the IRSBP-1 gene. The probe is exposed to nucleic acid of a tissue sample; and the hybridization of the probe to the sample
nucleic acid is detected. In certain embodiments, detection of the lesion comprises
utilizing the probe/primer in, for example, a polymerase chain reaction (PCR) (see,
e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), or, alternatively, in a ligation chain
reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241: 1077-1080; and
Nakazawa et al. (1994) Proc. Natl. Acad. Sci. 91: 360-364), the later of which can be
particularly useful for detecting even point mutations in the IRSBP-1 gene and which
are incorporated herein in their entirety. Alternatively, or additionally, the level of
IRSBP-1 protein can be detected in an immunoassay.
The IRSBP-1 polypeptides of the invention can be used in therapeutic
applications. Since IRSBP-1 increases the transcription of IGFBP-3, IRSBP-1 can be
used to treat diseases (e.g., diabetes) associated with low levels of IGFBP-3. Further,
many diseases are associated with an excess of circulating IGF-1 or IGF-II, for
example, some cancers and type II diabetes. IRSBP-1 can be used in patients with low
levels of IGFBP-3 or high levels of IGF. Introduction of the gene encoding IRSBP-1
(or a functional derivative) into cells using either retroviral vectors or liposomes
results in increased production of IGFBP-3. Many methods of delivering expressible
coding sequences to cells are known in the art. Methods for gene therapy are
described in U.S. Patent No. 5,399,346, issued to Anderson et al. and U.S. Patent No.
5,766,899, issued to Kuo et al. describes methods for gene delivery into liver cells.
The use of amphipathic compounds to deliver DNA is described in U.S. Patent No.
5,744,335 issued to Wolf et al. and which are incorporated herein in their entirety. Adenovirus vectors and adeno-associated virus vectors are useful in gene delivery in
vivo.
Further, IRSBP-1 encoding sequences of the invention are useful in increasing
production of recombinant IGFBP-3 for treatment of the aforementioned diseases,
including GH deficiencies and complications caused by increased unbound IGF, can
be accomplished by administration of recombinant IGFBP-3 (for example, produced
in cell culture) via pharmaceutical compositions. Production of IGFBP-3 from
recombinant cells can be increased by transfecting such cells with an IRSBP-1
encoding sequence either under the control of its own or a heterologous promoter.
IRSBP-1 polypeptides of the present invention are also useful in the treatment
of growth hormone disorders, especially those where IGFBP-3 levels are below
normal. IRSBP-1 is formulated into a pharmaceutical composition for parenteral
administration, and a therapeutical dose is administered, with the result of raising
IGFBP-3 and IRSBP-1 levels in the treated patient.
The presence of micro-satellite DNA downstream of the IRSBP-1 coding
sequence is also further noted. Expression of the IRSBP-1 coding sequence is greater
in the presence than absence of this micro-satellite DNA. Probes and/or primers for
analysis of this region may allow the identification of genetic diseases associated with
aberrant IRSBP-1 expression.
In a diagnostic embodiment of the present invention, therefore the nucleotide
sequence of the isolated DNA molecule of the present invention may be used as a
probe in nucleic acid hybridization assays for the detection of the IRSBP-1 gene in various patient body fluids. The nucleotide sequence of the present invention may be
used in any nucleic acid hybridization assay system known in the art, including, but
not limited to, Southern blots (Southern, E.M. (1975) J. Mol. Biol. 98: 508; Northern
blots (Thomas et al. (1980) Proc. Natl. Acad. Sci. 77: 5201-05); Colony blots
(Grunstein et al, (1975) Proc. Natl. Acad. Sci. 72: 3961-65, which are hereby
incorporated by reference). Alternatively, the isolated DNA molecule of the present
invention can be used in a gene amplification detection procedure such as a
polymerase chain reaction (Erlich et al. (1991) "Recent Advances in the Polymerase
Chain Reaction" Science 252: 1643-51, which is hereby incorporated by reference) or
in restriction fragment length polymorphism (RFLP) diagnostic techniques, as
described in Watson et al, (2d ed. 1992), Recombinant DNA, Scientific American
Books, 519-522, 545-547, which is hereby incorporated by reference.
Specifically, for example, the DNA molecules of the invention can be used in
prenatal or postnatal diagnosis of the human diseases associated with defects in
response to variation in the level of insulin. A probe for the DNA encoding IRSBP-1
can be designed using the DNA molecule of the invention, and used to probe the DNA
obtained from amniotic fluid or chorionic tissue and amplified by PCR, LCR or any
other known amplification technique for the presence of the IRSBP-1 gene or a variant
thereof, as noted above. Similar procedures can be used in postnatal diagnostic work,
as, for example, to diagnose the source of an IRSBP-1 deficiency in a person who is
diabetic. Another potentially useful application of the DNA molecule of the present
invention is the possibility of increasing the amount of IRSBP-1 protein present in a
mammal by gene transfer (so-called "gene therapy"). Of course, in most instances,
this gene would be transferred into the animal host along with promoters, inducers,
and the like (which are well known and recognized techniques in the field of genetic
engineering, as noted supra) to allow the cell to initiate and continue production of the
genetic product protein. The DNA molecule of the present invention can be
transferred into the extra-chromosomal or genomic DNA of the host.
Another aspect of the invention relates to the use of the isolated nucleic acid in
"antisense" therapy. An antisense construct of the present invention can be delivered,
for example, as an expression plasmid which, when transcribed in the cell, produces
RNA which is complementary to at least a unique portion of the cellular mRNA
which encodes a IRSBP-1 -protein, e.g. the rat or human IRSBP-1 nucleic acid
sequences represented in SEQ ID NOS: 2, 5 - 8. Alternatively, the antisense construct
can be an ohgonucleotide probe which is generated ex vivo and which, when
introduced into the cell causes inhibition of expression by hybridizing with the mRNA
and or genomic sequences encoding one of the subject IRSBP-1 proteins. Such
ohgonucleotide probes are preferably modified oligonucleotides that are resistant to
endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore
stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides
are phosphoramidate, phosphorothioate and methylphosphonate analogs of DNA (see
also U.S. Patent Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed,
for example, by van der Krol et al (1988) Biotechniques 6: 958-976; and Stern et al.
(1988) Cancer Res. 48: 2659-2668 and which are incorporated herein in their entirety.
Accordingly, the modified oligomers of the invention are useful in therapeutic,
diagnostic, and research contexts. Inhibition of cell proliferation may result, but this
condition may be desirable where, for example, proliferation may lead to a
pathological condition such as, but not limited to a blockage of a blood vessel after
angioplasty, or proliferation of endothelial cells for angiogenesis in tumor formation.
An increase in cell regulation may result, but this condition may be desirable where,
for example, a deterioration or deficiency in the number of cells results in a
pathological condition such as, but not limited to, a progressive decrease in neural
cells, or muscular atrophy. In therapeutic applications, the oligomers are utilized in a
manner appropriate for antisense therapy in general. For such therapy, the oligomers
of the invention can be formulated for a variety of loads of administration, including
systemic and topical or localized administration. Techniques and formulations
generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing
Co., Easton, Pa. For systemic administration, injection is preferred, including
intramuscular, intravenous, intraperitoneal, and subcutaneous for injection, the
oligomers of the invention can be formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or Ringer's solution. In
addition, the oligomers may be formulated in solid form and redissolved or suspended
immediately prior to use. Lyophilized forms are also included. Systemic administration can also be by transmucosal or transdermal means, or
the compounds can be administered orally. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and include, for
example, for transmucosal administration bile salts and fusidic acid derivatives. In
addition, detergents may be used to facilitate permeation. Transmucosal
administration may be through nasal sprays or using suppositories. For oral
administration, the oligomers are formulated into conventional oral administration
forms such as capsules, tablets, and tonics. For topical administration, the oligomers
of the invention are formulated into ointments, salves, gels, or creams as generally
known in the art.
In addition to use in therapy, the oligomers of the invention may be used as
diagnostic reagents to detect the presence or absence of the target DNA or RNA
sequences to which they specifically bind. Such diagnostic tests are described in
further detail below.
It is further contemplated to be within the scope of the present invention for
IRSBP-1 -expression vectors to be used as a part of a gene therapy protocol to
reconstitute IRSBP-1 function in a cell in which IRSBP-1 is mis-expressed, or
alternatively, to provide an antagonist of the naturally-occurring IRSBP-1 or an
antisense construct. For instance, expression constructs of the subject IRSBP-1 -
proteins may be administered in any biologically effective carrier, e.g. any
formulation or composition capable of effectively transfecting cells in vivo with a recombinant IRSBP-1 -gene. Approaches include insertion of the subject gene in viral
vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and
herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. Viral
vectors can be used to transfect cells directly; plasmid DNA can be delivered with the
help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody
conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other
such intracellular carriers, as well as direct injection of the gene construct or CaPO4
precipitation carried out in vivo. It will be appreciated that because transduction of
appropriate target cells represents the critical first step in gene therapy, choice of the
particular gene delivery system will depend on such factors as the phenotype of the
intended target and the route of administration, e.g. locally or systemically.
A preferred approach for in vivo introduction of nucleic acid encoding one of
the subject proteins into a cell is by use of a viral vector containing nucleic acid, e.g. a
cDNA, encoding the gene product. Infection of cells with a viral vector has the
advantage that a large proportion of the targeted cells can receive the nucleic acid.
Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in
the viral vector, are expressed efficiently in cells that have taken up viral vector
nucleic acid.
Retrovirus vectors and adeno-associated virus vectors are generally understood
to be the recombinant gene delivery system of choice for the transfer of exogenous
genes in vivo, particularly into humans. These vectors provide efficient delivery of
genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to
ensure the safety of their use, particularly with regard to the possibility of the spread
of wild-type virus in the cell population. The development of specialized cell lines
(termed "packaging cells") that produce only replication-defective retroviruses has
increased the utility of retroviruses for gene therapy, and defective retroviruses are
well characterized for use in gene transfer for
gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76: 271). Thus,
recombinant retrovirus can be constructed in which part of the retroviral coding
sequence (gag, pol, env) has been replaced by nucleic acid encoding an IRSBP-1
proteins, thereby rendering the retrovirus replication defective. The replication
defective retrovirus is then packaged into virions that can be used to infect a target cell
through the use of a helper virus by standard techniques. Protocols for producing
recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses
can be found in Current Protocols in Molecular Biology, Ausubel et al. (1989) (eds.)
