+

WO1999046373A1 - IDENTIFICATION DE PACTOLUS: UNE PROTEINE DE TYPE INTEGRINE $g(b) - Google Patents

IDENTIFICATION DE PACTOLUS: UNE PROTEINE DE TYPE INTEGRINE $g(b) Download PDF

Info

Publication number
WO1999046373A1
WO1999046373A1 PCT/US1999/005454 US9905454W WO9946373A1 WO 1999046373 A1 WO1999046373 A1 WO 1999046373A1 US 9905454 W US9905454 W US 9905454W WO 9946373 A1 WO9946373 A1 WO 9946373A1
Authority
WO
WIPO (PCT)
Prior art keywords
pactolus
seq
protein
nucleic acid
ligand
Prior art date
Application number
PCT/US1999/005454
Other languages
English (en)
Inventor
Yiyou Chen
John H. Weis
Original Assignee
University Of Utah Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Utah Research Foundation filed Critical University Of Utah Research Foundation
Priority to AU30843/99A priority Critical patent/AU3084399A/en
Publication of WO1999046373A1 publication Critical patent/WO1999046373A1/fr

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • 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)

Definitions

  • DD differential display
  • Any transcript product that is specific for the cell type in question can, with this protocol, be identified, isolated, and sequenced.
  • the advantages of this protocol are many and include the requirement of much less RNA, the simplicity of PCR (polymerase chain reaction) amplification, and the ease of product resolution (Liang and Pardee, 1992; Liang et al., 1993; Bauer et al., 1993).
  • the primary difficulties with DD have been the large number of false positives generated, and the requirement of closely matched cell types with which to compare.
  • Mast cells arise from the multipotent bone marrow stem cells.
  • tissue mast cells There are two types of tissue mast cells in the mouse (mucosal mast cells and connective tissue mast cells) which are believed to be derived from the same precursor cells.
  • IL-3 interleukin 3
  • SCF stem cell factor
  • This gene is most pronounced in the murine bone marrow.
  • the expression of Pactolus in cells derived in SCF, but not the IL-3 cells suggests that the SCF-derived cells may represent a more immature mast cell type than those derived in IL-3 culture.
  • the present invention is directed to the finding of a new gene, named Pactolus, which encodes a protein which has homology with ⁇ -integrin.
  • the function of the encoded protein is two fold: retention of maturing cells within the marrow site and facilitation of cellular signal transduction to help regulate cellular maturation and differentiation.
  • Bone marrow precursor cells which express the Pactolus protein recognize a ligand within the stroma of bone marrow. This anchors the cells in situ until their differentiation allows for release.
  • the marrow By using either a soluble Pactolus or a soluble ligand, the marrow can be depleted of these precursor cells which will then flood the blood stream. These precursor cells, which are often used in bone marrow transplantations, can then be easily harvested from the blood rather than requiring a bone marrow harvest via needle aspiration of the pelvis/femur of the donor.
  • the Pactolus protein can be used to ensure receptor/ligand contact in tissue culture and thereby enhance recovery and growth of bone marrow precursor cells from the tissue culture, because growth of the precursor cells relies upon ligation of Pactolus with its ligand.
  • FIG. 1 Differential display analysis of two distinct bone marrow derived mast cell types: resolution of differential display products.
  • CTMC connective tissue mast cells
  • CTMC+IL-3 represents mast cells grown exclusively in SCF for 21 days followed by 24 hrs in IL-3 alone
  • MMC micosal mast cells
  • MMC+SCF represents mast cells grown exclusively in IL-3 for 21 days followed by 24 hrs in SCF alone.
  • Arrows A and B indicate two differentially expressed products.
  • FIG. 4 Homology matrix analysis between murine Pactolus and murine ⁇ 2.
  • cDNA sequences for murine Pactolus and murine ⁇ 2 were plotted against each other using the Mac Vector software with Pactolus on the x-axis and ⁇ 2 on the y-axis. The arrows point out the breakpoints in the homology matrix. These breakpoints are termed Gap#l and Gap#2, respectively.
  • Figures 5A-B RT-RPCR analysis with Pactolus specific primers predicts two discrete gene products. Pactolus specific PCR primers were designed to span Gap#l and Gap #2 on the cDNA sequences. Standard RT-RPCR was performed as described in Example 3. Shown in Figure 5 A is the result from primers spanning Gap#l.
  • Figure 5B shows the result from primers for Gap#2.
  • ⁇ -actin was amplified for 16 cycles and Pactolus transcripts were amplified for 26 cycles.
  • Two distinct PCR bands are readily identifiable with the primer set spanning Gap#2.
  • the lower band agrees with the predicted size from the Pactolus- 1 cDNA sequence.
  • the upper band is approximately 40 bp larger than the lower form.
  • TM stands for transmembrane form of Pactolus.
  • FIGS. 6A-E Alternative spliced forms of Pactolus predict a truncated and membrane bound form.
  • the amino acid sequence corresponding to the smaller fragment is SEQ ID NO:24.
  • the nucleic acid sequence of the smaller fragment is SEQ ID NO:25.
  • the nucleic acid sequence of the larger fragment is SEQ ID NO:26.
  • the amino acid sequence corresponding to the larger fragment is SEQ ID NO:27.
  • the full length Pactolus encodes a 738 amino acid transmembrane protein.
  • the full length nucleic acid sequence is shown in the SEQUENCE LISTING as SEQ ID NO:28.
  • the encoded 738 amino acid sequence is shown in Figure 6B and as SEQ ID NO:29.
  • the predicted transmembrane domain is shown in bold characters.
  • the truncated form Pactolus has only 540 amino acid residues.
  • the truncated nucleic acid sequence is shown in the SEQUENCE LISTING as SEQ ID NO:22 and the truncated amino acid sequence is SEQ ID NO:23.
  • Pactolus protein sequence (Pact) and the murine ⁇ 2 sequence (Wilson et al., 1989).
  • the Pactolus sequence shown is identical to amino acid residues 1-631 of SEQ ID NO:29.
  • the ⁇ 2 amino acid sequence is SEQ ID NO:31.
  • the vertical bar indicates identical amino acids, the dotted line represents sequences missing in the Pactolus sequence (Gap #1) to conserve alignment. These two proteins are 63% identical to each other for the region shown: there is no significant sequence homology between the two following residue 631 of the Pactolus sequence.
  • the mouse Pactolus sequence is SEQ ID NO:32; the mouse ⁇ 2 is SEQ ID NO:33; the mouse ⁇ l is SEQ ID NO:34.
  • the consensus sequences for the MIDAS motif is shown below the murine sequences. Spacer amino acids are shown either as x (DxSxS) or by sequence length (12 amino acids, etc.). Residues demonstrated to be mutated in the human ⁇ 2 sequence and thus causative for the Lymphocyte Adhesion Deficiency syndrome (Wardlaw et al., 1990) are underlined.
  • FIGS 7A-B Different forms of Pactolus transcripts are generated via the use of alternative acceptor sites.
  • FIGS 8A-D Pactolus transcript expression.
  • the first lane (next to bone marrow) was a water PCR control (no cDNA added).
  • RNA derived from Cell aliquots were taken from the culture at different time points (as shown in the figure) and RNA derived. Pactolus transcripts (24 cycles) were visualized using the primer set specific for Gap#2: actin controls (16 cycles) were included to ensure equivalent quantities of cDNA were amplified. cDNA derived from total bone marrow was included as a positive control: water (H20) was a negative control (no cDNA added).
  • FIGS 9A-B Identification of Pactolus protein.
  • Pactolus cytoplasmic peptide The molecular weight markers are shown to the left.
  • Pactolus refers to the river in which Midas lost his golden touch
  • cytoplasmic domain that shares no obvious homology to similar domains of the other ⁇ integrins.
  • the Pactolus sequence was first identified in immature mast cell samples, screening of murine tissues indicated the highest level of Pactolus expression was found in the bone marrow indicating that the expression of Pactolus is confined to immature and maturing bone marrow derived cells, and that the SCF-derived mast cells are more representative of this state than are the IL-3 derived mast cells.
  • Immunoprecipitation of Pactolus revealed a cell surface protein with an apparent molecular weight of about 95 kDa.
  • the present invention employs the following definitions.
  • PCR polymerase chain reaction
  • LCR ligation amplification
  • SDA strand displacement amplification
  • thermophilic thermophilic
  • Primers useful to amplify sequences from the Pactolus region are preferably complementary to, and hybridize specifically to sequences in the Pactolus region or in regions that flank a target region therein.
  • Pactolus sequences generated by amplification may be sequenced directly.
  • the amplified sequence(s) may be cloned prior to sequence analysis.
  • a method for the direct cloning and sequence analysis of enzymatically amplified genomic segments has been described by Scharf, 1986.
  • analyte polynucleotide and “analyte strand” refer to a single- or double-stranded polynucleotide which is suspected of containing a target sequence, and which may be present in a variety of types of samples, including biological samples.
  • Antibodies The present invention also provides polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, which are capable of specifically binding to the Pactolus polypeptide and fragments thereof or to polynucleotide sequences from the Pactolus region.
