WO1991019007A1 - Sequences d'adn impliquees dans la degenerescence neuronale, organismes multicellulaires contenant ces sequences et utilisations de ces sequences - Google Patents

Sequences d'adn impliquees dans la degenerescence neuronale, organismes multicellulaires contenant ces sequences et utilisations de ces sequences Download PDF

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WO1991019007A1
WO1991019007A1 PCT/US1991/003826 US9103826W WO9119007A1 WO 1991019007 A1 WO1991019007 A1 WO 1991019007A1 US 9103826 W US9103826 W US 9103826W WO 9119007 A1 WO9119007 A1 WO 9119007A1
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nucleic acid
acid molecule
gene
dna
detecting
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Martin Chalfie
Eve Wolinsky
Monica Driscoll
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The Trustees Of Columbia University In The City Of New York
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    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C07K14/43545—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Neuronal cell death is a major feature of a variety of human neurological disorders, including the neurodegenerative diseases (such as Alzheimer's, Parkinson's, Huntington's and amyotrophic lateral sclerosis), stroke and trauma (67).
  • Alzheimer's Disease afflicts about 4 million people in the United States, primarily the elderly. It is characterized by progressive memory loss, disorientation, depression and eventual loss of bodily functions.
  • Amyotrophic lateral sclerosis afflicts about 30,000 Americans. It begins after age 40 and results in progressive weakness and paralysis.
  • Huntington's Disease which afflicts an estimated 25,000 patients in the United States, usually begins between the ages of 30 and 50 and includes violent, involuntary movements.
  • Cell death occurs not only as a disease process, but also as a normal aspect of development and of tissue homeostasis (68, 69, 70).
  • tissue homeostasis 68, 69, 70.
  • Studies of both normal and abnormal cell deaths that occur in organisms as diverse as mammals, insects and nematodes have suggested that many of the distinct primary events that initiate the process of cell death act by triggering one of only a few general mechanisms that cause cells to die (71). If so, an understanding of cell death processes gained from any of these experimental systems might help reveal aspects of the cell death processes that occur in human diseases.
  • C . elegans The mechanisms of cell death of one free-living nematode, Caenorhabditis elegans can be studied. In C . elegans, cell death can be observed in living animals at the level of resolution of single, identified cells (11). In addition, C. elegans is easily studied genetically, which not only allows the isolation of mutants with neurodegenerative genetic disorders that might serve as models for human disease, but more generally allows the identification of the genes and proteins that function in cell death. The mechanisms of cell death in wild-type and/or in mutant C . elegans may be similar to those involved in human disease. Furthermore, the genes and gene products involved in C . elegans cell death processes may be sufficiently conserved to allow the identification of corresponding molecules that cause human nerve cell deaths.
  • C . elegans programmed cell death a dying cell shrinks, becomes engulfed by a neighboring cell and eventually disappears (10, 11, 73).
  • the chromatin forms granular aggregates underlying the nuclear envelop, a cluster of electron-dense particles appears in the center of the nucleus, the nuclear envelop dilates, and both the nucleus and the cytoplasm become more electron dense.
  • the chromatin condenses further (so that very little euchromatin remains visible), the nucleus becomes pycnotic, mitochondria become electron-lucent, and parts of the dying cell split off into membrane-bound fragments. Later, the nuclear membrane becomes highly convoluted, nuclear membrane-bound structures (some containing chromatin-like material) are formed, mitochondria appear distorted (and frequently are found within vacuoles), the cytoplasm appears less granular than before, both internal and plasma membranes display increased whorling, and the cellular outline becomes irregular. Finally, as the dying cell is shrinking, the nuclear membrane breaks down completely, and chromatin-like fragments appear within the cytoplasm.
  • ced-3, ced-4 and ced-9 Three genes function in the killing of cells during programmed cell death: ced-3, ced-4 and ced-9. Mutations that eliminate the activity of either ced-3 or ced-4 prevent the deaths of all 131 cells that normally die (19).
  • ced-3 or ced-4 animals the "undead" cells not only survive, but they also can differentiate and express characteristics of other cells normally present in the animal; different surviving cells differentiate into different cell types.
  • a surviving cell can be sufficiently normal that it is functional: one surviving cell in the animal's pharynx has been shown to acquire characteristics like those normally expressed by its sister, the M4 motor neuron; if the M4 neuron is killed
  • ced-3 and ced-4 normally act to convert live, potentially functional cells into non-functional cell corpses.
  • ced-3 and ced-4 cause cells to die.
  • killer genes ced-3 and ced-4, may act within the dying cells themselves or within other cells that function to cause dying cells to die (75). To determine this, the technique of genetic mosaic analysis was used.
  • ced-9 The third gene that acts in the killing step of programmed cell death is ced-9.
  • the original ced-9 mutant strain is phenotypically similar to the ced-3 and ced-4 mutants described above: all programmed cell deaths are blocked.
  • the ced-9 mutation in this strain is opposite in nature to the ced-3 and ced-4 mutations that have been studied. Specifically, cell death is prevented by mutations that cause a loss of ced-3 and ced-4 gene function or a gain of ced-9 gene function.
  • programmed cell death can be regarded as a cell fate, analogous to any differentiated fate, such as becoming a muscle cell or a dopaminergic neuron.
  • all cells that undergo programmed cell death display the same sequence of morphological changes and require the same set of genes, and hence proteins.
  • the same physiological processes seem to act in all programmed cell deaths in C . elegans.
  • programmed cell death seems likely to involve functions responsible both for determination -- the specification of which cells will and which cells will not die -- and for differentiation -- the expression of the cell death fate itself.
  • the 11 genes discussed above are involved in this latter step, as they carry out programmed cell death.
  • Genes that act in the determinative aspect of programmed cell death would be recognized by their altering patterns of cell death without affecting the machinery necessary for the cell death process. Such genes would include those that could mutate to cause specific cells that normally survive instead to undergo programmed cell death.
  • This phenotype would constitute a degenerative genetic disorder of C . elegans.
  • One neurodegenerative genetic disorder of this class has been identified in C . elegans.
  • egl-1 mutants egl, egg-laying abnormal
  • the serotonergic HSN motor neurons which normally innervate the egg-laying musculature and drive egg laying, die (19). No other cells have been found to be abnormal in egl-1 animals.
  • mec-4 mec, mechanosensorv abnormal
  • deg-1 degener- ation
  • mec-4 and deg-1 mutations cause cells to die independently of the ced genes discussed above.
  • mec-4 mutants a specific set of six touch receptor neurons die.
  • deg-1 mutants another small set of neurons, including both sensory and inter- neurons, die.
  • the cells that die in these degenerative deaths swell and lyse. The remains of the nucleus and cytoplasmic debris can be seen within a large vacuole many cell diameters in size.
  • This invention provides an isolated nucleic acid molecule encoding a wild-type animal protein associated with neuronal degeneration.
  • the invention further provides an isolated nucleic acid molecule encoding a mutated animal protein associated with neuronal degeneration.
  • the wild-type animal protein may be encoded by the deg-1 gene.
  • the mutated animal protein may be encoded by a mutant of the deg- 1 gene, the deg-1 gene having the cDNA sequence shown in Figure 7. Examples of mutants of the deg-1 gene are designated u38 or ulnl.
  • the wild-type animal protein in Caenorhabditia elegans may be encoded by the mec-4 gene.
  • the mutated animal protein may be encoded by a mutant of the mec-4 gene, the mec-4 gene having the cDNA sequence shown in Figure 9 . Examples of mutants of the mec-4 gene are designated e1611, u214, or u231.