Greene Publishing Associates, Sections 9.10-9.14 and other standard laboratory
manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which
are well known to those skilled in the art. Examples of suitable packaging virus lines
for preparing both ecotropic and amphotropic retroviral systems include psiCrip,
psiCre, psi2 and psiAm. Retroviruses have been used to introduce a variety of genes
into many different cell types, including neural cells, epithelial cells, endothelial cells,
lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see
for example Eglitis, et al. (1985) Science 230: 1395-1398; Danos & Mulligan (1988) Proc. Natl. Acad. Sci. 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. 85:
3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. 87: 6141-6145; Hwber et
al. (1991) Proc. Natl. Acad. Sci. 88: 8039-8043; Eerry et al. (1991) Proc. Natl. Acad.
Sci. 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van
Beusechem et al. (1992) Proc. Natl. Acad. Sci. 89: 7640-7644; Kay et al. (1992)
Human Gene Therapy 3: 641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. 89: 10892-
10895; Hwu et al. (1993) J. Immunol. 150: 4104-4115; U.S. Patent No. 4,868,116;
U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO
89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573), and
which are incorporated herein in their entireties.
Furthermore, it has also been shown that it is possible to limit the infection
spectrum of retroviruses and consequently of retroviral-based vectors, by modifying
the viral packaging proteins on the surface of the viral particle (see, for example PCT
publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies
for the modification of the infection spectrum of retroviral vectors include: coupling
antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989)
Proc. Natl. Acad. Sci. 86: 9079-9083; Julan et al. (1992) J. Gen. Virol. 73: 3251-
3255; and Goud et al (1983) Virology 163: 251-254); or coupling cell surface ligands
to the viral env proteins (Neda et al. (1991) J. Biol. Chem. 266: 14143-14146), and
which are incorporated herein in their entireties. Coupling can be in the form of the
chemical cross-linking with a protein or other variety (e.g. lactose to convert the env
protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single- chain antibody/env fusion proteins). This technique, while useful to limit or
otherwise direct the infection to certain tissue types, and can also be used to convert
an ecotropic vector into an amphotropic vector. Moreover, use of retroviral gene
delivery can be further enhanced by the use of tissue- or cell-specific transcriptional
regulatory sequences that control expression of the IRSBP-1 -gene of the retroviral
vector.
Another viral gene delivery system useful in the present invention utilizes
adenovirus-derived vectors. The genome of an adenovirus can be manipulated such
that it encodes a gene product of interest, but is inactivated in terms of its ability to
replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988)
BioTechniques 6: 616; Rosenfeld et al. (1991) Science 252: 43 1434; and Rosenfeld et
al. (1992) Cell 68: 143-155), and which are incorporated herein in their entirety.
Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or
other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled
in the art. Recombinant adenoviruses can be advantageous in certain circumstances in
that they are not capable of infecting nondividing cells and can be used to infect a
wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited
supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. 89: 6482-
6486), hepatocytes (Herz & Gerard (1993) Proc. Natl. Acad. Sci. 90: 2812-2816) and
muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. 89: 2581-2584), and which
are incorporated herein in their entireties. Furthermore, the virus particle is relatively
stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA
(and foreign DNA contained therein) is not integrated into the genome of a host cell
but remains episomal, thereby avoiding potential problems that can occur as a result
of insertional mutagenesis in situations where introduced DNA becomes integrated
into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the
adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene
delivery vectors (Berkner et al. supra; Haj-Ahmand & Graham (1986) J. Virol.
57:267). Most replication-defective adenoviral vectors currently in use and therefore
favored by the present invention are deleted for all or parts of the viral El and E3
genes but retain as much as 80%> of the adenoviral genetic material (see, e.g., Jones et
al. (1979) Cell 16:683; Berkner et al, supra; and Graham et al. in Methods in
Molecular Biology, E. J. Murray, (1991) Ed. (Humana, Clifton, N.J.) vol. 7. pp. 109-
127), and which are incorporated herein in their entirety. Expression of the inserted
IRSBP-1-gene can be under control of, for example, the E1A promoter, the major late
promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously
added promoter sequences.
Yet another viral vector system useful for delivery of, for example, the subject
IRSBP-1 -gene, is the adeno-associated virus (AAV). Adeno-associated virus is a
naturally occurring defective virus that requires another virus, such as an adenovirus
or a heφes virus, as a helper virus for efficient replication and a productive life cycle.
(For a review see Muzyczka et al. (1992) Curr. Topics in Micro, and Immunol.
158:97-129). It is also one of the few viruses that may integrate its DNA into non- dividing cells, and exhibits a high frequency of stable integration (see for example
Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989)
J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973).
Vectors containing as little as 300 base pairs of AAV can be packaged and can
integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such
as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used
to introduce DNA into cells. A variety of nucleic acids have been introduced into
different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc.
Natl. Acad. Sci. 81 :6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;
Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.
51 :611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790), and which are
incorporated herein in their entirety.
Other viral vector systems that may have application in gene therapy have
been derived from such as, but not limited to, heφes virus, vaccinia virus, and several
RNA viruses. In particular, heφes virus vectors may provide a unique strategy for
persistence of the recombinant IRSBP-1 gene in cells of the central nervous system.
In addition to viral transfer methods, such as those illustrated above, non-viral
methods can also be employed to cause expression of an IRSBP-1 -protein, or an
IRSBP-1 antisense molecule, in the tissue of an animal. Most non- viral methods of
gene transfer rely on normal mechanisms used by mammalian cells for the uptake and
intracellular transport of macromolecules. In preferred embodiments, non- viral gene
delivery systems of the present invention rely on endocytic pathways for the uptake of the subject IRSBP-1 gene by the targeted cell. Exemplary gene delivery systems of
this type include liposomal derived systems, poly-lysine conjugates, and artificial
viral envelopes.
In a representative embodiment, a gene encoding one of the subject IRSBP-1
proteins can be entrapped in liposomes bearing positive charges on their surface (e.g.,
lipofectins) and (optionally) which are tagged with antibodies against cell surface
antigens of the target tissue (Mizuno et al. (1992) NO Shinkei Geka 20:547-551; PCT
publication WO91/06309; Japanese patent application 1047381; and European patent
publication EP-A-43075), and which are incoφorated herein in their entireties. For
example, lipofection of papilloma- virus infected epithelial cells can be carried out
using liposomes tagged with monoclonal antibodies against, for example, squamous
cells.
In similar fashion, the gene delivery system comprises an antibody or cell
surface ligand that is cross-linked with a gene binding agent such as polylysine (see,
for example, PCT publications WO93/04701, WO92/22635, WO92/20316,
WO92/19749, and WO92/06180), and which are incoφorated herein in their
entireties. For example, an IRSBP-1 gene construct encoding an antagonistic form of
the protein, e.g. a dominant negative mutant, can be used to transfect HPV-infected
squamous cells in vivo using a soluble polynucleotide carrier comprising an HPV
viral coat protein conjugated to a polycation, e.g. poly-lysine (see U.S. Patent No.
5,166,320). It will also be appreciated that effective delivery of the subject nucleic
acid constructs via receptor-mediated endocytosis can be improved using agents which enhance escape of gene from the endosomal structures. For instance, whole
adenovirus or fusogenic peptides of the influenza HA gene product can be used as part
of the delivery system to induce efficient disruption of DNA-containing endosomes
(Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) Proc. Natl. Acad. Sci.
89:7934; and Christiano et al. (1993) Proc. Natl. Acad. Sci. 90:2122), and which are
incoφorated herein in their entirety.
In clinical settings, the gene delivery systems can be introduced into a patient
by any of a number of methods, each of which is familiar in the art. For instance, a
pharmaceutical preparation of the gene delivery system can be introduced
systemically, e.g. by intravenous injection, and specific transduction of the gene into
the target cells relies predominantly on the specificity of transfection provided by the
gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional
regulatory sequences controlling expression of the gene, or a combination thereof. In
other embodiments, initial delivery of the recombinant gene is more limited with
introduction into the animal being quite localized. For example, the gene delivery
vehicle can be introduced by catheter (see U.S. Patent No. 5,328,470) or by
stereotactic injection (e.g. Chen et al. (1994) Proc. Natl. Acad. Sci. 91: 3054-3057),
both of which references are incoφorated herein in their entireties.
Moreover, the pharmaceutical preparation can consist essentially of the gene
delivery system in an acceptable diluent, or can comprise a slow release matrix in
which the gene delivery vehicle is imbedded. Alternatively, where the complete gene
delivery system can be produced intact from recombinant cells, e.g. retroviral packages, the pharmaceutical preparation can comprise one or more cells which
produce the gene delivery system. In the case of the latter, methods of introducing the
viral packaging cells may be provided by, for example, rechargeable or biodegradable
devices. The generation of such implants is generally known in the art. See, for
example, Concise Encyclopedia of Medical & Dental Materials, ed. by David
Williams (MIT Press: Cambridge, Mass., 1990); Sabel et al. U.S. Patent No.
4,883,666; Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et al. U.S. Patent
No. 5,106,627; Lim U.S. Patent No. 4,391,909; Sefton U.S. Patent No. 4,353,888; and
Aebischer et al. (1991) Biomaterials 12:50-55), and which are incoφorated herein in
their entireties.
Another aspect of the present invention concerns transgenic animals, such as,
but not limited to animal models for diabetes, obesity, mood disorders, developmental
and, proliferative diseases, that are comprised of cells (of that animal) which contain a
transgene of the present invention and which preferably (though optionally) express
the subject IRSBP-1 in one or more cells in the animal. In embodiments of the
present invention, therefore, the expression of the transgene is restricted to specific
subsets of cells, tissues or developmental stages utilizing, for example, cis-acting
sequences that control expression in the desired pattern. In the present invention, such
mosaic expression of the subject IRSBP-1 proteins can be essential for many forms of
lineage analysis and can additionally provide a means to assess the effects of IRSBP-1
mutations or overexpression that might grossly alter development in small patches of
tissue within an otherwise normal embryo. Toward this end, tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of
the transgene in certain spatial patterns. Moreover, temporal patterns of expression
can be provided by, for example, conditional recombination systems or prokaryotic
transcriptional regulatory sequences.
