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Mung bean nuclease exhibits an exon-excision activity upon the Plasmodium falciparum SERA gene
Molecular and Biochemical Parasitology, 56 (1992) 185 188 L3 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
185
MOLBIO 01859
Short Communication Mung bean nuclease exhibits an exon-excision activity upon the Plasmodium falciparum SERA gene D a v i d J. Bzik, J u a n i t a Y. P e c k a n d B a r b a r a A. F o x Department of Microbiology, Dartmouth Medical School, Hanover, NH, USA (Received 23 July 1992; accepted 23 August 1992)
Key words: Plasmodium falciparum; Mung bean nuclease; Selective intron cleavage; Exonase activity
Introduction
Mung bean nuclease (MBN) selectively cleaves duplex genomic D N A at sites 5' and 3' of Plasmodium falciparum genes in the presence of formamide [1,2]. This activity is described as a gene-excision, or genease, activity [2]. M B N exhibits a similar activity upon other protozoan parasites such as Babesia [3], Giardia [4], Leishmania [5,6], Toxoplasma [7], and Trypanosoma [8]. Noncoding D N A is highly A + T rich in P. falciparum in contrast to coding D N A [9]. Since selection of cleavage sites can be influenced by high A + T content of the target DNA, but is not absolutely dependent on high A + T content, altered D N A structural features near gene boundaries are proposed to promote recognition and/or cleavage by MBN [1,2]. On the question of whether selective cleavage occurs within introns, only 1 of 4 P. falciparum introns tested was found to be selectively cleaved in the presence of 32.5%, or 35%, formamide [2]. To determine whether
Correspondence address: David J. Bzik, Department of Microbiology, Dartmouth Medical School, Hanover, NH 03755-3842, USA. Tel.: (603) 650-1769. Abbreviations." MBN, mung bean nuclease; SERA, serinerepeat antigen.
MBN generally fails to cleave within introns because introns lack a selective cleavage site or because conditions were not identified for selective cleavage, we undertook a study of MBN cleavage of the SERA gene. SERA was chosen as a model gene to study selective cleavage of introns because it contains 3 wellcharacterized introns of different size, base composition, and A + T content [10,11]. Here we describe conditions that result in selective MBN cleavage within each of the 3 SERA gene introns and its 5' and 3' flanking DNA. The sizes and locations of SERA exons, introns, and exon-specific oligonucleotide probes are shown in Fig. 1F. At 35% formamide we observed dihydrofolate reductase-thymidylate synthase (DHFR-TS)-specific bands of 1.9 kb and 2.1 kb (Fig. 1E) in agreement with the study of Vernick et al. [2]. Sharp DHFR-TS-speciflc bands suggest selective cleavage and demonstrate the previously described genease activity [2]. Blots that gave this D H F R - T S pattern were stripped and sequentially probed with exon-specific oligonucleotides. The exon 1 probe hybridized to sharp bands of 3.75 kb and 1.40 kb at 32.5% and 35% formamide (Fig. 1A). The exon 2 probe hybridized to sharp bands of 3.75 kb and 1.40 kb at 32.5% and 35% formamide, to bands of 0.95 kb, 1.15 kb, and 3.75 kb at 37.5% formamide, and to a band of 0.95 kb at
186 1
2
3
4
1
3,75-
1.40-
2
3
4
5
1
3.75-
3.75-
1.401.15 ~
1.15 t"
2
4
5
O,95-
O. 2 4 -
1
2
3
4
5
1
D ~5:
C
B
A 2
3
4
5
E 175
12g
e×or] ~D
F
Fig. 1. Mung bean nuclease cleavage pattern of the P. falciparum SERA gene. Panels A-E: genomic D N A (strain Honduras-1) was digested with mung bean nuclease in 32.5% (lane 1), 35% (lane 2), 37.5% (lane 3), 40% formamide (lane 4), or was treated with 40% formamide under the same conditions without MBN (lane 5). The Southern blot of digestion products was sequentially hybridized with labeled P. falciparum D H F R - T S or SERA exon-specific probes. The methods used were previously described by Vernick et al. [2]. A, exon 1; B, exon 2; C, exon 3a and 3b; D, exon 4; E, 5' D H F R (5' = 5'T T T C T C C T T T T T A T G A T G G ) or 3' TS ( 5 ' - G A A C T G C G C A G G T T G C ) [17]. Panel F (top): structure of the SERA gene and location of exon-specific oligonucleotide probes; (bottom) MBN cleavage map; (top) exon/intron structure of the SERA gene is shown with arrows showing the location and size (in bp) of exons and introns (above), or the location of exon-specific oligonucleotide probes (below). 5' and 3' flanking D N A (shaded), exons ( I ) , introns (r-l). The complete SERA gene sequence is detailed in GenBank accession number JO4000 [10,11 ], and the drawing is to scale. The SERA exon-specific oligonucleotide (5' to 3') probes used in this work were as follows: exon 1 ( C A A T A T G A A A A A C A A G G A A A T A T A T G A ) , exon 2 ( T G A A T C A A A C A G T G G T T C A T T A G ) , exon 3a (5' end) ( G T T G A T G T A A G A A A G T A T T T G A T A A A T G ) , exon 3b (3' end) ( C G C A T T A G G A T C A G A C A T T C C A ) , exon 4 ( G C A A T T G G A A G C T A A G G T A T C A ) . (bottom) MBN cleavage map. Sites of MBN cleavage at different formamide concentrations are shown by arrowheads and resulting D N A fragment sizes are shown in kb.
