WO1995007349A1 - Dosage biologique du selenium - Google Patents

Dosage biologique du selenium Download PDF

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WO1995007349A1
WO1995007349A1 PCT/US1994/009856 US9409856W WO9507349A1 WO 1995007349 A1 WO1995007349 A1 WO 1995007349A1 US 9409856 W US9409856 W US 9409856W WO 9507349 A1 WO9507349 A1 WO 9507349A1
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selenium
coli
plasmid
derivative
sample
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PCT/US1994/009856
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Hanna Engelberg-Kulka
Myriam Reches
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Yissum Research Development Company Of The Hebrew University Of Jerusalem
Kohn, Kennedy, I.
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01043—Formate dehydrogenase (NADP+) (1.2.1.43)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to a bio-assay of selenium in selenium derivatives, transformed micro-organisms therefor, and plasmids suitable for generating said transformed microorganisms.
  • the trace element selenium is an. essential component of several enzymes from various prokaryotic and eukaryotic organisms [for review see Stadtman, T.C. (1991) J. Biol. Cheat. 266, 16257-16260]. These enzymes have been shown to contain selenium in the form of a single selenocysteine residue in the active site of the enzyme [for reviews see Stadtman, T.C. (1990) Ann. Rev. Biochem 59, 111-127; Sunde, R.A. (1990) Ann. Rev. Nutr. 10, 451-474]].
  • the seleno-containing proteins the best known is the peroxide-destroying enzyme glutathione peroxidase (GPX) [Rotruck, J.T. et al.
  • selenium is also involved in the form of selenocysteine in two additional human selenium-containing proteins.
  • the first is the thyroid enzyme type 1 iodothyronine 5' deiodinase (5' deiodinase) [Berry, M.J. et al. Nature 349, 438-440] that converts thyroxine to the active thyroid hormone.
  • the second is the plasma protein selenoprotein P [Read, R. et al. (1990) J. Biol. Chem. 265, 17899-17905; Kill, K.E. et al., J. Biol. chem.
  • the assays currently available for determining selenium concentrations in various compounds are mainly physical or chemical methods by which the element selenium is determined directly [Ihnat, M. et al. (1986) Acta Pharmacol Toxicol. 59, 566-572; Neve, J. (1991) J. Trace Elem Electrolytes Health Dis. 5, 1-17 ⁇ .
  • the known prior art methods for quantitative determination of selenium require sophisticated and expesive equipment, are not very specific and are also not sensitive to very low concentrations of selenium.
  • the present invention offers a simple, relatively inexpensive bio-assay that relates linearly and specifically to very low concentrations of selenium in several simple selenium derivatives.
  • the bio-assay of the invention is carried out in E. coli and employs specific novel recombinant DNA plasmid constructs built for this specific purpose and measures only selenium compounds which are included in pathways of "real" selenoproteins.
  • the bio-assay of the inven tion is suitable for the determination of selenium in blood and also other biological materials and has several advantages over procedures currently used for this purpose.
  • the invention relates to plasmids carrying a seleniumspecifying DNA sequence of the E. coli fdhF gene upstream the E. coli lac'Z gene which permits the incorporation of selenocystein into ß-galactosidase.
  • the plasmids according to the invention comprise at their beginning a lacZ derivative carrying at least the -9 to +47 nucleotide bases of the TGA codon region of E. coli fdhF gene.
  • Preferred plasmids according to the invention comprise at their beginning a lacZ derivative consisting of nucleotide bases -9 to 4-47 or -1 to +47 of the TGA codon region of E. coli fdhF gene.
  • the invention relates to micro-organisms transformed with a plasmid according to the invention, having selenium-dependent ß-galactosidase activity, E. coli being preferred.
  • the invention provides a method for quantitative determination of selenium in selenium derivatives in a biological sample comprising incubating transformed micro-organisms according to the invention in a suitable medium also containing said sample and measuring the level of ß-galactosidase activity.
  • the method of the invention may further comprise a preliminary step in which said biological sample is subjected to treatment with acid vapor prior to being added to incubation medium.
