EP0871722A1 - Post-transkriptionelle genregulation durch spurenelemente - Google Patents

Post-transkriptionelle genregulation durch spurenelemente

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Publication number
EP0871722A1
EP0871722A1 EP94919139A EP94919139A EP0871722A1 EP 0871722 A1 EP0871722 A1 EP 0871722A1 EP 94919139 A EP94919139 A EP 94919139A EP 94919139 A EP94919139 A EP 94919139A EP 0871722 A1 EP0871722 A1 EP 0871722A1
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European Patent Office
Prior art keywords
nucleic acid
cell
stem
loop
codon
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EP94919139A
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English (en)
French (fr)
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EP0871722A4 (de
Inventor
Jack L. Leonard
Peter E. Newburger
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University of Massachusetts Medical Center
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University of Massachusetts Medical Center
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Publication of EP0871722A4 publication Critical patent/EP0871722A4/xx
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the invention relates to post-transcriptional control of heterologous gene expression.
  • One of the major goals of the biotechnology industry is to stably transfect genes encoding proteins of medical and commercial value into cellular and animal systems under conditions which allow control of the expression of the transfected gene.
  • the methods most widely used involve controlling gene expression at the transcriptional level by placing the gene of interest under the control of an inducible promoter.
  • inducible promoters allow the production of significant background levels of gene expression under non-inducing conditions, thus limiting the usefulness of these methods only to applications where low levels of the transfected gene product do not have a significant effect on the cell.
  • most inducible promoters are capable of producing only a limited increase in gene expression (usually about 3- fold) under inducing conditions.
  • the invention features a method of controlling the production of a heterologous polypeptide in a eukaryotic cell which includes the steps of (a) providing a cell containing a first nucleic acid encoding the heterologous polypeptide wherein at least one codon of the mRNA transcribed from the first nucleic acid has been replaced by the codon UGA, operably linked to a second nucleic acid which is capable of directing the translation of the UGA codon as selenocysteine; and (b) growing the cell under conditions wherein the production of the heterologous polypeptide is controlled by the level of selenium available to the cell.
  • the method of the invention may be carried out in vitro in any eukaryotic cell type which is capable of being maintained in cell culture.
  • the cell is a eukaryotic cell such as a mammalian tissue culture cell, (e.g., COS-1, HL-60, CV-1, C-6, LLC/PK-1, 3T3L1 or CHO cells) or a yeast cell, e.g., Saccharomyces cerevisiae .
  • the cells used do not contain a native protein which is substantially homologous to the recombinant polypeptide.
  • the recombinant polypeptide may be distinguished from the native protein by the increased reactivity of the recombinant polypeptide to nucleophilic reagents due to the presence of the selenocysteine residue, or alternatively, by radiolabeling with the radioisotope 75 Se.
  • the first and second nucleic acids may be introduced into and maintained in the cell in a recombinant vector which is capable of autonomously replicating in the cell, or stably integrated into the genome of the cell according to standard techniques.
  • the production of the heterologous polypeptide is controlled by the amount of the trace mineral, selenium, in the medium in which the cells are cultured.
  • the cells are maintained in a medium which is substantially deficient in available selenium, i.e., the concentration of selenium in the medium is less than 1 ng/ml and preferably less than 0.1 ng/ml.
  • the cell culture medium typically contains between 1 and 50 ng/ml, preferably 2 to 40 ng/ml, and most preferably 5 to 25 ng/ml.
  • the method of the invention may be carried out in vivo by stably incorporating the first and second nucleic acids into the genome of an embryonal cell derived from a non-human mammal, and obtaining transgenic progeny of the non-human mammal.
  • Transgenic as used herein means a mammal which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the animal which develops from that cell. Such a transgene may be partly or entirely heterologous to the transgenic animal.
  • Embryonal cells as used herein include embryonic stem (ES) cell and fertilized oocytes.
  • ES embryonic stem
  • the preferred method of transgene introduction is by microinjection, whereas for ES cells, the preferred method is electroporation.
  • other methods including viral delivery systems such as retroviral infection, or liposomal fusion can be used.