  • the term “antibody” is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Polypeptides may be prepared synthetically in a peptide synthesizer and coupled to a carrier molecule (e.g., keyhole limpet hemocyanin) and injected over several months into rabbits. Rabbit sera is tested for immunoreactivity to the Pactolus polypeptide or fragment. Monoclonal antibodies may be made by injecting mice with the protein polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with Pactolus polypeptide or fragments thereof. See, Harlow and Lane, 1988.
  • a carrier molecule e.g., keyhole limpet hemocyanin
  • antibodies will be useful in assays as well as pharmaceuticals. Once a sufficient quantity of desired polypeptide has been obtained, it may be used for various purposes. A typical use is the production of antibodies specific for binding. These antibodies may be either polyclonal or monoclonal, and may be produced by in vitro or in vivo techniques well known in the art. For production of polyclonal antibodies, an appropriate target immune system, typically mouse or rabbit, is selected. Substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and by other parameters well known to immunologists. Typical sites for injection are in footpads, intramuscularly, intraperitoneally, or intradermally. Of course, other species may be substituted for mouse or rabbit. Polyclonal antibodies are then purified using techniques known in the art, adjusted for the desired specificity.
  • An immunological response is usually assayed with an immunoassay.
  • immunoassays involve some purification of a source of antigen, for example, that produced by the same cells and in the same fashion as the antigen.
  • a variety of immunoassay methods are well known in the art. See, e.g., Harlow and Lane, 1988, or Goding, 1986. Monoclonal antibodies with affinities of 10 "8 M “1 or preferably 10 "9 to 10 "10 M “1 or stronger will typically be made by standard procedures as described, e.g., in Harlow and Lane, 1988 or Goding, 1986. Briefly, appropriate animals will be selected and the desired immunization protocol followed.
  • the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supematants of each clone tested for their production of an appropriate antibody specific for the desired region of the antigen.
  • Binding partner refers to a molecule capable of binding a ligand molecule with high specificity, as for example, an antigen and an antigen-specific antibody or an enzyme and its inhibitor.
  • the specific binding partners must bind with sufficient affinity to immobilize the analyte copy/complementary strand duplex (in the case of polynucleotide hybridization) under the isolation conditions.
  • Specific binding partners are known in the art and include, for example, biotin and avidin or streptavidin, IgG and protein A, the numerous, known receptor-ligand couples, and complementary polynucleotide strands.
  • the partners are normally at least about 15 bases in length, and may be at least 40 bases in length. It is well recognized by those of skill in the art that lengths shorter than 15 (e.g., 8 bases), between 15 and 40, and greater than 40 bases may also be used.
  • the polynucleotides may be composed of DNA, RNA, or synthetic nucleotide analogs.
  • a “biological sample” refers to a sample of tissue or fluid suspected of containing an analyte polynucleotide or polypeptide from an individual including, but not limited to, e.g., bone marrow, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents.
  • Encode A polynucleotide is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
  • the anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • nucleic acid e.g., an RNA, DNA or a mixed polymer
  • An “isolated” or “substantially pure” nucleic acid is one which is substantially separated from other cellular components which naturally accompany a native mouse sequence or protein, e.g., ribosomes, polymerases, many other mouse genome sequences and proteins.
  • the term embraces a nucleic acid sequence or protein which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • Actolus Allele refers to normal alleles of the Pactolus locus as well as alleles of Pactolus carrying variations. 11
  • Polynucleotide each refers to polynucleotides, all of which are in the Pactolus region.
  • Pactolus locus is intended to include coding sequences, intervening sequences and regulatory elements controlling transcription and/or translation.
  • the Pactolus locus is intended to include all allelic variations of the DNA sequence.
  • nucleic acid when applied to a nucleic acid, refer to a nucleic acid which encodes a mouse Pactolus polypeptide, fragment, homolog or variant, including, e.g., protein fusions or deletions.
  • the nucleic acids of the present invention will possess a sequence which is either derived from, or substantially similar to a natural Pactolus-encoding gene or one having substantial homology with a natural Pactolus-encoding gene or a portion thereof.
  • the polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • pendent moieties
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • the present invention provides recombinant nucleic acids comprising all or part of the Pactolus region.
  • the recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosomal DNA of the host cell.
  • a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation, 1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; 2) is linked to a polynucleotide other than that to which it is linked in nature; or 3) does not occur in nature.
  • nucleic acids comprising sequences otherwise not naturally occurring are provided by this invention.
  • wild-type sequence may be employed, it will often be 12 altered, e.g., by deletion, substitution or insertion.
  • cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR.
  • the choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences.
  • the DNA sequences used in this invention will usually comprise at least about five codons (15 nucleotides), more usually at least about 7-15 codons, and most preferably, at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with a Pactolus- encoding sequence.
  • nucleic acid manipulation is described generally, for example, in Sambrook et al. , 1989 or Ausubel et al. , 1992.
  • Reagents useful in applying such techniques such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega, U. S. Biochemicals, New England Nuclear, and a number of other sources.
  • the recombinant nucleic acid sequences used to produce fusion proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health.
  • a "portion" of the Pactolus locus or region or allele is defined as having a minimal size of at least about eight nucleotides, or preferably about 15 nucleotides, or more preferably at least about 25 nucleotides, and may have a minimal size of at least about 40 nucleotides. This definition includes all sizes in the range of 8-40 nucleotides as well as greater than 40 nucleotides.
  • Pactolus protein or “Pactolus polypeptide” refers to a protein or polypeptide encoded by the Pactolus locus, variants or fragments thereof.
  • polypeptide refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, .and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural 13
  • polypeptides will be at least about 50%) homologous to the native Pactolus sequence, preferably in excess of about 90%, and more preferably at least about 95% homologous.
  • proteins encoded by DNA which hybridize under high or low stringency conditions, to Pactolus-encoding nucleic acids and closely related polypeptides or proteins retrieved by antisera to the Pactolus protein(s).
  • the length of polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.
  • "Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • Polynucleotide polymorphisms associated with Pactolus alleles are detected by hybridization with a polynucleotide probe which forms a stable hybrid with that of the target sequence, under highly stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes will be perfectly complementary to the target sequence, high stringency conditions will be used. Hybridization stringency may be lessened if some mismatching is expected, for example, if variants are expected with the result that the probe will not be completely complementary. Conditions are chosen which rule out nonspecific/adventitious bindings, that is, which minimize noise. (It should be noted that throughout this disclosure, if it is simply stated that
  • Probes for Pactolus alleles may be derived from the sequences of the Pactolus region or its cDNA.
  • the probes may be of any suitable length, which span all or a portion of the Pactolus region, and which allow specific hybridization to the region. If the target sequence contains a sequence identical to that of the probe, the probes may be short, e.g., in the range of about 8-30 base pairs, since the hybrid will be relatively stable under even highly stringent conditions. If some degree of mismatch is expected with the probe, i.e., if it is suspected that the probe will hybridize to a variant region, a longer probe may be employed which hybridizes to the target sequence with the requisite specificity.
  • the probes will include an isolated polynucleotide attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences, having sequence similarity by standard methods. For techniques for preparing and labeling probes see, e.g., Sambrook et al., 1989 or 14
  • Probes comprising synthetic oligonucleotides or other polynucleotides of the present invention may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labeled by nick translation, Klenow fill-in reaction, or other methods known in the art.
  • Portions of the polynucleotide sequence having at least about eight nucleotides, usually at least about 15 nucleotides, and fewer than about 6 kb, usually fewer than about 1.0 kb, from a polynucleotide sequence encoding Pactolus are preferred as probes.