  • the method according to the invention is particularly suitable for the determination of selenium in biologically active selenium derivatives and can be employed for the determination of selenium derivative in blood samples, food samples and in other biological materials.
  • the invention relates to a diagnostic kit for the quantitative determination of selenium in selenium derivatives in accordance with the method of the invention.
  • Fig. 1 The synthesis of a selenium-dependent fused protein directed by plasmids pRM2 and pBM4
  • E. coli strains MC4100 (A and C) and its selc derivative RM1 (B and D) were transformed by plasmids pMRl (slot 1), pRM2 (slot 2), pRM4 (slot 3), and pMR1 (TGAC) (slot 4).
  • Freshly transformed cells were grown in M9 minimal medium, labeled either with [ 35 S]-methionine (A and B) or with [ 75 Se]-selenite (c and D).
  • the labeled cells were lysed and subjected to electrophoresis on 7.5% SDS-polyacrylamide gels which were subsequently autoradiographed.
  • the [ 35 S]-methionine labeled lysates were treated with antibodies against ß-galactosidase and immuno- precipitated.
  • the position of the 140kD fused protein product of genes ⁇ cI-lac'I"Z is indicated in the gel by an arrow.
  • Fig. 2 The selenium-dependent synthesis of ß-galactosidase directed by plasmids pRM2 and pRM4 in E. coli.
  • Fig. 3 Determining the concentrations of several simple selenium-containing compounds: The bio-assay (A-D) versus Atomic Absorption Spectrophotmetry (E). E. coli strain YN3230 ( ⁇ ) and its selC derivative RM2 (- ⁇ -) were transformed by pRM4. The transformed cells were grown to mid-log phase in M9 medium in the presence of various concentrations of selenite 3 (A), selenocysteine (B), selenate (C), and selenomethio nine (D). The levels of ß-galactosidase activity are presented in Miller Units and were determined as described in Fig. 2. The results represent the average of 4 experiments.
  • A-D Atomic Absorption Spectrophotmetry
  • Fig. 3E concentrations of the selenium-containing derivatives (as indicated) were determined by Atomic Absorption Spectrophotoraetry (AAS) using the Varians graphite furnace SpectrAA 300 Zeeman Atomic Spectrophotometer.
  • AAS Atomic Absorption Spectrophotoraetry
  • This Spectrophotometer has an automatic background correcting system; a solution of o. ⁇ % nitric acid and palladium was used as a chemical modifier (10 ⁇ l of 500 ⁇ g/ml).
  • Fig. 4 The effect of HCl-vapor treatment on simple Se derivatives used for the bio-assay.
  • the assay described in Fig. 3 was used to determine levels of selenite (A), selenocysteine (B), selenate (C), and selenomethionine (D) which were applied either directly to E. coli strain YN3230 carrying the SelC gene ( ⁇ ), or after HCl-vapor treatment to either strain YN3230 ( ⁇ ) or to its SelC derivative ( ⁇ ).
  • the selC E. coli derivative RM2 was used as a control.
  • the Se-containing compounds (selenite, selenocysteine, selenate, and selenomethionine) were used either directly or after HCl-vapor-hydrolysis treatment. Only compounds treated with HCl vapor were used with the selc derivative RM2 control strain.
  • E Determination of Se concentration in several se-containing compounds by AAS, carried out as in Fig. 3. Fig. 5 Se status in blood serum.
  • A Determination by the bio-assay as described in Fig. 3. 50-100 ⁇ l blood serum samples were subjected to HCl-vapor hydrolysis prior to the assay. Before determining Se levels by AAS, the blood serum samples were diluted 1/40 to prevent viscosity.
  • the present invention relates to a specific and sensitive bio-assay for the quantitative determination of selenium in selenium derivatives in biological materials, to transformed micro-organisms to be used therewith and to plasmids for generating these transformed microorganisms.
  • Recombinant DNA technology has been used to provide reporter systems than can be used generally to detect cis-acting elements like regulatory sites or trans-acting elements like proteins, RNA, and other large biological molecules. This approach has now been used in a new way to measure the presence and concentration of the essential chemical trace element selenium.