  • This invention further provides a Caenorhabditis elegans strain designated TU38 and deposited with the ATCC under Accession No.40818; a Caenorhabditis elegans strain designated TU1191 and deposited with the ATCC under Accession No. 40817; a Caenorhabditis elegans strain designated CB1611 and deposited with the ATCC under Accession No. 4 0820; a Caenorhabditis elegans strain designated TU214 and deposited with the ATCC under Accession No.40819; and a Caenorhabditis elegans strain designated TU231 and deposited with the ATCC under Accession No. 40821.
  • the invention further provides a method for detecting a nucleic acid molecule encoding a wild-type protein associated with a degenerative disorder in a human subject which comprises obtaining a sample of DNA or mRNA from the subject, contacting the DNA or mRNA with a nucleic acid molecule encoding a wild-type human protein associated with neuronal degeneration, such nucleic acid molecule being labeled with a detectable moiety, under suitable conditions permitting hybridization of the DNA or mRNA and the nucleic acid molecule, and detecting the hybridized nucleic acid molecules, thereby detecting the nucleic acid molecule encoding the wild-type protein associated with the degenerative disorder.
  • a method for detecting a mutation associated with a degenerative disorder in a human subject which comprises obtaining a sample of DNA or mRNA from the subject, contacting the DNA or mRNA with the nucleic acid molecule encoding a mutated human protein associated with neuronal degeneration, such nucleic acid molecule being labeled with a detectable moiety, under suitable conditions permitting hybridization of the DNA or mRNA and the nucleic acid molecule, and detecting the hybridized nucleic acid molecules, thereby detecting the mutation associated with the degenerative disorder.
  • a method of diagnosing degenerative disease in a human subject which comprises detecting the presence of a mutation associated with a degenerative disorder using the methods disclosed above for detecting a mutation associated with a degenerative disorder.
  • the subject invention further provides a method of treating a degenerative disease in a human subject which comprises introducing into the human subject an amount of a nucleic acid molecule encoding a wild-type human protein associated with neuronal degeneration effective to suppress neuronal degeneration caused by mutants of the nucleic acid molecule, thereby decreasing neuronal degeneration in the human subject and treating the degenerative disease.
  • the subject invention also provides a method of causing a diseased human cell to degenerate which comprises introducing a nucleic acid molecule encoding a mutated human protein associated with neuronal degeneration into the diseased human cell so as to cause neuronal degeneration of the diseased human cell, thereby causing the diseased human cell to degenerate.
  • the strains of Caenorhabditis ele ⁇ ans provided by the subject invention can also be used in a method of screening drugs to identify drugs which prevent or decrease neuronal degeneration which comprises contacting a Caenorhabditis elegans strain with a plurality of drugs, determining those drugs which prevent or decrease neuronal degeneration of the strain, and thereby identifying drugs which prevent or decrease neuronal degeneration.
  • the invention further provides a protein encoded by any one of the nucleic acid molecules disclosed herein.
  • FIG. 1 Onset of the deg-1 touch-insensitivity (Tab) phenotype at various temperatures.
  • the abscissa records time in 25° hour equivalents (C . elegans grows at approximately twice the 15°C rate at 25°C).
  • Methods Strains were maintained on Escherichia coli strain OP50-1 as described previously (14, 57). Animals from strains grown at the indicated temperatures for at least two generations were synchronized at hatching by washing hatched animals and bacteria from the plates with M9 buffer
  • deg-1 (u38) mutation maps within 0.014 map units of the mec-7 gene on the X chromosome by the following criteria: no wild-type progeny were found among 3533 total progeny from +[+deg- 1(u38)]/lon-2(e678) [mec-7 (el506)+]heterozygotes nor among 5334 and 5960 heterozygote progeny from, respectively, +[+deg-1(u38)]dpy-6(e14)/dpy-7(e88) [mec-7(e1506)+]+ and dpy-7[+deg-1(u38)]+/+[mec-7
  • deg-1 loss-of-function mutations complement the recessive mec-7 allele el506 for touch insensntivity. an indication that the two loci are different genes.
  • FIG. 1 Defects in deg-1 mutants.
  • the pattern in u38 males does not differ substantially from that of hermaphrodites.
  • the ced-1 mutation el735 delays the engulfment of cells dying by programmed cell death.
  • the two types of deaths are morphologically distinct.
  • the cells here and in subsequent photomicrographs have been tentatively identified by criteria given below.
  • IL1 cells die one to three IL1 cells (wild-type have six) were seen in six deg-1 adults examined by serial section electron microscopy (58, 59). Magnification x800.
  • IL1 Cells with the characteristic rootlet (59) of the IL1 cells were missing in serial sections of the tip of the nose of adult animals.
  • AVG A single degeneration is seen in the retrovesicular ganglion just posterior to the rear bulb of the pharynx in many newly hatched animals. AVG is the only unpaired neuron in this region (60).
  • AVD This pair of interneurons helps mediate anterior touch sensitivity (9), but since the anterior touch circuit also utilizes other interneurons, loss of the AVD cells in the deg-1 mutants does not produce a Tab phenotype.
  • FIG. 3 Onset of the Tab phenotype in deg-1(u38) and two partially reverted strains [deg-1 (u38u354) and deg-1(u38u352)]. Animals were grown from synchronized populations at 25°C ( Figure 1). At least 100 animals of each strain were examined. The Tab phenotype, as in u38 mutants, is tempera ture dependent in u38u352 and u38u354 animals (data not shown). Not all deg-1-dependent deaths seem to be affected the same way by these suppressor mutations.
  • Figure 4 Onset of the Tab phenotype in animals with different doses of the wild-type (+) and mutant (u38) alleles of deg-1. Progeny of hermaphrodites with the genotype stDp2/+; deg-1 (u38) + dpy-6(el4)/unc- 18(e81) + dpy-6(el4) were synchronized at hatching ( Figure 1 legend). At the indicated times after hatching animals that were Tab were plated individually so that their progeny could be examined so as to determine their genotypes.
  • All animals that have three copies of the region [u38/u38/+ (filled diamonds), u38/+/+ (open diamonds), and +/+/+ (open circle)] contain stDp2, a duplication of the dpy-6 deg-1 region of the X chromosome that has been translocated to chromosome II and is lethal when homozygous. All these animals are wild-type in length. At least 50 animals of each of these genotypes were examined. Animals with two copies of the region (u38/u38 (open bars) and U38/+ (filled bars) are homozygous for the dpy-6 mutation and are shorter in length (Dpy).
  • FIG. 5 Southern blot analysis of deg-1 DNAs.
  • a Genomic DNA digested with EcoRI and Xbal and probed with cosmid C47C12.
  • Lane 1 deg-1(u38);
  • Lane 2 a mut-2-derived revertant of deg-1(u38) containing a Tc1 insertion in the 3.0 kb EcoRI- Xbal fragment (fragment I in Figure 6; the resuiting fragment with the insertion comigrates with a second fragment and increases the intensity of the band at 4.6 kb);
  • Lane 6 an EMS-derived revertant missing the 4.5 kb EcoRI fragment (fragment D in Figure 6).
  • C C . elegans genomic DNA was prepared essentially as described (61). General molecular methods were as described (62) with final washes of blots in 0.1X SSC at 65°C. Initially deg-1 DNA was cloned from the u38u476 strain. Southern blots of DNA from this strain, when probed with DNA from the transposon Tel (61), revealed a single, novel Tc1-containing EcoRI/Xbal fragment that cosegregated with the non-Tab phenotype in recombinants with u38. This fragment was isolated from a plasmid library constructed of size-fractionated EcoRI/Xbal fragments from u38u476 DNA in plasmid pUC18. The resulting plasmid was digested with EcoRV to remove the Tel element and religated to produce plasmid TU#8.
