Nucleotide sequence encoding carbamoyl phosphate synthetase II

Abstract

The present invention provides a nucleotide sequence encoding carbamoyl phosphate synthetase II of Plasmodium falciparum. Carbamoyl phosphate synthetase II catalyses the first committed and rate-limiting step in the de novo pyrimidine biosynthetic pathway. P. falciparum relies exclusively on pyrimidine synthesis de novo because of its inability to salvage pyrimidines. Mature human red blood cells, however, have no recognized requirement for a pyrimidine nucleotide. Accordingly, this enzyme represents a prime chemotherapeutic locus. The present invention relates to the use of the sequence encoding carbamoyl phosphate synthetase II in the recombinant production of carbamoyl phosphate synthetase II and to antisense molecules, ribozymes and other gene inactivation agents designed from this sequence.

Description

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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 (CPSU, 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 (Gln) 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., 1935). 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 cpa1 and cpa2 in yeast) as well as rat mitochondrial CPSI arose by defusion from the pyrimidine chimera.

DESCRIPTION OF THE INVENTION

The present inventors have isolated and characterised the complete gene encoding the CPSII enzyme from P. falciparum (pfCPSII). Reported here is the 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, of 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 falciparum [SEQ ID NOS:1 and 2]
	.         .         .         .         .         .
−1225	GAATTCCTTCAGCCAAAAAAAATGACAACGCAAATTTTAAGAAAAGAAAAACAATCGACT	−1156
	.         .         .         .         .         .
−1165	CGTCTTTGAATGAGGTTAGAAATTCGATACGTGAAAGGGACTTAAGAAGGCTTAACAGAG	−1106
	.         .         .         .         .         .
−1105	AAAAGAGTAAAATCTTATAAGCATTTGAAGGAAAAAATAATAAAATAAAAAAATAAAAAG	−1046
	.         .         .         .         .         .
−1045	ATAAAAAATATTTATATTTGATATGTAGTATATATAATGATTATTCATATTAATAACATA	−986
	.         .         .         .         .         .
−985	GATAAAAAACTTTTTTTTTTTTTTTTTTTCTTTATATTTATTAACAATACATTTAAGTTA	−926
	.         .         .         .         .         .
−925	TTTTATATATATATATATATATATATATATATATATATATATATATGTTTGTGTGTTCAT	−866
	.         .         .         .         .         .
−865	TTGTTTATAAAATTACTTGAAATATAAAACTTATTAATATATTTCCAATTAATATGAATA	−806
	.         .         .         .         .         .
−805	CAATTATTAATATTTTCATGTGTACACATTAATATAGTTTTACACTTCTTATAATAAAAC	−746
	.         .         .         .         .         .
−745	CATCCTATATATTATACACAATATATAATACTCCCCAATATTGTGGTTCCTATAATTTTA	−686
	.         .         .         .         .         .
−685	TTTATATATTTATTTATTAATTTATTCATTTATTTATTTTTTTTCTTAGTTTATAAAATA	−626
	.         .         .         .         .         .
−625	GTAATTCTACTAATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAATT	−566
	.         .         .         .         .         .
−565	TACATATGAAAAATGAACTTGTATATGTAAATTTATAAATATTTTAAACATAAATATAAA	−506
	.         .         .         .         .         .
−505	TGTATAAAAAAAAAAAAGAAAAATGGGAAAAAATAATATAGATATATATATAAATATATA	−446
	.         .         .         .         .         .
−445	TATATATATAATTATTGGGGATATTCTCTGAATCATAGGTCTTAAACAGTTTTATTCTTT	−385
	.         .         .         .         .         .
−385	TAACATCACAAAGTTGTTATTAAAAGTATATATATCTTATTGGTTCCTATATAAAACTAT	−326
	.         .         .         .         .         .
−325	AGTATTCTATAATATATTCTGTATATTTCATTTTATCATTTGTAAGCAATCCCTATTTAT	−266
	.         .         .         .         .         .
−265	TATAATTATTATTTTTTTTTTTATAAAAGAGGTATAAAACAGTTTATTCAATTTTTTTCC	−206
	.         .         .         .         .         .
−205	TAAAGGAGCAACCTTCAGTCAATTTACATTTTCCACCGGTTGGTTGGCACAACATAATGT	−146
	.         .         .         .         .         .
−145	TACAGCTAAAAAAAGAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATAT	−86
	.         .         .         .         .         .
−85	ATATATATATATATACATAATATGTACAATGCTACCATACAAGTATATAAATTTTTCAAC	−26
	.         .
−25	ATTGTTGTGATGTTGCATTTTTCTT	−1
	.         .         .         .         .	.
1	ATGTATATTTCTTTTAAATATAATTTATATATATATATATATATATATATATATATATTT	60
1	M  Y  I  S  F  K  Y  N  L  Y  I  Y  I  Y  I  Y  I  Y  I  F	20
	.         .         .         .         .         .
61	GTTCTTATAGATTTTAAAACAGTTGGGAGGTTAATTCTTGAAGATGGTAACGAATTTGTA	120
21	V  L  I  D  F  K  T  V  G  R  L  I  L  E  D  G  N  E  F  V	40
	.         .         .         .         .         .
121	GGGTACAGTGTAGGTTACGAAGGGTCTAAAGGAAATAATAGTATATCATGTCATAAGGAG	180
41	G  Y  S  V  G  Y  E  G  C  K  G  N  N  S  I  S  C  H  K  E	60
	.         .         .         .         .         .
181	TATAGAAATATTATTAATAATGATAATAGCAAGAATAGTAATAATTCATTTTGTAATAAT	240
61	Y  R  N  I  I  N  N  D  N  S  K  N  S  N  N  S  F  C  N  N	80
	.         .         .         .         .         .
241	GAACAAAACAATTTGAAAGATCATTTATTATATAAAAATAGTCGATTAGAAAATGAAGAT	300
81	E  E  N  N  L  K  D  D  L  L  Y  K  N  S  R  L  E  N  E  D	100
	.         .         .         .         .         .
301	TTTATTGTTACAGGTGAAGTTATATTTAATACAGCTATGGTTGGATATCCTGAAGCTTTA	360
101	F  X  V  T  G  E  V  Z  F  N  T  A  M  V  G  Y  P  E  A  L	120
	.         .         .         .         .         .
361	ACGGACCCAAGTTATTTTGGTCAAATATTAGTTTTAACATTTCCTTCTATTGGTAATTAT	420
121	T  D  P  S  Y  F  G  Q  I  L  V  L  T  F  P  S  I  G  N  Y	140
	.         .         .         .         .         .
421	GGTATTGAAAAAGTAAAACATGATGAAACGTTTGGATTAGTACAAAATTTTGAAAGTAAT	480
141	G  I  E  K  V  K  H  D  E  T  F  G  L  V  Q  N  F  E  S  N	160
	.         .         .         .         .         .
481	AAAATTCAAGTACAAGGTTTAGTTATTTGTGAATATTCGAAGCAATCATATCATTACAAT	540
161	K  X  Q  V  Q  G  L  V  X  C  E  Y  S  K  Q  S  Y  H  Y  N	180
	.         .         .         .         .         .
541	TCTTATATTACCTTAAGTGAATGGTTAAAGATTTATAAAATTCCATGTATAGGTGCTATA	600
181	S  Y  I  T  L  S  E  W  L  K  I  Y  K  I  P  C  I  G  G  I	200
	.         .         .         .         .         .
601	GATACAAGAGCCTTAACAAAACTTTTAAGAGAAAAAGGTAGTATGTTAGGTAAAATACTT	660
201	D  T  R  A  L  T  K  L  L  R  E  K  G  S  M  L  G  K  I  V	220
	.         .         .         .         .         .
661	ATATATAAAAACAGACAACATATTAATAAATTATATAAAGAAATTAATCTTTTTGATCCT	720
221	I  Y  K  N  R  Q  H  I  N  K  L  Y  K  E  I  N  L  F  D  P	240
	.         .         .         .         .         .
721	GGTAATATAGATACTCTAAAATATGTATGTAATCATTTTATACGTGTTATTAAGTTGAAT	780
241	G  N  I  D  T  L  K  Y  V  C   N  H  F  I  R  V  I  K  L  N	260
	.         .         .         .         .         .
781	AATATTACATATAATTATAAAAATAAGGAAGAATTTAATTATACCAATGAAATGATTACT	840
261	N  I  T  Y  N  Y  K  N  K  E  E  F  N  Y  T  N  E  N  I  T	280
	.         .         .         .         .         .
841	AATGATTCTTCAATGGAAGATCATGATAATGAAATTAATGGTAGTATTTCTAATTTTAAT	900
281	N  D  S  S  M  E  D  H  D  N  Z  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	TTGTTAAGAGATAAAATGAACCTAATAACTTCATCTGAAGAATATCTGAAACATCTTCAT	1020
321	L  L  R  D  K  M  N  L  I  T  S  S  E  E  Y  L  K  D  L  H	340
	.         .         .         .         .         .
1021	AATTGTAATTTTAGTAATAGTAGTGATAAAAATGATTCTTTTTTTAAGTTATATGGTATA	1080
341	N  C  N  F  S  N  S  S  D  K  N  D  S  F  F  K  L  Y  G  I	360
	.         .         .         .         .         .
1021	TGTGAATATGATAAATATTTAATTGACCTTGAAGAAAATGCTAGCTTTCATTATAATAAT	1140
351	C  E  Y  D  K  Y  L  I  D  L  E  E  N  A  S  F  H  Y  N  N	380
	.         .         .         .         .         .
1141	GTAGATGAATATGGATATTATGATGTTAATAAAAATACAAATATTCTATCTAATAATAAA	1200
381	V  D  E  Y  G  Y  Y  D  V  N  K  N  T  N  I  L  S  N  N  K	400
	.         .         .         .         .         .
1201	ATAGAACAAAACAACAATAACGAAAATAACAAAAATAACAAAAATAACAACAATAACGAG	1260
401	I  E  Q  N  N  N  N  Z  N  N  K  N  N  K  N  N  N  N  N  E	420
	.         .         .         .         .         .
1261	GTTGATTATATAAAGAAAGATGAGGATAATAATGTCAATAGTAAGGTCTTTTATAGCCAA	1320
421	V  D  Y  I  K  K  D  E  D  N  N  V  N  S  K  V  F  Y  S  Q	440
	.         .         .         .         .         .
1321	TATAATAATAATGCACAAAATAATGAACATACCGAATTTAATTTAAATAATGATTATTCT	1380
441	Y  N  N  N  A  Q  N  N  E  H  T  E  F  N  L  N  N  D  Y  S	460
