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WO1999058671A2 - Homologues du gene d'assurance de longevite des levures, lag1, d'origine humaine ou issus de nematodes - Google Patents

Homologues du gene d'assurance de longevite des levures, lag1, d'origine humaine ou issus de nematodes Download PDF

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WO1999058671A2
WO1999058671A2 PCT/US1999/010160 US9910160W WO9958671A2 WO 1999058671 A2 WO1999058671 A2 WO 1999058671A2 US 9910160 W US9910160 W US 9910160W WO 9958671 A2 WO9958671 A2 WO 9958671A2
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isolated
nucleic acid
seq
cell
human
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PCT/US1999/010160
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WO1999058671A3 (fr
WO1999058671A9 (fr
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S. Michal Jazwinski
Paul Kirchman
James Jiang
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Research Corporation Technologies, Inc.
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Publication of WO1999058671A2 publication Critical patent/WO1999058671A2/fr
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Publication of WO1999058671A3 publication Critical patent/WO1999058671A3/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/43504—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
    • 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 present invention is directed to human and nematode LAGl nucleic acids and the polypeptides encoded and expressed thereby, including appropriate vectors and host cells.
  • the present invention contemplates antisense RNA and DNA.
  • the invention also contemplates transgenic animals.
  • This invention further provides methods for increasing the longevity and the tolerance to stress of eu aryotic cells by providing to the cell human and/or nematode LAGl polypeptides. Methods for providing the cell with such LAGl polypeptides include overexpressing the endogenous LAGl gene, or providing the cell with an expression vector containing the appropriate LAGl coding region. Such methods can be practiced, for example in mammalian cells, such as human cells.
  • the present invention is also directed to pharmaceutical compositions to effect such methods.
  • mice In mammals, studies with mice have focused on immune function since the immune system is the first line of defense against environmental insults and stress in mammals. Mutant mouse strains have been used to define a role for the H-2 locus in determining longevity. High immune responsiveness was positively correlated with life-span. Covelli, et al . (1989) J. Immunol . 142 : 1224. Immune function has also been implicated in the genetics of aging in humans in studies of the HLA locus. Proust, et al . (1982) Tissue Antigens 9_:168; Takata, et al . (1987) Lancet .11:824. The apolipoprotein E gene has also been associated with human longevity in a study of French centenarians.
  • apoE4 isoform of apolipoprotein E is correlated with an increased susceptibility to both coronary heart disease and Alzheimer's disease. Thus the presence of apoE4 is apparently associated with shorter life expectancy in humans. Jazwinski (1996) Science 273:54-59.
  • the present invention provides human and nematode homologues of the Saccharomvces cerevisiae longevity-assurance gene LAGl .
  • the human and nematode homologues of yeast LAGl increase the longevity and tolerance to cellular stress of eukaryotic cells, including human cells .
  • the present invention is directed to isolated nucleic acids encoding Longevity Assurance Gene 1 (LAGl) polypeptides such as human and nematode homologues of yeast LAGl which increase the longevity and tolerance to cellular stress of eukaryotic cells and organisms.
  • LAGl Longevity Assurance Gene 1
  • Preferred nucleic acids include:
  • nucleic acids capable of hybridizing to a DNA having SEQ ID N0:1 under moderate to stringent conditions
  • nucleic acids the complement of which selectively hybridize to a DNA having a SEQ ID N0:1 under moderate to stringent conditions
  • nucleic acids differing from any one of the nucleic acids of (a) or (b) in codon sequence due to the degeneracy of the genetic code;
  • nucleic acids encoding various species of LAGl polypeptides such as, for example, mammals including human (LAGlHs) , bovine, murine; nematode (LAGlCel) Podospora, galago, and salmon.
  • mammals including human (LAGlHs) , bovine, murine; nematode (LAGlCel) Podospora, galago, and salmon.
  • the present LAGl nucleic acids preferably have at least 50% homology to SEQ ID N0:1 or SEQ ID NO: 3.
  • the present invention provides LAGl polypeptides, i.e. isolated or synthesized polypeptides or derivatives thereof which increase the longevity and tolerance to cellular stress of eukaryotic cells, including e.g. mammalian including human, bovine and mouse; nematode, galago, salmon and the like.
  • the present invention is drawn to an isolated human LAGl polypeptide (termed LAGlHs) having, for example, SEQ ID NO: 2.
  • these polypeptides have at least about 25% to 30% homology to SEQ ID NO: 2.
  • the present invention is drawn to isolated (____ elegans (LAGlCel) polypeptides, having for example, SEQ ID NO: 4.
  • LAGlCel isolated polypeptides
  • these polypeptides have at least about 25% to 30% homology to SEQ ID NO: 4.
  • the present invention is also directed to an isolated LAGl antisense RNA or DNA having sufficient length and sufficient complementarity to selectively hybridize to a nucleic acid having SEQ ID N0:1 or SEQ ID NO: 3.
  • the antisense RNA or DNA has sufficient length and sufficient complementarity to selectively hybridize to a nucleic acid.
  • the length of an antisense RNA and DNA is at least about 10 to about 2500 nucleotides.
  • the present invention provides isolated replication and expression vectors containing the present LAGl nucleic acids as well as host cells for these vectors.
  • Another embodiment of the present invention provides a method for increasing the longevity, reproductive capacity and/or tolerance to stress of a eukaryotic cell. Such methods include administering mutant or wild type LAGlHs or LAGlCel polypeptides to the cell of interest.
  • a further embodiment of the present invention provides a method for increasing the longevity, reproductive capacity and tolerance to acidic pH of a eukaryotic cell or organism which includes expressing a mutant or wild type LAGlHs or LAGlCel polypeptide in the cell.
  • the present invention provides pharmaceutical compositions containing at least one of the present LAGl nucleic acids, oligonucleotides or polypeptides, and a pharmaceutically acceptable carrier.
  • these LAGl nucleic acids, oligonucleotides or polypeptides include, for example, human, nematode, bovine, mouse, galago, salmon and related eukaryotic nucleic acids, oligonucleotides or polypeptides.
  • Preferred compositions include LAGlHs or LAGlCel nucleic acids having at least 50% homology to SEQ ID NO:l or SEQ ID NO : 3.
  • Preferred compositions also include polypeptides having at least about 25% to 30% homology to SEQ ID NO: 2 or SEQ ID NO: 4.
  • Figure 1 depicts a multiple sequence comparison of yeast LAGl (LAGlSc, SEQ ID NO: 6) and its homologues yeast LAClSc (SEQ ID NO:5), nematode LAGlCel (SEQ ID NO: 4) and human LAGl (LAGlHs, SEQ ID NO: 2) .
  • Figure 2 depicts hydrophobicity plots for LAGlSc, LAClSc, LAGlCel and LAGlHs.
  • Figure 3 depicts an autoradiogram of RT-PCR from C. elecrans RNA.
  • Lane 1 molecular marker
  • Lane 2 RNA from N2 (wild type) strain
  • Lane 3 RNA from Jig 23 strain (sterile male)
  • Lane 4 RNA from el490 strain (sterile male) .
  • Figure 4 depicts a comparison between the predicted LAGlCelD amino acid sequence (SEQ ID NO: 7) and the actual LAGlCel amino acid sequence (SEQ ID NO: 4) .
  • Figure 5 depicts expression of LAGlHs in transformed yeast cells by Northern analysis. RNA was extracted from transformed cells, then Northern analysis was performed.
  • Figure 6A depicts tetrad dissection of LAGlHs transformants.
  • Figure 6B depicts tetrad dissection of LAGlCel transformants.
  • Figures 7A and B depict genotype verification of the deletion of LAGlSc and LAClSc by PCR amplification.
  • Figure 8A depicts the effect of the deletion of LAGlSc and LAClSc on the viability of yeasts, confirming that LAGlCel is needed to complement the deletion of LAGlSc and LAClSc.
  • Figure 8B depicts the effect of the deletion of LAGlSc and LAClSc on the viability of yeasts, confirming that LAClHs is needed to complement the deletion of LAGlSc and LAClSc.
  • Figure 8C depicts the effect of the deletion of LAGlSc and LAClSc on the viability of yeast confirming that LAGlHs is needed for viability of yeasts .
  • Figure 9 depicts hybridization of a LAGlHs probe to a Northern blot of human mRNA from several human tissues, including adult heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes.
  • the LAGlHs probe was also hybridized to tissue from fetal brain, lung, liver and kidney.
  • Figure 10 depicts a cytogenetic mapping of LAGlHs .
