[go: up one dir, main page]

WO1992013077A1 - ADNc CODANT LA IODOTHYRONINE 5' DEIODINASE DE TYPE I - Google Patents

ADNc CODANT LA IODOTHYRONINE 5' DEIODINASE DE TYPE I Download PDF

Info

Publication number
WO1992013077A1
WO1992013077A1 PCT/US1992/000740 US9200740W WO9213077A1 WO 1992013077 A1 WO1992013077 A1 WO 1992013077A1 US 9200740 W US9200740 W US 9200740W WO 9213077 A1 WO9213077 A1 WO 9213077A1
Authority
WO
WIPO (PCT)
Prior art keywords
deiodinase
type
sequence
selenocysteine
dna
Prior art date
Application number
PCT/US1992/000740
Other languages
English (en)
Inventor
P. Reed Larsen
Marla A. Berry
Original Assignee
Brigham And Women's Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Brigham And Women's Hospital filed Critical Brigham And Women's Hospital
Publication of WO1992013077A1 publication Critical patent/WO1992013077A1/fr

Links

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to the cloning, characterization and uses of both human and rat Type I iodothyronine 5 ' deiodinase, a selenocysteine-containing enzyme involved in the conversion of thyroxine to 3,3 ',5-triiodothyronine.
  • the invention further relates to one or more mutant forms of the enzyme and the use of genes coding for such mutant forms as reporter genes.
  • T 3 3,3 ',5-triiodothyronine
  • T 4 thyroxine
  • Iodothyronine 5 '-deiodination is catalyzed by two general classes of enzymes (Leonard, et al, In: Hennermann, G. (ed.), Thyroid Hormone Metabolism, Marcel Dekker, New York, pp. 289-229 (1986)) distinguished by their tissue distribution, physiological roles, K m for substrate, and sensitivity to propylthiouracil (PTU).
  • PTU propylthiouracil
  • Type I deiodinase present predominantly in liver and kidney, provides most of the plasma T 3 in the rat.
  • This class of enzyme exhibits a K m for T 4 of ⁇ 2 ⁇ M and is sensitive to inhibition by PTU.
  • Type II deiodinase found in pituitary, cerebral cortex, and brown adipose, functions primarily to provide an intracellular source of T 3 for these tissues.
  • This enzyme exhibits a K m for T 4 of ⁇ 2 nm and is PTU resistant. Many attempts at elucidating the molecular structure of these enzymes are in progress, but these efforts have to date been unsuccessful. Furthermore, the purification of these deiodinases has not been reported. (Berry, et al, Mol. Endocrin. 4:743-748 (1990).)
  • Type I deiodinase which requires reduced thiols for maximal enzyme activity, is closely related to rat protein disulfide isomerase (PDI) (Boada, et al, Biochem. Biophys. Res. Comm. 155:1297-1304 (1988)).
  • PDI rat protein disulfide isomerase
  • Type I iodothyronine 5' deiodinase has not been well-characterized.
  • the need for a DNA sequence encoding Type I iodothyronine 5' deiodinase is clearly recognized in the art. Summary of the Invention
  • the present invention meets the needs for a DNA sequence encoding Type I iodothyronine 5 ' deiodinase and for a method of achieving synthesis of 5 ' deiodinase, now discovered to be a selenocysteme-containing enzyme.
  • Type I iodothyronine 5' deiodinase is disclosed.
  • the product of this DNA sequence, and antibodies reacting with the product, are useful in relation to diagnosis and treatment of disease states related to thyroid function.
  • the invention also relates to the discovery that 5' deiodinase contains selenocysteine, an amino acid encoded by the termination codon TGA and previously identified in only one mammalian enzyme. According to the invention, a 3 ' untranslated segment of 5 ' deiodinase cDNA is essential for successful expression of the active selenocysteinecontaining enzyme.
  • the invention further relates to the characterization of the 3' untranslated region of 5' deiodinase, and selenocysteine-insertion sequences. Such sequences are useful for incorporation of selenocysteine into peptides or proteins to study the effects of the presence of selenocysteine on the properties of such proteins.
  • the invention further relates to mutant gene sequences of iodothyronine 5' deiodinase.
  • Such sequences including for example the gene encoding the cysteine-126 mutant, as well as wild-type sequences are useful, for example, as "reporter" genes for monitoring transfection efficiencies or in the study of heterologous promoter function in transient expression assays.
  • the invention yet further relates to genetic constructs useful for the expression of selenocysteine-containing proteins, and methods of producing selenocysteine-containing proteins.
  • Such methods including the introduction of selenocysteine at a desired site into a polypeptide or protein when the native protein does not contain selenocysteine allow production of peptides or proteins with altered biochemical properties. These alterations provide insight into biochemical mechanisms, or result in proteins with properties that are advantageous over the native protein.
  • Previous studies have shown that either chemical conversion of cysteine to selenocysteine in an intact protein (Wu and Hilvert, J. Am. Chem. Soc.
  • the invention further relates to methods of measuring the responsiveness of a cell to thyroid hormone and characterizing thyroid-cell containing tissue, and kits useful for detecting 5' deiodinase. Such methods and kits are useful for determining whether a malignant thyroid tumor has spread to other tissue and for the diagnosis of thyroid cancer.
  • Figure 1 illustrates the DNA sequence and predicted amino acid sequence of rat liver Type I iodothyronine 5' deiodinase.
  • Figure 2 illustrates a Northern blot analysis of Type I iodothyronine 5 ' deiodinase mRNA in rat tissues.
  • Lanes 1-6 total RNA from kidney, liver, spleen, heart, lung and small intestine.
  • Lane 7 poly(A) + RNA from thyroid of methimazoletreated rats.
  • Lane 8 poly(A) + RNA from rat kidney.
  • Lanes 9 and 10 poly(A) + RNA from pituitary and brown adipose tissue.
  • Figure 3 illustrates the effect of thyroid states on Type I iodothyronine 5 ' deiodinase mRNA levels.
  • Liver and kidney poly(A) + RNA from hypothyroid (-), euthyroid (Eu) and hyperthyroid (+) rats were probed with G21 cRNA.
  • Figure 4A illustrates partial restriction maps of the human liver and kidney clones. Relevant restriction sites are shown at the corresponding nucleotide position of the cDNA and the vector. The TGA codon is also indicated. The curved line indicates that the exact border of the exonic sequence is not known.
  • Figure 4B depicts the DNA and predicted amino acid sequence of the human Type I 5'-deiodinase. Nucleotides are numbered as described in the text. The amino acid selenocysteine is noted as SeC.
  • Figure 5 illustrates the DNA sequence comparison between the human and rat Type I 5' deiodinase coding regions. Only the nucleotides of the rat cDNA that differ from those of the human are shown. Both the ATG initiation codon and the TGA codon encoding selenocysteine are marked with asterices. Nucleotide 32, the 5' end of the human liver clone is marked with a #. The rat coding sequence is 24 nucleotides longer than that of the human protein.
  • Figure 6 is a Northern Blot analysis of Type I 5'-deiodinase mRNA.
  • Poly(A)+ RNA was isolated from human liver (HL1, HL2), kidney (HK), and thyroid (HT), and from hyperthyroid rat liver (RL) as described in Example IX. Each lane contains 2 ug of the indicated sample.
  • Figure 7 illustrates the expression of Type I iodothyronine 5' deiodinase from G21 wild-type and deletion constructs, in oocytes and JEG cells.
  • Figure 8 illustrates the expression of Type I iodothyronine 5' deiodinase from G21 wild-type and mutant constructs, in oocytes.
  • Figure 9 is an illustration of a polyacrylamide gel analysis of in vitro translation products of clone G21, substitution mutants and the HindIII internal deletion.
  • In vitro transcribed RNA was translated in rabbit reticulocyte lysate using 35 S methiomne.
  • Figure 10 illustrates the kinetics of inhibition of rT 3 deiodination by gold thioglucose (GTG). Double-reciprocal plots of deiodination rate vs. rT 3 concentration at varying GTG concentrations are shown. Reaction conditions were as described in Example IX.
  • FIG 11 illustrates the inhibition of T 4 to T 3 deiodination.
  • the products of T 4 deiodination, T 3 and I are shown as percent of total T 4 present under the various conditions. Reactions were performed as described in Example IX.
  • Figure 12 illustrates bromoacetyl affinity labeling of human and rat transiently expressed 5'-deiodinases. Transfection with CDM-8 vector alone or vector containing the human and rat 5' DI cDNA is indicated under the corresponding lane. Concentrations of the various added competitors are shown.
  • Figure 13A is an illustration of deletion and inversion mutations of rat 5' deiodinase cDNA 3' untranslated region. Wild-type and mutant rat 5' deiodinase constructs were assayed for production of 5' deiodinase activity following transient transfection in JEG-3 or COS-7 cells. Deiodinase activity at the level of the wild-type rat 5' deiodinase construct is defined as 100%, and was equivalent to 5' deiodination of 2 pmol reverse T 3 /min/mg protein for TGA-containing constructions and 1 pmol reverse T 3 /min/mg protein for TGT-containing constructs, in JEG cell extracts.
  • Figure 13B is an illustration of rat 5' deiodinase constructs containing 3' untranslated sequences from rat or human 5' deiodinase or rat GPX cDNAs. Constructs containing either rat or human 5' deiodinase or rat GPX 3'ut sequences adjacent to rat 5' deiodinase coding sequences were assayed for production of 5' deiodinase activity as above.
  • Figure 14 is an illustration of predicted secondary structures in the 3' untranslated regions of selenocysteine-encoding RNAs. Sequences from the 3' untranslated regions of the rat 5' deiodinase (Zinoni et al, Proc. Natl Acad. Sci. USA 87:4660-4664 (1990)), human 5' deiodinase and rat GPX (Ho et al, J. Nucl. Acids Res. 16:5207 (1988)) are shown. The positions of deletions which resulted in partial or complete loss of function are indicated. Structure analysis was performed using the FOLD program of the Univ. of Wisconsin Genetics Computer Group (UWGCG) software (Devereux et al, Nucl. Acids Res. 12:387-395 (1984)).
  • UWGCG Univ. of Wisconsin Genetics Computer Group
  • Figure 15 is an illustration of deletion mutations in the stem-loop regions of rat 5' deiodinase and GPX mRNAs.
  • PCR deletions were generated as described in PRC protocols (Higuchi, R. "Recombinant PCR, in PCR Protocols, Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. Academic Press, 177-183 (1990), and cloned into the vector fragment described in Example VIIA.
  • Deiodinase assays were performed as described in Example VIIA.
  • Figure 16 is an illustration of sequence similarities in the stem-loop regions of the rat and human 5' deiodinase, and mammalian GPX cDNAs. Analysis was performed using the LINEUP program of the UWGCG software (Devereux et al, Nucl Acids Res. 12:387-395 (1984)).
  • FIG 17, Seq. ID No. 3, illustrates the DNA sequence of the human selenocysteine insertion sequence, 5' deiodinase cDNA from nucleotide 1572 to 1893 (corresponding to nucleotides 1-322 of Seq. ID No. 3).
  • Figure 18, Seq. ID No. 4 illustrates the DNA sequence of rat GPX cDNA from nucleotide 922 to 1155 (corresponding to nucleotides 1-234 of Seq. ID No. 4).
  • T 4 thyroxine
  • T 3 3,3',5-triiodothyronine
  • PTU propylthiouracil
  • a 2.1 kb cDNA for this deiodinase has now been isolated from a rat liver cDNA library by expression cloning in the Xenopus oocyte.
  • the kinetic properties of the protein expressed in transient assay systems, the tissue distribution of the mRNA, and its changes with thyroid status confirm its identity.
  • the cDNA for rat deiodinase was then used to probe human cDNA libraries for the analogous human gene.
  • Human cRNA hybridizes to a 2.4 kb mRNA in human liver, kidney and thyroid.
  • the gene for human Type I iodothyrinone-5'-deiodinase was constructed from human liver and kidney cDNAs. This gene was expressed in COS-7 cells and its kinetic properties were studied.