  • the cell After introduction of the transgene into an embryonal cell, the cell is introduced into pseudo-pregnant females and progeny is obtained which is heterozygous for the transgene.
  • a stable line of heterozygous animals may then be maintained by appropriate backcrossing to the original animal line, or the heterozygous progeny may be mated to obtain homozygous animals.
  • the transgenic animals When it is desirable to inhibit the expression of the heterologous polypeptide, the transgenic animals are maintained on a diet containing less than 0.02 mg/kg of food, and induction of expression is triggered by supplementing the diet with selenium to a concentration 0.1 mg/kg or higher.
  • the polypeptide encoded by the first nucleic acid may be any desired polypeptide for which the nucleotide sequence is known. Methods of modifying the polypeptide to incorporate a selenocysteine amino acid residue are well known and described herein. Preferably, the selenocysteine residue is substituted for an amino acid at a position in the polypeptide which does not abolish the normal biological activity of the naturally occurring protein, e.g., a nonessential amino acid.
  • Such amino acids may be identified by means well known to those skilled in the art and will usually occur at positions which are not involved in the catalytic or binding activity of the protein (as determined for example by mutational analysis) , or at positions which are considered critical for the structural integrity of the polypeptide (e.g., as predicted by computer analysis or crystallography) . Most often, the selenocysteine will be inserted at a position in the polypeptide which normally carries a cysteine residue.
  • the second nucleic acid includes a contiguous sequence of nucleotides capable of forming a stem-loop secondary structure in the mRNA transcribed from the second nucleic acid, wherein the stem-loop formed by the mRNA is capable of directing the translation of said UGA codon as selenocysteine.
  • the second nucleic acid is derived from approximately 90 contiguous nucleotides from the 3' untranslated region of a gene encoding a naturally occurring mammalian selenoprotein.
  • the second nucleic acid comprises a nucleotide sequence substantially homologous to nucleotides 654 to 740 of the human selenoprotein, glutathione peroxidase, shown in Figure 8.
  • the second nucleic acid is synthetically derived, and is capable of forming a stem-loop containing the sequence 5'-
  • NAAAUNNUAAAN-3' in the loop at the apex of the stem-loop contains at least 12, and preferably at least 14, non-complementary nucleotides which form a bubble containing the sequence 5'-NUAGUN-3' symmetrically opposed on each half of the bubble; preferably, the stem- loop contains approximately 90 nucleotides, the bubble is placed approximately 17 nucleotides from the base of the stem, and the loop is placed approximately 11 nucleotides from the bubble at the apex of the stem-loop structure.
  • the invention also features a single- stranded nucleic acid containing a contiguous stretch of nucleotides capable of forming a stem-loop secondary structure, wherein the loop of the stem-loop the sequence 5 , -NAAAUNNUAAAN-3 , , and the stem contains at least 12, and preferably at least 14, non-complementary nucleotides which form a bubble containing the sequence S-'NUAGUN-S' symmetrically opposed on each half of the bubble.
  • the nucleic acid is capable of directing the translation of the codon, UGA, as selenocysteine when the nucleic acid is operably linked to an mRNA molecule which contains a UGA codon.
  • the nucleic acid of the stem-loop contains approximately 90 nucleotides
  • the bubble is placed approximately 17 nucleotides from the base of the stem and the loop is placed approximately 11 nucleotides from the bubble at the apex of the stem-loop structure.
  • each half of the bubble is approximately 7 nucleotides and the loop contains approximately 12 nucleotides.
  • a double-stranded nucleic acid which contains DNA encoding the single-stranded nucleic acid of the invention.
  • heterologous nucleic acid is meant a nucleic acid which is partly or entirely foreign to the cell or animal in which it is introduced, or a nucleic acid which is homologous to an endogenous gene of the cell or animal with the exception that the heterologous protein contains selenocysteine substituted at least one amino acid.
  • operably linked is meant that the contiguous stretch of nucleotides which form the stem-loop secondary structure is in sufficient proximity with the nucleic acid encoding the protein to allow translation of any UGA codon in the protein to be translated as selenocysteine.