  • FIG. 6 DNAs of the deg-1 region.
  • the top line represents a partial restriction map of genomic DNA.
  • Triangles denote the approximate positions of transposon insertions in the indicated revertants (all insertions are 1.6 kb, the size of the transposon Tel, but the presence of this transposon has been confirmed only in strain u38u476).
  • the boxes under the restriction map indicate fragments that harbor DNA rearrangements in u38 revertants or that hybridize to deg-1 cDNAs; D is deleted in U38U421; I contains the Tel insertion in u38u476; IC contains the transposon insertion in u38u478 and hybridizes to the deg-1 cDNAs (the transposon resides in a 187 bp EcoRI/Ndel fragment at the 5' end of IC; the sequence of this fragment is identical with the deg-1 cDNAs, except for a 50 bp intron 160 bp from the EcoRI site); and C is a fragment that also hybridizes to the cDNAs.
  • C47C12 and R02A8 are cosmids containing wild-type DNA (24).
  • TU#3 is a cosmid clone of the BamHI fragment from deg-1(u38); when transformed into wild-type animals, TU#3 produces the Deg and Tab phenotypes.
  • TU#4 is a derivative of TU#3 that lacks the 3' Smal/BamHI fragment and does not generate the Deg or Tab phenotypes on transformation into wild-type.
  • B BamHI
  • E EcoRI
  • K
  • Genomic subclones of wild-type DNA from C47C12 were made by ligation into pUC18.
  • the TU#3 cosmid was obtained by isolating large fragments from BamHI-digested deg-1(u38) DNA from a sucrose gradient (64) and ligating them to the BamHI- Hindlll and SalI-BamHI fragments of the cosmid vector pJB8 (63).
  • This library was screened with all four genomic fragments (D, I, IC, and C) individually as probes.
  • TU#3 DNA Animals were transformed with TU#3 DNA by co-injection of the DNA into wild-type oocytes (64) with either pPD10.41 or pGB3.5, plasmids that contain an antisense construct of the C . elegans unc-22 gene that produces a twitching phenotype. About half of the twicher transformants also segregated animals that were Tab and Deg. ulnl resulted from co-injection of TU#3 and pGB3.5 and showed linkage with unc-18(e81) on the X chromosome. Transformations with TU#4 were done similarly; southern blotting confirmed that TU#4 was present in the transformed animals.
  • FIG. 7 DNA and predicted protein sequence of a deg-1 cDNA. The sequence of the larger cDNA is shown.
  • the smaller cDNA lacks the DNA encoding residues 176 to 199 (bracketed). Potential glycosylation sites (26) are underlined, cysteine residues are asterisked, and the hydrophobic region is double underlined.
  • ced-3 and ced-4 act to cause cells to die; mutations that block the activity of either ced-3 or ced-4 prevent cell death.
  • the gene ced-9 appears to antagonize the action of ced-3 and ced-4; mutations that cause a gain of ced-9 function prevent programmed cell death.
  • the activities of the genes ced-1, ced-2, ced-5, ced- 6, ced-7, ced-8 and ced-10 are necessary for the phagocytosis of dying cells by their neighbors. If these genes fail to function, phagocytosis is blocked, but cell death still occurs.
  • the gene nuc-1 acts to degrade the DNA of dying cells.
  • Figure 9 DNA and predicted protein sequence of a sec-4 cDNA.
  • Figure 10 Autoradiogram of mec-4 homology to various species. Southern blot where DNA from different species (i.e., human, monkey, rat, mouse, dog, cow, rabbit, chicken, and yeast) are probed with the mec-4 gene. Both the mouse and human lanes shown cross-hybridization with mec-4 indicating they may contain genes which are members of a gene family.
  • species i.e., human, monkey, rat, mouse, dog, cow, rabbit, chicken, and yeast
  • FIG. 11 mec-4-deg-1 homology. This figure shows the mec- 4-deg-1 gene homology. The mec-4 dominant alleles change Ala at position 438 to Val or Thr as indicated.
  • the Caenorhabditis elegans strains designated TU38, TU1191, CB1611, TU214, and TU231 were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 40818, 40817 40820, 40819, and 40821 , respectively.
  • Nematode The term "nematode” is intended to refer generally to the class Nematoda or Nematoidea and comprises those animals of a slender cylindrical or thread-like form commonly called worms.
  • Mutant is intended to refer generally to a nematode which contains a stably altered genotype. The altered genotype results from a mutation not generally found in the genome of the wild-type nematode.
  • a "library" of nematodes is a collection of different nematodes and may include both wild-type and mutant nematodes.
  • Drug The term “drug” is intended to refer to a chemical entity, whether in the solid, liquid, or gaseous phase which is capable of providing a desired therapeutic effect when administered to a subject.
  • the term “drug” should be read to include synthetic compounds, natural products and macromolecular entities such as polypeptides, polynucleotides, or lipids and also small entities such as neurotransmitters, ligands, hormones or elemental compounds.
  • drug is meant to refer to that compound whether it is in a crude mixture or purified and isolated.
  • cell death By the term “cell death” is meant the process through which cells die. As referred to for the purposes of this invention, cell death is meant to exclude those processes wherein cells die as an immediate result of acute physical injury.
  • late-occurring cell death is meant cell death which occurs after the cell has become functional.
  • late-occurring neuronal cell death is meant cell death which occurs after the neurons form synapses and become functional nerve cells.
  • Cloning vehicle By “cloning vehicle” is meant a plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about its replication and cloning.
  • the cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicillin resistance.
  • the word “vector” is sometimes used for "cloning vehicle.”
  • expression vehicle is meant a vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene which has been cloned into it, after transformation into a host.
  • the cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
  • Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a procaryotic or eucaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • a “functional derivative” of the proteins of the invention is a protein which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of such proteins.
  • a functional derivative may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional derivative” is intended to include the “fragments,” “variants,” “analogues,” or “chemical derivatives” of a molecule.
  • Fragment A "fragment" of a protein of the invention is meant to refer to any portion of the protein of the invention which contains less than the complete amino acid sequence of the protein.
  • Variant A “variant” of a protein of the invention is meant to refer to a protein substantially similar in structure and biological activity to either the entire protein of the invention or to a fragment thereof. Thus, provided that two proteins possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.
  • Analog of the proteins of the invention is meant to refer to a protein substantially similar in function to either the native protein of the invention or to a fragment thereof.
  • a protein is said to be a "chemical derivative" of another protein when it contains additional chemical moieties not normally a part of the protein. Such moieties may improve the protein's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the protein, eliminate or attenuate any undesirable side effect of the protein, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a protein are well-known in the art.
  • the subject invention identifies defective genes in round- worms that cause mature, functioning nerve cells to die. Such late-occurring nerve cell death is seen in several human disorders such as Huntington's, Lou Gehrig's (amyotrophic lateral sclerosis) and Alzheimer's diseases.
  • the subject invention provides scientists an animal model with which to study the causes of neurodegenerative disease.
  • the findings will lead to ways to prevent and treat the human disorders, which afflict millions of people and have no known cures.
  • the invention discloses a gene called deg-1 (degeneration).
  • a rare mutation in this gene causes a small set of cells in the nervous system of the microscopic roundworm Caenorhabditis elegans to die. Similar deaths, but of different nerve cells, are caused by rare mutations in a second gene called mec-4 (mechanosensory abnormal).
  • mec-4 mechanosensory abnormal
  • mec-6 a third gene, is required for the abnormal cell deaths caused by the deg-1 and mec-4 mutations. If the mec-6 gene is defective, neurodegeneration does not occur; in other words, it is suppressed.
