GB2221910A - DNA encoding a uricase gene - Google Patents

Description

2rI Ct ZZ 19 10 DNA SEQUENCE FOR URICASE AND MANUFACTURING PROCESS OF

URICASE

BACKGROUND OF THE INVENTION

The present invention relates to DNA sequence for uricase and the production of uricase. More particularly, this invention relates to DNA containing a gene encoding uricase, a plasmid having said DNA, a transformant containing said plasmid, and a process for producing uricase by using said transformant.

Uricase (EC 1, 7, 3, 3) is an enzyme which catalyzes the reaction in which uric acid is hydrolyzed into allantoin, hydrogen peroxide and carbon dioxide, and is useful for assaying uric acid in blood or urine.

Uricase has heretofore been prepared by cultivating an uricase producing microorganism, for example microorganism belonging to Genus Canadida in the presence of uric acid in the culture medium and recovering uricase from the culture broth (Japanese Patent Publication No. 519211967).

Since this process gives a relatively low yield of uricase, it is desirable to develop a process for producing uricase effectively.

The present inventors have investigated on DNA containing a gene encoding uricase, a plasmid having said DNA, a tran'sformant containing said plasmid and a process for producing uricase by using said transformant. At first, some of the present inventors cloned uricase gene of thermophilic microorganism, Bacillus sp. TB-90 (FERM BP795, Japanese Patent Kokai 61-280272) being capable of producing thermally stable uricase, which was isolated from nature by the present inventors, and determined DNA sequence of said gene. Then, they prepared a plasmid having said DNA, then a transformat containing said plasmid and investigated a rpocess for producing uricase by using said transformant. Thus, the present invention has been established. SUMMARY OF THE INVENTION

The present invention relates to DNA containing a gene encoding uricase, a plasmid having said DNA, a transformant having said plasmid and a process for preparing uricase by using said transformant. BRIEF DESCRIPTION OF THE DRAWING

Figure 1 (parts 1 and 2) shows DNA sequence of a gene encoding uricase originated from Bacillus sp. TB-90 and the corresponding amino acid sequence.

Figure 2 shows a drawing for constructing expression plasmids pUOD316 and pKU1 from a recombinant plasmid pUOD31 of E. coli having DNA sequence encoding uricase originated from Bacillus sp. TB-90. The black and white boxes show each DNA fragment containing uricase gene and the region of lac or tac promoter. The ligation means a ligating reaction of DNA fragments by T4 DNA ligase.

Figure 3 shows a drawing for constructing a recombinant plasmid pEB2 of Bacillus subtilis having DNA sequence encoding uricase originated from. Bacillus sp. TB-90. The black and white boxes and ligation have the same meanings as described in Figure 2. DETAILED DESCRIPTION OF THE INVENTION

The gene encoding uricase in the present invention illustratively includes a gene encoding the following amino acid sequence or having the following nucleotide sequence.