  • the stem-loop is inserted in the 3' untranslated region of the mRNA molecule encoding the polypeptide; preferably within 2000 nucleotides of the UGA codon, more preferably within 400 to 1500 nucleotides, and most preferably within 500 to 1200 nucleotides.
  • functionally active is meant possessing any in vivo or in vitro activity which is characteristic of the naturally occurring protein.
  • homologous refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same nucleotide base or amino acid subunit, then the molecules are homologous as that position. Thus, by “substantially homologous” is meant a nucleotide or amino acid sequence that is largely but not wholly homologous.
  • heterologous nucleic acid is meant a nucleic acid which is partly or entirely foreign to the animal in which it is transfected, or a nucleic acid which is homologous to an endogenous gene of the transgenic animal, but which is inserted into the animal's genome at a location which differs from that of the natural gene.
  • the methods of the present invention provide several advantages over currently used methods of gene expression.
  • a transfected gene product which contains SeCys can be readily distinguished from native cellular proteins via its heightened reactivity toward nucleophilic reagents, or by 75 Se incorporation.
  • FIG. 1A is a schematic diagram of the human cellular glutathione peroxidase cDNA constructs.
  • the open reading frame (ORF) and 3'UTR are indicated by a wide bar; plasmid elements and 5'UTR are indicated by flanking lines. Nucleotide numbering starts at the beginning of the open reading frame; the ATG initiation codon is at nt 1-3, the TGA selenocysteine codon is at nt 142-144, and the TAG termination codon is at nt 607-609. Arrows indicate the positions of restriction endonuclease sites. Lines below the diagram represent the positions of the indicated deletions. The hatched bar below the diagram shows the position at which the epitope tagging sequence was inserted, and the region of cDNA replaced.
  • Figure IB is a schematic diagram of the potential secondary structure immediately downstream of the UGA 142 selenocysteine codon in the coding region of the human Gpx mRNA, and diagrams the positions of deletions ORF-D1 , ORF-D2, ORF-D3, and ORF-D4.
  • Figure 1C is a schematic diagram of an alternative potential secondary structure in the coding region of the human Gpx mRNA wherein the UGA 142 selenocysteine codon is within a hairpin structure.
  • the deletion ORF-D5 is also indicated.
  • Figure 2A is an autoradiograph of an SDS- polyacrylamide gel of immunoprecipitated 75 Se-labelled COS-1 cell extracts after transfection with pCMV4 (lane
  • Figure 2B is an autoradiograph of an SDS- polyacrylamide gel of immunoprecipitated 75 Se-labelled COS-1 cell extracts after transfection with pCMV4 vector
  • Figure 3 is an autoradiograph of an SDS- polyacrylamide gel of immunoprecipitated 75 Se-labelled COs-1 cell extracts after transfection with pCMV4 vector
  • Figure 4 is an autoradiograph of an SDS- polyacryla ide gel of immunoprecipitated 75 Se-labelled COS-1 cell extracts after transfection with pCMV4 vector
  • UTR-D4 (lane 3) , or deletion mutant UTR-D5 (lane 4) .
  • Figure 5 is a schematic diagram of the potential secondary structure of the 3'UTR of human Gpx mRNA.
  • Figure 6 is an autoradiograph of a polyacrylamide gel of the products of an RNase protection assay using a labeled riboprobe. Lane 1, undigested probe; lane 2, probe hybridized with RNA from untransfected COS-1 cells; lane 3, probe hybridized with epitope-tagged GPx COS-1 transfectants; lane 4, probe hybridized with UTR-D4 COS-1 transfectants; lane 5, probe hybridized with UTR-D5 COS-1 transfectants.
  • Figure 7 is an autoradiograph of an SDS- polyacrylamide gel of immunoprecipitated 35 S-labeled (lanes 1-4) and 75 -Se-labeled (lanes 5-8) COS-1 cells transfected with rab ⁇ b opal mutants and fusion constructs. Lanes 1 and 5, pCMV4 vector; lanes 2 and 6, rab5b(opal)GPx3'UTR; lanes 3 and 7, rab ⁇ b(opal) ; lanes 4 and 8, rab5b(wt)GPx3'UTR.