  • the LAGlHs gene mapped to chromosome 19 (19pl2) .
  • the inset shows chromosome 19 homologues enlarged.
  • Figure 11 depicts a genomic map of LAGlHs .
  • the present invention is directed to isolated nucleic acids encoding human and nematode LAGl polypeptides, respectively, which can increase the longevity, reproductive capacity and tolerance to cellular stress of eukaryotic cells.
  • Preferred nucleic acids include nucleic acids having SEQ ID NO:l or SEQ ID NO: 3.
  • the present invention provides nucleic acids capable of hybridizing to a DNA having SEQ ID NO:l or SEQ ID NO: 3 under moderate to high stringency conditions.
  • the present invention also provides nucleic acids, the complement of which selectively hybridize to a DNA having SEQ ID N0:1 or SEQ ID NO: 3.
  • nucleic acids encode various species of LAGl polypeptides such as, for example, Podospora, galago, salmon and mammalian species including human, bovine, murine and the like.
  • Preferred nucleic acids of this invention are those which encode the human LAGl polypeptide (LAGlHs , SEQ ID NO: 2) and nematode LAGl polypeptide (LAGlCel, SEQ ID NO: 4) .
  • the present invention provides nucleic acids which encode a LAGl polypeptide which differs from the aforementioned nucleic acids in codon sequence due to the degeneracy of the genetic code.
  • the present LAGl nucleic acids have at least 50% homology to SEQ ID NO:l or SEQ ID NO: 3.
  • the present invention provides a nucleic acid having SEQ ID NO:l which encodes a full length human LAGl polypeptide (LAGlHs) .
  • pJJll A plasmid, identified as pJJll, which contains a 1.1 kb fragment encoding the LAGlHs obtained from total human brain mRNA by amplification using RT-PCR was deposited with the American Type Culture Collection (10801 University Boulevard., Manassas, VA 20110) on April 29, 1999. This pJJll Plasmid was provided ATCC No. 203962.
  • the present invention is directed to any nucleotide sequence which can encode the present LAGlHs or LAGCel polypeptides. Therefore, for example, while the LAGlHs polypeptide sequence of SEQ ID NO: 2 is encoded by a nucleic acid having SEQ ID N0:1, there are alternative nucleic acid sequences which can encode the same SEQ ID NO: 2 polypeptide sequence.
  • the present invention is also directed to any of the present isolated LAGl nucleic acids which have such alternative nucleic acid sequences .
  • the present invention provides nucleic acids capable of hybridizing to a DNA having SEQ ID N0:1 or SEQ ID NO: 3.
  • the present invention also provides nucleic acids, the complement of which selectively hybridize to a DNA having SEQ ID N0:1 or SEQ ID N0:3.
  • a human LAGl DNA probe detects a nucleotide sequence in the mouse genome under moderate to high stringency hybridization conditions.
  • the human LAGl is expressed in human tissues as a transcript of about 2.5 kb.
  • the Longevity Assurance Gene I has been detected in the genomic DNA of other eukaryotes including bovine, mouse, galago and salmon.
  • the LAGl gene is highly conserved across the spectrum of eukaryotes and LAGl homologues from other species are isolated by techniques readily available to the skilled artisan.
  • a yeast homologue of the S. cerevisiae LAGl gene can be identified in the GenBank database.
  • the product of this homologous gene, termed LACl is about 70% identical to the LAGl protein (SEQ ID NO: 6) at the amino acid level.
  • LACl SEQ ID NO: 5
  • LAGl protein SEQ ID NO: 6
  • Both the yeast LAGl and its homologue LACl can be used to identify homologous genes from other species including humans and nematodes in the GenBank database for example.
  • a LAGl genomic DNA can be isolated from another eukaryotic species, e.g. a mammal such as a mouse, by hybridization methods.
  • This method includes providing a LAGlHs probe to a eukaryotic chromosomal library for a time and under conditions sufficient for hybridization; identifying a hybridization complex containing the probe and the eukaryotic LAGl genomic DNA; and isolating the eukaryotic LAGl genomic DNA.
  • a LAGlHs probe is selected from SEQ ID NO:l such that the LAGlHs probe has sufficient length and sufficient complementarity to hybridize to the eukaryotic LAGl genomic DNA.
  • a LAGlHs probe has sufficient length and sufficient complementarity to hybridize to SEQ ID NO:l.
  • the LAGlHs probe has at least about 50% complementarity to SEQ ID NO:l.
  • the LAGlHs probe is preferably at least about 10 nucleotides to about 2500 nucleotides.
  • This method is also used for isolating a eukaryotic LAGl cDNA when a eukaryotic cDNA library is used.
  • the present invention provides a method of isolating a eukaryotic, preferably human, LAGl nucleic acid by in vitro nucleic acid amplification.
  • Such methodology includes contacting a sample containing the LAGl nucleic acid with at least one appropriate oligonucleotide and an amplification enzyme for a time and under conditions sufficient to produce RNA or DNA copies of the LAGl nucleic acid. These copies are identified and isolated.
  • the oligonucleotides used for in vitro amplification are selected from SEQ ID N0:1 to have sufficient complementarity and sufficient length to hybridize to the eukaryotic LAGl nucleic acid and permit amplification.
  • the oligonucleotide has sufficient complementarity and sufficient length to hybridize to SEQ ID N0:1.
  • a preferred oligonucleotide has at least 50% complementarity to SEQ ID NO:l.
  • Preferred oligonucleotides have at least about 10 to about 2500 nucleotides .
  • Complementarity between nucleic acids is the degree to which the bases in one nucleic acid strand can hydrogen bond, or base pair, with the bases in a second nucleic acid strand.
  • sufficient complementarity means that a sufficient number of the nucleotides in SEQ ID NO:l form base pairs with nucleotides in a nucleic acid, e.g. a eukaryotic LAGl nucleic acid from a mammal, a LAGl oligonucleotide or a LAGl probe, to generate a stable hybridization complex at about room temperature (i.e. at about 20°C to about 25°C) .
  • Complementarity can sometimes be conveniently described by the percentage, i.e. proportion, of nucleotides which can form base pairs between two nucleic acid strands or within a specific region or domain of the two strands.
  • the degree of complementarity can range from at least about 50% to full, i.e. 100% complementarity.
  • the overall degree of complementarity between a eukaryotic LAGl nucleic acid, oligonucleotide or probe and SEQ ID NO:l is at least about 50%, and preferably about 60% or higher.
  • the term "homology”, as used herein, is the degree of sequence identity between two nucleic acid strands or two polypeptide sequences.
  • a target is a double- stranded nucleic acid
  • one target strand is complementary, and the other target strand is homologous, to a probe or oligonucleotide of the present invention.
  • sequence listing recites the sequence of only one nucleic acid strand.
  • some of the sequences described in the sequence listing are intended to be double- stranded. Accordingly, when a double- stranded target is identified by sequence number a probe or oligonucleotide can also be homologous to the recited sequence and hence can hybridize to the strand not recited in the sequence listing.
  • the degree of homology can also be described by the percentage of identical nucleotides or amino acids in two nucleotide or polypeptide sequences, respectively.
  • the degree of homology between a target nucleic acid and a probe or oligonucleotide of the present invention can vary so long as selective hybridization is attained, and can range from at least about 50% to about 100% homology.
  • the overall degree of homology between a eukaryotic LAGl nucleic acid, oligonucleotide or probe and SEQ ID NO:l is preferably about 60% or higher.
  • the degree of homology between a LAGl polypeptide and SEQ ID NO: 2 or SEQ ID NO:4 can range preferably from at least about 25% to 30% to about 100% homology and is more preferably about 30% or higher.
  • eukaryotic nucleic acids homologous to human LAGl nucleic acids are readily isolated, for example, from nematode, bovine, mouse, rat, galago, salmon and related eukaryotes.
  • Hybridization and in vitro amplification conditions for such isolation can readily be ascertained by the skilled artisan. See, e.g., Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual , Vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY.
  • a LAGl nucleic acid is copied using at least one appropriate oligonucleotide and an appropriate amplification enzyme.
  • the oligonucleotide is selected such that it is sufficiently complementary to hybridize to the eukaryotic LAGl nucleic acid to permit amplification.
  • the oligonucleotide is at least about 10 nucleotides long and at least 50% homologous to nucleic acid of SEQ ID NO:l.
  • the oligonucleotide includes a nucleotide sequence of at least about 10 nucleotides which is about 70% homologous to SEQ ID N0:1.