  • a unidirectional, size-fractionated rat liver cDNA library for expression screening in Xenopus oocytes was constructed. Plasmid DNA was transcribed in vitro, the resulting RNA injected into oocytes, and oocyte homogenates assayed for deiodination of 3,3 ',5 '-triiodothyronine ("reverse" T 3 ; rT 3 ). This strategy resulted in isolation of a single positive clone, G21.
  • the DNA sequence and predicted amino acid sequence of rat liver Type I 5 ' deiodinase clone G21 are shown in Figure 1.
  • the Km for rT 3 was 130 nM in the presence of 5 mM dithiothreitol (DTT). There was no deiodinase activity in cells transfected with CDM-8 vector alone.
  • DTT dithiothreitol
  • PTU was a competitive inhibitor of DTT, with greater than 50% inhibition by 0.5 ⁇ M PTU.
  • T 4 was a competitive inhibitor of rT 3 deiodination, and was converted to T 3 by microsomal protein from transfected, but not control, cells.
  • G21 cRNA hybridized to a single band of ⁇ 2 kb in mRNA from thyroid, kidney, liver, and pituitary, but not in mRNA from spleen, heart, lung, small intestine, or brown fat (Figure 2).
  • This tissue distribution is in agreement with previous studies using enzyme assays in tissue homogenates (Leonard et al, Biochemistry of Deiodination. In: Thyroid Hormone Metabolism (Hennemann, G., ed.) 189-229 (1986)).
  • rat cDNA encoding Type I 5'-deiodinase (nucleotides 1 to 745 of Figure 1) was used to screen human liver and kidney cDNA libraries for human Type 1 5'-deiodinase.
  • Initial screening of human liver library in the CDM-8 vector yielded a 2188 base pair clone with a 5' boundary corresponding to nucleotide 32 of the rat sequence ( Figure 1).
  • a 417 base pair Nco I to Pst I fragment of this liver cDNA (nucleotides 134 to 551) was then used to screen a human kidney cDNA library in ⁇ -gt 10 vector. This method successfully isolated the remainder of the human gene.
  • a single cDNA of 2222 nucleotides was then constructed from the liver and kidney cDNAs that encoded the entire gene based on the partial restriction map of Figure 4A.
  • the sequence of human 5'-deiodinase is shown in Figure 4B.
  • Figure 5 is a DNA sequence comparison between the human and rat Type I 5'-deiodinase coding regions.
  • the coding regions are 82% homologous.
  • the putative amino acid sequences are 88% identical.
  • the cDNA encodes a functional 5'-deiodinase
  • it was transiently expressed in COS-7 cells, which contain no endogenous iodinase.
  • the transiently expressed enzyme was identified by its capacity to deiodinate rT 3 in a saturable fashion
  • Figure 6 shows that human cRNA hybridizes to a 2.4 kb mRNA in human liver, kidney and thyroid. This mRNA is approximately 200 nucleotides longer than the rat liver 5'-deiodinase, which is shown for comparison on the same blot.
  • the DNA sequence of clone G21 predicts a protein of ⁇ 14 kD. initiating at nucleotide 7 and terminating at nucleotide 382 (TGA, a known termination codon). Deletions from the 5 ' or 3 ' ends, an internal deletion, and frameshift insertion were constructed to identify regions essential for deiodinase activity. The locations of these mutations and their effects on activity in both oocytes and transfected JEG cells are shown in Figure 7. The absence of activity with the Pst I 5' deletion confirmed that sequences 5' to nucleotide 56 are required for production of active enzyme, indicating that the ATG at position 7 is indeed the initiation codon.
  • the corresponding TGA was converted to either the stop codon, TAA, the leucine codon, TTA, or the cysteine codon, TGT, by oligonucleotide directed mutagenesis.
  • In vitro synthesized mRNAs from these constructs were assayed for expression of Type I 5 ' deiodinase in oocytes ( Figure 8) and translated with 35 S methionine in vitro in reticulocyte lysates ( Figure 9).
  • Table 1 demonstrates another similarity to glutathione peroxidase. sensitivity to inhibition by gold, which is believed to complex with the selenolate group in the active site of this enzyme (Cliaudiere et al, J. Inorganic Biochem. 20:313-325 (1984)).
  • the activity of the transiently expressed wild type deiodinase protein is inhibited ⁇ 50% by 10 nM gold thioglucose (GTG).
  • GTG gold thioglucose
  • Substitution of cysteine for selenocysteine resulted in an enzyme with ⁇ 20% of the intrinsic activity of the wild type protein. in agreement with the oocyte studies. This mutant protein was much less sensitive to inhibition by GTG than the native enzyme.
  • a UGA codon is also present at position 382 of the human 5'- deiodinase sequence -- the same site as in the rat sequence.
  • deiodination is inhibited by gold thioglucose with an apparent Ki of 4.7 nM ( Figure 10).
  • the enzyme also catalyzes T 4 to T 3 conversion by a PTU-sensitive mechanism with the production of equimolar quantities of T 3 and I-, albeit at a much slower rate (Figure 11).
  • Bromoaceryl (BrAcT 3 ) labeling of the human and rat transiently expressed deiodinase was performed to establish that the in vitro expressed protein was of the size predicted by the deduced amino acid sequence presuming that the UGA encodes selenocysteine.
  • the CDM-8 vector alone Figure 12, lanes 1 and 2
  • several discrete labeled bands are present (64, 46, 34, and 16 kDa). Labeling of the 16 kDa band is completely, and that of the 64 and 46 kDa bands partially, blocked by excess unlabeled BrAcT 3 .
  • Type I iodothyronine 5 ' deiodinase No significant homology was found between Type I iodothyronine 5 ' deiodinase and glutathione peroxidase. Furthermore, 5 ' deiodinase was not significantly homologous to any other protein sequence in GenBank or EMBL (Devereux et al. Nucleic Acids Res. 12:387-395 (1984)). This includes protein disulfide isomerase, another thiol-requiring protein which has been speculated to be related to the Type I deiodinase (Boada et al, Biochem. Biophys. Res. Commun. 155:1297-1304 (1988)).
  • Type I deiodinase The lack of relationship between type I deiodinase and protein disulfide isomerase was further demonstrated by the following experiments.
  • no Type I deiodinase activity was detected in oocytes injected with PDI mRNA.
  • PDI cRNA hybridized to a 2.8 kb mRNA present in total poly(A) + RNA from liver and to the 2.2 - 3.2 kb fraction.
  • the PDI cRNA was not detectable in the 3.2 - 4.6 kb fraction or the 1.7 - 2.2 kb fraction.
  • the Type I deiodinase mRNA falls within the 1.9 - 2.4 kb region of rat liver poly(A)+ RNA. (Berry, M. J. et al, Mol Endocrin. 4:743-748 (1990)).
  • the mechanism which allows the eukaryotic cell to incorporate the amino acid selenocysteine into a protein, as opposed to terminating translation at the UGA codon, has been elucidated for the first time.
  • the requirements for successful translation of the active deiodinase protein in Xenopus oocytes and in transfected JEG cells have also been analyzed.
  • sequences between about nucleotide 1360 and 1615 in the 3 '-untranslated region of the cDNA must be present, with sequences between 1440 and 1615 being essential.
  • nucleotides can be inserted immediately 3 ' of the coding sequences and retain the ability to induce the translation of completely active enzyme. However, if these nucleotides are removed or if the sequence is inverted, there is no expression of the active enzyme. Characterization of the 3' Untranslated Sequence
  • UGA is recognized as a selenocysteine codon rather than a stop codon due to the presence of a segment of about 200-255 nucleotides, with 200 nucleotides being essential, of a 3' untranslated sequence. This segment is located greater than a kilobase downstream from the UGA codon.
  • the present inventors have surprisingly discovered that the mechanism by which this recognition occurs involves a stem-loop structure in the 3 ' untranslated region of the mRNA.
  • 5' deiodinase was used to investigate selenoprotein synthesis in eukaryotes.
  • the present inventors have discovered that successful incorporation of selenocysteine into this enzyme requires a specific 3' untranslated segment of about 200 nucleotides, which is found in both rat and human 5' deiodinase mRNAs. These sequences are not required for expression of a cysteine-mutant deiodinase. While little primary sequence similarity exists between the 3' untranslated regions of these mRNAs and those encoding GPX, the 3' untranslated sequences of rat GPX can substitute for the 5' deiodinase sequences in directing selenocysteineinsertion.
  • the human deiodinase gene contains a similar SECIS motif at nucleotides 1573 to 1894 of Figure 4. Comparison of the 321 nucleotide sequence with the corresponding rat sequence shows a 66% homology. Mutant Sequences of Iodothyronine 5' deiodinase.
  • Mutant sequences of 5' deiodinase are useful as "reporter” genes for monitoring transfection efficiencies or in the study of heterologous promoter function in transient expression assays. Cysteine-126 is useful as an internal control for transfection efficiency in DNA transfer studies.
  • the rate of removal of 123 I from the 3 ' or 5 ' position of reverse 3,3 ',5 '-triiodothyronine is measured as a measure of the activity of the enzyme coded for by the mutant cysteine-126 sequence.
  • cysteine-126 and functional equivalents thereof.
  • a functional equivalent of cysteine-126 is also a mutant of iodothyronine 5 ' deiodinase wherein the mutant is readily expressed by a number of different cell lines, is easy to measure accurately with a minimum of manipulation of cell extracts or medium.
  • reporter genes can be used to assess the function of various heterologous promoters or to determine the transfection efficiency of plasmids introduced into cells by various DNA transfer techniques.
  • G-5 cys-126 mutant enzyme
  • a plasmid can be constructed, using known techniques, in which the cDNA coding for the reporter enzyme is cloned into a plasmid in which either incorporates a constitutive promoter (for example, TK) or a poly cloning sequence 5' to the reporter enzyme sequence.
  • a constitutive promoter for example, TK
  • the plasmid is used as an internal control.
  • a poly cloning site either immediately upstream of the reporter gene or upstream of an amputated heterologous promoter such as TK, such a plasmid is used to study the influence of various DNA sequences of interest on the expression of the reporter gene.
  • deiodinase as a reporter gene is that the assay for deiodinase uses a readily available, low-cost substrate which can be labeled with 125 I to extremely high specific activity. Thus, only a small amount of cell sonicate is necessary to obtain a signal, for example, about 1-2 ⁇ l of cell sonicate.
  • Use of the cysteine-126 mutant that is, substitution of cysteine for selenocysteine in 5 ' deiodinase. removes the requirement that a cell have the appropriate selenocysteine-insertion machinery. Thus, there is a broad repertoire of cells which can be subjected to this technique.
  • cysteine mutant is assayed by quantitation of 5' deiodinase catalyzed by the cell sonicates, as described previously (Berry et al, J. Biol. Chem. 266:14155-14158 (1991)). Using this procedure, one can screen large numbers of cell lines at the same time. Transfection of 20 different cell lines can easily be performed on day 1 (2 hours), followed by DMSO-treatment on day 2 (2 hours), and assay on day 3 (4 hours). The cells to be tested are transfected with either the cys mutant (G-5) or wild-type (G-21) construct directed by the CMV promoter together with TKGH (or TKCAT) plasmid (see Table II).
  • TK-directed, reporter genes allows ascertainment that successful transfection of DNA has occurred.
  • Cells which expressed deiodinase activity after transfection with either G-5 or G-21 are those which possess the appropriate selenocysteine insertion machinery.
  • Cells which express deiodinase after transfection with G-5, but not with G-21 are those which can synthesize deiodinase but which are unable to incorporate selenocysteine into the wild-type protein.
