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:
This application is a continuation-in-part of application Ser. No. 08/446,885, filed Jul. 6, 1995, now U.S. Pat. No. 5,849,573. 
    
    
     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&#39;s population—approximately 500 million people—are thought to live in malaria areas (Miller, 1989). It ranks eighth in the World Health Organization&#39;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&#39; 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&#39;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&#39;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&#39;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&amp;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 &amp; 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. &amp; Gerlach, W. L. (1988) Nature 334. 585; Kruger, K., Grabowski, P. J., Zaug, A. J., Sands, J., Gottschling, D. E. &amp; 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. 
     REFERENCES 
     Cox, F. E. G. (1991) Malaria vaccines: while we are waiting. Parasitology Today 7: 189-190 
     Gero, A. M. and O&#39;Sullivan, W. J. (199)) Purines and pyrimidines in malarial parasites. Blood Cells 16: 467-498 
     Hammond, D. J. Burchell, J. R. and Pudney, M. (1965) Inhibition of pyrimidine biosynthesis de novo in Plasmodium falciparum by 2.(4.t-butylcyclohexyl)-3-hydroxy-1, 4-naphthoquinine in vitro. Mol. Biochem. Parasitol 14: 97-109 
     Haseloff, J. And Gerlach, W. L. (1988) Simple RNA enzymes with new and highly specific endoribonuclease activity. Nature 334:585-591 
     Hill, B., Kilsby, J. Rogerson, G. W., McIntosh, R T. and Ginger, C. D. (1981). The Enzymes of pyrimidine biosynthesis in a range of parasitic protozoa and helminths. Mol. Biochem. Parasitol. Z: 123-134. 
     Johnson, C. Malaria back to plague us. Sydney Morning Herald, Nov. 13, 1991. 
     Jones, M. E. (1980) Pyrimidine nucleotide biosynthesis in animals: genes, enzymes and regulation of UMP biosynthesis. Annu. Rev. Biochem. 49: 253-279. 
     Krungkrai, J. Cerami, A. and Henderson, G. B. (1990) Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata. Biochemistry 29: 6270-6275. 
     Krungkrai, J. Krungkrai, S. R. and Phakanont, K. (1992) Antimalarial activity of orotate analogs that inhibit dihydrootase and dihydroorotate dehydrogenase. Biochem. Pharmacol. 43: 1295-1301. 
     Marshal, E. (1991) Malaria parasite gaining ground against science. Science 2: 190, 
     Nyunoya, H., Broglie, K. E., Widgren, W. E. and Lusty C. J (1985) Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat. J. Biol. Chem. 260: 9346-9356. 
     Prapunwattana, P., O&#39;Sullivan, W. J. and Yuthavong, Y. (1988) Depression of Plasmodium falciparum dihydroorotate dehydrogenase activity in in vitro culture by tetracycline. Mol. Biochemi. 27: 119-124. 
     Queen, S. A., Vander Jagt, D. L. and Reyes, P. (1990) In vitro susceptibilities of Plasmodium falciparum to compounds which inhibit nucleotide metabolism. Antimicrob. Agents Chemother. 34: 1393-1398. 
     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. 
     Rubino S. D., Nyunoya, H. and Lusty, C. J. (1986) JBC 261(24):11320-11327. 
     Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis K. B. and Erlich H. A. (1958) Science 239:487-491, 
     Scott, H. V., Gero, A. M. and O&#39;Sullivan, W. J. (1986) In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo. Mol Biochem. Parasitol. 18: 3-15. 
     Sherman, I. W. (1979) Biochemistry of Plasmodium (malarial parasites) Microbiol. Rev. 43: 453-495. 
     Simmer, J. P., Kelly, R. E., Rinker, Jr., A. G., Scully, J. L. and Evans D. R. (1990) Mammalian carbamyl phosphate synthetase (CPS), J. Biol. Chem 265: 10395-10402. 
     Simmer, J. P., Kelly, R. E., Austin, G. R., Jr., Scully, J. L. and Evans, D. R. (1990) JBC 285(18):10395-10402. 
     Souciet, J. L., Nagy, M., Le Gouar, M., Lacroute, F. and Potier, S. (1989) Gene (Amst.) 79: 59-70. 
     Triglia, T., Peterson, M. G. and Kemp, D. J. (1988) PNAS 16:8186. 
     Werner, M., Feller, A. and Pierard, A. (1985) Nucleotide sequence of yeast genie CPA1 encoding the small subunit of arginine-pathway carbamoyl-phosphate synthetase. Eur. J. Biochem. 146: 371-381. 
     
       
         