187
40% formamide (Fig. 1B). The exon 3b probe hybridized to sharp bands of 3.75 kb and 1.40 kb at 32.5% and 35% formamide, to bands of 0.24 kb, 1.15 kb, 2.20 kb, and 3.75 kb at 37.5% formamide, and to a band of 0.24 kb at 40% formamide (Fig. 1C). The exon 3a probe gave identical results (data not shown). The exon 4 probe hybridized to sharp bands of 3.75 kb and 2.35 kb at 32.5% and 35% formamide, and to a band of 2.10 kb at 37.5% and 40% formamide (Fig. 1D). The results were used to construct a MBN cleavage map (Fig. 1F). Selective cleavages occurred within the 3 introns and the 5' and 3' flanking DNA, and resulted in the excision of bands containing exons 1 + 2 + 3 + 4 (3.75 kb), exons 1 + 2 + 3 (1.40 kb), exons 2 + 3 (1.15 kb), exon 4 only (2.35 or 2.10 kb), exon 3 only (0.24 kb), exon 2 only (0.95 kb), and exon 1 only (not resolved). At 32.5% and 35% formamide, selective cleavage only occurred within the 5' and 3' flanking DNA, and intron 3 (Fig. 2F). The 5' and 3' flanking cleavages yield the 3.75-kb band, while cleavage within intron 3 split the 3.75-kb band into the 2.35-kb and 1.40-kb bands. The ratio of the 3.75/1.40/2.35-kb bands is approximately 1:1:1 at 32.5% and 35% formamide, suggesting that the cleavage rate of intron 3 is one-half the cleavage rate of the flanking sites. Alternatively, the cleavage rate of all 3 sites would be the same if the 3.75kb band were inert to further cleavage. At 37.5% and 40% formamide, selective cleavage occurred within the 5' and 3' flanking DNA, intron 1, intron 2, and intron 3 (Fig. 1F). Selective cleavage in intron 1 and intron 2 was only observed at 37.5% and 40% formamide. Selective cleavages at 37.5% and 40% formamide produced sharp bands containing exon 4 only, exon 3 only, exon 2 only, and exon 1 only. Of the 5 selective cleavage domains, intron 1 is the least A + T rich (83%) [10]. Since intron 2 (87% A + T rich) is nearly identical in A + T content to intron 3 (88%) and to the 3' flanking 279 bp (87%) [10], A + T content itself cannot fully explain cleavage site selection. Interestingly, all 5 cleavage domains contained at least 1 stretch of 100% A + T of
41 bases, or more. At a 10-fold lower enzyme concentration, MBN cleaves P. knowlesi D N A at sites of tandemly repeated purine/pyrimidine bases ( A p T dimers) [13]. Although several examples of possible intron cleavage were previously reported when MBN was used to create D N A libraries to clone P. falciparum genes for H P G R T [14,15], HRPII and HRPIII [16], SERA [10], and GBP-130 [17], none of these reported cleavages was shown to be selective. A previous study found that only 1 of 4 introns was selectively cleaved at 32.5% and 35% formamide [2]. Our data also show that only 1 of 3 SERA introns was selectively cleaved at 32.5% and 35% formamide. However, at 37.5% and 40% formamide we observed that all 3 SERA introns were selectively cleaved, as well as the 5' and 3' flanking DNA. In summary, this report describes conditions for selective MBN cleavage within SERA introns. Our observation of selective MBN cleavage within SERA introns suggests that intron cleavage may be a general result. Since MBN is presently considered to only have a genease activity upon P. falciparum genes, rather than a gene-excision and exon-excision ("exonase") activity, individual MBN derived clones should be further examined for possible intron cleavages. We speculate that MBN may be a useful reagent to isolate exons from intron-containing P. falciparum genes.