  • the subject invention is based on genetic studies of a microscopic roundworm called Caenorhabditis elegans.
  • C. elegans is a particularly useful model for studying neuro- degeneration because it allows researchers to observe changes in cells within a living organism over the three days that it takes to develop from a single-cell zygote to a mature adult. These kinds of observations are extremely difficult in other animals and impossible in humans.
  • C. elegans is one of the most thoroughly understood of all multicellular organisms. The biology of its nervous system, which contains 302 neurons, has been well documented. There are several similarities between the C. elegans and human nervous systems. For example, many of the C. elegans' neurotransmitters, chemicals that nerve cells use to communicate with each other, are the same as human neurotransmitters. In addition, many C. elegans genes used both inside and outside of the nervous system have counterparts in mammals. Approximately half of the C. elegans genes and proteins that have been characterized to date have structures and functions similar to mammalian genes. These include enzymes, proteins necessary for cell structure, cell surface receptors and genetic regulatory molecules.
  • the subject invention discloses and characterizes a rare, altered (mutant) form of a gene called deg-1 (degeneration gene-1) that results in an abnormal form of nerve cell death.
  • deg-1 degeneration gene-1
  • the deg-1 gene is not involved in the normal process of cell death that ordinarily occurs as part of the animal's growth and development.
  • the degeneration produced by the deg-1 mutation has parallels to nerve cell loss that occurs in human diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease. As in these disorders, the degeneration in some nerve cells is late-onset; something goes awry after neurons develop and mature in apparently normal fashion. Further, like several human neurodegenerative diseases, the disorder can be inherited from only one parent.
  • the deg-1 gene is one of three C. elegans genes identified disclosed in the subject application that are implicated in neurodegeneration. Three mutations of another gene, called mec-4 (mechanosensory abnormal gene-4), produce similar, neurodegenerative effects on a different set of nerve cells.
  • mec-4 mechanosensory abnormal gene-4
  • These C. elegans genes provide tools for understanding the molecular process of neurodegeneration. Genetic studies to characterize the molecular defect in the deg-1 and mec-4 genes provide an understanding of how the mutant genes cause abnormal neurodegeneration. One can also determine why the product of the mec-6 gene is needed for the degeneration induced by the other genes and what other cellular components may be necessary for this neurodegeneration. With the DNA structure of the deg-1 gene in hand, scientists can search for similar genes and proteins in humans.
  • This invention provides an isolated nucleic acid molecule encoding a wild-type animal protein associated with neuronal degeneration.
  • the invention further provides an isolated nucleic acid molecule encoding a mutated animal protein associated with neuronal degeneration.
  • the animal is preferably a human being, or the animal may be a nematode, such as Caenorhabditis elegans.
  • the wild-type animal protein may be encoded by the deg-1 gene.
  • the mutated animal protein may be encoded by a mutant of the deg-1 gene, the deg-1 gene having the cDNA sequence shown in Figure 7. Examples of mutants of the deg-1 gene are designated u38 or ulnl.
  • the wild-type animal protein in Caenorhabditis elegans may be encoded by the mec-4 gene.
  • the mutated animal protein may be encoded by a mutant of the mec-4 gene, the mec-4 gene having the cDNA sequence shown in Figure 9. Examples of mutants of the mec-4 gene are designated e1611, u214, or u231.
  • This invention also provides an isolated genomic DNA encoding a wild-type animal protein associated with neuronal degeneration, and an isolated genomic DNA encoding a mutated animal protein associated with neuronal degeneration.
  • This invention further provides a Caenorhabditis elegans strain designated TU38 and deposited with the ATCC under Accession No.40818; a Caenorhabditis elegans strain designated TU1191 and deposited with the ATCC under Accession No. 40817; a Caenorhabditis elegans strain designated CB1611 and deposited with the ATCC under Accession No. 40820; a Caenorhabditis elegans strain designated TU214 and deposited with the ATCC under Accession No. 40819 ; and a Caenorhabditis ele ⁇ ans strain designated TU231 and deposited with the ATCC under Accession No. 40821.
  • a vector which comprises a nucleic acid molecule encoding a wild-type animal protein associated with neuronal degeneration or a nucleic acid molecule encoding a mutated animal protein associated with neuronal degeneration.
  • Suitable vectors includes plasmids, cosmids, and phages.
  • the isolated nucleic acid molecules of the subject invention may be labeled with a detectable moiety, preferably a radioactive moiety.
  • the invention further provides a method for detecting a nucleic acid molecule encoding a wild-type protein associated with a degenerative disorder in a human subject which comprises obtaining a sample of DNA or mRNA from the subject, contacting the DNA or mRNA with a nucleic acid molecule encoding a wild-type human protein associated with neuronal degeneration, such nucleic acid molecule being labeled with a detectable moiety, under suitable conditions permitting hybridization of the DNA or mRNA and the nucleic acid molecule, and detecting the hybridized nucleic acid molecules, thereby detecting the nucleic acid molecule encoding the wild-type protein associated with the degenerative disorder.
  • a method for detecting a mutation associated with a degenerative disorder in a human subject which comprises obtaining a sample of DNA or mRNA from the subject, contacting the DNA or mRNA with the nucleic acid molecule encoding a mutated human protein associated with neuronal degeneration, such nucleic acid molecule being labeled with a detectable moiety, under suitable conditions permitting hybridization of the DNA or mRNA and the nucleic acid molecule, and detecting the hybridized nucleic acid molecules, thereby detecting the mutation associated with the degenerative disorder.
  • This invention also provides a method of detecting a nucleic acid molecule encoding a wild-type protein associated with a degenerative disorder in a human subject which comprises isolating a human subject's mRNA molecules, separating the mRNA molecules, immobilizing the mRNA molecules on a suitable solid support, contacting the immobilized mRNA molecules with a nucleic acid molecule encoding a wild-type human protein associated with neuronal degeneration, such nucleic acid molecule being labeled with a detectable moiety, under suitable conditions permitting hybridization of complementary molecules, detecting the presence of molecules hybridized to sequences on both the first and second nucleic acid sequences and thereby detecting the nucleic acid molecule encoding the wild-type protein associated with the degenerative disorder.
  • Also provided is a method of detecting a mutation associated with a degenerative disorder in a human subject which comprises isolating a human subject's mRNA molecules, separating the mRNA molecules, immobilizing the mRNA molecules on a suitable solid support, contacting the immobilized mRNA molecules with a nucleic acid molecule encoding a mutated human protein associated with neuronal degeneration, such nucleic acid molecule being labeled with a detectable moiety, under suitable conditions permitting hybridization of complementary molecules, detecting the presence of molecules hybridized to sequences on both the first and second nucleic acid sequences and thereby detecting the mutation associated with the degenerative disorder.
  • the invention provides a method of detecting a mutation associated with a degenerative disorder in a human subject which comprises isolating a human subject's mRNA molecules, separating the mRNA molecules so obtained by gel electrophoresis, immobilizing the separated mRNA molecules on a suitable solid support, contacting the immobilized mRNA molecules with a nucleic acid molecule encoding a mutated human protein associated with neuronal degeneration, such nucleic acid molecule being labeled with a detectable moiety, under suitable conditions permitting hybridization of complementary molecules, detecting the presence of molecules hybridized to sequences on both the first and second nucleic acid sequences and thereby detecting abnormalities in the subject's mRNA caused by the mutation associated with the degenerative disorder.
  • a method of diagnosing degenerative disease in a human subject which comprises detecting the presence of a mutation associated with a degenerative disorder using the methods disclosed above for detecting a mutation associated with a degenerative disorder.