           
             15 
           
           
             1 
             8920 
             DNA 
             Plasmodium falciparum 
           
            1
gaattccttc agccaaaaaa aatgacaacg caaattttaa gaaaagaaaa acaatcgact     60
cgtctttgaa tgaggttaga aattcgatac gtgaaaggga cttaagaagg cttaacagag    120
aaaagagtaa aatcttataa gcatttgaag gaaaaaataa taaaataaaa aaataaaaag    180
ataaaaaata tttatatttg atatgtagta tatataatga ttattcatat taataacata    240
gataaaaaac tttttttttt tttttttttc tttatattta ttaacaatac atttaagtta    300
ttttatatat atatatatat atatatatat atatatatat atatatgttt gtgtgttcat    360
ttgtttataa aattacttga aatataaaac ttattaatat atttccaatt aatatgaata    420
caattattaa tattttgatg tgtacacatt aatatagttt tacacttctt ataataaaac    480
catcctatat attatacaca atatataata ctccccaata ttgtggttcc tataatttta    540
tttatatatt tatttattaa tttattcatt tatttatttt ttttcttagt ttataaaata    600
gtaattctac taatttaaaa aaaaaaaaaa aaaaaaaaaa aaaaagaaaa aaaaaaaatt    660
tacatatgaa aaatgaactt gtatatgtaa atttataaat attttaaaca taaatataaa    720
tgtataaaaa aaaaaaagaa aaatgggaaa aaataatata gatatatata taaatatata    780
tatatatata attattgggg atattctctg aatcataggt cttaaacagt tttattcttt    840
taacatcaca aagttgttat taaaagtata tatatcttat tggttcctat ataaaactat    900
agtattctat aatatattct gtatatttca ttttatcatt tgtaagcaat ccctatttat    960
tataattatt attttttttt ttataaaaga ggtataaaac agtttattca atttttttcc   1020
taaaggagca accttcagtc aatttacatt ttccaccggt tggttggcac aacataatgt   1080
tacagctaaa aaaagaaaga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa atatatatat   1140
atatatatat atatacataa tatgtacaat gctaccatac aagtatataa atttttcaac   1200
attgttgtga tgttgcattt ttcttatgta tatttctttt aaatataatt tatatatata   1260
tatatatata tatatatata tatttgttct tatagatttt aaaacagttg ggaggttaat   1320
tcttgaagat ggtaacgaat ttgtagggta cagtgtaggt tacgaagggt gtaaaggaaa   1380
taatagtata tcatgtcata aggagtatag aaatattatt aataatgata atagcaagaa   1440
tagtaataat tcattttgta ataatgaaga aaacaatttg aaagatgatt tattatataa   1500
aaatagtcga ttagaaaatg aagattttat tgttacaggt gaagttatat ttaatacagc   1560
tatggttgga tatcctgaag ctttaacgga cccaagttat tttggtcaaa tattagtttt   1620
aacatttcct tctattggta attatggtat tgaaaaagta aaacatgatg aaacgtttgg   1680
attagtacaa aattttgaaa gtaataaaat tcaagtacaa ggtttagtta tttgtgaata   1740
ttcgaagcaa tcatatcatt acaattctta tattacctta agtgaatggt taaagattta   1800
taaaattcca tgtataggtg gtatagatac aagagcctta acaaaacttt taagagaaaa   1860
aggtagtatg ttaggtaaaa tagttatata taaaaacaga caacatatta ataaattata   1920
taaagaaatt aatctttttg atcctggtaa tatagatact ctaaaatatg tatgtaatca   1980
ttttatacgt gttattaagt tgaataatat tacatataat tataaaaata aggaagaatt   2040
taattatacc aatgaaatga ttactaatga ttcttcaatg gaagatcatg ataatgaaat   2100
taatggtagt atttctaatt ttaataattg tccaagtatc tctagttttg ataaaagtga   2160
atcgaaaaat gttattaatc atacattgtt aagagataaa atgaacctaa taacttcatc   2220
tgaagaatat ctgaaagatc ttcataattg taattttagt aatagtagtg ataaaaatga   2280
ttcttttttt aagttatatg gtatatgtga atatgataaa tatttaattg accttgaaga   2340
aaatgctagc tttcattata ataatgtaga tgaatatgga tattatgatg ttaataaaaa   2400
tacaaatatt ctatctaata ataaaataga acaaaacaac aataacgaaa ataacaaaaa   2460
taacaaaaat aacaacaata acgaggttga ttatataaag aaagatgagg ataataatgt   2520
caatagtaag gtcttttata gccaatataa taataatgca caaaataatg aacataccga   2580
atttaattta aataatgatt attctactta tattagaaag aaaatgaaaa atgaagaatt   2640
ccttaatttg gtaaacaaaa gaaaagtaga ccataaagaa aaaattattg ttattgttga   2700
ttgtggtatt aaaaatagta taatcaaaaa tttaataaga cacggtatgg atcttccatt   2760
aacatatatt attgtacctt attattacaa ttttaatcat atagattatg atgcagttct   2820
tttatctaat ggtcctggag atcctaaaaa gtgtgatttc cttataaaaa atttgaaaga   2880
tagtttaaca aaaaataaaa ttatatttgg tatttgttta ggtaatcaac tattaggtat   2940
atcattaggt tgtgacacat ataaaatgaa atatggtaat agaggtgtta atcaacccgt   3000
aatacaatta gtagataata tatgttacat tacctcacaa aatcatggat actgtttaaa   3060
gaaaaaatca attttaaaaa gaaaagagct tgcgattagt tatataaatg ctaatgataa   3120
atctatagaa ggtatttcac ataaaaatgg aagattttat agtgtccagt ttcatcctga   3180
gggtaataat ggtcctgaag atacatcatt tttatttaag aattttcttt tagatatctt   3240
taataagaaa aaacaatata gagaatattt aggatataat attatttata taaaaaagaa   3300
agtgcttctt ttaggtagtg gtggtttatg tataggacaa gcaggagaat tcgattattc   3360
aggaacacaa gcaattaaaa gtttaaaaga atgtggtata tatgttatat tagttaatcc   3420
taacatagca actgttcaaa catcaaaagg tttggcagat aaggtatact ttttaccagt   3480
taattgtgaa tttgtagaaa aaattattaa aaaggaaaaa cctgatttta ttttatgtac   3540
atttggtggt cagacagctt taaattgtgc tttaatgtta gatcaaaaaa aagtattgaa   3600
aaagaataat tgtcaatgtt taggtacatc tttagaatct ataagaataa cagaaaatag   3660
aacattattt gctgaaaaat taaaagaaat taatgaaaga atagctccat atggtagtgc   3720
aaaaaatgtt aatcaagcta ttgatatagc taataaaata ggatatccaa tattagtacg   3780
tacaacattt tcgttaggag gattaaatag tagtttcata aataatgaag aagaacttat   3840
cgaaaaatgt aataaaatat ttttacaaac tgataatgaa atatttatag ataaatcatt   3900
acaaggatgg aaagaaatag aatatgaatt attaagagat aataaaaata attgtatagc   3960
tatatgtaat atggaaaata tagatccatt aggtatacat acaggagata gtatagttgt   4020
tgcaccttca caaacattaa gtaattatga atattataaa tttagagaaa tagcattaaa   4080
ggtaattaca catttaaata ttataggaga atgtaatata caatttggta taaatccaca   4140
aacaggagaa tattgtatta ttgaagttaa tgctaggctt agtagaagtt cagcattagc   4200
ttctaaagct actggttatc cacttgctta tatatcagca aaaatagcct tgggatatga   4260
tttgataagt ttaaaaaata gcataactaa aaaaacaact gcctgttttg aaccctctct   4320
agattacatt acaacaaaaa taccacgatg ggatttaaat aaatttgagt ttgcttctaa   4380