20 MetThrlystlisLysGiuArSVatMetTyr TyrGlyLysGiyAspValPheAlaTyrArg 40 ThrTyrLeuLysProLeuThrGlyValArs ThrileProGluSerProPheSerGlyArs 60 AspHislieLeuPheGlyValAsnValLys lieSerVaiGlyGlyThrLysleuLeuThr 80 SerPheThrLysGIyAspAsnSerLeuVal VaiAlaThrAspSerNetLysAsnPhelle GinLysHisLeuAlaSerTyrThrGlyThr --- 110 ThrSerPheLcuLyslysTyrSerflistle ProPheGluThrThrPheAlaValLysAn LysLysSerArgAsnGluTyrAlaThrAla ThrLeuAsnileThrGluGinGinSerGly GIVAsnSerPheVaiGlyPhelleArgAsp 210 ArSProLeuPheVaiTyrLeuAsniteLys 230 GlyThrAsnProGluAsnTyrYalAlaAla 250 PheNisGIuThrGluThrLeuSerfleGin 270 LeuGluArgPheProGinLeuGinGluVal 290 LysileVaiGluGlulleProGluSerGlu 310 TyrGlyPheGinCysPheThrValThrGin 330 PheSerAspGluProAspHisLysGIyAla - 4 ThrileGluGlyPheleuGluTyrValAta GlulysileSerLeulleGLyGluGlu Ife GlyAsnArgAlaAlaSerGluleuValPhe TyrLeuAsnMetYalArgAsnGluAspAsn LeuAlaGlyLeuGinLeulleLysValSer GluTyrThrThrLeuProGluAspSerAn 220 TrPLYsTyrLysAsnThrGluAspSerPhe 240 GluG&ntleArsAspiteAlaThrSerVal 260 HisLeulleTyrLeulleGlyArgArgile 280 TyrPheGluSerGinAsnUisThrTrpAsp 300 GlyLysValTyrThrGluProArSProPro 320 GluAspleuProNisGiuAsnileLeuMet 1 i 1 ATGACCAW ACAAAWAC AGTGATGTAT TATWAMAG GTGACCTATT TCUTATCGC ACCTATTUA AACCACTTAC TGGAGTTAGA ACGATTCCTC AATUCCATT MCWTCCA GATCATATTC TTTTTGGAGT AAATGUAAA ATMAGTAG GAGUACAAA ATTGUGACC MMACGA AAWGGATAA CAGMAGR GMCAKAG AMGATGAA AAACTTTATA 300 CAAAAACATT TAGUAGTTA TACAGGAKA AGATAGMG GMMAGA ATATGTACCT AUMMT TGAAWATA TTUCATATT GAMAGAT7 CGTTGATAGG AGAGGAAATT 400 CCUTTGAAA CAACTMCC AGMAGAAT GGAWAGAG CAGWAGTGA GWAGTAM 1 AAAAAATCAC GAAATGAATA MCCACCWT TATTTGAM TGGTTCGTAA MAGATAK 500 ACCCTAAACA TTACTUACA ACAAAGCGGA MCCTWC TTCAATTAAT AMACTCAGC 600 GGAAATTCCT TTGTCGGTTT TATTMGAC GAMCACAA CTUTWAGA GGATTCAAAC CCUCTUAT TTGTTTACTT AAACATCAAA TWAGTACA AAAACACGGA AGACTCATTT 700 GUACUATC CAGAAAATTA TGTTGCAGCT GAACAAATTC GWACATCGC CACTCUTA MCATGAAA CCAGACWT MCATCCAA CATTRATTT ATTUATCGG WGAWATA 800 TTAGAMGAT TCUTCAACT TCAWAGTT TAMMAT UCAAAATCA TACATGWAT 900 AAAATAMG AGGAAAMC TUATCAGAA GGGAAAGTAT ATACAGAAW GCGACCGCCA TATGGAMC AATGUTTAC MCACCCAA GAMACTTGC CACACAAAA CATTMUG 999 TMUGATG AACCCUTCA WAGGAWA CTTAAATGA The plasmid containing uricase gene in the present invention can be prepared by construction of a gene library from the chromosomal DNA of said Bacillus sp. TB-90, screening the library with rabbit anti-uricase antibody, isolating DNA fragment containing uricase gene from phase DNA containing uricase gene of the present invention and ligating it with a vector plasmid.

It is well known that many amino acids are coded by more than one codon. Such base sequence cannot be single but possibly many base sequences may exist. In case of the gene encoding amino acid sequence of uricase originated from Bacillas sp. TB-90 found by the present inventors, there is a possibility of many DNA base sequences other than the base sequence of naturally existing gene. Thus, DNA sequence of the present invention is not only limited to naturally occurring DNA base sequence but also includes other DNA sequences encoding the amino acid sequence of uricase identified by the present invention.

According to recombinant DNA technique, any artificial variations may take place at a specific region of a fundamental DNA without changing the fundamental properties of what said DNA encodes or so as to improve the properties. Concerning DNA having the naturally existing base sequence or DNA having different sequence from the naturally existing one, artificial insertion, deficiency or substitution thereof can be similarly made to provide with some genes having properties equivalent to or improved from those of the naturally existing gene. Thus, the present invention includes modified genes.

