AP429A - Nucleotide sequence encoding carbamoyl phosphate synthetase 11. - Google Patents
Nucleotide sequence encoding carbamoyl phosphate synthetase 11. Download PDFInfo
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- AP429A AP429A APAP/P/1994/000607A AP9400607A AP429A AP 429 A AP429 A AP 429A AP 9400607 A AP9400607 A AP 9400607A AP 429 A AP429 A AP 429A
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- acid molecule
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- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/05—Carbon-nitrogen ligases with glutamine as amido-N-donor (6.3.5)
- C12Y603/05005—Carbamoyl-phosphate synthase (glutamine-hydrolysing) (6.3.5.5)
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Abstract
The present invention provides a nucleotide sequence encoding
Description
The present invention relates to nucleotide sequences encoding carbamoyl phosphate synthetase II of Plasmodium falciparum, to methods of producing this enzyme using recombinant DNA technology and to the use of this sequence and enzyme in the design of therapeutics.
The urgency for the design of novel chemotherapeutic agents for the treatment of malaria has been renewed in recent times due to the evolution of human malarial , parasites, primarily Plasmodium falciparum T which are resistant to traditional drugs. Research into a vaccine seems a very plausible alternative, but after years of investigation, no clinically acceptable product has come to date. At the same time, there is also an increasing decline in the efficacy of insecticides against mosquito vectors. At present, more than two-thirds of the world's population - approximately 500 million people - are thought to live in malaria areas (Miller, 1989). It ranks eighth in the World Health Organization's (WHO) list of ten most prevalent diseases of the world (270 million infections a year) and ranks ninth of the ten most deadly diseases, claiming over 2 million lives a year (Cox, 1991; Marshall, 1991). Though chiefly confined to poor nations, there are recent reports of infections in the United States (Marshall, 1991) and Australia (Johnson, 1991), and ever increasing cases of travellers' malaria (Steffen and Behrens, 1992).
Comparative biochemical studies between the malaria parasite, P. falciparum and its host have revealed differences in a number of metabolic pathways. One such distinction is that the parasite relies exclusively on pyrimidine synthesis de novo because of its inability to salvage preformed pyrimidines (Sherman, 1973). Moreover, the mature human red blood cell has no recognised requirement for pyrimidine nucleotides (Gero and
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AP 0 0 0 4 2 9
O'Sullivan, 199Q). Major efforts have been directed towards the development of inhibitors of the pyr5mi dine biosynthetic pathway (Hammond et al·, 1985; Scott et al., 1986; Prapunwattana et al., 1988; Queen et al., 1990;
Krungkrai et al., 1992), confirming its potential as a chemotherapeutic locus. Current research into the molecular biology of the key pyrimidine enzymes is envisioned as a powerful tool, not only to get a better understanding of the parasite's biochemistry, but also to io explore specific differences between the parasite and the C mammalian enzymes.
Glutamine-dependent carbamoyl phosphate synthetase (CPSII, EC 6.3.5.5) catalyses the first committed and rate-limiting step in the de novo pyrimidine biosynthetic pathway of eukaryotic organisms (Jones, 1980). Moreover, because it catalyzes a complex reaction involving three catalytic units and several substrates and intermediates, it is a very interesting enzyme to study from a biochemical point of view. The structural relationship of
CPSii to other pyrimidine enzymes varies in different organisms, making it a good subject for evolutionary studies.
The paucity of material that can be obtained from malarial cultures has hampered the isolation of adequate amounts of pure protein for analysis. The difficulty in purifying CPS is further augmented by its inherent instability. Studies using crude extracts from P. berghei (a rodent malaria) revealed a high molecular weight protein containing CPS activity, which was assumed to be so associated with ATCase (Hill et al., 1981), a situation also found in yeast (Makoff and Radford, 1978). However, recent analysis by Krungkrai and co-workers (1990) detected separate CPSH and ATCase activities in P. berghei. Although CPS activity has been detected in
P· falclpaxvza (Reyes et al., 1982) until this current
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AP 0 0 0 4 2 9 study there is no indication of its size nor its linkage with other enzymes in the pathway.
The glutamine-dependent activity of CPSII can be divided into two steps: (1) a glutaminase (GLNase) reaction which hydrolyzes glutamine (Gin) and transfers ammonia to the site of the carbamoyl phosphate synthetase; and (2) a synthetase reaction, where carbamoyl phosphate is synthesised from two molecules of adenosine triphosphate (ATP), bicarbonate and ammonia. The second activity involves three partial reactions: (a) thej activation of bicarbonate by ATP; (b) the reaction of the activated species carboxyphasphate with ammonia to form carbamate; and (c) the ATP-dependent phosphorylation of carbamate to form carbamoyl phosphate (powers and Meister,
1978). Hence, there are two major domains in CPSII, the glutamine amidotransferase domain (GAT) and the carbamoyl phosphate synthetase domain (CPS) or simply synthetase domain. The glutaminase domain (GLNase) is a subdomain of GAT, while there are two ATP-binding subdomains in the synthetase domain.
In view of the similarities between the glutamine amidotransferase domain of CPS and other amidotransf erases, it has been proposed that these subunits arose by divergent evolution from a common ancestral gene (= 20 kDa) representing the GLNase domain and that particular evolution of the CPS GAT domain (= 42 kDa which includes the putative structural domain only present in CPS) must have involved fusions and/or insertions of other sequences (Werner et al., 1985). The
GAT of mammalian CPS I gene has been proposed to be formed by a simple gene fusion event at the 5' end of this ancestral gene with an unknown gene (Nyunoya et al.,
1985).
The genes for the larger synthetase domains of various organisms were postulated to have undergone a gene duplication of an ancestral kinase gene resulting in a
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AP 0 0 0 4 2 9 polypeptide with, two homologous halves (Simmer et al., 1990). Unlike the subunit structure of E. coli and arginine-specific CPS of yeast, a further fusion of the genes encoding GAT and the synthetase domains was suggested to have formed the single gene specific for pyrimidine biosynthesis in higher eukaryotes. Conversely, Simmer and colleagues (1990) proposed that the argininespecific CPS's (like cpal and cpa2 in yeast) as well as rat mitochondrial CPSI arose by defusioa from the pyrimidine chimera.
The present inventors have isolated and characterised the complete gene encoding the CPSII enzyme from P. falciparum (pfCPSII). Reported here is the cDNA sequence including 5' and 3' untranslated regions. In so is doing, the present inventors have identified the respective glutaminase and synthetase domains. Unlike CPSII genes in yeast, D. discoideum, and mammals, there is no evidence for linkage to the subsequent enzyme, aspartate transcarbamoylase (ATCase). This is in contrast to the report by Hill et al., (1981) for the enzymes from p. berghei. The present inventors have, however, found two large inserts in the P. falciparum gene of a nature that does not appear to have been previously described.