tacaatgaat agtagtatga aaagtgtagg agaagttatg tctataggta gaacctttga   4440
agaatctata caaaaatctt taagatgtat tgatgataat tatttaggat ttagtaatac   4500
gtattgtata gattgggatg aaaagaaaat tattgaagaa ttaaaaaatc catcaccaaa   4560
aagaattgat gctatacatc aagctttcca tttaaatatg cctatggata aaatacatga   4620
gctgacacat attgattatt ggttcttaca taaattttat aatatatata atttacaaaa   4680
taagttgaaa acgttaaaat tagagcaatt atcttttaat gatttgaagt attttaagaa   4740
gcatggtttt agtgataagc aaatagctca ctacttatcc ttcaacacaa gcgataataa   4800
taataataat aataatatta gctcatgtag ggttacagaa aatgatgtta tgaaatatag   4860
agaaaagcta ggattatttc cacatattaa agttattgat accttatcag ccgaatttcc   4920
ggctttaact aattatttat atttaactta tcaaggtcaa gaacatgatg ttctcccatt   4980
aaatatgaaa aggaaaaaga tatgcacgct taataataaa cgaaatgcaa ataagaaaaa   5040
agtccatgtc aagaaccact tatataatga agtagttgat gataaggata cacaattaca   5100
caaagaaaat aataataata ataatatgaa ttctggaaat gtagaaaata aatgtaaatt   5160
gaataaagaa tcctatggct ataataattc ttctaattgt atcaatacaa ataatattaa   5220
tatagaaaat aatatttgtc atgatatatc tataaacaaa aatataaaag ttacaataaa   5280
caattccaat aattctatat cgaataatga aaatgttgaa acaaacttaa attgtgtatc   5340
tgaaagggcc ggtagccatc atatatatgg taaagaagaa aagagtatag gatctgatga   5400
tacaaatatt ttaagtgcac aaaattcaaa taataacttt tcatgtaata atgagaatat   5460
gaataaagca aacgttgatg ttaatgtact agaaaatgat acgaaaaaac gagaagatat   5520
aaatactaca acagtattta tggaaggtca aaatagtgtt attaataata agaataaaga   5580
gaatagttct ttattgaaag gtgatgaaga agatattgtg atggtaaatt taaaaaagga   5640
aaataattat aatagtgtaa ttaataatgt agattgtagg aaaaaggata tggatggaaa   5700
aaatataaat gatgaatgta aaacatataa gaaaaataaa tataaagata tgggattaaa   5760
taataatata gtagatgagt tatccaatgg aacatcacat tcaactaatg atcatttata   5820
tttagataat tttaatacat cagatgaaga aatagggaat aataaaaata tggatatgta   5880
tttatctaag gaaaaaagta tatctaataa aaaccctggt aattcttatt atgttgtaga   5940
ttccgtatat aataatgaat acaaaattaa taagatgaaa gagttaatag ataacgaaaa   6000
tttaaatgat gaatataata ataatgttaa tatgaattgt tctaattata ataatgctag   6060
tgcatttgta aatggaaagg atagaaatga taatttagaa aatgattgta ttgaaaaaaa   6120
tatggatcat acatacaaac attataatcg tttaaacaat cgtagaagta caaatgagag   6180
gatgatgctt atggtaaaca atgaaaaaga gagcaatcat gagaagggcc atagaagaaa   6240
tggtttaaat aaaaaaaata aagaaaaaaa tatggaaaaa aataagggaa aaaataaaga   6300
caaaaagaat tatcattatg ttaatcataa aaggaataat gaatataata gtaacaatat   6360
tgaatcgaag tttaataatt atgttgatga tataaataaa aaagaatatt atgaagatga   6420
aaatgatata tattatttta cacattcgtc acaaggtaac aatgacgatt taagtaatga   6480
taattattta agtagtgaag aattgaatac tgatgagtat gatgatgatt attattatga   6540
tgaagatgaa gaagatgact atgacgatga taatgatgat gatgatgatg atgatgatga   6600
tggggaggat gaggaggata atgattatta taatgatgat ggttatgata gctataattc   6660
tttatcatct tcaagaatat cagatgtatc atctgttata tattcaggga acgaaaatat   6720
atttaatgaa aaatataatg atataggttt taaaataatc gataatagga atgaaaaaga   6780
gaaagagaaa aagaaatgtt ttattgtatt aggttgtggt tgttatcgta ttggtagttc   6840
tgtagaattt gattggagtg ctatacattg tgtaaagacc ataagaaaat taaaccataa   6900
agctatatta ataaattgta acccagaaac tgtaagtaca gattatgatg aaagtgatcg   6960
tctatatttt gatgaaataa caacagaagt tataaaattt atatataact ttgaaaatag   7020
taatggtgtg attatagctt ttggtggaca aacatcaaat aatttagtat ttagtttata   7080
taaaaataat gtaaatatat taggatcagt gcacaaagtg ttgattgttg tgaaaatagg   7140
aataaatttt cgcacttatg tgattcttaa aattgatcaa ccgaaatgga ataaatttac   7200
aaaattatcc aaggctatac aatttgctaa tgaggtaaaa tttcctgtat tagtaagacc   7260
atcgtatgta ttatctggtg cagctatgag agttgtaaat tgttttgaag aattaaaaaa   7320
ctttttaatg aaggcagcta ttgttagtaa agataatcct gttgtaatat caaaatttat   7380
tgagaatgct aaagaaatag aaatagattg tgttagtaaa aatggtaaaa taattaatta   7440
tgctatatct gaacatgttg aaaatgctgg tgtacattca ggtgatgcaa cattaatatt   7500
acctgcacaa aatatatatg ttgaaacaca taggaaaata aagaaaatat ccgaaaagat   7560
ttcaaaatca ttaaatatat ctggtccatt taatatacaa tttatatgtc atcaaaatga   7620
aataaaaatt attgaatgta atttaagagc atctagaact tttccattta tatcaaaagc   7680
tctaaatcta aactttatag atttagctac aaggatatta atgggttatg acgtcaaacc   7740
aattaatata tcattaattg atttagaata tacagctgta aaagcaccga ttttctcatt   7800
taatagatta catggatcag attgtatact aggtgtagaa atgaaatcta caggtgaagt   7860
agcatgtttt ggtttaaata aatatgaagc tttattaaaa tcattaatag ctacaggtat   7920
gaagttaccc aaaaaatcaa tacttataag tattaaaaat ttaaataata aattagcttt   7980
tgaagaaccg ttccaattat tatttttaat gggatttaca atatatgcga ctgaaggtac   8040
gtatgatttc tactctaaat ttttagaatc ttttaatgtt aataaaggtt ctaaatttca   8100
tcaaagactt attaaagttc ataataaaaa tgcagaaaat atatcaccaa atacaacaga   8160
tttaattatg aatcataaag ttgaaatggt tattaatata actgatacat taaaaacaaa   8220
ggttagttca aatggttata aaattagaag attagcatca gatttccagg ttcctttaat   8280
aactaatatg aaactttgtt ctctttttat tgactcatta tatagaaaat tctcaagaca   8340
aaaggaaaga aaatcattct ataccataaa gagttatgac gaatatataa gtttggtata   8400
agcaagaaat tattcaataa attcgattta acattactta tttatgtatt tattaacttt   8460
cattccataa caacatgaaa agtataaata tataaatagt aatatataat atataatata   8520
tatatatata tatatatata tatttattta tttaattata tttacgttta aatattaata   8580
aatgttttta ttaaatatga tcattaattt atattgattt atttttttat aaatttttgt   8640
tatatataca aattttattt attcactcat atgtataaac caaaatggtt ttttcaattt   8700
acaaataatt ttataatttt aataaattta ttaattataa aaaaaataaa aatatataaa   8760
cattaaaatg tataaattct tttaattata taataattta taaatgttat gattttttta   8820
aaaaattcaa cgaaaaaaaa gaggaactgt atatacaaaa gggactatat atatgtatat   8880
atatatatat atatatatgt ttttttttcc ttattctaga                         8920 
           
