WO2002030978A1 - Polypeptides, procedes d'elaboration, et utilisations - Google Patents

Polypeptides, procedes d'elaboration, et utilisations Download PDF

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Publication number
WO2002030978A1
WO2002030978A1 PCT/US2000/027797 US0027797W WO0230978A1 WO 2002030978 A1 WO2002030978 A1 WO 2002030978A1 US 0027797 W US0027797 W US 0027797W WO 0230978 A1 WO0230978 A1 WO 0230978A1
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human
oghbetal
polypeptide
nucleic acid
sequence
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PCT/US2000/027797
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English (en)
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Andrew J. Murphy
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Regeneron Pharmaceuticals, Inc.
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Priority to PCT/US2000/027797 priority Critical patent/WO2002030978A1/fr
Priority to AU2000280019A priority patent/AU2000280019A1/en
Publication of WO2002030978A1 publication Critical patent/WO2002030978A1/fr

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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
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the field of this invention is polypeptides which regulate cell function, and in particular, HUMAN OGHbeta14, as well as methods of making said polypeptides and methods of using said polypeptides.
  • the target cell In order for a particular hormone to have an effect on a particular cell, the target cell must have a way in which to recognize the hormone.
  • One way that cells do this is by having membrane-bound receptors on their cell surface.
  • the receptors are able to recognize and bind hormones with a high degree of specificity, thus eliminating inappropriate response to hormones by non-target cells.
  • Many hormones utilize receptors that belong to a class of receptors known as the G: jotein coupled receptors (GPCR).
  • GPCR jotein coupled receptors
  • hormones that signal via GPCRs include, but are not limited to, the tropic adenohypophyseal hormones f ollicle stimulating hormone (FSH), Luteinizing hormone (LH), and thyroid stimulating hormone (TSH).
  • FSH tropic adenohypophyseal hormone
  • LH Luteinizing hormone
  • TSH thyroid stimulating hormone
  • CG chorionic gonadotropin
  • All four hormones are glycoproteins and belong to the glycoprotein hormone-beta family.
  • Each hormone is a heterodimer comprised of a common glycoprotein hormone alpha ( ) subunit and a beta ( ⁇ ) subunit. All four family members share the same ⁇ subunit, but each has its own unique ⁇ subunit.
  • the ⁇ subunit provides for binding specificity to each hormone's cognate GPCR: LH/CGR for both LH and CG, FSHR for FSH and TSHR for TSH.
  • LH/CGR for both LH and CG
  • FSHR for FSH
  • TSHR TSHR
  • FSH, LH, and TSH are considered tropic hormones in that they regulate the activity of other endocrine glands.
  • FSH is primarily responsible for stimulating development of the oocyte during the female ovarian cycle. A deficiency in this hormone is responsible for thousands of cases of infertility.
  • LH is primarily responsible for triggering ovulation of the mature oocyte during the female ovarian cycle. As with FSH, a deficiency of this hormone can also lead to infertility.
  • LH also known as interstitial cell stimulating hormone, ICSH
  • ICSH is primarily responsible for stimulating production of testosterone by the interstitial cells of Leydig which are located in the seminiferous tubules of the testes.
  • CG is primarily responsible for signaling the corpus luteum to secrete progesterone to support a developing embryo until a placenta can form and assume this responsibility.
  • TSH is involved is stimulating the normal growth, development, and functioning of the thyroid gland, an endocrine gland whose secretions, the thyroid hormones thyroxine (T4) and triiodothyronine (T3), regulate many cellular activities in almost every cell in the body.
  • T4 thyroid hormones thyroxine
  • T3 triiodothyronine
  • the thyroid gland also produces the hormone calcitonin which is involved in regulating blood calcium levels and bone homeostasis.
  • this family of polypeptide hormones represents an important class of molecules with functions ranging from reproduction, cellular metabolism, and growth and development to blood calcium and bone homeostasis.
  • the identification of additional family members would be an important step in further understanding hormone action in general and, in particular, helping to develop tests for diagnosing diseases that may be caused by a lack of or a malfunctioning of a family member as well as developing therapies to treat such diseases.
  • these novel molecules can be used to design screening assays to identify their receptor.
  • the subject invention provides for isolated HUMAN OGHbeta14 polypeptides.
  • the subject invention also provides for isolated HUMAN OGHbeta14 polypeptides, comprising the amino acid sequence as set forth in Figure 1 or Figure 3.
  • the subject invention further provides for the isolated HUMAN OGHbeta14 polypeptides encoded by the nucleic acid molecule as set forth in Figure 1 or Figure 3.
  • Another embodiment of the invention is a vector which comprises nucleic acid molecules as set forth in Figure 1 or Figure 3.