  • This definition therefore includes probes of sizes 8 nucleotides through 6000 nucleotides. The probes may also be used to determine whether mRNA encoding Pactolus is present in a cell or tissue.
  • Protein modifications or fragments are provided by the present invention for Pactolus polypeptides or fragments thereof which are substantially homologous to primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art.
  • a variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as 32 P, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand.
  • radioactive isotopes such as 32 P
  • ligands which bind to labeled antiligands e.g., antibodies
  • fluorophores e.g., chemiluminescent agents
  • enzymes chemiluminescent agents
  • antiligands which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation.
  • Methods of labeling polypeptides are well known in the art. See Sambrook et al. , 1989
  • the present invention provides for biologically active fragments of the polypeptides.
  • Significant biological activities include ligand-binding, immunological activity and other biological activities characteristic of Pactolus polypeptides.
  • Immunological activities include both immunogenic function in a target immune system, as well 15 as sharing of immunological epitopes for binding, serving as either a competitor or substitute antigen for an epitope of the Pactolus protein.
  • epitope refers to an antigenic determinant of a polypeptide.
  • An epitope could comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of such amino acids are known in the art.
  • tandem-repeat polypeptide segments may be used as immunogens, thereby producing highly antigenic proteins.
  • polypeptides will serve as highly efficient competitors for specific binding. Production of antibodies specific for Pactolus polypeptides or fragments thereof is described below.
  • the present invention also provides for fusion polypeptides, comprising Pactolus polypeptides and fragments.
  • Homologous polypeptides may be fusions between two or more Pactolus polypeptide sequences or between the sequences of Pactolus and a related protein.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. For example, ligand-binding or other domains may be
  • Fusion partners include immunoglobulins, bacterial ⁇ -galactosidase, trpE, protein A, ⁇ - lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. See Godowski et al, 1988.
  • Fusion proteins will typically be made by either recombinant nucleic acid methods, as described below, or may be chemically synthesized. Techniques for the synthesis of polypeptides are described, for example, in Merrifield, 1963.
  • Protein purification refers to various methods for the isolation of the Pactolus polypeptides from other biological material, such as from cells transformed with recombinant nucleic acids encoding Pactolus, and are well known in the art.
  • polypeptides may be purified by immunoaffinity chromatography employing, e.g., the antibodies provided by the present invention.
  • Various methods of protein purification are well known in the art, and include those described in Deutscher, 1990 and Scopes, 1982.
  • isolated isolated
  • substantially pure and “substantially homogeneous” are used interchangeably to describe a protein or polypeptide which has been separated from components which accompany it in its natural state.
  • a monomeric protein is substantially pure when at least 16 about 60 to 75% of a sample exhibits a single polypeptide sequence.
  • a substantially pure protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%>, .and preferably will be over about 99%> pure.
  • Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art which are utilized for purification.
  • a Pactolus protein is substantially free of naturally associated components when it is separated from the native contaminants which accompany it in its natural state.
  • a polypeptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • a polypeptide produced as an expression product of an isolated and manipulated genetic sequence is an "isolated polypeptide," as used herein, even if expressed in a homologous cell type.
  • Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules.
  • Recombinant nucleic acid is a nucleic acid which is not naturally occurring, or which is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.
  • regulatory sequences refers to those sequences normally within 100 kb of the coding region of a locus, but they may also be more distant from the coding region, which affect the expression of the gene (including tr.anscription of the gene, and translation, splicing, stability or the like of the messenger RNA). "Substantial homology or similarity”.
  • a nucleic acid or fragment thereof is “substantially homologous" ("or substantially similar") to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there 17 is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases.
  • substantial homology or (similarity) exists when a nucleic acid or fragment thereof will hybridize to another nucleic acid (or a complementary strand thereof) under selective hybridization conditions, to a strand, or to its complement.
  • Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa, 1984.
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
  • Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37 C, and preferably in excess of 45°C.
  • Stringent salt conditions will ordinarily be less than 1000 mM, typically less than
  • Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes.
  • the preparation of such probes and suitable hybridization conditions are well known in the art.
  • substantially homology when referring to polypeptides, indicate that the polypeptide or protein in question exhibits at least about 30% identity with an entire naturally-occurring protein or a portion thereof, usually at least about 70% identity, and preferably at least about 95% identity.
  • substantially similar function refers to the function of a modified nucleic acid or a modified protein, with reference to the wild-type Pactolus nucleic acid or wild-type Pactolus polypeptide.
  • the modified polypeptide will be substantially homologous to the wild-type Pactolus 18 polypeptide .and will have substantially the same function.
  • the modified polypeptide may have an altered amino acid sequence and/or may contain modified amino acids.
  • the modified polypeptide may have other useful properties, such as a longer half-life.
  • the similarity of function (activity) of the modified polypeptide may be substantially the same as the activity of the wild-type Pactolus polypeptide.
  • the similarity of function (activity) of the modified polypeptide may be higher than the activity of the wild-type Pactolus polypeptide.
  • the modified polypeptide is synthesized using conventional techniques, or is encoded by a modified nucleic acid and produced using conventional techniques.
  • the modified nucleic acid is prepared by conventional techniques. A nucleic acid with a function substantially similar to the wild-type Pactolus gene function produces the modified protein described above.
  • homology for polypeptides, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • a polypeptide "fragment,” “portion” or “segment” is a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids.
  • polypeptides of the present invention may be coupled to a solid-phase support, e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates.
  • a solid-phase support e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates.
  • Such supports may take the form, for example, of beads, wells, dipsticks, or membranes.
  • Target region refers to a region of the nucleic acid which is amplified and/or detected.
  • target sequence refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, and immunology. See, e.g., Maniatis et al, 1982; Sambrook et al, 1989; Ausubel et al, 1992; Glover, 19
  • polynucleotides of the present invention may be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments coding for a desired fragment will be incorporated into recombinant polynucleotide constructs, usually DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the polynucleotide constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to (with and without integration within the genome) cultured mammalian or plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention are described, e.g., in Sambrook et al., 1989 or Ausubel et al., 1992.
  • the polynucleotides of the present invention may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Carruthers, 1981 or the triester method according to Matteucci .and Caruthers, 1981, and may be performed on commercial, automated ohgonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment.
  • Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences.
  • ARS origin of replication or autonomously replicating sequence
  • Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al., 1989 or Ausubel et al., 1992. 20
  • An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may include, when appropriate, those naturally associated with the Pactolus gene. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al, 1989 or Ausubel et al., 1992; see also, e.g., Metzger et al., 1988. Many useful vectors are known in the art and may be obtained from such vendors as Stratagene,
  • Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts.
  • Useful yeast promoters include promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3 -phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others.
  • Vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al., EP 73,675A.