             2 
             2391 
             PRT 
             Artificial Sequence 
             
               Description of Artificial Sequence protein 
             
           
            2
Met Tyr Ile Ser Phe Lys Tyr Asn Leu Tyr Ile Tyr Ile Tyr Ile Tyr
  1               5                  10                  15
Ile Tyr Ile Phe Val Leu Ile Asp Phe Lys Thr Val Gly Arg Leu Ile
             20                  25                  30
Leu Glu Asp Gly Asn Glu Phe Val Gly Tyr Ser Val Gly Tyr Glu Gly
         35                  40                  45
Cys Lys Gly Asn Asn Ser Ile Ser Cys His Lys Glu Tyr Arg Asn Ile
     50                  55                  60
Ile Asn Asn Asp Asn Ser Lys Asn Ser Asn Asn Ser Phe Cys Asn Asn
 65                  70                  75                  80
Glu Glu Asn Asn Leu Lys Asp Asp Leu Leu Tyr Lys Asn Ser Arg Leu
                 85                  90                  95
Glu Asn Glu Asp Phe Ile Val Thr Gly Glu Val Ile Phe Asn Thr Ala
            100                 105                 110
Met Val Gly Tyr Pro Glu Ala Leu Thr Asp Pro Ser Tyr Phe Gly Gln
        115                 120                 125
Ile Leu Val Leu Thr Phe Pro Ser Ile Gly Asn Tyr Gly Ile Glu Lys
    130                 135                 140
Val Lys His Asp Glu Thr Phe Gly Leu Val Gln Asn Phe Glu Ser Asn
145                 150                 155                 160
Lys Ile Gln Val Gln Gly Leu Val Ile Cys Glu Tyr Ser Lys Gln Ser
                165                 170                 175
Tyr His Tyr Asn Ser Tyr Ile Thr Leu Ser Glu Trp Leu Lys Ile Tyr
            180                 185                 190
Lys Ile Pro Cys Ile Gly Gly Ile Asp Thr Arg Ala Leu Thr Lys Leu
        195                 200                 205
Leu Arg Glu Lys Gly Ser Met Leu Gly Lys Ile Val Ile Tyr Lys Asn
    210                 215                 220
Arg Gln His Ile Asn Lys Leu Tyr Lys Glu Ile Asn Leu Phe Asp Pro
225                 230                 235                 240
Gly Asn Ile Asp Thr Leu Lys Tyr Val Cys Asn His Phe Ile Arg Val
                245                 250                 255
Ile Lys Leu Asn Asn Ile Thr Tyr Asn Tyr Lys Asn Lys Glu Glu Phe
            260                 265                 270
Asn Tyr Thr Asn Glu Met Ile Thr Asn Asp Ser Ser Met Glu Asp His
        275                 280                 285
Asp Asn Glu Ile Asn Gly Ser Ile Ser Asn Phe Asn Asn Cys Pro Ser
    290                 295                 300
Ile Ser Ser Phe Asp Lys Ser Glu Ser Lys Asn Val Ile Asn His Thr
305                 310                 315                 320
Leu Leu Arg Asp Lys Met Asn Leu Ile Thr Ser Ser Glu Glu Tyr Leu
                325                 330                 335
Lys Asp Leu His Asn Cys Asn Phe Ser Asn Ser Ser Asp Lys Asn Asp
            340                 345                 350
Ser Phe Phe Lys Leu Tyr Gly Ile Cys Glu Tyr Asp Lys Tyr Leu Ile
        355                 360                 365
Asp Leu Glu Glu Asn Ala Ser Phe His Tyr Asn Asn Val Asp Glu Tyr
    370                 375                 380
Gly Tyr Tyr Asp Val Asn Lys Asn Thr Asn Ile Leu Ser Asn Asn Lys
385                 390                 395                 400
Ile Glu Gln Asn Asn Asn Asn Glu Asn Asn Lys Asn Asn Lys Asn Asn
                405                 410                 415
Asn Asn Asn Glu Val Asp Tyr Ile Lys Lys Asp Glu Asp Asn Asn Val
            420                 425                 430
Asn Ser Lys Val Phe Tyr Ser Gln Tyr Asn Asn Asn Ala Gln Asn Asn
        435                 440                 445
Glu His Thr Glu Phe Asn Leu Asn Asn Asp Tyr Ser Thr Tyr Ile Arg
    450                 455                 460
Lys Lys Met Lys Asn Glu Glu Phe Leu Asn Leu Val Asn Lys Arg Lys
465                 470                 475                 480
Val Asp His Lys Glu Lys Ile Ile Val Ile Val Asp Cys Gly Ile Lys
                485                 490                 495
Asn Ser Ile Ile Lys Asn Leu Ile Arg His Gly Met Asp Leu Pro Leu
            500                 505                 510
Thr Tyr Ile Ile Val Pro Tyr Tyr Tyr Asn Phe Asn His Ile Asp Tyr
        515                 520                 525
Asp Ala Val Leu Leu Ser Asn Gly Pro Gly Asp Pro Lys Lys Cys Asp
    530                 535                 540
Phe Leu Ile Lys Asn Leu Lys Asp Ser Leu Thr Lys Asn Lys Ile Ile
545                 550                 555                 560
Phe Gly Ile Cys Leu Gly Asn Gln Leu Leu Gly Ile Ser Leu Gly Cys
                565                 570                 575