Accordingly, in a first aspect, the present invention consists In a nucleic acid molecule encoding carbamoyl phosphate synthetase II of Plasmodium falciparum, the nucleic acid molecule including a sequence substantially as shown in Table 1 from 1 to 7176, or from 1 to 750, or from 751 to 1446, or from 1447 to 2070, or from 2071 to 3762, or from 3763 to 5571, or from 5572 to 7173, or from 1 to 3360, or from 2071 to 6666, or from 2071 to 7173, or a functionally equivalent sequence.
In a preferred embodiment of the present invention, the nucleic acid molecule includes a sequence shown in
Table 1 from -1225 to 7695 or a functionally equivalent sequence.
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In a second aspect, the present invention consists in an isolated polypeptide, the polypeptide including an amino acid sequence substantially as shown in Table 1 from 1 to 2391, from 483 to 690, from 691 to 1254, 1858 to
2391, from 1 to 1120, from 691 to 2222, or from 691 to
2391.
As used herein the term functionally equivalent sequence is intended to cover minor variations in the nucleic acid sequence which, due to degeneracy in the code, do not result in the sequence encoding a different C polypeptide.
In a third aspect the present invention consists in a method of producing Plasmodium falciparum carbamoyl phosphate synthetase II, the method comprising culturing a
IS cell transformed with the nucleic acid molecule of the first aspect of the present invention under conditions which allow expression of the nucleic acid sequence/ and recovering the expressed carbamoyl phosphate synthetase
II.
The cells may be either bacteria or eukaryotic cells. Examples of preferred cells include E.ccli, yeast, ( and Dictyostalium discoideom.
As will be readily understood by persons skilled in this field, the elucidation of the nucleotide sequence for
CPSII enables the production of a range of therapeutic agents. These include antisense nucleotides, ribozymes, and the targeting of RNA and DNA sequences using other approaches, e.g., triplex formation.
As can be seen from a consideration of the sequence set out in Table 1 the Plasmodium falciparum CPSII gene includes two inserted sequences not found in other carbamoyl phosphate synthetase genes. The first inserted sequence separates the putative structural domain and the glutininase domain whilst the second inserted sequence separates the two ATP binding subdomains of the synthetase subunit CPSa and CPSb.
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TABLE 1. Nucleotide and Deduced Amino Acid Sequence of the Carbamoyl Phosphate Synthetase II Gene from Plasmodium falcipanun
| -1225 | « · · · · · GAATTCCTTCAGCCAAAAAAAATGACAACGCAAATTTTAAGAAAAGAAAAACAAICGACT -1166 |
| -1165 | » · · · - · CGTCTrTGAATGAGGTTAGAAATTCGATACCTGAAAGGGACTTAAGAAGGCTTAACAGAG -1106 |
| -1105 | ( · · · · · AAAAGAGTAAAATCTTAIAAGCATTTGAAGGAAAAAATAATAAAATAAAAAAATAAAAAG -1046 |
| -1045 | AIAAAAAATAITIATATTTCATATGTAGTAIATATAATGAITAI TCATATTAAIAACATA - 93 6 |
| -985 | a · · a a · GATAAAAAACTTTTTTTTTTTTTTTTTTTCTTTATATTTATTAACAATACATTTAAGIIA -926 |
| -925 | • · · · · ' * TTTTATATATATATAIATATATATaIAIATAIATATATATATATATGTTTGTGTGTTCAT -866 |
| -865 | • · · · ♦ · TTGTTTATAAAATTACTTGAAATATAAAACTTATTAATATATTTCCAATTAATATGAATA -806 |
| -805 | a · · · · * CAATTATTAATATTTTGATGTGTACACATTAATATAGTTTTACACTTCTTAIAATAAAAC - 746 |
| -745 | CATCCTATAIATTATACACAATATAIAATACTCCCCAAIATTGTGGTTCCTATAATTTTA -686 |
| -685 | *«··*· TTTAIATATTTATTTATTAATTIATTCATTTaTTTATTTTTTTTCTTAGTTTATAAAATA -626 |
| -625 | a · · « « * GTAATTCTACTAATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAaTT -566 |
| -565 | • · · · · * tacatatgaaaaatgaacttgtataigtaaatttataaatattitaaacataaatataaa - 5 0 6 |
| -505 ( ' -445 | TGTATaAAAAAAAAAAAGAAAAATGGGAAAAAATAATAZAGATATATATaTAAATATATA -446 • · · * · · TATATATaTAATTATTCCGCATAITCTCTGAAICATAGGTCTTAAACAGTTTTATTCTTT -386 |
| -385 | TAACATCACAAAGTTGTTATTAUAGTAIATATATCTTATTSGTTCCTATATAAAACTAT -326 |
| -325 | « « a · · · AGTATTCTaTAATATAITCTCTATaTTTCATTTTATCATTTGIAAGCAATCCCTATTTAT -266 |
| -265 | TATAATTATTATTTTTTTTTTTATAAAAGAGGTATAAAACAGTTTAITCAATTTTTTTCC -206 |
| -205 | • * · · · · TAAAGGAGCAACCTTCAGTCAAiTTACATTTTCCACCGGTTeGTTGGCACAACATAATGT -146 |
| -145 | a » · · ♦ TACAGCTAAAAAAAGAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATAT -86 |
| -85 | • · a · · » ATATATATAIATATACATAAlAIGIlACAATGeTACCAIACAAGTATATAAATTTTTCAAC -26 |
| -25 | ATTGTTGTGATGTTGCATTTTTCTt -1 |
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TAJLE 1 (eont)
ΑΤΟΤΑΤΑΤΤΤΟΤΤΤΤΑΔΑΙΑΤΑΑΤΤΤΛΪΑΤΑΤΑΤΑΤΑΓΑΤΑΤΑΤΑΊΑΤΑΤΑΤΑΙΑΤΑΤΤΤ 60 lMYISFKYNLYIYlYIYXTI P 20
GTTCTIATAGATTTTAAAACAGTTCGGAGGTTAATTCTTGAAGATGGTAACGAATTTGTA 120 21VLIDFKTVC&1ILEDGNEF V 40 • · · · ·
121 GGGTACAGTGTAGGTTACGAACGGTGTAAAGGAAAIAATAGTAIATCATGTCATAAGGAG 180
41GYSVCY BGCKGNNSISCHKE 60
181 TATaGAAAIATTATTAAIAATGATAATAGCAAGAATAGTAATAATTCATTTTGTAAIAAT 240 61YRKIINNDNSKNSNNSFCNN 80
241 GAAGAAAACAATTTGAAACATGATTTATTATATAAAAATAGTCGATTAGAAAATGAAGAT a 00 81EENNLKDDLLYKMSRLENED 100 • » » · · *
301 TTTATTGTTACAGCTGAAGTTATATTTAATACAGCTATGGTTGGAIATCCTGAAGCTTTA 360 101 F I V T C E V I F Μ I A Μ V 6 Y B Ε A L 120
361 ACGGACCXAAGTTaTTTTGGTCAAATATIAGTTTTAACATTTCCTTCIATTGGTAATTaT 420 121 I D B 5 Y F G Q I L V L T F F S I G N Y 140 • # · · · ·
421 GGTATTGAAAAAGIAAAACATGATGAAACGTTTGGATTAGIACAAAATTTTGAAAGTAAT 480 141 G I ϊ K V K H D E T F G L V Q H F E 5 N 160 ί ......