  • Non-native mammalian promoters might include the early and late promoters from SV40 (Fiers et al., 1978) or promoters derived from murine Molony leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma.
  • the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made.
  • DHFR e.g., DHFR
  • Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene ensures growth of only those host cells which express the inserts.
  • Typical selection genes encode proteins that a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc., b) complement auxotrophic deficiencies, or c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for
  • the vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection (see, Kubo et al, 1988), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other 21 substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome); and other methods. See generally, Sambrook et al., 1989 and Ausubel et al., 1992.
  • the introduction of the polynucleotides into the host cell by any method known in the art, including, inter alia, those described above, will be referred to herein as "transformation.”
  • the cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.
  • nucleic acids and polypeptides of the present invention may be prepared by expressing the Pactolus nucleic acid or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells.
  • prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or
  • Pseudomonas may also be used.
  • Mammalian or other eukaryotic host cells such as those of yeast, filamentous fungi, plant, insect, or amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. See, Jakoby and Pastan (eds.), 1979. Examples of commonly used mammalian host cell lines are VERO and
  • HeLa cells Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features.
  • Clones are selected by using markers depending on the mode of the vector construction.
  • the marker may be on the same or a different DNA molecule, preferably the same DNA molecule.
  • the transformant may be selected, e.g., by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
  • Prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention, but also, for example, in studying the characteristics of Pactolus polypeptide.
  • the probes and primers based on the Pactolus gene sequence disclosed herein are used to identify homologous Pactolus gene sequences and proteins in other species. These gene sequences and proteins are used in the therapeutic and drug screening methods described herein for the species from which they have been isolated. 22
  • This invention is particularly useful for screening compounds by using the Pactolus polypeptide or binding fragment thereof in any of a variety of drug screening techniques.
  • the Pactolus polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface.
  • One method of drug screening utilizes eucaryotic or procaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may measure, for example, for the formation of complexes between a Pactolus polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a Pactolus polypeptide or fragment and a known ligand is interfered with by the agent being tested.
  • the present invention provides methods of screening for drugs comprising contacting such an agent with a Pactolus polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the Pactolus polypeptide or fragment, or (ii) for the presence of a complex between the Pactolus polypeptide or fragment and a ligand, by methods well known in the art.
  • the Pactolus polypeptide or fragment is typically labeled. Free Pactolus polypeptide or fragment is separated from that present in a proteimprotein complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to Pactolus or its interference with Pactolus: ligand binding, respectively.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the Pactolus polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with Pactolus polypeptide and washed. Bound Pactolus polypeptide is then detected by methods well known in the art.
  • Purified Pactolus can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the Pactolus polypeptide on the solid phase.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the Pactolus polypeptide compete with a test compound for binding to the Pactolus polypeptide or fragments thereof. In this manner, the 23 antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the Pactolus polypeptide.
  • Rational Drug Design The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, 1991.
  • peptides e.g., Pactolus polypeptide
  • an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined.
  • Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • Pactolus polypeptide activity e.g., improved Pactolus polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of Pactolus polypeptide activity.
  • drugs which have, e.g., improved Pactolus polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of Pactolus polypeptide activity.
  • sufficient amounts of the Pactolus polypeptides may be made available to perform such analytical studies as x-ray crystallography.
  • the knowledge of the Pactolus protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to, x-ray crystallography. 24
  • a method is also provided of supplying wild-type Pactolus function to a cell which carries a mutant Pactolus allele. Supplying such a function should allow normal functioning of the recipient cells.
  • the wild-type gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. More preferred is the situation where the wild-type gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant gene present in the cell. Such recombination requires a double recombination event which results in the correction of the gene mutation.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used.
  • Methods for introducing DNA into cells such as electroporation, calcium phosphate co-precipitation and viral transduction are known in the art, and the choice of method is within the competence of the practitioner.
  • the Pactolus gene or fragment may be employed in gene therapy methods in order to increase the amount of the expression products of such gene in cells. It may also be useful to increase the level of expression of the Pactolus gene even in those persons in which the mutant gene is expressed at a "normal" level, but the gene product is not fully functional.
  • Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman, 1991.
  • Cells from a patient would be first analyzed by the diagnostic methods described above, to ascertain the production of Pactolus polypeptide in the cells.
  • a virus or plasmid vector (see further details below), containing a copy of the Pactolus gene linked to expression control elements .and capable of replicating inside the cells, is prepared.
  • Suitable vectors are known, such as disclosed in U.S. Patent 5,252,479 and PCT published application WO 93/07282.
  • the vector is then injected into the patient. If the transfected gene is not permanently incorporated into the genome of each of the targeted cells, the treatment may have to be repeated periodically.
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods.
  • viruses have been used as gene transfer vectors, including papovaviruses (e.g., SV40, Madzak et al.,
  • adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vaccinia 25 virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990), and retroviruses of avian (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller vac
  • Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical techniques, for example microinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster et al.,
  • plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector.
  • the trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged.
  • Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is nonspecific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992). Gene transfer techniques which target DNA directly to brain tissue is preferred. Receptor- mediated gene transfer, for example, is accomplished by the conjugation of DNA (usually in the form of covalently closed supercoiled plasmid) to a protein ligand via polylysine. Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type.
  • ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA- protein complex occurs.
  • coinfection with adenovirus can be included to disrupt endosome function.
  • the therapy is as follows: patients who carry a Pactolus susceptibility allele are treated with a gene delivery vehicle such that some or all of their brain precursor cells receive at least one additional copy of a functional normal Pactolus allele.
  • Peptides which have Pactolus activity can be supplied to cells which carry mutant or missing Pactolus alleles.
  • Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors.
  • Pactolus polypeptide can be extracted from Pactolus-producing mammalian cells.
  • the techniques of synthetic chemistry can be employed to synthesize Pactolus protein. Any of such techniques can provide the preparation of the present invention which comprises the Pactolus protein. The preparation is substantially free of other human proteins. This is most readily accomplished by synthesis in a microorganism or in vitro.
  • Active Pactolus molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Other molecules with Pactolus activity (for example, peptides, drugs or organic compounds) may also be used. Modified polypeptides having substantially similar function are also used for peptide therapy.
  • Pactolus or its biological equivalent may also be administered to a person with wild-type Pactolus.
  • the purpose is not to replace an activity, rather it is to cause an overabundance of Pactolus activity thereby causing bone marrow precursor cells to be released from the marrow into the bloodstream.