Asp Thr Tyr Lys Met Lys Tyr Gly Asn Arg Gly Val Asn Gln Pro Val
            580                 585                 590
Ile Gln Leu Val Asp Asn Ile Cys Tyr Ile Thr Ser Gln Asn His Gly
        595                 600                 605
Tyr Cys Leu Lys Lys Lys Ser Ile Leu Lys Arg Lys Glu Leu Ala Ile
    610                 615                 620
Ser Tyr Ile Asn Ala Asn Asp Lys Ser Ile Glu Gly Ile Ser His Lys
625                 630                 635                 640
Asn Gly Arg Phe Tyr Ser Val Gln Phe His Pro Glu Gly Asn Asn Gly
                645                 650                 655
Pro Glu Asp Thr Ser Phe Leu Phe Lys Asn Phe Leu Leu Asp Ile Phe
            660                 665                 670
Asn Lys Lys Lys Gln Tyr Arg Glu Tyr Leu Gly Tyr Asn Ile Ile Tyr
        675                 680                 685
Ile Lys Lys Lys Val Leu Leu Leu Gly Ser Gly Gly Leu Cys Ile Gly
    690                 695                 700
Gln Ala Gly Glu Phe Asp Tyr Ser Gly Thr Gln Ala Ile Lys Ser Leu
705                 710                 715                 720
Lys Glu Cys Gly Ile Tyr Val Ile Leu Val Asn Pro Asn Ile Ala Thr
                725                 730                 735
Val Gln Thr Ser Lys Gly Leu Ala Asp Lys Val Tyr Phe Leu Pro Val
            740                 745                 750
Asn Cys Glu Phe Val Glu Lys Ile Ile Lys Lys Glu Lys Pro Asp Phe
        755                 760                 765
Ile Leu Cys Thr Phe Gly Gly Gln Thr Ala Leu Asn Cys Ala Leu Met
    770                 775                 780
Leu Asp Gln Lys Lys Val Leu Lys Lys Asn Asn Cys Gln Cys Leu Gly
785                 790                 795                 800
Thr Ser Leu Glu Ser Ile Arg Ile Thr Glu Asn Arg Thr Leu Phe Ala
                805                 810                 815
Glu Lys Leu Lys Glu Ile Asn Glu Arg Ile Ala Pro Tyr Gly Ser Ala
            820                 825                 830
Lys Asn Val Asn Gln Ala Ile Asp Ile Ala Asn Lys Ile Gly Tyr Pro
        835                 840                 845
Ile Leu Val Arg Thr Thr Phe Ser Leu Gly Gly Leu Asn Ser Ser Phe
    850                 855                 860
Ile Asn Asn Glu Glu Glu Leu Ile Glu Lys Cys Asn Lys Ile Phe Leu
865                 870                 875                 880
Gln Thr Asp Asn Glu Ile Phe Ile Asp Lys Ser Leu Gln Gly Trp Lys
                885                 890                 895
Glu Ile Glu Tyr Glu Leu Leu Arg Asp Asn Lys Asn Asn Cys Ile Ala
            900                 905                 910
Ile Cys Asn Met Glu Asn Ile Asp Pro Leu Gly Ile His Thr Gly Asp
        915                 920                 925
Ser Ile Val Val Ala Pro Ser Gln Thr Leu Ser Asn Tyr Glu Tyr Tyr
    930                 935                 940
Lys Phe Arg Glu Ile Ala Leu Lys Val Ile Thr His Leu Asn Ile Ile
945                 950                 955                 960
Gly Glu Cys Asn Ile Gln Phe Gly Ile Asn Pro Gln Thr Gly Glu Tyr
                965                 970                 975
Cys Ile Ile Glu Val Asn Ala Arg Leu Ser Arg Ser Ser Ala Leu Ala
            980                 985                 990
Ser Lys Ala Thr Gly Tyr Pro Leu Ala Tyr Ile Ser Ala Lys Ile Ala
        995                1000                1005
Leu Gly Tyr Asp Leu Ile Ser Leu Lys Asn Ser Ile Thr Lys Lys Thr
   1010                1015                1020
Thr Ala Cys Phe Glu Pro Ser Leu Asp Tyr Ile Thr Thr Lys Ile Pro
1025               1030                1035                1040
Arg Trp Asp Leu Asn Lys Phe Glu Phe Ala Ser Asn Thr Met Asn Ser
               1045                1050                1055
Ser Met Lys Ser Val Gly Glu Val Met Ser Ile Gly Arg Thr Phe Glu
           1060                1065                1070
Glu Ser Ile Gln Lys Ser Leu Arg Cys Ile Asp Asp Asn Tyr Leu Gly
       1075                1080                1085
Phe Ser Asn Thr Tyr Cys Ile Asp Trp Asp Glu Lys Lys Ile Ile Glu
   1090                1095                1100
Glu Leu Lys Asn Pro Ser Pro Lys Arg Ile Asp Ala Ile His Gln Ala
1105               1110                1115                1120
Phe His Leu Asn Met Pro Met Asp Lys Ile His Glu Leu Thr His Ile
               1125                1130                1135
Asp Tyr Trp Phe Leu His Lys Phe Tyr Asn Ile Tyr Asn Leu Gln Asn
           1140                1145                1150
Lys Leu Lys Thr Leu Lys Leu Glu Gln Leu Ser Phe Asn Asp Leu Lys
       1155                1160                1165
Tyr Phe Lys Lys His Gly Phe Ser Asp Lys Gln Ile Ala His Tyr Leu
   1170                1175                1180