	.         .         .         .         .         .
1381	ACTTATATTAGAAAGAAAATGAAAAATCAAGAATTCCTTAATTTGGTAAACAAAAGAAAA	1440
461	T  Y  I  R  K  K  M  K  N  E  E  F  L  N  L  V  N  K  R  K	480
	.         .         .         .         .         .
1441	GTAGACCATAAAGAAAAAATTATTGTTATTGTTGATTGTGGTATTAAAAATAGTATAATC	1500
481	V  D   H  K  E  K  I  I  V  I  V  D  C  G  I  K  N  S  I  I	500
	.         .         .         .         .         .
1501	AAAAATTTAATAAGACACGGTATGGATCTTCCATTAACATATATTATTGTACCTTATTAT	1560
501	K  N  L  I  R  H  G  M  D  L  P  L  T  Y  I  I  V  P  Y  Y	520
	.         .         .         .         .         .
1561	TACAATTTTAATCATATAGATTATGATGCAGTTCTTTTATCTAATGGTCCTGGAGATCCT	1620
521	Y  N  F  N  H  I  D  Y  D  A  V  L  L  S  N  G  P  G  D  P	540
	.         .         .         .         .         .
1621	AAAAAGTGTGATTTCCTTATAAAAAATTTGAAAGATAGTTTAACAAAAAATAAAATTATA	1680
541	K  K  C  D  F  L  I  K  N  L  K  D  S  L  T  K  N  K  I  I	560
	.         .         .         .         .         .
1681	TTTGGTATTTGTTTAGGTAATCAACTATTAGGTATATCATTAGGTTGTGACACATATAAA	1740
561	F  G  I  C  L  G  N  Q  L  L  G  I  S  L  G  C  D  T  Y  K	580
	.         .         .         .         .         .
1741	ATGAAATATGGTAATAGAGGTGTTAATCAACCCGTAATACAATTAGTAGATAATATATGT	1800
581	M  K  Y  G  N  R  G  V  N  Q  P  V  I  Q  L  V  D  N  I  C	600
	.         .         .         .         .         .
1801	TACATTACCTCACAAAATCATGGATACTGTTTAAAGAAAAAATCAATTTTAAAAAGAAAA	1860
601	Y  I  T  S  Q  N  H  G  Y  C  L  K  K  K  S  I  L  K  R  K	620
	.         .         .         .         .         .
1861	GAGCTTGCGATTAGTTATATAAATGCTAATGATAAATCTATACAAGGTATTTCACATAAA	1920
621	E  L  A  I  S  Y  I  N  A  N  D  I  S  I  E  G  I  S  H  K	640
	.         .         .         .         .         .
1921	AATGGAAGATTTTATAGTGTCCAGTTTCATCCTCAGGGTAATAATGGTCCTGAAGATACA	1980
641	N  G  R  F  Y  S  V  Q  F  H  F  E  G  N  N  G  P  E  D  T	660
	.         .         .         .         .         .
1981	TCATTTTTATTTAACAATTTTCTTTTAGATATCTTTAATAAGAAAAAACAATATAGAGAA	2040
661	S  F  L  F  K  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	TTATGTATAGGACAAGCAGCACAATTCGATTATTCAGGAACA(AAGCAATTAAAAGTTTA	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  F  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	ATTAAAAAGGAAAAACCTGATTTTATTTTATGTACATTTGGTGGTCACACAGCTTTAAAT	2340
761	I  K  K  E  K  P  D  F  I  L  C  T  F  G  G  Q  T  A  L  N	780
2341	TGTGCTTTAATGTTAGATCAAAAAAAAGTATTGAAAAAGAATAATTGTCAATGTTTAGGT	2400
781	C  A  L  M  L  D  Q  K  K  V  L  K  K  N  N  C  C  C  L  G	800
	.         .         .         .         .         .
2401	ACATCTTTAGAATCTATAAGAATAACAGAAAATAGAACATTATTTGCTGAAAAATTAAAA	2460
801	T  S  L  E  S  I  R  I  T  E  N  R  T  L  F  A  E  K  L  K	820
	.         .         .         .         .         .
2461	GAAATTAATGAAAGAATAGCTCCATATGGTAGTGCAAAAAATGTTAATCAAGCTATTGAT	2520
821	E  I  N  E  R  I  A  P  Y  G  S  A  K  N  V  N  Q  A  I  D	840
	.         .         .         .         .         .
2521	ATAGCTAATAAAATAGGATATCCAATATTAGTACGTACAACATTTTCGTTAGGAGGATTA	2580
841	I  A  N  K  I  G  Y  P  I  L  V  R  T  T  F  S  L  G  G  L	860
	.         .         .         .         .         .
2581	AATAGTAGTTTCATAAATAATGAAGAAGAACTTATCGAAAAATGTAATAAAATATTTTTA	2640
861	N  S  S  F  I  N  N  E  E  E  L  I  E  K  C  N  K  I  F  L	880
	.         .         .         .         .         .
2641	CAAACTGATAATGAAATATTTATAGATAAATCATTACAAGGATGGAAAGAAATAGAATAT	2700
881	Q  T  D  N  E  T  F  I  D  K  S  L  Q  G  W  K  E  I  E  Y	900
	.         .         .         .         .         .
2701	GAATTATTAAGAGATAATAAAAATAATTGTATAGCTATATGTAATATGGAAAATATAGAT	2760
901	E  J  L  R  D  N  K  N  N  C  I  A  I  C  N  M  E  N  I  D	920
	.         .         .         .         .         .
2761	CCATTAGGTATACATACAGGAGATAGTATAGTTGTTGCACCTTCACAAACATTAAGTAAT	2820
921	P  L  G  I  H  T  G  D  S  I  V  V  A  P  S  Q  T  L  S  N	940
	.         .         .         .         .         .
2821	TATGAATATTATAAATTTAGAGAAATAGCATTAAAGGTAATTACACATTTAAATATTATA	2880
941	Y  L  Y  Y  K  F  R  E  I  A  L  K  V  I  T  H  L  N  I  I	960
	.         .         .         .         .         .
2881	GGAGAATGTAATATACAATTTGGTATAAATCCACAAACAGGAGAATATTGTATTATTGAA	2940
961	G  E  C  N  I  Q  F  G  I  N  P  Q  T  G  E  Y  C  I  I  E	980
	.         .         .         .         .         .
2941	GTTAATGCTAGGCTTAGTAGAAGTTCAGCATTACCTTCTAAAGCTACTGGTTATCCACTT	3000
981	V  N  A  R  L  S  R  S  S  A  L  A  S  K  A  T  G  Y  P  L	1000
	.         .         .         .         .         .
3001	GCTTATATATCAGCAAAAATAGCCTTGGGATATGATTTGATAAGTTTAAAAAATAGCATA	3060
1001	A  Y  I  S  A  K  I  A  L  G  Y  D  L  I  S  L  K  N  S  I	1020
	.         .         .         .         .         .
3061	ACTAAAAAAACAACTGCCTGTTTTGAACCCTCTCTAGATTACATTACAACAAAAATACCA	3120
1021	T  K  K  T  T  A  C  F  E  P  S  L  D  Y  I  T  T  K  I  P	1040
	.         .         .         .         .         .
3121	CGATCGGATTTAAATAAATTTGAGTTTGCTTCTAATACAATGAATAGTAGTATGAAAAGT	3180
1041	R  W  D  L  N  K  F  E  F  A  S  N  T  M  N  S  S  M  K  S	1060
	.         .         .         .         .         .
3181	GTAGGAGAAGTTATGTCTATAGGTAGAACCTTTGAAGAATCTATACAAAAATCTTTAAGA	3240
1061	V  G  E  V  M  S  I  G  R  T  F  E  E  S  I  Q  K  S  L  R	1080
	.         .         .         .         .         .
3241	TGTATTGATGATAATTATTTAGGATTTAGTAATACGTATTGTATAGATTGGGATGAAAAG	3300
1081	C  I  D  D  N  Y  L  G  F  S  N  T  Y  C  I  D  W  D  E  K	1100
	.         .         .         .         .         .
3301	AAAATTATTCAAGAATTAAAAAATCCATCACCAAAAAGAATTGATGCTATACATCAAGCT	3360
1101	K  I  I  E  E  L  K  N  P  S  P  K  R  I  D  A  I  H  Q  A	1120
	.         .         .         .         .         .
3361	TTCCATTTAAATATGCCTATGGATAAAATACATGAGCTGACACATATTGATTATTGGTTC	3420
1121	F  H  L  N  M  P  M  D  K  I  H  E  L  T  H  I  D  Y  W  F	1140
	.         .         .         .         .         .
3421	TTACATAAATTTTATAATATATATAATTTAGAAAATAAGTTGAAAACGTTAAAATTAGAG	3480
1141	L  H  K  F  Y  N  I  Y  N  L  Q  N  K  L  K  T  L  K  L  E	1160
	.         .         .         .         .         .
3481	CAATTATCTTTTAATGATTTGAAGTATTTTAAGAAGCATGGTTTTAGTGATAAGCAAATA	3540
1161	Q  L  S  F  N  D  L  X  Y  F  K  K  W  G  F  S  D  K  Q  I	1180
	.         .         .         .         .         .
3541	GCTCACTACTTATCCTTCAACACAAGCGATAATAATAATAATAATAATAATATTAGCTCA	3600
1181	A  H  Y  L  S  F  N  T  S  D  N  N  N  N  N  N  N  I  S  S	1200
	.         .         .         .         .         .
3601	TGTAGGGTTACAGAAAATGATGTTATGAAATATA&AGAAAAGCTAGGATTATTTCCACAT	3660
1201	C  R  V  T  E  N  D  V  M  K  Y  R  E  K  L  G  L  F  P  H	1220
	.         .         .         .         .         .
3661	ATTAAAGTTATTGATACCTTATCAGCCGAATTTCCGGCTTTAACTAATTATTTATATTTA	3720
1221	I  K  V  I  D  T  L  S  A  E  F  P  A  L  T  N  Y  L  Y  L	1240
	.         .         .         .         .         .
3721	ACTTATCAAGGTCAAGAACATGATGTTCTCCCATTAAATATGAAAAGGAAAAAGATATGC	3780
1241	T  Y  Q  G  Q  E  H  D  V  L  P  L  N  M   K  R  K  K  I  C	1260
	.         .         .         .         .         .
3781	ACGCTTAATAATAAACGAAATGCAAATAAGAAAAAAGTCCATGTCAAGAACCACTTATAT	3840
1261	T  L  N  N  K  R  N  A  N  K  K  K  V  R  V  K  N  X  L  Y	1280
	.         .         .         .         .         .
3841	AATGAAGTAGTTGATGATAAGGATACACAATTACACAAAGAAAATAATAATAATAATAAT	3900
1281	N  E  V  V  D  D  K  D  T  Q  L  H  K  E  N  N  N  N  N  N	1300
	.         .         .         .         .         .
3901	ATGAATTCTGGAAATGTAGAAAATAAATGTAAATTGAATAAAGAATCCTATGGCTATAAT	3960
1301	M  N  S  G  N  V  E  N  K  C  K  L  N  K  E  S  Y  G  Y  N	1320
	.         .         .         .         .         .
3961	AATTCTTCTAATTGTATCAATACAAATAATATTAATATAGAAAATAATATTTGTCATGAT	4020
1321	N  S  S  N  G  I  N  T  N  N  I  N  I  E  N  N  I  C  H  D	1340
	.         .         .         .         .         .
4021	ATATCTATAAACAAAAATATAAAAGTTACAATAAACAPLTTCCAATAATTCTATATCAAT	4080
1341	I  S  I  N  K  N  I  K  V  T  I  N  N  S  N  N  S  I  S  N	1360
	.         .         .         .         .         .
4081	AATGAAAATGTTGAAACAAACTTAAATTGTGTATCTGAAAGGGCCGGTAGCCATCATATA	4140
1361	N  E  N  V  E  T  N  L  N  C  V  S  E  R  A  G  S  H  H  I	1380
	.         .         .         .         .         .
4141	TATGGTAAAGAAGAAAAGAGTATAGGATCTGATGATACAAATATTTTAAGTGCACAAAAT	4200