  • This treatment would be given to a person who will be a bone marrow donor.
  • the cells which are normally collected from bone marrow can more simply be collected from the blood of such a treated person, thereby preventing the necessity of having to perform a needle aspiration of the pelvis or femur of the donor.
  • Pactolus a novel murine gene which shares a high degree of homology with the family of adhesion molecules known as the ⁇ integrins.
  • the Pactolus sequence was obtained from a differential display (DD) analysis of murine bone marrow derived mast cells in which transcripts derived from cells cultured in IL-3 were compared to those derived in SCF. Based upon our data and the data of others, those cells derived solely in IL-3 possess a phenotype associated with mast cells found in the intestinal mucosa, while those derived in SCF possess characteristics of mast cells found in the skin and peritoneal cavity of the animal (Tsai et 27 al., 1991; Marietta et al., 1996).
  • the protein predicted by the Pactolus sequence is related to the ⁇ integrins but is clearly divergent in two important domains.
  • the ⁇ integrin chains share two regions of highest homology. The first of these is an I (inserted) domain-like structure (also termed A for Activation domain) which is also present within the majority of the a integrin chains (reviewed in Humphries, 1996).
  • This region resides at the amino terminus of the protein (thus extracellular) and is implicated in ligand binding, heteroduplex formation and metal ion binding. Single amino acid mutations in this site abrogate stable expression of the integrin heterodimers and block ligand binding.
  • Structural analysis of the I domain of the CD1 lb chain of the CR3 integrin complex demonstrated the presence of a MIDAS motif (metal ion-dependent adhesion site) that was formed in a three dimensional fold utilizing a conserved DxSxS--65 amino acids ⁇ T— 25 amino acids ⁇ D sequence, where the x residues and spacer amino acids are not conserved (Lee et al., 1995).
  • the I domain sequence present in Pactolus differs from that of the ⁇ and ⁇ integrins. Not only does Pactolus lack the amino terminal D residue but the spacing between the S and T residues, which presumably is critical for the ternary folds of the protein, is 21 amino acids shorter than the consensus sequence. Of the more than 40 gene sequences which possess the conserved MIDAS motif, the shortest distance between the analogous S and T residues is 61 amino acids, compared to 44 for Pactolus. This deletion would, with respect to the proposed MIDAS motif structure, apparently delete the ⁇ 2, ⁇ 3 helices, thus placing the T residue in opposite orientation to the Mg 2+ ion within the MIDAS structure. These two alterations in the MIDAS motif of the I domain of Pactolus suggest that this site may not be functionally similar to those of the ⁇ integrins.
  • cytoplasmic domain The other site of high homology within the ⁇ integrin family is the cytoplasmic domain. Sequences have been defined in the cytoplasmic tail of these subunits which are implicated in the binding of the ⁇ chain to cytosolic proteins including ⁇ -actinin, talin, paxillin, and others (reviewed in Dedhar and Hannigan, 1996). Pactolus is lacking these conserved residues in its proposed cytoplasmic domain suggesting it may bind to a different set of cytoplasmic proteins than those described for the ⁇ integrins. 28
  • the Pactolus gene transcripts also differ from members of the ⁇ integrin family in predicting two distinct forms of the protein, plus or minus the transmembrane and cytoplasmic domains. Attempts to generate an antisera specific for the truncated form of the Pactolus protein have so far been unsuccessful thus we cannot conclude whether the protein produced by the truncated transcript is stably expressed in mammalian cells. It should be noted that the transcript analyses described were done with cell populations, either from primary tissues or after in vitro culture for up to 8 weeks. Thus it is not clear if each cell produces the same ratio of truncated to membrane forms of the Pactolus gene, or if some cells in the culture exclusively produce one form.
  • Some ⁇ integrins utilize alternative splicing to produce variant isoforms of the proteins.
  • at least 4 distinct alternative cytoplasmic domains have been described for ⁇ 1 which alter the functional characteristics of the protein (Zhidkova et al., 1995; Altruda et al., 1990; Languino and Ruoslahti, 1992; Meredith et al., 1995).
  • ⁇ integrin subunit that would, like Pactolus, predict a secreted form of the protein.
  • the expression of the Pactolus gene appears to be limited to immature cells of bone marrow derivation.
  • the murine tissue demonstrating the highest level of expression is the mouse marrow.
  • the quantity of Pactolus transcripts in the splenic sample was 10% or less that of the bone marrow. Since the spleen is primarily populated by mature cells of bone marrow origin (B and T cells and macrophages), the absence of appreciable Pactolus transcripts in the spleen indicates the down regulation of this gene during cellular maturation.
  • SCF allows for the proliferation of mast cell precursors while the addition of IL-3 leads to their differentiation into more mature cell types. If IL-3 derived cells do represent more mature cells than their SCF-derived counterparts, then the relative loss of Pactolus transcripts in the IL-3 derived cells would be expected if the expression of the gene is limited to immature bone marrow derived cells.
  • the protein is expressed on the surface of the bone marrow cells with an apparent molecule weight of 95 kDa.
  • Pactolus does not function as the typical ⁇ integrin subunit despite its sequence homology with ⁇ 2 29 and ⁇ 7. Therefore placing Pactolus within the integrin gene/protein family would imply a functionality of the protein that it may not possess.
  • the expression pattern of Pactolus shows it is expressed by immature and maturing cells of bone marrow derivation. Its similarity in structure to the ⁇ integrin gene family indicates it can act as a receptor mediating adhesion of such cells within the marrow stroma. Previously the 4 integrin has been shown to be critical in marrow maintenance. Reconstitution of RAG- 1 or RAG-2 deficient blastocysts with ⁇ 4 deficient ES cells created chimeric mice with marked deficiencies in lymphoid cell development in the bone marrow (Arroyo et al., 1996). Additional studies in which anti- 4 .antibodies were introduced into animals demonstrated a transitory loss of cells within the marrow (Papayannopoulou and Nakamoto, 1993).
  • Pactolus can also play a signaling role for cells within the marrow.
  • one of the alternative cytoplasmic domains for the ⁇ l integrin ( ⁇ lc) acts to directly inhibit cell cycle progression (Meredith et al., 1995).
  • Mouse cells transfected with this protein were arrested in the late GI phase of the cell cycle.
  • the marrow consists of many cell types, some of which are held in a state of low replicative activity.
  • the two different forms of the Pactolus protein can, upon ligation with ligand, send two quite distinct signals into the cell.
  • RNA Preparation and cDNA Synthesis Total RNA from various cells and tissues was isolated using the CsCl/guanidine method
  • cDNA was synthesized by mixing: 5 ⁇ g of RNA, 10 ⁇ L of 5X first str.and buffer, 5 ⁇ L 5 mM dNTP, 5 ⁇ L 0.1 M DDT, 2 ⁇ L perspective .anchoring primer (Liang and Pardee, 30).
  • EXAMPLE 3 Differential Display PCR Amplification PCR amplification was done by mixing: 200 ng cDNA (reverse transcribed with anchoring primers), 1 ⁇ L 500 mM dNTP, 1 ⁇ L lOx PCR buffer, 0.5 ⁇ L 1 ⁇ g/ ⁇ L decamer, 0.15 ⁇ L Taq DNA polymerase (GIBCO-BRL), 0.25 ⁇ L of P 32 -dCTP, and water to a total volume of 10 ⁇ L per reaction. Each reaction was done in triplicate. The reaction mixture was then put into a capillary tube (Idaho Technology) and amplified under the following conditions.
  • the amplification was carried out at 94°C for 1 second, 40°C for 1 second, 72°C for 10 seconds.
  • the following 35 cycles were carried out at 94°C for 1 second, 50°C for 1 second and 72°C for 10 seconds.
  • the reaction was then quenched by adding 10 ⁇ L of stop buffer and then 5 ⁇ L of the reactions was loaded onto a 6% sequencing gel. After 2 hours of electrophoresis, the gel was dried followed by autoradiography.
  • Random decamer 5' TGGATTGGTC 3' (SEQ ID NO:4)
  • RT-RPCR was performed as previously described (Tan and Weis, 1992; Weis et al., 1992). Pactolus specific primer sets are shown below:
  • Gap #1 5' CTC TGG CTC TGC GCA AGG CC 3' (SEQ ID NO:5) and 5' AAG CAC CAG AAA TCG GGT CC 3' (SEQ IDNO:6)
  • Gap #2 5' TAG TAC TCG GAG CAG CGA TGG 3' (SEQ ID NOJ) and 5' CGA GTG CGA CAA TGT CAA CTG 3' (SEQ ID NO:8) 31
  • EXAMPLE 6 In Vitro Transcription and Translation In vitro transcription was performed according to the manufacturer's manual of RNA Transcription Kit (Stratagene). mRNA was then translated in vitro using the rabbit reticulocyte lysate system (Amersham) in the presence of S 35 labeled methionine.
  • EXAMPLE 7 Generation of Pactolus-Specific Antisera Two rabbits were injected with a peptide derived from the Pactolus cytoplasmic domain (CGTQKAAKLPRKG (SEQ ID NO:21 )) using the KLH/BSA conjugation protocol and reagents from Pierce. The first injection was done with an equal volume of Freund's complete adjuvant, while the subsequent injections were with Freund's incomplete adjuvant. Antisera from one of the rabbits was utilized.