Ser Phe Asn Thr Ser Asp Asn Asn Asn Asn Asn Asn Asn Ile Ser Ser
1185               1190                1195                1200
Cys Arg Val Thr Glu Asn Asp Val Met Lys Tyr Arg Glu Lys Leu Gly
               1205                1210                1215
Leu Phe Pro His Ile Lys Val Ile Asp Thr Leu Ser Ala Glu Phe Pro
           1220                1225                1230
Ala Leu Thr Asn Tyr Leu Tyr Leu Thr Tyr Gln Gly Gln Glu His Asp
       1235                1240                1245
Val Leu Pro Leu Asn Met Lys Arg Lys Lys Ile Cys Thr Leu Asn Asn
   1250                1255                1260
Lys Arg Asn Ala Asn Lys Lys Lys Val His Val Lys Asn His Leu Tyr
1265               1270                1275                1280
Asn Glu Val Val Asp Asp Lys Asp Thr Gln Leu His Lys Glu Asn Asn
               1285                1290                1295
Asn Asn Asn Asn Met Asn Ser Gly Asn Val Glu Asn Lys Cys Lys Leu
           1300                1305                1310
Asn Lys Glu Ser Tyr Gly Tyr Asn Asn Ser Ser Asn Cys Ile Asn Thr
       1315                1320                1325
Asn Asn Ile Asn Ile Glu Asn Asn Ile Cys His Asp Ile Ser Ile Asn
   1330                1335                1340
Lys Asn Ile Lys Val Thr Ile Asn Asn Ser Asn Asn Ser Ile Ser Asn
1345               1350                1355                1360
Asn Glu Asn Val Glu Thr Asn Leu Asn Cys Val Ser Glu Arg Ala Gly
               1365                1370                1375
Ser His His Ile Tyr Gly Lys Glu Glu Lys Ser Ile Gly Ser Asp Asp
           1380                1385                1390
Thr Asn Ile Leu Ser Ala Gln Asn Ser Asn Asn Asn Phe Ser Cys Asn
       1395                1400                1405
Asn Glu Asn Met Asn Lys Ala Asn Val Asp Val Asn Val Leu Glu Asn
   1410                1415                1420
Asp Thr Lys Lys Arg Glu Asp Ile Asn Thr Thr Thr Val Phe Met Glu
1425               1430                1435                1440
Gly Gln Asn Ser Val Ile Asn Asn Lys Asn Lys Glu Asn Ser Ser Leu
               1445                1450                1455
Leu Lys Gly Asp Glu Glu Asp Ile Val Met Val Asn Leu Lys Lys Glu
           1460                1465                1470
Asn Asn Tyr Asn Ser Val Ile Asn Asn Val Asp Cys Arg Lys Lys Asp
       1475                1480                1485
Met Asp Gly Lys Asn Ile Asn Asp Glu Cys Lys Thr Tyr Lys Lys Asn
   1490                1495                1500
Lys Tyr Lys Asp Met Gly Leu Asn Asn Asn Ile Val Asp Glu Leu Ser
1505               1510                1515                1520
Asn Gly Thr Ser His Ser Thr Asn Asp His Leu Tyr Leu Asp Asn Phe
               1525                1530                1535
Asn Thr Ser Asp Glu Glu Ile Gly Asn Asn Lys Asn Met Asp Met Tyr
           1540                1545                1550
Leu Ser Lys Glu Lys Ser Ile Ser Asn Lys Asn Pro Gly Asn Ser Tyr
       1555                1560                1565
Tyr Val Val Asp Ser Val Tyr Asn Asn Glu Tyr Lys Ile Asn Lys Met
   1570                1575                1580
Lys Glu Leu Ile Asp Asn Glu Asn Leu Asn Asp Glu Tyr Asn Asn Asn
1585               1590                1595                1600
Val Asn Met Asn Cys Ser Asn Tyr Asn Asn Ala Ser Ala Phe Val Asn
               1605                1610                1615
Gly Lys Asp Arg Asn Asp Asn Leu Glu Asn Asp Cys Ile Glu Lys Asn
           1620                1625                1630
Met Asp His Thr Tyr Lys His Tyr Asn Arg Leu Asn Asn Arg Arg Ser
       1635                1640                1645
Thr Asn Glu Arg Met Met Leu Met Val Asn Asn Glu Lys Glu Ser Asn
   1650                1655                1660
His Glu Lys Gly His Arg Arg Asn Gly Leu Asn Lys Lys Asn Lys Glu
1665               1670                1675                1680
Lys Asn Met Glu Lys Asn Lys Gly Lys Asn Lys Asp Lys Lys Asn Tyr
               1685                1690                1695
His Tyr Val Asn His Lys Arg Asn Asn Glu Tyr Asn Ser Asn Asn Ile
           1700                1705                1710
Glu Ser Lys Phe Asn Asn Tyr Val Asp Asp Ile Asn Lys Lys Glu Tyr
       1715                1720                1725
Tyr Glu Asp Glu Asn Asp Ile Tyr Tyr Phe Thr His Ser Ser Gln Gly
   1730                1735                1740
Asn Asn Asp Asp Leu Ser Asn Asp Asn Tyr Leu Ser Ser Glu Glu Leu
1745               1750                1755                1760
Asn Thr Asp Glu Tyr Asp Asp Asp Tyr Tyr Tyr Asp Glu Asp Glu Glu
               1765                1770                1775
Asp Asp Tyr Asp Asp Asp Asn Asp Asp Asp Asp Asp Asp Asp Asp Asp