  • Cells which do not express deiodinase from either construct and yet have been successfully transfected i.e., showing suitable expression of CAT or hGH
  • Cell lines that express transfected cysteine mutant include, for example. COS-7 cells and the JEG choriocarcinoma cell lines. These cell lines will express transfected cysteine mutant, under the influence of the cytomegalovirus (CMV) promoter in the plasmid CDM8. Other suitable cell lines can be readily determined by routine experimentation by one of ordinary skill in the art.
  • CMV cytomegalovirus
  • Reporter genes are also used to evaluate whether cells are capable of successfully translating selenocysteine-containing proteins. This is accomplished using mutant clones (for example, cysteine-126) and wild- type selenocysteine clones. The level of deiodinase produced by the cysteine mutant is compared to the level of deiodinase produced by cells in which the gene containing the wild-type selenocysteine enzyme has been introduced. A higher ratio of selenocysteine to cysteine activity indicates a more efficient, better, selenocysteine-insertion mechanism.
  • the term “5 ' deiodinase” includes the Type I iodothyronine 5 ' deiodinase molecule.
  • the term “5 ' deiodinase” additionally includes the functional derivatives of such molecules.
  • the term “5 ' deiodinase” additionally includes both glycosylated and unglycosylated forms of any of the above-described molecules.
  • a "functional derivative" of 5' deiodinase is a compound which possesses a biological activity that is substantially similar to the biological activity of 5 ' deiodinase.
  • the term “functional derivatives” is intended to include the “fragments,” “variants,” “analogs,” or “chemical derivatives” of 5' deiodinase.
  • fragment is meant to refer to any polypeptide subset of 5' deiodinase.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire 5 ' deiodinase molecule, or to a fragment thereof.
  • a molecule is said to be "substantially similar” to 5 ' deiodinase if both molecules have substantially similar structures or if both molecules possess a similar biological activity.
  • two molecules possess a similar activity they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequences of amino acid residues are not identical.
  • analog is meant to refer to a protein that differs structurally from the wild type enzyme 5' deiodinase, but possesses biological activity that is substantially similar to that of 5 ' deiodinase.
  • DNA segment refers to a sequence of
  • DNA segment denotes an untranslated DNA sequence located 3 ' to the rat cDNA sequence encoding Type I iodothyronine 5 ' deiodinase.
  • stem-loop structure denotes a stem loop structure located in the 3' untranslated region of mRNA of a selenocysteine containing protein which allows a UGA codon to be recognized as a selenocysteine codon rather than a UGA stop codon.
  • Suitable structures include, for example, specific sequences located in the 3' untranslated region of the wild-type selenocysteine containing construct, for example, the sequence located between nucleotides 1440 and 1615 in the wild-type 5' deiodinase construct; and functional equivalents thereof.
  • a functional equivalent is defined as a stem-loop structure which allows a UGA codon to be recognized as a selenocysteine codon and not a stop codon.
  • SECIS senocysteine-insertion sequence
  • reporter gene is meant to refer to both mutant sequences of iodothyronine 5' deiodonese, including for example cysteine ⁇
  • a plasmid is said to be an "internal control" if the plasmid is such that cDNA coding for a reporter enzyme is cloned into a plasmid having incorporated therein a constitutive promoter, including for example TK.
  • the present invention relates in part to the cloning of the gene which encodes Type I iodothyronine 5' deiodinase (5 ' deiodinase).
  • a first step for obtaining a gene sequence which encodes the rat 5 ' deiodinase comprises obtaining DNA from cells which contain such gene sequences. This DNA is used to prepare a genomic library. Alternatively. cDNA is obtained using cells expressing 5 ' deiodinase and a cDNA library is prepared. Techniques for preparing such libraries are disclosed by Maniatis, et al. (In: Molecular Cloning, A Labonitory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor. NY ( 1982)). A cDNA library can be conveniently prepared using rat liver poly(A) + RNA.
  • the above-described library is then screened for gene sequences which hybridize to a probe sequence of either the entire rat liver 5' deiodinase encoding sequence, a sequence complementary to such 5' deiodinase-encoding sequence, or a fragment of either of such sequences.
  • a probe sequence of either the entire rat liver 5' deiodinase encoding sequence, a sequence complementary to such 5' deiodinase-encoding sequence, or a fragment of either of such sequences are then screened for gene sequences which hybridize to a probe sequence of either the entire rat liver 5' deiodinase encoding sequence, a sequence complementary to such 5' deiodinase-encoding sequence, or a fragment of either of such sequences.
  • human 5 ' deiodinase expressing cells are used to produce a DNA (or cDNA) library.
  • the members of this library are screened for their ability to hybridize with the above-described rat 5 ' deiodinase probe sequence using techniques, such as those disclosed by Maniatis, et al. (In: Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), or by Haymes, et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)).
  • a preferred method for preparing the desired sequence is to obtain a 1.9 to 2.4 kb fraction of rat liver pory(A) + RNA using the methods described in Berry, et al, Molec Endo. 4:743-748 (1990), and St. Germain, et al, J. Biol. Chem. 264:3054-3056 (1989). Briefly, the poly(A) + RNA was injected into Xenopus oocytes. Plasmid DNA from the resulting cDNA was transcribed in vitro, the resulting RNA injected into oocytes, and oocyte homogenates assayed for deiodination of 3,3 ',5 '-triiodothyronine. This strategy resulted in isolation of a clone designated G21.
  • the DNA probe for identifying and isolating DNA encoding 5 ' deiodinase may be labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassays and in general most any label useful in such methods can be applied to the present invention.
  • Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem. 22: 1243 ( 1976)): enzyme substrates (see British Pat. Spec. 1,548,741 ): coenzymes (see U.S. Pat. Nos. 4,230,797 and 4,238,565); enzyme inhibitors (see U.S. Pat. No.
  • Such labels and labeling pairs are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).
  • a cofactor-labeled probe can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme.
  • an enzyme which acts upon a substrate to generate a product with a measurable physical property examples of the latter include, but are not limited to, beta-galactosidase, alkaline phosphatase and peroxidase.
  • hybridization of the probe to the DNA sequences of the library may be accomplished under a variety of conditions of stringency so as to permit either a stable hybrid to form only between two gene sequences which have very similar sequences (high stringency) or to permit such a hybrid to form between two gene sequences having more divergent sequences (low stringency).
  • Conditions of high stringency employ high temperatures (such as 50-65°C) and high concentrations of agents such as formamide (for example 50% formamide).
  • Conditions of low stringency employ lower temperatures (approximately 42°C) and lower concentrations of agents such as formamide (for example 20-40% formamide) ((Lawler, et al, Bone Marrow Transpl.
  • the gene sequence can be introduced into a suitable host cell, expressed, and the expressed protein tested for its ability to deiodinate 3,3 ',5'-triiodothyronine (rT 3 ).
  • rT 3 deiodinate 3,3 ',5'-triiodothyronine
  • a gene sequence which expresses a protein that is capable of catalyzing this reaction encodes 5 ' deiodinase.
  • the expressed molecule can be tested for its ability to bind to antibody (prepared as described below) that is reactive with 5 ' deiodinase.
  • the isolated sequence encodes only a fragment of the desired gene sequence. Accordingly, the isolated gene sequence is used to identify and isolate any missing fragments of the desired gene sequence (Bender, et al, J. Supramolec. Struc. 10(suppl):32 (1979); Chinault, et al, Gene 5:111 ( 1979); Clarke, el al, Nature 287:504 (1980)). Once any such sequences have been identified and isolated, it is possible to construct a single gene sequence which is capable of encoding the entire desired enzyme using well known methods of recombinant DNA technology.
  • the expressed enzyme should possess at least one selenocysteine residue(s), preferably at site 126.
  • Selenocysteine is encoded by the codon UGA, which generally functions as a termination codon (the "opal" codon).
  • incorporation of selenocysteine at the appropriate UGA-encoded site requires that a 3 ' untranslated segment of DNA be operably linked to the 5 ' deiodinase-encoding region.
  • this DNA segment is found between nucleotides 1360 and 1615. more essentially between nucleotides 1440 and 1615. of the 3 ' untranslated region, and is approximately 200-255 nucleotides in length, more essentially 200 nucleotides in length.
  • the approximately 200-255 nucleotide segment can also be inserted immediately 3 ' to the 5' deiodinase coding region to achieve expression of the active enzyme.
  • UGA is recognized as a termination signal, resulting in expression of an incomplete and inactive form of 5' deiodinase.
  • the invention is also related to 5' deiodinase enzymes which retain activity but differ from the native enzyme by at least one amino acid.
  • Amino acid sequence variants of 5' deiodinase can be prepared by mutations in the DNA. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence shown in Figure 1. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
  • the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see EP Patent Application Publication No. 75,444).
  • these variants ordinarily are prepared by site-directed mutagenesis of nucleotides in the DNA encoding the 5 ' deiodinase molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring analog.
  • the mutation per se need not be predetermined.
  • random mutagenesis may be conducted at the target codon or region and the expressed 5 ' deiodinase variants screened for the optimal combination of desired activity.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, site-specific mutagenesis.
  • Preparation of a 5 ' deiodinase variant in accordance herewith is preferably achieved by site-specific mutagenesis of DNA that encodes an earlier prepared variant or a nonvariant version of the protein.
  • Site-specific mutagenesis allows the production of 5 ' deiodinase molecule variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by publications such as Adelman et al, DNA 2: 183 ( 1983), the disclosure of which is incorporated herein by reference.
  • the site-specific mutagenesis technique typically employs a phage vector that exists in both single-stranded and double-stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage, for example, as disclosed by Messing et al, Third Cleveland Symposium on Macromotecules and Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam ( 1981), the disclosure of which is incorporated herein by reference. These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • plasmid vectors that contain a single-stranded phage origin of replication may be employed to obtain single-stranded DNA.
  • site-directed mutagenesis can be performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein.
  • An oligonucleotide prime, bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al., Proc. Natl. Acad. Sci. (USA) 75:5765 (1978).
  • This primer is then annealed with the single- stranded protein-sequence-containing vector, and subjected to DNA- porymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA- porymerizing enzymes