1381	Y  G  K  E  E  K  S  I  G  S  D  D  T  N  I  L  S  A  C  N	1400
	.         .         .         .         .         .
4201	TCAAATAATAACTTTTCATGTAATAATGAGAATATGAATAAAGCAAACGTTGATGTTAAT	4260
1401	S  N  N  N  F  S  C  N  N  E  N  M  N  K  A  N  V  D  V  N	1420
	.         .         .         .         .         .
4261	GTACTAGAAAATGATACGAAAAAACGAGAAGATATAAATACTACAACAGTATTTATGGAA	4320
1421	V  L  E  N  D  T  K  K  R  E  D  I  N  T  T  T  V  F  N  E	1440
	.         .         .         .         .         .
4321	GGTCAAAATAGTGTTATTAATAATAAGAATAAAGAGAATAGTTCTTTATTGAAAGGTGAT	4380
1441	G  Q  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  N  V  N  L  K  K  E  N  N  Y  N  S  V  Y  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
	.         .         .         .         .         .
4681	AATAAAAACCCTGGTAATTCTTATTATGTTGTAGATTCCGTATATAATAATGAATACAAA	4740
1561	N  K  N  P  G  N  S  Y  Y  V  V  D  S  V  Y  N  N  E  Y  K	1580
	.         .         .         .         .         .
4741	ATTAATAAGATGAAAGAGTTAATAGATAACGAAAATTTAAATGATGAATATAATAATAAT	4800
1581	I  N  K  M  K  E  L  I  D  N  E  N  L  N  D  E  Y  N  N  N	1600
	.         .         .         .         .         .
4801	GTTAATATGAATTGTTCTAATTATAATAATGCTAGTGCATTTGTAAATGGAAAGGATAGA	4860
1601	V  N  M  N  C  S  N  Y  N  N  A  S  A  F  V  N  G  K  D  K	1620
	.         .         .         .         .         .
4861	AATGATAATTTAGAAAATGATTGTATTGAAAAAAATATGGATCATACATACAAACATTAT	4920
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  C  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	AATACTGATGAGTATGATGATGATTATTATTATGATGAACATGAAGAAGATGACTATGAC	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	5400
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
	.         .         .         .         .         .
5461	GTATCATCTGTTATATATTCAGGGAACGAAAATATATTTAATGAAAAATATAATGATATA	5520
1821	V  S  S  V  I  Y  S  G  N  E  N  T  F  N  E  K  Y  N  D  I	1840
	.         .         .         .         .         .
5521	GGTTTTAAAATAATCGATAATAGGAATGAAAAAGAGAAAGAGAAAAAGAAATGTTTTATT	5580
1841	G  F  K  I  I  D  N  R  N  E  K  E  K  E  K  K  K   C  F  I	1860
	.         .         .         .         .         .
5581	GTATTAGGTTGTGGTTGTTATCGTATTGGTAGTTCTGTAGAATTTGATTGGAGTGCTATA	5640
1861	V  L  G  C  G  C  Y  R  I  Q  S  S  V  E  F  D  W  S  A  I	1880
	.         .         .         .         .         .
5641	CATTGTGTAAAGACCATAAGAAAATTAAACCATAAAGCTATATTAATAAATTGTAACCCA	5700
1881	H  C  V  K  T  I  R  K  L  N  H  K  A  I  L  T  N  C  N  F	1900
	.         .         .         .         .         .
5701	GAAACTGTAAGTACAGATTATGATGAAAGTGATCGTCTATATTTTGATGAAATAACAACA	5760
1901	E  T  V  S  T  D  Y  D  E  S  D  R  L  Y  F  D  E  I  T  T	1920
	.         .         .         .         .         .
5761	GAAGTTATAAAATTTATATATAACTTTGAAAATAGTAATGGTGTGATTATAGCTTTTGGT	5820
1921	E  V  I  K  F  I  Y  N  F  E  N  S  N  G  V  I  I  A  F  G	1940
	.         .         .         .         .         .
5821	GGACAAACATCAAATAATTTAGTATTTAGTTTATATAAAAATAATGTAAATATATTACGA	5880
1941	G  Q  T  S  N  N  L  V  F  S  L  Y  K  N  N  V  N  I  L  G	1960
	.         .         .         .         .         .
5881	TCAGTGCACAAAGTGTTGATTCTTGTGAAAATAGGAATAAATTTTCGCACTTATGTGATT	5940
1961	S  V  H  K  V  L  I  V  V  K  I  G  I  N  F  R  T  Y  V  I	1980
	.         .         .         .         .         .
5941	CTTAAAATTGATCAACCGAAATGGAATAAATTTACAAAATTATCCAAGGCTATACAATTT	6000
1981	L  K  I  D  Q  P  K  W  N  K  F  T  K  L  S  K  A  I  Q  F	2000
	.         .         .         .         .         .
6001	GCTAATGAGGTAAAATTTCCTGTATTAGTAAGACCATCGTATGTATTATCTGGTGCAGCT	6060
2001	A  N  E  V  K  F  F  V  L  V  R  P  S  Y  V  L  S  G  A  A	2020
	.         .         .         .         .         .
6061	ATGAGAGTTGTAAATTGTTTTGAAGAATTAAAAAACTTTTTAATGAAGGCAGCTATTGTT	6120
2021	M  R  V  V  N  C  F  E  E  L  K  N  F  L  M  K  A  A  I  V	2040
	.         .         .         .         .         .
6121	AGTAAAGATAATCCTGTTCTAATATCAAAATTTATTGAGAATGCTAAAGAAATAGAAATA	6180
2041	S  K  D  N  P  V  V  I  S  K  F  T  E  N  A  K  E  I  E  I	2060
	.         .         .         .         .         .
6181	GATTGTGTTAGTAAAAATGGTAAAATAATTAATTATGCTATATCTGAACATGTTGAAAAT	6240
2061	D  C  V  S  K  N  G  K  I  I  N  Y  A  I  S  E  H  V  E  N	2080
	.         .         .         .         .         .
6241	CCTGGTGTACATTCAGGTGATCCAACATTAATATTACCTGCACAAAATATATATGTTGAA	6300
2081	A  G  V  H  S  C  D  A  T  L  I  L  P  A  Q  N  I  Y  V  E	2100
	.         .         .         .         .         .
6301	ACACATAGGAAAATAAAGAAAATATCCGAAAACATTTCAAAATCATTAAATATATCTGGT	6360
2101	T  H  R  K  I  K  K  I  S  E  K  I  S  K  S  L  N  I  S  G	2120
	.         .         .         .         .         .
6361	CCATTTAATATACAATTTATATCTCATCAAAATGAAATAAAAATTATTGAATGTAATTTA	6420
2121	P  F  N  I  Q  F  I  C  H  Q  N  E  I  K  I  I  E  C  N  L	2140
	.         .         .         .         .         .
6421	AGAGCATCTAGAACTTTTCCATTTATATCAAAAGCTCTAAATCTAAACTTTATAGATTTA	6480
2141	R  A  S  R  T  F  P  F  I  S  K  A  L  N  L  N  F  I  D  L	2160
	.         .         .         .         .         .
6481	GCTACAAGGATATTAATGGGTTATGACGTCAAACCAATTAATATATCATTAATTGATTTA	6540
2161	A  T  R  I  L  N  G  Y  D  V  K  P  I  N  I  S  L  I  D  L	2180
	.         .         .         .         .         .
6541	GAATATACAGCTGTAAAAGCACCGATTTTCTCATTTAATAGATTACATGGATCAGATTGT	6600
2181	E  Y  T  A  V  K  A  P  I  F  S  F  N  R  L  H  G  S  D  C	2200
	.         .         .         .         .         .
6601	ATACTAGGTGTAGAAATGAAATCTACAGGTGAAGTAGCATGTTTTGGTTTAAATAAATAT	6660
2201	I  L  C  V  E  M  K  S  T  G  E  V  A  C  F  G  L  N  K  Y	2220
	.         .         .         .         .         .
6661	GAAGCTTTATTAAAATCATTAATAGCTACAGGTATGAAGTTACCCAAAAAATCAATACTT	6720
2221	E  A  L  L  K  S  L  I  A  T  G  M  K  L  F  K  K  S  I  L	2240
	.         .         .         .         .         .
6721	ATAAGTATTAAAAATTTAAATAATAAATTAGCTTTTGAAGAACCGTTCCAATTATTATTT	6780
2241	I  S  I  K  N  L  N  N  K  L  A  F  E  E  P  F  C  L  L  F	2260
	.         .         .         .         .         .
6781	TTAATGGGATTTACAATATATGCGACTCAAGGTACGTATCATTTCTACTCTAAATTTTTA	6840
2261	L  N  G  F  T  I  Y  A  T  E  G  T  Y  D  F  Y  S  K  F  L	2280
	.         .         .         .         .         .
6841	CAATCTTTTAATCTTAATAAAGGTTCTAAATTTCATCAAACACTTATTAAAGTTCATAAT	6900
2281	E  S  F  N  V  N  K  G  S  K  F  H  Q  R  L  I  K  V  H  N	2300
	.         .         .         .         .         .
6901	AAAAATGCAGAAAATATATCACCAAATACAACAGATTTAATTATGAATCATAAAGTTGAA	6960
2301	K  N  A  E  N  I  S  P  N  T  T  D  L  I  M  N  H  K  V  E	2320
	.         .         .         .         .         .
6961	ATGGTTATTAATATAACTGATACATTAAAAACAAAGGTTAGTTCAAATGGTTATAAAATT	7020
2321	M  V  I  N  I  T  D  T  L  K  T  K  V  S  S  N  G  Y  K  I	2340
	.         .         .         .         .         .
7021	AGAAGATTAGCATCAGATTTCCAGGTTCCTTTAATAACTAATATGAAACTTTGTTCTCTT	7080
2341	R  R  L  A  S  D  F  Q  V  P  L  I  T  N  M  K  L  C  S  L	2360
	.         .         .         .         .         .
7081	TTTATTGACTCATTATATAGAAAATTCTCAAGACAAAAGGAAAGAAAATCATTCTATACC	7140
2361	F  I  D  S  L  Y  R  K  F  S  R  Q  K  E  R  K  S  F  Y  T	2380
	.         .         .
7141	ATAAAGAGTTATGACGAATATATAAGTTTGGTATAA	7176
2381	I  K  S  Y  D  E  Y  I  S  L  V  *	2392
	.         .         .         .         .         .
7177	GCAAGAAATTATTCAATAAATTCGATTTAACATTACTTATTTATGTATTTATTAACTTTC	7235
	.         .         .         .         .         .
7237	ATTCCATAACAACATGAAAAGTATAAATATATAAATAGTAATATATAATATATAATATAT	7296
	.         .         .         .         .         .
7297	ATATATATATATATATATATATTTATTTATTTAATTATATTTACGTTTAAATATTAATAA	7356
	.         .         .         .         .         .
7357	ATGTTTTTATTAAATATGATCATTAATTTATATTGATTTATTTTTTTATAAATTTTTGTT	7416
	.         .         .         .         .         .
7417	ATATATACAAATTTTATTTATTCACTCATATGTATAAACCAAAATGGTTTTTTCAATTTA	7476
	.         .         .         .         .         .
7477	CAAATAATTTTATAATTTTAATAAATTTATTAATTATAAAAAAAATAAAAATATATAAAC	7536
	.         .         .         .         .         .
7537	ATTAAAATGTATAAATTCTTTTAATTATATAATAATTTATAAATGTTATGATTTTTTTAA	7596
	.         .         .         .         .         .
7597	AAAATTCAACGAAAAAAAAGAGGAACTGTATATACAAAAGGGACTATATATATGTATATA	7656
	.         .         .
7657	TATATATATATATATATGTTTTTTTTTCCTTATTCTAGA	7695