Genetic techniques that allow for the expression of transgenes can be regulated
via site-specific genetic manipulation in vivo are well known to those skilled in the
art. For instance, genetic systems are available which allow for the regulated
expression of a recombinase that catalyzes the genetic recombination a target
sequence. As used herein, the phrase "target sequence" refers to a nucleotide
sequence that is genetically recombined by a recombinase. The target sequence is
flanked by recombinase recognition sequences and is generally either excised or
inverted in cells expressing recombinase activity. Recombinase catalyzed
recombination events can be designed such that recombination of the target sequence
results in either the activation or repression of expression of the subject receptor. For
example, excision of a target sequence that interferes with the expression of the
receptor can be designed to activate expression of that protein. This interference with
expression of the subject protein can result from a variety of mechanisms, such as
spatial separation of the IRSBP-1 gene from the promoter element or an internal stop
codon. Moreover, the transgene can be made wherein the coding sequence of the
IRSBP-1 gene is flanked by recombinase recognition sequences and is initially
transfected into cells in a 3' to 5' orientation with respect to the promoter element. In
such an instance, inversion of the target sequence will reorient the subject IRSBP-1 gene by placing the 5' end of the coding sequence in an orientation with respect to the
promoter element that allow for promoter driven transcriptional activation.
In an illustrative embodiment, either the cre/loxP recombinase system of
bacteriophage PI (Lakso et al. (1992) Proc. Natl. Acad. Sci. 89:6232-6236; Orb an et
al. (1992) Proc. Natl. Acad. Sci. 89:6861-6865) or the FLP recombinase system of
Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCT
publication WO 92/15694), and which are incoφorated herein in their entireties, can
be used to generate in vivo site-specific genetic recombination systems. Cre
recombinase catalyzes the site-specific recombination of an intervening target
sequence located between loxP sequences. loxP sequences are 34 base pair nucleotide
repeat sequences to which the Cre recombinase binds and are required for Cre
recombinase mediated genetic recombination. The orientation of loxP sequences
determines whether the intervening target sequence is excised or inverted when Cre
recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514);
catalyzing the excision of the target sequence when the loxP sequences are oriented as
direct repeats and catalyzes inversion of the target sequence when loxP sequences are
oriented as inverted repeats.
Accordingly, genetic recombination of the target sequence is dependent on
expression of the Cre recombinase. Expression of the recombinase can be regulated
by promoter elements which are subject to regulatory control, e.g., tissue-specific,
developmental stage-specific, inducible or repressible by externally added agents.
This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element. Thus,
the activation of expression of the recombinant UBC9 gene can be regulated via
regulation of recombinase expression.
Use of the these recombinase system to regulate expression of, for example, a
dominant negative IRSBP-1 gene, or an antisense gene, requires the construction of a
transgenic animal containing transgenes encoding both the Cre recombinase and the
subject gene. Animals containing both the Cre recombinase and the IRSBP-1 genes
can be provided through the construction of "double" transgenic animals. A
convenient method for providing such animals is to mate two transgenic animals each
containing a transgene, e.g., one harboring the IRSBP-1 gene, and the other harboring
the recombinase gene.
One advantage derived from initially constructing transgenic animals
containing a IRSBP-1 transgene in a recombinase-mediated expressible format derives
from the likelihood that the subject IRSBP-1 protein, whether antagonistic or
agonistic, will be deleterious upon expression in the transgenic animal. In such an
instance, a founder population, in which the subject transgene is silent in all tissues,
can be propagated and maintained. Individuals of this founder population can be
crossed with animals expressing the recombinase in, for example, one or more tissues,
or in a developmentally restricted pattern. Thus, the creation of a founder population
in which, for example, an antagonistic IRSBP-1 transgene is silent will allow the study
of progeny from that founder in which disruption of IRSBP-1 -mediated insulin responsiveness in a particular tissue or at certain developmental stages could result in,
for example, a lethal phenotype.
Similar conditional transgenes can be provided using prokaryotic promoter
sequences which require prokaryotic proteins to be simultaneous expressed in order to
facilitate expression of the transgene. Operators present in prokaryotic cells have
been extensively characterized in vivo and in vitro and can be readily manipulated to
place them in any position upstream from or within a gene by standard techniques.
Such operators comprise promoter regions and regions which specifically bind
proteins such as activators and repressors. One example is the operator region of the
lexA gene of E. coli to which the LexA polypeptide binds. Other exemplary
prokaryotic regulatory sequences and the corresponding trans-activating prokaryotic
proteins are given in U.S. Patent No. 4,833,080. Thus, as described above for the
recombinase-mediated activation, silent transgenic animals can be created which
harbor the subject transgene under transcriptional control of a prokaryotic sequence
that is not appreciably activated by eukaryotic proteins. Breeding of this transgenic
animal with another animal that is transgenic for the corresponding prokaryotic trans-
activator, can permit activation of the IRSBP-1 transgene. Moreover, expression of
the conditional transgenes can be induced by gene therapy-like methods (such as
described above) wherein a gene encoding the trans-activating protein, e.g. a
recombinase or a prokaryotic protein, is delivered to the tissue and caused to be
expressed, such as in a cell-type specific manner. By this method, the IRSBP-1 transgene could remain silent into adulthood until "turned on" by the introduction of
the trans-activator.
Additionally, inducible promoters can be employed, such as the tet operator
and the metallothionein promoter which can be induced by treatment with tetracycline
and zinc ions, respectively (Gossen et al. (1992) Proc. Natl. Acad. Sci. 89:5547-5551;
and Walden et al. (1987) Gene 61:317-327), and which are incoφorated herein in
their entirety.
Methods of making knock-out or disruption transgenic animals are also
generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent
knockouts can also be generated, e.g. by homologous recombination to insert
recombinase target sequences flanking portions of an endogenous IRSBP-1 gene, such
that tissue specific and/or temporal control of inactivation of an IRSBP-1 allele can be
controlled as above. Furthermore, the present invention, by making available purified
and recombinant forms of the subject IRSBP-1 proteins, will allow the development
of assays which can be used to screen for drugs which either agonize or antagonize the
function of IRSBP-1 in vivo.
Assays for the measurement of IRSBP-1 can be generated in many different
forms, and include assays based on cell-free systems, e.g. purified proteins or cell
lysates, as well as cell-based assays which utilize intact cells. Such agents can be
used, for example, in the treatment of diabetic or feeding disorders, proliferative
and/or differentiative disorders, and to modulate cellular metabolism. In many drug screening programs which test libraries of compounds and
natural extracts, high throughput assays are desirable in order to maximize the number
of compounds surveyed in a given period of time. Assays which are performed in
cell-free systems, such as may be derived with purified or semi-purified proteins or
with lysates, are often preferred as "primary" screens in that they can be generated to
permit rapid development and relatively easy detection of an alteration in a molecular
target which is mediated by a test compound. Moreover, the effects of cellular
toxicity and/or bioavailability of the test compound can be generally ignored in the in
vitro system, the assay instead being focused primarily on the effect of the drug on the
molecular target as may be manifest in an alteration of binding affinity with other
proteins or change in enzymatic properties of the molecular target. Accordingly,
potential inhibitors of IRSBP-1 function can be detected in a cell-free assay generated
by constitution of a functional IRSBP-1 /target nucleic acid sequence in a cell lysate.
Another aspect of the present invention concerns three-dimensional molecular
models of the subject IRSBP-1 proteins, and their use as templates for the design of
agents able to inhibit at least one biological activity of the IRSBP-1 protein. An
integral step to designing inhibitors of the subject IRSBP-1 involves construction of
computer graphics models of the IRSBP-1 that can be used to design pharmacophores
by rational drug design. For instance, for an inhibitor to interact optimally with the
subject protein, it will generally be desirable that it have a shape which is at least
partly complimentary to that of a particular binding site of the protein, as for example
those portions of the human IRSBP-1 that are involved in recognition of a particular region of a nucleic acid sequence. Additionally, other factors, including electrostatic
interactions, hydrogen bonding, hydrophobic interactions, desolvation effects, and
cooperative motions of ligand and enzyme, all influence the binding effect and should
be taken into account in attempts to design bioactive inhibitors.
A computer-generated molecular model of the subject protein can be created
by homology modeling, and then calculate the structure of the protein and velocities
of each atom at a simulation temperature. Computer programs for performing energy
minimization routines are commonly used to generate molecular models. For
example, both the CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and
AMBER (Weiner et al (1981) J. Comput. Chem. 106: 765) algorithms handle all of
the molecular system setup, force field calculation, and analysis (see also, Eisenfield
et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J Pharm. Belg. 46:49-54;
Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111;
Pedersen (1985) Environ Health Perspect. 61:185-190; and KM et al. (1991) J.
Biomol. Struct. Dyn. 9:475-488), and which are incoφorated herein in their entirety.
Moreover, a number of programs are presently available for virtual design of
IRSBP-1 protein inhibitors. For instance, the increasing availability of
biomacromolecule structures of potential pharmacophoric molecules that have been
solved crystallographically has prompted the development of a variety of direct
computational methods for molecular design, in which the steric and electronic
properties of substrate binding sites are used to guide the design of potential inhibitors
(Cohen et al. (1990) J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol. 161: 269-288; Desjarlais (1988) J. Med. Cam. 31 : 722-729; Bartlett et al. (1989) Spec.
Publ., Roy. Soc. Chem. 78: 182-196; Goodford et al. (1985) J. Med. Cam. 28: 849-
857; Desjarlais et al. J. Med. Cam. 29: 2149-2153), and which are incoφorated herein
in their entireties. Most algorithms of this type provide a method for finding a wide
assortment of chemical structures that are complementary to the shape of a binding
site of the subject protein. Each of a set of small molecules from a particular data¬
base, such as the Cambridge Crystallographic Data Bank (CCDB) (Allen et al. (1973)
J. Chem. Doc. 13: 119), is individually docked to a nucleic acid or other ligand
binding site of the IRSBP-1 protein in a number of geometrically permissible
orientations with use of a docking algorithm. In an illustrative embodiment, a set of
computer algorithms called DOCK, can be used to characterize the shape of
invaginations and grooves that form the active sites and recognition surfaces of the
subject protein (Kuntz et al. (1982) J. Mol. Biol. 161: 269-288). The program can also
search a database of small molecules for templates whose shapes are complementary
to particular binding sites of the protein (Desjarlais et al. (1988) J. Med. Chem. 31:
722-729). These templates normally require modification to achieve good chemical
and electrostatic interactions (Desjarlais et al. (1989) ACS Symp. Ser. 413: 60-69).
However, the program has been shown to position accurately known cofactors for
inhibitors based on shape constraints alone.
Other exemplary virtual drug design programs include GRID (Goodford
(1985, J. Med. Chem. 28:849-857); Boobbyer et al. (1989) J. Med. Chem. 32:1083-
1094), CLIX Lawrence et al. (1992) Proteins 12:31-41), GROW (Moon et al. (1991) Proteins 11:314-328), the multiple copy simultaneous search method (MCSS)
(described by Miranker et al. (1991) Proteins 11 : 29-34), and NEWLEAD (Tschinke
et al. (1993) J. Med. Chem. 36: 3863,3870), which are incoφorated herein in their
entireties.