  • This heteroduplex vector is then used to transform appropriate cells such as JEG-3 cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • a preferred mutagenesis method is that developed by Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, employing the pSELECT TM -1 vector system.
  • One of skill will choose an appropriate system for use.
  • the mutated protein region may be removed and placed in an appropriate vector for protein production.
  • an expression vector of the type that may be employed for transformation of an appropriate host.
  • Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably 1 to 10 residues, and typically are contiguous.
  • Amino acid sequence insertions include amino and/or carboxyl-terminal fusions of from one residue to polypeptides of essentially unrestricted length, as well as intrasequence insertions of single or multiple amino acid residues.
  • Intrasequence insertions i.e., insertions within the complete 5' deiodinase molecule sequence
  • An example of a terminal insertion includes a fusion of a signal sequence, whether heterologous or homologous to the host cell, to the N-terminus of the 5 ' deiodinase molecule to facilitate the secretion of mature 5 ' deiodinase molecule from recombinant hosts.
  • the third group of variants are those in which at least one amino acid residue in the 5 ' deiodinase molecule, and preferably, only one, has been removed and a different residue inserted in its place.
  • deletions and insertions, and substitutions in particular are not expected to produce radical changes in the characteristics of the 5 ' deiodinase molecule.
  • substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
  • a variant typically is made by site-specific mutagenesis of the native 5 ' deiodinase molecule-encoding nucleic acid, expression of the variant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity adsorption on a polyclonal anti-5 ' deiodinase molecule column (to adsorb the variant by binding it to at least one remaining immune epitope).
  • the activity of the cell lysate or purified 5 ' deiodinase molecule variant is then screened in a suitable screening assay for the desired characteristic. For example, a change in the immunological character of the 5 ' deiodinase molecule, such as affinity for a given antibody, is measured by a competitive type immunoassay. Changes in immunomodulation activity are measured by the appropriate assay. Modifications of such protein properties as red ox or thermal stability, hydrophobicity. susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.
  • DNA or cDNA molecules which encode 5' deiodinase can be operably linked to an expression vector and introduced into a host cell to enable the expression of the 5 ' deiodinase molecule by that cell.
  • Two DNA sequences (such as a promoter region sequence and a desired 5 ' deiodinase molecule encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired 5' deiodinase molecule encoding gene sequence, or (3) interfere with the ability of the desired 5 ' deiodinase molecule gene sequence to be transcribed by the promoter region sequence.
  • the DNA or cDNA molecule is preferably operably linked to a 3 ' untranslated region necessary for the incorporation of selenocysteine at the appropriate UGA codon
  • a DNA sequence encoding a 5 ' deiodinase molecule may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or stagger-ended termini for ligation, restriction digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases.
  • the present invention encompasses the expression of 5 ' deiodinase in either prokaryotic or eukaryotic cells.
  • Preferred eukaryotic hosts include yeast (especially Saccharomyces), or mammalian cells (such as, for example, human or primate cells).
  • Yeast and mammalian cells are preferred hosts of the present invention.
  • the use of such hosts provides substantial advantages in that they can also carry out post-translational peptide modifications including glycosylation.
  • Yeast recognize leader sequences on cloned mammalian gene products and secrete peptides bearing leader sequences (i.e., pre-peptides). Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites.
  • Mammalian cells which may be useful as hosts include cells such as JEG-3 human choriocarcinoma cells, and their derivatives. Liver, kidney or pituitary cell lines may also be suitable host cells.
  • a mammalian host several possible vector systems are available for the expression of the desired protein molecule.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, etc.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.
  • eukaryotic regulatory regions Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer. ct al. J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, et al, Nature (London) 290:304-310 (1981)); the yeast gal4 gene promoter (Johnston, et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, et al, Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).
  • the vector system contains the 3'-untranslated region necessary for incorporation of selenocysteine into the polypeptide at a site corresponding to the appropriate UGA codon site in the 5 ' deiodinasecoding region.
  • procaryotic hosts include bacteria such as E. coli, Bacillus. Streptomyces, Pseudomonas, Salmonella, Setralia, etc.
  • the most preferred prokaiyotic host is E. coli.
  • Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X I 776 (ATCC 31537), E. coli W3110 (F-, lambda-, prototrophic (ATCC 27325)). and other enterobacteria (such as Salmonella typhimurium or Serratia marcescens). and various Pseudomonas species.
  • the prokaiyotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • 5 ' deiodinase in a prokaryotic cell (such as. for example. E. coli. B. subtilis, Pseudomonas. Streptomyces, etc.). it is neces- sary to operably link the 5' deiodinase encoding sequence to a functional prokaryotic promoter.
  • promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage ⁇ , and the bla promoter of the ⁇ -lactamase gene of pBR322.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ), the trp, recA, lacZ, lad, gal, and tac promoters of E. coli, the ⁇ -amylase (Ulmanen, et al, J. BacterioL 762:176-182 (1985)), the ⁇ -28-specific promoters of B.
  • subtilis (Gilman, et al., Gene 32:11-20 (1984)), the promoters of the bacteriophages oi Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, et al, Mol. Gen. Genet. 203:468-478 (1986)).
  • Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)).
  • ribosome binding sites are disclosed, for example, by Gold, et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).
  • the 5 ' deiodinase encoding sequence may be introduced into a recipient prokaryotic or eukaiyotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the desired protein molecule may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome
  • a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may complement an auxotrophy in the host (such as leu2, or ura3, which are common yeast auxotrophic markers), biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by cotransfection.
  • the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • yeast gene expression systems can be utilized.
  • expression vectors include the yeast 2-micron circle, the expression plasmids YEP13, YCP and YRP, etc., or their derivatives.
  • Such plasmids are well known in the art (Botstein, et al, Miami Wntr. Symp. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981): Broach. J.R., Cell 28:203-204 (1982)).
  • vectors For a mammalian host, several possible vector systems are available for expression.
  • One class of vectors utilize DNA elements which provide autonomously replicating extra-chromosomal plasmids. derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, or SV40 virus.
  • a second class of vectors relies upon the integration of the desired gene sequences into the host chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes may be selected by also introducing one or more markers which allow selection of host cells which contain the expression vector.
  • the marker may provide for prototropy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like.
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • the cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol. Cell. Biol. 5:280 (1983), and others.
  • the preferred expression vector is the CDM-8 mammalian expression vector (Aruffo et al, Proc. Null Acad. Sci. USA 54:8573-8577 (1987)).
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli such as, for example, pBR322, ColE1, pSC101, pACYC 184, ⁇ VX.
  • plasmids are, for example, disclosed by Maniatis, et al. (In: Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • Bacillus plasmids include pC194. pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, NY ( 1982), pp. 307-329).
  • Suitable Streptomyces plasmids include pIJ 101 (Kendall. et al, J. Bacietiol. 169:4177-4183 (1987)), and Streptomyces bacteriophages such as ⁇ C31 (Chater, et al., In: Sixth International Symposium on Actinomycetales Biology. Akademiai Kaido, Budapest, Hungary ( 1986). pp. 45-54). Pseudomonas plasmids are reviewed by John, et al. (Rev. Infect. Dis. 8:693-704 ( 1986)). and Izaki. K. (Jpn. J. Bacterial. 33:729-742 ( 1978)).
  • the DNA constructs may be introduced into an appropriate host.
  • Various techniques may be employed, such as protoplast fusion, calcium phosphate precipitation, electroporation or other conventional techniques. After the fusion, the cells are grown in media and screened for appropriate activities. Expression of the sequence results in the production of the protein molecule.
  • the 5' deiodinase molecules of the invention may be isolated and purified from the above-described recombinant molecules in accordance with conventional methods, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like. Such conventional methods can yield 5' deiodinase in substantially pure form.
  • the molecules in the substantially purified fractions are recovered by any suitable method. Most preferably, for example, such recovery is accomplished by affinity chromatography, followed by concentration of sample, and resolution by gel electrophoresis. The recovered molecules may then be sequenced, preferably using an automated sequenator, and the amino acid sequence of the molecule thereby determined.
  • the sequence of the 5' deiodinase molecule may be determined using the microsequencing methods of Rodriguez (J. Chromatog. 350:217 (1985)).
  • the 5' deiodinase molecule may be purified by electrophoresis and, after electroelution, cleaved by cyanogen bromide or lysyl-C endopeptidase. The fragments may then be resolved, preferably by HPLC or by tricine gels (H. Shagger et al. Anal. Biochem. 166:368 (1987)) followed by electroblotting and gas-phase microsequencing. The sequence of the complete molecule can then be determined and compared with that deduced from the cDNA sequence of 5 ' deiodinase.
  • kits containing the elements necessary to carry out the methods of the invention.
  • a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means, such as tubes or vials.
  • One of the container means may contain an unlabeled or detectably labeled polynucleotide sequence, such as for example the radioactively labeled DNA or RNA encoding Type I iodothyronine 5 ' deiodinase.
  • the labeled polynucleotide sequence may be present in lyophilized form, or in an appropriate buffer as necessary.
  • One or more container means may contain one or more endonuclease enzymes to be utilized in digesting the nucleic acids from the cells or tissues under analysis. These enzymes may be present by themselves or in admixtures, in lyophilized form or in appropriate buffers.
  • the kit may also contain in one container probe RNA for probe synthesis, in another container radiolabeled deoxyribonucleoside triphosphate, and in another container primer. In this manner the user can prepare probe cDNA.