The GAT domain is made up of two subdomains: a putative structural domain (1-750) and a glutaminase domain (1447-2070). These two subdomains are separated by a first inserted sequence (751-1446, underlined). The two ATP 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 U.S. Pat. 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 334. 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, U.S. Pat. No. 4,987,071 and U.S. Pat. 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 Examples and Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : A summary of a “gene walking” strategy used to clone and sequence the full length P. falciparum carbamoyl phosphate synthetase II gene.

FIG. 2 : P. falciparum carbamoyl phosphate synthetase II (pfCPSII) gene sequence with the 21 consensus GUC (GTC) ribozyme cleavage sites identified (underlined) (SEQ ID NO:1).

FIG. 3 : Output of RNA mfold analysis showing the GUC sites from CPSRz1/M10 a and CPSRz4/M15 more accessible than the M17 and M18 sites (Seq. Id. No:1).

FIGS. 4 A-C: A. Sequences for the phosphorothioated antisense DNA used in inhibition studies of P. Falciparum in culture (SEQ ID NOS:3-15); B. Map of the positions of the antisense sequences within the pfCPSII gene; C. Growth supression of P. Falciparum in vitro after a 24 hr incubation with the oligonucleotides.

FIG. 5 . Cultures of P. Falciparum and selected mammalian cell lines were incubated with 2.5 and 5.0 μM CPSRz1, CPSRz4 and 60-mer random oligonucleotide. Cell viability was assessed by measuring the % incorporation of tritiated hypoxanthine.

EXAMPLES

Cloning of the P. falciparum Carbamoyl Phosphate Synthetase II (pfCPSII) Gene

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 pfCPSII 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 FIG. 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 HindIII/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 HindIII recombinant containing CPS3 as well as a related 3.3 kb XBaI clone (not presented in FIG. 1 ) were isolated by the mini-library technique. Using a 200 bp XbaI/HindIII fragment from the 3′ end of CPS 3, a 1.3 kb XbaI 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.

Design of Ribozymes

Our first generation ribozymes were designed using the consensus sequences cleavage site, GUC, found in naturally occurring hammerhead ribozymes (see Haseloff and Gerlach, 1988, the entire contents of which are incorporated herein by reference). In total, there are 21 GUC sites in the entire 7.1 kb coding region of pfCPSII (FIG. 1 and Table 2). To select the putative sites that are relatively more accessible to binding, the pfCPSII mRNA folding pattern was analysed using mfold Program (ANGIS/GCG). This was done in windows of 500 nucleotides, with 250 nucleotide overlaps between each window.

To screen for the best (most accessible) sites within the CPS II mRNA, a series of antisense DNA oligonucleotides were designed and synthesised from sequences around the selected (by mfold) GUC sequences mentioned above. Also included were two antisense oligonucleotides (M17 and M18) where the mfold program had indicated intramolecular base pairing of the GUC sequences may be occurring (FIG. 2 ). As seen from the bio-assays of suppression of malarial growth by the antisense oligonucleotides, there appears to be a correlation between the predicted accessibility of each site and the effectiveness as judged by the % inhibition of growth (FIG. 3 ). The antisense oligonucleotides M10 (Rz1 site) and M15 (Rz4 site) were much more effective in growth inhibition than M17 and M18 where the mfold program predicted internal base pairing.

These results led to the synthesis of ribozymes based on the Rz1 and Rz4 sites (ribozymes CPSRz1 and CPSRz4).