  • Figure 8 depicts the nucleotide sequence of the human glutathione peroxidase gene including the 3 , UTR.
  • Figure 9 depicts the sequence and secondary structure of the "optimized" selenocysteine insertion sequence (SECIS) .
  • eukaryotic and prokaryotic proteins including bacterial formate dehydrogenases, the mammalian glutathione peroxidase (GPx) family (Mullenbach et al.. Nucleic Acids ites.15;5484. 1987; Chambus et al., EMBO J. 5:1221, 1986; Esworthy et al.. Arch . Biochem . Biophy ⁇ . 286:330, 1991; Takahashi et al.. Blood 68: 640, 1986) , type I iodothyronine 5'deiodinase (Berry et al.
  • GPx mammalian glutathione peroxidase
  • selenoprotein P (Read et al. (1990) J. Biol . Chem . 265, 17899-17905), belong to a unique group polypeptides which contain the unusual amino acid selenocysteine.
  • the production of selenoproteins has been reported to be strictly regulated by the level of exogenous selenium. For example. Knight et al. (J. Nutr. 117:732, 1987) reported that glutathione peroxidase activity decreased to undetectable levels in rates given a selenium deficient diet ( ⁇ 0.02 ppm, 0.016mg/kg). Chanoine et al. (Endocrinology 131:1787.
  • Genomics 6, 268-271 instead of the CAG observed by Mullenbach et al. (Mullenbach et al. (1987) Nucleic Acids Res . 15, 5484) (GenBank accession numbers Y00369 and M21304) .
  • the former insertion is a polymorphism we have observed in other normal GPX1 sequences.
  • GPx deletion subclones were constructed by overlap extension polymerase chain reaction (PCR) according to standard methods (Ho et al. (1989) Gene 77, 51-59), using a Perkin-Elmer Cetus thermal cycler and reagents. This PCR method required two flanking primers defining the size of the final product and two mutually complementary primers directing the desired mutation in the target sequence. The sequences of the flanking primers and of one of each pair of complementary mutagenesis primers are listed in Table 1. The final PCR products were inserted back into pBluescript KS, and the sequences were confirmed by standard methods.
  • PCR overlap extension polymerase chain reaction
  • each mutant GPx sequence was subcloned into the eukaryotic expression vector pCMV4 (Andersson et al. (1989) J. Biol . Chem . 264, 8222-8229) for transfection into COS-1 cells as described below.
  • Epitope tagging of GPx was performed (as diagrammed in figure 1) by replacing the first 12 nucleotides (nt) of the open reading frame of GPx with a 30 nt sequence encoding an ATG start codon followed by 27 bases encoding a nine amino acid epitope of human influenza hemagglutinin protein (Chada et al. (1989) Blood 74, 2535-2541).
  • the two oligonucleotides listed in Table 1 were annealed, then the resulting short double- stranded fragment was inserted into GPx wild-type or mutant subclones in pBluescript KS and/or pCMV4 via the Clal and Nhel restriction sites.
  • subclone UTR-D3 in which the entire GPx 3'UTR was deleted, was constructed by excision of a 250 nt Avrll-Spel fragment from the epitope-tagged GPx subclone GPxEPI in pBluescript KS, followed by religation of the remaining large fragment.
  • Subclone UTR-D2 was constructed by excision of the Avrll-X ol fragment followed by religation of the remaining large fragment in the GPx 3'UTR sequence from GPxEPI-containing pBluexcript KS with the plasmid Xho ⁇ site eliminated.
  • the subclone UTR-D1 was obtained by inserting a GPxEPI containing fragment with a sticky Clal end and a end-filled Xhol end, excised from the construct GPxEPI in pBluescript KS, into the expression vector pCMV4 via the Clal and Smal polylinker restriction sites.
  • the overlap extension PCT method was also used to construct mutant and fusion subclones of the rab5b gene, which encodes a member of Ras-related GTPase superfamily (Wilson et al. (1992) J. Clin . Invest . 89, 996-1005).