Expression vector capable of producing uricase of Bacillus sp. TB-90 in the cells of Escherichia coli can be constituted by ligating a gene of uricase of Bacillus sp. TB-90 with appropriate expression vector of Escherichia coli such as expression vector pUC18 (Toyobo) retaining lac promoter, expression vector pKK223-3 (Pharmacia) retaining a potent promoter of Escherichia coli, naturally tac promoter and terminater of rrnB ribosome RNA, expression vector pDR720 (Pharmacia) retaining trp promoter, inducible expression vector pPLLambda (Pharmacia) or the like. Furthermore, it is possible to make a recombinant plasmid capable of producing uricase of Bacillus sp. TB-90 in the cells of Bacillus subtilis or in the culture broth by ligating it with vectors, for example, shuttle vector pHY300PLK (Toyobo) between Bacillus subtilis and Escherichia coli, plasmid vector pUB110(J. Bacteriol., 134, 318-329, 1978) or the like.

The transformant capable of producing uricase intracellularly or extracellularly can be prepared by introducing the recombinant plasmid retaining uricase gene of the present invention into a host cells such as Escherichia coli, Bacillus subtilis or etc.

Uricase can be produced in a large scale by cultivating the transformed microorganism thus obtained in appropriate medium under appropriate conditions. In this case, uricase can be produced effectively for example, by adding an inducer isopropyl thiogalactoside (IPTG) or the like at the early stage of cultivation.

After cultivation, uricase can be isolated, for example, by treating the cells with lysozyme or lysing the cells with supersonic wave or the like means or by extraction, separation and purification of the culture broth.

Moreover, not only Escherichia coli or Bacillus subtilis host-vector system but also Saccharomyces sp., Pseudomonas sp. or Streptomyces sp. host-vector system may be available. Mass production of uricase can be carried out, depending upon the specific advantage of the respective hostvector systems.

As described in detail above, DNA sequence and plasmid encoding uricase can be prepared by the present invention, and uricase can be produced by gene engineering technique. For example, DNA sequence encoding uricase which is more stable than the uricase conventionally obtained by the known process, plasmid having such DNA sequence, and the production of uricase by using the transformant containing such plasmid in gene engineering technique have been available, and so this invention contributes to industrial developments.

The present invention will be explained with reference to the following examples. Any conventional modifications in the technical field of the invention are included within the scope of the present invention. Example 1 Cloning of uricase gene. Step 1 Preparation of rabbit anti-uricase antibody

Anti-rabbit anti-uricase antibody antiserum was prepared by administering uricase,obtained by extracting and purifying from the culture broth of Bacillus sp. TB-90 (FERM BP-795), to a rabbit for immunization. Titer of this antiserum was 10 2 to 10 3 measured by ELISA method and 16 fold by Ouchterlony method. Then, antiserum was purified by applying 10 ml of antiserum to Protein A Sepharose column chromatography (4 ml), whereby 8. 9 ml of anti-uricase antibody Ig G was obtained. Step 2 Preparation of phage DNA library of Bacillus sp. TB-90 Chromosomal DNA was prepared from 2.5 g of cell body of Bacillus sp. TB- 90 cultivated in the bouillon medium (liquid medium, pH7.2 made by adding 5 g of meat extract, 10 g of peptone and 5 g of sodium chloride and diluting to a volume of lL), according to Doi, R.H (Recombinant Techniques, ed. Rodriquez et al., p162, Addison-Wesley Publishing Company, 1983) or Koizumi, J. et al., (Biotech. Bioeng., 27, 721-728, 1985).

As the result, about 900 pg of considerably pure (OD 260 /OD 280 about 1. 8) chromosomal DNA was obtained. Next, said DNA was partially digested with restriction enzyme Sau3AI in a conventional manner and subjected to 5-20 % sucrose density gradient centrifugation to give 2-20 kb fractions of DNA.

One pg of X phage cloning vector EMBL3 arms (Toyobo) was mixed with 0.4 pg of Sau3AI partially digested chromosomal DNA, ligated with 1 unit of T4 DNA ligase (Toyobo), subjected to packaging with X DNA using an in vitro packaging kit (Gigapack Gold, Toyobo), to infected.E. coli Q359 (Toyobo) plated so as to give about 2000 plagues per plate. Step 3 Selection of recombinant phages containing the gene for uricase (Isolation of uricase gene clone by plaque hybridization) Said purified IgG was mixed with horseradish peroxidase (HRPO) to give IgG-HRPO conjugate. Uricase gene clone was isolated by using Gene Expression kit (Boehringer Manheim) with the conjugate. In this case the detection sensitivity was 100 pg DNA. Said phage DNA library was screened to give bluish green-colored positive clones, and then strongly colored clone was selected and phage was purified until all the plaques colored. As the result, phages 1 and 3 were selected, infected with E. coli Q359, and the supernatant of each culture broth was asayed for uricase activity, allowing 7 mU/ml and 9 mU/ml, respectively. Identification of uricase gene of Bacillus sp. TB-90 Phage DNAs of isolated positive clones 1 and 3 were prepared in a conventional method (Molecular Cloning, ed. Maniatis et al., p. 85, Cold Spring Harbor Laboratory U.S.A., 1982), digested with restriction enzymes BamHI and SalI and analyzed by 0.8% agarose gel electrophoresis, showed that 18kb and 15kb SalI DNA fragments were inserted in DNAs from phages 1 and 3, respectively.