  • the LAGl target nucleic acid for in vitro amplification is that segment of nucleic acid which is copied during amplification.
  • the oligonucleotide (s) employed for amplification hybridize to only a portion of the target nucleic acid. This portion is the oligonucleotide binding site.
  • An oligonucleotide binding site can define the 3 ' or 5 ' end of the target nucleic acid. Therefore, when copies of the target nucleic acid are made during amplification, the actual 3 ' or 5 ' ends of such copies are composed of oligonucleotides which, e.g. act as primers for synthesis of the copy.
  • a portion or the whole of an oligonucleotide sequence is copied during the amplification procedure or the oligonucleotide sequence is not to be copied at all but instead forms a recognition site for binding the amplification enzyme.
  • the methods of amplifying LAGl target sequences are methods of in vitro nucleic acid amplification which include any procedure using an oligonucleotide to direct synthesis of a nucleic acid copy of the target sequence.
  • In vitro nucleic acid amplification thus allows selective synthesis of a specific DNA or RNA target relative to the complex bulk of nucleic acid present in a sample.
  • the specificity of the process is determined by the oligonucleotide, e.g. the oligonucleotide primers, capable of hybridizing with LAGl nucleic acids to the exclusion of other nucleic acids.
  • Conditions for in vitro nucleic acid amplification generally include temperature and salt concentrations permitting selective hybridization between the oligonucleotide and target.
  • preferred hybridization temperature is about 5°C to about 10°C below the melting temperature of the target: oligonucleotide hybrid (Sambrook, et al . ) .
  • This hybridization temperature can readily be varied to accommodate other considerations such as the thermal instability of the amplification enzyme.
  • the skilled artisan can readily ascertain appropriate temperature and salt conditions for hybridization of a LAGl oligonucleotides to LAGl nucleic acids from other eukaryotes .
  • Conditions for in vitro nucleic acid amplification also include those salt, cation, pH, nucleotide subunit concentration and temperature conditions required for enzymatic activity of the amplification enzyme.
  • some amplification enzymes require a cation such as magnesium for optimal activity.
  • copying of LAGl target nucleic acids requires that the appropriate nucleotide subunits be added to the amplification mixture, e.g. ATP, CTP, GTP, UTP, dATP, dCTP, dGTP or dTTP .
  • amplification enzymes like the Thermus aquaticus or Thermococcus litoralis DNA polymerases are stable for extended periods of time at 98°C, others such as the SP6 or T7 RNA polymerases are rapidly denatured at a temperature of about 65°C.
  • Optimal salt, cation, pH and temperature conditions for obtaining amplification enzyme activity are readily available to the skilled artisan, e.g. from a commercial manufacturer of these enzymes .
  • DNA polymerases can only copy DNA from a single- stranded target. Therefore the present methods can include at least one denaturing step for double- stranded target nucleic acids. Such methods can further include at least one denaturing step for separating a DNA or RNA copy from a target nucleic acid.
  • In vitro nucleic acid amplification techniques are known in the art. A review of such techniques can be found in Kwoh, et al . (1990) Am. Biotechnol . Lab. 8:14.
  • In vitro nucleic acid amplification techniques include polymerase chain reaction (PCR) , transcription-based amplification system (TAS) , self - sustained sequence replication system (3SR) , ligation amplification reaction (LAR) , ligase-based amplification system (LAS) , Q ⁇ RNA replication system and run-off transcription.
  • PCR polymerase chain reaction
  • TAS transcription-based amplification system
  • 3SR self - sustained sequence replication system
  • LAR ligation amplification reaction
  • LAS ligase-based amplification system
  • Q ⁇ RNA replication system Q ⁇ RNA replication system and run-off transcription.
  • PCR is a method for primer-directed enzymatic amplification of target nucleic acids.
  • PCR synthesis occurs by repeated cycles of heat denaturation of the target, primer annealing and primer extension. These cycles can be performed manually or, preferably, automatically.
  • Thermal cyclers such as the Perkin-Elmer Cetus cycler are specifically designed for automating the PCR process, and are preferred. The number of cycles per round of synthesis can be varied from 2 to more than 50, and is readily determined by considering the source and amount of the nucleic acid template, the desired yield and the procedure for detection of the synthesized DNA fragment.
  • PCR techniques and many variations of PCR are known. Basic PCR techniques are described by Saiki, et al . (1988) Science 239:487-491 and by U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, which are incorporated herein by reference.
  • the conditions generally required for PCR include temperature, salt, cation, pH and related conditions needed for efficient copying of the target.
  • PCR conditions include repeated cycles of heat denaturation (i.e. heating to at least about 95°C) and incubation at a temperature permitting target: oligonucleotide hybridization and copying of the target by the amplification enzyme.
  • Heat stable amplification enzymes like the Thermus aquaticus or Thermococcus litoralis DNA polymerases are commercially available which eliminate the need to add enzyme after each denaturation cycle.
  • the salt, cation, pH and related factors needed for amplification enzyme activity are available from commercial manufacturers of amplification enzymes.
  • the transcription-based amplification system utilizes a sample (sense) RNA template from which a double stranded complementary DNA (i.e. cDNA) is made.
  • a sample (sense) RNA template from which a double stranded complementary DNA (i.e. cDNA) is made.
  • One or more of the oligonucleotides used for synthesis of the cDNA contains an RNA polymerase recognition site.
  • An RNA polymerase capable of recognizing and synthesizing RNA starting at that recognition site is then added to produce many RNA copies of the cDNA.
  • additional rounds of cDNA synthesis can be performed using the synthesized RNA as template and this additional cDNA can be used to make even more RNA product .
  • RNA polymerases which can be used for TAS include, for example, SP6, T3, T7 and other RNA polymerases. TAS techniques are described by Kwoh, et al .
  • Conditions for TAS amplification are generally determined by the temperature, salt, cation and pH requirements of the RNA polymerase employed. These conditions are readily available to the skilled artisan, e.g. as provided by commercial manufacturers of such RNA polymerases .
  • the subject oligonucleotides can contain an additional sequence which encodes a recognition or binding site for an RNA polymerase, e.g. a T7 , T3 or SP6 RNA polymerase recognition sequence.
  • RNA polymerase recognition sequences are well known in the art and are readily incorporated into the present oligonucleotides by the skilled artisan.
  • the self -sustained sequence replication (3SR) procedure involves continuous cycling of reverse transcriptase and RNA polymerase synthesis.
  • 3SR utilizes RNase H enzymatic degradation of the RNA in an RNA:cDNA duplex, an innovation which eliminates thermal denaturation and repetitive addition of reagents.
  • the 3SR procedure involves synthesis of a double stranded cDNA wherein the oligonucleotide used for synthesis of either the first or second cDNA strand, has an RNA polymerase recognition site.
  • the double- stranded cDNA then acts as target for synthesis of either an antisense or sense RNA, depending on whether the first or second cDNA strand, respectively, has the RNA polymerase recognition site.
  • Conditions for 3SR amplification are generally determined by the temperature, salt, cation and pH requirements of the reverse transcriptase and the RNA polymerase employed. These conditions are readily available to the skilled artisan, e.g. as provided by commercial manufacturers of such enzymes .
  • the 3SR procedure has some advantages over PCR or TAS in that all reagents are placed in a single tube and incubation is at a single temperature. Accordingly, no thermal cycling or repeated addition of reagents is required. 3SR is also more rapid than many other in vitro nucleic acid amplification procedures since an approximate 10 6 -fold amplification of a desired DNA or RNA is achieved in about an hour.
  • DNA ligase is used to synthesize DNA by repeatedly joining oligonucleotides hybridized to a template nucleic acid. Such procedures have been termed ligation amplification (LAR) and ligase-based amplification systems (LAS) .
  • LAR or LAS utilizes four oligonucleotides wherein two oligonucleotides hybridize to one strand of the target DNA and the other two hybridize to the complementary sequences. The adjacently hybridizing oligonucleotides are then joined by DNA ligase. After thermal denaturation, an additional cycle of hybridization and ligation can be performed. Each round of denaturation, hybridization and ligation increases the ligated product by about two- fold. Blunt end ligation of oligonucleotides hybridizing to complementary oligonucleotides can be controlled by adjusting the temperature of the ligation step.
  • Conditions for LAS include temperature, salt, cation, pH and the like needed for repeated rounds of denaturation, hybridization and ligation.
  • Denaturation is generally performed at about 95°C to about 100°C.