  • Bone marrow cells were harvested from mice and red blood cells (RBC) were lysed with RBC lysis solution (0.15 M NH 4 C1, 1.0 mM KHC0 3 , and 0.1 mM EDTA, pH 7.2 ) for 5 minutes at room temperature.
  • RBC lysis solution (0.15 M NH 4 C1, 1.0 mM KHC0 3 , and 0.1 mM EDTA, pH 7.2 ) for 5 minutes at room temperature.
  • EL-4 cells were maintained in RPMI media supplemented with 5% fetal bovine serum. Before labeling, cells were washed two times with PBS. Cells were labeled by incubating with 0.5 mg/mL Sulfo-NHS-LC-Biotin (Pierce) for 30 minutes at room temperature.
  • lysis buffer (0.5% NP-40, 0.5% NaDOC, 50 mM NaCl, 2 mM CaCl 2 , 25 mM Tris pH7.5, 1% BSA, 0.2 mM PMSF, 2 ⁇ g/mL aprotinin, and 1 ⁇ g/mL pepstatin A) at 5xl0 7 cells/mL, and the reactions were incubated on ice for 1 hour. Ly sates were immunoprecipitated with either polyclonal rabbit antisera against Pactolus cytoplasmic peptide or anti-mouse ⁇ 2 monoclonal antibody (PharMingen).
  • the protein-antibody complex was absorbed with either protein A- Sepharose (for rabbit antibody) or protein G-Sepharose (for rat antibody). After the absorption, the Sepharose beads were washed 4 times: once with lysis buffer, twice with lysis buffer with 150 mM NaCl, and once with 0.05 M Tris pH 6.8. The samples were boiled in lx SDS loading buffer for
  • RNA to be analyzed was isolated from four different mast cell sources. Bone marrow cells were cultured in the presence of either IL-3 or SCF for 21 days to develop into mast cells phenotypically similar to immature mucosal (MMC) or connective tissue mast cells (CTMC), respectively. RNA was isolated from such cells. Additionally, RNA was isolated from IL-3 derived cells that had been transferred into SCF without IL-3 for 24 hrs (MMC + SCF) and SCF derived cells transferred into IL-3 without SCF for 24 hours (CTMC + IL-3). The isolated total RNA was reverse transcribed with a specific anchoring primer and expanded by PCR-based amplification with randomly designed decamers.
  • MMC immature mucosal
  • CTMC connective tissue mast cells
  • the DNA sequence obtained from the A and B fragments was used to search GenBank for homologous sequences. The sequences derived from these fragments did not match any sequence in GenBank. Additional analyses of the Fragment A sequence are in progress and will not be reported here, however, they were clearly not derived from the same gene as Fragment B. The sequence from Fragment B was utilized to design nested primers to confirm the specificity of expression, and to generate a probe with which to screen a murine mast cell cDNA library.
  • EXAMPLE 10 Identification of a Novel ⁇ -Inte rin-Like Sequence
  • the nested primers derived from the Fragment B sequence were used to re-analyze the primary mRNA samples (using the standard RT-RPCR protocol) the expression of the Fragment B gene was primarily confined to the SCF-derived cells, not those initially derived in IL-3 (Fig 3). Accordingly a cDNA library constructed with RNA isolated from mast cells derived in SCF was screened with the fragment B probe. Twelve clones were isolated from this screening. The sequence of the largest of these cDNA clones possessed an insert with 2,585 nucleotides (shown in the SEQUENCE LISTING as SEQ ID NO:22).
  • a single large open reading frame was determined to start at an ATG at base 64 and terminate at a stop codon at base 1684, encoding a protein with 540 amino acids (shown in the SEQUENCE LISTING as SEQ ID NO:23).
  • This sequence thus predicted a long 3' untranslated sequence of 901 nucleotides.
  • This predicted amino acid sequence suggested a secreted protein possessing a signal sequence but lacking a transmembrane domain for membrane anchoring (see below).
  • This cDNA sequence has been submitted to GenBank under accession number Bankltl75798 AF051367. The gene and gene product was named Pactolus-1.
  • the oligonucleotides spanning Gap #2 generated two distinct products differing in size by about 40 bp (Figure 5B).
  • the size of the smaller of the two was predicted from the Pactolus cDNA sequence. Both fragments were excised from the gel, cloned and sequenced. As expected, the shorter of the two was identical to the Pactolus sequence. The larger fragment possessed the expected Pactolus sequences plus a 43 bp insertion ( Figure 6A). When this additional sequence was inserted within the Pactolus coding sequence, the reading frame was altered such that the predicted stop codon was shifted to base 2,286, predicting the inclusion of an additional 223 amino acids.
  • the 36 cytoplasmic domain of Pactolus does not demonstrate significant homology with any of the eight characterized ⁇ integrin chains (data not shown) (Wilson et al., 1989; Argraves et al., 1987; Fitzgerald et al., 1987; Hogervorst, et al., 1990; Ramaswamy and Hemler, 1990; Sheppard et al., 1990; Gurish et al., 1992b; Moyle et al, 1991). All characterized ⁇ integrin chains possess the MIDAS motif (metal ion-dependent adhesion site) at about residue 130 (Figure 6E).
  • This site is characterized by a conserved DxSxS sequence followed by a threonine (about 65 amino acids carboxyl to the DxSxS sequence) and an aspartate residue (about 25 amino acids carboxyl of the threonine residue).
  • This site within the ⁇ integrin chain has been demonstrated to be critical for ligand binding (Bajt and Loftus, 1994), chain association (at least two mutations in the ⁇ 2 chain at this site are responsible for the Lymphocyte
  • Adhesion Deficiency syndrome and are underlined in the ⁇ 2 sequence) (Wardlaw et al., 1990), and Mg 2+ ion binding (conversion of the amino terminal aspartate of the MIDAS motif to alanine abolishes metal binding at this site) (Michishita et al, 1993; Ueda et al., 1994; Rieu et al., 1994). All of the eight known ⁇ integrins demonstrate conservation of this site, as demonstrated by the comparison between the mouse ⁇ 1 and ⁇ 2 sequences ( Figure 6E). Interestingly Pactolus does not as it lacks the critical aspartate residue as part of the DxSxS sequence.
  • EXAMPLE 12 mRNA Expression of Pactolus
  • Pactolus transcripts are evident in bone marrow derived mast cells (cultured in SCF), and in bone marrow and spleen. Due to the extreme sensitivity of RT- PCR (reverse transcriptase-rapid polymerase chain reaction), the expression of Pactolus transcripts in the bone marrow was further confirmed by Northern blot analysis.
  • RT- PCR reverse transcriptase-rapid polymerase chain reaction
  • FIG. 8 A a 2.8 to 3.0 kb band was evident when 5 ⁇ g of total RNA from mouse bone marrow was hybridized with the Pactolus cDNA sequence. The size differential between the two Pactolus mRNA isoforms is too small to detect two distinct bands with this type of assay.
  • the tissue distribution of Pactolus transcripts was further analyzed by using a semi- quantitative RT-RPCR assay. These assays were done with the oligo set which discriminates between the truncated and full length Pactolus transcript (thus covering Gap #2) and were done at low cycle number to inhibit saturation of the product. As shown in Figure 8B, the mouse bone marrow clearly possesses the highest level of Pactolus transcripts. The high amount of transcripts for the truncated form (compared to the full length product) may be due to the competitive advantage of the smaller fragment over that of the larger fragment during PCR amplification. When such an analysis was done with higher cycle numbers, the presence of Pactolus transcripts in the spleen and lung samples was evident (data not shown).
  • RT-RPCR analysis was done using oligo sets specific for Pactolus, integrins ⁇ l, ⁇ 2, ⁇ L, ⁇ M, and L-selectin.
  • the Pactolus- specific product is that which would only be found within the transcript encoding the full length protein product. All of the reactions were done for the same number of cycles (and produce products of approximately the same size) for this comparison.
  • Pactolus is expressed at a lower level than ⁇ 1, ⁇ 2, and ⁇ M, but in comparable quantities as those seen for ⁇ L and L-selectin.
  • Pactolus with another subunit is so weak that the partner can not be resolved using the standard approaches.
  • Segments of Pactolus coding sequence are expressed as fusion protein in E. coli.
  • the overexpressed protein is purified by gel elution and used to immunize rabbits and mice using a procedure similar to the one described by Harlow and Lane, 1988. This procedure has been shown to generate Abs against various other proteins (for example, see Kraemer et al., 1993). Briefly, a stretch of Pactolus coding sequence is cloned as a fusion protein in plasmid PET5A
  • fusion protein with the expected molecular weight is verified by SDS/PAGE. Fusion protein is purified from the gel by electroelution. Identification of the protein as the Pactolus fusion product is verified by protein sequencing at the N-terminus. Next, the purified protein is used as an immunogen in rabbits. Rabbits are immunized with 100 ⁇ g of the protein in complete Freund's adjuvant and boosted twice in 3 week intervals, first with 100 ⁇ g of immunogen in incomplete Freund's adjuvant followed by 100 ⁇ g of immunogen in PBS. Antibody containing serum is collected two weeks thereafter.
  • EXAMPLE 15 Generation of Monoclonal Antibodies Specific for Pactolus Monoclonal antibodies are generated according to the following protocol. Rats are immunized with immunogen comprising intact Pactolus or Pactolus peptides (wild type or mutant) conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC as is well known.
  • the immunogen is mixed with an adjuvant.
  • Each rat receives four injections of 10 to 100 ⁇ g of immunogen and after the fourth injection blood samples are taken from the rats to determine if the serum contains antibody to the immunogen.