  • the subject invention further provides a method of treating a degenerative disease in a human subject which comprises introducing into the human subject an amount of a nucleic acid molecule encoding a wild-type human protein associated with neuronal degeneration effective to suppress neuronal degeneration caused by mutants of the nucleic acid molecule, thereby decreasing neuronal degeneration in the human subject and treating the degenerative disease.
  • the nucleic acid molecule may be introduced into the human subject by any method known to those skilled in the art, preferably by a vector or in a suitable carrier.
  • the subject invention also provides a method of causing a diseased human cell to degenerate which comprises introducing a nucleic acid molecule encoding a mutated human protein associated with neuronal degeneration into the diseased human cell so as to cause neuronal degeneration of the diseased human cell, thereby causing the diseased human cell to degenerate.
  • diseased human cells include any diseased cells which one would wish to cause to degenerate, such as a cancer cell or a human cell which is infected with the AIDS virus.
  • the nucleic acid molecule may be introduced into the human subject by any method known to those skilled in the art, preferably by a vector or in a suitable carrier.
  • the strains of Caenorhabditis elegans provided by the subject invention can also be used in a method of screening drugs to identify drugs which prevent or decrease neuronal degeneration which comprises contacting a Caenorhabditis elegans strain with a plurality of drugs, determining those drugs which prevent or decrease neuronal degeneration of the strain, and thereby identifying drugs which prevent or decrease neuronal degeneration.
  • the Caenorhabditis elegans strain is designated TU38, TU1191, CB1611, TU214, or TU231.
  • the invention further provides a protein encoded by any one of the nucleic acid molecules disclosed herein.
  • the dominant mutation deg-1 results in a toxic gene product that leads to the late-onset degeneration of a small number of neurons in the nematode Caenorhabditis elegans. Both intragenic and extragenic mutations as well as changes in wild-type gene dosage can delay or block the time of onset of the neuronal deaths.
  • the deg-1 gene has been cloned and a partial complementary (cDNA) reveals that the gene encodes a novel protein with an amino acid sequence similar to that of a membrane receptor. Because the late-onset loss of specific sets of neurons, often as a result of dominant mutations, is characteristic of several human neurodegenerative diseases, the analysis of the deg-1 gene and its suppressors provides a means of understanding the mechanisms underlying some of these human diseases.
  • the selective degeneration of specific classes of nerve cells is characteristic of the cell death mechanisms of the invention and of many inherited human disorders (1) such as Huntington's disease (2, 3), familial amyotrophic lateral sclerosis (4), familial Alzheimer's Disease (5) and several cerebellar ataxias (6). Many of these diseases are caused by dominant alleles that result in a late-onset degeneration of the affected nerve cells similar to that of the nematode mutants of the invention but their molecular bases are not known.
  • the DNA sequences, nematode mutants and methods of the invention provide unique tools which allow, for the first time, a detailed analysis of neuronal degenerative mechanisms which are characterized by a late-onset degeneration of selective nerve cells.
  • u38 a dominant mutation of the C. elegans gene deg-1 (degeneration), that results in the degeneration of a small set of neurons.
  • u38 a dominant mutation of the C. elegans gene deg-1 (degeneration)
  • the deg-1(u38) mutation results in an abnormal, toxic form of a gene product that is normally non-essential for neuron viability and function.
  • the affected cells swell to many cell diameters and lyse. Without intending to be held to this theory, one possibility for this result is that the deg-1 gene encodes a membrane component, and cell lysis results from compromised membrane integrity.
  • the time of the neuronal degeneration depends upon the level of deg-1(u38) mutant gene activity, which is consistent with an accumulation of a toxic product(s). Mutations in the mec-6 (mechanosensory abnormal) gene suppress the deg-1(u38) neurodegeneration, as well as similar neuronal deaths produced by abnormal, toxic forms of the mec-4 gene product. An understanding of how the mutant deg-1 and mec-4 gene products result in neurodegeneration, and of how normal mec-6 activity is required for this degeneration will provide a basis for methods for preventing or curing neurodegenerative disorders in humans.
  • the deg-1 (u38) mutant was identified in a screen for touchinsensitive mutants following ethyl methanesulfonate (EMS) mutagenesis at 25oC (7).
  • the gene is located on the X chromosome (see legend to Figure 1).
  • the deg-1 (u38) animals differ from other touch-insensitive mutants (7, 8) in that although both types of mutants are insensitive to the gentle touch of a hair, only the deg-1 mutants are insensitive to the more severe prod of a thin wire. They are also insensitive only at the tail.
  • This more severe phenotype suggests a defect in the two PVC interneurons, which receive synaptic inputs from posterior touch receptor neurons (9).
  • the PVC cells degenerate in these mutants.
  • a similar behavioral abnormality results from laser ablation of the PVC cells in wild-type animals.
  • deg-1 (u38) degeneration phenotype (termed Deg) has been seen in animals with dominant mutations of the gene mec-4 [mec-4 (d)].
  • mec-4 (d) the gene mec-4 [mec-4 (d)].
  • the deg-1 mutants also contain empty vacuoles (vacuoles not associated with nuclei) at the tip of the nose, along the ventral cord, and in the tail (Figure 2), especially in newly hatched animals. They may be located in the hypodermis (epidermis).
  • the u38 animals can readily be reverted to a wild-type phenotype.
  • non-Tab (touch sensitive) revertants obtained by EMS sutagenesis have not segregated Tab animals, these reverting mutations are presumed to be intragenic.
  • the remaining revertant strain produces some Tab progeny but with an unusual segregation pattern and has not been studied further.
  • Three additional intragenic revertants arose spontaneously from a mut-2; deg-1 double mutant (the mut-2 mutation often activates transposons in C. elegans (16)).
  • the mutant deg-1 revertant of the invention does not produce a detectable phenotype.
  • the mutant deg-1 gene encodes an abnormal gene product.
  • the abnormal deg-1 gene product interferes with normal cell homeostasis.
  • the invention is meant to include wild-type and mutant deg-1 gene products and alleles thereof and other genes which may be described as a deg-1 gene family, that is, genes which are highly homologous to deg-1 and encode proteins which have similar amino acid sequences and functions to the deg-1 gene cloned herein.
  • the invention further includes the proteins encoded by such genes. Two of the intragenic suppressor mutations, u352 and u354, only partially suppress the effects of u38.
  • Late-onset degeneration of the PVC cells may be triggered by larval cell interactions, including, but not limited to, the initation of aberrant differentation and/or the toxic stimulation of the cells, as in, for example, glutamate neurotoxicity (21).
  • Many neurons arise post-embryonically in C. elegans, but they do not seem to be required for the PVC deaths, as the double mutant lin-6(e1466); deg-1 (u38), in which none of these cells are made (22, 23), is Tab.
  • the elimination of the touch receptor neurons, which synapse onto the PVC cells, by the addition of the unc- 86(e1416) mutation (8) had no effect in the deg-1 deaths.
  • the deg-1(u38) mutation may act in a cell-autonomous fashion.
  • the cell interactions trigger a deg-1-dependent degeneration.
  • mec-6 mutations Suppression by mec-6 mutations Mutations in the mec-6 gene suppress the degenerations caused by both the mec-4 (d) and deg-1 (u38) mutations. This result not only indicates an underlying genetic similarity in the deaths caused by these mutations, but also identifies a gene, mec-6, whose activity is required for the neurodegenerative proccss.
  • the effect on the mec-4 (d) mutations was discovered through the construction of a set of double mutants, each containing a mec-4 (d) mutation and a mutation in one of the other genes required for touch receptor function (7) to determine whether any of these mutations could prevent the mec-4 (d) deaths.