  • Hybridization is preferably performed at about 5°C to about 10°C below the melting temperature of the target :oligonucleotide hybrid, however slow cooling from the denaturation temperature can also lead to selective hybridization between the target and the oligonucleotide (s) .
  • the conditions needed for efficient ligation are well known to the skilled artisan (e.g., see Sambrook, et al . ) .
  • the LAS technique is also described by Kwoh, et al .
  • RNA can be synthesized from a nucleic acid by employing a Q ⁇ replicase RNA replication system in which a first strand of a cDNA is made having a Q ⁇ replicase 5 ' -recognition site lying on the 3' -side of an RNA polymerase recognition site. This is done with an oligonucleotide capable of hybridizing to an RNA target which also encodes the 5'-Q ⁇ and RNA polymerase recognition sites in the correct positions. A second cDNA strand is then synthesized using an oligonucleotide encoding a Q ⁇ 3 ' -recognition site.
  • RNA polymerase can then use the double-stranded cDNA as a template for synthesis of antisense RNA having, as 5' and 3' ends, the respective 5'- and 3 ' -Q ⁇ replicase recognition sites.
  • This antisense RNA can then serve as a template for Q ⁇ replicase synthesis of sense and antisense RNA.
  • This Q ⁇ replicase technique is described by Kwoh, et al .
  • the subject oligonucleotides can contain additional nucleotide sequences which encode an RNA polymerase recognition site, the 5 1 Q ⁇ replicase recognition site and the 3' Q ⁇ replicase recognition site as necessary to conduct Q ⁇ replicase RNA replication. Such sites are well known in the art and can readily be incorporated in the oligonucleotides of the present invention.
  • a suitable amount of each oligonucleotide for in vitro nucleic acid amplification to enable isolation of a eukaryotic LAGl RNA or DNA is about 0.01 ⁇ mole to about 5 ⁇ mole, and preferably about 0.05 ⁇ mole to about 1.0 ⁇ mole.
  • reagents as needed are added to the amplification reaction mixtures.
  • Such reagents include nucleotides, additional enzymes, a source of a high-energy phosphate (e.g. ATP) , and the like.
  • the target nucleic acid can be either DNA, RNA or both and depends on the in vitro nucleic acid amplification system selected. In many of these procedures DNA is the preferred template.
  • an amplification enzyme is any enzyme which can be used for in vitro nucleic acid amplification, e.g. by the above-described procedures.
  • amplification enzymes include Escherichia coli DNA polymerase I, Klenow fragment of - coli DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus DNA polymerase, Thermococcus litoralis DNA polymerase, SP6 RNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 polynucleotide kinase, Avian Myeloblastosis Virus reverse transcriptase, Moloney Murine Leukemia Virus reverse transcriptase, T4 DNA ligase, E. coli DNA ligase or Q ⁇ replicase.
  • oligonucleotides for the present amplification methods include oligonucleotides which can selectively hybridize to LAGl from the selected eukaryotic species, e.g. as observed by Southern or Northern analysis of nucleic acids from that species.
  • oligonucleotides are selected to be of sufficient length and complementarity to provide detectable hybridization to one to two bands on a Southern or Northern blot of that eukaryote's DNA or RNA.
  • oligonucleotides each have a nucleotide sequence with at least about 50% complementarity to either strand of SEQ ID NO:l.
  • Preferred oligonucleotides have at least about 70%, and more preferably 80%, sequence homology to SEQ ID NO:l. Sequence homology may be calculated by conventional algorithms including but not limited to BLAST (NCBI) , FAST (NCBI) , BEAUTY (Baylor College of Medicine, Texas) , and WEBB-MILLER (Penn State, College Station, Pennsylvania) .
  • oligonucleotides can have additional sequences which encode RNA polymerase recognition sites or Q ⁇ replicase recognition sites in the configuration necessary to practice in vitro nucleic acid amplification.
  • the length of the nucleic acids or oligonucleotides for use in isolating e.g. human LAGl nucleic acids depends on several factors including the nucleotide sequence and the temperature at which these nucleic acids are hybridized or used during in vitro nucleic acid amplification. The considerations necessary to determine a preferred length for a nucleic acid or oligonucleotide are well known to the skilled artisan (Sambrook, et al . ) . For example, as is known to the skilled artisan, the length of a short nucleic acid or oligonucleotide can relate to its hybridization selectivity. When a test sample contains complex mixtures of nucleic acids, e.g.
  • oligonucleotides which have less than about 14 nucleotides may hybridize to more than one site in the mammalian genome. These short oligonucleotides accordingly would not have sufficient hybridization selectivity for detecting a single target nucleic acid.
  • sequence of a nucleic acid which is at least about 14-15 nucleotides is generally represented only once in a mammalian genome (Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual, Vol. 2, Cold Spring Harbor Press, Cold Spring Harbor, NY; pp. 11.7-11.8). Accordingly, to eliminate cross hybridization with mammalian genomic DNA, the nucleic acid probes and oligonucleotides of the present invention are generally at least about 14 nucleotides long.
  • nucleic acids or oligonucleotides which are shorter than 14 nucleotides are specific for a given target. Therefore the term at least "about” is used to include any such nucleic acids and oligonucleotides which are less than 14 nucleotides long but which can specifically hybridize to a eukaryotic LAGl nucleic acid such as human LAGl .
  • the present nucleic acids and oligonucleotides are at least 10 nucleotides in length. More preferred nucleic acids and oligonucleotides are at least 17 nucleotides in length (Sambrook, et al . , pp. 11.7-11.8).
  • Nucleic acid probes of the present invention contain at least about 10 nucleotides to about 2500 nucleotides. Oligonucleotides of the present invention typically contain at least about 14 to about 150 or more nucleotides.
  • a further embodiment of the present invention includes isolated nucleic acids having an antisense nucleotide sequence of an RNA transcribed from any of the present nucleic acids. These antisense oligonucleotides are used to bind to LAGl mRNA, e.g. as described in Uhlmann, et al . (1990) Chemical Reviews 9.0:544-584.
  • the present antisense nucleic acids preferably have sufficient length and complementarity to hybridize to a non- template strand of one of the present isolated nucleic acids, e.g. SEQ ID NO:l.
  • the template strand is the DNA strand read by RNA polymerase; such a template strand is complementary to an RNA synthesized therefrom.
  • the non- template strand of the DNA has a sequence similar to the transcribed RNA.
  • the present antisense nucleic acids are at least about 50% complementary to the non- template strand of SEQ ID NO:l or SEQ ID NO: 3.
  • These antisense oligonucleotides preferably are at least about 14 to about 17 or more nucleotides in length, thereby permitting selective hybridization to a target nucleic acid.
  • Sense or antisense oligonucleotides and nucleic acids can be chemically synthesized by available synthetic procedures for nucleic acids. Chemical synthesis of nucleic acids is well known in the art and is achieved by solution or solid phase techniques. Moreover, oligonucleotides or nucleic acids of defined sequence are purchased commercially or are made by any of several different synthetic procedures including the phosphormidite, phosphite triester, H-phosphonate and phosphotriester methods, typically by automated synthesis methods. Modified bases can also be incorporated into the nucleic acid, e.g. inosine.
  • Enzymatic methods are also available for DNA, RNA or oligonucleotide synthesis.
  • DNA and oligodeoxyribonucleotide synthesis these methods frequently employ Klenow, T7 , T4 , Taq or E. coli DNA polymerases, e.g. as described in Sambrook, et al .
  • Enzymatic methods of RNA or oligoribonucleotide synthesis frequently employ SP6, T3 or T7 RNA polymerase as described, for example, in Sambrook, et al .
  • Reverse transcriptase can also be used to synthesize DNA from RNA.
  • nucleic acid or oligonucleotide enzymatically requires a template nucleic acid which can either be synthesized chemically, or be obtained as mRNA, genomic DNA, cloned genomic DNA, cloned cDNA or recombinant DNA.
  • a template nucleic acid which can either be synthesized chemically, or be obtained as mRNA, genomic DNA, cloned genomic DNA, cloned cDNA or recombinant DNA.
  • Some enzymatic methods of DNA or oligodeoxyribonucleotide synthesis can require a short primer oligonucleotide; this primer is obtained or synthesized by any available procedure.
  • nucleic acids and oligonucleotides are purified by polyacrylamide gel electrophoresis, or by any of a number of chromatographic methods, including gel, ion- exchange and high pressure liquid chromatography.