  • kit may contain all of the additional elements necessary to carry out the methods of the invention, such as buffers, media, enzymes, pipettes, plates, nucleic acids, nucleoside triphosphates, filter paper, gel materials, transfer materials, autoradiography supplies, and the like.
  • the 5 ' deiodinase molecules of the present invention may be used to induce the formation of anti-5 ' deiodinase antibodies.
  • Such antibodies may either be polyclonal or monoclonal antibodies, or antigen binding fragments of such antibodies (such as for example, F(ab) or F(ab) 2 fragments).
  • Suitable polyclonal antibodies can be obtained by immunizing an animal with an immunogenic amount of the 5' deiodinase molecule (preferably with an adjuvant, such as Freund's adjuvant).
  • monoclonal antibodies may be prepared, such as by immunizing splenocytes with 5' deiodinase and then fusing an immunized cell with a myeloma cell (Kohler et al, Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al, Eur. J. Immunol. 6:292 (1976); Hammerling et al, In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)) in order to obtain a hybridoma cell that secretes an anti-5' deiodinase antibody.
  • antibodies which are produced in humans, or are "humanized” (i.e. non-immunogenic in a human) by recombinant or other technology such that they will not be antigenic in humans, or will be maintained in the circulating serum of a recipient for a longer period of time.
  • Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies)
  • a corresponding, but non-immunogenic portion i.e. chimeric antibodies
  • the anti-5 ' deiodinase antibodies of the present invention may be used for diagnostic purposes such as to measure the expression and function of a patient's 5 ' deiodinase.
  • the anti-5 ' deiodinase antibodies can also be used in imaging in order to characterize tissue, or to define the presence and site of metastasized 5 ' deiodinase-expressing cells.
  • the 5 ' deiodinase and anti-5 ' deiodinase antibodies can be used in accordance with immunoassay technology.
  • immunoassays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970.
  • 5' deiodinase molecules can be detectably labeled and incubated with a sample, and the amount of 5 ' deiodinase molecule bound to the sample can be ascertained.
  • antibody to the 5' deiodinase can be used in order to create a "pseudo-sandwich immunoassay.”
  • a sample suspected of containing 5 ' deiodinase can be incubated in the presence of an immobilized anti-5 ' deiodinase antibody. Solubilized. detectably labeled, 5 ' deiodinase molecules can be added to the reaction mixture, and the amount of 5 ' deiodinase determined by measuring the amount of bound label.
  • the assay may be a simple ''yes/no" assay to determine whether 5 ' deiodinase is present or may be made quantitative by comparing the measure of labeled molecule with that obtained for a standard sample containing known quantities of 5 ' deiodinase.
  • “simultaneous” and “reverse” assays are used.
  • a simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled molecules associated with the solid support is then determined as it would be in a conventional sandwich assay.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the simultaneous and forward assays.
  • the principal reagents can be packaged in kit form for a particular assay together with any additional components needed or desired, such as a set of standard analyte solutions which mimics or covers the anticipated concentration range for the 5' deiodinase.
  • a buffer for dilutions of reconstituted reagents or for pH adjustment may be included.
  • the kit may include labeled antibody, and unlabeled antibody bound to a solid support.
  • the kit may also contain labeled Type I 5' deiodinase molecules.
  • the various components can be packaged in the kit in solution or lyophilized form, depending upon the stability, shipping and other requirements.
  • Quantitation of nucleic acid molecules which encode the 5 ' deiodinase molecule (or a fragment thereof) can be used to determine the extent and rate of the expression of the 5 ' deiodinase in the cells of a patient.
  • a sample of a patient's cells is treated, via in situ hybridization, or by other suitable means, and analyzed to determine whether the sample contains mRNA molecules capable of hybridizing with the nucleic acid molecule.
  • Type I 5 ' deiodinase The elucidation of the amino acid sequence of Type I 5 ' deiodinase is useful in the development and testing of compounds that inhibit the conversion of T 4 to T 3 . Such compounds have therapeutic value in the treatment of certain forms of hyperthyroidism.
  • Type I 5 ' deiodinase contains the rare amino acid selenocysteine.
  • a protein preparation can be obtained from a cell that normally expresses Type I 5' deiodinase or from a host cell, such as a JEG-3 cell, transfected with cDNA encoding the enzyme.
  • 5' deiodinase is a second mammalian selenocysteine-containing enzyme. Furthermore, substitution of selenocysteine with cysteine at site 126 reduces enzyme activity by at least 90%. Thus, selenocysteine is essential for normal activity of this enzyme.
  • the first step is the identification of a protein as containing at least one selenocysteine moiety. It is then preferable to identify a cell type which naturally synthesizes this protein, in order to obtain a total poly(A) + RNA population.
  • Initial recovery of the RNA encoding the desired protein can be enhanced if the cell or tissue is subject to manipulations to increase production of the protein.
  • Type I 5 ' deiodinase activity is elevated in the hyperthyroid state, and poly(A) + RNA can be isolated from tissues of rats made hyperthyroid by treatment with injection of T 4 .
  • the RNA can be size-fractionated and introduced into a suitable cell for transcription.
  • rat liver poly(A) + can conveniently be translated in Xenopus oocytes.
  • the cell homogenates are tested for the presence of the desired protein.
  • detection can be by measuring enzymatic activity, such as deiodination of 3,3 ',5 '-triiodothyronine in the case of 5 ' deiodinase.
  • the protein can be detected using a binding molecule capable of specifically binding the protein.
  • a preferred method is the use of an antibody directed against the protein.
  • the protein may be detected by its ability to bind a suitably labeled molecule which is capable of binding to the receptor.
  • a cDNA library for further screening in cells capable of translating the mRNA, such as Xenopus oocytes. After one or more clones representing the cDNA encoding the desired protein have been identified, these clones can be used to identify and locate regions of the cDNA that are important for co-translational incorporation of selenocysteine into the protein.
  • the DNA sequence of a cDNA clone capable of directing expression of the desired protein is determined. Putative initiation and termination codons can be identified, as can restriction endonuclease sites.
  • the next step is the identification of sequences necessary for expression of the protein. It is preferable to construct cDNA molecules having terminal and internal deletions, as well as a frameshift mutation or insertion. The effect of these various alterations on the ability of the cDNA to express the protein is determined using a suitable cell type. The presence of expressed protein indicates that the deleted or mutated sequence was not essential for expression. According to the present invention, this sequence of steps was successfully used to identify a 3' untranslated sequence in the cDNA, the presence of which was necessary for successful expression of the protein 5' deiodinase. The importance of a particular sequence of the cDNA for the successful translation of other selenocysteine-containing proteins can therefore be determined in a similar manner, using the steps disclosed above.
  • the present invention now discloses for the first time the importance of an untranslated region for selenocysteine incorporation.
  • the flanking nucleotides of the TGA codon, or the intracellular environment effected the co- translational incorporation of selenocysteine at a TGA-encoded site (Engelberg-Kulka, et al, Trends in Biochem. Sci. 13:419-421 (1988)).
  • the present invention further provides methods and genetic constructs for achieving expression of a selenocysteine-containing protein such as 5' deiodinase.
  • a selenocysteine-containing protein such as 5' deiodinase.
  • Expression of most mammalian proteins can be accomplished by the transfection of a suitable host cell with DNA consisting of the structural gene for the protein, operably linked to a suitable promoter region and, optionally, a region encoding a secretion signal. Such methods are described above in detail.
  • the genetic construct should additionally contain a 200-255 nucleotide 3' untranslated region, with a 200 nucleotide sequence being essential, having the sequence of nucleotides 1360-1615 of Figure 1, with nucleotides 1440- 1615 being essential, or a functional equivalent thereof.
  • the approximately 200-255 nucleotide untranslated segment can be located 1-582 nucleotides from the 3 ' end of the structural gene for 5 ' deiodinase.
  • Successful expression can equally be accomplished by locating the approximately 200-255 nucleotide untranslated segment immediately 3 ' to the structural gene.
  • An additional aspect of the present invention relates to the introduction of one or more selenocysteine residues into a polypeptide or protein which, in its native state, does not contain selenocysteine.
  • Such modification of a protein may be desired in order to alter or enhance the function of the polypeptide or protein.
  • the selenium moiety of the selenocysteine residue would further provide a highly conserved, isomorphic reference atom for X-ray crystallographic analysis.
  • the TGA codon intended to encode selenocysteine can be introduced into the DNA encoding the polypeptide or protein by means known in the art, discussed fully above in Section II.
  • the DNA having the TGA codon is then used to construct an expression vector having a suitable operably linked promoter, and a 3 ' untranslated segment, in the presence of which selenocysteine is co-translationally incorporated into the protein or polypeptide at a site corresponding to the TGA codon.
  • the preferred locations of the untranslated segment are more fully discussed above.
  • Benign and malignant tumors can develop in the thyroid, and malignant thyroid tumors can spread, for example to lung or bone tissue.
  • the presence of Type I 5 ' deiodinase mRNA in a tissue can aid in characterizing the presence of thyroid-derived cells.
  • the absence of Type I 5' deiodinase mRNA in a sample of thyroid tissue would suggest that the tissue is non-functioning and indicate the possible presence of carcinoma.
  • the presence of type I 5' deiodinase mRNA can be detected by contacting the tissue with detectably labeled DNA containing a sequence complementary to the mRNA.
  • an RNA preparation from the tissue can be introduced into a cell, such as a Xenopus laevis oocyte, and expression of Type 15' deiodinase assayed.
  • the amount of mRNA can be compared with that of normal thyroid tissue, or with normal tissue similar to that suspected of containing malignant thyroid-derived cells, in order to evaluate the status of the tissue in question.
  • a unidirectional, size-fractionated rat liver cDNA library for expression screening in Xenopus oocytes was constructed using methods as described in Berry, et al, Mol. Endocrin. 4:743-748 ( 1990).
  • cDNA synthesis was catalyzed by AMV reverse transcriptase (Life Sciences) (Gubler et al, Gene, 25:263-269 (1983)). Double stranded cDNA was size-fractionated on low melting temperature agarose (Sea Plaque, FMMC) and the region corresponding to 1.8 to 2.5 kb isolated. The resulting cDNA was ligated to adaptors (In Vitrogen), inserted into lambda Zap II (Stratagene). and packaged in vitro.