  • the mec-6 mutation u247 results in a temperature-sensitive phenotype for touch sensitivity (7). Suppression of the mec- 4(d) degenerations by U247 is also temperature sensitive; deaths were seen in newly-hatched larvae at 15oC, but not at 25oC (it was not possible to delay the onset of these deaths in temperature-shift experiments). Thus, in a preferred embodiment the loss of mec-6 activity results in suppression. In another embodiment, the production of an allele-specific product results in the suppression. As other features of the touch receptors seem unaffected in mec-6 mutants (7, 8), the wild-type mec-6 product is probably required for neurodegen- eration induced by the abnormal products of the mec-4 (d) alleles.
  • mec-6 Mutations in mec-6 (u41, u247 and u450 were used) also suppress the effects of deg-1 (u38). Double mutants are insensitive to the touch of hair at both head and tail (the mec-6 phenotype), but they are not Tab. Moreover, the mec-6 mutations seem to suppress all of the deg-1-induced deaths (no vacuolated cells were seen in any newly-hatched larvae) as well as the appearance of the presumptive hypodermal vacuoles (Figure 2D). As with the suppression of the mec- 4(d) deaths, suppression only occurred in mec-6 homozygotes.
  • deg-1 The deg-1 gene was cloned by transposon tagging, utilizing the mut-2-derived revertants ( Figure 5). DNA flanking the insertion site of the transposon Tel hybridized to C47C12 ( Figure 6), a cosmid clone adjacent to a cosmid containing the mec-7 gene (24, 25), a position consistent with the genetic mapping of deg-1 ( Figure 1 legend). Hybridization with C47C12 revealed insertions in one other mut-2-derived revertant and a possible deletion in one EMS-derived revertant ( Figure 5).
  • cDNA or genomic library construction may also be obtained commercially (for example, with Promega Corporation, Madison, Wisconsin).
  • transposon is inserted in the revertant u38u478 resides in a 187-bp region that contains sequences in the cDNAs surrounding a 50-bp intron ( Figure 6 legend), it is likely that these cDNAs represent deg-1 transcripts.
  • the larger cDNA encodes an open reading frame of 884 base pairs (bp) ( Figure 7) followed by 141 bp and a poly(A) tail.
  • the smaller cDNA lacks 72-bp from within the open reading frame.
  • the inferred polypeptide sequence of 294 amino acids from the larger cDNA which is not strongly homologous to other DNA and protein database entries, has three notable features: (1) a hydrophobic region from residues 215-255; (2) two possible sites for N-linked glycosylation (26) located at positions 8 and 122; and (3) a cysteine-rich region, containing 13 of the 14 cysteines in the sequence, between positions 66 and 187.
  • the 72 bp absent in the smaller cDNA encode 24 amino acids in the cysteine-rich region (including two cysteines) and could represent an alternatively-spliced variant.
  • the structure of the deg-1 product is consistent with its being a membrane protein.
  • the hydrophobic domain has an uninterrupted stretch of 17 amino acids, which is sufficient to span the lipid bylayer (27), and an additional 24 amino acids, only three of which are charged. This structure but not sequence, is also found in the predicted membrane spanning domain of the ⁇ subunit of the T-cell receptor (28, 29). It is possible that the extended hydrophobic region of the deg-1 product (including the charged residues) is submerged in the cell membrane, and may interact with analogous domains of other membrane proteins. The localization of the large number of cysteines, a feature of several receptor proteins (30-33), and the two N-linked glycosylation sites in the N-terminal region, is consistent with this region being an extracellular domain (Figure 7).
  • deg-1 may be a member of a gene family.
  • the cDNAs as well as a genomic DNA fragment to which they bind hybridize to several genomic fragments ( Figure 5b).
  • the wild-type deg-1 gene as defined by the restriction fragments that differ in the u38 revertants or that hybridize with the cDNAs, is contained within a 35 kilobase (kb) BamHI fragment ( Figure 6).
  • the corresponding fragment cloned from the dominant allele deg-1(u38) Figure 6, TU#3
  • when transformed into wild-type animals produces both the Tab and Deg phenotypes ( Figure 6 legend).
  • stable cell lines are identified which possess copies of the transformed DNA attached to, or inserted within, the genomic DNA in a manner which is stably inherited from generation to generation. It is highly desirable to identify such stable transformant cell lines.
  • Transformant lines may be unstable if the DNA is not attached or otherwise integrated into the host's chromosomal DNA. Especially, those transformants which possess extrachromosomal arrays of the injected DNA may be genetically unstable.
  • the mutant phenotypes are stably inherited, and karyotype and Southern blot analysis indicate that at least ten intact copies of the transformed DNA have attached to the X chromosome.
  • the ulnl animals resemble u38 mutants, but display some differences.
  • the extra copies of deg-1 (u38) DNA or its misexpression in the transformants may cause additional cells to die or change the onset of some cell degeneration.
  • the Deg and Tab phenotypes of ulnl animals are not completely suppressed by mec-6 mutations u450 and el342.
  • deg-1 and mec-4 cause the degeneration of a small number of neurons. Although different cells die in the deg-1 and mec-4 mutants, these mutations cause an apparently similar cell death that differs morphologically and genetically from programmed cell death. The degeneration results from the expression of alleles that seem to encode abnormal products; the wild-type genes are not needed for cell viability. The dominant mutations are suppressed by mec-6 mutations, suggesting that similar molecular processes may underlie these neuronal degenerations.
  • deg-1(u38) phenotype An interesting feature of the deg-1(u38) phenotype is the late onset of cell degeneration of many of the affected cells. Because the loss of the PVC cells results in a detectable phenotype, the behavioral defect in these animals also seems to be of late onset, even though other cells have died earlier.
  • the PVC degenerations can occur in animals of different ages (from second stage larvae to egg-laying adults) depending on the nature of the mutation, the growth temperature, and the dosage of the wild-type gene.
  • Membrane proteins which have similar structures to the deg-1 protein of the invention include ion channels and membrane- bound receptors.
  • An abnormal product from a dominant mutation might prove toxic if, directly or indirectly, it compromised membrane integrity.
  • Several observations suggest that chemically open membrane channels can lead to cell lethality.
  • the continued opening of acetylcholine channels at the neuromuscular junction leads to localized degeneration of the endplate because of the activation of calcium-dependent proteases (37), and sustained opening of capsaicin-sensitive channels in putative pain receptor neurons can cause lysis through osmotic imbalance and calcium influx (38).
  • the osmotic disruption and calcium entry that result from glutamate neurotoxicity (39) may also result from a similar mechanism.
  • the partial sequence of the deg-1 product is consistent with a role in membrane function, although it is not homologous to known channel proteins.
  • the deg-1 product could affect membrane integrity indirectly.
  • the mutant product might bind to a channel or other membrane component and modify its activity.
  • Of relevance may be a mammalian protein with only one apparent transmembrane domain that confers novel channel properties on Xenopus oocytes (40, 41).
  • deg-1 may encode a ligand-activated receptor.
  • the cysteine-rich extracellular domain suggests such a function.
  • the u38 phenotype could then arise from an inappropriate activation of a second messenger pathway that alters the activity of membrane proteins.
  • the deg-1(u38) product interferes with essential cell functions, as hypothe sized for the abnormal catabolism of the ⁇ -amyloid precursor protein in Alzheimer' s disease (42) .
  • mec-6 mutations The suppression of deaths by mec-6 mutations indicates an important role for mec-6 in neurodegeneration.
  • This mec-6 activity could be required for the mec-4 (d) and deg-1 degenerations either because it activates the products of these genes (perhaps by regulating their synthesis or subcellular localization) or because it is a necessary target (such as a channel) or cof actor for their action. In all these cases , loss of mec-6 activity would prevent the neuronal degeneration.