  • a further embodiment is a vector wherein the nucleic acid molecule is operatively linked to an expression control sequence capable of directing its expression in a host cell.
  • the vector is a plasmid.
  • Another embodiment of the invention is a host-vector system for the production of HUMAN OGHbeta14 which comprises a vector in a host cell wherein the host cell is a bacterial, yeast, insect or mammalian cell.
  • the invention further contemplates a method of producing HUMAN OGHbeta14 which comprises growing cells of a host-vector system of under conditions permitting production of the HUMAN OGHbeta14, and recovering the HUMAN OGHbeta14 so produced.
  • the invention further contemplates a method of producing a stable heteromer of HUMAN OGHbeta14 and the common glycoprotein subunit which comprises growing cells of a host-vector system of under conditions permitting production of the HUMAN OGHbeta14/common glycoprotein hormone alpha ( ) subunit heteromer, and recovering the HUMAN OGHbeta14/common glycoprotein hormone alpha ( ) subunit so produced.
  • the invention further contemplates a method of producing a single chain HUMAN OGHbeta14/a common glycoprotein hormone alpha ( ) subunit heteromer which comprises growing cells of a host-vector system of under conditions permitting production of a single chain HUMAN OGHbeta14/common glycoprotein hormone alpha ( ⁇ ) subunit heteromer, and recovering the a single chain HUMAN OGHbeta14/common glycoprotein hormone alpha ( ) subunit heteromer so produced.
  • Still another embodiment of the invention provides for an antibody which specifically binds the HUMAN OGHbeta14 polypeptide.
  • the antibody may be a monoclonal antibody.
  • the invention provides for a composition comprising HUMAN OGHbeta14 polypeptide and a carrier as well as a composition comprising an antibody and a carrier wherein said compositions are for use in a method of treatment of the human or animal body, or in a method of diagnosis.
  • OGHbeta14 fused to an immunoglobulin constant region including a ligandbody wherein the immunoglobulin constant region is the Fc portion of human lgG1.
  • the ligandbodies may be used in a method of treatment of the human or animal body, or in a method of diagnosis.
  • Another embodiments of the invention provides a method of identifying a HUMAN OGHbeta14 binding target comprising
  • the subject invention provides for unique polypeptides called HUMAN OGHbeta14 encoded by nucleic acids as set forth in Figure 1 or Figure 3 which were identified by screening the NCBI genomic database with sequences obtained from known family members.
  • the invention comprises nucleic acids which are complementary to the HUMAN OGHbetal 4 sequences as set forth in Figure 1 or Figure 3.
  • the invention also comprises the use of HUMAN OGHbetal 4 sequences to identify and obtain a full length HUMAN OGHbetal 4 cDNA.
  • the invention further comprises the use of oligomers from the HUMAN OGHbetal 4 sequence in a HUMAN OGHbetal 4 kit which can be used to identify a disorder or disease with altered HUMAN OGHbetal 4 expression and provide a method for monitoring progress of a patient during drug therapy.
  • the invention comprises the use of HUMAN OGHbetal 4- specific antibodies in assays to identify a disorder or disease with altered HUMAN OGHbetal 4 expression and provides a method to monitor the progress of a patient during drug therapy.
  • Figure 1 The nucleic acid sequences (sense and antisense) and deduced amino acid sequence of HUMAN OGHbetal 4.
  • Figure 2 A sequence comparison of the novel ⁇ subunit polypeptide identified as HUMAN OGHbetal 4 with the ⁇ subunits of the other family members: rLH-beta, hCG-beta-e, rTSH-beta, and rFSH-beta. Asterisks (*) indicate amino acid identity between all family members.
  • Figure 3 The nucleic acid sequences (sense and antisense) and deduced amino acid sequence of HUMAN OGHbetal 4 including additional 5' sequence.
  • Figure 4 Expression of HUMAN OGHbetal 4 using TaqMan analysis.
  • the analysis shows weak expression of HUMAN OGHbetal 4 in pituitary, testis, thymus and trachea.
  • the expression levels are normalized to molecules of mRNA per cell assuming each cell contains 20pg of total RNA or 1 pg of poly a+ RNA.
  • FIG. 5 Expression of HUMAN OGHbetal 4 using Northern analysis. The results of this analysis reveal the presence of a 1.3 kb transcript in the pituitary. Since the pituitary is the site of expression of the homologous glycoprotein hormone family members LH, FSH and TSH, it seems likely that HUMAN OGHbetal 4 is also a circulating hormone.
  • Figure 6. Subunit structure of HUMAN OGHbetal 4.