  • the present invention is based on the finding that (i) selenium is incorporated into polypeptides in the form of selenocysteine; and ii) in E. coli this incorporation is permitted by the presence of a TGA codon within a specific codon context [Zinoni et al. (1990) ibid.; Heider et al. (1992) ibid.].
  • a selenium-specific TGA codon context is present in the E. coli fdhF gene which specifies for the selenium-containing enzyme formate dehydrogenase [Zinoni et al. (1990) ibid.].
  • These selenium-specifying sequences of the fdhF gene were inserted upstream from the E.
  • plasmids carrying the inserted fdhF sequences directed selenium incorporation into a fused polypeptide product (Figs. 1A and 1C) which has ß-galactosidase activity that is selenium-dependent (Fig. 2). Since neither the fused protein nor ß-galactosidase activity are obtained in selC derivatives lacking the gene for tRNA Sec it is suggested that selenium is incorporated in the form of selenocysteine (Figs. 1B, 1D and 2).
  • the level of ß-galactosidase is proportionally and specifically related to the simple selenium derivatives selenite and selenocysteine (Figs. 3A and 3B).
  • the present system of plasmids in appropriate E. coli strains can be used as a bio-assay for determining the selenium concentrations in these compounds.
  • Either plasmid pRM2 or plasmid pRM4 can be used.
  • plasmid pRM4 is preferred since it directs slightly higher levels of the selenium-dependent ß-galactosidase activity (Fig. 2). This is probably because, unlike pRM2, pRM4 carries the nine additional nucleotides which precede the 47 nucleotides following the TGA of E. coli gene fdhF (see Table 1B).
  • the invention therefore relates to plasmids carrying a selenium-specifying DNA sequence of the E. coli fdhF gene upstream the E. coli lac'Z gene which permits the incorporation of selenocystein into ß-galactosidase.
  • Specific plasmids according to the invention are those comprising at their beginning a lacZ derivative carrying at least the -9 to +47 nucleotide bases of the TGA codon region of E. coli fdhF gene.
  • a preferred plasmid according to the invention is a plasmid comprising at its beginning a lacZ derivative consisting of nucleotide bases -9 to +47 of the TGA codon region of E. coli fdhF gene.
  • Another preferred plasmid comprises at its beginning a lacZ derivative consisting of nucleotide bases -1 to +47 of the TGA codon region of E. coli fdhF gene.
  • the invention relates to microorganisms transformed with a plasmid according to the invention, having selenium-dependent ß-galactosidase activity, E. coli strains being preferred.
  • micro-organisms are E. coli strains transformed with plasmid pRM2 or with plasmid pRM4.
  • the invention relates to a method for the quantitative determination of selenium in selenium derivatives in a biological sample comprising incubating transformed micro-organisms according to the invention in a suitable medium also containing said sample and measuring the level of ß-galactosidase activity.
  • the method according to the invention employs transformed E. coli strains.
  • the method according to the invention is particularly suitable for the determination of biologically active selenium derivatives, for example sodium selenite or selenocystein.
  • selenium derivatives can be quantitatively assayed different biological materials such as blood, food, and others.
  • biological materials such as blood, food, and others.
  • the sample is first subjected to acid-vapor treatment, and are then incubated with transformed micro-organisms according to the invention in a suitable medium in accordance with the method of the invention, follwed by measuring the level of ß-galactosidase activity.
  • HCl vapor is used.
  • a diagnostic kit for the quantitative determination of selenium derivatives, employing the tranformed microorganisms and methods of the invention is also within the scope of this application.
  • the assays currently available for determining selenium concentrations in various compounds are mainly physical or chemical methods by which the element selenium is determined directly [ Ihnat et al. (1986) ibid.; Neve (1991) ibid.]. since one of the best method for this purpose is direct Atomic Absorption Spectrophotometry, the atomic absorption of selenium in various simple selenium derivatives including sodium selenite, sodium selenate, selenocysteine, and selenomethionine, using selenium dioxide solutions as a standard (Fig. 3E) was measured.