Acknowledgements This work was supported.by NIH Grant AI26651 (D.J.B.), and in part by Hitchcock Foundation Grant No. 250-1 (D.J.B.). We are grateful to Dr. Joseph Inselburg for providing us with D H F R - and TS-specific oligonucleotide probes. We would like to thank Dr. Stephen Holloway for helpful discussions.
References 1 McCutchan, T.F., Hanson, J.L., Dame, J.B. and Mullins, J.A. (1984) Mung bean nuclease cleases Plasmodium genomic DNA at sites before and after
188 genes. Science 225, 625 628. 2 Vernick, K.D., Imberski, B. and McCutchan, T.F. (1988) Mung bean nuclease exhibits a generalized geneexcision activity upon purified Plasmodium falciparum genomic DNA. Nucl. Acids Res. 16, 6883 6896. 3 Tripp, C.A., Wagner, G.G. and Rice-Ficht, A.C. (1989) Babesia boris: gene isolation and characterization using a mung bean nuclease derived expression library. Exp. Parasitol. 69, 211-225. 4 Adam, R.G., Aggarwal, A., Lal, A.A., de la Cruz, V.F., McCutchan, T.F. and Nash, T.E. (1988) Antigenic variation of a cysteine-rich protein in Giardia lamblia. J. Exp. Med. 167, 109 118. 5 Sim, B.K.L., Landfear, S.M. and Wirth, D.F. (1984) Digestion of alpha-tubulin genes from Leishmania enrietti with mung bean nuclease. Biol. Bull. 167, 537. 6 Muhich, M.L. and Simpson, L. (1986) Specific cleavage of kinetoplast rninicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes. Nucl. Acids Res. 14, 5531 5556. 7 Johnson, A.M., Illana, S., Dubey, J.P. and Dame, J.B. (1987) Toxoplasma gondii and Hammondia hammondi: DNA comparisons using cloned rRNA gene probes. Exp. Parasitol. 63, 272-278. 8 Brown, K.H., Brentano, S.T. and Donelson, J.E. (1986) Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein transpositions in Trypanosome DNA. J. Biol. Chem. 261, 10352 10358. 9 Weber, J.L. (1988) Molecular biology of malaria parasites. Exp. Parasitol. 66, 143-170. 10 Li, W.B., Bzik, D.J., Horii, T. and Inselburg, J. (1989) Structure and expression of the Plasmodium falciparum
SERA gene. Mol. Biochem. Parasitol. 33, 13-26. I 1 Bzik, D.J., Li, W.B., Horri, T. and Inselburg, J. (1988) Amino acid sequence of the serine-repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA. Mol. Biochem. Parasitol. 30, 279-288. 12 Bzik, D.J., Li, W.B., Horii, T. and Inselburg, J. (1987) Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc. Natl. Acad. Sci. USA 84, 836~8364. 13 Szafranski, R. and Godson, G.N. (1990) Hypersensitive mung bean nuclease cleavage sites in Plasmodium knowlesi DNA. Gene 88, 141 147. 14 Sullivan, M.A., Lloyd, D.B. and Holland, L.E. (1987) Isolation and characterization of the gene encoding hypoxanthine-guanine-phosphoribosyltransferase from Plasmodiumfalciparum. In: (Agabian, N., Goodman, H. and Nogueira, N., eds), Molecular Strategies of Parasite Invasion, Vol. 42, pp. 575 584, Alan R. Liss, New York. 15 King, A. and Melton, D.W. (1987) Characterisation of cDNA clones for hypoxanthine-guanine-phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum: comparisons to the mammalian gene and protein. Nucl. Acids Res. 15, 10469 10481. 16 Wellems, T.E. and Howard, R.J. (1986) Homologous genes encode two distinct histidine-rich proteins in a clonal isolate of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 83, 6065-6069. 17 Bonnefoy, S. and Mercereau-Puijalon, O. (1989) Plasmodium falciparum: An intervening sequence in the GBP 130/96 tR gene. Exp. Parasitol. 69, 37~43.