  • nucleic acids and oligonucleotides are subjected to DNA sequencing by available procedures, including Maxam and Gilbert sequencing, Sanger sequencing, capillary electrophoreses sequencing or by using selective chemical degradation of oligonucleotides bound to Hybond paper. Sequences of short oligonucleotides can also be analyzed by plasma desorption mass spectroscopy or by fast atom bombardment (McNeal, et al. (1982) J.
  • RNA oligonucleotides sequences for use as probes to detect a target LAGl nucleic acid.
  • Labeled probes have utility in diagnostic and analytical hybridization procedures for localizing, quantitating or detecting a target nucleic acid in tissues, chromosomes or in mixtures of nucleic acids .
  • Labeling of a nucleic acid or an oligonucleotide is accomplished by incorporating a "reporter molecule" into the subject nucleic acids and oligonucleotides by known procedures, e.g. as provided in Sambrook, et al . or Beaucage, et al . (1993) Tetrahedron 49.: 1925- 1963.
  • a "reporter molecule”, as defined herein, is a molecule or atom which, by its chemical nature, provides an identifiable signal allowing detection of the nucleic acid or the oligonucleotide. Detection is either qualitative or quantitative.
  • the present invention contemplates using any commonly used reporter molecule including, for example, radionuclides, enzymes, fluorophores, biotins, digoxigenin, chemiluminescent molecules, bioluminescent molecules, avidin, streptavidin, psoralens, chelated heavy metals, and luciferin.
  • the most commonly used reporter molecules are either enzymes, digoxigenin, radionuclides or fluorophores which are linked to nucleotides either before or after nucleic acid or oligonucleotide synthesis.
  • Commonly used enzymes include horseradish peroxidase, alkaline phosphatase, glucose oxidase and ⁇ -galactosidase, among others.
  • Enzymes can be conjugated to avidin or streptavidin for use with a biotinylated probe.
  • nucleic acid probes can be conjugated to avidin or streptavidin for use with a biotinylated enzyme.
  • the substrates to be used with the specific enzymes are generally chosen because a detectably colored product is formed by the enzyme acting upon the substrate.
  • p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for horseradish peroxidase, 1, 2 -phenyl nediamine, 5-aminosalicylic acid or toluidine are commonly used.
  • a digoxigenin reporter molecule is detected by binding an anti-digoxigenin antibody which has conjugated thereto a second reporter molecule, e.g. an enzyme.
  • the antibody- conjugated enzyme is then detected by application of a substrate for the enzyme.
  • Radionuclides are commonly used reporter molecules to form nucleic acid or oligonucleotide probes. Radionuclides can be incorporated into the present nucleic acids and oligonucleotides either during synthesis or by end- labeling after synthesis as described in Sambrook, et al . To incorporate radionuclides during nucleic acid synthesis radioactively labeled nucleotides are used, e.g. nucleotides with an ⁇ - 32 P moiety. Radionuclides can be incorporated after oligonucleotide synthesis by end labeling either the 3' or 5 ' end of the present nucleic acids and oligonucleotides.
  • Such end labeling can be done enzymatically, e.g. a 5 '-phosphate can be enzymatically removed with alkaline phosphatase and then replaced with a radioactively labeled 5'- phosphate, e.g. the ⁇ - 32 P from adenosine 5'-[ ⁇ - 32 P] triphosphate, using T4 polynucleotide kinase. Radioactively labeled nucleotide triphosphates are readily available commercially.
  • Fluorophores that are readily available and suitable for the methods of the present invention include fluorescein isothiocyanate (FITC) , rhodamine red and the like. Such fluorophores can be covalently linked to the present nucleic acids and oligonucleotide by readily available procedures, e.g. as provided in Beaucage, et al . (1993) Tetrahedron 49:1925-1963.
  • FITC fluorescein isothiocyanate
  • rhodamine red rhodamine red
  • nucleic acid and oligonucleotide probes linked to reporter molecules are used as herein described in the detection and isolation of a LAGl DNA or RNA from other eukaryotic species, e.g. from human, nematode, rat, bovine, mouse, galago, salmon and related eukaryotes.
  • these probes are also used to detect LAGl nucleic acids from a variety of eukaryotes by solution hybridization, Southern analysis, Northern analysis, in situ hybridization to tissue sections or chromosomal squashes and other analytical and diagnostic procedures. The methods of using such hybridization are well known. Some examples of such methodology are provided by Sambrook, et al.
  • the present invention provides isolated eukaryotic LAGl polypeptides which are encoded by any of the present LAGl nucleic acids identified herein.
  • the LAGl polypeptides provided herein include polypeptides having SEQ ID NO: 2 or SEQ ID NO: 4 and derivatives thereof.
  • the present isolated eukaryotic polypeptides have at least about 25% to 30% homology to SEQ ID NO: 2 or SEQ ID NO: 4.
  • LAGl polypeptides from eukaryotic species such as, for example, human, rat, bovine, murine, Podospora, nematode, galago, salmon and related eukaryotes whether prepared synthetically or recombinantly.
  • LAGl polypeptides are isolated by recombinant techniques readily available to the skilled artisan.
  • these polypeptides have at least about 25% to 30% homology to SEQ ID NO: 2 or SEQ ID NO: 4.
  • the present polypeptides include derivatives and fragments of the full length LAGl polypeptides of the present invention which confer the biological activity of the polypeptides of for example, SEQ ID NO: 2 or SEQ ID NO : 4.
  • fragments and derivatives as defined below include those polypeptides which increase the longevity, tolerance to stress and reproductive capacity of, for example, human, bovine, murine, Podospora, galago, salmon, yeast and other eukaryotic cells.
  • Derivatives of the present polypeptides include all modifications to the subject polypeptides, including, for example, substitutions, deletions and insertions where the modified polypeptide retains the biological activity of the unaltered polypeptide having for example, SEQ ID NO: 2 and SEQ ID NO: 4 with respect to increasing longevity, tolerance to stress or improving reproductive capacity in the narrow respective species of eukaryotes.
  • Fragments of the present polypeptides include peptides having at least about 8 contiguous amino acids which retain the biological activity of the full length polypeptide having for example, SEQ ID NO: 2 and SEQ ID NO: 4 with respect to increasing longevity tolerance to stress or comprising reproductive capacity in eukaryotic cells and organisms .
  • LAGl polypeptides are isolated from a variety of eukaryotic species, for example, by first isolating a nucleic acid encoding a LAGl polypeptide using the hybridization and in vitro amplification methods described hereinabove. These isolated LAGl nucleic acids are placed in an expression vector and the encoded polypeptides are expressed as described hereinbelow. These procedures are known to the skilled artisan and are readily adapted as needed to produce the desired eukaryotic isolated LAGl polypeptides. See, Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY and Goeddel, D.V. (Ed. 1990) Gene Expression Technology, Methods in Enzvmology 185, Academic Press; Perbal, B. (1988) A Practical Guide to Molecular Cloning, John Wiley & Sons, Inc.
  • LAGl polypeptides are identified by polyacrylamide gel electrophoresis or other procedures. These polypeptides are then isolated or purified by known procedures, e.g. detergent solubilization, salt precipitation, differential centrifugation, gradient centrifugation, column chromatographic procedures using gel filtration, ion exchange or reversed phase resins, and immunoaffinity or immunoprecipitation procedures.
  • the present invention provides methods for increasing the longevity, reproductive capacity, or tolerance to stress of a eukaryotic cell and/or organism, which includes administering to a cell or organism an effective amount of a LAGl polypeptide.
  • such polypeptides can have SEQ ID NO: 2 or SEQ ID NO: 4.
  • Another embodiment of the present invention is directed to a method for increasing the longevity or reproductive capacity of a eukaryotic cell which includes expressing a LAGl polypeptide in the cell.
  • a eukaryotic cell which includes expressing a LAGl polypeptide in the cell.
  • expression can increase the longevity of eukaryotic cells, particularly when the LAGl polypeptide is expressed late in the life of the target cell.
  • Such cells include mammalian such as for example, human, bovine, murine, and Podospora, galago and salmon.
  • such methods can also increase the tolerance of a eukaryotic cell to stress, e.g. starvation and acidic pH, where an acidic pH is about 5.0 to about 5.5.
  • expression of full length LAGl polypeptide in a eukaryotic cell is from the natural LAGl promoter endogenous to the cell.
  • LAGl is expressed from a natural promoter which has been mutated or modulated, e.g. by binding origins of replication, selectable markers, transcription or termination signals, centromeres, autonomous replication sequences, and the like.
  • an expression vector is a replicable or a non-replicable expression vector.