481 AAAATTCAAGTACAAGGTTTAGTTATTTGTGAATATTCGAAGCAATCATATCATTACAAT 340 ¢( 161 K I Q V Q G L V I C E Y S K Q S Y Η Y N 180
541 ICTIAIAITACCTTAAGTGAATGGTTAAAGATTTATAAAATTCCATGTATAGGTGGIAIA 600 181 $ Y I T L S E V L K I Y K I P C I G G I 200 « · » · · ·
601 GAIACAAGAGCCTTAACAAAACTTTTAAGACAAAAAGGIAGTATGTTAGGTAAAATAGTT 660 201DTRALTXLLREKGSMLGK1V 220 • · · · · ♦
661 ATATATAAAAACAGACAACATATTAAIAAAITATATAAAGAAATTAATCTTTTTGATCCT 720 221 I Y K N R Q S X N K L Y X £ I H L F D ? 240 • . · · · · ·
721 GGTAAIAIAGATACTCTAAAATATGIATGTAATCATTTTATACGTGTTATXAAGTTGAAT 780 241 GKIDTLKYVC NHFIRV IKLX 260
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TABLE 1 (cont)
840
283
900
300
960
320
1020
340
1080
360
1140
380
1200
400
1260
420
1320
440
1380
460
1440
460
1500
500
1560
520
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IAKJC i (cent)
1561 TACAATTTTAATCATAiAGATIATGATCGAGITCTTTTATCTAATGGTCCTGGAGAXCCT 1620
521 Y N F Ν Η I P Y P A V L L S N G P G D P
540
1621 AAAAASTGTGATTTCCTTATAAAAAATTTGAAAGATAGTTTAACAAAAAATAAAATTATA 1680 541 K K C D F L I R N L K D S L T K Ν K I I 560
1681 TTTGGTATTiGTTIAGGTAATCAACTATTACCTAlATCATTACGTTGTGACACATATAAA 1740
561PGICLGNQLLCXSLGCDTY K
580
1741 ATGAAATAIGGTAATAGaCGTCTTAATCAACCCGTAATACAATTAGTAGATAATATATGT 1800
581MKYGHR GVNQP VIQLVDHIC
600
1801 TACATTACCTCACAAAATCAieGATACTGiTTAAAGAAAAAATCAAITTIAAAAAGAAAA 1860 601YITSQHHGYCLKKKSILKR.K 620
1861 GAGCTTGCGATTAGXIAIAIAAATGCIAATCAXAAAICTATAGAAGGTATTTCACaIAAA 1920 621 E L A I S Y I N A R D K S I E G I S H K 640
1921 AATGGAAGATTTTATaSTGTCCAGTTXCATCCTGAGGGTAATAATGGTCCTGAAGATAGA 1980 641NGRFYSVQFHPEGSNGPEDT 660
1981 TCATTTTTATTTAAGAAIITTCTTTTAGATATCTTTAATAAGAAAAAACAATATAGAGAA 2040 661SFLFKNFLLDIFKKKRQYRE 660
2041 tatttacgataiaatattatttatataaaaaagaaagtgcttcttttaggtagtggtggt 2100 681YLGYNIXYIKXKVLLLGSGG 700
2101 TTAIGTATAGGACAAGCAGGAGAATTCGaTTATTCAGGAACACAAGCAATTAAAAGTITA 2160 701 L C I G Q A G £ F P Y S G T Q A I K S L 720
2161 AAAGAATGTGGTAIAIATGTTATATTAGTTAATCCTAACATAGCAACTGTTCAAACATCA 2220 721 K E C G I Υ V I L V Η P Ν I A T V Q T S 740
2221 AAAGGTTTGGCAGATAAGGTATACTTTTTACCAGTTAaTTGIGAATTTGXAGAAAAAATT 2280 741RGLADK VYPLPVNCEFVEKI 760
2281 ATTAAAAACGAAAAACCTGATTTTATTTTATGIACATTTGGTGGTCAGACAGCTTIAAAT 2340
761 IKKEXPDFILCTFGGQTAt.il 780
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TABLE 1 (coat) » « · · · ·
2341 TGTGCTTTAAIGTTACATCAAAAAAAAGTATTGAAAAAGAATAATTGTCAATGTTTAGGT 2400 781 C A L M L D Q K K V L K X N N C Q C L , G 800
2401 ACATCTTIAGAATCTATAAGAAIAACAGAAAATAGAACATTATTTGCTGAAAAATTAAAA 2460 801 T S L E S I R I T E H R T L F A E K L R 820 • · · * · J ·
2461 GAAATTAAIGAAAGAATAGCTGCATAIGCTAGTGGAAAAAATGTTAATCAAGCTATTCAT 2520 821 E I IT E R 1 A Ρ Y G S A R N V M Q A I D 840 «*«···
2521 ATAGCTaaTAAAATAGGATATCCAATATTAGTACGTACAACATTTTCGTTAGGAGGATTA 2580
841 lANKIGYPILVRTTFSLGG'L 860 • · « · · ·
2581 AAIAGTAGITTCATAAAIAATGAAGAAGAACTTATCGAAAAATCTAATAAAATATTTTTA 2640
861 NSSFIHKEEELIEKCNKIP L 880 « « « · · ·
2641 CAAACTGATAATCAAATATTTATAGAIAAATCAITACAAGGATGGAAAGAAAIAGAATAT 2700 881 Q T D H E I F I D K S L Q G V K E I £ Y 900 » . » * · · «
2701 GAATTATTAAGAGATAAIAAAAAIAATTGTAIAGCTATATGTAATATGGAAAAIATAGAT 2760
901 E L L R D N K Ν Ν C I A I C R Μ £ N I D 920 a * « · · ·
2761 CCATIAGGTATACATACAGGAGATAGTATAGTTGTTGCACCITCACAAACATTAAGTAAT 2820 921 P L G Σ Η T G D S I V V A P S Q T L 5 N 940
.......
2821 TATGAATAIIATAAATTTAGAGAAATAGCATTAAAGGTAATTACACATTTAAATATTATA 28 BO - 941 YBYYKFRIXALKVJTHLNI'I 960 • · · · · ·
2831 GGAGAATGTAATATACAATTTGGTATAAATCCACAAACAGGAGAATATTGTATTATTGAA 2940
961 G E C N X Q F G I N P Q T G E Y C 1 I E 980
2941 GTTAATGCTAGGCTTAGTAGAAGTTCAGCATTAGCTTCTAAAGCTACTGGTTATCCACTT 3000
981 7 IT A R L S R S S A L A S KATGYPL 1000 • « » · »
3001 GCTTAtATATCAGCAAAAATAGCCTTCGGATAIGATTTGATAAGTTTAAAAAATAGCATA 3060 1001 AYXSAKIALGYDL I S L K N S I 1020 • · 4 · · ·
3061 actaaaaaaacaactgcctgttttgaaccctctciagatiacattacaacaaaaatacca 3120 1021 TKKTTACFEPSLDYITTKIP 1040
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II
TABLE 1 (coat) • * · · · ·
3121 CGATGGGATTTAAATAAATTTGAGTTTGCTTCTAATACAATGAATAGTAGTATGAAAAGT 3180 1041 RVDLNKFEFASNTMNSSMKS 1060 • « · · · ·
3181 GTAGGACAAGTTaTGTCTATAGGTAGAACCTTTGAAGAAICTATACAAAAATCTTTAAGA 3240 1061 VCEVMS1GR.TFEES1QESLR 1080
3241 TGTATTGATGAIAATTATTTAGGATTTAGTAATACGTATTGTATAGAITGGGATGAAAAG 3300 1081 CTDDNYLGFSNTYCXDWDE K 1100 • · · · ·
3301 AAAATTATTGAAGAATTAAAAAATCCATCACCAAAAAGAATTGATGCTATACATCAAGCT 3350 1101 KT IEELKNB SPKR.IDAIHQA 1120
3361 TTCCATTTMAIAIGCCTATCGATAAAAIACATGAGCTGACACATAITGATIATTGGTIC 3420 1121 FHLNMPMDKXBEL THIDYW F 1140
3421 TTACATAAATTTIAIAAIATATATAATTIACAAAATAAGTTGAAAACGTTAAAATiAGAG 1141 LHKFYK IYNLQNKLKTLKL
3480
1160
3481 CAATTATCTTTTAATGAinGAAGTATTTTAAGAAGCATGGTTTTAGTGATAAGCAAATA 3540 1161 QLSFHDLKYFKXHGFSDKQ I 1180
3341 GCTCACTACTTATCCTTCAACACAAGCGATAATAATAATAATAATAATAATATIAGCTCA. 3600 1181 ABYLSFNTSDNNHNKHIIIS S 1200 *···»·
3601 TGTAGGGTTACAGAAAATGATGTTATGAAATATAGAGAAAAGCTAGGATTATTTCCACAT 3660 1201 CRVTENDVMKYREKLGLFPH 1220
3661 AITAAACTTATTGATACCTTATCAGCCGAATTTCCGGCTTTAACTAATTATTTAIATTTA 3720 1221 IKVIDTLSAEFFALTNYLYL 1240 • · · · · ·
3721 ACTTATCAAGGTCAAGAACATGATGTTCTCCCATTAAATAIGAAAAGGAAAAAGATAIGC 3780 1241 TYQGQESDVLPLNM K _R. K K I C 1260 ···«··
3781 ACGCTTAATAATAAACGAAATGCAAATAAGAAAAAAGTCCATGTCAAGAACCACTTATAT 3B40 1261 T L N K K R N A N K.....K K V Η V ,K N H L Y 1280
3841 AATGAAGTAGTTGATGATAAGGATACACAATTACACAAAGAAAATAATAATAATAATAAT 3900 1281 N E V__V DDKDTOLg KEWWyNNy 1300
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TABLE 1 (contJ
3960
1320
4080
1360
4140
1380
4200
1400
4260
1420
4320
1440
4380
1460
4440
1480
4500
1500
4S60
1S20
4620
1540
4680
1560
4020
1340
BAD ORIGINAL
30/11-'93 09:55 ©44 534 70933
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Af* 0 0 0 4 2 9
TABLE 1 (cont)
4740
1580
4800
1600
4860
1620
4920
1640
4980
1660
5040
1680
5100
1700
5160
1720
5220
174Q
5280
1760
5340
1780
5400
1600
5460
1820
BAD ORIGINAL Ά
30/11/ 93 09:56 ©44 534 70933
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AP 0 0 0 4 2 9
TABLE 1 (COEX) ······
3461 GTATCaTCTGTTATATATTCAGGGAACGAAAATATATTTAAIGAAAAATATAATGATATA 5520 1821 V S -S . V . I, T . $ G N E N I E N I K Y K , D _I. . 1840 ··»·«·
5521 GGTTTTAAAATAAICGATAATAGGAATGAAAAAGAGAAAGAGAAAAAGAAATGTTTTATT 5580 1841 G F K , I I D S R S E K g R E_ K K R C F I 1860
5581 GTATTaGGITCTCCTTGTTATCCTATTGGTAGTTCTGTAGAATTTGATTGGAGTGCTATA 5640 1861 VLGCGCYRIGSSVEFDWSAI 1880
5641 CATTGTGTAAAGACCATAAGAAAATTAAACCAIAAAGCTATATTAATAAAITGTAACCCA 5700 1881 BCVRTIRKLilHKAILINCHP 1900 • · · * »
5701 GAAACTGTAAGTACAGAITATGATGAAAGTGATCGTCTATATTTTCATGAAATAACAACA 5760 1901 ETV’STD TDESDRLYFDEIT T 1920 • · · · « «
5761 CAAGTTATAAAATTTATATAIAACTTTGAAAAIAGTAATGGTGTGATTATAGCTTTTGGT 5820 1921 E Y I K F 1YNFEKSN GVI I A F G 1940 • · · · · ·
5821 GGACAAACATCAAATAAiTTAGTATTTAGTTTAIATAAAAAIAAieTAAATATATTAGGA 5880 1941 GQTSNN LVFSLYKNNVNXl/G 1960 « . « · t · ·
5881 TCAGTGCAGAAAGTGTTGATTGTTGTGAAAATAGGAATAAATTTTCGCACTTATCTCATT 5940 1961 SVHRVLIVVKIOINFRTYVX 1980 • >····
5941 CTTAAAATTGATCAACCGAAATGGAATAAATTTACAAAATIATCGAAGGCTATACAATTT 6000 1981 LRIDQPKWNRFTRLSRAIQF 2000
6001 GCTAATGAGGTAAAATTTCCTGTATTaGTAAGACCATCGIATGTATTATCTGGTGCAGCT 6060 2001 AN EVKFPVLVRP.S Y V L S G A A 2020 » · · w · «
6061 ATGAGAGTTGTAAATTGTTTTGAAGAATTAAAAAACTTTTTAATGAAGGCAGCTATTGTT 6120 2021 HRVVNCFEELRNPLMKAAIV 2040
6121 AGTAAAGATAATCCTGTTGTAATATCAAAATTTATTGaGAATCCTAAAGAAAIAGAAATA 6180 2041 SRDNPV VISKFIENAREIEI 2060
6181 GaITGTGTTAGTAAAAAIGGTAAAAIAATTAATTATGCTAIATCTGAACATGTTCAAAAT 6240 2061 DCVSKNGKIXNYAISBHVEN 2080
BAD ORIGINAL ft
| ύθ/il -93 09:57 | 044 534 70933 . S&F JOHANNESBURG @018/032 AP 0 P 0 4 2 9 |
| 6241 2061 | 15 TASLE 1 (cont) • * · « GCTGGTGTACATTCACGTGAIGCAACATTAATaTIACCTGCACAAAAIAIATATGTTGAA 6300 AGVHSGDATLILPAQNIVVE 2100 |
| 6301 2101 | ♦ ····· ACACATACGAAAAIAAAGAAAATATCCGAAAAGATTTCAAAATCATTAAATATATCTCGT 6360 THRKIKK1SERI SKSLNIS G 2120 |
| 6361 2121 | • « · · · * CCATTIAATATACAATTIATAIGTCATCAAAATGAAAIAAAAATTATTCAATCTAATMA 6420 PFNIQF ICBQNEIK.IIECR L 2140 |
| 6421 r 2141 | • · · · · - · ΑσΑΟΟΑΤεΤΑΟΑΑεΤΤΤΤεεΑΤΤΤΑΤΑΤαΑΑΑΑΟΰΤεΤΑΑΑΤΟΤΑΑΑεΤΤΐΑΙΑαΑΤΤΕΑ 6480 RASRTF PFI SKALKLHFID L 2160 |
| 6481 2161 | Λ · « · · · GCTaCAAGGATATTAATGCGTTATGACGTCAAACCAATTAATATATCATTAATTGATTTA 6540 ATRILMGYDVKPIBI S L ID L 2180 |
| 6541 2181 | GAATAIACAGCTGTAAAAGCACCGATTTTCTCATTTAATASATTACATGGATCA5ATTGT 6600 EYTAVKAPIFSFHRLHGSDC 2200 |
| 6601 2201 | • · · * · · ATACTAGGTGTAGAAATGAAATCTACAGGTGAAGTAGCATGTTTTGGTTTAAAIAAAIAT 6660 ILCVEKKSTGEVACFGLNKY . 2220 |
| 6661 2221 | * . · « · · - GAAGCTTTaTTAAAATCATTAATAGCIACAGCTATGAAGTIACCCAAAAAATCAATACTT 6720 EALLKSLIATGMKLPRKSIL 2240 |
| 6721 2241 | • · · · · · ATAAGTATTAAAAATTTAAATAATAAATTAGCTTTTGAAGAACCGTTCCAATTATTATTT 6780 ISIKNLBNKLAFEEPFQLLF 2260 |
| 6781 2261 | • · · « · · τΤΑΑΤΟασΑΤΤΙΑΟΑΑΤΑΤΑΤΟεΟΑΟΤΘΑΙβΟΤΑεΟΤΑΐσΑΤΤΤΟΤΑΟΤΟΤΔΑΑΤΤΤΤΤΛ 6840 LKGFTIYATEGTYDFYSKFL 2280 |
| 6841 2281 | • · · · » · GAATCTTTTAATGTTAATAAAGGTTCTAAATTTCATCAAAGACTTATTAAAGTTCATAAT 6900 ESFNVN KGSKPHQRLIKVHN 2300 |
| 6901 2301 | AAAAATGCAGAAAATAIATCACCAAATACAACAGATTTAATTATGAATCAIAAAGTTGAA 6960 KNAENI SPNTTDLIMNHKVE 2320 |
| 6961 2321 | • · · · · · ATGGTTATTAATATAACTGATACATTAAAAACAAAGGTTAGTTCAAATGGTTATAAAATT 7020 KVINITDTLKTKVSSNGYKI 2340 |
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S&F JOHANNESBURG © 019/-3 3 2’
044 534 70933
AP 0 0 0 4 2 9
TABLE 1 (cant)
7021 AGAAGATTAGCATCAGATTTCCAGGTTCCTTrAATAACIAATATGAAACTTTGTTCTCTT 7080
2341 H.RLASDFQVPL1TKKKLCSL 2360
7081 TTTATTGACTCATTATATAGAAAATTCTCAAGACAAAAGGAAAGAAAATCATTCTATACC 7140
2361 FlDSLYRKrSRQKtRKSFYT 2380 • · »
7141 aTaAAGAGTTATGACGAAIATATAAGTTTGGTATAA 7176
2381 IKSYDEYISLV* 2392 ···»«·
7177 GCAAGAAATTATTCAAIAAATTCGATTTAACATTACTTATTIATGTATTTATTAACTTTC 7236 • . · · · · ·
7237 ATTCCaTAACAACATGAAAAGTATAAATAIATAAATACTAATATATAATATATAAIATAT 7296
7297 AIaIaTATATATATATAIAIAITTATTTATTTAATTATATTTACGTTTAAATATTAATAA 7356 • · » · · ·
7357 ATGTTTTTaTTAAATATGATCATTAATTTAIATTGATTTATTTTTTTATAAATTTTTGTT 7416 • · · · · ·
7417 ATATATACAAAITTTATTTATTCACTCAIATCTATAAACCAAAATGGTTTTTTCAATTIA 7476 ···»*·
7477 CAAATAATTTTATAATtTTAATAAATTTATTAATTATAAAAAAAATAAAAAIATATAAAC 7536 • · » « · ·
7537 ATTAAAATGTATAAATTCTTTTAATTATATAAIAATTTATAAATGTTATGATTTTTTTAA 7596 «·«·«·
7597 AAAATTCAACGAAAAAAAAGAGGAACTGTAXATACAAAAGGGACTAIATATATGTAIATA 7656
7657 TATATATATATAIATATGTTTTTTTTTCCTTATTCTAGA 7695
The GAT domain is made op of two subdomains: a putative structural domain (1-750) and a glutaminase domain (1447-2070). These tve subdomains are separated by a first inserted sequence (751-1446, underlined). The two AT? binding subdomains of the synthetase subunit. CPSa (2071-3762) and CPSb (5572-5173) are separated by a second inserted sequence (3763-5571, underlined).
BAD ORIGINAL
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ΑΡ Π Ο Ο 429
As these inserted sequences are not found in other carbamoyl phosphate synthetase genes they represent prime targets for therapies including, but not limited to, antisense nucleotides, ribosymes and triplex forming nucleotides as there is a decreased likelihood of deleterious reaction with host homologues of the gene.
Antisense RNA molecules are known to be useful for regulating gene expression within the cell. Antisense RNA molecules which axe complementary to portion(s) of CPSII can be produced from the CPSII sequence. These antisense molecules can be used as either diagnostic probes to determine whether or not the CPSII gene is present in a cell or can be used as a therapeutic to regulate expression of the CPSII gene. Antisense nucleotides prepared using the CPSII sequence include nucleotides having complementarity to the CPSII mRNA and capable of interfering with its function in vivo and genes containing CPSII sequence elements that can be just transcribed in living cells to produce antisense nucleotides. The genes may include promoter elements from messenger rna (polymerase II) from cells, viruses, pathogens or structural RNA genes (polymerase I & III) or synthetic ( promoter elements. A review of antisense design is provided in “Gene Regulation: Biology of Antisense RNA and
DNA R.P. Erickson and J.G. Isant, Raven Press 1992.
}
Reference may also be had to US Patent No. 5,208,149 which includes further examples on the design of antisense nucleotides. The disclosure of each of these references is incorporated herein by reference.
As used herein the term nucleotides include but are not limited to oligomers of all naturally-occurring deoxyribonucleotides and ribonucleotides as well as any nucleotide analogues. Nucleotide analogues encompass all compounds capable of forming sequence-specific complexes (eg duplexes or hetroduplexes) with another nucleotide including methylphosphonates or phosphorothioates but may
BAD ORIGINAL Λ
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044 534 70933
S&F JOHANNESBURG @021/^32
AP Ο Ο Ο 4 2 9 r~ have advantageous diffusion or stability properties. The definition of nucleotides includes natural or analogue bases linked by phosphodiester bonds, peptide bonds or any other covalent linkage. These nucleotides may be synthesised by any combination of in vivo in living cells, enzymatically in vitro or chemically.
Ribozymes useful in regulating expression of the CPSII gene include nucleotides having CPSII sequence for specificity and catalytic domains to promote the cleavage of CPSII mRNA in vitro or in vivo. The catalytic domains include hammerheads, hairpins, delta-virus elements, ribosome RNA introns and their derivatives. Further information regarding the design of ribozymes can be found in Haseloff, J. fi Gerlach, W.L. (1988) Nature 3,34. 585;
Kruger, K., Grabowski, P.J., Zaug, A.J., Sands,J., Gottschliag, D.E. & Cech, T.R. (1982) Cell 31, 147; International Patent Application No. W0 88/04300,
US 4,987,071 and US Patent No. 5,254,678. The disclosure of each of these references is incorporated herein by reference. The catalytic elements may enhance the artificial regulation of a CPSII target mRNA by accelerating degradation or some other mechanism.
Triple helix oligonucleotides can be used to inhibit transcription from the genome. Given the sequence provided herein for the CPSII gene it will now be possible to design oligonucleotides which will form triplexes thereby inhibiting transcription of the CPSII gene. Information regarding the generation of oligonucleotides suitable for triplex formation can be found in a Griffin et al (Science 245:967-971 (1989)) this disclosure of this reference is incorporated herein by reference.
Triplex agents include all nucleotides capable of binding to the CPSII gene through formation of the complex with DNA or chromatin. The interaction can be through formation of a triple-stranded Hoogsteen structure or
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I
09.39 034 70933
AP 0 0 0 4 2 9 :>&! JUtiA.\.\tbDL KG liJUZZ/UJi other mechanisms such as strand invasion that relies on the CPSII sequence information.
Accordingly/ in a fourth aspect the present invention consists in a ribozyme capable of cleaving carbamoyl phosphate synthetase II mRNA, the ribozyme including sequences complementary to portions of mRNA obtained from the nucleic acid molecule of the first aspect of the present invention.
In a preferred embodiment of this aspect of the present invention the ribozyme includes sequences complementary to mKNA obtained from the first or second inserted sequences of the nucleic acid molecule of the first aspect of the present invention.
In a fifth aspect the present invention consists in an antisense oligonucleotide capable of blocking expression of the nucleic acid molecule of the first aspect of the present invention.
As stated above, in one aspect the present invention relates to a method of producing CPSII by recombinant, technology. The protein produced by thia method and the polypeptides of the present invention will be useful in in vitro drug binding studies in efforts to develop other antl-ma T arial therapeutics .
In order that the nature of the present invention may be more clearly understood the method by which the P. falciparum CPSII gene was cloned will now be described with reference to the following example and Figures. EXAMPLE
The conventional way to screen for genes of which the amino acid sequence had not been previously determined is via heterologous probing, i.e. with gene fragments of the target enzyme from closely related organisms. This has proved to be unsuccessful for several workers with Plasmodium falciparum largely due to the unusually high A-T content of its genome. After initial unfruitful attempts to isolate the CPSH gene in Plasmodium
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AP 0 0 0 4 2 9 io falciparum using a yeast ura2 gene fragment (Souciet et al., 1989), the present inventors opted to amplify part of the CPSii gene using the polymerase chain reaction (PCR) (Saiki et al., 1988) with a view to use the amplified product as probe for screening.
The present inventors isolated and cloned a PCR product using oligonucleotides designed from conserved sequences from the amino terminal GAT domain and the first half of the synthetase domain of the CPS gene. Nucleotide sequencing confirraed that a portion of the CPSII gene had been obtained. Total parasite DNA was fragmented with a restriction enzyme and subjected to Southern analysis using the CPSII-specific gene probe. The sizes of DNA fragments hybridizing to the gene probe were determined then the DNA in the corresponding bands were used for the construction of a mini-library. In this way a smaller population of clones were screened for the pf CPSII gene.
To isolate the full length pf CPSII gene, a series of mini-libraries were constructed utilising different segments of known sequence to gain information of the unknown flanking regions both towards the 5' and 3· termini of the gene using gene-walking. The strategy employed is summarised in Figure 1.
In the first Southern analysis, total P. falciparum DNA was digested with HindiII and KeoRI and hybridisation was carried out using the pfCPSII 453 bp PCR product.
A 3.0 kb Kindi 11 and a smaller EcoH.1 fragment hybridised to the probe. Subsequent screening of a Hindi 11 pTZ18U mini-library resulted in the isolation of a recombί nant that contained a 3.0 kb pfCPSII gene fragment, CPS2- The 453 bp PCR product was localised in the middle of this segment.
Two regions from both the 5' and 3' ends of CPS2 were used to isolate neighbouring sequences at either end in order to obtain the further gene sequences. A Hiadlll/EccRl fragment from the 5' end of CPS2 was
BAD ORIGINAL
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S&F JOHANNESBURG ®024/032
AP 0 0 0 4 2 9 21 · instrumental in isolating a further 1.5 kb fragment, CPS1 consisting of the complete 5' region of the gene and some non-encoding sequences.
A 550 bp inverse PCR (IPCR; Triglia et al., 1988) product was obtained with the aid of known sequences from the 3' end of CPS2.
This IPCR product was used to screen for the 3' region flanking CPS2. A 3.3 kb fCindlll recombinant containing CPS3 as well as a related 3.3 kb JCSal clone (not presented in Figure 1) were isolated by the minilibrary technique. Using a 200 bp Xbal/Rindlll fragment from the 3' end of CPS3, a 1.3 kb Xbal segment, CPS4 was cloned which contained the putative stop codon and some 3' non-coding region.
Combining these four gene fragments (CPS1, CPS2,
CPS3 and CPS4) excluding their overlaps, gives a total of 8.8 kb consisting of approximately 7.0 kb coding and 1.8 flanking sequences.
The complete nucleotide sequence of the CPS1I gene in P. falciparum, together with its 5' and 3* f lanki ng sequences, is presented in Table 1.
As will be readily appreciated by those skilled in the art the isolation of this gene and its sequencing by the present inventors opens up a range of new avenues for treatment of Plasmodium falciparum infection. The present invention enables the production of quantities of the Plasmodium falciparum carbamoyl phosphate synthetase II enzyme using recombinant DNA technology. Characterisation of this enzyme may enable its use as a chemotherapeutic loci.
The isolation, of this gene also will enable the production of antisense molecules, ribozymes or other gene inactivation agents which can be used to prevent the multiplication of the parasite in infected individuals.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made
BAD ORIGINAL
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-- S&F JOHANNESBURG @OF5/c*32
AP 0 0 0 4 2 9 to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
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AP 0 0 0 4 2 9
REFERENCES
Cox, F.E.G. (1991) Malaria vaccines: while we are waiting. Parasitology Today 2i 189-190
Gero, A.M. and O'Sullivan, W.J. (199)) Purines and pyrimidines in malarial parasites. Blood Cells ifi.: 467498
Hammond, D. J. Burchell, J.R. and Pudney, M. (1985) Inhibition of pyrimidine biosynthesis de novo in
Plasmodium falciparum by 2.(4.t-butylcyclohexyl)-3hydroxy-1, 4-naphthoquinine in vitro. Mol. Biochem.
Parasitol AA: 97-109
Hill, B-, Kilsby, J. Rogerson, G.W., McIntosh, RT. and Ginger, C.D. (1981). The Enzymes of pyrimidine biosynthesis in a range of parasitic protozoa and helminths. Mol. Biochem. Parasitol. £: 123-134.
Johnson, C. Malaria back to plague us. Sydney Morning Herald, November 13, 1991Jones, M.E. (1980) Pyrimidine nucleotide biosynthesis in animals: genes, enzymes and regulation of DMP biosynthesis. Annu. Rev. Biochem. 42.: 253-279.
Krungkrai, J. Cerami, A. and Henderson, G.B. (1990) Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from
Plasmodium berghei and Crithidia fasciculata. Biochemistry 22.: 6270-6275.
Krungkrai, J. Krungkrai, S.R. and Phakanont, K.
(1992) Antimalarial activity of oxotate analogs that inhibit dihydrootase and dihydroorotate dehydrogenase.
Biochem. Pharmacol. 42.: 1295-1301.
Marshal, E. (1991) Malaria parasite gaining ground against science. Science 2.· 190.
Nyunoya, H., Broglie, K.E., Widgren, W.E. and Lusty C.J,. (1985) Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat. J. Biol. Chem. 260: 9346-9356.
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AP 0 0 0 4 2 9
S&F JOHANNESBURG @10U7/ύ32 c
(
Prapunwattana, P., O'Sullivan, W.J. and Yuthavong, Y. (1988) Depression of Plasmodium falciparum dihydroorotate dehydrogenase activity in in vitro culture by tetracycline. Mol. Biochem. 27.: 119-12420 c
(
Queen, S.A., Vander Jagt, D.L. and Reyes, P. (1990) In vitro susceptibilities of Plasmodium falciparum to compounds which inhibit nucleotide metabolism. Antimicrob. Agents Chemother. 34¾ 1393-1398.
Reyes, P., Rathod, P.K., Sanchez, D.J. Krema,
J.E.K., Rieckmann, K.H. and Heidrich, H.G. (1982) Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol.
275-290.
Rubino 5.D., Nyunoya, H. and Lusty, c.J. (198S) JBC 261(24)i11320-11327.
Saiki, R.K., Gelfand, D.E., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis K.B. and Erlich H.A. (1988) Science 23,9:487-491.
Scott, H.v., Gexo, A.M. and O'Sullivan, W.J. (1986) in vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo. Mol. Biochem. Parasitol. 18.: 3-15.
Sherman, I.w. (1979) Biochemistry of Plasmodium (malarial parasites) Microbiol. Rev, 43.: 453-495Simmer, J.P., Kelly, R.E., Rinker, Jr., A.G.,
Scully, J.L. and Evans D.R. (1990) Mammalian carbamyl phosphate synthetase (CPS)- J- Biol. Chem 265: 1039510402.
Simmer, J.P., Kelly, R.E., Austin, G.R., Jr-,
Scully, J.L. and Evans, D.R. (1990) JBC 285(18):10395-10402.
Souciet, J-L., Nagy, M., Le Gouar, M., Lacroute, f. and Potier, S. (1989) Gene (Amst.) 79.: 59-70.
Triglia, T-, Peterson, M.G. and Kemp, D.J- (1988) PNAS 16:8186.
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3': '11.
'93 10:04 ©44 534 70933 s&F JOHANNESBURG 0028/032
AP 0 0 U 4 2 9
Werner, M., Feller, A. and Pierard, A. (1985) Nucleotide sequence of yeast gene CPA1 encoding the small subunit of arginine-pathway carbamoyl-phosphate synthetase. Eur. J. Biochem. 146? 371-381.
Claims (9)
- CLAIMS:1. A nucleic acid molecule encoding carbamoyl phosphate synthetase II of Plasmodium falciparum or a portion thereof, the nucleic acid molecule including a sequence5 substantially as shown in Table 1 from residue 1 to 7176, or from 1 to 750, or from 751 to 1446, or from 1447 to 2070, or from 2071 to 3762, or from 3763 to 5571, or from 5572 to 7173, or from 1 to 3360, or from 2071 to 6666, or from 2071 to 7173, or a functionally equivalent sequence.10
- 2. A nucleic acid molecule as claimed in claim 1 in which, the nucleic acid molecule includes a sequence as shown in Table 1 from -1225 to 7695 or a functionally equivalent sequence.
- 3. An isolated polypeptide, the polypeptide including15 an amino acid sequence substantially as shown in Table 1 from 1 to 2391, from 483 to 690, from 691 to 1254, 1858 to 2391, from 1 to 1120, from 691 tc 2222, or from 691 to 2391.
- 4. A method of producing Plasmodium falciparum20 carbamoyl phosphate synthetase II or a portion thereof, the method comprising culturing a cell transformed withk. the nucleic acid molecule as claimed in claim 1 or claim 2 . under conditions which allow expression of the nucleic acid sequence, and recovering the expressed carbamoyl25 phosphate synthetase II.
- 5. Method as claimed in claim 4 in which the cell isE.coli, yeast or Dictyostelium discoideum.
- 6. A ribozyme capable of cleaving carbamoyl phosphate synthetase II mRNA, the ribozyme including sequences30 complementary to portions of iqRNA obtained from the nucleic acid molecule as claimed in claim 1 or claim 2.
- 7. A ribozyme as claimed in claim 6 in which the ribozyme includes sequences complementary to portions of itlRNA obtained from the first ox second inserted sequences35 of the nucleic acid molecule as claimed in claim 1 or claim 2.BAD ORIGINAL l: 93 10:05 ©44 534 70933S&F JOHANNESBURG @030/032AP 0 0 0 4 2 9
- 8. An antisense oligonucleotide capable of blocking expression of the nucleic acid molecule as claimed in claim 1 or claim 2.
- 9. A polynucleotide construct which produces in a cell 5 the ribo2yme as claimed in claim 5 or 6 or the antisense oligonucleotide as claimed in claim 7.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPL620692 | 1992-12-03 | ||
| AUPL638092 | 1992-12-16 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AP9400607A0 AP9400607A0 (en) | 1994-01-31 |
| AP429A true AP429A (en) | 1995-11-09 |
Family
ID=25644386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| APAP/P/1994/000607A AP429A (en) | 1992-12-03 | 1993-12-01 | Nucleotide sequence encoding carbamoyl phosphate synthetase 11. |
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| Country | Link |
|---|---|
| US (1) | US5849573A (en) |
| EP (1) | EP0675958A4 (en) |
| JP (1) | JPH08503607A (en) |
| KR (1) | KR950704495A (en) |
| CN (1) | CN1041535C (en) |
| AP (1) | AP429A (en) |
| BR (1) | BR9307584A (en) |
| CA (1) | CA2150572A1 (en) |
| FI (1) | FI952697A (en) |
| HU (2) | HUT71930A (en) |
| IL (1) | IL107845A0 (en) |
| MX (1) | MX9307662A (en) |
| MY (1) | MY109022A (en) |
| NO (1) | NO952175L (en) |
| OA (1) | OA10163A (en) |
| PL (1) | PL309261A1 (en) |
| WO (1) | WO1994012643A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6468765B1 (en) * | 1995-04-21 | 2002-10-22 | Human Genome Sciences, Inc. | Selected Haemophilus influenzae Rd polynucleotides and polypeptides |
| AU3984497A (en) * | 1996-08-16 | 1998-03-06 | Innovir Laboratories, Inc. | Phenotypic conversion of cells mediated by external guide sequences |
| US6610478B1 (en) | 1996-08-16 | 2003-08-26 | Yale University | Phenotypic conversion of cells mediated by external guide sequences |
| EP1082414A1 (en) * | 1998-06-01 | 2001-03-14 | Incyte Pharmaceuticals, Inc. | Carbamoyl phosphate synthase homolog |
| CN100510079C (en) * | 2001-04-04 | 2009-07-08 | 中国医学科学院基础医学研究所 | Malignant malarial parasite dynein gene |
| CN100510080C (en) * | 2001-04-04 | 2009-07-08 | 中国医学科学院基础医学研究所 | Malignant malarial parasite new antigen candidate gene pfMAg |
| CN1316015C (en) * | 2001-08-03 | 2007-05-16 | 味之素株式会社 | New-mutant carbamyl-phosphate synthesized enzyme and method for producing compound derivated from carbamyl-phosphate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5585479A (en) * | 1992-07-24 | 1996-12-17 | The United States Of America As Represented By The Secretary Of The Navy | Antisense oligonucleotides directed against human ELAM-I RNA |
-
1993
- 1993-12-01 AP APAP/P/1994/000607A patent/AP429A/en active
- 1993-12-02 PL PL93309261A patent/PL309261A1/en unknown
- 1993-12-02 WO PCT/AU1993/000617 patent/WO1994012643A1/en not_active Application Discontinuation
- 1993-12-02 BR BR9307584-7A patent/BR9307584A/en not_active Application Discontinuation
- 1993-12-02 HU HU9501583A patent/HUT71930A/en unknown
- 1993-12-02 US US08/446,855 patent/US5849573A/en not_active Expired - Fee Related
- 1993-12-02 IL IL10784593A patent/IL107845A0/en unknown
- 1993-12-02 EP EP94900652A patent/EP0675958A4/en not_active Ceased
- 1993-12-02 MY MYPI93002571A patent/MY109022A/en unknown
- 1993-12-02 JP JP6512592A patent/JPH08503607A/en not_active Ceased
- 1993-12-02 KR KR1019950702245A patent/KR950704495A/en not_active Application Discontinuation
- 1993-12-02 CA CA002150572A patent/CA2150572A1/en not_active Abandoned
- 1993-12-03 MX MX9307662A patent/MX9307662A/en unknown
- 1993-12-03 CN CN93121697A patent/CN1041535C/en not_active Expired - Fee Related
-
1995
- 1995-06-01 NO NO952175A patent/NO952175L/en unknown
- 1995-06-02 OA OA60671A patent/OA10163A/en unknown
- 1995-06-02 FI FI952697A patent/FI952697A/en not_active Application Discontinuation
- 1995-06-20 HU HU95P/P00284P patent/HU211526A9/en unknown
Non-Patent Citations (2)
| Title |
|---|
| Journal of Biological Chemistry, (Aug. 1985), Vol. 260, No. 16, page 9346-9356 NYUNOYA H. * |
| Journal of Biological Chemistry, Volume 258, No. 23, pp 14466-14472, issued 10 December 1983, LUSTY C.J. * |
Also Published As
| Publication number | Publication date |
|---|---|
| OA10163A (en) | 1996-12-18 |
| CN1090327A (en) | 1994-08-03 |
| BR9307584A (en) | 1999-08-31 |
| HU211526A9 (en) | 1995-11-28 |
| US5849573A (en) | 1998-12-15 |
| NO952175D0 (en) | 1995-06-01 |
| FI952697A0 (en) | 1995-06-02 |
| HUT71930A (en) | 1996-02-28 |
| JPH08503607A (en) | 1996-04-23 |
| EP0675958A1 (en) | 1995-10-11 |
| CA2150572A1 (en) | 1994-06-09 |
| IL107845A0 (en) | 1994-04-12 |
| CN1041535C (en) | 1999-01-06 |
| PL309261A1 (en) | 1995-10-02 |
| NO952175L (en) | 1995-06-01 |
| KR950704495A (en) | 1995-11-20 |
| WO1994012643A1 (en) | 1994-06-09 |
| FI952697A (en) | 1995-07-14 |
| AP9400607A0 (en) | 1994-01-31 |
| HU9501583D0 (en) | 1995-08-28 |
| EP0675958A4 (en) | 1997-10-22 |
| MY109022A (en) | 1996-11-30 |
| MX9307662A (en) | 1994-06-30 |
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