  • the further molecular analysis of the deg-1 , mec-4 , and mec-6 genes as well as the localization of their products will be important in elucidating the ways these genes act.
  • the selective degeneration of particular sets of neurons is characteristic of a number of human genetic diseases (1) .
  • Many late-onset human neurodegenerative diseases are expressed as dominant traits (1) , and although many more dominant human genetic diseases are known than recessive or X- linked ones (43) , the proportion of dominant, late-onset diseases of the nervous system is particularly high.
  • Another similarity between the C . elegans degenerations and those in human diseases is their appearance. It is difficult to determine whether the pathologies described for the human diseases are identical to the vacuolated deaths seen in the C. elegans mutants .
  • vacuolated cortical cells (ballooned neurons") occur in some patients with neurodegenerative diseases, including instances of Alzheimer' s disease (44) , and vacuolated cells have been reported in two cases of motor neuron disease (45, 46) . Vacuolization is also seen in genetically induced neuronal death in several mouse mutants (47-49) .
  • Such a process may occur in the death of the subplate neurons of the mammalian cerebral cortex die with a vacuolated appearance after making functional embryonic synapses (54 , 55) . It seems likely that cell death during normal development occurs through more than one mechanism, one of which could utilize a deg-1 (u38) - or mec-4 (d) -like product.
  • This invention comprises genetic sequences coding for the deg-1 gene, the mec-6 gene and the mec-4 gene, mRNA or antisense mRNA, expression vehicles containing the genetic sequences for these genes, hosts transformed therewith and recombinant protein and antisense RNA produced by such transformed host expression.
  • the invention further comprises antibodies directed against the proteins .
  • genetic sequences are intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences which are capable of encoding the proteins of the invention are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.
  • the DNA which encodes the proteins of the invention may or may not include naturally-occurring introns. Moreover, such genomic DNA may be obtained in association with the 5' promoter region of the gene sequences and/or with the 3' transcriptional termination region. Further, such genomic DNA may be obtained in association with the genetic sequences which encode the 5' non-translated region of the mRNA and/or with the genetic sequences which encode the 3' non-translated region.
  • Genomic DNA can be extracted and purified from any cell of the species by means well-known in the art (for example, see Guide to Molecular Cloning Techniques, S.L.
  • mRNA which encodes the proteins of the invention can be isolated from any cell which produces or expresses such proteins and used to produce cDNA by means well-known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al., eds., Academic Press (1987).)
  • the mRNA preparation used will be enriched in mRNA coding for the proteins of the invention, either naturally, by isolation from cells which are producing large amounts of the protein, or in vitro , by techniques commonly used to enrich mRNA preparations for specific sequences, such as sucrose gradient centrifugation, or both.
  • suitable DNA preparations for cloning into a vector, such suitable DNA preparations
  • a DNA sequence encoding the proteins of the invention or functional derivatives thereof may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T. , et al . , supra , and are well-known in the art.
  • Libraries containing clones encoding the proteins of the invention may be screened and a desired clone identified by any means which specifically selects for that protein ' s DNA such as, for example, (a) by hybridization with an appropriate nucleic acid probe (s) containing a sequence specific for the DNA of this protein; or (b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized; or (c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated protein product produced by the host containing the clone. Oligonucleotide probes specific for the proteins of the invention which can be used to identify clones to these proteins can be designed from knowledge of the amino acid sequence of the protein.
  • the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., in: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357).
  • the peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.
  • amino acid sequence may be encoded by only a single oligonucleotide sequence
  • amino acid sequence may be encoded by any of a set of similar oligonucleotides.
  • all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment
  • only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene.
  • this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
  • oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding the proteins of the invention.
  • the probability that a particular oligonucleotide will , in fact, constitute the actual protein sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eucaryotic cells.
  • cognid usage rules are disclosed by Lathe, R. , et al . , J. Molec . Biol . 183 : 1-12 (1985) . Using the "codon usage rules" of Lathe, a single oligonucleotide sequence, or a set of oligonucleotide sequences, that contain a theoretical "most probable" nucleotide sequence capable of encoding the desired sequence is identified.
  • oligonucleotide or which is complementary to such an oligonucleotide, or set of oligonucleotides
  • the above- described DNA probe is labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention.
  • Particularly useful are radioactive labels, such as 32 P, 3 H, 14 C, 35 S, 125 I, or the like. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life.
  • the oligonucleotide may be radioactively labeled, for example, by "nick- translation" by well-known means, as described in, for example, Rigby, P.J.W., et al., J. Mol. Biol.113: 237 (1977) and by T4 DNA polymerase replacement synthesis as described in, for example, Deen, K.C., et al., Anal. Biochem. 135: 456 (1983).
  • polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • oligonucleotide complementary to this theoretical sequence or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides, one obtains a DNA molecule (or set of DNA molecules) , capable of functioning as a probe (s) for the identification and isolation of clones containing the gene .
  • a library is prepared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing the desired protein, into an expression vector.
  • the library is then screened for members which express the desired protein, for example, by screening the library with antibodies to the protein.
  • the above discussed methods are, therefore, capable of identifying genetic sequences which are capable of encoding the proteins of the invention or fragments of this protein.
  • Such expression identifies those clones which express proteins possessing characteristics of the proteins of the invention. Such characteristics may include the ability to specifically bind antibody to the proteins , the ability to elicit the production of antibody which is capable of binding to the protein, the ability to provide a protein-specific function to a recipient cell , among others.
  • the cloned protein encoding sequences obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a host cell , either procaryote or eucaryote, to produce recombinant protein or a functional derivative thereof.
  • a host cell either procaryote or eucaryote
  • protein antisense RN ⁇ or a functional derivative threof it is also possible to express protein antisense RN ⁇ or a functional derivative threof.
  • the present invention encompasses the expression of the protein, or a functional derivative thereof, in eucaryotic cells, and especially mammalian, insect and yeast cells.
  • eucaryotic hosts are mammalian cells neural cells, either in vivo or in tissue cultue. Mammalian cells provide post-translational modifications to recombinant proteins which include folding and/or glycosylation at sites similar or identical to that found for the native protein.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are “operably linked” to the nucleotide sequence which encodes the polypeptide.
  • An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence.
  • Two DNA sequences are said to be operably linked if inducation of promoter function results in the transcription of the protein encoding sequence mRNA and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation; (2) interfere with the ability of the expression regulatory sequences to direct the expression of the protein's mRNA, antisense RN ⁇ , or protein; or (3) interfere with the ability of the protein's template to be transcribed by the promote region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • regulatory regions needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing control sequences will include a region which contains a promoter for transcriptional control of the operably linked gene.
  • transcriptional and translational regulatory signals can be employed, depending upon the nature of the eucaryotic host.
  • the transcriptional and translational regulatory signals can also be derived from the genomic sequences of viruses which infect eucaryotic cells, such as adenovirus, bovine papilloma virus. Simian virus, herpes virus, or the like. Preferably, these regulatory signals are associated with a particular gene which is capable of a high level of expression in the host cell.
  • Vectors may also be designed which possess the ability to target certain cells. such as neuronal cells, and which cross the blood-brain barrier. For example, a mutant retroviral vector say be used if such a vector is designed to by non-transforming or otherwise harmful to the host cell, and if it retains an ability to transport across the blood-brain barrier and infect neuronal target cells.
  • control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such a methionine.
  • Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell. Promoters from heterologous mammalian genes which encode mRNA product capable of translation are preferred, and especially, strong promoters such as the promoter for actin, collage, myosin, etc., can be employed provided they also function as promoters in the host cell.