           1780                1785                1790
Gly Glu Asp Glu Glu Asp Asn Asp Tyr Tyr Asn Asp Asp Gly Tyr Asp
       1795                1800                1805
Ser Tyr Asn Ser Leu Ser Ser Ser Arg Ile Ser Asp Val Ser Ser Val
   1810                1815                1820
Ile Tyr Ser Gly Asn Glu Asn Ile Phe Asn Glu Lys Tyr Asn Asp Ile
1825               1830                1835                1840
Gly Phe Lys Ile Ile Asp Asn Arg Asn Glu Lys Glu Lys Glu Lys Lys
               1845                1850                1855
Lys Cys Phe Ile Val Leu Gly Cys Gly Cys Tyr Arg Ile Gly Ser Ser
           1860                1865                1870
Val Glu Phe Asp Trp Ser Ala Ile His Cys Val Lys Thr Ile Arg Lys
       1875                1880                1885
Leu Asn His Lys Ala Ile Leu Ile Asn Cys Asn Pro Glu Thr Val Ser
   1890                1895                1900
Thr Asp Tyr Asp Glu Ser Asp Arg Leu Tyr Phe Asp Glu Ile Thr Thr
1905               1910                1915                1920
Glu Val Ile Lys Phe Ile Tyr Asn Phe Glu Asn Ser Asn Gly Val Ile
               1925                1930                1935
Ile Ala Phe Gly Gly Gln Thr Ser Asn Asn Leu Val Phe Ser Leu Tyr
           1940                1945                1950
Lys Asn Asn Val Asn Ile Leu Gly Ser Val His Lys Val Leu Ile Val
       1955                1960                1965
Val Lys Ile Gly Ile Asn Phe Arg Thr Tyr Val Ile Leu Lys Ile Asp
   1970                1975                1980
Gln Pro Lys Trp Asn Lys Phe Thr Lys Leu Ser Lys Ala Ile Gln Phe
1985               1990                1995                2000
Ala Asn Glu Val Lys Phe Pro Val Leu Val Arg Pro Ser Tyr Val Leu
               2005                2010                2015
Ser Gly Ala Ala Met Arg Val Val Asn Cys Phe Glu Glu Leu Lys Asn
           2020                2025                2030
Phe Leu Met Lys Ala Ala Ile Val Ser Lys Asp Asn Pro Val Val Ile
       2035                2040                2045
Ser Lys Phe Ile Glu Asn Ala Lys Glu Ile Glu Ile Asp Cys Val Ser
   2050                2055                2060
Lys Asn Gly Lys Ile Ile Asn Tyr Ala Ile Ser Glu His Val Glu Asn
2065               2070                2075                2080
Ala Gly Val His Ser Gly Asp Ala Thr Leu Ile Leu Pro Ala Gln Asn
               2085                2090                2095
Ile Tyr Val Glu Thr His Arg Lys Ile Lys Lys Ile Ser Glu Lys Ile
           2100                2105                2110
Ser Lys Ser Leu Asn Ile Ser Gly Pro Phe Asn Ile Gln Phe Ile Cys
       2115                2120                2125
His Gln Asn Glu Ile Lys Ile Ile Glu Cys Asn Leu Arg Ala Ser Arg
   2130                2135                2140
Thr Phe Pro Phe Ile Ser Lys Ala Leu Asn Leu Asn Phe Ile Asp Leu
2145               2150                2155                2160
Ala Thr Arg Ile Leu Met Gly Tyr Asp Val Lys Pro Ile Asn Ile Ser
               2165                2170                2175
Leu Ile Asp Leu Glu Tyr Thr Ala Val Lys Ala Pro Ile Phe Ser Phe
           2180                2185                2190
Asn Arg Leu His Gly Ser Asp Cys Ile Leu Gly Val Glu Met Lys Ser
       2195                2200                2205
Thr Gly Glu Val Ala Cys Phe Gly Leu Asn Lys Tyr Glu Ala Leu Leu
   2210                2215                2220
Lys Ser Leu Ile Ala Thr Gly Met Lys Leu Pro Lys Lys Ser Ile Leu
2225               2230                2235                2240
Ile Ser Ile Lys Asn Leu Asn Asn Lys Leu Ala Phe Glu Glu Pro Phe
               2245                2250                2255
Gln Leu Leu Phe Leu Met Gly Phe Thr Ile Tyr Ala Thr Glu Gly Thr
           2260                2265                2270
Tyr Asp Phe Tyr Ser Lys Phe Leu Glu Ser Phe Asn Val Asn Lys Gly
       2275                2280                2285
Ser Lys Phe His Gln Arg Leu Ile Lys Val His Asn Lys Asn Ala Glu
   2290                2295                2300
Asn Ile Ser Pro Asn Thr Thr Asp Leu Ile Met Asn His Lys Val Glu
2305               2310                2315                2320
Met Val Ile Asn Ile Thr Asp Thr Leu Lys Thr Lys Val Ser Ser Asn
               2325                2330                2335
Gly Tyr Lys Ile Arg Arg Leu Ala Ser Asp Phe Gln Val Pro Leu Ile
           2340                2345                2350
Thr Asn Met Lys Leu Cys Ser Leu Phe Ile Asp Ser Leu Tyr Arg Lys
       2355                2360                2365
Phe Ser Arg Gln Lys Glu Arg Lys Ser Phe Tyr Thr Ile Lys Ser Tyr
   2370                2375                2380
Asp Glu Tyr Ile Ser Leu Val
2385               2390 
           