  • Flag-tagged HUMAN OGHbetal 4 immunoprecipitated both HA-tagged common glycoprotein hormone alpha ( ) subunit (Lane 1 , Panel C) and HA-tagged HUMAN OGHbetal 4 (Lane 2, Panel C) with efficiencies roughly comparable to that at which FLAG-tagged hCG immunoprecipitated common glycoprotein hormone alpha ( ) subunit (Lane 7, Panel C). These results indicate that HUMAN OGHbetal 4 can form homo-dimers (or higher order structures) as well as hetero-dimers (or higher order structures) with the common glycoprotein hormone alpha ( ) subunit.
  • oligonucleotide or “oligomer” is a stretch of nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). These short sequences are based on (or designed from) genomic or cDNA sequences and are used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and may be used as probes.
  • Probes are nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies.
  • Reporter molecules are chemical moieties used for labeling a nucleic or amino acid sequence. They include, but are not limited to, radionuclides, enzymes, fluorescent, chemi-luminescent, or chromogenic agents. Reporter molecules associate with, establish the presence of, and may allow quantification of a particular nucleic or amino acid sequence.
  • a "portion" or “fragment” of a nucleic acid or nucleic acid comprises all or any part of the nucleic acid sequence having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb which can be used as a probe.
  • probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. After pretesting to optimize reaction conditions and to eliminate false positives, nucleic acid probes may be used in Southern, northern or in situ hybridizations to determine whether DNA or RNA encoding the protein is present in a biological sample, cell type, tissue, organ or organism.
  • Recombinant nucleotide variants are nucleic acids which encode a protein. They may be synthesized by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce specific restriction sites or codon usage- specific mutations, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic host system, respectively.
  • Control elements or "regulatory sequences” or “expression control sequences” are those nontranslated regions of the gene or DNA such as enhancers, promoters, introns and 3 1 untranslated regions which interact with cellular proteins to carry out replication, transcription, and translation. They may occur as boundary sequences or even split the gene. They function at the molecular level and along with regulatory genes are very important in development, growth, differentiation and aging processes.
  • Chimeric or “fusion” molecules are nucleic acids or polypeptides which are created by combining one or more of nucleotide sequences of this invention (or their parts) with additional nucleic acid sequence(s). Such combined sequences may be introduced into an appropriate vector and expressed to give rise to a chimeric polypeptide which may be expected to be different from the native molecule in one or more of the following characteristics: cellular location, distribution, ligand- binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • “Active” is that state which is capable of being useful or of carrying out some role. It specifically refers to those forms, fragments, or domains of a polypeptide sequence which display the biologic and/or immunogenic activity characteristic of the naturally occurring HUMAN OGHbetal 4.
  • “Naturally occurring HUMAN OGHbetal 4" refers to a polypeptide produced by cells which have not been genetically engineered or which have been genetically engineered to produce the same sequence as that naturally produced. Specifically contemplated are various polypeptides which arise from post-translational modifications. Such modifications of the polypeptide include but are not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Derivative refers to those polypeptides which have been chemically modified by such techniques as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Recombinant polypeptide variant refers to any polypeptide which differs from naturally occurring HUMAN OGHbetal 4 by amino acid insertions, deletions and/or substitutions, created using recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing characteristics of interest may be found by comparing the sequence of HUMAN OGHbetal 4 with that of related polypeptides and minimizing the number of amino acid sequence changes made in highly conserved regions.
  • Amino acid “substitutions” are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid "insertions” or “deletions” are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. The variation allowed in a particular amino acid sequence may be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the HUMAN OGHbetal 4 sequence using recombinant DNA techniques.
  • a “signal or leader sequence” or “signal peptide” is a short amino acid sequence which or can be used, when desired, to direct the polypeptide through a membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. It may be functionally equivalent to and either the same length as or considerably shorter than a "fragment ", "portion ", or “segment” of a polypeptide. Such sequences comprise a stretch of amino acid residues of at least about 5 amino acids and often about 17 or more amino acids, typically at least about 9 to 13 amino acids, and of sufficient length to display biologic and/or immunogenic activity.
  • inhibitor is a substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, antagonists and their derivatives.
  • a "standard” is a quantitative or qualitative measurement for comparison. Preferably, it is based on a statistically appropriate number of samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles. The samples of a particular standard may be normal or similarly abnormal.
  • Disorders or diseases in which altered HUMAN OGHbetal 4 activity have been implicated specifically include, but are not limited to, reproductive diseases, diseases related to cellular metabolism, growth, development, blood and bone homeostasis.
  • any undefined terms shall be construed to have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
  • the singular forms "a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.
  • reference to a “restriction enzyme” or a “high fidelity enzyme” may include mixtures of such enzymes and any other enzymes fitting the stated criteria, or reference to the method includes reference to one or more methods for obtaining cDNA sequences which will be known to those skilled in the art or will become known to them upon reading this specification.