  • Serum titer is determined by ELISA or RIA. Rats with sera indicating the presence of antibody to the immunogen are selected for hybridoma production. 40
  • Spleens are removed from immune rats and a single cell suspension is prepared (see Harlow and Lane, 1988). Cell fusions are performed essentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, MD) are fused with immune spleen cells using polyethylene glycol as described by Harlow and Lane, 1988. Cells are plated at a density of 2x10 5 cells/well in 96 well tissue culture plates. Individual wells are examined for growth and the supematants of wells with growth are tested for the presence of Pactolus specific antibodies by ELISA or RIA using wild type or mutant Pactolus target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.
  • Clones with the desired specificities are expanded and grown as ascites in rats or in a hollow fiber system to produce sufficient quantities of antibody for characterization and assay development.
  • EXAMPLE 16 Sandwich Assay for Pactolus Monoclonal antibody is attached to a solid surface such as a plate, tube, bead or particle.
  • the antibody is attached to the well surface of a 96-well ELISA plate. 100 ⁇ L sample
  • a second monoclonal antibody (to a different determinant on the Pactolus peptide/protein) is added to the solid phase.
  • This antibody is labeled with a detector molecule (e.g., 125 I, enzyme, fluorophore, or a chromophore) and the solid phase with the second antibody is incubated for two hrs at room temperature. The second antibody is decanted and the solid phase is washed with buffer to remove unbound material.
  • the amount of bound label which is proportional to the amount of Pactolus peptide/protein present in the sample, is quantified. Separate assays are performed using monoclonal antibodies which are specific for the wild-type Pactolus as well as monoclonal antibodies specific for any mutations identified in Pactolus.
  • Pactolus is expressed in immature cells in bone marrow and expression is decreased as the cells mature.
  • Pactolus is a cell surface protein and can bind to ligands thereby holding the cells 41 within the marrow.
  • the free Pactolus will compete with the Pactolus located on cell surfaces and will thereby displace the cells from the ligand.
  • the free ligand will compete with bound ligand which is bound to Pactolus and holding the cells in place within the bone marrow.
  • the immature cells When the Pactolus binds to the free ligand which has been added, the immature cells will be free to migrate to the bloodstream. This freeing of the cells allows the immature cells to leave the bone marrow and enter the bloodstream. These immature cells may then be collected directly from blood rather than from bone marrow. This will be useful because bone marrow transplants presently require that bone marrow be taken from the donor by extraction from the pelvis or femur. The purpose of collecting the bone marrow is to obtain the immature cells. By using the method of adding Pactolus or free ligand to the body and causing the immature cells to leave the marrow and flood the blood, these same immature cells can be obtained simply by collecting blood from the donor rather than having to collect bone marrow.
  • Binding assays are well known in the art. One method of screening for binding agents is to synthesize many different test ligands and to bind them to a solid support in a grid-like fashion.
  • the binding partner, here Pactolus can be labeled, added onto the solid support in solution and allowed to incubate with the ligands which are bound to the solid support.
  • this incubation would preferably be performed under conditions which simulate the physiological conditions found in bone marrow, i.e., the pH and ionic strength should preferably be close to or identical to that found in bone marrow.
  • the Pactolus containing solution is poured off and the solid support is washed, again using a solution simulating physiological conditions would be the most ideal wash solution, to remove unbound Pactolus.
  • the solid support is then assayed for the presence of bound Pactolus. This could be done by a variety of methods.
  • the Pactolus could be labeled prior to incubating it with the bound ligands.
  • the bound Pactolus could be located by treatment with, e.g., labeled antibodies which bind to Pactolus.
  • labeled antibodies which bind to Pactolus.
  • Those of skill in the art know a variety of methods for assaying for the bound Pactolus. The locations of bound Pactolus are determined, and those ligands which were at those locations, are selected as ligands to which Pactolus will bind. If desired, further assays may be performed to determine 42 binding affinity between the selected ligands and Pactolus. For actual use in medical procedures, those ligands with the highest affinity for Pactolus would be the most effective.
  • the assays for ligands which will bind to Pactolus can be performed by other methods than those outlined above.
  • the reverse type of experiment can be performed, wherein Pactolus can be bound to a solid support and the various ligands can then be incubated with the bound Pactolus, washed, and assayed for their presence.
  • the ligands can be prelabeled such as with a radioactive tracer, a fluorophore, or other label known to those of skill in the art.
  • An assay to determine ligands of Pactolus can also be performed according to the two-hybrid system. See Chien et al., 1991 and U.S. Patent Nos. 5,283,173, 5,468,614 and 5,667,973.
  • a hybrid nucleic acid encoding Pactolus is cointroduced into yeast along with another hybrid nucleic acid encoding a potential ligand.
  • This method can be performed as part of a cDNA library screening process. Positive clones can be selected and the hybrid nucleic acid can be recovered, cloned and sequenced to determine the nucleotide sequence and predicted amino acid sequence of the ligand.
  • EXAMPLE 18 Cloning of Human Pactolus It is well known to those of skill in the art that the genomes of different species have extensive homology. Species as different as humans and the slime mold Dictyostelium discoideum, yeast, and the worm C. elegans are known to have extensive homology to each other within their genomes. The more closely related that species are the greater the homology becomes. Humans and mice are both not only vertebrates but also mammals and their genomes show extensive homology to one another. It is common practice to first find a gene in one species and then to use that gene to locate the equivalent gene in a different species. The homology between closely related species tends to be high enough that the equivalent genes will cross-hybridize with one another under appropriate hybridization conditions.
  • the human Pactolus gene by screening human genomic or cDNA libraries with the mouse Pactolus gene which has been disclosed herein. Standard library screening techniques using moderately stringent hybridization conditions would preferably be used. To decrease the number of positive clones seen which would correspond to known ⁇ integrins rather than Pactolus, the library screen is preferably performed with probes which are homologous to those regions of Pactolus which are not homologous to the ⁇ integrins. 43
  • Pactolus amino acid sequence can be determined. Human Pactolus can then be obtained by using in vitro transcription and translation systems, it can be synthesized via an amino acid synthesizer, or it may be purified by traditional techniques or via antibody precipitation.
  • the immature blood cells of the bone marrow can be caused to leave the marrow and flood the bloodstream by the addition of either Pactolus or a ligand to which Pactolus binds to the bone marrow.
  • the free Pactolus will compete with Pactolus on the immature cells for the natural ligand in the marrow. This competition will result in the immature cells being freed once enough free Pactolus has reached the marrow.
  • free ligand if free ligand is added to the marrow this free ligand will compete with the naturally occurring bound ligand of the marrow, and the Pactolus on the immature blood cells will bind to the free ligand thereby freeing the cells to migrate to the bloodstream.
  • Pactolus or free ligand can be administered, e.g., by intravenous injection into the blood stream. This route will be effective in allowing the administered Pactolus or free ligand to reach the bone marrow. 44
  • Transferrin-polycation-mediated introduction of DNA into human leukemic cells stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. USA 87, 4033-4037.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention se rapporte à un nouveau gène, dénommé Pactolus, et à sa protéine codée. Le gène et la protéine présentent une homologie avec les intégrines β. Pactolus est une protéine de surface cellulaire qui est impliquée dans le maintien de cellules sanguines immatures dans la moelle osseuse par liaison à un ligand. L'épissage différentiel de l'ARNm produit deux formes de Pactolus, une forme membranaire relativement longue et une forme tronquée. L'administration de Pactolus ou d'un ligand qui se lie au gène Pactolus provoque une libération de cellules sanguines immatures provenant de la moelle osseuse dans la circulation sanguine où ces cellules peuvent être facilement prélevées.
PCT/US1999/005454 1998-03-13 1999-03-12 IDENTIFICATION DE PACTOLUS: UNE PROTEINE DE TYPE INTEGRINE $g(b) WO1999046373A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU30843/99A AU3084399A (en) 1998-03-13 1999-03-12 Identification of pactolus: a beta integrin-like protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7786898P 1998-03-13 1998-03-13
US60/077,868 1998-03-13