185
MOLBIO 01859
Short Communication Mung bean nuclease exhibits an exon-excision activity upon the Plasmodium falciparum SERA gene D a v i d J. Bzik, J u a n i t a Y. P e c k a n d B a r b a r a A. F o x Department of Microbiology, Dartmouth Medical School, Hanover, NH, USA (Received 23 July 1992; accepted 23 August 1992)
Key words: Plasmodium falciparum; Mung bean nuclease; Selective intron cleavage; Exonase activity
Introduction
Mung bean nuclease (MBN) selectively cleaves duplex genomic D N A at sites 5' and 3' of Plasmodium falciparum genes in the presence of formamide [1,2]. This activity is described as a gene-excision, or genease, activity [2]. M B N exhibits a similar activity upon other protozoan parasites such as Babesia [3], Giardia [4], Leishmania [5,6], Toxoplasma [7], and Trypanosoma [8]. Noncoding D N A is highly A + T rich in P. falciparum in contrast to coding D N A [9]. Since selection of cleavage sites can be influenced by high A + T content of the target DNA, but is not absolutely dependent on high A + T content, altered D N A structural features near gene boundaries are proposed to promote recognition and/or cleavage by MBN [1,2]. On the question of whether selective cleavage occurs within introns, only 1 of 4 P. falciparum introns tested was found to be selectively cleaved in the presence of 32.5%, or 35%, formamide [2]. To determine whether
Correspondence address: David J. Bzik, Department of Microbiology, Dartmouth Medical School, Hanover, NH 03755-3842, USA. Tel.: (603) 650-1769. Abbreviations." MBN, mung bean nuclease; SERA, serinerepeat antigen.
MBN generally fails to cleave within introns because introns lack a selective cleavage site or because conditions were not identified for selective cleavage, we undertook a study of MBN cleavage of the SERA gene. SERA was chosen as a model gene to study selective cleavage of introns because it contains 3 wellcharacterized introns of different size, base composition, and A + T content [10,11]. Here we describe conditions that result in selective MBN cleavage within each of the 3 SERA gene introns and its 5' and 3' flanking DNA. The sizes and locations of SERA exons, introns, and exon-specific oligonucleotide probes are shown in Fig. 1F. At 35% formamide we observed dihydrofolate reductase-thymidylate synthase (DHFR-TS)-specific bands of 1.9 kb and 2.1 kb (Fig. 1E) in agreement with the study of Vernick et al. [2]. Sharp DHFR-TS-speciflc bands suggest selective cleavage and demonstrate the previously described genease activity [2]. Blots that gave this D H F R - T S pattern were stripped and sequentially probed with exon-specific oligonucleotides. The exon 1 probe hybridized to sharp bands of 3.75 kb and 1.40 kb at 32.5% and 35% formamide (Fig. 1A). The exon 2 probe hybridized to sharp bands of 3.75 kb and 1.40 kb at 32.5% and 35% formamide, to bands of 0.95 kb, 1.15 kb, and 3.75 kb at 37.5% formamide, and to a band of 0.95 kb at
186 1
2
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3,75-
1.40-
2
3
4
5
1
3.75-
3.75-
1.401.15 ~
1.15 t"
2
4
5
O,95-
O. 2 4 -
1
2
3
4
5
1
D ~5:
C
B
A 2
3
4
5
E 175
12g
e×or] ~D
F
Fig. 1. Mung bean nuclease cleavage pattern of the P. falciparum SERA gene. Panels A-E: genomic D N A (strain Honduras-1) was digested with mung bean nuclease in 32.5% (lane 1), 35% (lane 2), 37.5% (lane 3), 40% formamide (lane 4), or was treated with 40% formamide under the same conditions without MBN (lane 5). The Southern blot of digestion products was sequentially hybridized with labeled P. falciparum D H F R - T S or SERA exon-specific probes. The methods used were previously described by Vernick et al. [2]. A, exon 1; B, exon 2; C, exon 3a and 3b; D, exon 4; E, 5' D H F R (5' = 5'T T T C T C C T T T T T A T G A T G G ) or 3' TS ( 5 ' - G A A C T G C G C A G G T T G C ) [17]. Panel F (top): structure of the SERA gene and location of exon-specific oligonucleotide probes; (bottom) MBN cleavage map; (top) exon/intron structure of the SERA gene is shown with arrows showing the location and size (in bp) of exons and introns (above), or the location of exon-specific oligonucleotide probes (below). 5' and 3' flanking D N A (shaded), exons ( I ) , introns (r-l). The complete SERA gene sequence is detailed in GenBank accession number JO4000 [10,11 ], and the drawing is to scale. The SERA exon-specific oligonucleotide (5' to 3') probes used in this work were as follows: exon 1 ( C A A T A T G A A A A A C A A G G A A A T A T A T G A ) , exon 2 ( T G A A T C A A A C A G T G G T T C A T T A G ) , exon 3a (5' end) ( G T T G A T G T A A G A A A G T A T T T G A T A A A T G ) , exon 3b (3' end) ( C G C A T T A G G A T C A G A C A T T C C A ) , exon 4 ( G C A A T T G G A A G C T A A G G T A T C A ) . (bottom) MBN cleavage map. Sites of MBN cleavage at different formamide concentrations are shown by arrowheads and resulting D N A fragment sizes are shown in kb.