  • the plasmid pMT2, carrying a 1.6 Kb rab ⁇ b cDNA clone, was obtained from D.B.
  • rab5b(opal)GPx3'UTR contained a fusion product of the rab5b coding region with an opal (UGA) mutation at codon 63, fused with the GPx 3'UTR sequence.
  • the oligonucleotide sequence of the flanking and mutagenesis primers are listed in Table 1.
  • the 3'PCR flanking primer sequence resulted in the removal of the native rabSb TGA termination codon, and substitution of the last 3 codons of the GPx open reading frame, including its TAG stop codon.
  • the resultant rab ⁇ b(opal) mutant was inserted into a pBluescript KS construct containing the entire GPx 3'UTR sequence derived from the Clal-Avrll double digestion of the native GPxR clone in pBluescript KS.
  • the gene fusion product was then subcloned into pCMV4 as described above.
  • the same strategy was also used to construct rab5b(WT)GPx3'UTR except, in this case, conventional PCR was applied using only the flanking primers, and the fusion product (WT, i.e. wild type without the opal mutation) was inserted into pCMV4.
  • rab ⁇ b(opal) which contains the coding region opal mutation but the native rab5b 3'UTR, was constructed by fusion of the approximately 900 nt N el-E ⁇ oRI fragment of the rab ⁇ b 3'UTR sequence with the rab5b(opal)Gpx3'UTR subclone, from which the GPx 3'UTR had been deleted as an Avrll-EcoKL fragment.
  • the resulting rab5b(opal) sequence was then inserted into pCMV4 as above.
  • COS-1 cells were transfected for transient expression of the GPx or rab5b constructs by modified calcium phosphate mediated or electroporation methods (Maniatis et al. (1990) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor) , and then cultured in DMEM medium supplemented with 10% fetal bovine serum, 5 ng/ml sodium selenite, 25 mM HEPES pH 7.4, and lx penicillin-streptomycin-fungizone (Gibco-BRL) . All experiments were performed 2-4 times.
  • COS-1 cells were cotransfected with 2 ⁇ g of plasmid pXGH5 included in a human growth hormone transient expression assay system supplied by Nichols Institute. Human growth hormone secreted into the medium was detected by radiommunoassay using the Crystal Multidetector RIA System (United Technologies Packard) .
  • a human growth hormone transient expression assay system supplied by Nichols Institute. Human growth hormone secreted into the medium was detected by radiommunoassay using the Crystal Multidetector RIA System (United Technologies Packard) .
  • 10 Ci of 75 Se as selenous acid diluted in nitric acid with an original specific activity of 750-1000 Ci/g (from the University of Missouri Research Reactor Facility) , was added to the transfected cells in each plate, and the cells were incubated at 37°C for an additional 2 hours.
  • the transfected cells in each plate were first incubated for 30 min in methionine- and glutamine-free DMEM medium (Gibco) , supplemented with 10% dialyzed calf serum, lx glutamine (Gibco) , and 25 mM HEPES. Then 250 Ci of Express 35 S protein labeling mix (NEN DuPont) , with a specific activity of 1140 Ci/mmole for methionine, was added to the plate, and the cells were incubated at 37°C for an additional 2 hours.
  • Immunoprecipitation utilized two rabbit antisera raised (by Berkeley Antibody Co., Richmond, CA) against synthetic peptide sequences from the GPx polypeptide chain, one from residues 26 to 46, and the other from residue 174 to residue 192. Fifteen ⁇ l of each antiserum, plus 20 ⁇ .1 of protein A-Sepharose CL-4B beads (Sigma) were added to each lysate, and the mixture was incubated at 4°C overnight with constant tumbling.
  • the beads were subsequently pellet, washed twice with washing buffer (50 mM HEPES pH 7.8, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) and once with 50 mM HEPES at pH 7.8, mixed with 30 ⁇ l SDS-gel loading buffer (50 mM Tris-HCl pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) , heated in boiling water for 3 minutes, and then pelleted in a microfuge. The supernatant was then collected for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .
  • washing buffer 50 mM HEPES pH 7.8, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS
  • 50 mM HEPES at pH 7.8 mixed with 30 ⁇ l SDS-gel loading buffer (50 mM Tri
  • Protein electrophoresis was performed by standard techniques (Maniatis et al. (1990) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor) on 12% SDS-polyacrylamide gels. RNase protection assay
  • RNase protection assays of hybridization mixtures of 3 ⁇ g total cell RNA, 10 ⁇ g yeast tRNA, and 6 ⁇ l of the riboprobe (400,000 TCA-precipitable cpm/ ⁇ -1) were performed by standard techniques (Maniatis et al. (1990) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor) .
  • One putative stem-loop structure immediately downstream of the UGA 142 , creates a stem-loop structure (shown in figure 1, panel B) similar to that found in the mRNA of the E coli formate dehydrogenases and related prokaryotic selenoenzyme genes (Zinoni et al. (1990) Proc. Natl . Acad. Sci . USA 87, 4660-4664) .
  • Another, which incorporates the UGA 1 2 codon at the tip of the "hairpin” (shown in figure 1C) , is conserved among several mammalian GPx mRNAs, as well as E. coli formate dehydrogenase mRNA sequences (Chada et al.
  • ORF-Dl lacks a sequence from codon 49 through codon 53; 0RF-D2 lack codons 65 through 69; ORF-D3 lacks codons 54 through 63; and ORF-D4 lacks codons 71 through 74.
  • the sequences deleted from ORF-Dl, ORF-D2, and ORF-D3 correspond, respectively to the 5' part of the stem, the 3' part of the stem, and most (29 of 31 nt) of the loop of the putative stem-loop structure (Zinoni et al. (1990) Pro ⁇ . Natl . Acad. Sci .
  • ORF-D4 represents a 12 nt sequence immediately downstream of the putative stem-loop structure which corresponds to a sequence which has been reported to be important to selenocysteine translation in E. coli formate dehydrogenases (Zinoni et al. (1990) Proc. Natl . Acad . Sci . USA 87, 4660-4664).
  • GPx subclone ORF-D5 contains a deletion of codon 47, located immediately upstream of the UGA 1 2 codon of the GPx mRNA, which forms part of the stem of the alternative, putative hairpin loop structure (Chada et al.
  • COS-1 cells transfected by the vector alone demonstrate a low background level of 75 Se-containing polypeptide (most likely the native monkey cellular GPx) with a 23 kD size similar to that of human GPx.
  • Transient expression of the native human GPx cDNA and of deletions ORF-Dl through 0RF-D4 (lane 2 and lanes 3-6, respectively) all show high levels of 75 Se incorporation into Gpx protein. These deletions appeared to exhibit a slight, but not substantial, decrease in GPx expression. Repeated experiments (including the creation of identical deletions in the epitope-tagged construct) also showed slightly diminished expression.
  • deletion 0RF-D5 produces little or no diminution of selenocysteine insertion into GPx.
  • putative loop structures in the open reading frame of the GPx mRNA may slightly modulate GPx expression, neither is absolutely necessary for translation of the UGA 142 codon as selenocysteine in human GPx.
  • the unambiguous discrimination of the transiently expressed, epitope-tagged GPx was possible because the tagged GPx migrated slowly enough on SDS-PAGE gels that its band resolved at a position detectably higher than that of the untagged GPx.
  • This difference of mobility permitted assessment of transient expression of transfected constructs without the need for the substantial overexpression necessary for evaluation of the coding region deletion constructs described above.
  • the epitope sequence was also inserted into the wild type GPx subclone GPxR to yield a new GPx subclone GPxEPI, which served as a positive control for the transient expression of the GPx 3'UTR deletion constructs. These deletions are also indicated in figure IA.
  • Lane 1 demonstrates the background GPx signal in cells transfected with vector alone.
  • the slightly larger epitope-tagged GPx is expressed by the GPxEPI construct with its 3'UTR intact (lane 2) and is easily distinguished from the endogenous COS-1 background. Deletion of the distal 100 nt of the 3'UTR (UTR-D1, lane 3) did not diminish expression of the transfected GPx.