  • the subject application provides for the identification of a novel member of the glycoprotein hormone family of hormones called HUMAN OGHbetal 4.
  • the invention further provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of HUMAN OGHbetal 4 modulatable cellular function.
  • these screening methods involve assaying for compounds which modulate HUMAN OGHbetal 4 interaction with a natural HUMAN OGHbetal 4 binding target.
  • assays for binding agents are provided including protein-protein binding assays, immunoassays, and cell based assays.
  • Preferred methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds.
  • In vitro binding assays employ a mixture of components including a HUMAN OGHbetal 4 polypeptide, which may be part of a fusion product with another peptide or polypeptide, e.g., a tag for detection or anchoring and a sample suspected of containing a natural HUMAN OGHbetal 4 binding target.
  • a variety of other reagents such as salts, buffers, neutral proteins, e.g., albumin, detergents, protease inhibitors, nuclease inhibitors, or antimicrobial agents may also be included.
  • the mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding.
  • the mixture is incubated under conditions whereby the HUMAN OGHbetal 4 specifically binds the suspected cellular binding target contained in the sample with a reference binding affinity. Incubation periods are chosen for optimal binding but are also minimized to facilitate rapid, high throughput screening.
  • the binding between the HUMAN OGHbetal 4 and the suspected binding target is detected by any convenient way.
  • a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation, immobilization, followed by washing by, e.g., membrane filtration or gel chromatography.
  • one of the components usually comprises or is coupled to a label.
  • the label may provide for direct detection as radioactivity, luminescence, optical or electron density, or indirect detection such as an epitope tag, or an enzyme.
  • a variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g., through optical or electron density, radiative emissions, nonradiative energy transfers, or indirectly detected with antibody conjugates, etc.
  • a difference in the binding affinity of the HUMAN OGHbetal 4 polypeptide to the suspected binding target as compared with the binding of the HUMAN OGHbetal 4 polypeptide in the absence of the suspected binding target indicates that the test sample contains a suitable binding target for the HUMAN OGHbetal 4 polypeptide.
  • a difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.
  • assays for binding targets for HUMAN OGHbetal 4 can be performed using Biacore technology. Examples of how to use this technology can be found in US Patent No. 5,641 ,640.
  • the nucleic acids, cDNAs, oligonucleotides, polypeptides and antibodies for the HUMAN OGHbetal 4, which are the subject of this invention, provide a plurality of tools for studying hormone action in various cells and tissues and for diagnosing diseases and selecting inhibitors or drugs with the potential to intervene in various disorders or diseases in which altered HUMAN OGHbetal 4 expression is implicated.
  • the disorders or diseases include, but are not limited to, reproductive diseases such as infertility, contraception, maintenance of pregnancy, menopause, dysmenhorrea; diseases related to thyroid malfunction such as thyroid deficiency or hyperactive thyroid leading to altered cellular metabolism; muscle atrophy, growth, development, blood and bone homeostasis.
  • HUMAN OGHbetal 4 These sequences were identified by their similarity to published or known open reading frames. Since hormones are associated with basic cellular processes such as cell proliferation, differentiation and cell signaling, these sequences are useful in the characterization and delineation of normal and abnormal processes.
  • HUMAN OGHbetal 4 nucleic acid sequences are useful in diagnostic assays used to evaluate the role of a specific HUMAN OGHbetal 4 in normal, diseased, or therapeutically treated cells. Purified HUMAN OGHbetal 4 nucleic acid sequences have numerous applications in techniques known to those skilled in the art of molecular biology.
  • nucleic acid sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleic acid sequences that are currently known, including but not limited to such properties as the triplet genetic code and specific base pair interactions.
  • HUMAN OGHbetal 4-encoding nucleic acid sequences may be produced and some of these will bear only minimal homology to the endogenous sequence of any known and naturally occurring HUMAN OGHbetal 4.
  • This invention has specifically contemplated each and every possible variation of nucleic acid sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleic acid sequence of naturally occurring HUMAN OGHbetal 4, and all such variations are to be considered as being specifically disclosed.
  • HUMAN OGHbetal 4 nucleic acid sequences and their derivatives or variants are preferably capable of identifying the nucleic acid sequence of the naturally occurring HUMAN OGHbetal 4 under optimized conditions, it may be advantageous to produce HUMAN OGHbetal 4-encoding nucleic acid sequences possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a longer half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleic acid sequences encoding a HUMAN OGHbetal 4 may be joined to a variety of other nucleic acid sequences by means of well established recombinant DNA techniques (Sambrook J., et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; or Ausubel F. M. et al (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York City).
  • Useful sequences for joining to the HUMAN OGHbetal 4 include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include vectors for replication, expression, probe generation, sequencing, and the like.
  • vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188 provides additional uses for oligonucleotides based upon the HUMAN OGHbetal 4 nucleic acid sequence.
  • oligomers are generally chemically synthesized, but they may be of recombinant origin or a mixture of both.
  • Oligomers generally comprise two nucleic acid sequences, one with sense orientation (5 1 to 3') and one with antisense (3' to 5') employed under optimized conditions for identification of a specific gene or diagnostic use. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification and/or quantitation of closely related DNA or RNA sequences.
  • Full length genes may be cloned utilizing partial nucleic acid sequence and various methods known in the art. Gobinda, et al. (1993; PCR
  • Methods Applic 2:318-22 disclose "restriction-site PCR" as a direct method which uses universal primers to retrieve unknown sequence adjacent to a known locus.
  • genomic DNA is amplified in the presence of primer to linker and a primer specific to the known region.
  • the amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Gobinda, et al. present data concerning Factor IX for which they identified a conserved stretch of 20 nucleotides in the 3' noncoding region of the gene.
  • Inverse PCR is the first method to report successful acquisition of unknown sequences starting with primers based on a known region (Triglia T., et al. (1988) Nucleic Acids Res 16:8186).
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. The multiple rounds of restriction enzyme digestions and ligations that are necessary prior to PCR make the procedure slow and expensive (Gobinda, et al. supra).
  • Capture PCR (Lagerstrom M., et al. (1991 ) PCR Methods Applic 1 :111 -19) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and YAC DNA. As noted by Gobinda, et al. ⁇ supra), capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before PCR. Although the restriction and Hgation reactions are carried out simultaneously, the requirements for extension, immobilization and two rounds of PCR and purification prior to sequencing render the method cumbersome and time consuming.
  • PromoterFinder.TM is a new kit available from Clontech (Palo Alto, Calif.) which uses PCR and primers derived from p53 to walk in genomic DNA. Nested primers and special PromoterFinder libraries are used to detect upstream sequences such as promoters and regulatory elements. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • a new method for analyzing either the size or the nucleic acid sequence of PCR products is capillary electrophoresis.
  • Systems for rapid sequencing are available from Perkin Elmer (Foster City Calif.), Beckman Instruments (Fullerton, Calif.), and other companies.
  • Capillary sequencing employs flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera.
  • Output/light intensity is converted to electrical signal using appropriate software (e.g. Genotyper.TM. and Sequence Navigators. M. from Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display is computer controlled.
  • Capillary electrophoresis provides greater resolution and is many times faster than standard gel based procedures. It is particularly suited to the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample. The reproducible sequencing of up to 350 bp of M13 phage DNA in 30 min has been reported (Ruiz-Martinez M. C, et al. (1993) Anal Chem 65:2851-8).
  • Another aspect of the subject invention is to provide for HUMAN OGHbetal 4 hybridization probes which are capable of hybridizing with naturally occurring nucleic acid sequences encoding HUMAN OGHbetal 4.
  • the stringency of the hybridization conditions will determine whether the probe identifies only the native nucleic acid sequence of that specific HUMAN OGHbetal 4 or sequences of closely related molecules.
  • Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5 x SSPE (0.18 M NaCl, 0.01 M NaP ⁇ 4, pH 7.7, 0.001 M
  • EDTA EDTA buffer at a temperature of 420C and remaining bound when subject to washing at 420C with 0.2 x SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5 x SSPE buffer at a temperature of 420C and remaining bound when subject to washing at 420C with 0.2 x SSPE buffer at 420C.
  • HUMAN OGHbetal 4 cDNA homologs can also be distinguished from one another using alignment algorithms, such as BLASTX (Altschul, et al., (1990) Basic Local Alignment Search Tool, J. Mol. Biol. 215:403-410).
  • HUMAN OGHbetal 4 nucleic acid sequences of the subject invention are used for the detection of related HUMAN OGHbetal 4 encoding sequences, they should preferably contain at least 50% of the nucleotides of the sequences presented herein.
  • Hybridization probes of the subject invention may be derived from the nucleic acid sequence of HUMAN OGHbetal 4, or from surrounding or included genomic sequences comprising untranslated regions such as promoters, enhancers and introns. Such hybridization probes may be labeled with appropriate reporter molecules.
  • Means for producing specific hybridization probes for HUMAN OGHbetal 4 include oligolabelling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • the cDNA sequence may be cloned into a vector for the production of cRNA probe.
  • vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • a number of companies (such as Pharmacia Biotech, Piscataway, NJ; Promega, Madison, Wl; US Biochemical Corp, Cleveland, OH.) supply commercial kits and protocols for these procedures.
  • DNA sequence or portions thereof, entirely by synthetic chemistry.