Further, a restriction map of inserted DNA using BamHI, SphI and KpnI, showed that phage 1 contained phage 3 and both had a common region. Then phage DNA of phage 1 was digested with restriction enzyme SalI, and 18kb inserted DNA fragment was extracted from agarose gel (Yoshiyuki Sakaki, Vector DNA, p.67, Kohdansha, Southern - 1 1 - I hybridization analysis, using the 18kb fragment as a probe (J.Mol, Biol., 98, 503-517, 1975), showed that 18kb DNA fragment of phage 1 was hybridized with not only 15kb DNA fragment of phage 3 but also with chromosomal DNA of TB-90 strain. This fact indicated that phage 1 DNA and phage 3 DNA, that had uricase activity, contained a common region and had an inserted DNA fragment derived from the chromosomal DNA of Bacillus sp. TB-90.

Then, 15kb DNA fragment was isolated from phage 3 DNA in the same method as described above, ligated with SalI-digested plasmid vector pUC18, subcloned and uricase gene region was specified in the insertion DNA fragment to give a recombinant plasmid named pUOD31, having 4.8kb BamHISphI fragment containing uricase gene. The restriction map of this plasmid is shown at the upper center of Fig. 2.

Subsequently, DNA fragment which was digested with various restriction enzymes was subcloned into vectors pUC18 and 19, and plasmid DNA was prepared according to Birnboim, and Doly (Nucleic Acids Res., 7, 15131523, 1979). The resulting DNA was suspended in 18 pl of TE (1OmM Tris?"hydr.oclllo_r-ic -acid (pH7. 4), lmM EDTA), mixed with 2 p 1 2N NaOH, allowed to stand at room temperature for 5 minutes, mixed with 8 pl of 5M ammonium acetate and mixed with 100 pl of cold ethanol for ethanol precipitation.

Determination of base sequence on these plasmid DNAs was performed with M13 sequencing kit (Toyobo) and [a-32p] dCTP (400 Ci/mmol, Amersham Japan).

Fig. 1 shows the determined sequence. The uricase gene originated from Bacillus sp TB-90 had a 999 bases coding region starting from an initiation codon ATG and ending in a stop codon TGA, which encoded 332 amino residues as shown in Fig. 1. Construction of expression plasmid pUOD316 and pKU1 for the purpose of expressing uricase gene of Bacillus sp. TB-90 in the cells of Escherichia coli About 10pg of recombinant plasmid pUOD31 containing uricase gene was mixed with EcoRI and HincII, allowed to react at 37C for 2 hours in 30P1 of M buffer (1OmM Tris-HCl (pH7.5), 1OmM MgCl 2. lmM dithiothreitol and SOmM NaCl), and the reaction mixture was subjected to electrophoresis with 0.8% agarose gel containing 0.1 pg/ml of ethidium bromide, to isolate 1.4kb of EcoRI-HincII DNA fragment.

Then, lpg of expression vectors pUC18 (Toyobo) or pKK223-3 (Pharmacia) were digested with EcoRI, HincII and with EcoRI, SmaI, respectively, to isolate 2.7kb and 4.6kb of DNA fragments were isolated in the same method as described above, respectively.

Subsequently, lpg of 1.4 kb EcoRI-HincII DNA fragment - 13 intially prepared was mixed with each lpg of expression vector pUC18 or pKK223-3, respectively mixed with 5 units of T4DNA ligase (Toyobo) and allowed to react at 16C for 6 hours in 45P1 of ligase reaction buffer (66mM Tris-HCl (pH 7.6), 6.6mM MgCl 2' 1OmM dithiothreitol and 1.OmM ATP).