TABLE 2
GUC sites present in the coding region of pfCPSII. Nucleotide
numbers indicate the position of the G of the triplet in the gene sequence.
1.	170
2.	281
3.	380
4.	905	CPSRz1 site/M10
5.	1294
6.	1306
7.	1607
8.	1939
9.	1967
10.	2324
11.	2387
12.	3195
13.	3731	CPSRz4 site/M15
14.	3817
15.	3823
16.	4013
17.	432Z
18.	5223	M17
19.	5735
20.	6359
21.	6383

CPSRz1 and CPSRz4 as Antimalarials

The ability of CPSRz1 and CPSRz4 to cleave a 550 base mRNA fragment of pfCPSII was assessed. Growth inhibition studies on P. falciparum cultures were initially conducted using higher concentrations of ribozymes, at 2.5 and 5.0 μM. Both ribozymes were shown to be very effective at these concentrations as shown in the marked decrease in incorporation of tritiated hypoxanthine (FIG. 4 ). In the same study, a series of mammalian cell lines in culture were treated with the same concentrations of the ribozymes, and no effect cell viability was observed. As a negative control, a 60-mer DNA oligonucleotide of random sequence was used.

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.

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15 1 8920 DNA Plasmodium falciparum 1gaattccttc agccaaaaaa aatgacaacg caaattttaa gaaaagaaaa acaatcgact 60cgtctttgaa tgaggttaga aattcgatac gtgaaaggga cttaagaagg cttaacagag 120aaaagagtaa aatcttataa gcatttgaag gaaaaaataa taaaataaaa aaataaaaag 180ataaaaaata tttatatttg atatgtagta tatataatga ttattcatat taataacata 240gataaaaaac tttttttttt tttttttttc tttatattta ttaacaatac atttaagtta 300ttttatatat atatatatat atatatatat atatatatat atatatgttt gtgtgttcat 360ttgtttataa aattacttga aatataaaac ttattaatat atttccaatt aatatgaata 420caattattaa tattttgatg tgtacacatt aatatagttt tacacttctt ataataaaac 480catcctatat attatacaca atatataata ctccccaata ttgtggttcc tataatttta 540tttatatatt tatttattaa tttattcatt tatttatttt ttttcttagt ttataaaata 600gtaattctac taatttaaaa aaaaaaaaaa aaaaaaaaaa aaaaagaaaa aaaaaaaatt 660tacatatgaa aaatgaactt gtatatgtaa atttataaat attttaaaca taaatataaa 720tgtataaaaa aaaaaaagaa aaatgggaaa aaataatata gatatatata taaatatata 780tatatatata attattgggg atattctctg aatcataggt cttaaacagt tttattcttt 840taacatcaca aagttgttat taaaagtata tatatcttat tggttcctat ataaaactat 900agtattctat aatatattct gtatatttca ttttatcatt tgtaagcaat ccctatttat 960tataattatt attttttttt ttataaaaga ggtataaaac agtttattca atttttttcc 1020taaaggagca accttcagtc aatttacatt ttccaccggt tggttggcac aacataatgt 1080tacagctaaa aaaagaaaga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa atatatatat 1140atatatatat atatacataa tatgtacaat gctaccatac aagtatataa atttttcaac 1200attgttgtga tgttgcattt ttcttatgta tatttctttt aaatataatt tatatatata 1260tatatatata tatatatata tatttgttct tatagatttt aaaacagttg ggaggttaat 1320tcttgaagat ggtaacgaat ttgtagggta cagtgtaggt tacgaagggt gtaaaggaaa 1380taatagtata tcatgtcata aggagtatag aaatattatt aataatgata atagcaagaa 1440tagtaataat tcattttgta ataatgaaga aaacaatttg aaagatgatt tattatataa 1500aaatagtcga ttagaaaatg aagattttat tgttacaggt gaagttatat ttaatacagc 1560tatggttgga tatcctgaag ctttaacgga cccaagttat tttggtcaaa tattagtttt 1620aacatttcct tctattggta attatggtat tgaaaaagta aaacatgatg aaacgtttgg 1680attagtacaa aattttgaaa gtaataaaat tcaagtacaa ggtttagtta tttgtgaata 1740ttcgaagcaa tcatatcatt acaattctta tattacctta agtgaatggt taaagattta 1800taaaattcca tgtataggtg gtatagatac aagagcctta acaaaacttt taagagaaaa 1860aggtagtatg ttaggtaaaa tagttatata taaaaacaga caacatatta ataaattata 1920taaagaaatt aatctttttg atcctggtaa tatagatact ctaaaatatg tatgtaatca 1980ttttatacgt gttattaagt tgaataatat tacatataat tataaaaata aggaagaatt 2040taattatacc aatgaaatga ttactaatga ttcttcaatg gaagatcatg ataatgaaat 2100taatggtagt atttctaatt ttaataattg tccaagtatc tctagttttg ataaaagtga 2160atcgaaaaat gttattaatc atacattgtt aagagataaa atgaacctaa taacttcatc 2220tgaagaatat ctgaaagatc ttcataattg taattttagt aatagtagtg ataaaaatga 2280ttcttttttt aagttatatg gtatatgtga atatgataaa tatttaattg accttgaaga 2340aaatgctagc tttcattata ataatgtaga tgaatatgga tattatgatg ttaataaaaa 2400tacaaatatt ctatctaata ataaaataga acaaaacaac aataacgaaa ataacaaaaa 2460taacaaaaat aacaacaata acgaggttga ttatataaag aaagatgagg ataataatgt 2520caatagtaag gtcttttata gccaatataa taataatgca caaaataatg aacataccga 2580atttaattta aataatgatt attctactta tattagaaag aaaatgaaaa atgaagaatt 2640ccttaatttg gtaaacaaaa gaaaagtaga ccataaagaa aaaattattg ttattgttga 2700ttgtggtatt aaaaatagta taatcaaaaa tttaataaga cacggtatgg atcttccatt 2760aacatatatt attgtacctt attattacaa ttttaatcat atagattatg atgcagttct 2820tttatctaat ggtcctggag atcctaaaaa gtgtgatttc cttataaaaa atttgaaaga 2880tagtttaaca aaaaataaaa ttatatttgg tatttgttta ggtaatcaac tattaggtat 2940atcattaggt tgtgacacat ataaaatgaa atatggtaat agaggtgtta atcaacccgt 3000aatacaatta gtagataata tatgttacat tacctcacaa aatcatggat actgtttaaa 3060gaaaaaatca attttaaaaa gaaaagagct tgcgattagt tatataaatg ctaatgataa 3120atctatagaa ggtatttcac ataaaaatgg aagattttat agtgtccagt ttcatcctga 3180gggtaataat ggtcctgaag atacatcatt tttatttaag aattttcttt tagatatctt 3240taataagaaa aaacaatata gagaatattt aggatataat attatttata taaaaaagaa 3300agtgcttctt ttaggtagtg gtggtttatg tataggacaa gcaggagaat tcgattattc 3360aggaacacaa gcaattaaaa gtttaaaaga atgtggtata tatgttatat tagttaatcc 3420taacatagca actgttcaaa catcaaaagg tttggcagat aaggtatact ttttaccagt 3480taattgtgaa tttgtagaaa aaattattaa aaaggaaaaa cctgatttta ttttatgtac 3540atttggtggt cagacagctt taaattgtgc tttaatgtta gatcaaaaaa aagtattgaa 3600aaagaataat tgtcaatgtt taggtacatc tttagaatct ataagaataa cagaaaatag 3660aacattattt gctgaaaaat taaaagaaat taatgaaaga atagctccat atggtagtgc 3720aaaaaatgtt aatcaagcta ttgatatagc taataaaata ggatatccaa tattagtacg 3780tacaacattt tcgttaggag gattaaatag tagtttcata aataatgaag aagaacttat 3840cgaaaaatgt aataaaatat ttttacaaac tgataatgaa atatttatag ataaatcatt 3900acaaggatgg aaagaaatag aatatgaatt attaagagat aataaaaata attgtatagc 3960tatatgtaat atggaaaata tagatccatt aggtatacat acaggagata gtatagttgt 4020tgcaccttca caaacattaa gtaattatga atattataaa tttagagaaa tagcattaaa 4080ggtaattaca catttaaata ttataggaga atgtaatata caatttggta taaatccaca 4140aacaggagaa tattgtatta ttgaagttaa tgctaggctt agtagaagtt cagcattagc 4200ttctaaagct actggttatc cacttgctta tatatcagca aaaatagcct tgggatatga 