  • the library was subdivided, amplified, and converted to Bluescript plasmid by in viva excision as described in the procedures of Stratagene. Plasmid DNA was linearized and transcribed in vivo using T7 RNA polymerase. Xenopus laevis oocytes were manually dissected, injected with in vitro transcribed RNA (0.5 to 20 ng per oocyte in 40 nl diethylpyrocarbonate-H 2 O (DEPC-H 2 O)), and incubated for 3 days at 18°C in 50% Leibovitz's L-15 media, 15 mM HEPES, 100 ⁇ g/ml gentamycin and 50 units/ml nystatin.
  • in vitro transcribed RNA 0.5 to 20 ng per oocyte in 40 nl diethylpyrocarbonate-H 2 O (DEPC-H 2 O)
  • Type I 5' deiodinase assays of oocyte homogenates were performed as described previously (Berry et al, Molec. Endo. 4:743-748 (1990)). Because Type I deiodinase exhibits a 1000-fold higher V max /K m ratio for rT 3 than for T 4 (Leonard, et al., In: Hennemann, G. (ed.), Thyroid Hormone Metabolism, Marcel Dekker, New York, pp. 189-229 (1986)), rT 3 was used as a substrate for type I deiodinase assays.
  • RNA-injected or uninjected oocytes were homogenized in 100 mM potassium phosphate (pH 6.9)-1 mM EDTA in microcentrifuge tubes, using a Teflon pestle. Homogenates were then divided into two or three replicate assays. Reaction volumes were adjusted to 100 ⁇ l/oocyte. Type I deiodinase reactions were initiated by the addition of 0.5 nM [ 125 I]rT 3 and 10 mM DTT.
  • DNA sequencing of both upper and lower strands was by the dideoxy method (Sanger et al, Proc. Natl. Acad. Sci. USA. 74:5463-5467 (1977)), using a T7 sequencing kit from Pharmacia.
  • RNA was isolated from rat tissues by standard guanidiniumthiocyanate methods as described previously (Berry et al., Molec. Endo. 4:743-748 (1990)). Briefly, rats were made hyperthyroid by five sc injections of T 4 (12 ⁇ g/100 g BW) over 3-5 days. Hypothyroidism was produced by giving rats 0.02% methimazole in drinking water for 3 weeks. Livers from six rats were used for each RNA preparation.
  • Liver was homogenized in 4.0 M guanidium thiocyanate, 20 mM sodium acetate, 10 mM vanadyl ribonucleoside complex, and 20 mM dithiothreitol (DTT) in a Brinkmann Polytron homogenizer (Westbury, NY), followed by three passages through a 20-gauge needle to shear chromosomal DNA.
  • the homogenate was layered onto 12-ml cushions of 5.7 M CsCl-0.1 M EDTA, pH 8.0, and centrifuged at 27,000 rpm for 18 h in a Beckman SW 28 rotor (Fullerton, CA) at 15°C.
  • RNA pellets were resuspended in 2 mM EDTA (pH 8.0)-0.1% sodium dodecyl sulfate (SDS) and extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), followed by ethanol precipitation. Precipitated RNA was resuspended in 2 mM EDTA (pH 8.0)-0.1%; SDS. Polyadenylated [poly(A) + ] RNA was obtained by two cycles of chromatography on oligo-(dT) cellulose (Collaborative Research,
  • Poly(A) + RNA was ethanol precipitated and resuspended in diethyl pyrocarbonate treated (DEPC)-H 2 O before injection into oocytes or agarose gel size fractionation. Either total or poly(A) + RNA was electrophoresed on 1.1% agarose formaldehyde gels. Blots were probed with clone G21 cRNA or ⁇ -actin cRNA, and washed at high stringency.
  • DEPC diethyl pyrocarbonate treated
  • Lanes 1-6 of Figure 2 contain
  • Lane 7 and 8 of Figure 2 contain 2 ⁇ g poly(A) + RNA, from the thyroids of methimazole treated rats (lane 7) and from kidney (lane 8). Lanes 9 and 10 contains 5 ⁇ g poly(A) + RNA from pituitary and brown adipose tissue, respectively. Autoradiography of lanes 1-6 was for 4 days, lanes 7 and 8 for 1 hr., and lanes 9 and 10 for 1 week.
  • mice were made hypothyroid by treatment for 3 weeks with 0.02% methimazole in the drinking water. Hyperthyroidism was induced by intraperitoneal injection of 50 ⁇ g T 3 daily for three days.
  • Deletions were constructed by restriction digestion at the indicated sites in clone G21 and convenient sites in the vectors, followed by agarose gel purification of the desired fragments, rehgation, and mapping of the resulting constructs.
  • the Ace I frameshift was constructed by digestion with Ace I, followed by conversion to blunt ends with DNA polymerase large fragment, and rehgation. All mutations were confirmed by DNA sequencing.
  • RNAs were transcribed in vitro and 0.1 to 20 ng injected per oocyte. DNA transfections (Brent et al, Molec. Endo 3:1996-2004) and deiodinase assays (Berry et al, Molec. Endo. 4:743-748 (1990)) were as described previously. Assay of human growth hormone (hGH) in the media from a cotransfected hGH expressing plasmid confirmed equal transfection efficiencies (Brent et al, Molec. Endo 3:1996-2004).
  • hGH human growth hormone
  • Oocyte activity of 100% is defined as deiodination of 30 to 40% of 2 nM 125 I rT 3 /hr with a homogenate of 4 oocytes injected with 0.1 ng G21 RNA per oocyte.
  • G21 RNA was at least 100-fold more active per nanogram than liver poly(A) + RNA.
  • JEG-3 cells were homogenized and 100 to 300 ⁇ g protein from homogenates or 20,000 ⁇ g pellets were incubated in a volume of 400 ⁇ l containing 25 mM DTT and 5 nM 125 I rT 3 for 1 hr.
  • 125 I was quantitated as described (Berry et al, Molec. Endo. 4:743-748 (1990)). Equal quantities of 125 I and 3.3 ' diiodothyronine are produced during this reaction. The results are shown in Figure 7; all assays were in duplicate. ND, not done.
  • the TGA codon at nucleotide 382 was replaced by the indicated codons (see Figure 8) using the P-select ® in vitro mutagenesis system of Promega. Briefly, the insert from clone G21 was ligated into P-select and single stranded phagemid DNA was obtained. Oligonucleotides corresponding to the desired changes were annealed to the single-stranded DNA and double-stranded DNA was synthesized. The entire coding regions of plasmids thus obtained were sequenced to confirm that these were the only mutations. Injection and assays were described above.
  • Wild type (TGA) or cysteine mutant (TGT) G21 cDNA in CDM-8 was transfected into JEG-3 as described in Example III.
  • Cell sonicate protein 435 ⁇ g (wild type) or 680 ⁇ g (cysteine mutant)
  • 125 I was quantitated as described (Berry et al., Molec. Endo. 4:743-748 ( 1990)).
  • 1- ⁇ -D-Thioglucose (5 ⁇ M) had no effect on enzyme activity. The results are presented in Table 1.
  • Rat Type I iodothyronine 5' deiodinase is encoded by a DNA sequence of approximately 778 nucleotides in length, as shown in
  • the cDNA there is a region of approximately 582 nucleotides between the 3 ' end of the 5' deiodinase coding region and the necessary 255-nucleotide untranslated region.
  • the 255 nucleotide segment can also be inserted immediately 3 ' of the coding region to achieve translation of a completely active enzyme. No expression of the enzyme is found if these 255 nucleotides are removed or if the sequence is inverted.
  • expression of enzymatically active 5' deiodinase is achieved by transfection of a host cell with a DNA sequence comprising the structural gene for 5' deiodinase, and a DNA segment consisting of nucleotides 1360-1615 of the untranslated region of 5' deiodinase cDNA.
  • the DNA segment can be located immediately 3 ' to the coding region. Alternatively, up to approximately 582 nucleotides can be present between the 3' end of the coding region and the 255 nucleotide segment.
  • Chimeric constructs were generated by PCR-amplification using 5' oligonucleotides encoding an XmaI site and 3' oligonucleotides encoding a NotI site adjacent to sequences specific for the region to be amplified. PCR products were cloned into sequences specific for the region to be amplified. PCR products were cloned into XmaI + NotI cut CDM-8 containing the 5' deiodinase cDNA. This vector fragment contains the 5' deiodinase coding region and 126 base pairs of 3' untranslated region, and is non-functional for deiodinase activity in transient transfection and oocyte injection assays.
  • JEG-3 or COS-7 cells were transfected with calcium phosphate-DNA precipitates as described previously (Brent et al., Molecular Endocrinol 3:1996-2004 (1989)). Transfection efficiencies were monitored by assay of human growth hormone in the media, produced by a cotransfected constitutive thymidine kinase promoter-directed human growth hormone-expressing plasmid (Brent et al, Molecular Endocrinol. 5:1996-2004 (1989)).
  • Type I iodothyronine deiodinase catalyzes the first step in thyroid hormone action, the monodeiodmation of the prohormone, thyroxine
  • T4 to form the active thyroid hormone, 3,5,3 '-triiodothyronine (T 3 ).
  • T 4 3,5,3 '-triiodothyronine
  • a cysteine mutant is also functional, albeit with a 10-fold higher apparent K m for the preferred substrate, 3,3',5' triiodothyronine (reverse T 3 ,7.10).
  • the open-reading frame of the 2.1 kb rat 5' deiodinase mRNA begins at nucleotide 7 and ends at 780, and the UGA (selenocysteine) codon is located at nucleotides 382-384 (Berry et al, Nature 349:438-440 ( 1991)
  • the relative 5' deiodinase activity produced by injection of these RNAs into Xenopus oocytes paralleled that produced by transfection, evidence that impaired translation causes the reduced expression by the 3' untranslated mutants.
  • 3' untranslated sequences of the cDNA for the rat 5' deiodinase were compared with the sequence of a cDNA for the human 5' deiodinase. Although the 3' untranslated sequences are overall about 55% conserved, a region of ⁇ 79% identity (nucleotides 1642 to 1819) corresponded to the essential 3' untranslated sequences (1440- 1615) identified in the rat 5' deiodinase cDNA.
  • Examination of the 3' untranslated region of the rat GPX mRNA revealed less than 38'v primary sequence similarity to the conserved 5' deiodinase sequence.
  • Reticulocyte lysates translate rat 5' deiodinase mRNA inefficiently, producing small amounts of full length ⁇ 27 kDa protein, with most of the translated product being the ⁇ 14 kDa protein predicted by termination at the UGA codon (Berry et al, Nature 349:438-440 (1991)). If the 3' untranslated sequences are involved in selenocysteine codon recognition, the ratio of 27 to 14 kDa protein should be reduced in in vitro translations of 3' untranslated mutant transcripts. In vitro transcripts of wild-type and mutant 5' deiodinase constructs were prepared.
  • RNA was translated in vitro and the translation products were immunoprecipitated with 5' deiodinase specific antisera generated against a rat 5' deiodinase amino-terminal peptide.
  • the 27 and 14 kDa 35 S methionine labelled products were quantitated after SDS-polyacrylamide gel electrophoresis.
  • the ratio of 27 to 14 kDa protein was 0117 ⁇ 0.03 for the Rat WT, 0.08 ⁇ 0.04 for the Human M1, and 0.013 ⁇ 0.004 for the Rat M2 construct.
  • GPX mRNAs identifies the conserved nucleotides shown in Figure 10.
  • SECIS senocysteine-insertion sequence
  • cysteine mutant (G-5) is proportional to DNA input into the transfection system (see Table II).
  • column 1 are shown the quantities of DNA co-transfected into COS cells (G5DI) together with 3 ⁇ g of TKGH.
  • the activity in the cell extracts (deiodinase-"DI") and hGH in the medium (hGH) are shown on the right side of the table.
  • the calculated ratio of deiodinase to hGH is shown in the far right column.
  • TKGH is a reporter gene for control of internal transfection efficiency. If the uptake and expression of G5 is proportional to DNA input into the transfection system then this will be reflected in the ratio of Dl to hGH.
  • the ratio of Dl to hGH parallels the ratio of input G5DI to TKGH DNA over a 10-fold range (5/3 to 0.5/3). Notable is the fact that this relationship is maintained even though the uptake and expression of the TKGH plasmid in plates 1 and 2 (1843 cpm) is less than half of that in plates 3 through 6 (5210 and 5690). Since the expression of G5 does not require the selenocysteine insertion sequence motif it should have a broader repertoire of utility for different cell lines than would the wild-type enzyme.
  • the activity of G5DI in this experiment is regulated by the CMV promoter in the construct CDM8.
  • a further useful modification is to attach a signal peptide to the amino terminus of the G-5 or wild-type enzyme to permit deiodinase secretion into the media similar to the transiently expressed human growth hormone (hGH) employed in the experiment shown in Table II.