187
40% formamide (Fig. 1B). The exon 3b probe hybridized to sharp bands of 3.75 kb and 1.40 kb at 32.5% and 35% formamide, to bands of 0.24 kb, 1.15 kb, 2.20 kb, and 3.75 kb at 37.5% formamide, and to a band of 0.24 kb at 40% formamide (Fig. 1C). The exon 3a probe gave identical results (data not shown). The exon 4 probe hybridized to sharp bands of 3.75 kb and 2.35 kb at 32.5% and 35% formamide, and to a band of 2.10 kb at 37.5% and 40% formamide (Fig. 1D). The results were used to construct a MBN cleavage map (Fig. 1F). Selective cleavages occurred within the 3 introns and the 5' and 3' flanking DNA, and resulted in the excision of bands containing exons 1 + 2 + 3 + 4 (3.75 kb), exons 1 + 2 + 3 (1.40 kb), exons 2 + 3 (1.15 kb), exon 4 only (2.35 or 2.10 kb), exon 3 only (0.24 kb), exon 2 only (0.95 kb), and exon 1 only (not resolved). At 32.5% and 35% formamide, selective cleavage only occurred within the 5' and 3' flanking DNA, and intron 3 (Fig. 2F). The 5' and 3' flanking cleavages yield the 3.75-kb band, while cleavage within intron 3 split the 3.75-kb band into the 2.35-kb and 1.40-kb bands. The ratio of the 3.75/1.40/2.35-kb bands is approximately 1:1:1 at 32.5% and 35% formamide, suggesting that the cleavage rate of intron 3 is one-half the cleavage rate of the flanking sites. Alternatively, the cleavage rate of all 3 sites would be the same if the 3.75kb band were inert to further cleavage. At 37.5% and 40% formamide, selective cleavage occurred within the 5' and 3' flanking DNA, intron 1, intron 2, and intron 3 (Fig. 1F). Selective cleavage in intron 1 and intron 2 was only observed at 37.5% and 40% formamide. Selective cleavages at 37.5% and 40% formamide produced sharp bands containing exon 4 only, exon 3 only, exon 2 only, and exon 1 only. Of the 5 selective cleavage domains, intron 1 is the least A + T rich (83%) [10]. Since intron 2 (87% A + T rich) is nearly identical in A + T content to intron 3 (88%) and to the 3' flanking 279 bp (87%) [10], A + T content itself cannot fully explain cleavage site selection. Interestingly, all 5 cleavage domains contained at least 1 stretch of 100% A + T of
41 bases, or more. At a 10-fold lower enzyme concentration, MBN cleaves P. knowlesi D N A at sites of tandemly repeated purine/pyrimidine bases ( A p T dimers) [13]. Although several examples of possible intron cleavage were previously reported when MBN was used to create D N A libraries to clone P. falciparum genes for H P G R T [14,15], HRPII and HRPIII [16], SERA [10], and GBP-130 [17], none of these reported cleavages was shown to be selective. A previous study found that only 1 of 4 introns was selectively cleaved at 32.5% and 35% formamide [2]. Our data also show that only 1 of 3 SERA introns was selectively cleaved at 32.5% and 35% formamide. However, at 37.5% and 40% formamide we observed that all 3 SERA introns were selectively cleaved, as well as the 5' and 3' flanking DNA. In summary, this report describes conditions for selective MBN cleavage within SERA introns. Our observation of selective MBN cleavage within SERA introns suggests that intron cleavage may be a general result. Since MBN is presently considered to only have a genease activity upon P. falciparum genes, rather than a gene-excision and exon-excision ("exonase") activity, individual MBN derived clones should be further examined for possible intron cleavages. We speculate that MBN may be a useful reagent to isolate exons from intron-containing P. falciparum genes.