4260tttgataagt ttaaaaaata gcataactaa aaaaacaact gcctgttttg aaccctctct 4320agattacatt acaacaaaaa taccacgatg ggatttaaat aaatttgagt ttgcttctaa 4380tacaatgaat agtagtatga aaagtgtagg agaagttatg tctataggta gaacctttga 4440agaatctata caaaaatctt taagatgtat tgatgataat tatttaggat ttagtaatac 4500gtattgtata gattgggatg aaaagaaaat tattgaagaa ttaaaaaatc catcaccaaa 4560aagaattgat gctatacatc aagctttcca tttaaatatg cctatggata aaatacatga 4620gctgacacat attgattatt ggttcttaca taaattttat aatatatata atttacaaaa 4680taagttgaaa acgttaaaat tagagcaatt atcttttaat gatttgaagt attttaagaa 4740gcatggtttt agtgataagc aaatagctca ctacttatcc ttcaacacaa gcgataataa 4800taataataat aataatatta gctcatgtag ggttacagaa aatgatgtta tgaaatatag 4860agaaaagcta ggattatttc cacatattaa agttattgat accttatcag ccgaatttcc 4920ggctttaact aattatttat atttaactta tcaaggtcaa gaacatgatg ttctcccatt 4980aaatatgaaa aggaaaaaga tatgcacgct taataataaa cgaaatgcaa ataagaaaaa 5040agtccatgtc aagaaccact tatataatga agtagttgat gataaggata cacaattaca 5100caaagaaaat aataataata ataatatgaa ttctggaaat gtagaaaata aatgtaaatt 5160gaataaagaa tcctatggct ataataattc ttctaattgt atcaatacaa ataatattaa 5220tatagaaaat aatatttgtc atgatatatc tataaacaaa aatataaaag ttacaataaa 5280caattccaat aattctatat cgaataatga aaatgttgaa acaaacttaa attgtgtatc 5340tgaaagggcc ggtagccatc atatatatgg taaagaagaa aagagtatag gatctgatga 5400tacaaatatt ttaagtgcac aaaattcaaa taataacttt tcatgtaata atgagaatat 5460gaataaagca aacgttgatg ttaatgtact agaaaatgat acgaaaaaac gagaagatat 5520aaatactaca acagtattta tggaaggtca aaatagtgtt attaataata agaataaaga 5580gaatagttct ttattgaaag gtgatgaaga agatattgtg atggtaaatt taaaaaagga 5640aaataattat aatagtgtaa ttaataatgt agattgtagg aaaaaggata tggatggaaa 5700aaatataaat gatgaatgta aaacatataa gaaaaataaa tataaagata tgggattaaa 5760taataatata gtagatgagt tatccaatgg aacatcacat tcaactaatg atcatttata 5820tttagataat tttaatacat cagatgaaga aatagggaat aataaaaata tggatatgta 5880tttatctaag gaaaaaagta tatctaataa aaaccctggt aattcttatt atgttgtaga 5940ttccgtatat aataatgaat acaaaattaa taagatgaaa gagttaatag ataacgaaaa 6000tttaaatgat gaatataata ataatgttaa tatgaattgt tctaattata ataatgctag 6060tgcatttgta aatggaaagg atagaaatga taatttagaa aatgattgta ttgaaaaaaa 6120tatggatcat acatacaaac attataatcg tttaaacaat cgtagaagta caaatgagag 6180gatgatgctt atggtaaaca atgaaaaaga gagcaatcat gagaagggcc atagaagaaa 6240tggtttaaat aaaaaaaata aagaaaaaaa tatggaaaaa aataagggaa aaaataaaga 6300caaaaagaat tatcattatg ttaatcataa aaggaataat gaatataata gtaacaatat 6360tgaatcgaag tttaataatt atgttgatga tataaataaa aaagaatatt atgaagatga 6420aaatgatata tattatttta cacattcgtc acaaggtaac aatgacgatt taagtaatga 6480taattattta agtagtgaag aattgaatac tgatgagtat gatgatgatt attattatga 6540tgaagatgaa gaagatgact atgacgatga taatgatgat gatgatgatg atgatgatga 6600tggggaggat gaggaggata atgattatta taatgatgat ggttatgata gctataattc 6660tttatcatct tcaagaatat cagatgtatc atctgttata tattcaggga acgaaaatat 6720atttaatgaa aaatataatg atataggttt taaaataatc gataatagga atgaaaaaga 6780gaaagagaaa aagaaatgtt ttattgtatt aggttgtggt tgttatcgta ttggtagttc 6840tgtagaattt gattggagtg ctatacattg tgtaaagacc ataagaaaat taaaccataa 6900agctatatta ataaattgta acccagaaac tgtaagtaca gattatgatg aaagtgatcg 6960tctatatttt gatgaaataa caacagaagt tataaaattt atatataact ttgaaaatag 7020taatggtgtg attatagctt ttggtggaca aacatcaaat aatttagtat ttagtttata 7080taaaaataat gtaaatatat taggatcagt gcacaaagtg ttgattgttg tgaaaatagg 7140aataaatttt cgcacttatg tgattcttaa aattgatcaa ccgaaatgga ataaatttac 7200aaaattatcc aaggctatac aatttgctaa tgaggtaaaa tttcctgtat tagtaagacc 7260atcgtatgta ttatctggtg cagctatgag agttgtaaat tgttttgaag aattaaaaaa 7320ctttttaatg aaggcagcta ttgttagtaa agataatcct gttgtaatat caaaatttat 7380tgagaatgct aaagaaatag aaatagattg tgttagtaaa aatggtaaaa taattaatta 7440tgctatatct gaacatgttg aaaatgctgg tgtacattca ggtgatgcaa cattaatatt 7500acctgcacaa aatatatatg ttgaaacaca taggaaaata aagaaaatat ccgaaaagat 7560ttcaaaatca ttaaatatat ctggtccatt taatatacaa tttatatgtc atcaaaatga 7620aataaaaatt attgaatgta atttaagagc atctagaact tttccattta tatcaaaagc 7680tctaaatcta aactttatag atttagctac aaggatatta atgggttatg acgtcaaacc 7740aattaatata tcattaattg atttagaata tacagctgta aaagcaccga ttttctcatt 7800taatagatta catggatcag attgtatact aggtgtagaa atgaaatcta caggtgaagt 7860agcatgtttt ggtttaaata aatatgaagc tttattaaaa tcattaatag ctacaggtat 7920gaagttaccc aaaaaatcaa tacttataag tattaaaaat ttaaataata aattagcttt 7980tgaagaaccg ttccaattat tatttttaat gggatttaca atatatgcga ctgaaggtac 8040gtatgatttc tactctaaat ttttagaatc ttttaatgtt aataaaggtt ctaaatttca 8100tcaaagactt attaaagttc ataataaaaa tgcagaaaat atatcaccaa atacaacaga 8160tttaattatg aatcataaag ttgaaatggt tattaatata actgatacat taaaaacaaa 8220ggttagttca aatggttata aaattagaag attagcatca gatttccagg ttcctttaat 8280aactaatatg aaactttgtt ctctttttat tgactcatta tatagaaaat tctcaagaca 8340aaaggaaaga aaatcattct ataccataaa gagttatgac gaatatataa gtttggtata 8400agcaagaaat tattcaataa attcgattta acattactta tttatgtatt tattaacttt 8460cattccataa caacatgaaa agtataaata tataaatagt aatatataat atataatata 8520tatatatata tatatatata tatttattta tttaattata tttacgttta aatattaata 8580aatgttttta ttaaatatga tcattaattt atattgattt atttttttat aaatttttgt 8640tatatataca aattttattt attcactcat atgtataaac caaaatggtt ttttcaattt 8700acaaataatt ttataatttt aataaattta ttaattataa aaaaaataaa aatatataaa 8760cattaaaatg tataaattct tttaattata taataattta taaatgttat gattttttta 8820aaaaattcaa cgaaaaaaaa gaggaactgt atatacaaaa gggactatat atatgtatat 8880atatatatat atatatatgt ttttttttcc ttattctaga 8920 2 2391 PRT Artificial Sequence Description of Artificial Sequence protein 2Met Tyr Ile Ser Phe Lys Tyr Asn Leu Tyr Ile Tyr Ile Tyr Ile Tyr 1 5 10 15Ile Tyr Ile Phe Val Leu Ile Asp Phe Lys Thr Val Gly Arg Leu Ile 20 25 30Leu Glu Asp Gly Asn Glu Phe Val Gly Tyr Ser Val Gly Tyr Glu Gly 35 40 45Cys Lys Gly Asn Asn Ser Ile Ser Cys His Lys Glu Tyr Arg Asn Ile 50 55 60Ile Asn Asn Asp Asn Ser Lys Asn Ser Asn Asn Ser Phe Cys Asn Asn 65 70 75 80Glu Glu Asn Asn Leu Lys Asp Asp Leu Leu Tyr Lys Asn Ser Arg Leu 85 90 95Glu Asn Glu Asp Phe Ile Val Thr Gly Glu Val Ile Phe Asn Thr Ala 100 105 110Met Val Gly Tyr Pro Glu Ala Leu Thr Asp Pro Ser Tyr Phe Gly Gln 115 120 125Ile Leu Val Leu Thr Phe Pro Ser Ile Gly Asn Tyr Gly Ile