  • hGH human growth hormone
  • the 26 amino acid signal peptide of hGH is attached to the G-5 sequence via synthesis of an oligonucleotide which is ligated to the cDNA by appropriate recombinant techniques before or after deletion of the two putative membrane-spanning domains of the enzyme located between amino acids 1 -21 and 56-76.
  • the deiodinase is assayed simply by sampling the media. This is advantageous in studies in which the time course of expression is of interest.
  • Two cDNA and one human genomic library were screened.
  • the first was a human liver cDNA library in a CDM-8 vector prepared according to the methods of Arrufo and Seed (Proc. Natl. Acad. Set USA 84:8573-8577 (1987)) and kindly provided by Dr. Brian Seed. Because we could not identify the complete coding sequence from this library, a second human kidney cDNA library in ⁇ gt10 was obtained through the courtesy of Dr. Graeme Bell.
  • a human genomic library in ⁇ FixII vector was purchased from Stratagene (La Jolla. CA).
  • the human liver library in the CDM-8 vector was plated on 400 cm 2 agar plates, colonies were immobilized on nylon filters (GeneScreen Plus. DuPont. New England Nuclear. Boston. MA), denatured in NaOH. neutralized in tris buffer, and the DNA cross-linked to the filter using a UV-Stratalinker 1800 (Stratagene). Filters were prehybridized for 3 hours at 65°C in 1 M NaCl. 1% SDS. 10% dextran sulfate. and denatured salmon sperm DNA ( 100 ug ml -1 ).
  • the human kidney cDNA library in the ⁇ gt10 vector was expressed in E. coli and phage DNA was transferred to nylon filters, denatured, neutralized. and cross-linked as above. Filters were prehybridized and hybridized at 65°C and washed as above.
  • This library was probed with a 417 base pair cDNA fragment (nucleotides 134 to 551, NcoI to PstI) from the 5 ' end of the isolated human liver cDNA clone. Both upper and lower strands were sequenced by the dideoxynucleotide chain termination method using T7 polymerase according to the instructions of the kit manufacturer (Pharmacia, Piscataway. NJ).
  • Poly(A)+ mRNA was subjected to gel electrophoresis in a 1% agarose gel containing 20 mM 3-(N-morpholino) propane sulfonicacid (MOPS) pH 7.0, 5 mM sodium acetate. 1 mM EDTA. and 1.3% (wt/vol) formaldehyde. Gels were rinsed in 10X SSC and blotted overnight in 20X SSC to a GeneScreen Plus nylon membrane (DuPont). RNA was cross-linked to the nylon with a UV Stratalinker. Following prehybridization with salmon sperm DNA and E. coli tRNA.
  • MOPS 3-(N-morpholino) propane sulfonicacid
  • the filter was hybridized with a 1.5 kb cRNA from the human liver clone (nucleotides 32 to 1516. XbaI). that was transcribed in vitro from a pBluescript KS vector (Stratagene) using T7 polymerase and UT P. Filters were washed in 1 X SSC, 0.1% SDS at 25°C followed by washes of increasing stringency with a final wash being 0.1X SSC. 0.1% SDS at 65°C according to standard techniques. DNA Transfections and Deiodinase Assays
  • Deiodinase reactions contained 10 to 55 ⁇ g of cell sonicate protein in 300 ⁇ l PE buffer and varying concentrations of DTT, 3,5,3'-triiodothyronine (rT 3 ). and other reagents as indicated. Deiodinase activity was monitored by the release of 125 I- from 125 I-rT 3 (DuPont, New England Nuclear) under conditions specific for each experiment. Incubations were for 30 minutes at 37°C and 125 I was quantitated as previously described (Berry, et al. Mol Endocrinol. 4:743-748 (1990)).
  • T 4 to T 3 conversion was measured by incubation of approximately 600 ⁇ g of COS-7 cell sonicates with 25 mM DTT, 100 nM 125 I-T 4 , and 200 nM or 10 ⁇ M rT 3 for 16 hours at 37°C in a total volume of 200 ⁇ l PE with or without 0.5 mM PTU.
  • T 4 , T 3 and I- were separated by paper chromatography and identified by staining of chromatographed unlabelled standards. The 125 I content of the products was quantitated by counting the paper strips in a gamma scintillation counter. All assays were performed in duplicate. Kinetic analyses were performed as previously described (Berry, et al. J. Biol. Chem.
  • Bromoacetylated 125 I labelled T 3 (BrAcT 3 ) was synthesized from 125 I T 3 (DuPont, New England Nuclear, specific activity 1200 uCi mg -1 ) and bromoacetyl chloride according to published methods (Mol, et al., Biochem. Biophys. Res. Commun. 124:475-483 (1984)). The final product in 2 ml of acidified 20% ethanol was diluted with 3 volumes of water and purified by chromatography on a column (2.5 ⁇ 0.8 cm) of Sephadex LH-20 (Pharmacia).
  • the initial screening of the human liver cDNA library identified two identical 2188 base pair clones from a total of approximately 600.000 recombinants. This sequence did not contain a polyadenylation signal or tail. By its close homology to the rat 5' deiodinase sequence, it had a 5' boundary at nucleotide number 32 ( Figure 5).
  • the library was re-screened using a liver cDNA fragment consisting of nucleotides 134 to 551 (NcoI to PstI). but no other recombinants were identified that extended 5' to nucleotide 32.
  • a human kidney cDNA library was screened using the same human cDNA Ncol to PstI fragment which identified an approximately 4 kb insert.
  • This clone contained sequences identical to the human liver cDNA between nucleotides 32 and 300 but diverged 3 ' to this region.
  • the insert also contained 1.8 kb of sequence 5' to nucleotide 32.
  • This recombinant kidney clone apparently contained an exon flanked on two sides by intronic sequences which had not undergone splicing (Figure 4A). This was confirmed by identifying and sequencing a similar fragment from a human genomic library. There were several consensus branch points and splice junctions at the 3 ' border of the upstream intron. There was no initiator methionine within the 150 base pairs 5' to the nucleotide designated number 7 based on the homology to the rat 5' deiodinase cDNA ( Figure 5).
  • a cDNA containing the coding and downstream sequences of the human 5'deiodinase was constructed from the liver (HL5) and kidney (HK5) recombinants as follows.
  • a 2.3 kb EcoRI fragment from the 4.4 kb insert in HK5 was subcloned into Bluescript and a 500 nucleotide PstI fragment of this subclone was then inserted into Bluescript (HK5Pst. Figure 4A).
  • the 1251 base pair NcoI fragment from HL5 ( Figure 4A) was inserted into HK5Pst at the NcoI site.
  • Hind3/NsiI fragment containing the initiator methionine from this new construct was then substituted for the shorter Hind3/NsiI fragment of the HL5 clone.
  • the numbering of this sequence is assigned arbitrarily by its homology to the rat 5 ' deiodinase sequence ( Figures 1. 4B and 5). This results in a cDNA of 2222 nucleotides. slightly shorter than the mRNA identified by Northern blotting ( Figure 4B). Of note is the fact that a UGA codon is present at the identical position (382) in both the human and rat 5' deiodinase sequences.
  • the enzyme was transiently expressed in COS-7 cells which contain no endogeneous deiodinase.
  • the transiently expressed enzyme was readily identified by its capacity to deiodinate rT 3 in a saturable fashion with an apparent Ka of 0.52 ⁇ 0.04 ⁇ M (Table III) and Vmax of 63.2 ⁇ 16.4 pmol min -1 mg - 1 . both at 10 mM DTT.
  • T 4 is a competitive inhibitor of rT 3 deiodination with an apparent Ki of 6.2 ⁇ M. This is about 16 times higher than the Ka for rT 3 , demonstrating that the latter is the preferred substrate.
  • the apparent Kb for DTT is 5.0 mM.
  • PTU is an uncompetitive inhibitor of rT 3 deiodination (Ki 0.17 ⁇ M) and is competitive with respect to DTT (Ki 0.014 ⁇ M) as would be expected from the ping-pong kinetics of the Type I reaction.
  • deiodination is competitively inhibited by gold thioglucose (GTG) with an apparent Ki of 4.7 nM ( Figure 10).
  • GTG gold thioglucose
  • Figure 10 The enzyme also catalyzes T 4 to T 3 conversion by a PTU-sensitive mechanism with the production of equimolar quantities of T 3 and I- ( Figure 11), albeit at a much slower rate.
  • Bromoacetyl affinity labelling of the human and rat transiently expressed proteins was performed to establish that the in vitro expressed protein was of the size predicted by the deduced amino acid sequence presuming that the UGA encodes selenocysteine.
  • cells transfected with the CDM-8 vector alone Figure 12, lanes 1 and 2
  • several discrete labelled bands are present (64. 46. 34. and 16 kDa). Labelling of the 16 kDa band is completely and that of the 64 and 46 kDa bands partially blocked by excess unlabelled BrAcT 3 .
  • the present results demonstrate that the human 5' deiodinase gene and protein are highly homologous to those of the rat.
  • the coding region nucleotide sequences of the two species are 82% homologous ( Figure 5) and their putative amino acid sequences are 88 % identical.
  • the cDNA we have isolated is approximately 200 nucleotides shorter than the mRNA in the liver, kidney and thyroid. It is lacking sequences at both the 5 ' and 3 ' extremes since it does not contain a poly A tail and the 5 '-untranslated portion is of unknown length due to the presence of a long unspliced intronic sequence in the kidney cDNA clone.
  • the human cDNA encodes a functional 5' deiodinase.
  • the deduced human protein sequence is 7 amino acids or 0.7 kDa shorter than that of the rat.
  • the SECIS motif (Selenocsteine Incorporation Sequence), recently identified in the 3 '-untranslated regions of these two mRNAs as well as in the selenoenzyme glutathione peroxidase, bears a high degree of homology with the rat sequence (Berry, et al., Nature 353:273-276 (1991)).
  • the secondary structure of the mRNA in this region suggests that there is RNA/protein or RNA/RNA interaction involved in the mechanism by which suppression of the UGA stop codon functions and insertion of selenocysteine occurs.
  • Comparison of the 321 nucleotide sequence (1573 to 1894) with the corresponding rat SECIS motif shows 66% homology.
  • Bromoacetyl T 3 labelling of a 27 kDa protein has been correlated with the activity of 5 ' deiodinase in liver and kidney microsomes (Schoenmakers. et al., Biochem. Biophys. Res. Commun. 162:857-868 ( 1989); Safran. et al., Endoamology 126:826-831 (1990): Kohrle. et al., J. Biol. Chem. 265:6146-0154 (1990): Kohrle. el al.,J. Biol. Chem. 265:6155-6163 ( 1990)) and more recently in microsomes from human liver (Schoenmakers and Pigmans.
  • hyperthyroidism increases the hepatic and renal 5 ' deiodinase in the rat by increasing the mRNA (Berry, et al., Nature 349:438-440 (1991 ): Berry, et al., Mol. Endocrinol. 4:743-748 (1990)). It seems likely that a similar effect would occur in hyperthyroid man, making the Type I enzyme a more important source of T 3 in hyperthyroid than in euthyroid individuals. Secondly.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne le clonage, le séquençage et des utilisations d'une séquence de nucléotides codant l'enzyme mammifère iodothyronine 5' déiodidase de type I ainsi que des séquences mutantes de celles-ci. L'enzyme catalyse la conversion de thyroxine en triiodothyronine, produisant la forme active de l'hormone thyroÏdienne. L'invention concerne également la découverte qu'une séquence nucléotide non traduite est nécessaire à la reconnaissance d'un codon (de terminaison) TGA dans la région codante de la 5' déiodinase codant la sélénocystéine, un acide aminé essentiel à l'activité totale de la 5' déiodinase. Le mécanisme par lequel cette reconnaissance a lieu fait intervenir une structure tige-boucle dans la région non traduite 3' de l'ARNm. La séquence non traduite peut être utilisée afin d'introduire la sélénocystéine dans des protéines et dans des polypeptides ne contenant pas de sélénocystéine.
PCT/US1992/000740 1991-01-29 1992-01-29 ADNc CODANT LA IODOTHYRONINE 5' DEIODINASE DE TYPE I WO1992013077A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US64765791A 1991-01-29 1991-01-29
US647,657 1991-01-29
US75702491A 1991-09-03 1991-09-03
US757,024 1991-09-03