  • a replicable expression vector can replicate either independently of host cell chromosomal DNA or with the host cell chromosomal DNA by virtue of integration therein.
  • such an expression vector can lose some structural elements but retains the nucleic acid encoding the LAGl polypeptide and segment which can effect expression of the polypeptide. Therefore, the expression vectors of the present invention are chromosomally integrating or chromosomally nonintegrating expression vectors.
  • the present expression vectors can replicate in one host cell type, e.g., Escherichia coli, and undergo little or no replication in another host cell type, e.g., a eukaryotic host cell, so long as an expression vector permits expression of the present LAGl polypeptides in a selected host cell type.
  • Expression vectors as described herein include DNA or RNA molecules engineered for controlled expression of a desired gene product which include nucleic acid segments operably linked to nucleic acids encoding such gene products. Operably linked in this context means that such segments can effect expression of nucleic acids encoding the present gene products.
  • nucleic acid segments include promoters, enhancers, upstream control elements, transcription factors or repressor binding sites, termination signals and other elements which can control gene expression in the contemplated host cell.
  • the vectors are plasmids, bacteriophages, cosmids, artificial chromosomes or viruses.
  • Expression vectors of the present invention function in eukaryotic cells, including yeast and mammalian cells.
  • Mammalian vectors can include SV40 based vectors, polyoma based vectors, retrovirus based vectors, Epstein-Barr virus based vectors, papovavirus based vectors, bovine papilloma virus (BPV) vectors, vaccinia virus vectors, baculovirus insect vectors and the like.
  • Muzyczka (Ed. 1992) Curr. Top. Microbiol . Immunol . 158:97-129) provides a comprehensive review of eukaryotic expression vectors. Control of gene expression as used herein includes the ability to regulate expression both positively and negatively (i.e., turning gene expression on or off) to obtain the desired level of expression.
  • Elements or nucleic acid segments capable of effecting expression of a gene product include promoters, enhancer elements, upstream activating sequences, transcription termination signals and polyadenylation regulatory elements, singly or in combination, are contemplated for use in the present expression vectors. Moreover, genetically-engineered and mutated regulatory sequences are also contemplated herein.
  • Promoters are DNA sequence elements for controlling gene expression.
  • promoters specify transcription initiation sites and can include a TATA box and upstream promoter elements.
  • Higher eukaryotic promoters which are useful in the present expression vectors include promoters of viral origin, such as the baculovirus polyhedron promoter, the vaccinia virus hemagglutinin (HA) promoter, SV40 early and late promoter, the herpes simplex thymidine kinase promoter, the Rous sarcoma virus LTR, the Moloney Leukemia Virus LTR, and the Murine Sarcoma Virus (MSV) LTR.
  • the present invention contemplates cellular promoters which may be cell specific or developmentally regulated. Sambrook, et al. (1989) and Goeddel (1990) review higher eukaryote promoters .
  • Preferred promoters of the present invention include inducible promoters, i.e. promoters which direct transcription at an increased or decreased rate upon binding of a transcription factor.
  • Transcription factors as used herein include any factor that can bind to a regulatory or control region of a promoter and thereby affect transcription.
  • the synthesis or the promoter binding ability of a transcription factor within the host cell can be controlled by exposing the host to an inducer or removing an inducer from the host cell medium. Accordingly to regulate expression of an inducible promoter, an inducer is added or removed from the growth medium of the host cell.
  • inducers can include sugars, phosphate, alcohol, metal ions, hormones, heat, cold and the like.
  • commonly used inducers in yeast are glucose, galactose, and the like.
  • the expression vectors of the present invention can also encode selectable markers.
  • Selectable markers are genetic functions that confer an identifiable trait upon a host cell so that cells transformed with a vector carrying the selectable marker are distinguished from non- transformed cells. Inclusion of a selectable marker into a vector can also be used to ensure that genetic functions linked to the marker are retained in the host cell population.
  • Such selectable markers can confer any easily identified dominant trait, e.g. drug resistance, the ability to synthesize or metabolize cellular nutrients and the like.
  • Higher eukaryotic selectable markers can include genetic functions encoding an enzyme required for synthesis of a required nutrient, e.g.
  • tk thymidine kinase
  • DHFR dihydrofolate reductase
  • CAD uridine
  • ADA adenosine deaminase
  • AS asparagine synthetase
  • APH aminoglycoside phosphotransferase
  • hyg hygromycin B phosphotransferase confers resistance to hygromycin in higher eukaryotes.
  • Some of the foregoing selectable markers can also be used to amplify linked genetic functions by slowly adding the appropriate inhibitor for the enzyme encoded by marker such as DHFR, CAD, ADA, AS and others .
  • the present expression vectors can encode selectable markers which are useful for identifying and maintaining vector-containing host cells within a cell population present in culture. In some circumstances selectable markers can also be used to amplify the copy number of the expression vector.
  • RNA messenger RNA
  • mRNA messenger RNA
  • expression in mammalian cells generally does not require specific number of nucleotides between a ribosomal -binding site and an initiation codon, as is sometimes required in prokaryotic expression systems.
  • the first AUG codon in an mRNA is preferably the desired translational start codon.
  • nucleic acids encoding LAGl gene products generally include the natural ribosomal -binding site and initiation codon because, while the number of nucleotides between transcription and translational start sites can vary, such variability does not greatly affect the expression of the polypeptide in a mammalian host.
  • the first AUG codon in an mRNA is preferably the desired translational start codon.
  • factors which can effect the level of expression obtained include the copy number of a replicable expression vector.
  • the copy number of a vector is generally determined by the vector's origin of replication and any cis-acting control elements associated therewith.
  • the skilled artisan has available many choices of expression vectors.
  • commercially available higher eukaryotic expression vectors include pSVL, pMSG, pKSV-10, pSVN9 and the like.
  • expression vectors which include the above-described sequences by combining DNA fragments from available vectors, by synthesizing nucleic acids encoding such regulatory elements or by cloning and placing new regulatory elements into the present vectors. Methods for making expression vectors are well-known. Overexpression methods are found in any of the myriad of standard laboratory manuals on genetic engineering (Sambrook, et al . (1989); Goeddel (1990) and Romanos, et al . (1992).
  • Such vectors are transformed into host cells where the LAGl gene product is expressed.
  • Methods for transforming higher eukaryotic cells with expression vectors are well known and readily available to the skilled artisan.
  • mammalian host cells can be transformed with the present expression vectors by a variety of techniques including transfection, infection and other transformation procedures.
  • transformation procedures include calcium phosphate-mediated, DEAE-dextran-mediated or polybrene-mediated transformation, protoplast or liposomal fusion, electroporation, direct icroinjection into nuclei and the like. Such procedures are provided in Sambrook, et al . and the references cited therein.
  • Tissue culture cells that are used with eukaryotic expression vectors can include primary cells and cell lines as well as immortalized cell lines.
  • cell lines contemplated by the present invention include WI38 cells, IMR9 cells, VERO cells, MRC-5 cells, SCV-1 cells, COS-1 cells, CV-1 cells, LCC-MK 2 cells, NIH3T3 cells, CH0-K1 cells, mouse L cells, HeLa cells, Antheraea eucalypti moth ovarian cells, Aedes aegypti mosquito cells, S. cerevisiae cells, S. frugiperda cells and other cultured cell lines known to one skilled in the art.
  • host cells are also obtained from the American Type Culture Collection (12301 Parklawn Drive, Rockville, MD 20852, hereinafter ATCC) .
  • a further aspect of this invention provides pharmaceutical compositions containing the subject LAGl nucleic acids, oligonucleotides or polypeptides with a pharmaceutically acceptable carrier.
  • these nucleic acids, oligonucleotides and polypeptides are provided in a therapeutically effective amount of about 0.1 ⁇ g to about 100 mg per kg of body weight per day, and preferably of about 0.1 ⁇ g to about 10 mg per kg of body weight per day. Dosages are readily determined by one of ordinary skill in the art and formulated into the subject pharmaceutical compositions.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the subject nucleic acids, oligonucleotides and polypeptides may be administered topically or parenterally by, for example, osmotic pump, intravenous, intramuscular, intraperitoneal subcutaneous or intradermal route.
  • the present nucleic acids, oligonucleotides and polypeptides can be orally administered.
  • topical administration is contemplated the subject nucleic acids, oligonucleotides and polypeptides may be incorporated into a cream, solution or suspension.
  • nucleic acids, oligonucleotides and polypeptides may be protected by enclosure in a gelatin capsule.