  • the atomic absorption method can be used to measure the selenium concentrations in each of these compounds, and in the range of 2-100 ng Se/ml the results are linear and are similar for all the compounds.
  • the bio-assay according to the invention which reflects UGA directed selenium incorporation, is more specific because it responds to only a few simple selenium derivatives.
  • sodium selenite (Fig. 3A) and selenocysteine (Fig. 3B) can penetrate the cells and are included in the biochemical pathway of E. coli UGA-directed selenocysteine incorporation, the levels of ß-galactosidase activity are a linear function of low concentrations of these compounds.
  • the bio-assay cannot be used to measure selenium in either sodium selenate (Fig. 3C) or selenomethionine (Fig. 3D).
  • the bio-assay is sensitive to sodium selenite and ⁇ elenocy6tein at concentrations (0.07-1.0 ng Se/ml) (Fig. 3A) about 40 times lower than those can be measured by Atomic Absorption Spectrophotometry (2-100 ng Se/ml) (Fig. 3E).
  • the bio-assay of the invention is suitable for determining selenium concentrations either in the inorganic form selenite or in the simple organic form selenocysteine.
  • Bacteria cannot use selenium when it is in complicated organic forms.
  • the bio-assay to measure the selenium in proteins like GPX, 5' deiodinase or selenoprotein P, the protein must first be converted to a simple inorganic or organic form by proteolysis.
  • the basic method of the invention may be modified such that samples are first subjected to acid-vapor treatment, conditions known to cause hydrolysis in proteins. Fig.
  • the levels of ßGal were found to be linearly proportional to the concentrations of sodium selenite, sodium selenate, or selenocysteine in the sample solutions.
  • the bio-assay according to the invention has several advantages over currently available physical assays.
  • the physical assays require sophisticated instruments not always available in clinical laboratories.
  • the present recombinant DNA bio-assay preferably carried out in E. coli, is simple and relatively inexpensive.
  • the present bio-assay is sensitive at lower concentrations than is Atomic Absorption Spectrophotometry (Fig. 3E), one of the best available physical methods.
  • a major advantage of the bio-assay of the invention may be that, unlike the physical methods, it is specific only for certain simple selenium derivatives like selenite or selenocysteine.
  • Fig. 3E Measuring the total selenium status of a biological sample (like blood) may be misleading.
  • the physical methods available are also sensitive to selenium when it is in the form of selenomethionine which is incorporated into proteins as a random substitute for methionine [Cowie, D.B. et al. (1957) Biochem. Biophys. Acta 26, 252-261; Sliwkowski, M.X. and Stadtman, T.C. (1985) J. Biol. Chem. 260, 3140-3144; Frank, P. et al. (1985) J. Biol. Chem. 260, 5518-5525; Beilstein, M.A. and Whanger, P.D. (1986) J.
  • the modified bio-assay was tested by using it to determine the Se status in samples of rat blood serum.
  • the results of the bio-assay were compared with measurements by two well established methods.
  • Plasma serum samples from two groups of rats (i) Rats fed their usual diet [Se(+)] and (ii) Rats fed a Se-deficient diet [Se(-)] for a 6 week period was used.
  • the bio-assay was used to determine the Se status after the serum samples were subjected to acid hydrolysis (Fig. 5). As shown in Fig.
  • Bacteria were grown in LB or in M9 minimal medium [Miller (1972) ibid.] (pH 7.0) supplemented with 10 ⁇ M Na 2 MoO 4 and a mixture of amino acids each at a final concentration of 20 ⁇ g/ml. The final concentration of cysteine was 200 ⁇ g/ml. Unless otherwise stated, methionine was missing from the media. All the reagents added to M9 medium were analytically pure, and the media were prepared in water double distilled in a Corning water purification system. Ampicillin (100 ⁇ g/ml) was added to media in which the plasmid carrying strains were grown.