Publications (1)

Publication Number Publication Date
WO1999046373A1 true WO1999046373A1 (fr) 1999-09-16

Family

ID=22140522

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/005454 WO1999046373A1 (fr) 1998-03-13 1999-03-12 IDENTIFICATION DE PACTOLUS: UNE PROTEINE DE TYPE INTEGRINE $g(b)

Country Status (2)

Country Link
AU (1) AU3084399A (fr)
WO (1) WO1999046373A1 (fr)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GUMLEY T. P., ET AL.: "SEQUENCE AND STRUCTURE OF THE MOUSE THB GENE.", IMMUNOGENETICS, SPRINGER VERLAG, BERLIN, DE, vol. 42., no. 03., 1 January 1995 (1995-01-01), DE, pages 221 - 224., XP002920550, ISSN: 0093-7711, DOI: 10.1007/BF00191229 *
LAW S. K. A., ET AL.: "THE PRIMARY STRUCTURE OF THE BETA-SUBUNIT OF THE CELL SURFACE ADHESION GLYCOPROTEINS LFA-1, CR3 AND P150,95 AND ITS RELATIONSHIP TO THE FIBRONECTIN RECEPTOR.", EMBO JOURNAL., OXFORD UNIVERSITY PRESS, SURREY., GB, vol. 06., no. 04., 1 January 1987 (1987-01-01), GB, pages 915 - 919., XP002920552, ISSN: 0261-4189 *
LEE J.-O., ET AL.: "CRYSTAL STRUCTURE OF THE A DOMAIN FROM THE ALPHA SUBUNIT OF INTEGRIN CR3 (CD11B/CD18).", CELL, CELL PRESS, US, vol. 80., 24 February 1995 (1995-02-24), US, pages 631 - 638., XP002920554, ISSN: 0092-8674, DOI: 10.1016/0092-8674(95)90517-0 *
MEREDITH J., ET AL.: "INHIBITION OF CELL CYCLE PROGRESSION BY THE ALTERNATIVELY SPLICED INTEGRIN BETA1C.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 269., 15 September 1995 (1995-09-15), US, pages 1570 - 1572., XP002920553, ISSN: 0036-8075, DOI: 10.1126/science.7545312 *
ZEGER D. L., ET AL.: "MOUSE MACROPHAGE BETA SUBUNIT (CD11B) CDNA FOR THE CR3 COMPLEMENT RECEPTOR/MAC-1 ANTIGEN.", IMMUNOGENETICS, SPRINGER VERLAG, BERLIN, DE, vol. 31., 1 January 1990 (1990-01-01), DE, pages 191 - 197., XP002920551, ISSN: 0093-7711, DOI: 10.1007/BF00211555 *

Also Published As

Publication number Publication date
AU3084399A (en) 1999-09-27

Similar Documents

Publication Publication Date Title
US6124104A (en) Chromosome 13-linked breast cancer susceptibility gene
JP5328603B2 (ja) 良性の家族性新生児痙攣（ｂｆｎｃ）および他の癲癇において突然変異されたｋｃｎｑ２およびｋｃｎｑ３−カリウムチャンネル遺伝子
EP1260520A2 (fr) Gène BRCA2 lié au chromosome 13, de la susceptibilité au cancer du sein
US7297489B2 (en) Mutations in and genomic structure of HERG—a long QT syndrome gene
CA2337491C (fr) Mutations geniques mink humaines associees a l'arythmie
CA2416545C (fr) Polymorphisme commun dans scn5a a l'origine d'arythmie cardiaque medicamenteuse
US6441156B1 (en) Calcium channel compositions and methods of use thereof
AU758048B2 (en) KVLQT1 - a long QT syndrome
WO1999046373A1 (fr) IDENTIFICATION DE PACTOLUS: UNE PROTEINE DE TYPE INTEGRINE $g(b)
WO2001023561A2 (fr) Nouveaux polynucleotides codant des proteines contenant des sequences repetees de thrombospondine type 1 (tsrx)
US6844189B1 (en) Chromosome 17P-linked prostate cancer susceptibility gene
CA2712809C (fr) Kncq2 et kncq3, genes du canal potassium ayant subi une mutation dans des convulsions neonatales familiales benignes (bfnc) et d'autres epilepsies
WO2003025197A2 (fr) Compositions a base de proteine mutante de liaison a sh3 et methodes associees
AU1118301A (en) Tumour suppressor genes from chromosome 16

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 09623565

Country of ref document: US

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载