  • rab5b encodes a 25 kD GTP-binding protein which is a member of Ras-related GTPase superfamily (Wilson et al. (1992) J. Clin . Invest . 89, 996-1005). This gene was used for three constructs: rab5b(opal.
  • rab5b (opa1)Gpx3'UTR consisted of the rab ⁇ b(opal) coding sequence fused to a 3' portion of GPx cDNA incorporating the last three codons of the GPx coding region, including its stop codon (UAG) , and the entire GPx 3'UTR; and rab5b(wt)Gpx3'UTR was also a rab5b-GPx fusion product but carried the wild type codon 63 rather than the opal mutation.
  • the fusion constructs placed the UGU (cysteine) or UGA (potential selenocysteine) codon the same number of nt upstream from the GPx 3'UTR as in native GPx transcripts.
  • Figure 7 presents the results of a representative transient expression experiment of these constructs in COS-1 cells.
  • the expression of rab ⁇ b was detected by an affinity-purified rabbit antibody against a synthetic peptide sequence, following either 35 S (lanes 1-4) or 75 Se (lanes 5-8) radioisotope labeling.
  • COS-1 cells transfected with the vector alone (lanes 1 and 5) showed no detectable immunoreactive protein at the appropriate 25 kD molecular mass for rab ⁇ b.
  • the essential 3-4 nucleotide targeting elements are positioned in mirror image on the appropriate bubble and balloon regions of the artificial stem loop.
  • the structure of this optimized element is shown in Fig. 9.
  • MRS denotes a multiple restriction site for ease of insertion of the element into any appropriate cloning vector
  • N indicates any nucleotide: N x denotes a stretch of two or more nucleotides of any sequence; N:N denotes complementary base pairs.
  • a nucleotide sequence containing the elements of this optimized stem-loop may be constructed by standard techniques known to those skilled in the art of molecular biology.
  • oligonucleotide comprising the loop-bubble-balloon using an Applied Biosystems DNA synthesizer. After gel purification, this single-stranded oligonucleotide served as the template for PCR amplification using 22-mer sense and antisense PCR primers containing 12 and/or 6 nucleotide overlapping sequences: 1) template oligonucleotide:
  • the PCR reaction was carried out using 0.1 ⁇ g/ ⁇ l template oligonucleotide, 50 pmole/ ⁇ l of each PCR primer according to standard methods for 10 cycles of 95°C for 1 min, 50°C for 1 min, and 70°C for 1 min.
  • the double-stranded PCR product was then ligated into the pCRII vector (InVitrogen, San Diego, CA) using the TA cloning system (InVitrogen) and transformed into INV ⁇ F' cells.
  • the sequence of the construct was confirmed by nucleotide sequencing using fmol PCR sequencing from Promega (Madison, WI) . Construction of Recombinant Selenocysteine containing Polypeptides.
  • Any desired polypeptide for which the DNA sequence is known may be used in the method of the invention by substitution of codon encoding any amino acid which is not essential for the natural activity of the polypeptide.
  • the approach to the preparation of these "TGA" mutants may be generally accomplished by site- directed or oligonucleotide based mutagenesis techniques, e.g., using commercially available kits (Promega) .
  • the cDNA encoding the human thyroid hormone receptor-01 was cloned into the multiple cloning site of the vector p-alter (Promega) and the first cysteine codon was mutated to TGA by oligonucleotide based mutagenesis.
  • Polypeptides according to the invention may be produced by the expression from a recombinant nucleic acid having a sequence encoding the polypeptide linked to a recombinant nucleic acid containing the stem-loop structure required for translation of selenocysteine, using any appropriate expression system: e.g., transformation of a suitable eukaryotic host cell with the recombinant nucleic acid in a suitable expression vehicle such as those described above.
  • any of a wide variety of expression systems may be used to provide a selenocysteine containing recombinant protein of the invention.
  • the precise host cell used is not critical to the invention and includes Saccharomyces cerevisiae or mammalian cells (e.g., COS-1, HL-60, CV-1, LLC/PK-1, C-6, 3T3L1, and CHO cells). Such cells are available from a wide range of sources (e.g., the .American Type Culture collection, Rockland, MD) .