Glu Lys 130 135 140Val Lys His Asp Glu Thr Phe Gly Leu Val Gln Asn Phe Glu Ser Asn145 150 155 160Lys Ile Gln Val Gln Gly Leu Val Ile Cys Glu Tyr Ser Lys Gln Ser 165 170 175Tyr His Tyr Asn Ser Tyr Ile Thr Leu Ser Glu Trp Leu Lys Ile Tyr 180 185 190Lys Ile Pro Cys Ile Gly Gly Ile Asp Thr Arg Ala Leu Thr Lys Leu 195 200 205Leu Arg Glu Lys Gly Ser Met Leu Gly Lys Ile Val Ile Tyr Lys Asn 210 215 220Arg Gln His Ile Asn Lys Leu Tyr Lys Glu Ile Asn Leu Phe Asp Pro225 230 235 240Gly Asn Ile Asp Thr Leu Lys Tyr Val Cys Asn His Phe Ile Arg Val 245 250 255Ile Lys Leu Asn Asn Ile Thr Tyr Asn Tyr Lys Asn Lys Glu Glu Phe 260 265 270Asn Tyr Thr Asn Glu Met Ile Thr Asn Asp Ser Ser Met Glu Asp His 275 280 285Asp Asn Glu Ile Asn Gly Ser Ile Ser Asn Phe Asn Asn Cys Pro Ser 290 295 300Ile Ser Ser Phe Asp Lys Ser Glu Ser Lys Asn Val Ile Asn His Thr305 310 315 320Leu Leu Arg Asp Lys Met Asn Leu Ile Thr Ser Ser Glu Glu Tyr Leu 325 330 335Lys Asp Leu His Asn Cys Asn Phe Ser Asn Ser Ser Asp Lys Asn Asp 340 345 350Ser Phe Phe Lys Leu Tyr Gly Ile Cys Glu Tyr Asp Lys Tyr Leu Ile 355 360 365Asp Leu Glu Glu Asn Ala Ser Phe His Tyr Asn Asn Val Asp Glu Tyr 370 375 380Gly Tyr Tyr Asp Val Asn Lys Asn Thr Asn Ile Leu Ser Asn Asn Lys385 390 395 400Ile Glu Gln Asn Asn Asn Asn Glu Asn Asn Lys Asn Asn Lys Asn Asn 405 410 415Asn Asn Asn Glu Val Asp Tyr Ile Lys Lys Asp Glu Asp Asn Asn Val 420 425 430Asn Ser Lys Val Phe Tyr Ser Gln Tyr Asn Asn Asn Ala Gln Asn Asn 435 440 445Glu His Thr Glu Phe Asn Leu Asn Asn Asp Tyr Ser Thr Tyr Ile Arg 450 455 460Lys Lys Met Lys Asn Glu Glu Phe Leu Asn Leu Val Asn Lys Arg Lys465 470 475 480Val Asp His Lys Glu Lys Ile Ile Val Ile Val Asp Cys Gly Ile Lys 485 490 495Asn Ser Ile Ile Lys Asn Leu Ile Arg His Gly Met Asp Leu Pro Leu 500 505 510Thr Tyr Ile Ile Val Pro Tyr Tyr Tyr Asn Phe Asn His Ile Asp Tyr 515 520 525Asp Ala Val Leu Leu Ser Asn Gly Pro Gly Asp Pro Lys Lys Cys Asp 530 535 540Phe Leu Ile Lys Asn Leu Lys Asp Ser Leu Thr Lys Asn Lys Ile Ile545 550 555 560Phe Gly Ile Cys Leu Gly Asn Gln Leu Leu Gly Ile Ser Leu Gly Cys 565 570 575Asp Thr Tyr Lys Met Lys Tyr Gly Asn Arg Gly Val Asn Gln Pro Val 580 585 590Ile Gln Leu Val Asp Asn Ile Cys Tyr Ile Thr Ser Gln Asn His Gly 595 600 605Tyr Cys Leu Lys Lys Lys Ser Ile Leu Lys Arg Lys Glu Leu Ala Ile 610 615 620Ser Tyr Ile Asn Ala Asn Asp Lys Ser Ile Glu Gly Ile Ser His Lys625 630 635 640Asn Gly Arg Phe Tyr Ser Val Gln Phe His Pro Glu Gly Asn Asn Gly 645 650 655Pro Glu Asp Thr Ser Phe Leu Phe Lys Asn Phe Leu Leu Asp Ile Phe 660 665 670Asn Lys Lys Lys Gln Tyr Arg Glu Tyr Leu Gly Tyr Asn Ile Ile Tyr 675 680 685Ile Lys Lys Lys Val Leu Leu Leu Gly Ser Gly Gly Leu Cys Ile Gly 690 695 700Gln Ala Gly Glu Phe Asp Tyr Ser Gly Thr Gln Ala Ile Lys Ser Leu705 710 715 720Lys Glu Cys Gly Ile Tyr Val Ile Leu Val Asn Pro Asn Ile Ala Thr 725 730 735Val Gln Thr Ser Lys Gly Leu Ala Asp Lys Val Tyr Phe Leu Pro Val 740 745 750Asn Cys Glu Phe Val Glu Lys Ile Ile Lys Lys Glu Lys Pro Asp Phe 755 760 765Ile Leu Cys Thr Phe Gly Gly Gln Thr Ala Leu Asn Cys Ala Leu Met 770 775 780Leu Asp Gln Lys Lys Val Leu Lys Lys Asn Asn Cys Gln Cys Leu Gly785 790 795 800Thr Ser Leu Glu Ser Ile Arg Ile Thr Glu Asn Arg Thr Leu Phe Ala 805 810 815Glu Lys Leu Lys Glu Ile Asn Glu Arg Ile Ala Pro Tyr Gly Ser Ala 820 825 830Lys Asn Val Asn Gln Ala Ile Asp Ile Ala Asn Lys Ile Gly Tyr Pro 835 840 845Ile Leu Val Arg Thr Thr Phe Ser Leu Gly Gly Leu Asn Ser Ser Phe 850 855 860Ile Asn Asn Glu Glu Glu Leu Ile Glu Lys Cys Asn Lys Ile Phe Leu865 870 875 880Gln Thr Asp Asn Glu Ile Phe Ile Asp Lys Ser Leu Gln Gly Trp Lys 885 890 895Glu Ile Glu Tyr Glu Leu Leu Arg Asp Asn Lys Asn Asn Cys Ile Ala 900 905 910Ile Cys Asn Met Glu Asn Ile Asp Pro Leu Gly Ile His Thr Gly Asp 915 920 925Ser Ile Val Val Ala Pro Ser Gln Thr Leu Ser Asn Tyr Glu Tyr Tyr 930 935 940Lys Phe Arg Glu Ile Ala Leu Lys Val Ile Thr His Leu Asn Ile Ile945 950 955 960Gly Glu Cys Asn Ile Gln Phe Gly Ile Asn Pro Gln Thr Gly Glu Tyr 965 970 975Cys Ile Ile Glu Val Asn Ala Arg Leu Ser Arg Ser Ser Ala Leu Ala 980 985 990Ser Lys Ala Thr Gly Tyr Pro Leu Ala Tyr Ile Ser Ala Lys Ile Ala 995 1000 1005Leu Gly Tyr Asp Leu Ile Ser Leu Lys Asn Ser Ile Thr Lys Lys Thr 1010 1015 1020Thr Ala Cys Phe Glu Pro Ser Leu Asp Tyr Ile Thr Thr Lys Ile Pro1025 1030 1035 1040Arg Trp Asp Leu Asn Lys Phe Glu Phe Ala Ser Asn Thr Met Asn Ser 1045 1050 1055Ser Met Lys Ser Val Gly Glu Val Met Ser Ile Gly Arg Thr Phe Glu 1060 1065 1070Glu Ser Ile Gln Lys Ser Leu Arg Cys Ile Asp Asp Asn Tyr Leu Gly 1075 1080 1085Phe Ser Asn Thr Tyr Cys Ile Asp Trp Asp Glu Lys Lys Ile Ile Glu 1090 1095 1100Glu Leu Lys Asn Pro Ser Pro Lys Arg Ile Asp Ala Ile His Gln Ala1105 1110 1115 1120Phe His Leu Asn Met Pro Met Asp Lys Ile His Glu Leu Thr His Ile 1125 1130 1135Asp Tyr Trp Phe Leu His Lys Phe Tyr Asn Ile Tyr Asn Leu Gln Asn 1140 1145 1150Lys Leu Lys Thr Leu Lys Leu Glu Gln Leu Ser Phe Asn Asp Leu Lys 1155 1160 1165Tyr Phe Lys Lys His Gly Phe Ser Asp Lys Gln Ile Ala His Tyr Leu 1170 1175 1180Ser Phe Asn Thr Ser Asp Asn Asn Asn Asn Asn Asn Asn Ile Ser Ser1185 1190 1195 1200Cys Arg Val Thr Glu Asn Asp Val Met Lys Tyr Arg Glu Lys Leu Gly 1205 1210 1215Leu Phe Pro His Ile Lys Val Ile Asp Thr Leu Ser Ala Glu Phe Pro 1220 1225 1230Ala Leu Thr Asn Tyr Leu Tyr Leu Thr Tyr Gln Gly Gln Glu His Asp 1235 1240 1245Val Leu Pro Leu Asn Met Lys Arg Lys Lys Ile Cys Thr Leu Asn Asn 1250 1255 1260Lys Arg Asn Ala Asn Lys Lys Lys Val His Val Lys Asn His Leu Tyr1265 1270 1275 1280Asn Glu Val Val Asp Asp Lys Asp Thr Gln Leu His Lys Glu Asn Asn 1285 1290 1295Asn Asn Asn Asn Met Asn Ser Gly Asn Val Glu Asn Lys Cys Lys Leu 1300 1305 1310Asn Lys Glu Ser Tyr Gly Tyr Asn Asn Ser Ser Asn Cys Ile Asn Thr 1315 1320 1325Asn Asn Ile Asn Ile Glu Asn Asn Ile Cys His Asp Ile Ser Ile Asn 1330 1335 1340Lys Asn Ile Lys Val Thr Ile Asn Asn Ser Asn Asn Ser Ile Ser Asn1345 1350 1355 1360Asn Glu Asn Val Glu Thr Asn Leu Asn Cys Val Ser Glu Arg Ala Gly 1365 1370 1375Ser His His Ile Tyr Gly Lys Glu Glu Lys Ser Ile Gly Ser Asp Asp 1380 1385 1390Thr Asn Ile Leu Ser Ala Gln Asn Ser Asn Asn Asn Phe Ser Cys Asn 1395 1400 1405Asn Glu Asn Met Asn Lys Ala Asn Val Asp Val Asn Val Leu Glu Asn 1410 1415 1420Asp