Then, Escherichia coli JM109 (Takara Shuzo) was transformed with the reaction mixture of ligation according to Hanahan's method, (J. Mol. Biol. , 166, 557, 1983). Ampicillin resistant colonies appeared were cultivated in L-broth solid medium [10g of trypton (Difco), 5g of yeast extract (Difco), 5g of NaCl, and a medium (pH7.2) prepared by dissolving 15g of powdery agar in IL of distilled water] containing 50pg/m1 of am15.icillin, and plasmid DNA was prepared by the method of Birnboim and Doly and digested with various restriction enzymes. The restriction fragment analysis on agalose gel electrophoresis showed correct insertion of 1.4 kb EcoRI-HincII DNA fragments into the respective expression vectors. Recombinant plasmid ligated with pUC18 was called pUOD316, and those ligated with pKK223-3 was called pKU1. Fig. 2 shows the construction method of expression plasmids pUOD316 and pKU1 from recombinant plasmid pUOD31.

3 1 Production of uricase in Escherichia coli Each expression plasmid pUOD316 or pKU1 thus constructed was introduced into Escherichia coli JM109 according to Hanahan's method, and each uricase produced by recombinant E. Coli JM109/pUOD316 and JM109/pKU1, respectively was identified and analyzed as shown below.

Each recombinant E. coli was cultivated overnight at 37'C in L broth liquid medium. 0.1 ml of culture broth was transferred to 10 ml of L broth liquid medium and cultivated at 370C. When OD 660 reached at 0.2, isopropylthiogalactoside (IPTG) was added to a final concentration of lmM. After the cultivation was further continued for 16 hours, 1.0m1 of the culture broth was separated, mixed with 0.5m1 of extraction buffer [SOmM borate buffer (pH8.0), 1OmM EDTA.3Na, 0.3% Triton X-100, and 0.3% lysozyme], incubated at 37'C for 10 minutes and centrifuged at 12,000 rpm for 10 minutes to give a bacteriolyticlysate (supernatant). Twenty pl of this lysate was suspended in the same amount of sample loading buffer [62. 5mM Tris-HCl (pH6.8), 2% SDS, 10% glycerol, 5% 2-melcaptoethanol, and 0. 001% BPB], heated at 1000C for 5 minutes and subjected to SDSpolyacryldmide gel electrophoresis according to Laemmli et al., (Nature, 227, 680-685, 1970). After the electrophoresis, the gel was stained with Coomassie - -1 brilliant blue, destained, dried and fixed on a filter paper. As the result, an uricase band of about 35K molecular weight was detected on E. coli JM109 containing expression plasmid, and this protein band showed specific binding with anti-uricase antibody (IgG). When each protein band on the gel was measured with a densitometer, it was found that each 1% and 3% of uricase per whole intracellular protein was produced by E. coli JM109/pUOD316 and JM109/pKU1. These results showed that these recombinant E. coli produced efficiently uricase of Bacillus sp. TB-90. E. coli JM109/pUOD316 was deposited as FERM BP-1979 and E. coli JM109/pKU1 as FERM BP-1980 in Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, respectively. Uricase thus obtained above has the following characteristics. (1) Reactivity: Uricase catalyzes the reaction in which uric acid is decomposed oxidatively to release hydrogen peroxide. (2) Optimum pH 5-10 (3) Stable pH 5-9 Optimum temperature 45-SOOC.