The present invention is further illustrated by the following examples, which
are provided by way of illustration and should not be construed as limiting. The
contents of all references, published patents and patents cited throughout the present
application are hereby incoφorated by reference in their entirety.
Example 1: Cloning of cDNAs containing an IRSBP coding sequence.
An isolated nucleic acid with the IRE associated with the IGFBP-3 gene and
comprising the nucleotide sequence SEQ ID NO: 1 was multimerized as follows.
Two antiparallel oligonucleotides, one representing the sense strand of SEQ ID NO: 1,
and the other its antisense complement were annealed. The resulting double-stranded
DNA was phosphorylated with T4 polynucleotide kinase, concatemerized with T4
DNA ligase at 22°C for 5 minutes, and electrophoretically fractionated on a
polyacrylamide gel. A fragment containing three contiguous copies of the annealed
oligonucleotides was inserted into the pHISi reporter vector (Clontech, Palo Alto,
CA), and transformed into the yeast Saccharomyces cerevisiae strain YM4271
(Clontech, Palo Alto, CA).
Southern blotting confirmed the integration of the multimerized IRE nucleic
acid sequence into the yeast genome. Southern blotting was carried out using prehybridization and hybridization buffers containing 1%> w/v BSA, 1 mM EDTA. 0.5
M NaHPO4, pH 7.2, and 1% w/v SDS. Hybridization was done with the radiolabeled
multimerized IRE of IGFBP-3 with 32P as the radiolabel. The radioactive probe was
added at a concentration of 1-2 x 106 cpm/ml. After hybridization, blots were washed
twice with 2X SSC, 0.1% SDS for 30 mins., followed by a 30 min. wash with 0.1X
SSC, 0.1%) SDS at 50°C, and autoradiography. The procedure was as described in
Ausubel et al. (1993) incoφorated herein by reference in its entirety
After confirming the integration of the IGFBP-3 IRE nucleic acid sequence
(SEQ ID NO: 1) into the yeast genome, a rat liver cDNA library was screened using a
yeast one-hybrid system. The yeast one-hybrid system is an in vivo genetic assay that
uses growth selection based on reconstruction and activation of the nutritional reporter
gene HIS3. To this aim, a nucleic acid fragment comprising three contiguous repeats
of the IGFBP-3 IRE (SEQ ID NO: 1) was inserted in the region 5' upstream of a H7S3
reporter under the control of a G-___ -responsive promoter. The construct was
transformed into yeast cells.
The one-hybrid screening procedure used herein is described by Chong et al,
(1995) Cell 80: 949-957 and Li & Herskowitz, (1993) Science 1252: 1870-1873,
incoφorated herein by reference in their entireties. To search for genes encoding
insulin-responsive binding proteins, yeast containing the IRE target nucleic acid
sequence (SEQ ID NO: 1) were transformed with DNA purified from an activation
domain (AD) library that contained fusions between a target-independent activation
domain (GAL4 AD) and cDNA derived from a normal rat liver. Colonies of yeast were selected on His'Leu" plates and the plasmids were isolated from the yeast.
Positive clones were confirmed by retransformation of the cDNA into yeast
containing a Lac Z reporter gene with tandem repeats of the IRE target nucleic acid,
and tested for transcriptional activation of the GAL4 promoter.
Seventy-nine clones were found which grew on His'Leu" plates containing 15
mM 3-AT (3-amino-l,2,4-triazole, Sigma Chemical Co., St. Louis, MO). The
plasmids from those clones were isolated as described by Hoffman & Winsten, (1987)
Gene 57: 267-272 and transformed into E. coli according to Kaiser & Auser (1993)
Biotechniques 14: 552. The isolated cDNAs were sequenced using automated
sequencing. The sequences were identified by an NCBI BLAST search for similarity
to sequences reported in GenBank.
Sixty-six out of the seventy-nine clones were identified as secretory or
structural proteins, membrane proteins, or enzymes. Two of the clones contained
cDNAs encoding the known transcription factors NFkB p65 and HBPl. Eleven out of
seventy-nine clones contained novel sequences.
Gel shift mobility assays.
cDNAs from the 11 novel clones were subcloned into the plasmid pSPUTK
(Stratagene). The coding regions of the cDNA clones were translated into protein
using coupled transcriptional in vitro translation as described by Hook et al. (1996)
Peptide Research 9: 183-187 and incoφorated herein in its entirety. After translation,
the proteins were tested for their ability to bind to the IRE element of IGFBP-3 (SEQ
ID NO: 1) by gel mobility shift analysis. Gel mobility shift assays were done essentially as described in Villafuerte et
al, (1997) J. Biol. Chem. 272: 5024-5030. 32P-ATP-labeled oligonucleotides
corresponding to nucleotide positions -1150 to -1117 bp-fragment of the rat IGFBP-3
(SEQ ID NO: 1) gene were incubated with the proteins derived from the cDNA clones
at concentrations of approximately 20 ng protein per lane in 25 μl of binding buffer
containing 10 mM Tris, pH 7.6, 50 mM KCI, ImM EDTA, 0.5 mM dithiothreitol,
0.2%) Nonidet P-40, 20 μg of bovine serum albumin, 36 μg of salmon sperm DNA,
and 10%) glycerol at 25°C for 20 mins. Incubations were carried out with or without
unlabeled competitor DNA. Protein-DNA complexes were separated from free probe
on 6% polyacrylamide gels in 0.25.x TBE at 12 V/cm for 2-3 hours, and visualized by
autoradiography.
One clone of the eleven, clone 52 (SEQ ID NO: 2, shown in Fig. 1) contained
a 952 bp cDNA insert that encoded a polypeptide (SEQ ID NO: 3, shown in Fig 2)
that formed a DNA-protein complex. No other clones contained cDNA sequences
that after translation with reticulocyte lysate, could produce the gel shift in the gel
mobility shift experiments as shown in Fig. 12. The clone 52 nucleic acid sequence
(SEQ ID NO: 2) comprises 952 bp of sequence capable of hybridizing to a second
IRSBP-1 nucleic acid sequence (SEQ ID NO: 5 shown in Figs. 4A, B) and to a region
of the human genomic sequence having the ATCC accession number AC005237.
The specificity of binding was established by using labeled or unlabeled (25
pmoles) IGFBP-3 IRE (SEQ ID NO: 1) as shown in Fig. 14. Excesses of unlabeled
IGFBP-3 IRE and NFKB (Stratagene,La Jolla, CA) were incubated with about 20 ng protein per lane. The double-stranded DNA competitors were added at molar
concentrations between about 10-fold and about 100-fold greater than labeled
oligonucleotides, or 2.5 pmole, 12.5 pmole and 25 pmole, and electrophoresed on a
6%> polyacrylamide gel.
cDNA coding sequences were also expressed as thioredoxin (Trx) fusion
proteins in E. coli. The clone 52 cDNA (SΕQ ID NO: 2) was subcloned in-frame into
a prokaryotic expression vector (pΕT-32a from Novagen, Madison, WT), transformed
into the AD494(DE3) strain of E. coli, and grown in culture until OD6000.6. IPTG
(isopropyl-β-thiogalactopyranoside) was added to a final concentration of 1 mM three
hours before harvest. The thioredoxin-clone 52 fusion protein was purified by affinity
chromatography on immobilized His-bound metal chelation resin (Novagen), and
used in a gel-shift assays, as shown in Figs. 13 and 14. The fusion proteins were
tested in additional gel mobility shift experiments with the IGFBP-3 IRE nucleic acid
(SEQ ID NO: 1) as describe above. While not wishing to be bound by any particular
theory, since the IRE of IGFBP-3 (SEQ ID NO: 1) includes an
AGGAAAGTCTCCTT palindrome, and the leucine zipper encourages dimerization,
gel shift bands seen in Figs. 12-14 reflect binding of IRSBP-1 to the IGFBP-3 IRE as
a homodimer and monomer, respectively. Competiton assays demonstrate that IREs
associated with other insulin-responsive genes compete with the IRE of IGFBP-3
(SEQ ID NO: 1) for binding to IRSBP-1, as shown in Fig. 15. Example 2: Sequencing of cDNA clones
Single-strand sequencing of cDNA clones was with an Applied Biosystems
Automated DNA Sequencer (Applied Biosystems,, Foster City, CA) and a PCR-based
fluorescent dideoxy method, according to the recommendations of the manufacturer.
The partial rat IRSBP-1 cDNA clone 52 (SEQ ID NO: 2) is shown in Fig. 1. The
longest open-reading frame amino acid sequence derived from SEQ ID NO: 2 is SEQ
ID NO: 3, as shown in Fig.2.
Example 3: Expression of clone 52 mRNAs using Northern blot analysis
Total RNA was isolated from cultured hepatic non-parenchymal cells using a
Tri-Reagent Kit (Molecular Research Center, Cincinnati, OH) according to the
manufacturer's protocol. Clone 52 cDNA (SEQ ID NO: 2) containing the 952 bp
cDNA nucleic acid obtained from the yeast one-hybrid screen as described in
Example 1, was random primer labeled with [32P] dCTP and used as a probe to
hybridize with RNA electrophoresed on 1.2% formaldehyde-agarose gel using
protocols described by Ausubel
et al. (1993) and incoφorated herein by reference in its entirety.
Northern analysis showed that hepatic cells expressed an mRNA species of at
least 3.5 kb in length. Example 4: Interactions with other insulin-response binding proteins
CHO cells were co-transfected with IGFBP-1 IRE reporter or PEPCK reporter
constructs and combinations of vectors containing an IRSBP-1 cDNA (SEQ ID NO:
2) or DNA encoding for HBPl. Cells were prepared and transfected as described in
Example 6. Luciferase activity was measured as described in Example 6. The IREs
identified from the IGFBP-land PEPCK genes are described in Cichy et al (1998) J.
Biol. Chem. 273: 6483-6487; O'Brien et al. (1994) J. Biol. Chem. 269: 30419-30428
and incoφorated herein in their entireties. HBP-1 induced the formation of luciferase
in the absence of IRSBP-1 or insulin, (as shown in Fig. 16). Cotransfection with the
IRSBP-1 -encoding cDNA reduced the activation of the IGFBP-1 IRE and decreased
the luciferase expression. Induction of cellular IRSBP-1 by insulin reduced luciferase
levels still further.