  • Plasmid pFM20 carries the E. coli fdhF [Zinoni et al. (1986) ibid.]. Using the required primers and with plasmid pFM20 as a template, PCR technique was used to amplify two over-lapping regions of the fdhF gene flanked by restriction sites HindIII and BamHI: i) the DNA from nucleotides -1 to +47 (plasmid pRM2); and ii) the DNA from nucleotides -9 to +47 (plasmid pRM4). The nucleotides are numbered relative to the TGA codon of gene fdhF and the positions are indicated by arrows in Fig. 1E.
  • Freshly transformed cells were grown in M9 medium at 37oC overnight in the presence of either l.o Ci/ml [ 75 Se]-selenite or 15 ⁇ Ci/ml [ 35 S]-methionine. During labeling with [ 75 Se]-selenite, cold sodium selenite was added to a final concentration of 1.5 ⁇ M. Cells were lysed and proteins were extracted and than immunoprecipitated with antibodies to ß-galactosidase as described previously [Schoulaker-Schwartz,
  • Samples to be treated with HCl vapor were transferred to a 6 x 50mra Pyrex tube, dried by vacuum, and sealed into a large vessel to which 6N HCl had been added at the bottom. This procedure permits the acid vapor only to be in contact with the sample. Vapor-phase was carried out under vacuum at 110oC for 48 h. The dried samples were suspended in M9 minimal medium and added to the bacterial culture.
  • GPX activity was quantitated by the coupled enzyme procedure of Lawrence and Burk [ibid.].
  • the reaction mixture contained 50 mM potassium phosphate buffer (pH7) lmM EDTA/lmM NaN 3 / ImM GSH/ and 1 E.U./ml glutathione reductase per ml.
  • the serum sample (0.1 ml) was added to 0.6 ml of reaction mixture and pre-incubated for 5 min at 25oC before the reaction was initiated by the addition of 0.1 ml H 2 O 2 (2.5 mM). Abosrbance at 340 nm was recorded for 5 min.
  • One unit of activity catalyzes the oxidation of 1.0 ⁇ mol of NADH reduced per min. In order to correct for background, distilled water was used instead of the sample.
  • Recombinant DNA technology had been previously used by the present inventor ⁇ to construct a reporter ⁇ ystem for sensitively detecting UGA readthrough by tryptophanyl-tRNA p in E. coli [Kopelowitz (1992) ibid.].
  • the present system has been constructed for detecting UGA readthrough by selenocy ⁇ teinyl-tRNA Sec .
  • plasmid pMRl which carries the fused genes XcI'-lac'I"Z [Kopelowitz (1992) ibid.; Gray (1982) ibid.] was u ⁇ ed. At the junction of these genes, regions surrounding the TGA codon of the E.
  • coli fdhF gene were inserted; these sequences have been found to permit selecocystein incorporation into polypeptides [Zinoni (1990) ibid.].
  • the plasmid carrying the region of fdhF from -1 to +47 is called pRM2, and that carrying region -9 to +47 is called pRM4 (and see Materials and Methods and Fig. 1E).
  • Release factor 2 competes with natural or mutated UGA suppressors for the recognition of the UGA codon [Caskey, C.T. (1980) TIBS. 5, 234-237; Curran J.F. and Jarus, M. (1986) Proc. Natl. Acad. Sci. USA 83, 6538-6542]]; mutation prfBl in the gene specifying for RF2 increased UGA suppression by a UGA suppressor tRNA and UGA readthrough by tRNA Trp [Kopelowitz (1992) ibid.; Kawakami, K. et al. (1988) J. Bactriol. 170, 5378-5381; Roesser, J.R. et al. (1989) J.
  • the bio-assay for determining the presence and concentration of selenium in various simple selenium derivatives The bio-assay for determining the presence and concentration of selenium in various simple selenium derivatives.
  • the selenium-dependent ß-galactosidase activity directed by the gene fusion ⁇ cl'-lac'I"Z of pRM4 in YN3230 was examined as to whether it could be used as a measure for determining the concentrations of simple selenium derivatives in ⁇ olution ⁇ .