  • Nucleic acids, oligonucleotides and polypeptides may be incorporated into liposomes or liposomes modified with polyethylene glycol for parenteral administration. Incorporation of additional substances into the liposome, for example, antibodies reactive against membrane proteins found on specific target cells, can help target LAGl nucleic acids, oligonucleotides and polypeptides to specific cell types.
  • the present invention contemplates administering the subject nucleic acids, oligonucleotides and polypeptides with an osmotic pump providing continuous infusion, for example, as described in Ratajczak, et al . (1992) Proc. Natl. Acad. Sci. USA 89 . : 11823 - 11827) .
  • osmotic pumps are commercially available, e.g., from Alzet, Inc. (Palo Alto, CA) .
  • Parenteral administration e.g. in a liposomal carrier, is preferred.
  • the present invention contemplates transgenic ("knockout” and “knockin”) animals comprising germ cells and somatic cells which contain the LAGlHs gene.
  • the transgenic animals of the present invention may be conventionally designed and prepared using established techniques. See e.g. Hogan, et al . Manipulating the Mouse Embryo, A Laboratory Manual : Cold Spring Harbor Laboratory Press (1994) Second Edition, pp. 275-281, incorporated herein by reference.
  • the animal is preferably a rodent.
  • Preferred rodents include rats and mice.
  • a preferred transgenic mouse comprises germ cells containing a LAGlHs gene which is operably linked to a promoter effective for overexpression of LAGlHs .
  • Such mice are conventionally known as "knockin" transgenic mice.
  • the LAGlHs gene is preferably introduced into the mouse at an embryonic stage.
  • a preferred embryonic stage is the one- cell or fertilized oocyte stage.
  • Introduction of the recombinant gene at the fertilized oocyte stage ensures that the LAGlHs gene sequence will be present in all of the germ cells and somatic cells of the transgenic "founder” animal.
  • a transgenic "founder” animal is herein defined as the animal into which the LAGlHs gene is introduced at the one cell mouse embryo stage.
  • the animals of the invention can advantageously be used as a source of cells for cell culture.
  • Cells from the animals may exhibit desired properties of both normal and transformed cultured cells, i.e. the cells will be normal or nearly normal morphologically and physiologically, but can, like N1H3T3 cells, for example, be cultured for extended periods of time.
  • a yeast homologue of the Saccharomvces cerevisiae longevity- assurance gene LAGl was identified in the GenBank database.
  • the product of this gene displayed 70% identity in amino acid sequence to the yeast LAGl protein. Deletion of this homologue in yeast did not affect viability, but it resulted in an increase in the replicative life span of the yeast. Similarly, deletion of LAGl itself increased the replicative life span of the yeast as well.
  • the yeast homologue was named LACl .
  • the availability of both the LAGl and the LACl sequences facilitated searches in the GenBank database. These searches revealed three homologous genes, two in Caenorhabditis elegans and one in human. The C. elegans genes were deposited in the database by the C.
  • ORFs putative open reading frames
  • the human sequence was a potential ORF identified on a bicistronic mRNA upstream of GDFl in a cDNA clone from human brain. This ORF was designated UOG1, indicating that this sequence was upstream of GDFl.
  • a 52 amino acid stretch was present in all the proteins. The 52 amino acid stretch showed a sequence identity of 28% and overall similarity plus identity of 66% in a four way comparison. This 52 amino acid stretch was named the "LAGl motif".
  • the similarities indicated in the sequence comparison in Figure 1 were likely based on evolutionary conservation of protein sequences.
  • LAGlSc, LAGlCel , LAClSc and LAGlHs are membrane proteins.
  • a transmembrane prediction for each of these proteins is shown in Figure 2. Although the plots are not identical in all their details, all of these proteins showed substantial overall similarity in their respective transmembrane domain profiles. These similarities indicated that all of these proteins are homologous .
  • a C. elegans homologue LAGlCel was cloned.
  • RT-PCR was performed using RNA from C. elegans (strain N2) males.
  • the primers were designed using a predicted exon sequence from the GenBank database. They were: 5 ' -AGTCGGAACCTTGATTCTTC- 3 ' (SEQ ID NO: 8) and 5 ' -CCCAAAGTATGTAATC- 3 ' (SEQ ID NO: 9).
  • This RT-PCR produced a predicted 244 bp DNA fragment ( Figure 3) . This fragment was sequenced to confirm its identity (data not shown) . The 244 bp fragment was labeled and used to screen a C.
  • the human homologue, LAGlHs was cloned by RT-PCR of total RNA extracted from human fetal brain.
  • the primers were designed on the basis of the cDNA sequence in the database to amplify the entire ORF. They were: 5 ' -GCGGGCGAGCGGGCGGTATG- 3 ' (SEQ ID NO: 10) and 5 ' -CCGAGGGGTTCAGAAGCGCTTGT- 3 ' (SEQ ID NO: 11).
  • This RT-PCR reaction routinely resulted in the amplification of a product missing 235 bp, as determined by DNA sequencing. This was judged to be the result of the high GC content (80% GC) that resulted in the generation of a stem- loop structure.
  • a full length cDNA was amplified when GC Melt in the Clontech Advantage GC Klen Taq polymerase kit was used to eliminate secondary structure in the RNA. This full length cDNA was cloned and sequenced. Several clones were obtained in this way. All clones showed the same sequence which agreed with that found in the database.
  • LAGlCel and LAGlHs were each cloned into the plasmid pBM150 behind the GAL1 promoter.
  • This vector contained the ARS1 -CEN4 sequences for stable maintenance in low copy number in yeast and the selectable yeast marker URA3.
  • the yeast strain was transformed with the two clones to generate a strain containing LAGlCel and one containing LAGlHs .
  • the strain was transformed with pBM150 alone or with pBM150 containing LAGlHs in the antisense orientation alone. Expression of the cloned genes was induced by adding 2% galactose to the growth medium. This expression was verified by determining mRNA levels on Northern blots ( Figure 5) .
  • LAGlCel complements the deletion of LAGl (and LACl)
  • YPK4.7 diploid yeast strain that possessed a deletion in one copy of the LAGl gene and one copy of LACl gene was generated from YPK4.7. These deletions were marked by TRPl and LEU2 , in the case of LAGl and LACl , respectively.
  • TRPl and LEU2 in the case of LAGl and LACl , respectively.
  • both copies of LEU2 and TRPl were mutated at their normal locus.
  • YPK4.7 was ura3 " .
  • the presence of the LAGl deletion could be identified by growth in the absence of tryptophan, while the presence of the LACl deletion could be identified by growth in the absence of leucine.
  • LAGlCel and LAGlHs were each subcloned into the plasmid pBM150 behind the GAL1 promoter.
  • This vector contained the ARS1-CEN4 sequence for stable maintenance in low copy number in yeast and the selectable yeast marker URA3.
  • the yeast strain described above was transformed with each of these plasmids individually to generate one strain containing LAGlCel and another strain containing LAGlHs .
  • YPK4.7 was transformed with pBM150 alone or with pBM150 containing LAGlHs in the antisense orientation.
  • the transformants were grown on ura “ , trp " and leu " medium for 16 hours, then streaked on sporulation plates (carbon source 1% potassium acetate). After 5-6 days, a sample was removed from the plates and examined microscopically to identify the formation of tetrads (asci) containing four spores.
  • the tetrads were treated with 1% glusulase (NEN Research products) for 8-10 minutes to digest the ascus wall. 10 ⁇ l of digestion mixture were spotted in the surface of a ura- plate containing 2% galactose and 1% raffinose as carbon sources and allowed to form a stream across the plate. The plate was then allowed to stand 10 minutes to absorb excess liquid. Four spores from each tetrad were dissected and spotted on a line by microdissection. The spores containing PBM150-LAG1 homologue plasmids were grown to form colonies on the ura- plates for 5 days. Fully grown tetrad colonies (i.e.
  • the genotypes of the haploids that were leu + , trp + and ura + were verified for the absence of the LAGlSc and LAClSc genes. This was carried out by PCR amplification from genomic DNA. For transformants containing LAGlCel , primer pairs to detect the presence of LAGlSc, LAClSc and ASF2, a positive control, were used ( Figure 7A) . For transformants containing LAGlHs , the same primers (identified in Example 2) were used, and the presence of LAGlHs was also verified by the PCR procedure ( Figure 7B) . In all cases, the transformants were deleted for both LAGlSc and LAClSc confirming that growth was due to complementation by LAGlCel or LAGlHs , respectively.