The IREs were used to construct multimers as described in Example 1,
inserted into the pGL3 promoter vector (Promega., Madison, WI) and transfected into
CHO cells. Such cells showed an increase in luciferase reporter activity augmented
by the addition of insulin to the media, as shown in Fig. 17.
Example 5: Screening of bacteriophage human and rat cDNA libraries
Because the 952 bp IRSBP-1 -encoding clone 52 cDNA (SEQ ID NO: 2)
encodes a protein (SEQ ID NO: 3) that contains a binding domain with limited
transactivating properties, at least one longer cDNA sequence (SEQ ID NOS: 5 and 6)
were obtained by screening a lambda bacteriophage rat brain cDNA library (Uni-Zap XP library, Stratagene, La Jolla, CA) and then extending the cDNAs thus obtained by
5' RACE.
The 952 bp clone 52 cDNA of SEQ ID NO: 2 was used as the nucleic acid
probe to screen about 108 plaques. The phage and host E. coli were spread on agarose
plates and incubated to form plaques, nitrocellulose filters were applied, and the phage
particles and unpackaged DNA were adsorbed to the filter to produce a replica of the
plate surface. The filters were treated with NaOH to denature the phage DNA, which
was then hybridized with the cDNA probe. After isolation of positive plaques that
hybridized to the probe, the pBluescript phagemid was rescued with VCSM12 helper
phage. The final product is a double-stranded pBluescript phagemid with an inserted
DNA. Subsequent rescreening of the library combined with 5' RACE extensions
yielded isolated nucleic acids comprising the nucleotide sequence of SEQ ID NO: 5,
as shown in Figs. 4A and 4B, or fragments, variants or derivatives thereof, such as
SEQ ID NO: 6. Automated sequencing confirmed regions within SEQ ID NOS: 5 and
6 having substantially similarity to the nucleotide sequence of clone 52 (SEQ ID NO:
2).
To obtain an isolated nucleic acid encoding a region of the human IRSBP-1, a
3404 bp rat cDNA (SEQ ID NO: 14) was used to screen a human lambda phage
cDNA library (Uni-Zap XR human liver cDNA library, Stratagene, La Jolla, CA).
Two clones hybridizing to the 3404 bp rat IRSBP-1 -specific probe were obtained.
One was about 2480 bp long and another clone was about 1700 bp long. Subsequent
5' extensions were obtained by 5' RACE techniques, using the SMART RACE cDNA amplification system from Clontech, Inc, and polyA mRNA isolated from the human
cerebellum, until the 4584 bp sequence (SEQ ID NO: 7, shown in Fig 6) was
obtained. Comparison of the human IRSBP-1 nucleic acid sequence (SEQ ID NO: 7)
the rat IRSBP-1 sequence (SEQ ID NO: 5) showed at least 75% similarity when
aligned.
Comparison of the human IRSBP-1 nucleic acid sequence (SEQ ID NO: 7)
with the human genomic DNA sequence Accession No. AC005237 showed that the
human cDNA sequence SEQ ID NO: 7 was derived from at least 26 exons (SEQ ID
NOS: 16 - 41, as shown in Table 1).
Table 1. Exon positions of the human IRSBP-1 nucleic acid sequence SEQ ID
NO: 7
Figure imgf000101_0001
Figure imgf000102_0001
Example 6: Metabolic Activity of the IRSBP-1 Protein
To investigate the biological effects of IRSBP-1, the partial cDNA sequence
encoding IRSBP-1 (SEQ ID NO: 14 shown in Fig. 11) was subcloned into the pCMV-
Tag epitope tagging mammalian expression vector (Stratagene, La Jolla, CA) and
transfected into L6 myoblasts to establish IRSBP-1 -stably transfected cell lines.
G418-resistant clones were isolated and tested for [3H] 2-deoxyglucose uptake (Fig.
17).
The insulin induction of the expression of the IRSBP-1 gene was shown by
Northern analysis wherein from about 10"9 M to about 10"7 M insulin increased the
formation of IRSBP-1 -specific mRNA of about 3.5 kb, as shown in Fig. 18 A. Normal
liver parenchymal cells have elevated levels of the IRSBPl -specific mRNA compared
to cells from a rat having diabetes mellitus, also as shown in Fig. 18 A.
In addition, the 3.4 kb cDNA region (SEQ ID NO: 14 as shown in Fig. 11)
(ATCC ) and derived from SEQ ID NO: 5 and obtained from the lambda
bacteriophage cDNA library screening, was also tested for the ability to induce a
luciferase reporter gene expressed under the regulatory control of the IGFBP-3 IRE
(Fig. 18B). cDNAs found to produce proteins that bound to IRE sequences were subcloned into plasmid pTARGET, a mammalian expression vector containing Kozak
initiation sequences (Promega, Madison, WI). cDNAs subcloned into pTARGET and
a chimeric construct containing the IGFBP-3 promoter region (SEQ ID NO: 1)
attached to the firefly luciferase reporter gene (pGL2-Basic, Promega) were co-
transfected into Chinese hamster ovary (CHO) cells. The chimeric construct contains
three tandem copies of the IRE region of the IGFBP-3 promoter sequence (SEQ ID
NO: 1) attached to the pGL3 promoter vector (Promega, Madison, WI). The cloning
of the IGFBP-3 promoter region into pGL2 -Basic was as described in Villafuerte et
al, (1997) J. Biol. Chem. 272: 5024-5030.
Transient transfections of the IGFBP-3 IRE-luciferase construct together with
the cDNA clones in pTARGET were undertaken with CHO cells when the cells
reached 60-70%> confluence. Lipofectin (Life Technologies, Rockvillc, MD) and
DNA complexes were mixed at a 15 μg to 2.5 μg ratio and incubated with the cells
overnight. Medium was replaced with serum-free DMEM medium, with or without
the addition of 10"6M human recombinant insulin (Life Technologies, Rockville, MD)
for 24 hours, and cell extracts were assayed for gene activity using a luciferase assay
system (Promega, Madison WI, and following the manufacturer's recommended
protocol) and measured using a Microsure 100 luminometer (LKB Wallac, Turku,
Finland). All readings were within the linear range of the instrument when compared
with known luciferase concentrations.
The 3.4 kb cDNA region (SEQ ID NO: 14) increased IGFBP-3 IRE-induced
reporter activity 4-fold, and addition of 10"6 M insulin increased the activity further by 12-fold (Fig. 17). There was a 110% increase in glucose uptake in cells stably
transfected with nucleic acid SEQ ID NO 14, as compared to wild type cells as shown
in Fig. 18. With the addition of 10'6 M insulin, there was a 30%> further increase in
glucose uptake in wild type cells and a 16%> further increase in clone 52-transfected
cells. These experiments indicate that IRSBP-1 is functionally insulin-mimetic.
Example 7: Determination of IRSBP-1 biological activity
To examine the ability of clone 52 to allow storage of substrates related to
insulin action, wild type L6 myoblasts and the clone 52-stably transfected cells were
stained with the periodic acid-Schiff base stain specific for glycogen. As shown in
Fig 19, the IRSBP-1 -stably transfected cell line showed intense periodic acid Schiff
base (+) materials in the cytoplasm, consistent with glycogen accumulation in the
clone 52-transfected cells. Thus, clone 52 mediates storage of ingested substrates, and
it can act as an insulin substitute at the target organ level.
Example 8 : IRSBP-1 expression correlates to tissue-specific glucose utilization
In vivo disposition of glucose in various organs and tissues was studied.
Following oral glucose load, tracer and forearm catheterization techniques showed
that glucose is taken up by splanchnic tissues, including liver and gut (29%), muscle
(26%), brain (23%), kidney (7%). heart (4%), fat (3%) and others (8%).
The RNase protection assay used a Kpnl-Xhol fragment (SEQ ID NO: 4,
shown in Fig. 3) from clone 52 cDNA (SEQ ID NO: 1) inserted in pGem7Z and transcribed in vitro to give a riboprobe (antisense probe). The assay was carried out
using the Hybspeed RPA Kit (Ambion, Inc., Austin, TX) according to the
manufacturer's directions. Briefly, RNA extracted from various tissues of the rat were
hybridized with the [32P] UTP-labeled clone 52 probe-derived RNA at 45°C in the
presence of 40 mM PIPES and 80%> formamide. The unhybridized probe was
degraded with RNase A and T, and protected RNA was purified and resolved on a
sequencing gel.
Using an ribonuclease protection assay, both insulin-dependent (muscles,
adipose tissue, liver) and non-insulin dependent tissues or organs in which insulin
were shown not to be required for utilization of glucose (brain, kidney, gut) expressed
IRSBP-1 mRNA (Figs. 20 and 21). Thus, the common denominator for IRSBP-1
expression is the dependence of the organ or tissues on glucose for energy utilization.
At the tissue level, studies have shown that when comparing the glucose
metabolic rates of different adipose regions, measured as the sum of glucose
converted to CO2, triglycerides and lactate, the mesenteric fat cells metabolized
significantly more glucose per cell than other fat depots. The hierarchy of the glucose
metabolic rate in the different adipose depot is as follows: mesenteric >
retroperitoneal > epididymal > subcutaneous fat. As shown in Figs. 20 and 21,
IRSBP-1 expression is highest in mesenteric fat, followed by retroperitoneal,
epididymal and subcutaneous fat. Thus, the mRNA abundance of IRSBP-1 in adipose
tissues reflects the glucose utilization rates in those tissues. Since diabetes is
characterized by resistance to insulin action on glucose uptake and utilization in adipocytes and skeletal muscle, the studies illustrated in Figs. 18 - 21 indicate that
over-expression of IRSBP-1 may overcome the problems of both poor glucose uptake
and poor glucose utilization.
Example 9: IRSBP-1 and its role in the onset and maintenance of obesity
IRSBP-1 could also be used for the treatment of obesity and complications
associated with obesity. The organ systems and the specific diseases associated with
obesity include the following: (1) cardiovascular system - hypertension, congestive
heart failure, cor pulmonale, varicose veins, pulmonary embolism, coronary heart
disease; (2) Endocrine-insulin resistance, glucose intolerance, type II diabetes
mellitus, dyslipidemia, polycystic ovary syndrome, infertility, amenorrhea; (3)
Musculoskeletal - immobility, degenerative arthritis, low back pain; (4) Integument -
venous stasis of legs, cellulitis, intertrigo, carbuncles; (5) Respiratory system -dyspnea
and fatigue, obstructive sleep apnea, hypoventilation (pickwickian) syndrome; (6)
Gastrointestinal - gastroesophageal reflux disease, hepatic steatosis, nonalcoholic
steatohepatitis, cholelithiasis, hernias, colon cancer; (7) Psychosocial - work
disability, depression; (8) Genitourinary - urinary stress incontinence, hypogonadism,
breast and uterine cancer; (9) Neurologic - stroke, meralgia paresthetica, idiopathic
interacranial hypertension. Any of the above conditions, when associated with
obesity, could be used as indications for the effective use of IRSBP-1 agonists or
antagonists.