  • Preferred eucaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al., J.
  • TK promoter of herpes virus McKnight, S., Cell 31: 355-365 (1982)
  • SV40 early promoter Boist, C, et al., Nature (London) 290: 304-310 (1981)
  • yeast ga14 gene promoter Johnston, S.A., et al., Proc. Natl. Acad. Sci. (USA) 79: 6971-6975 (1982); Silver, P.A., etal., Proc. Natl. Acad. Sci. (USA) 81: 5951-5955 (1984) or a glycolytic gene promoter may be used.
  • a fusion product of the protein may be constructed.
  • the sequence coding for the protein may be linked to a signal sequence which will allow secretion of the protein from, or the compartmentalization of the protein in, a particular host.
  • signal sequences may be designed with or without specific protease sites such that the signal peptide sequence is amenable to subsequent removal.
  • the native signal sequence for this protein may be used.
  • Transcriptional initiation regulatory signals can be selected which allow for repression or activation, so that expression of the operably linked genes can be modulated.
  • modulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.
  • Translational signals are not necessary when it is desired to express protein antisense RNA sequences.
  • the non-transcribed and/or non-translated regions 3' to the sequence coding for the desired protein can be obtained by the above-described cloning methods.
  • the 3'-non-transcribed region may be retained for its transcriptional termination regulatory sequence elements; the 3-non-translated region may be retained for its translational termination regulatory sequence elements, or for those elements which direct polyadenylation in eucaryotic cells.
  • sequences functional in the host cell may be substituted.
  • the vectors of the invention may further comprise other operably linked regulatory elements such as enhancer sequences, or DNA elements which confer tissue or cell-type specific expression on an operably linked gene.
  • DNA encoding sequence and an operably linked promoter is introduced into a recipient eucaryotic cell as a non- replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule which is incapable of autonomous replication, the expression of the protein may occur through the transient expression of the introduced sequence.
  • genetically stable transformants may be constructed with vector systems, or transformation systems, whereby a desired protein's DNA is integrated into the host chromosome.
  • Such integration may occur de novo within the cell or, in a most preferred embodiment, be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with retroviral vectors, transposons or other DNA elements which promote integration of DNA sequences in chromosomes.
  • a vector is employed which is capable of integrating the desired gene sequences into a mammalian host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by cotransfection.
  • the introduced sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose, as outlined below.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred eucaryotic plasmids include those derived from the bovine papilloma virus, vaccinia virus, SV40, and, in yeast, plasmids containing the 2-micron circle, etc., or their derivatives.
  • Such plasmids are well-known in the art (Botstein, D., et al., Miami Wntr. Symp. 12: 265-274 (1982); Broach, J.R., in The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp.
  • mammalian expression vector systems which utilize the MSV-LTR promoter to drive expression of the cloned gene, and in which it is possible to cotransfect with a helper virus to amplify plasmid copy number, and, integrate the plasmid into the chromosomes of host cells have been described (Perkins, A.S. et al., Mol. Cell Biol. 3: 1123 (1983); Clontech, Palo Alto, California).
  • the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transfection. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner, for example, expression which follows induction of differentiation of the transformed cells (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).
  • the expressed protein is isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like, by means well-known in the art.
  • the protein encoding sequences of the invention will provide sequences which by definition, encode a desired protein and which may then be used to obtain antisense RNA genetic sequences as the antisense RNA sequence will be that sequence found on the opposite strand of the strand transcribing the peptide sequence's mRNA.
  • the antisense DNA strand may also be operably linked to a promoter in an expression vector such that transformation with this vector results in a host capable of expression of an antisense RNA in the transformed cell.
  • Antisense RNA and its expression may be used to interact with an endogenous DNA or RNA in a manner which inhibits or represses transcription or translation of the gene or mRNA respectively in a highly specific manner. Use of antisense RNA probes to block gene expression is discussed in Lichtenstein, C., Nature 333: 801-802 (1988). Such antisense RNA may be useful in the therapeutic treatments of the invention.

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Abstract

Molécule d'acide nucléique isolée codant une protéine animale de type sauvage associée à la dégénérescence neuronale et molécule d'acide nucléique isolée codant une protéine animale mutée associée à la dégénérescence neuronale. On décrit aussi des souches du nématode Caenorhabditis elegans contenant les molécules d'acide nucléique codant une protéine C. elegans mutée associée à la dégénérescence neuronale. On présente en outre des procédés servant à détecter de telles molécules d'acide nucléique, à diagnostiquer des maladies de dégénérescence, à déterminer les causes pour lesquelles une cellule humaine malade dégénère, et à sélectionner les médicaments qui permettent de prévenir ou d'atténuer la dégénérescence neuronale.
PCT/US1991/003826 1990-05-30 1991-05-30 Sequences d'adn impliquees dans la degenerescence neuronale, organismes multicellulaires contenant ces sequences et utilisations de ces sequences WO1991019007A1 (fr)

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US5962301A (en) * 1992-06-12 1999-10-05 Massachusetts Institute Of Technology Relatedness of human interleukin-1β convertase gene to a C. elegans cell death gene, inhibitory portions of these genes and uses therefor
EP0996708A1 (fr) * 1997-07-11 2000-05-03 The General Hospital Corporation Modele de nematode transgenique de maladies neurologiques a repetition par triplet
US6939850B2 (en) 1992-06-12 2005-09-06 Massachusetts Institute Of Technology Altered human interleukin-1β convertase (ICE), NEDD-2, and C. elegans ced-3 polypeptides and uses therefor
US7071302B1 (en) 1992-06-12 2006-07-04 Massachusetts Institute Of Technology Cloning sequencing and characterization of two cell death genes and uses therefor
US8314067B1 (en) 1992-06-12 2012-11-20 Massachusetts Institute Of Technology Relatedness of human interleukin-1β convertase gene to a C. elegans cell death gene, inhibitory portions of these genes and uses therefor
US8524891B2 (en) 2005-07-14 2013-09-03 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Tetraazaporphyrin-based compounds and their uses

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GENETICS, Vol. 77, issued May 1974, BRENNER, "The Genetics of Caenorhabditis Elegans", pages 78-94. *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962301A (en) * 1992-06-12 1999-10-05 Massachusetts Institute Of Technology Relatedness of human interleukin-1β convertase gene to a C. elegans cell death gene, inhibitory portions of these genes and uses therefor
US6939850B2 (en) 1992-06-12 2005-09-06 Massachusetts Institute Of Technology Altered human interleukin-1β convertase (ICE), NEDD-2, and C. elegans ced-3 polypeptides and uses therefor
US7071302B1 (en) 1992-06-12 2006-07-04 Massachusetts Institute Of Technology Cloning sequencing and characterization of two cell death genes and uses therefor
US7138510B1 (en) 1992-06-12 2006-11-21 Massachusetts Institute Of Technology Cloning sequencing and characterization of two cell death genes and uses therefor
US8314067B1 (en) 1992-06-12 2012-11-20 Massachusetts Institute Of Technology Relatedness of human interleukin-1β convertase gene to a C. elegans cell death gene, inhibitory portions of these genes and uses therefor
EP0996708A1 (fr) * 1997-07-11 2000-05-03 The General Hospital Corporation Modele de nematode transgenique de maladies neurologiques a repetition par triplet
EP0996708A4 (fr) * 1997-07-11 2004-09-08 Gen Hospital Corp Modele de nematode transgenique de maladies neurologiques a repetition par triplet
US8524891B2 (en) 2005-07-14 2013-09-03 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Tetraazaporphyrin-based compounds and their uses

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