  • the source of information for producing this sequence comes from the known homologous sequence from closely related organisms.
  • the nucleic acid sequence can be used alone or joined with a preexisting sequence and inserted into one of the many available DNA vectors and their respective host cells using techniques well known in the art.
  • synthetic chemistry may be used to introduce specific mutations into the nucleic acid sequence.
  • a portion of sequence in which a mutation is desired can be synthesized and recombined with a portion of an existing genomic or recombinant sequence.
  • the HUMAN OGHbetal 4 nucleic acid sequences can be used individually, or in panels, in a diagnostic test or assay to detect disorder or disease processes associated with abnormal levels of HUMAN OGHbetal 4 expression.
  • the nucleic acid sequence is added to a sample (fluid, cell or tissue) from a patient under hybridizing conditions. After an incubation period, the sample is washed with a compatible fluid which optionally contains a reporter molecule which will bind the specific nucleic acid. After the compatible fluid is rinsed off, the reporter molecule is quantitated and compared with a standard for that fluid, cell or tissue. If HUMAN OGHbetal 4 expression is significantly different from the standard, the assay indicates the presence of disorder or disease.
  • the form of such qualitative or quantitative methods may include northern analysis, dot blot or other membrane based technologies, dip stick, pin or chip technologies, PCR, ELISAs or other multiple sample format technologies.
  • This same assay combining a sample with the nucleic acid sequence, is applicable in evaluating the efficacy of a particular therapeutic treatment regime. It may be used in animal studies, in clinical trials, or in monitoring the treatment of an individual patient. First, standard expression must be established for use as a basis of comparison. Second, samples from the animals or patients affected by the disorder or disease are combined with the nucleic acid sequence to evaluate the deviation from the standard or normal profile. Third, an existing therapeutic agent is administered, and a treatment profile is generated. The assay is evaluated to determine whether the profile progresses toward or returns to the standard pattern. Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.
  • the nucleic acid sequence for any particular HUMAN OGHbetal 4 can also be used to generate probes for mapping the native genomic sequence.
  • the sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in situ hybridization to chromosomal spreads (Verma, et al. (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, NY, NY), flow-sorted chromosomal preparations, or artificial chromosome constructions such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions or single chromosome cDNA libraries.
  • telangiectasia AT
  • AT ataxia telangiectasia
  • any sequences mapping to that area may represent genes for further investigation.
  • the nucleic acid sequences of the subject invention may also be used to detect differences in the chromosomal location of nucleic acid sequences due to translocation, inversion, etc. between normal and carrier or affected individuals.
  • the partial nucleic acid sequence encoding a particular HUMAN OGHbetal 4 may be used to produce an amino acid sequence using well known methods of recombinant DNA technology.
  • Goeddel (1990, Gene Expression Technology, Methods and Enzymology, Vol 185, Academic Press, San Diego, Calif.) is one among many publications which teach expression of an isolated, purified nucleic acid sequence.
  • the amino acid or polypeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleic acid sequence was derived or from a different species.
  • Advantages of producing an amino acid sequence or polypeptide by recombinant DNA technology include obtaining adequate amounts for purification and the availability of simplified purification procedures.
  • Cells transformed with a HUMAN OGHbetal 4 nucleic acid sequence may be cultured under conditions suitable for the expression and recovery of polypeptide from cell culture.
  • the polypeptide produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence itself and/or the vector used. In general, it is more convenient to prepare recombinant proteins in secreted form, and this is accomplished by ligating HUMAN OGHbetal 4 to a recombinant nucleic acid sequence which directs its movement through a particular prokaryotic or eukaryotic cell membrane.
  • Direct peptide synthesis using solid-phase techniques is an alternative to recombinant or chimeric polypeptide production.
  • Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer in accordance with the instructions provided by the manufacturer.
  • a particular HUMAN OGHbetal 4 sequence or any part thereof may be mutated during direct synthesis and combined using chemical methods with other HUMAN OGHbetal 4 sequence(s) or a part thereof.
  • This chimeric nucleic acid sequence can also be placed in an appropriate vector and host cell to produce a variant polypeptide.
  • HUMAN OGHbetal 4 used to induce specific antibodies may have a polypeptide sequence consisting of at least five amino acids and preferably at least 10 amino acids. Short stretches of amino acid sequence may be fused with those of another protein or polypeptide such as keyhole limpet hemocyanin, and the chimeric polypeptide used for antibody production. Alternatively, the polypeptide may be of sufficient length to contain an entire domain.
  • Antibodies specific for HUMAN OGHbetal 4 may be produced by inoculation of an appropriate animal with an antigenic fragment of the polypeptide.
  • An antibody is specific for HUMAN OGHbetal 4 if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant protein.