Using in-situ hybridization to localize IRSBP-1 mRNA in the brain, IRSBP-1 expression was detected in the areas of the brain known to be involved in ingestive,
autonomic and neuroendocrine functions of feeding and satiety, as described in the
Examples 10 - 12 below. Regulation of body weight requires a balance among energy
intake, expenditure, and storage. The brain appears to define the set point around
which body weight is regulated. The levels of IRSBP-1 mRNA in the lateral
hypothalamus and the nucleus of the solitary tract are differentially regulated in obese
as compared to lean Zucker rats, showing a significant interactive role of IRSBP-1 in
modulating body weight.
Example 10: IRSBP-1 expression is detected in many areas of the brain,
including areas associated with feedings and satiety.
In-situ hybridization of rat brains using * S-labeled IRSBP-1 riboprobe.
Rat brains were obtained and fixed by immersion in 4%> paraformaldehyde in
0.1 M NaPO4, sectioned on a cryostat to 5-10 μM thickness, and mounted on slides.
Sense and antisense 35S-labeled IRSBP-1 riboprobes were generated by in vitro
transcription with 35S-UTP, and derived from the linearized fragment SEQ ID NO: 4
of IRSBP-1 cDNA. Following proteinase K treatment, prehybridization of the various
brain sections was performed for 3 hrs at 42 C, in a buffer containing 10 mM DTT,
0.3 M NaCl, 20 mM Tris pH8, 5 mM EDTA, lx Denhardt's, 10% Dextran sulfate, and
50% formamide. This was followed by addition of 3SS-labeled probe (600,000
cpm/slide) and tRNA (200 μg/ml) for hybridization. Hybridization was done
overnight at 55 C; slides were then washed, treated with RNAse A, dehydrated, and coated with photographic emulsion. The slides were exposed and developed after 4-
12 weeks. Both sagittal (Fig. 22) and coronal (Fig. 23) sections of brains from normal
Sprague-Dawley rats, obese fa/fa Zucker rats and lean fa/+ Zucker rats were
compared.
Example 11 : IRSBP-1 is expressed in the hypothalamus and nucleus of the
solitary tract
Information about the qualities of food are relayed by the primary senses of
smell, sight, and taste to the nucleus of the solitary tract in the medulla. The nucleus
of the solitary tract (NTS) integrates afferent and efferent information and connects
with nearby vagal and sympathetic centers that control metabolism in peripheral
organs. The NTS communicates rostrally with the central nucleus of the amygdala.
The central nucleus is integrated into the limbic and autonomic systems throughout
the brain, including the hypothalamus (paraventricular nucleus - PVN, lateral
hypothalamus - LH, ventromedial hypothalamus - VMH) and brainstem. Stimulation
of PVN, VMH or LH alters sympathoadrenal and vagal activities
Brain lesions of the ventromedial hypothalamus produced hypeφhagic
obesity. Lesions of the lateral hypothalamus caused hypophagia and weight loss. The
central administration of insulin also changed the level of defended body weight
rather than a simple suppression of food intake. Thus, the hypothalamic pathways
that are sensitive to adiposity signals have anatomical connections with caudal
brainstem neurons (solitary tract nucleus) that respond to meal-related signals and regulate meal size.
The results from in-situ hybridization with a IRSBP-1 riboprobe indicated that
IRSBP-1 mRNA is highly expressed in multiple areas of the hypothalamus. As
shown in Figs.. 22 - 25, a sagittal cut through the thalamic portion of the diencephalon
of a normal Sprague Dawley rat showed that IRSBP-1 is expressed in the
ventromedial and dorsomedial hypothalamus, arcuate nucleus and periventricular
nucleus (Fig. 22). A coronal cut through the thalamic region also showed that IRSBP-
1 mRNA is expressed in the lateral hypothalamus (Fig. 23). Furthermore, comparison
of the brain sections from obese and lean Zucker rats showed that the number of silver
grains, representing IRSBP-1 mRNA, is higher in the obese than the lean rats (Fig.
24), showing that IRSBP-1 is regulated in the feeding center of the brain, and has a
potential role in regulating weight of animals.
Since all of the information from the neural pathways related to feeding and
satiety are integrated in the nucleus of the solitary tract (NTS) of the hindbrain, the
expression of IRSBP-1 in the NTS was investigated. As shown in Fig. 25, a sagittal
cut through the medullary section of the brain showed that IRSBP-1 mRNA is higher
in the NTS of the lean rats, compared to the obese Zucker rats. This further shows
that IRSBP-1 is involved not only in altering sympathoadrenal and vagal activities
throughout the hypothalamus, but that it has also a potential role in integrating the
limbic and autonomic systems involved in maintenance of energy balance.
Differential IRSBP-1 expression was also seen in the in situ hybridization,
with labeled antisense IRSBP-1 probe, of the pyramidal tract and decussations of the pyramidal tract of obese and lean Zucker rats (Figs. 26 and 27).
Example 12: Role of IRSBP-1 in the glucose and insulin regulation of food
intake and body weight
IRSBP-1 mRNA is highly expressed in the olfactory bulb and amygdala of
normal rats. The glucostatic hypothesis proposed that short-term changes in plasma
glucose levels can be detected by the brain and will lead to alterations in food intake.
Although virtually all neurons require glucose, only select populations in various
areas of the brain respond to changes in glucose concentration by changes in their
firing rates. An increase in plasma glucose leads to increased plasma norepinephrine
levels and sympathetic nervous system activation, and this is mediated by the
glucoresponsive neurons in the hypothalamus.
Since IRSBP-1 -specific mRNA appears to be concentrated in selected areas of
the hypothalamus, and acts to increase glucose uptake and utilization, as shown in
Example 12 above, it is likely that IRSBP-1 has a critical role in mediating the
autonomic nervous system activation associated with food intake. With a diet high in
fat and sucrose, the plasma norepinephrine response to glucose is predictive of later
weight gain, with a high responder becoming obese and a low responder becoming
resistant to obesity. IRSBP-1 appears to be a factor that determines basal glucose
metabolism in the peripheral tissues, and is concomitantly expressed in regions of the
brain that modulate food intake. The efficiency of cellular functions related to
IRSBP-1 activity will have a significant impact on overall energy homeostasis. In addition to the roles of IRSBP-1 in affecting glucose metabolism, synthesis
of IRSBP-1 is stimulated by addition of insulin; therefore we also need to consider the
effect of insulin action on glucose utilization of the brain. Insulin receptors have been
localized to the olfactory bulb, hypothalamus, hippocampus, cerebellum, cerebral
cortex, and hindbrain. At the cellular level, insulin modulates expression of
hypothalamic neuropeptides, inhibits reuptake of norepinephrine, and enhances
endogenous β-adrenergic activity. Central administration of insulin decreases food
intake and body weight.
The results showed that IRSBP-1 mRNA is highly expressed in the olfactory
bulb and amygdala of normal rats (Figs. 28 and 29), and is also expressed in the
cerebral cortex, cerebellum and coφus callosum. Lesions of the posterodorsal aspects
of the amygdala have been associated with hyperinsulinemia, hypeφhagia, and
obesity without the preference for particular food that characterized other brain lesion-
induced obesity. The involvement of the olfactory system with high levels of
expressed IRSBP-1 (Figs. 28 and 29) in the primary sense of smell, and the affective
component associated with eating under the control of the limbic system, including
the amygdala and coφus callosum. IRSBP-1 therefore may affect multiple aspects of
brain function associated with feeding and satiety.
Example 13: IRSBP-1 is targeted to pancreatic beta cells of the islets of
Langerhans
IRSBP-1 appears to mediate a significant repertoire of insulin's cellular effects. Glucose is the principal regulator of insulin secretion from pancreatic beta
cells, and the kinetic response of insulin to glucose is biphasic in nature. A rapid
secretory burst begins within 1 min and decreases over the next 3 to 5 mins. The
second phase is characterized by a gradual increase in insulin levels over 5-10 mins,
which continues for the next hour. Many type II diabetics have a marked reduction in
first phase insulin secretion.
A polyclonal antibody raised against the epitopic region (SEQ ID NO: 13) of
the rat IRSBP-1 polypeptide SEQ ID NO: 11 and capable of detecting the presence of
the IRSBP-1 protein of at least rat or human, was used in immunohistochemical
staining of the pancreas to detect the cellular location of IRSBP-1. As shown in Fig.
30, IRSBP-1 expression is strongest in the beta cells of the islets of Langerhans.
Insulin acts on beta cells to regulate insulin secretion, insulin synthesis, and
glucose sensing/utilization. Functional insulin receptor and IRS-1 have been
identified in beta cells. IRSBP-1 is also present in the beta cells of the islets of
Langerhans (Fig. 30). Insulin secretion in isolated beta cells is mediated via the
insulin receptor. Mice with knockout of the beta cell insulin receptor lose their first-
phase insulin response to glucose, but not to other secretagogue like arginine, similar
to type II diabetes. It has been shown that glucose stimulation of beta cells activates
the insulin receptor in a similar fashion as insulin itself. Thus, the presence of insulin
receptors and normal insulin signaling are necessary for the physiological response of
beta cell secretion to occur.
A defect in insulin action may be the common molecular defect that links impaired insulin secretion in beta cells and insulin resistance at the peripheral tissues.
Since IRSBP-1 is an effector molecule by which the major signal transduction
pathways of insulin converge, and IRSBP-1 appears to mediate a significant repertoire
of insulin's cellular effects. IRSBP-1 may increase insulin synthesis (through
stimulation of protein synthesis), and increase glucose sensing and utilization in beta
cells (through increased uptake), leading to restoration of physiological insulin
response to glucose in diabetes and insulin resistance.
Example 14: Immunodetection of IRSBP-1 expresson in pancreatic renal,
vascular and neural tissues
Since glucose is the principal regulator of insulin secretion from pancreatic
beta cells, and clone 52 mimics insulin action on glucose transport and metabolism,
we also determined the expression of IRSBP-1 in the pancreas.