  • the method of transformation or transfection, and the choice of expression vehicle will depend on the nature of the polypeptide to be expressed and the host system selected.
  • Transformation and transfection methods are described, e.g., in Ausebel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989) ; expression vehicles may be chosen from those well-known in the art, e.g., in Cloning Vectors: A Laboratory Manual (P.H. Pouwels et al., 1985, Suppl. 1987).
  • the cDNA encoding a desired polypeptide is inserted into the eukaryotic expression vectors pcDNAl/neo and pRC/CMV (InVitrogen) which are especially preferred as parent vectors for the selenocysteine expression system in an orientation designed to allow expression.
  • pcDNAl/neo and pRC/CMV InVitrogen
  • selenocysteine containing polypeptides according to the invention may be produced by a stably-transfected mammalian cell line.
  • a number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al., supra; methods for constructing such cell lines are also publicly available, e.g., in Ausebel et al., supra .
  • the production of the polypeptide may be controlled by the content of the selenium in the medium.
  • the recombinant polypeptide may be isolated according to methods well known in the art and the functional activity may be determined by assays appropriate for the particular polypeptide, e.g., enzymatic activity or binding affinity.
  • the selenopolypeptide may be distinguished from the native protein by its higher reactivity with nucleophilic agents due to the selenocysteine moiety as described (Leonard et al., Biochim. Biophys . Acta 787:122. 1984), or alternatively by radiolabeling with 75 Se, as described herein.
  • the gene for any desired polypeptide which has been modified according to the methods described herein to encode a selenocysteine amino acid residue may be used to produce a transgenic animal wherein production of the polypeptide is controlled by the selenium content in the diet of animal.
  • Methods for producing transgenic animals are well known (e.g., see Hogan et al., Manipulating the Mouse Embryo: A laboratory manual , CSH Press, Cold Spring Harbor, NY, 1986; Leder et al., U.S. Patent No. 4,736,866).
  • selenopolypeptide in a transgenic animal will be inhibited when the animal is given a diet containing less than 0.016 mg/kg selenium, whereas high levels of the protein will be produced when the animal is given a diet containing 0.1 mg/kg or more selenium (e.g., as Na 2 Se0 3 , Sigma) .
  • selenium e.g., as Na 2 Se0 3 , Sigma
  • the methods of the invention may also be used to produce high levels of any commercially desirable selenopolypeptide.
  • the presence of available selenium produces a 30 to 50 fold increase in the expression of a selenopolypeptide over the level produced under selenium deficient conditions.
  • This level may be further increased by cotransfecting the cell with the gene encoding the selenocysteine tRNA in an expression vehicle which will allow overexpression of the tRNA under the appropriate conditions, e.g., when selenium is present.
  • this may be accomplished by putting the gene encoding the tRNA under the control of an inducible promoter and then supplying the factor required for induction of the gene at the same time, or before, the medium is supplemented with selenium.

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WO1992013077A1 (en) * 1991-01-29 1992-08-06 Brigham And Women's Hospital A cDNA ENCODING THE TYPE I IODOTHYRONINE 5' DEIODINASE

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EP0220679A2 (de) * 1985-10-24 1987-05-06 Danisco A/S Verfahren zur Expression von Genen in Hefe, DNS-Fragmente und Plasmide die letztere Fragmente enthalten und zur Durchführung des Verfahrens verwendet werden
WO1992013077A1 (en) * 1991-01-29 1992-08-06 Brigham And Women's Hospital A cDNA ENCODING THE TYPE I IODOTHYRONINE 5' DEIODINASE

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Title
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SHEN Q ET AL: "Sequences in the 3' untranslated region of the human cellular glutathione peroxidase gene are necessary and sufficient for selenocysteine incorporation at the UGA codon." JOURNAL OF BIOLOGICAL CHEMISTRY, (1993 MAY 25) 268 (15) 11463-9. JOURNAL CODE: HIV. ISSN: 0021-9258., XP002068428 *

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