  • the levels of ß-galactosidase are linearly related to the concentrations of both sodium selenite and selenocysteine.
  • linearity is in the range of 0.06-1.0 ng Se/ml (Fig. 3A) and for selenocysteine in the range of 2-30 ngSe/ml (Fig. 3B).
  • ß-galactosidase activity are a function of the logarithm of the concentration of sodium selenate only at very high concentrations (10 3-105 ngSe/ml) (Fig. 3C); no ß-galactosidase activity at all was found in the presence of selenomethionine with (data not shown) or without added methionine (Fig. 3D).
  • Fig. 4 shows the effects of acid treatment of various Se-containing compounds on their ability to stimulate the expression of the Se-reporter system.
  • the levels of ⁇ Gal are found to be linearly proportional to the concentrations of sodium, selenite, sodium selenate, or selenocysteine in the sample solutions.
  • the response of the basic bio-assay to these compounds is also dependent on the presence of the E. coli SelC gene (Fig.
  • coli cells nor is utilized by them, is converted under the acidic conditions to selenite, which does penetrate the cells and is included in the UGA-directed selenocysteine pathway. It may be noted that without the acid-vapor step the bio-assay is not at all sensitive to selenomethionine, which is not included in the UGA directed selenocysteine pathway, even after acid hydrolysis treatment (Fig. 4D).
  • the modified bio-assay was tested by using it to determine the Se status in samples of rat blood serum.
  • the results of the bio-assay were compared with measurements by two well established methods, AAS (see above) and the level of the activity of the se-containing enzyme GPX.
  • Blood serum samples from two groups of rats was used: (i) Rats fed their usual diet [Se(+)] and (ii) Rats fed a Se-deficient diet [Se(-)] for a 6 week period.
  • the bio-assay was used to determine the Se status after the serum samples were subjected to acid hydrolysis (Fig. 5). As shown in Fig.

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Abstract

L'invention concerne un plasmide portant une séquence d'ADN spécifiant le sélénium, du gène fdhF de E. coli en amont du gène lac'Z de E. Coli, ce qui permet l'incorporation de sélénocysteine dans la β-galactosidase. Les plasmides mentionnés par l'invention sont pRM2, enregistré auprès de l'ATCC sous le No. 75594 et pRM4, enregistré auprès de l'ATCC sous le No. 75595. L'invention concerne également des micro-organismes transformés au moyen d'un plasmide selon l'invention et qui possède une activité de β-galactosidase dépendant de sélénium. L'invention concerne un procédé de détermination quantitative de sélénium dans des dérivés de sélénium dans un échantillon biologique comprenant l'incubation de micro-organismes dans un milieu approprié contenant également ledit échantillon et la mesure du niveau d'activité de la β-galactosidase. L'échantillon biologique peut être, par exemple, un échantillon de sang ou un échantillon d'aliment.
PCT/US1994/009856 1993-09-06 1994-09-02 Dosage biologique du selenium WO1995007349A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IL106925A IL106925A0 (en) 1993-09-06 1993-09-06 Bioassay of selenium
IL106,925 1993-09-06

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WO1995007349A1 true WO1995007349A1 (fr) 1995-03-16

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001012657A2 (fr) * 1999-08-16 2001-02-22 Karolinska Innovations Ab Procedes et moyens destines a l'expression de la selenoproteine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF MOLECULAR BIOLOGY, Volume 225, issued 1992, KOPELOWITZ et al., "Influence and Codon Context on UGA Suppression and Readthrough", pages 261-269. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 87, issued June 1990, ZINONI et al., "Features of the Formate Dehydrogenase mRNA Necessary for Decoding of the UGA Codon as Selenocysteine", pages 4660-4664. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001012657A2 (fr) * 1999-08-16 2001-02-22 Karolinska Innovations Ab Procedes et moyens destines a l'expression de la selenoproteine
WO2001012657A3 (fr) * 1999-08-16 2001-08-23 Karolinska Innovations Ab Procedes et moyens destines a l'expression de la selenoproteine

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IL106925A0 (en) 1993-12-28

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