(4) - 16 1 (5) Stable temperature range No less than uricase activity is observed when it treated at 500C for 10 minutes. (6) Substrate specificity Uricase shows substrate specificity to uric acid. Example 2 Construction of recombinant plasmid pEB2 for expression of uricase gene in Bacillus subtilis 3.Okb BamHI-Bg1II fragment containing uricase gene was isolated and extracted from E. coli recombinant plasmid pUOD31 as prepared in the same method as in Example 1. Then, 2pg of E. coli-Bacillus subtilis, shuttle vector pHY300 PLK(Toyobo) was digested with restriction enzyme BamHI, ligated with 2pg of 3.Okb BamHI-Bg1II fragment containing uricase gene by using 2 units of T4 DNA ligase, and E. coli C600 was transformed according to Hanahan's method. Ampicillion resistant strain was selected on L broth solid medium. Plasmid DNA was prepared from C600 strain showing uricase activity among the transformed strains according to the method of Birnboim, et al named pEB2. Fig. 3 shows a constructing method of this plasmid. Then competent cells of Bacillus subtilis ISW 1214 (Toyobo) were transformed with this plasmid according to Rodriguez et al., ed., (Recombinant DNA Techniques, p.184-186, 17 - is R Addison-Wesley Publishing Company, 1983). These cells were plated on L broth solid medium containing 15pg/ml of tetracycline and 0.2% glucose and cultivated at 371C overnight. As the result, Bacillus subtilis transformed with recombinant plasmid pEB2 was obtained by selecting a tetracycline resistant colonies. This transformant was cultivated at 371C overnight in L broth liquid medium containing 15pg/ml of tetracycline and 0.2% glucose, and the plasmid was isolated and extracted according to Rodriguez et al., (Recombinant DNA Techniques, ed., p. 164-165, Addison- Wesley Publishign Company, 1983). The plasmid of this transformant was digested with various restriction enzymes and subjected to electrophoresis on agarose gel, whereby it was confirmed that this plasmid retained the recombinant plasmid pEB2 in which 3.Okb BamHI-Bq1II fragment containing uricase gene was inserted. Production of uricase in the cells of Bacillus subtilis Uricase, produced by recombinant Bacillus subtilis ISW1214/PEB2 (FERM BP1981) which was obtained by introducing recombinant plasmid pEB2 containing uricase gene into Bacillus subtilis ISW 1214, was identified and analyzed as shown below.

Recombinant Bacillus subtilis was cultivated at 37C overnight in L broth liquid medium containing 15pg/mI of 1 -1 tetracycline and 0.2% glucose. After cultivation, 1.0m1 of the culture broth was separated and centrifuged at 8,000 rpm for 5 minutes, to separate supernatant from the cells. The cells were suspended in 1.0 ml of extraction buffer, incubated at 37'C for 10 minutes and centrifuged at 12,000 rpm for 10 minutes to give a cell lysate. Then, each 20 pl of the culture supernatant and the cell lysate was suspended in the same amount of said sample loading buffer, heated at 1OVC for 5 minutes and subjected to electrophoresis on SDS-polyacrylamide gel according to Laemmli et al. method. After electrophoresis, the gel was stained with Coomassie brilliant blue, destained, dried and fixed on a filter paper. As the result, it was found that a band of uricase in about 35K of molecular weight was detected in both the culture supernatant and the cell lysate of Bacillus subtilis ISW 1214 transformant containing pEB2, and this protein band showed a specific cross reaction with anti-uricase antibody (IgG). When each protein band was assayed by a densitometer, Bacillus subtilis ISW 1214/pEB2 produced 0.6% of uricase per whole cellular protein in the cells. Further, 40% of uricase produced in the cells was found to be secreted to the culture supernatant, namely extracellularly. Accordingly, it was found that recombinant Bacillus subtilis ISW 1214/pEB2 produced uricase intracellularly or extra cellularly.

Claims (9)

WHAT IS CLAIMED IS

1. DNA sequence containing a gene encoding uricase.

2. DNA sequence according to claim 1, in which the gene encoding uricase is a microorganism-origin gene.

3. DNA sequence according to claim 1, in which the gene encoding uricase is originated from Bacillus sp. TB-90.

4. Plasmid having DNA containing a gene encoding uricase.

5. Transformant containing a plasmid which has DNA containing a gene encoding uricase.

6. Transformant according to claim 5, in which the gene encoding uricase is foreign to hosts and the trans formant belongs to Escherichia coli, Bacillus subtilis,, Saccharomyces Pseudomonas 2. or Streptomyces sp..

7. Transformant according to claim 5, in which the transformant belongs to Escherichia coli or Bacillus subtilis.

8. Transformant according to claim 5, in which the transformant is Escherichia coli JM109 (pUOD316), Escherichia coli JM109(pKU1), Bacillus subtilis, ISW 1214 (pEB2) and variants thereof.

9. Process for producing uricase which comprises cultivating in a medium a transformant containing a plasmid which has DNA containing a gene encoding uricase to produce said uricase and recovering said uricase thus produced.

Published 1990atThe Patent office. State House, 6671 High Holborn. London WC1R4TP. Further copies maybe obtained from The Patent 0Mce.

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