Publications (1)

Publication Number Publication Date
WO1992013077A1 true WO1992013077A1 (fr) 1992-08-06

Family

ID=27095216

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/000740 WO1992013077A1 (fr) 1991-01-29 1992-01-29 ADNc CODANT LA IODOTHYRONINE 5' DEIODINASE DE TYPE I

Country Status (2)

Country Link
AU (1) AU1337092A (fr)
WO (1) WO1992013077A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996008568A3 (fr) * 1994-09-12 1996-05-17 Us Health Clonage et expression d'adn complementaire codant pour la dihydropyrimidine deshydrogenase humaine
US5700660A (en) * 1993-05-24 1997-12-23 University Of Masachusetts Medical Center Positional control of selenium insertion in polypeptides for X-ray crystallography
EP0871722A4 (fr) * 1993-05-24 1998-10-21
US5849520A (en) * 1993-05-24 1998-12-15 University Of Massachusetts Medical Center Post transcriptional gene regulation by selenium
EP1396544A1 (fr) * 2002-09-06 2004-03-10 Academisch Ziekenhuis Bij De Universiteit Van Amsterdam Acides nucléiques codant pour des déhydrogénases thyroidiennes humaines

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4727138A (en) * 1981-10-19 1988-02-23 Genentech, Inc. Human immune interferon
US4857467A (en) * 1986-07-23 1989-08-15 Phillips Petroleum Company Carbon and energy source markers for transformation of strains of the genes Pichia
US4910141A (en) * 1984-08-31 1990-03-20 Cetus Corporation 3'-expression enhancing fragments and method
US4935339A (en) * 1985-05-07 1990-06-19 Nichols Institute Diagnostics Delayed solid phase immunologic assay
US4959318A (en) * 1985-06-27 1990-09-25 Zymogenetics, Inc. Expression of protein C
US4975369A (en) * 1988-04-21 1990-12-04 Eli Lilly And Company Recombinant and chimeric KS1/4 antibodies directed against a human adenocarcinoma antigen

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4727138A (en) * 1981-10-19 1988-02-23 Genentech, Inc. Human immune interferon
US4910141A (en) * 1984-08-31 1990-03-20 Cetus Corporation 3'-expression enhancing fragments and method
US4935339A (en) * 1985-05-07 1990-06-19 Nichols Institute Diagnostics Delayed solid phase immunologic assay
US4959318A (en) * 1985-06-27 1990-09-25 Zymogenetics, Inc. Expression of protein C
US4857467A (en) * 1986-07-23 1989-08-15 Phillips Petroleum Company Carbon and energy source markers for transformation of strains of the genes Pichia
US4975369A (en) * 1988-04-21 1990-12-04 Eli Lilly And Company Recombinant and chimeric KS1/4 antibodies directed against a human adenocarcinoma antigen

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 162, No. 2, issued 31 July 1989, C.H.H. SCHOENMAKERS et al., "Rat Liver Type I Iodothyronine Deiodinase Is Not Identical to Protein Disulfide Isomerase", pages 857-868. *
ENDOCRINE REVIEUWS, Volume 2, No. 1, issued January 1981, P.R. LARSEN et al., "Relationships between Circulating and Intracellular Thyroid Hormones: Physiological and Clinical Implications", pages 87-102. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 10, No. 5, issued May 1990, B.J. LEE et al., "Selenocysteine tRNA(Ser)Sec Gene Is Ubiquitous within the animal Kingdom", pages 1940-1949. *
MOLECULAR ENDOCRINOLOGY, Volume 4, No. 5, issued 31 May 1990, M.J. BERRY et al., "Thyroid Hormone Regulates Type I Deiodinase Messenger RNA in Rat Liver", pages 743-747. *
PROTEIN ENGINEERING, Volume 2, No. 3, issued 21 September 1988, G.T. MULLENBACH et al., "Selenocysteine's Mechanism of Incorporation and Evolution Revealed in cDNAs of Three Glutathione Peroxidases", pages 239-246. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 265, No. 33, issued 25 November 1990, D.L. ST. GERMAIN et al., "Molecular Cloning by Hybrid Arrest of Translation in Xenopus laevis Oocytes", pages 20087-20090. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5700660A (en) * 1993-05-24 1997-12-23 University Of Masachusetts Medical Center Positional control of selenium insertion in polypeptides for X-ray crystallography
EP0871722A4 (fr) * 1993-05-24 1998-10-21
US5849520A (en) * 1993-05-24 1998-12-15 University Of Massachusetts Medical Center Post transcriptional gene regulation by selenium
WO1996008568A3 (fr) * 1994-09-12 1996-05-17 Us Health Clonage et expression d'adn complementaire codant pour la dihydropyrimidine deshydrogenase humaine
US5856454A (en) * 1994-09-12 1999-01-05 The United States Of America As Represented By The Department Of Health And Human Services CDNA for human and pig dihydropyrimidine dehydrogenase
EP1396544A1 (fr) * 2002-09-06 2004-03-10 Academisch Ziekenhuis Bij De Universiteit Van Amsterdam Acides nucléiques codant pour des déhydrogénases thyroidiennes humaines

Also Published As

Publication number Publication date
AU1337092A (en) 1992-08-27

Similar Documents

Publication Publication Date Title
Tontonoz et al. Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR7 and RXRa
JP3981416B2 (ja) Ｐｃａ３タンパク質、ｐｃａ３遺伝子、及びこれらの用途
US5942398A (en) Nucleic acid molecules encoding glutx and uses thereof
JP2000157286A (ja) アミノ酸輸送蛋白及びその遺伝子
JPH08510897A (ja) 細胞外シグナル制御キナーゼ、配列、製造法および用途
Raver et al. Large-scale preparation of biologically active recombinant chicken obese protein (leptin)
US5272078A (en) CDNA encoding the type I iodothyronine 5'deiodinase
JP2000500971A (ja) 肥満遣伝子の転写プロモーター
Wheeler et al. Stat5 phosphorylation status and DNA-binding activity in the bovine and murine mammary glands
US5614609A (en) Serine threonine kinase receptor
EP0714438B1 (fr) Nouveau facteur d'homeosequence stimulant l'expression de l'insuline dans les cellules des ilots pancreatiques
US20140088024A1 (en) Vesiculins
Mountjoy et al. Prolactin receptors in the rat kidney
Kashiwagi et al. Cloning, properties and tissue distribution of natriuretic peptide receptor‐A of euryhaline eel, Anguilla japonica
US7115728B1 (en) Human peroxisome proliferator activated receptor γ
WO1992013077A1 (fr) ADNc CODANT LA IODOTHYRONINE 5' DEIODINASE DE TYPE I
JPH11502402A (ja) レチノイドｘ受容体相互作用性ポリペプチドならびに関連する分子および方法
JPH07502883A (ja) 新規なシクロフィリン類、関連タンパク質類および用途
CA2399378C (fr) Petits transporteurs d'aminoacide neutre independants du sodium
EP2612678A1 (fr) Procédé de criblage d'agent antidiabétique mettant en oeuvre un facteur de régulation de la sécrétion d'insuline nouvellement identifié
CA2289903A1 (fr) Proteine-1 d'adhesion vasculaire a activite monoamine-oxydase
WO1999048920A1 (fr) Nouveaux peptides a activite physiologique et leur utilisation
WO1993021330A1 (fr) Glutamine:fluctose-6-phosphate amidotransferase humaine
US5284999A (en) DNA encoding a pituitary-specific thyroid hormone receptor
WO2001021795A2 (fr) Proteines de transport d'acides gras

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

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

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA