AU669413B2 - Nucleotide sequence encoding carbamoyl phosphate synthetase II - Google Patents

Description

"Nucleotide Sequence Encoding Carbamoyl Phosphate Synthetase II"

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, 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, 1979). Moreover, the mature human red blood cell has no recognised requirement for pyrimidine nucleotides (Gero and O'Sullivan, 1990). Major efforts have been directed towards the development of inhibitors of the pyrimidine 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 explore specific differences between the parasite and the 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 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 CPSII and ATCase activities in P. berghei . Although CPS activity has been detected in P. falciparum (Reyes et al . , 1982) until this current 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) the activation of bicarbonate by ATP; (b) the reaction of the activated species carboxyphosphate 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 amidotransferases, 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 CPSI 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 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 arginine- specific CPS's (like cpal and cpa2 in yeast) as well as rat mitochondrial CPSI arose by defusion from the pyrimidine chimera.

The present inventors have isolated and characterised the complete gene encoding the CPSII enzyme from P. falciparum (p CPSII). Reported here is the cDNA sequence including 5' and 3' untranslated regions. In so 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. 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 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 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. coli , yeast, and Dictyostelium discoideum.

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 glutiminase domain whilst the second inserted sequence separates the two ATP binding subdomains of the synthetase subunit CPSa and CPSb. TABLE 1 . Nucleotide and Deduced Amino Acid Sequence of the Carbamoyl Phosphate Synthetase II Gene from Plasmodium fal ciparum

-1225 GAATTCCTTCAGCCAAAAAj^AATGAC CGCAAATTTTAAGAAAAGAAAAACAATCGACT -11

-1165 CGTCTTTGAATGAGGTTAGAAATTCGATACGTGAAAGGGACTTAAGAAGGCTTAACAGAG -11

-1105 AAAAGAGTAAAATCTTATAAGCATTTGAAGGAAAAAATAATAAAATAAAAAAATAAAAAG -10

-1045 ATAAAAAATATTTATATTTGATATGTAGTATATATAATGATTATTCATATTAATAACATA -98

-985 GATAAAAAACTTTTTTTTTTTTTTTTTTTCTTTATATTTATTAACAATACATTTAAGTTA -92

-925 TTTTATATATATATATATATATATATATATATATATATATATATATGTTTGTGTGTTCAT -86

-865 TTGTTTATAAAATTACTTGAAATATAAAACTTATTAATATATTTCCAATTAATATGAATA -80

-805 CAATTATTAATATTTTGATGTGTACACATTAATATAGTTTTACACTTCTTATAATAAAAC -74

-745 CATCCTATATATTATACACAATATATAATACTCCCCAATATTGTGGTTCCTATAATTTTA -68

-685 TTTATATATTTATTTATTAATTTATTCATTTATTTATTTTTTTTCTTAGTTTATAAAATA -62

-625 GTAATTCTACTj^ATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAATT -56

• • • • • •

-565 TACATATGAAAAATGAACTTGTATATGTAAATTTATAAATATTTTAAACATAAATATAAA -50

-505 TGTATAAAAJ^VAAAAAGAAAAATGGGAAAAAATAATATAGATATATATATAAATATATA -44

-445 TATATATATAATTATTGGGGATATTCTCTGAATCATAGGTCTTAAACAGTTTTATTCTTT -38

-385 TAACATCACAAAGTTGTTATTAAAAGTATATATATCTTATTGGTTCCTATATAAAACTAT -32

-325 AGTATTCTATAATATATTCTGTATATTTCATTTTATCATTTGTAAGCAATCCCTATTTAT -26

-265 TATAATTATTATTTTTTTTTTTATAAAAGAGGTATAAAACAGTTTATTCAATTTTTTTCC -20

-205 TAAAGGAGCAACCTTCAGTCAATTTACATTTTCCACCGGTTGGTTGGCACAACATAATGT -14

-145 TACAGCTAAAJW JAGAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATAT -86

-85 ATATATATATATATACATAATATGTACAATGCTACCATACAAGTATATAAATTTTTCAAC -26

-25 ATTGTTGTGATGTTGCATTTTTCTT -1 TABLE 1 (cont) ATGTATATTTCTTTTAAATATAATTTATATATATATATATATATATATATATATATATTT 60 M Y I S F K Y N L Y I Y I Y I Y I Y I F 20

GTTCTTATAGATTTTAAAACAGTTGGGAGGTTAATTCTTGAAGATGGTAACGAATTTGTA 120 V L I D F K T V G R L I L E D G N E F V 40

GGGTACAGTGTAGGTTACGAAGGGTGTAAAGGAAATAATAGTATATCATGTCATAAGGAG 180 G Y S V G Y E G C K G N N S I S C H K E 60

TATAGAAATATTATTAATAATGATAATAGCAAGAATAGTAATAATTCATTTTGTAATAAT 240 Y R N I I N N D N S K N S N N S F C N N 80

GAAGAAAACAATTTGAAAGATGATTTATTATATAAAAATAGTCGATTAGAAAATGAAGAT 300 E E N N L K D D L L Y K N S R L E N E D 100

TTTATTGTTACAGGTGAAGTTATATTTAATACAGCTATGGTTGGATATCCTGAAGCTTTA 360 F I V T G E V I F N T A M V G Y P E A 120

ACGGACCCAAGTTATTTTGGTCAAATATTAGTTTTAACATTTCCTTCTATTGGTAATTAT 420 T D P S Y F G Q I L V L T F P S I G N Y 140

GGTATTGAAAAAGTAAAACATGATGAAACGTTTGGATTAGTACAAAATTTTGAAAGTAAT 480 G I E K V K H D E T F G L V Q N F E S N 160

AAAATTCAAGTACAAGGTTTAGTTATTTGTGAATATTCGAAGCAATCATATCATTACAAT 540 K I Q V Q G L V I C E Y S K Q S Y H Y N 180

TCTTATATTACCTTAAGTGAATGGTTAAAGATTTATAAAATTCCATGTATAGGTGGTATA 600 S Y I T L S E W L K I Y K I P C I G G I 200

GATACAAGAGCCTTAACAAAACTTTTAAGAGAAAAAGGTAGTATGTTAGGTAAAATAGTT 660 D T R A L T K L L R E K G S M L G K I V 220

ATATATAAAAACAGACAACATATTAATAAATTATATAAAGAAATTAATCTTTTTGATCCT 720 I Y K N R Q H I N K L Y K E I N L F D P 240

GGTAATATAGATACTCTAAAATATGTATGTAATCATTTTATACGTGTTATTAAGTTGAAT 780 G N I D T L K Y V C N H F I R V I K L N 260 TABLE 1 (cont)

781 AATATTACATATAATTATAAAAATAAGGAAGAATTTAATTATACCAATGAAATGATTACT 840 261 N I T Y N Y K N K E E F N Y T N E M I T 280

841 AATGATTCTTCAATGGAAGATCATGATAATGAAATTAATGGTAGTATTTCTAATTTTAAT 900 281 N D S S M E D H D N E I N G S I S N F N 300

901 AATTGTCCAAGTATCTCTAGTTTTGATAAAAGTGAATCGAAAAATGTTATTAATCATACA 960 301 N C P S I S S F D K S E S K N V I N H T 320

961 TTGTTAAGAGATAAAATGAACCTAATAACTTCATCTGAAGAATATCTGAAAGATCTTCAT 102 321 L L R D K M N L I T S S E E Y L K D L H 340

1021 AATTGTAATTTTAGTAATAGTAGTGATAAAAATGATTCTTTTTTTAAGTTATATGGTATA 108 341 N C N F S N S S D K N D S F F K L Y G I 360

1081 TGTGAATATGATAAATATTTAATTGACCTTGAAGAAAATGCTAGCTTTCATTATAATAAT 114 361 C E Y D K Y L I D L E E N A S F H Y N N 380

1141 GTAGATGAATATGGATATTATGATGTTAATAAAAATACAAATATTCTATCTAATAATAAA 120 381 V D E Y G Y Y D V N K N T N I L S N N K 400

1201 ATAGAACAAAACAACAATAACGAAAATAACAAAAATAACAAAAATAACAACAATAACGAG 126 401 I E Q N N N N E N N K N N K N N N N N E 420

1261 GTTGATTATATAAAGAAAGATGAGGATAATAATGTCAATAGTAAGGTCTTTTATAGCCAA 132 421 V D Y I K K D E D N N V N S K V F Y S O 440

1321 TATAATAATAATGCACAAAATAATGAACATACCGAATTTAATTTAAATAATGATTATTCT 138 1 Y N N N A O N N E H T E F N L N N D Y S 460

1381 ACTTATATTAGAAAGAAAATGAAAAATGAAGAATTCCTTAATTTGGTAAACAAAAGAAAA 144 461 T Y I R K K M K N E E F L N L V N K R K 480

1441 GTAGACCATAAAGAAAAAATTATTGTTATTGTTGATTGTGGTATTAAAAATAGTATAATC 150 481 V D H K E K I I V I V D C G I K N S I I 500

1501 AAAAATTTAATAAGACACGGTATGGATCTTCCATTAACATATATTATTGTACCTTATTAT 156 501 K N L I R H G M D L P L T Y I I V P Y Y 520 TABLE 1 (cont)

1561 TACAATTTTAATCATATAGATTATGATGCAGTTCTTTTATCTAATGGTCCTGGAGATCCT 162 521 Y N F N H I D Y D A V L L S N G P G D P 540

1621 AAAAAGTGTGATTTCCTTATAAAAAATTTGAAAGATAGTTTAACAAAAAATAAAATTATA 168 541 K K C D F L I K N L K D S L T K N K I I 560

1681 TTTGGTATTTGTTTAGGTAATCAACTATTAGGTATATCATTAGGTTGTGACACATATAAA 174 561 F G I C L G N Q L L G I S L G C D T Y K 580

1741 ATGAAATATGGTAATAGAGGTGTTAATCAACCCGTAATACAATTAGTAGATAATATATGT 180 581 M K Y G N R G V N Q P V I Q L V D N I C 600

1801 TACATTACCTCACAAAATCATGGATACTGTTTAAAGAAAAAATCAATTTTAAAAAGAAAA 186 601 Y I T S Q N H G Y C L K K K S I L K R K 620

1861 GAGCTTGCGATTAGTTATATAAATGCTAATGATAAATCTATAGAAGGTATTTCACATAAA 192 621 E L A I S Y I N A N D K S I E G I S H K 640

1921 AATGGAAGATTTTATAGTGTCCAGTTTCATCCTGAGGGTAATAATGGTCCTGAAGATACA 198 641 N G R F Y S V Q F H P E G N N G P E D T 660

1981 TCATTTTTATTTAAGAATTTTCTTTTAGATATCTTTAATAAGAAAAAACAATATAGAGAA 2040 661 S F L F R N F L L D I F N K K K Q Y R E 680

2041 TATTTAGGATATAATATTATTTATATAAAAAAGAAAGTGCTTCTTTTAGGTAGTGGTGGT 2100 681 Y L G Y N I I Y I K K K V L L L G S G G 700

2101 TTATGTATAGGACAAGCAGGAGAATTCGATTATTCAGGAACACAAGCAATTAAAAGTTTA 2160 701 L C I G Q A G E F D Y S G T Q A I K S L 720

2161 AAAGAATGTGGTATATATGTTATATTAGTTAATCCTAACATAGCAACTGTTCAAACATCA 2220 721 K E C G I Y V I L V N P N I A T V Q T S 740

2221 AAAGGTTTGGCAGATAAGGTATACTTTTTACCAGTTAATTGTGAATTTGTAGAAAAAATT 2280 741 K G L A D K V Y F L P V N C E F V E K I 760

2281 ATTAAAAAGGAAAAACCTGATTTTATTTTATGTACATTTGGTGGTCAGACAGCTTTAAAT 2340 761 I K K E K P D F I L C T F G G Q T A L N 780 TABLE 1 (cont)

2341 TGTGCTTTAATGTTAGATCAAAAAAAAGTATTGAAAAAGAATAATTGTCAATGTTTAGGT 240 781 C A L M L D Q K K V L K K N N C Q C L G 800

2401 ACATCTTTAGAATCTATAAGAATAACAGAAAATAGAACATTATTTGCTGAAAAATTAAAA 246 801 T S L E S I R I T E N R T L F A E K L K 820

2461 GAAATTAATGAAAGAATAGCTCCATATGGTAGTGCAAAAAATGTTAATCAAGCTATTGAT 252 821 E I N E R I A P Y G S A K N V N Q A I D 840

2521 ATAGCTAATAAAATAGGATATCCAATATTAGTACGTACAACATTTTCGTTAGGAGGATTA 258 841 I A N K I G Y P I L V R T T F S L G G L 860

2581 AATAGTAGTTTCATAAATAATGAAGAAGAACTTATCGAAAAATGTAATAAAATATTTTTA 264 861 N S S F I N N E E E L I E K C N K I F L 880

2641 CAAACTGATAATGAAATATTTATAGATAAATCATTACAAGGATGGAAAGAAATAGAATAT 270 881 Q T D N E I F I D K S L Q G W K E I E Y 900

2701 GAATTATTAAGAGATAATAAAAATAATTGTATAGCTATATGTAATATGGAAAATATAGAT 276 901 E L L R D N K N N C I A I C N M E N I D 920

2761 CCATTAGGTATACATACAGGAGATAGTATAGTTGTTGCACCTTCACAAACATTAAGTAAT 282 921 P L G I H T G D S I V V A P S Q T L S N 940

2821 TATGAATATTATAAATTTAGAGAAATAGCATTAAAGGTAATTACACATTTAAATATTATA 288 941 Y E Y Y K F R E I A L K V I T H L N I I 960

2881 GGAGAATGTAATATACAATTTGGTATAAATCCACAAACAGGAGAATATTGTATTATTGAA 294 961 G E C N I Q F G I N P Q T G E Y C I I E 980

2941 GTTAATGCTAGGCTTAGTAGAAGTTCAGCATTAGCTTCTAAAGCTACTGGTTATCCACTT 300 981 V N A R L S R S S A L A S K A T G Y P L 100

3001 GCTTATATATCAGCAAAAATAGCCTTGGGATATGATTTGATAAGTTTAAAAAATAGCATA 306 1001 A Y I S A K I A L G Y D L I S L K N S I 102

3061 ACTAAAAAAACAACTGCCTGTTTTGAACCCTCTCTAGATTACATTACAACAAAAATACCA 312 1021 T K K T T A C F E P S L D Y I T T K I P 104 TABLE 1 (cont)

3121 CGATGGGATTTAAATAAATTTGAGTTTGCTTCTAATACAATGAATAGTAGTATGAAAAGT 318 1041 R W D L N K F E F A S N T M N S S M K S 106

3181 GTAGGAGAAGTTATGTCTATAGGTAGAACCTTTGAAGAATCTATACAAAAATCTTTAAGA 324 1061 V G E V M S I G R T F E E S I Q K S L R 108

3241 TGTATTGATGATAATTATTTAGGATTTAGTAATACGTATTGTATAGATTGGGATGAAAAG 330 1081 C I D D N Y L G F S N T Y C I D W D E K 110

3301 AAAATTATTGAAGAATTAAAAAATCCATCACCAAAAAGAATTGATGCTATACATCAAGCT 336 1101 K I I E E L K N P S P K R I D A I H Q A 112

3361 TTCCATTTAAATATGCCTATGGATAAAATACATGAGCTGACACATATTGATTATTGGTTC 342 1121 F H L N M P M D K I H E L T H I D Y W F 114

3421 TTACATAAATTTTATAATATATATAATTTACAAAATAAGTTGAAAACGTTAAAATTAGAG 348 1141 L H K F Y N I Y N L Q N K L K T L K L E 116

3481 CAATTATCTTTTAATGATTTGAAGTATTTTAAGAAGCATGGTTTTAGTGATAAGCAAATA 354 1161 Q L S F N D L K Y F K K H G F S D K Q I 118

3541 GCTCACTACTTATCCTTCAACACAAGCGATAATAATAATAATAATAATAATATTAGCTCA 360 1181 A H Y L S F N T S D N N N N N N N I S S 120

3601 TGTAGGGTTACAGAAAATGATGTTATGAAATATAGAGAAAAGCTAGGATTATTTCCACAT 366 1201 C R V T E N D V M K Y R E K L G L F P H 122

3661 ATTAAAGTTATTGATACCTTATCAGCCGAATTTCCGGCTTTAACTAATTATTTATATTTA 372 1221 I K V I D T L S A E F P A L T N Y L Y L 124

3721 ACTTATCAAGGTCAAGAACATGATGTTCTCCCATTAAATATGAAAAGGAAAAAGATATGC 378 1241 T Y Q G Q E H D V L P L N M K R K R I C 126

3781 ACGCTTAATAATAAACGAAATGCAAATAAGAAAAAAGTCCATGTCAAGAACCACTTATAT 384 1261 T L N N K R N A N K K K V H V K N H L Y 128

3841 AATGAAGTAGTTGATGATAAGGATACACAATTACACAAAGAAAATAATAATAATAATAAT 390 1281 N E V V D D K D T O L H K E N N N N N N 130 TABLE 1 (cont)

3901 ATGAATTCTGGAAATGTAGAAAATAAATGTAAATTGAATAAAGAATCCTATGGCTATAAT 396 1301 M N S G N V E N K C K L N K E S Y G Y N 132

3961 AATTCTTCTAATTGTATCAATACAAATAATATTAATATAGAAAATAATATTTGTCATGAT 402 1321 N S S N C I N T N N I N I E N N I C H D 134

4021 ATATCTATAAACAAAAATATAAAAGTTACAATAAACAATTCCAATAATTCTATATCGAAT 408 1341 I S I N K N I K V T I N N S N N S I S N 136

4081 AATGAAAATGTTGAAACAAACTTAAATTGTGTATCTGAAAGGGCCGGTAGCCATCATATA 414 1361 N E N V E T N L N C V S E R A G S H H I 138

4141 TATGGTAAAGAAGAAAAGAGTATAGGATCTGATGATACAAATATTTTAAGTGCACAAAAT 420 1381 Y G K E E K S I G S D D T N I L S A Q N 140

4201 TCAAATAATAACTTTTCATGTAATAATGAGAATATGAATAAAGCAAACGTTGATGTTAAT 426 1401 S N N N F S C N N E N M N K A N V D V N 142

4261 GTACTAGAAAATGATACGAAAAAACGAGAAGATATAAATACTACAACAGTATTTATGGAA 4320 1421 V L E N D T K K R E D I N T T T V F M E 1440

4321 GGTCAAAATAGTGTTATTAATAATAAGAATAAAGAGAATAGTTCTTTATTGAAAGGTGAT 4380 1441 G O N S V I N N K N K E N S S L L K G D 1460

4381 GAAGAAGATATTGTGATGGTAAATTTAAAAAAGGAAAATAATTATAATAGTGTAATTAAT 4440 1461 E E D I V M V N L K K E N N Y N S V I N 1480

4441 AATGTAGATTGTAGGAAAAAGGATATGGATGGAAAAAATATAAATGATGAATGTAAAACA 4500 1481 N V D C R K K D M D G K N I N D E C K T 1500

4501 TATAAGAAAAATAAATATAAAGATATGGGATTAAATAATAATATAGTAGATGAGTTATCC 4560 1501 Y K K N K Y K D M G L N N N I V D E L S 1520

4561 AATGGAACATCACATTCAACTAATGATCATTTATATTTAGATAATTTTAATACATCAGAT 4620 1521 N G T S H S T N D H L Y L D N F N T S D 1540

4621 GAAGAAATAGGGAATAATAAAAATATGGATATGTATTTATCTAAGGAAAAAAGTATATCT 4680 1541 E E I G N N K N M D M Y L S K E K S I S 1560 TABLE 1 (cont)

4681 AATAAAAACCCTGGTAATTCTTATTATGTTGTAGATTCCGTATATAATAATGAATACAAA 474 1561 N K N P G N S Y Y V V D S V Y N N E Y K 158

4741 ATTAATAAGATGAAAGAGTTAATAGATAACGAAAATTTAAATGATGAATATAATAATAAT 480 1581 I N K M K E L I D N E N L N D E Y N N N 160

4801 GTTAATATGAATTGTTCTAATTATAATAATGCTAGTGCATTTGTAAATGGAAAGGATAGA 486 1601 V N M N C S N Y N N A S A F V N G K D R 162

4861 AATGATAATTTAGAAAATGATTGTATTGAAAAAAATATGGATCATACATACAAACATTAT 492 1621 N D N L E N D C I E K N M D H T Y K H Y 1640

4921 AATCGTTTAAACAATCGTAGAAGTACAAATGAGAGGATGATGCTTATGGTAAACAATGAA 4980 1641 N R L N N R R S T N E R M M L M V N N E 1660

4981 AAAGAGAGCAATCATGAGAAGGGCCATAGAAGAAATGGTTTAAATAAAAAAAATAAAGAA 5040 1661 K E S N H E K G H R R N G L N K K N K E 1680

5041 AAAAATATGGAAAAAAATAAGGGAAAAAATAAAGACAAAAAGAATTATCATTATGTTAAT 5100 1681 K N M E K N K G K N K D K K N Y H Y V N 1700

5101 CATAAAAGGAATAATGAATATAATAGTAACAATATTGAATCGAAGTTTAATAATTATGTT 5160 1701 H K R N N E Y N S N N I E S K F N N Y V 1720

5161 GATGATATAAATAAAAAAGAATATTATGAAGATGAAAATGATATATATTATTTTACACAT 5220 1721 D D I N K K E Y Y E D E N D I Y Y F T H 1740

5221 TCGTCACAAGGTAACAATGACGATTTAAGTAATGATAATTATTTAAGTAGTGAAGAATTG 5280 1741 S S Q G N N D D L S N D N Y L S S E E L 1760

5281 AATACTGATGAGTATGATGATGATTATTATTATGATGAAGATGAAGAAGATGACTATGAC 5340 1761 N T D E Y D D D Y Y Y D E D E E D D Y D 1780

5341 GATGATAATGATGATGATGATGATGATGATGATGATGGGGAGGATGAGGAGGATAATGAT 5 00 1781 D D N D D D D D D D D D G E D E E D N D 1800

5401 TATTATAATGATGATGGTTATGATAGCTATAATTCTTTATCATCTTCAAGAATATCAGAT 5460 1801 Y Y N D D G Y D S Y N S L S S S R I S D 1820 TABLE 1 (cont)

5461 GTATCATCTGTTATATATTCAGGGAACGAAAATATATTTAATGAAAAATATAATGATATA 552 1821 V S S V I Y S G N E N I F N E K Y N D I 184

5521 GGTTTTAAAATAATCGATAATAGGAATGAAAAAGAGAAAGAGAAAAAGAAATGTTTTATT 558 1841 G F K I I D N R N E K E K E K K K C F I 186

5581 GTATTAGGTTGTGGTTGTTATCGTATTGGTAGTTCTGTAGAATTTGATTGGAGTGCTATA 564 1861 V L G C G C Y R I G S S V E F D W S A I 188

5641 CATTGTGTAAAGACCATAAGAAAATTAAACCATAAAGCTATATTAATAAATTGTAACCCA 570 1881 H C V K T I R K L N H K A I L I N C N P 190

5701 GAAACTGTAAGTACAGATTATGATGAAAGTGATCGTCTATATTTTGATGAAATAACAACA 576 1901 E T V S T D Y D E S D R L Y F D E I T T 192

5761 GAAGTTATAAAATTTATATATAACTTTGAAAATAGTAATGGTGTGATTATAGCTTTTGGT 582 1921 E V I K F I Y N F E N S N G V I I A F G 194

5821 GGACAAACATCAAATAATTTAGTATTTAGTTTATATAAAAATAATGTAAATATATTAGGA 588 1941 G Q T S N N L V F S L Y K N N V N I L G 196

5881 TCAGTGCACAAAGTGTTGATTGTTGTGAAAATAGGAATAAATTTTCGCACTTATGTGATT 594 1961 S V H K V L I V V K I G I N F R T Y V I 198

5941 CTTAAAATTGATCAACCGAAATGGAATAAATTTACAAAATTATCCAAGGCTATACAATTT 600 1981 L K I D Q P K W N K F T K L S K A I Q F 200

6001 GCTAATGAGGTAAAATTTCCTGTATTAGTAAGACCATCGTATGTATTATCTGGTGCAGCT 606 2001 A N E V K F P V L V R P S Y V L S G A A 202

6061 ATGAGAGTTGTAAATTGTTTTGAAGAATTAAAAAACTTTTTAATGAAGGCAGCTATTGTT 612 2021 M R V V N C F E E L N F L M K A A I V 204

6121 AGTAAAGATAATCCTGTTGTAATATCAAAATTTATTGAGAATGCTAAAGAAATAGAAATA 618 2041 S K D N P V V I S K F I E N A K E I E I 206

6181 GATTGTGTTAGTAAAAATGGTAAAATAATTAATTATGCTATATCTGAACATGTTGAAAAT 624 2061 D C V S K N G K I I N Y A I S E H V E N 208 TABLE 1 (cont)

6241 GCTGGTGTACATTCAGGTGATGCAACATTAATATTACCTGCACAAAATATATATGTTGAA 630 2081 A G V H S G D A T L I L P A Q N I Y V E 210

6301 ACACATAGGAAAATAAAGAAAATATCCGAAAAGATTTCAAAATCATTAAATATATCTGGT 636 2101 T H R K I K K I S E K I S K S L N I S G 212

6361 CCATTTAATATACAATTTATATGTCATCAAAATGAAATAAAAATTATTGAATGTAATTTA 642 2121 P F N I Q F I C H Q N E I K I I E C N L 214

6421 AGAGCATCTAGAACTTTTCCATTTATATCAAAAGCTCTAAATCTAAACTTTATAGATTTA 648 2141 R A S R T F P F I S K A L N L N F I D L 216

6481 GCTACAAGGATATTAATGGGTTATGACGTCAAACCAATTAATATATCATTAATTGATTTA 654 2161 A T R I L M G Y D V K P I N I S L I D L 218

6541 GAATATACAGCTGTAAAAGCACCGATTTTCTCATTTAATAGATTACATGGATCAGATTGT 660 2181 E Y T A V K A P I F S F N R L H G S D C 220

6601 ATACTAGGTGTAGAAATGAAATCTACAGGTGAAGTAGCATGTTTTGGTTTAAATAAATAT 666 2201 I L G V E M K S T G E V A C F G L N K Y 222

6661 GAAGCTTTATTAAAATCATTAATAGCTACAGGTATGAAGTTACCCAAAAAATCAATACTT 672 2221 E A L L K S L I A T G M K L P K K S I L 224

6721 ATAAGTATTAAAAATTTAAATAATAAATTAGCTTTTGAAGAACCGTTCCAATTATTATTT 678 2241 I S I K N L N N K L A F E E P F Q L L F 226

6781 TTAATGGGATTTACAATATATGCGACTGAAGGTACGTATGATTTCTACTCTAAATTTTTA 684 2261 L M G F T I Y A T E G T Y D F Y S K F L 228

6841 GAATCTTTTAATGTTAATAAAGGTTCTAAATTTCATCAAAGACTTATTAAAGTTCATAAT 690 2281 E S F N V N K G S K F H Q R L I K V H N 230

6901 AAAAATGCAGAAAATATATCACCAAATACAACAGATTTAATTATGAATCATAAAGTTGAA 696 2301 K N A E N I S P N T T D L I M N H K V E 232

6961 ATGGTTATTAATATAACTGATACATTAAAAACAAAGGTTAGTTCAAATGGTTATAAAATT 702 2321 M V I N I T D T L K T K V S S N G Y K I 234 TABLE 1 (cont)

7021 AGAAGATTAGCATCAGATTTCCAGGTTCCTTTAATAACTAATATGAAACTTTGTTCTCTT 708 2341 R R L A S D F Q V P L I T N M K L C S L 236

7081 TTTATTGACTCATTATATAGAAAATTCTCAAGACAAAAGGAAAGAAAATCATTCTATACC 714 2361 F I D S L Y R K F S R Q K E R K S F Y T 238

7141 ATAAAGAGTTATGACGAATATATAAGTTTGGTATAA 717 2381 I K S Y D E Y I S L V * 239

7177 GCAAGAAATTATTCAATAAATTCGATTTAACATTACTTATTTATGTATTTATTAACTTTC 723

7237 ATTCCATAACAACATGAAAAGTATAAATATATAAATAGTAATATATAATATATAATATAT 729

7297 ATATATATATATATATATATATTTATTTATTTAATTATATTTACGTTTAAATATTAATAA 735

7357 ATGTTTTTATTAAATATGATCATTAATTTATATTGATTTATTTTTTTATAAATTTTTGTT 741

7417 ATATATACAAATTTTATTTATTCACTCATATGTATAAACCAAAATGGTTTTTTCAATTTA 747

7477 CAAATAATTTTATAATTTTAATAAATTTATTAATTATAAAAAAAATAAAAATATATAAAC 753

7537 ATTAAAATGTATAAATTCTTTTAATTATATAATAATTTATAAATGTTATGATTTTTTTAA 759

7597 AAAATTCAACGAAAAAAAAGAGGAACTGTATATACAAAAGGGACTATATATATGTATATA 765

7657 TATATATATATATATATGTTTTTTTTTCCTTATTCTAGA 7695

The GAT domain is made up of two subdomains: a putative structural domai (1-750) and a glutaminase domain (1447-2070). These two subdomains are separated by a first inserted sequence (751-1446, underlined). The two A binding subdomains of the synthetase subunit, CPSa (2071-3762) and CPSb (5572-5173) are separated by a second inserted sequence (3763-5571, underlined) .

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, ribozymes 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 are 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. Izant, 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 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. & Gerlach, W.L. (1988) Nature 134, 585; Kruger, K., Grabowski, P.J., Zaug, A.J., Sands,J., Gottschling, D.E. & Cech, T.R. (1982) Cell 31, 147; International Patent Application No. WO 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 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 mRNA 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 this method and the polypeptides of the present invention will be useful in in vitro drug binding studies in efforts to develop other anti-malarial 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 CPSII gene in Plasmodium 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 confirmed 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 EcoRI and hybridisation was carried out using the pfCPSII 453 bp PCR product. A 3.0 kb HindiII and a smaller EcoRI fragment hybridised to the probe. Subsequent screening of a HindiII pTZ18U mini-library resulted in the isolation of a recombinant 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 Hindi11/.EcoRI fragment from the 5' end of CPS2 was 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 Hindlll recombinant containing CPS3 as well as a related 3.3 kb XBal clone (not presented in Figure 1) were isolated by the mini- library technique. Using a 200 bp Xbal/Hindlll 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 CPSII gene in P. falciparum, together with its 5' and 3' flanking 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 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.

REFERENCES

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Reyes, P., Rathod, P.K., Sanchez, D.J. Mrema, 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. 5 -. 275-290.

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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 sequence 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.

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 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.

4. A method of producing Plasmodium falciparum carbamoyl phosphate synthetase II or a portion thereof, the, method comprising culturing a cell transformed with 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 carbamoyl phosphate synthetase II.

5. Method as claimed in claim 4 in which the cell is E. coli , yeast or Dictyoεtelium discoideum.

6. 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 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 mRNA obtained from the first or second inserted sequences of the nucleic acid molecule as claimed in claim 1 or claim 2.

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 the ribozyme as claimed in claim 5 or 6 or the antisense oligonucleotide as claimed in claim 7.