Fixed and paraffin embedded tissue was deparaffinized, rehydrated, treated
with proteinase K at 50 μg/ml for 10 min at room temperature, washed with PBS, and
blocked with a l% gelatin/PBS mixture for 20 mins. The primary antibody, anti-rat
IRSBP-1 peptide antibody was added at 1:200 dilution in 1% BSA / lx PBS, and the
sample was incubated in a humid chamber for 1 hour at room temperature. After
washing, a biotinylated secondary antibody was added at 1:400 dilution, and
incubated with the sample for 30 min. Color development was performed with the
ABC-vector Red complex from an alkaline phosphatase standard kit. The slide was
subsequently counterstained with Gill's hematoxylin, dehydrated and mounted. As shown in Fig. 30, immunostain of pancreas showed intense accumulation
of IRSBP-1 is the cytoplasm of the islet of Langerhans. In particular, the insulin-
secreting β cells of the pancreas, which comprised 74% of the islet mass and is central
in location, expressed abundant IRSBP-1, as described in Example 13 above. IRSBP-
1 expression was also detected by immunodetection in the mesangium of the
glomerulus of the kidney (Fig. 31), in vascular endothelial cells (Fig. 32) and in
neuronal cells (Fig. 33).
A Western blot analysis of the expressed proteins from cultured human
vascular endothelial cells, treated or untreated with insulin, probed with a polyclonal
anti-rat IRSBP-1 antibody shows that insulin induces the formation of IRSBP-1 in
such cells (Fig. 34). This experiment further shows that a rabbit anti-rat IRSBP-1
antibody will cross-react with an IRSBP-1 of a different species. The localization of
IRSBP-1 to the endothelium, mesangium and neurons has implications on the
development of vascular, renal and neuropathic complications of diabetes. Insulin
causes endothelium-derived nitric oxide-dependent vasodilation and modulates
vascular tone. Mesangial cell proliferation and expansion is the initial event in the
development of diabetic nephropathy. Thus, the action of IRSBP-1 in mediating
insulin action in the endothelium will increase the vasodilatory capacity of the blood
vessels, and decrease blood pressure and the subsequent onset of atherosclerosis. The
antiproliferative action of IRSBP-1 will also decrease the capacity of mesangium to
expand and delay the development of diabetic nephropathy. Example 15: Determining the position of IRSBP-1 in the insulin signaling
pathway
Agarose conjugates of both anti-Aktl (PkB) and anti-Erk2 (MAP kinase)
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with nuclear
extracts from COS7 cells, then precipitated with the agarose, the protein eluted and
western blotted with anti-IRSBP-1 peptide polyclonal antibody. As shown in Fig. 33
(left panel), the 98 kDa IRSBP-1 protein co-immunoprecipitated with both Aktl and
Erk2, indicating that in vivo, the two major signaling pathways of insulin action
probably converge on IRSBP-1. To test for the specificity of the
immunoprecipitation, the blot was reprobed with an anti-Erk polyclonal antibody.
The results showed that the lanes that precipitated with agarose conjugates of Erk2
antibody and the COS7 extracts contained the 40 kDa Erk2 protein, but not the lanes
precipitated with Akt antibodies.
To confirm further the specificity of the immunoprecipitation, the blot was
stripped and reprobed with anti-Spl antibody. Spl is a ubiquitous transcription factor
in mammalian cells, and as shown in the right panel of Fig. 33, Spl protein is detected
in COS7 cell extracts, but not in lanes precipitated with either Aktl or Erk2
antibodies. Decreased Erk-mediated phosphorylation of IRSBP-1 in diabetic and
obese rats was also seen (Fig. 36). The locations of the IRSBP-1 protein in the signal
transduction pathway of insulin signaling are shown in Fig. 37. Example 16: Administration of IRSBP-1 sense and antisense
oligonucleotides to an animal
Methods & Materials. The methodology was adapted from that of Apostolakis
et al, J. Neurosci. 16: 4823-4834). Briefly, ovariectomized (OVX) female rats (200-
250 gm) were housed individually and maintained on a 12: 12 h lightidark cycle (lights
on at 0700 CST) with rat chow and water in excess ad libitum. After acclimation (7
days), females underwent stereotaxic implantation of third ventricle cannula guides
(26 gauge, Plastics One, Roanoke VA). The experiment started 7 days after surgery.
Females were randomly assigned identification numbers and weighed daily between
0900 and 1000 CST for 5 days immediately before experimental treatment, allowing
each animal to serve as its own control. Each animal (n = 5) received a single
intraventricular (icv) injection of antisense (AS, sequence 5'-
CTAACTCACAGGTGATGATGTAGAG-3', SEQ ID NO: 42) oligos (4 nM in 1 μl
vol. over 2 min) after weighing on the fifth day. Another group of animals (n = 4)
served as positive controls and received sense (S, sequence 5'-
CTCTACATCATCACCTGTGAGTTAG-3', SEQ ID NO: 43) oligos (4 nM in 1 μl
vol. over 2 min). Animals were euthanized under deep anesthesia 8 days after
ohgonucleotide treatment.
There was a difference in mean weight loss between those animals following
treatment with antisense DNA and sense DNA (Fig. 38A). For the five days prior to
ohgonucleotide treatment, the animals demonstrated stable weight. Within 24 hours
after treatment, animals that received sense DNA began to lose weight with the greatest loss (54+6.4 gm) being attained at 72-96 hours after treatment as shown in
Fig. 38A. Antisense DNA had no significant effect on weight (251+2 gm) while sense
DNA-treated animals lost weight (54.3+6.2 gm). Individually, the females receiving
sense DNA lost 27%> of their body weight as compared with either their initial weight,
their mean weight during the prefreatment time or mean weight of antisense-treated
animals as shown in Fig. 38B.
Example 17: Inhibitory effect of IRSBP-1 on cell proliferation
Transfected L6 cells are growth arrested when transfected with IRSBP-1 expressing
nucleic acid.
L6 cells stably transfected with the IRSBP-1 -encoding cDNA (SEQ ID NO:
14) exhibited significantly reduced proliferation rates when cultured in soft agar,
compared to transfection with the vector alone, as shown in Fig. 39.
Flow cytometry analysis of the cell cycle compartments of L6 cells with or without
over-expression of the IRSBP-1 nucleic acid sequence.
Flow cytometry was conducted to analyze DNA content and progression
through the cell cycle. Stable transfectants were fixed in ice cold 100%> ethanol and
debris was removed by centrifugation through a cushion of fetal bovine serum. Cell
pellets were treated with RNAse solution (500 units/ml of 1.12% (w/v) sodium
citrate) at 37 C for 15 mins, and DNA was stained with propidium iodide (5 mg/100
ml of 1.12% sodium citrate) for 30 minutes at room temperature before analyzing on
the flow cytometer. Stable cell lines transfected with the expression vector pCMV-Tag with and
without the IRSBP-1 nucleic acid sequence (SEQ ID NO: 14) were grown to
confluence, fixed with ethanol, and stained with propidium iodide. Cell cycle
compartment analysis was done by flow cytometry, measuring excitation at 488 nm
with argon lasers. The histograms illustrated in Fig. 40 show that cells transfected
with the vector alone, 58% were in the GO/GI phase, 12%> in the S phase and 20%> in
the G2/M phases. With cells transfected with the plasmid comprising the IRSBP-1
sequence (SEQ ID NO: 14), 86% of the cells were in the GO/G 1 phase, and only 11%
of the cells were in S or G2/M phases.
Example 18: Generation of IRSBP-1 knockout mice using the IRSBP-1
cDNA clones of the present invention
To investigate the function of IRSBP-1, transgenic mice will be generated in
which the IRSBP-1 gene is replaced by the neor gene. A DNA construct will first be
generated that contains neor linked to a constitutive promoter. This gene will be
flanked on either side by at least lkb of genomic IRSBP-1 sequence, which will allow
for homologous recombination and integration of neor into the endogenous IRSBP-1
gene. Finally, the Heφes Simplex Virus thymidine kinase (HSV-tk) gene will be
incoφorated into each end of the DNA construct, adjacent to the IRSBP-1 sequences,
to allow for selection of homologous recombinants.
Following linearization, the DNA construct described above will be
transfected into embryonic stem (ES) cells by electroporation, and these cells will be transfeπed to culture on gelatin-coated dishes. The addition of G418 will permit
selection for cells that contain integration of neor- while the nucleoside analog
gancyclovir will allow for selection of cells in which homologous recombination has
occurred; homologous but not heterologous recombination results in removal of the
HSV-tk genes from the transfected construct, thus preventing gancyclovir
cytotoxicity. ES cell colonies that are resistant to both G418 and gancyclovir will be
screened by PCR or Southern analysis for presence and copy number of the neor gene,
and positive colonies will be subcultured and amplified.
ES cells that have successfully integrated neor in place of the IRSBP-1 gene
will be used for morula aggregation with 8-cell embryos; aggregates will subsequently
be implanted into pseudopregnant female mice. Chimeric mice will be identifiable by
their coat color, since the aggregation of ES cells from the 129 strain of agouti mice
with 8-cell embryos from an albino strand such as CDI will result in chimeric mice
exhibiting white coats with brown splotches. Chimeras will be bred in order to look
for germline transmission of the transgene; transgenic offspring will have completely
brown coats and will be heterozygous for the transgene. These heterozygous mice
will be crossed, resulting in a homozygous line if mutation of the IRSBP-1 is not
lethal.
If inactivation of IRSBP-1 proves to be lethal, we will produce tissue-specific
knockouts. A transgenic line will be created encoding Cre recombinase in selected
tissues using the strategy of targeted transgene expression. A responder "knock-in"
mouse will also be created containing a targeting vector that is nearly an exact copy of a segment of genomic IRSBP-1 DNA, except that a critical exon will be flanked by
the sites binding the recombinanse (lox-P sites). The mice will be intercrossed, and
compound transgenic knockout mice will be produced in which the target gene is
excised by Cre recombinase only in the desired cell type (Cre+ + x IRSBP-1 "/" or Cre+/"
x IRSBP-1"'").
Once IRSBP-1 knockout mice (IRSBP-1"/") are obtained that survive
widespread tissue inactivation of IRSBP-1, we will look for tissue-specific,
developmental and metabolic changes. Although it is possible that other gene
products might be capable of functionally replacing IRSBP-1, our preliminary
experiments with stably transfected L6 myoblasts suggest that IRSBP-1 acts at the
distal end of the insulin activation pathway and is thus unlikely to be genetically
redundant. With tissue-specific knockouts (muscle Cre+ + x floxed IRSBP-1"/"'
adipose Cre+ + x floxed IRSBP-1 " "> and liver Cre+ + x floxed IRSBP-l"'"), serial crosses
of mice will be conducted to allow concurrent inactivation of IRSBP-1 in multiple
organs.