  • Antibody production includes not only the stimulation of an immune response by injection into animals, but also analogous processes such as the production of synthetic antibodies, the screening of recombinant immunoglobulin libraries for specific-binding molecules (Orlandi R., et al. (1989) PNAS 86:3833-3837, or Huse W. D., et al.
  • the protein sequence of the beta subunit of human FSH (gi accession number: 476441 ) was used as an electronic probe to search (using tBLASTn) HTGS, which is that fraction of the GenBank nucleic acid sequence database that contains "unfinished” (phase 1 and phase 2) genomic sequences. Homology was discovered to a region of chromosome 14, clone BAC R-696D21 (accession number: AL049871.1) the nucleic acid sequence of which is set forth in Figure 1. A conceptual translation of the homologous region revealed a polypeptide sequence termed HUMAN OGHbetal 4 ( Figure 1) that showed homology to each member of the glycoprotein hormone family.
  • the homologous region was also found to have a potential splice acceptor at a location precisely equivalent to introns in each of the other known glycoprotein hormone ⁇ subunits, further confirming HUMAN OGHbetal 4 as a member of this hormone family.
  • the C-terminus was found to be slightly truncated compared to the other known members, although a potential splice donor site was identified indicating that a longer version might be encoded after splicing to an additional unidentified coding exon.
  • FIG. 1 is sequence comparison of the novel ⁇ subunit polypeptide identified as HUMAN OGHbetal 4 with the ⁇ subunits of the other glycoprotein hormone family members: rLH-beta, hCG-beta-e, rTSH-beta, and rFSH-beta.
  • Asterisks (*) indicate amino acid identity between all family members.
  • the protein sequence obtained for HUMAN OGHbetal 4 corresponds to amino acid residues 42-103 of hCG-beta (gi accession no. NP_000789).
  • a polypeptide consisting of amino acid residues 38-57 of the hCG-beta (gi accession no. NP_000789) is capable of stimulating steroidogenesis in rat Leydig cells and can strongly inhibit the binding of intact hCG to its receptor (Keutmann, H.T. et al. Proc. Natl. Acad. Sci. USA. 84: 2038- 2042).
  • Example 2 Identification of additional HUMAN OGHbetal 4 5' sequence.
  • Additional 5' HUMAN OGHbetal 4 sequence was identified as follows.
  • a region of CNS0000U (Accession # AL0498D1.1 , Gl:4837626, human chromosome 14 bac R-696D21 submitted as phase2 hts sequence on May 14, 1999) corresponding to all unique sequence upstream from the initial predicted exon (Figure 1) was analyzed for predicted polypeptides using GENSCAN 1.0.
  • a potential upstream exon containing a predicted signal peptide was identified about 4.5 kb upstream of the initial predicted exon. However this predicted upstream exon contained a predicted splice donor in the wrong reading frame to be spliced directly to the initial predicted exon.
  • the region of the upstream exon was subcloned from a human BAC clone (325d23- Patent Deposit Designation PTA-787) and sequenced. It was determined that the published BAC sequence was in error. The correct sequence contained an additional C (nucleotide 156 in Figure 3). Changing the public sequence of ...CAGGGGCTTCGG... to the corrected sequence of ...CAGGGGCCTTCGG... aligned the putative splice donor and acceptors into the correct reading frame to produce the two exon coding sequence set forth in Figure 3. Sequencing of RT-PCR products from pituitary- derived polyA+ RNA confirmed the two exon structure of Figure 3.
  • nucleotide and deduced amino acid sequence of the extended HUMAN OGHbetal 4 sequence is set forth in Figure 3.
  • This novel protein may function as a heterodimer with the known common glycoprotein hormone alpha ( ⁇ ) subunit, a novel undiscovered alpha ( ⁇ ) subunit or as a monomer or homodimer.
  • ⁇ common glycoprotein hormone alpha
  • ⁇ novel undiscovered alpha
  • ⁇ novel undiscovered alpha
  • This method employs two oligonucleotides spaced relative close to each other to PCR amplify a portion of the message from cDNA and a third "probe" oligonucleotide co-labeled with a fluorophore and quencher at each end.
  • a significant fraction of the fluorophore is released by a "nick-translation" exonucleolytic activity of the polymerase.
  • the released fluorophore becomes highly fluorescent by being dissociated from the quenching moiety.
  • the abundance of a specific mRNA is determined by reading fluorescence during the course of the PCR reaction: samples containing more abundant messages taking fewer PCR cycles to release probe fluorescence, while samples containing the same message in lower abundance will require more cycles.
  • the TaqMan PCR reactions were run on a Perkin Elmer ABI PRISM 7700 Sequence Detection System instrument. MicroAmp (Perkin Elmer) optical 96-well plates and optical caps were used.