  • the diploid transformants, used to obtain the haploids were also spread on these plates. Loss of the plasmid in the diploids allowed some of the colonies to grow. In each case, growth was obtained on medium lacking uracil and FOA.
  • LAGlHs A different version of the same test was carried out for LAGlHs .
  • This gene was cloned into plasmid pCT263 behind the MET3 promoter, allowing induction of expression in medium lacking methionine.
  • This plasmid had the HIS3 selectable marker.
  • the plasmid construct was transformed into a haploid lacking LAGl and LACl and maintained viable through the expression of LAGl from its own promoter on the low copy vector YCp50.
  • the empty pCT263 vector served as a control. Transformants were selected on medium lacking histidine, tryptophan and leucine.
  • LAGlHs The tissue expression profile of LAGlHs was examined by hybridizing multitissue Northern blots using the LAGlHs gene as a probe ( Figure 9) .
  • Fluorescent in situ hybridization was used to define the chromosomal location of LAGlHs.
  • Human peripheral blood lymphocytes were arrested in metaphase using TC Chromosome Microtest kit (Difco Laboratories, Detroit, MI, USA) .
  • the cells were exposed to 0.075 M KC1 for 20 minutes at 37°C, were fixed three times in 3 : 1 methanol : acetic acid at -20°C for 30 minutes, and dropped onto microscope slides.
  • DNA from BAC clone 421113 (Research Genetics, Huntsville, Alabama) containing human DNA was labeled with biotin-16-dUTP (Boehringer Mannheim, Indianapolis, IN) using a Large Fragment DNA Labeling kit (Oncor, Inc., Gaithersburg, MD, USA) as instructed by the manufacturer using a 45 minute incubation time.
  • Biotin- labeled 142N3 DNA 300 ng was combined with digoxigenin- labeled 19ql3.1- specific DNA (Oncor), and blocked with 60 ⁇ g of human Cot-1 DNA (Boehringer Mannheim) .
  • the cytogenetic mapping of LAGlHs was performed using fluorescent in situ hybridization (FISH) .
  • FISH fluorescent in situ hybridization
  • BAC 421113 DNA was labeled with biotin as described above and detected with FITC-avidin (light blue signal).
  • a probe specific for 19ql3.1 (red signal) was used to identify the long arm of chromosome 19.
  • DAPI counterstaining revealed the outlines of the individual chromosomes (dark blue signal) . Both homologues of chromosome 19 were labeled in each case.
  • the LAGlHs gene mapped to 19pl2. The same results were obtained with the second BAC clone 157D6 (See Figure 10) .
  • Bacterial Artificial Chromosome 157D6 containing a nucleic acid encoding the LAGlHs genomic sequence was deposited with the American Type Culture Collection (10801 University Boulevard., Manassas, VA 20110) on April 27, 1999. This BAC clone 157D6 was provided ATCC No. .
  • LAGlHs The genomic structure of LAGlHs (SEQ ID NO: 12) was identified by sequencing the relevant region of human BAC clone 157 D6 (Research Genetics, Huntsville, Alabama) . Restriction fragments from BAC clone DNA were conventionally subcloned. Restriction enzymes EcoRI, Hindlll, BamHI were employed to digest BAC DNA. Southern Blots of the fragments that reacted with a cDNA probe were cloned into vector pUC119. Several regions of LAGlHs cDNA were used as probes to hybridize to positive colonies from among the fragments cloned. Identified fragments covered the entire region of LAGlHs including an upstream 4 kb promoter region. Primers for sequencing were designed according to cDNA sequence. Primer walking was employed to sequence the LAGlHs genome.
  • LAGlHs cDNA regions and boundaries between exons and introns were sequenced.
  • the LAGlHs genomic sequence is present in SEQ ID NO: 12.
  • LLNL Genome Center unanalyzed raw data

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Abstract

La présente invention se rapporte à des acides nucléiques de LAG1 d'origine humaine ou issus de nématodes et aux polypeptides codés et exprimés par ces acides nucléiques, ainsi qu'aux vecteurs et cellules hôtes associés. La présente invention concerne de l'ADN et de l'ARN antisens. Elle se rapporte également à des procédés permettant d'accroître la longévité et la tolérance au stress de cellules eucaryotes et consistant à fournir à ces cellules des polypeptides de LAG1 d'origine humaine ou issus de nématodes. Les procédés permettant de fournir à une cellule des polypeptides de LAG1 consistent à surexprimer le gène LAG1 endogène, ou à introduire dans la cellule un vecteur d'expression contenant la région appropriée de codage de LAG1. Il est possible de mettre en oeuvre ces procédés sur des cellules mammaliennes, et notamment sur des cellules humaines. La présente invention se rapporte également à des compositions pharmaceutiques permettant la mise en oeuvre de ces procédés.
PCT/US1999/010160 1998-05-08 1999-05-10 Homologues du gene d'assurance de longevite des levures, lag1, d'origine humaine ou issus de nematodes WO1999058671A2 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992000382A1 (fr) * 1990-06-15 1992-01-09 Carnegie Institution Of Washington Gdf-1
WO1995033834A1 (fr) * 1994-06-03 1995-12-14 Research Corporation Technologies, Inc. Lag1: un gene pour augmenter la longevite des cellules eucaryotiques
WO1998005761A1 (fr) * 1996-08-07 1998-02-12 The General Hospital Corporation Polypeptides age-1 et molecules et methodes associees
WO1998017823A1 (fr) * 1996-10-21 1998-04-30 Mcgill University CONSERVATION STRUCTURELLE ET FONCTIONNELLE DU GENE D'HORLOGE clk-1 CHEZ $i(C. ELEGANS)
WO1999006558A1 (fr) * 1997-07-30 1999-02-11 Incyte Pharmaceuticals, Inc. Homologues proteiques d'assurance de longevite humaine

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Publication number Priority date Publication date Assignee Title
WO1992000382A1 (fr) * 1990-06-15 1992-01-09 Carnegie Institution Of Washington Gdf-1
WO1995033834A1 (fr) * 1994-06-03 1995-12-14 Research Corporation Technologies, Inc. Lag1: un gene pour augmenter la longevite des cellules eucaryotiques
WO1998005761A1 (fr) * 1996-08-07 1998-02-12 The General Hospital Corporation Polypeptides age-1 et molecules et methodes associees
WO1998017823A1 (fr) * 1996-10-21 1998-04-30 Mcgill University CONSERVATION STRUCTURELLE ET FONCTIONNELLE DU GENE D'HORLOGE clk-1 CHEZ $i(C. ELEGANS)
WO1999006558A1 (fr) * 1997-07-30 1999-02-11 Incyte Pharmaceuticals, Inc. Homologues proteiques d'assurance de longevite humaine

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Title
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HEKIMI S ET AL: "Molecular genetics of life span in C. elegans: how much does it teach us?" TRENDS IN GENETICS,NL,ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, vol. 14, no. 1, January 1998 (1998-01), page 14-20 XP004101479 ISSN: 0168-9525 *
HODES R ET AL: "Longevity assurance genes: how do they influence aging and life span?" JOURNAL OF THE AMERICAN GERIATRICS SOCIETY,US,ELSEVIER SCIENCE PUBLISHING, NEW YORK, NY, vol. 44, no. 8, 1996, page 988-991 XP002084173 ISSN: 0002-8614 *
JIANG, JAMES C. ET AL: "Homologs of the yeast longevity gene LAG1 in Caenorhabditis elegans and human" GENOME RES. (1998), 8(12), 1259-1272 , XP002127187 *
KIRCHMAN, PAUL A. ET AL: "LAG1 and LAC1 are homologous genes involved in determining the replicative life span of the budding yeast, Saccharomyces cerevisiae." FASEB JOURNAL, (1996) VOL. 10, NO. 6, PP. A1517. MEETING INFO.: JOINT MEETING OF THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, THE AMERICAN SOCIETY FOR INVESTIGATIVE PATHOLOGY AND THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS NEW ORLEANS, , XP002127186 *
LEE S -J: "EXPRESSION OF GROWTH/DIFFERENTIATION FACTOR 1 IN THE NERVOUS SYSTEM: CONSERVATION OF A BICISTRONIC STRUCTURE" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA,US,NATIONAL ACADEMY OF SCIENCE. WASHINGTON, vol. 88, no. 10, 1991, page 4520-4524 XP000561931 ISSN: 0027-8424 *
WILSON R. ET AL.: "AC Q17870" EMBL DATABASE, 1 November 1996 (1996-11-01), XP002127184 Heidelberg *

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