Once IRSBP-1 knockout mice are obtained , they will used to test insulin
action, and investigate their susceptibility to diabetes and diabetes complications.
Growth curves, including intrauterine growth, size and weight of the viscera, and
necropsy at various ages to determine for gross histological differences will be
determined. We will define the phenotype of the animals in terms of blood glucose,
insulin, glucagon, cortisol and leptin measurements at fasted and fed levels.
Immunohistochemical analysis of the pancreas, kidneys, intestine and organs targeted for knockout studies will be done to confirm the effectiveness of gene ablation and
detect presence of changes associated with diabetes. Glucose tolerance tests will be
performed by intraperitoneal glucose injection (2 mg/gm body weight), and tail bleed
will be at 0, 30, 60 and 120 mins, after injection to check for glucose and insulin
levels. Finally, wild-type, IRSBP-1 -deficient and heterozygote littermates will be
subjected to a diet high in fat (50% of calories from fat) and monitored for parameters
of obesity- induced insulin resistance.
Although preferred embodiments of the invention have been described using
specific terms, devices, and methods, such description is for illustrative pmposes only.
The words used are words of description rather than of limitation. It is to be
understood that changes and variations may be made by those of ordinary skill in the
art without departing from the spirit or the scope of the present invention, which is set
forth in the following claims. In addition, it should be understood that aspects of the
various embodiments may be interchanged both in whole or in part.

Claims

What is Claimed Is:
1. An isolated nucleic acid comprising the nucleotide sequence selected from SEQ ID NOS: 2, 5 - 8, 13, and 16 - 41, or degenerate variants thereof.
2. The isolated nucleic acid of Claim 1 further selected from SEQ ID NOS: 5 and 7.
3. The isolated nucleic acid of Claim 1 further selected from SEQ ID NOS: 16 - 41.
4. The isolated nucleic acid of Claim 1 comprising a nucleotide sequence that encodes a polypeptide substantially identical to SEQ ID NOS: 11 or 12, or a degenerate variants thereof.
5. The isolated nucleic acid of Claim 4, wherein the polypeptide is a fragment of SEQ ID NOS: 11 or 12.
6. The isolated nucleic acid of Claim 4, wherein said polypeptide is capable of regulating the expression of an insulin responsive gene.
7. The isolated nucleic acid of Claim 1 comprising a sequence at least 75% identical to NOS: 5 or 7.
8. The isolated nucleic acid of Claim 1 comprising a sequence at least
90% identical to NOS: 5 or 7.
9. A vector comprising the nucleic acid of Claims 1 - 8.
10. The vector of Claim 9, wherein the vector is an expression vector.
11. The vector of Claim 9, wherein the vector is an expression vector comprising the nucleic acid of Claim 9 operably linked to an expression control sequence, wherein the expression control sequence directs production of a transcript that hybridizes under physiological conditions to SEQ ID NOS: 5 or 7 or the complement thereof.
12. An expression system comprising the isolated nucleic acid of Claims 1 - 8.
13. An isolated nucleic acid probe comprising a sequence that hybridizes under conditions that permit hybridization of the isolated nucleic acid probe with the nucleic acid of Claims 1 - 8.
14. The isolated nucleic acid of Claims 1 - 8 operably linked to a heterologous coding sequence.
15. The isolated nucleic acid of Claims 14, wherein the heterologous coding sequence is selected from the group consisting of promoters, enhancers, transcription, activators and the like.
16. A cultured cell comprising the vector of Claims 9 - 11.
17. The cultured cell of Claim 16, wherein the cell is transfected with the vector of Claims 9 - 11 , or a progeny of said cell, wherein said cell expresses a polypeptide.
18. The cultured cell of Claim 17, wherein said polypeptide comprises amino acid sequence SEQ ID NOS: 11 or 12, or a degenerate variant thereof.
19. A method of producing a protein, comprising culturing the cell of Claim 17 under conditions permitting expression under the control of the expression control sequence, and purifying the polypeptide from the cell or the medium of the cell.
20. A method of hybridization comprising providing a single-stranded ohgonucleotide at least eight nucleotides in length, the ohgonucleotide being complementary to a portion of a nucleic acid sequence selected from SEQ ID NOS: 2, 5-8, 13, or the complement of any sequence thereof; and contacting the ohgonucleotide with a nucleic acid comprising the sequence of SEQ ID NOS: 2, 5-8, 13, or the complement of any sequence thereof under conditions that permit hybridization of the ohgonucleotide with the nucleic acid.
21. A method for identifying a nucleic acid homolog comprising the steps of: a) obtaining a nucleic acid sequence probe molecule having a nucleotide sequence, said nucleotide sequence comprising the sequence selected from SEQ ID NO: 2, 5, 8, 13, or the complement of any sequence thereof, or a degenerate variant thereof; b) accessing a sequence database comprising a target nucleotide sequence; c) aligning said nucleic acid sequence probe molecule with said target nucleotide sequence; d) determining a percent homology between a nucleotide sequence of said nucleic acid sequence probe molecule and said target nucleotide sequence; and e) identifying said target nucleotide sequence in said sequence database wherein the percent homology between the nucleotide sequence of said nucleic acid sequence probe molecule and said target nucleotide sequence is at least 80% identical to SEQ ID NO: 2, 5, 8 and 13.
22. The method as in Claim 21 , wherein the percent homology between the nucleotide sequence of said nucleic acid sequence probe molecule and said target nucleotide sequence is at least 75%> identical to SEQ ID NOS: 5 or 7.
23. The method as in Claim 21, wherein the percent homology between the nucleotide sequence of said nucleic acid sequence probe molecule and said target nucleotide sequence is at least 90%> identical to SEQ ID NOS: 5 or 7.
24. A single-stranded nucleic acid that hybridizes under highly stringent
conditions to a nucleic acid of Claims 1 - 8 or a complement thereof.
25. The single-stranded nucleic acid of Claim 24, further comprising an
isolated nucleic acid comprising at least 10 consecutive nucleotides of the
complement of SEQ ID NOS: 5 or 7.
26. The single-stranded nucleic acid of Claim 24, wherein at least 10
consecutive nucleotides comprise an antisense or sense nucleic acid.
27. An antibody that binds specifically to a polypeptide encoded by the
nucleic acids as in Claims 1 - 8 or an immunogenic fragment thereof.
28. The antibody of Claim 27, wherein the immunogenic fragment has the
sequence SEQ ID NO: 13.
29. A method of determining whether a biological sample contains an
IRSBP-1 protein, comprising the steps of:
(a) obtaining a biological sample from a human or animal,
(b) extracting the biological sample with the antibody of
Claim 27; and
(c) determining whether the antibody specifically binds to
the sample, the binding indicating that the biological
sample contains IRSBP-1 or a fragment thereof.
30. The method of Claim 29, wherein the biological sample comprises at
least one cell and the binding of the antibody indicates the cell as
having IRSBP-1 or a fragment thereof.
31. A method of diagnosing a disease comprising the steps of providing a
biological sample from a human or animal; and selectively detecting a nucleic acid according to Claims 1-8, wherein the detection of a
nucleic acid according to Claims 1-8 indicates a disease.
32. A method of regulating the biological activity of IRSBP-1 by at least
one chemical or biological agonist and antagonist.
33. The method of regulating the biological activity of IRSBP-1 of Claim
28, wherein the biological agonist or antagonist is an antibody that
selectively binds to IRSBP-1 protein.
PCT/US2000/030096 1999-11-01 2000-11-01 Insulin-responsive sequence dna binding protein-1' (irsdbp-1), gene encoding it and uses thereof WO2001032873A1 (en)

Priority Applications (5)

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AT00976787T ATE312918T1 (en) 1999-11-01 2000-11-01 ßINSULIN DEPENDENT SEQUENCE DNA BINDING PROTEIN-1ß (IRSDBP-1), GENE CODING THEREOF AND USES THEREOF
CA002389615A CA2389615A1 (en) 1999-11-01 2000-11-01 Insulin-responsive sequence dna binding protein-1" (irsdbp-1), gene encoding it and uses thereof
EP00976787A EP1228209B1 (en) 1999-11-01 2000-11-01 "insulin-responsive sequence dna binding protein-1" (irsdbp-1), gene encoding it and uses thereof
DE60024862T DE60024862T2 (en) 1999-11-01 2000-11-01 "INSULIN-DEPENDENT SEQUENCE DNA BINDING PROTEIN-1" (IRSDBP-1), A GENERIC ENCODER AND ITS USES
AU14518/01A AU1451801A (en) 1999-11-01 2000-11-01 Insulin-responsive sequence DNA binding protein-1 (IRSDBP-1), gene encoding it and uses thereof

Applications Claiming Priority (2)

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US16268799P 1999-11-01 1999-11-01
US60/162,687 1999-11-01

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003018805A1 (en) * 2001-08-23 2003-03-06 Japan Science And Technology Agency Hematopoietic stem cell proliferation regulators and polynucleotides encoding the same
EP1497305A2 (en) * 2001-12-04 2005-01-19 Emory University Insulin-responsive dna binding protein-1 and methods to regulate insulin-responsive genes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL/GENBANK/DDBJ EBI; 2 July 1998 (1998-07-02), "Homo sapiens BAC clone RP11-556H17 from 2", XP002164028 *
VILLAFUERTE, B. C. ET AL.: "Identification of an Insulin-responsive Element in the Rat Insulin-like Growth Factor-binding Protein-3 Gene", J. BIOL. CHEM., vol. 272, no. 2, 21 February 1997 (1997-02-21), pages 5024 - 5030, XP002164027 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003018805A1 (en) * 2001-08-23 2003-03-06 Japan Science And Technology Agency Hematopoietic stem cell proliferation regulators and polynucleotides encoding the same
US7262026B2 (en) 2001-08-23 2007-08-28 Japan Science And Technology Agency Hematopoietic stem cell proliferation regulators and polynucleotides encoding the same
EP1497305A2 (en) * 2001-12-04 2005-01-19 Emory University Insulin-responsive dna binding protein-1 and methods to regulate insulin-responsive genes
EP1497305A4 (en) * 2001-12-04 2005-11-30 Univ Emory Insulin-responsive dna binding protein-1 and methods to regulate insulin-responsive genes

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EP1228209A1 (en) 2002-08-07
WO2001032873A8 (en) 2001-10-11
AU1451801A (en) 2001-05-14
ATE312918T1 (en) 2005-12-15
EP1228209B1 (en) 2005-12-14
CA2389615A1 (en) 2001-05-10
DE60024862T2 (en) 2006-08-17

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