Thr Lys Lys Arg Glu Asp Ile Asn Thr Thr Thr Val Phe Met Glu1425 1430 1435 1440Gly Gln Asn Ser Val Ile Asn Asn Lys Asn Lys Glu Asn Ser Ser Leu 1445 1450 1455Leu Lys Gly Asp Glu Glu Asp Ile Val Met Val Asn Leu Lys Lys Glu 1460 1465 1470Asn Asn Tyr Asn Ser Val Ile Asn Asn Val Asp Cys Arg Lys Lys Asp 1475 1480 1485Met Asp Gly Lys Asn Ile Asn Asp Glu Cys Lys Thr Tyr Lys Lys Asn 1490 1495 1500Lys Tyr Lys Asp Met Gly Leu Asn Asn Asn Ile Val Asp Glu Leu Ser1505 1510 1515 1520Asn Gly Thr Ser His Ser Thr Asn Asp His Leu Tyr Leu Asp Asn Phe 1525 1530 1535Asn Thr Ser Asp Glu Glu Ile Gly Asn Asn Lys Asn Met Asp Met Tyr 1540 1545 1550Leu Ser Lys Glu Lys Ser Ile Ser Asn Lys Asn Pro Gly Asn Ser Tyr 1555 1560 1565Tyr Val Val Asp Ser Val Tyr Asn Asn Glu Tyr Lys Ile Asn Lys Met 1570 1575 1580Lys Glu Leu Ile Asp Asn Glu Asn Leu Asn Asp Glu Tyr Asn Asn Asn1585 1590 1595 1600Val Asn Met Asn Cys Ser Asn Tyr Asn Asn Ala Ser Ala Phe Val Asn 1605 1610 1615Gly Lys Asp Arg Asn Asp Asn Leu Glu Asn Asp Cys Ile Glu Lys Asn 1620 1625 1630Met Asp His Thr Tyr Lys His Tyr Asn Arg Leu Asn Asn Arg Arg Ser 1635 1640 1645Thr Asn Glu Arg Met Met Leu Met Val Asn Asn Glu Lys Glu Ser Asn 1650 1655 1660His Glu Lys Gly His Arg Arg Asn Gly Leu Asn Lys Lys Asn Lys Glu1665 1670 1675 1680Lys Asn Met Glu Lys Asn Lys Gly Lys Asn Lys Asp Lys Lys Asn Tyr 1685 1690 1695His Tyr Val Asn His Lys Arg Asn Asn Glu Tyr Asn Ser Asn Asn Ile 1700 1705 1710Glu Ser Lys Phe Asn Asn Tyr Val Asp Asp Ile Asn Lys Lys Glu Tyr 1715 1720 1725Tyr Glu Asp Glu Asn Asp Ile Tyr Tyr Phe Thr His Ser Ser Gln Gly 1730 1735 1740Asn Asn Asp Asp Leu Ser Asn Asp Asn Tyr Leu Ser Ser Glu Glu Leu1745 1750 1755 1760Asn Thr Asp Glu Tyr Asp Asp Asp Tyr Tyr Tyr Asp Glu Asp Glu Glu 1765 1770 1775Asp Asp Tyr Asp Asp Asp Asn Asp Asp Asp Asp Asp Asp Asp Asp Asp 1780 1785 1790Gly Glu Asp Glu Glu Asp Asn Asp Tyr Tyr Asn Asp Asp Gly Tyr Asp 1795 1800 1805Ser Tyr Asn Ser Leu Ser Ser Ser Arg Ile Ser Asp Val Ser Ser Val 1810 1815 1820Ile Tyr Ser Gly Asn Glu Asn Ile Phe Asn Glu Lys Tyr Asn Asp Ile1825 1830 1835 1840Gly Phe Lys Ile Ile Asp Asn Arg Asn Glu Lys Glu Lys Glu Lys Lys 1845 1850 1855Lys Cys Phe Ile Val Leu Gly Cys Gly Cys Tyr Arg Ile Gly Ser Ser 1860 1865 1870Val Glu Phe Asp Trp Ser Ala Ile His Cys Val Lys Thr Ile Arg Lys 1875 1880 1885Leu Asn His Lys Ala Ile Leu Ile Asn Cys Asn Pro Glu Thr Val Ser 1890 1895 1900Thr Asp Tyr Asp Glu Ser Asp Arg Leu Tyr Phe Asp Glu Ile Thr Thr1905 1910 1915 1920Glu Val Ile Lys Phe Ile Tyr Asn Phe Glu Asn Ser Asn Gly Val Ile 1925 1930 1935Ile Ala Phe Gly Gly Gln Thr Ser Asn Asn Leu Val Phe Ser Leu Tyr 1940 1945 1950Lys Asn Asn Val Asn Ile Leu Gly Ser Val His Lys Val Leu Ile Val 1955 1960 1965Val Lys Ile Gly Ile Asn Phe Arg Thr Tyr Val Ile Leu Lys Ile Asp 1970 1975 1980Gln Pro Lys Trp Asn Lys Phe Thr Lys Leu Ser Lys Ala Ile Gln Phe1985 1990 1995 2000Ala Asn Glu Val Lys Phe Pro Val Leu Val Arg Pro Ser Tyr Val Leu 2005 2010 2015Ser Gly Ala Ala Met Arg Val Val Asn Cys Phe Glu Glu Leu Lys Asn 2020 2025 2030Phe Leu Met Lys Ala Ala Ile Val Ser Lys Asp Asn Pro Val Val Ile 2035 2040 2045Ser Lys Phe Ile Glu Asn Ala Lys Glu Ile Glu Ile Asp Cys Val Ser 2050 2055 2060Lys Asn Gly Lys Ile Ile Asn Tyr Ala Ile Ser Glu His Val Glu Asn2065 2070 2075 2080Ala Gly Val His Ser Gly Asp Ala Thr Leu Ile Leu Pro Ala Gln Asn 2085 2090 2095Ile Tyr Val Glu Thr His Arg Lys Ile Lys Lys Ile Ser Glu Lys Ile 2100 2105 2110Ser Lys Ser Leu Asn Ile Ser Gly Pro Phe Asn Ile Gln Phe Ile Cys 2115 2120 2125His Gln Asn Glu Ile Lys Ile Ile Glu Cys Asn Leu Arg Ala Ser Arg 2130 2135 2140Thr Phe Pro Phe Ile Ser Lys Ala Leu Asn Leu Asn Phe Ile Asp Leu2145 2150 2155 2160Ala Thr Arg Ile Leu Met Gly Tyr Asp Val Lys Pro Ile Asn Ile Ser 2165 2170 2175Leu Ile Asp Leu Glu Tyr Thr Ala Val Lys Ala Pro Ile Phe Ser Phe 2180 2185 2190Asn Arg Leu His Gly Ser Asp Cys Ile Leu Gly Val Glu Met Lys Ser 2195 2200 2205Thr Gly Glu Val Ala Cys Phe Gly Leu Asn Lys Tyr Glu Ala Leu Leu 2210 2215 2220Lys Ser Leu Ile Ala Thr Gly Met Lys Leu Pro Lys Lys Ser Ile Leu2225 2230 2235 2240Ile Ser Ile Lys Asn Leu Asn Asn Lys Leu Ala Phe Glu Glu Pro Phe 2245 2250 2255Gln Leu Leu Phe Leu Met Gly Phe Thr Ile Tyr Ala Thr Glu Gly Thr 2260 2265 2270Tyr Asp Phe Tyr Ser Lys Phe Leu Glu Ser Phe Asn Val Asn Lys Gly 2275 2280 2285Ser Lys Phe His Gln Arg Leu Ile Lys Val His Asn Lys Asn Ala Glu 2290 2295 2300Asn Ile Ser Pro Asn Thr Thr Asp Leu Ile Met Asn His Lys Val Glu2305 2310 2315 2320Met Val Ile Asn Ile Thr Asp Thr Leu Lys Thr Lys Val Ser Ser Asn 2325 2330 2335Gly Tyr Lys Ile Arg Arg Leu Ala Ser Asp Phe Gln Val Pro Leu Ile 2340 2345 2350Thr Asn Met Lys Leu Cys Ser Leu Phe Ile Asp Ser Leu Tyr Arg Lys 2355 2360 2365Phe Ser Arg Gln Lys Glu Arg Lys Ser Phe Tyr Thr Ile Lys Ser Tyr 2370 2375 2380Asp Glu Tyr Ile Ser Leu Val2385 2390 3 21 DNA Plasmodium falciparum 3ttttctaatc gactattttt a 21 4 21 DNA Plasmodium falciparum 4gacatacttg gacaattatt a 21 5 21 DNA Plasmodium falciparum 5ccttactatt gacattatta t 21 6 21 DNA Plasmodium falciparum 6ggctataaaa gaccttacta t 21 7 21 DNA Plasmodium falciparum 7ggatctccag gaccattaga t 21 8 21 DNA Plasmodium falciparum 8cctaaacatt gacaattatt c 21 9 21 DNA Plasmodium falciparum 9tcatgttctt gaccttgata a 21 10 21 DNA Plasmodium falciparum 10gatatatcat gacaaatatt a 21 11 21 DNA Plasmodium falciparum 11gttaccttgt gacgaatgtg t 21 12 21 DNA Plasmodium falciparum 12tcattttgat gacatataaa t 21 13 29 DNA Plasmodium falciparum 13aacatcatgt tcttgacctt gataagtta 29 14 21 DNA Plasmodium falciparum 14cgtcggaact gaatttggct c 21 15 21 DNA Plasmodium falciparum 15cctcagtgct gacagcccat c 21

Claims

1. 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 shown in SEQ ID NO:1.

2. The ribozyme as claimed in claim 1 in which the ribozyme includes sequences complementary to portions of mRNA obtained from inserted sequence one or two of the nucleic acid molecule as shown by nucleotides 1976-2671 or 4988-6796 of SEQ. ID. NO: 1.

3. An antisense oligonucleotide capable of blocking expression of the nucleic acid molecule as shown in SEQ ID No:1.

4. A polynucleotide construct which produces in a cell the ribozyme as claimed in claim 1 or the antisense oligonucleotide as claimed in claim 3.