             3 
             21 
             DNA 
             Plasmodium falciparum 
           
            3
ttttctaatc gactattttt a                                               21 
           
             4 
             21 
             DNA 
             Plasmodium falciparum 
           
            4
gacatacttg gacaattatt a                                               21 
           
             5 
             21 
             DNA 
             Plasmodium falciparum 
           
            5
ccttactatt gacattatta t                                               21 
           
             6 
             21 
             DNA 
             Plasmodium falciparum 
           
            6
ggctataaaa gaccttacta t                                               21 
           
             7 
             21 
             DNA 
             Plasmodium falciparum 
           
            7
ggatctccag gaccattaga t                                               21 
           
             8 
             21 
             DNA 
             Plasmodium falciparum 
           
            8
cctaaacatt gacaattatt c                                               21 
           
             9 
             21 
             DNA 
             Plasmodium falciparum 
           
            9
tcatgttctt gaccttgata a                                               21 
           
             10 
             21 
             DNA 
             Plasmodium falciparum 
           
            10
gatatatcat gacaaatatt a                                               21 
           
             11 
             21 
             DNA 
             Plasmodium falciparum 
           
            11
gttaccttgt gacgaatgtg t                                               21 
           
             12 
             21 
             DNA 
             Plasmodium falciparum 
           
            12
tcattttgat gacatataaa t                                               21 
           
             13 
             29 
             DNA 
             Plasmodium falciparum 
           
            13
aacatcatgt tcttgacctt gataagtta                                       29 
           
             14 
             21 
             DNA 
             Plasmodium falciparum 
           
            14
cgtcggaact gaatttggct c                                               21 
           
             15 
             21 
             DNA 
             Plasmodium falciparum 
           
            15
cctcagtgct gacagcccat c                                               21