  • Each reaction had a final volume of 25 /I and the following concentrations of components: 1X TaqMan buffer A, 4 mM MgCl2, 200 mM of each of dATP, dCTP, dGTP, and 400 mM dUTP, 300 nM of each of forward (hOGH(B)for) and reverse (hOGH(B)rev) primers, 200 nM of hOGH(B)TaqMan probe, 5% DMSO, 10% glycerol, 0.025 U/ml AmpliTaq Gold, 1 U/ml AmpErase UNG and oligo- dT-primed cDNA derived from either 50ng of total RNA or the indicated amount of poly a+ RNA.
  • the PCR cycling conditions were as follows: 2 min. at 50°C, 10 min. at 95°C, followed by 40 two-step cycles of 15 sec at 95°C and 1 min. at 60°C.
  • the TaqMan probe had a 6-FAM 5'- Fluorescent label and TAMRA 3'-label that acts as a quencher.
  • the analysis shown in Figure 4, shows weak expression of HUMAN OGHbetal 4 in pituitary, testis, thymus and trachea.
  • the expression levels are normalized to molecules of mRNA per cell assuming each cell contains 20pg of total RNA or 1 pg of poly A+ RNA.
  • Example 4 Subunit structure of HUMAN OGHbetal 4
  • Epitope tagged versions of the indicated proteins HUMAN OGHbetal 4, hCG (human Chorionic Gonadotropin beta subunit; Genebank acc#NP_000728) and alpha ( ⁇ ) (the human, common alpha subunit of the glycoprotein hormones; Genebank acc#NP_000726) were expressed in COS cells using standard procedures known to the skilled artisan.
  • Media supematants obtained from the COS transfectants described supra were either run directly (15 ⁇ per lane, Panel A and Panel B) on reducing, denaturing acrylamide gels (4-20% gradient, Novex) or run after immunoprecipitation (Panel C) as follows: One ml of culture supernatant from each transfection was chilled on ice and mixed with 0.5 ml of cold TBS, 2.2 ⁇ g of M2 anti-FLAG monoclonal antibody (Sigma) and 0.05 ml of protein G sepharose beads (Pharmacia). The mixture was gently mixed at 4°C for 2.5 hours. The beads were collected by centrifugation, washed 3 times with TBS plus 1 % NP40 and proteins were eluted from pelleted beads with 30 ⁇ l of loading buffer. 15 ⁇ l of the recovered proteins were loaded per gel lane.
  • Flag-tagged HUMAN OGHbetal 4 immunoprecipitated both co-expressed HA-tagged common glycoprotein hormone alpha ( ) subunit (lane 1 , Panel C) and HA-tagged HUMAN OGHbetal 4 (lane 2, Panel C) with efficiencies roughly comparable to that at which FLAG-tagged hCG immunoprecipitated common glycoprotein hormone alpha ( ) subunit (lane 7, Panel C).
  • HUMAN OGHbetal 4 can form homo-dimers (or higher order homomeric structures) as well as hetero-dimers (or higher order heteromeric structures) with the common glycoprotein hormone alpha ( ⁇ ) subunit.
  • the homo-multimeric and hetero-multimeric forms are likely to have different biological activities perhaps binding to different receptors or acting as an agonist/antagonist pair on the same receptor or group of receptors.
  • ATCC ® American Type Culture Collection

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Abstract

L'invention concerne des polypeptides du type OGHbeta 14 HUMAIN et des acides nucléiques connexes, y compris des homologues naturels de ces polypeptides correspondant à plusieurs espèces et des polypeptides renfermant un domaine OGHbeta 14 HUMAIN à activité spécifique. On peut élaborer lesdits polypeptides par recombinaison à partir de cellules hôtes transformées, au moyen des acides nucléiques considérés. L'invention concerne également des sondes d'hybridation et de amorces oligonucléotidiques isolés capables d'hybridation spécifique avec les gènes décrits, des agents de liaison spécifiques et des procédés relatifs à l'élaboration et à l'utilisation des compositions visées.
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EP1239039A1 (fr) * 1999-12-17 2002-09-11 Takeda Chemical Industries, Ltd. Nouveau polypeptide et son adn

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1239039A1 (fr) * 1999-12-17 2002-09-11 Takeda Chemical Industries, Ltd. Nouveau polypeptide et son adn
EP1239039A4 (fr) * 1999-12-17 2004-05-06 Takeda Chemical Industries Ltd Nouveau polypeptide et son adn
WO2002014348A2 (fr) * 2000-08-11 2002-02-21 Applied Research Systems Ars Holding N.V. Glycoproteines et procedes d'utilisation
WO2002014348A3 (fr) * 2000-08-11 2003-05-15 Applied Research Systems Glycoproteines et procedes d'utilisation

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