Acknowledgements This work was supported.by NIH Grant AI26651 (D.J.B.), and in part by Hitchcock Foundation Grant No. 250-1 (D.J.B.). We are grateful to Dr. Joseph Inselburg for providing us with D H F R - and TS-specific oligonucleotide probes. We would like to thank Dr. Stephen Holloway for helpful discussions.
References 1 McCutchan, T.F., Hanson, J.L., Dame, J.B. and Mullins, J.A. (1984) Mung bean nuclease cleases Plasmodium genomic DNA at sites before and after
188 genes. Science 225, 625 628. 2 Vernick, K.D., Imberski, B. and McCutchan, T.F. (1988) Mung bean nuclease exhibits a generalized geneexcision activity upon purified Plasmodium falciparum genomic DNA. Nucl. Acids Res. 16, 6883 6896. 3 Tripp, C.A., Wagner, G.G. and Rice-Ficht, A.C. (1989) Babesia boris: gene isolation and characterization using a mung bean nuclease derived expression library. Exp. Parasitol. 69, 211-225. 4 Adam, R.G., Aggarwal, A., Lal, A.A., de la Cruz, V.F., McCutchan, T.F. and Nash, T.E. (1988) Antigenic variation of a cysteine-rich protein in Giardia lamblia. J. Exp. Med. 167, 109 118. 5 Sim, B.K.L., Landfear, S.M. and Wirth, D.F. (1984) Digestion of alpha-tubulin genes from Leishmania enrietti with mung bean nuclease. Biol. Bull. 167, 537. 6 Muhich, M.L. and Simpson, L. (1986) Specific cleavage of kinetoplast rninicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes. Nucl. Acids Res. 14, 5531 5556. 7 Johnson, A.M., Illana, S., Dubey, J.P. and Dame, J.B. (1987) Toxoplasma gondii and Hammondia hammondi: DNA comparisons using cloned rRNA gene probes. Exp. Parasitol. 63, 272-278. 8 Brown, K.H., Brentano, S.T. and Donelson, J.E. (1986) Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein transpositions in Trypanosome DNA. J. Biol. Chem. 261, 10352 10358. 9 Weber, J.L. (1988) Molecular biology of malaria parasites. Exp. Parasitol. 66, 143-170. 10 Li, W.B., Bzik, D.J., Horii, T. and Inselburg, J. (1989) Structure and expression of the Plasmodium falciparum
SERA gene. Mol. Biochem. Parasitol. 33, 13-26. I 1 Bzik, D.J., Li, W.B., Horri, T. and Inselburg, J. (1988) Amino acid sequence of the serine-repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA. Mol. Biochem. Parasitol. 30, 279-288. 12 Bzik, D.J., Li, W.B., Horii, T. and Inselburg, J. (1987) Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc. Natl. Acad. Sci. USA 84, 836~8364. 13 Szafranski, R. and Godson, G.N. (1990) Hypersensitive mung bean nuclease cleavage sites in Plasmodium knowlesi DNA. Gene 88, 141 147. 14 Sullivan, M.A., Lloyd, D.B. and Holland, L.E. (1987) Isolation and characterization of the gene encoding hypoxanthine-guanine-phosphoribosyltransferase from Plasmodiumfalciparum. In: (Agabian, N., Goodman, H. and Nogueira, N., eds), Molecular Strategies of Parasite Invasion, Vol. 42, pp. 575 584, Alan R. Liss, New York. 15 King, A. and Melton, D.W. (1987) Characterisation of cDNA clones for hypoxanthine-guanine-phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum: comparisons to the mammalian gene and protein. Nucl. Acids Res. 15, 10469 10481. 16 Wellems, T.E. and Howard, R.J. (1986) Homologous genes encode two distinct histidine-rich proteins in a clonal isolate of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 83, 6065-6069. 17 Bonnefoy, S. and Mercereau-Puijalon, O. (1989) Plasmodium falciparum: An intervening sequence in the GBP 130/96 tR gene. Exp. Parasitol. 69, 37~43.