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Isolation of a single messenger RNA and of the corresponding gene in Plasmodium berghei
Cell Biology
International
Reports,
Vol. 8, No. 3, March
1984
257
ISOLATION OF A SINGLE MESSENGERRNA AND OF THE CORRESPONDING GENE IN Plasmodium berghei C. Birago, M. Ponzi, P.A. Battaglia Laboratorio di Biologia Cellulare Istituto Superiore di Sanita, Rome, Italy
SUMMARY A genomic library of Plasmodium berghei DNA was constructed using 147.1 as a vector. It represents 90% of Plasmodium genome. Genes expressed during the intraerythrocytic stage of P. berghei were isolated among the recombinant clones of +he library using labelled cDNA complementary to the polyA Plasmodium mRNA extracted during this stage. The purified coding strand of an expressed clone was utilized to catch the corresponding mRNA(s). The hybridized mRNA fraction was eluted and in vitro translated. Translation products were analyzed by gel electrophoresis; the gel fluorography revealed a single protein band of 32.500 daltons of molecular weight, corresponding to a 900bp coding region in the examined clone. INTRODUCTION Malaria parasites undergo several differentiative stages during their life cycle, some of which have a fundamental role in parasite's transmission. For instance, Dore et al, (1980) evidenced genome rearrangements in Plasmodium berghei strains which had lost their ability to infect the vector as they did gametocytes any longer. The understanding of not produce viable the mechanisms involved in differential gene expression, which are at the basis of cellular differentiation, is therefore required to study the parasite's biology as well as to carry out an effective disease control. The isolation of expressed genes constitutes the first step in the study of their structure and expression; molecular cloning of the parasite DNA into procaryotic vectors makes the isolation of single genes feasible. Genomic libraries of Plasmodium falciparum have first been constructed at the Department of Molecular Biology of the University of Edinburgh (Goman et al., 1982). These libraries were screened for expression of different cloned genes without success (Goman et al., 1982). 0309-1651W4/030257-08/$03.0010
@ 1984 Academic
Press Inc. (London)
Ltd.
258
Cell Biology
International
Reports,
Vol. 8, No. 3, March
R.S. Nussenzweig and ~011. have recently constructed a cDNA library of Plasmodium knowlesi and succeeded in isolating a recombinant clone expressing a sporozoite surface antigen under the control of a procaryotic promoter (Ellis et al., 1983). The different strategies adopted indicate that expression in procaryotes has, so far, been successful only when cDNA libraries have been used. This, in turn, suggests that the Plasmodium gene structure is probably complex and not directly translatable in a procaryotic system. To overcome this problem, we used a cloned gene from P. berghei total DNA to catch the complementary mRNA fractionrthe mRNA which hybridized with the gene was eluted and translated in vitro. This approach allows direct investigation of both eucaryotic gene structure and gene product. RESULTS -~ Construction of Plasmodium berghei library The P. berghei strain used NK65 obtained from Wellcome Research Laboratories was serially transmitted by syringe infection in our Institute. We purified Plasmodia from contaminating leucocytes as described by Dore et al. (1980) and extracted high molecular weight DNA ( -3OKb as determined by agarose gel electrophoresis) (Gross-Bellard et al. 1973). The absence of contaminating host DNA was verified by analytical CsCl density gradient. Parasite DNA was digested with BamHI restriction enzyme. Restriction fragments distribution ranged from 5-15Kb as determined by gel electrophoresis of the digest. These fragments were used to construct a genomic library 1978) in BamBI digested A47.1 (Loenen and (Maniatis et al., Brammar, 1980). Following the method of Maniatis (Maniatis et 1982), we calculated that our genomic library was al., representative of about 90% of Plasmodium genome, assuming 1OKb as the average molecular weight of the inserted fragments. Screening of transcribed genes To isolate and characterize expressed genes of P. berghei cloned DNA in the genomic library, we extracted mRNA polyAt from intraerythrocytic stages of the parasites (Auffray and Rougeon, 1980; Aviv and Leder, 1972). As a first step we thoroughly purified parasites from 1980), before lysis in order to leucocytes (Dore et al., eliminate the presence of contaminating mRNA and to avoid nuclease activity. Total RNA was then passed twice through oligo (dT)-column (Aviv and Leder, 1972) to isolate polyadenylate mRNA fraction, which represents about 1% of total RNA. Labelled single stranded DNA, complementary to the mRNA fraction, was synthesized with AMV reverse transcriptase using oligo (dT) as a primer (Efstradiatis et al., 1975). Only
1984
Cell Biology
Fig.
1:
International
Reports,
Vol. 8, No. 3, March
1984
Agarose gel electrophoresis of .il recombinant phage DNA (4PL), digested with BamHI (a). The arrow indicates the 5Kb inserted fragment; the other bands correspond to 1 DNA fragments. The molecular weight of the inserted fragment was calculated by comparison double digested (EcoRI-HindIII), as marker (b).
polyadenylate mRNA will be primed by oligo (dT) and will function as a template, thus eliminating any contamination of residual r-RNA. This cDNA was used as a radioactive probe to screen the genomic library for the presence of expressed genes as described by Benton and Davies (Benton and Davies, 1977). Twelve hybridization positive plaques were found among 1000 recombinant phages tested; hence, approximatively, 1% of cloned DNA fragments were transcribed into mRNAs. Isolation of a single mRNA and of its translation product A first step towards the study of gene expression is the identification of the protein product of a single cloned gene in the pool of parasite proteins, in a cell free system, see fig. 3, lane a. To this aim, we purified DNA from one of the hybridization-positive clones, which was named 4PL. The BamHI digestion pattern showed a 5Kb insert, as shown in fig. 1. The identification of the mRNA-complementary DNA strand was performed by separation of the heavy and light strands of 4PL clone through CsCl gradients after hybridization of denatured DNA with polyUG, which binds only to one DNA strand, making it heavier than the untreated single stranded DNA (Miller, 1972).
259
Cell Biology
260
Fig.
2.
International
Reports,
Vol. 8, No. 3, March
Dot-blot of purified heavy and light strand of 4PL recombinant phage DNA hybridized with labelled cDNA complementary to mKNA as a probe; 1) Millipore filter spotted with light strand of 4PL recombinant phage DNA, in which no hybridization signal appears; 2) Millipore filter spotted with heavy strand of 4PL a recombinant phage DNA: the positive hybridization appears as a dark spot.
0.5 pg of either purified DNA strands were bound to Millipore filters and incybated overnight with 1 !hg of labelled single stranded cDNA ( 10 cpmlyg) complementary of the Plasmodium mBNA. Only the heavy strand gave a positive signal, namely a dark radioactive spot, fig. 2: therefore the light strand was subsequently used to catch the complementary mBNA fraction. 5 pg of the light strand were spotted onto a HAWP Millipore filter and incubated with 5 pg of mKNA for 18 hs (Kafatos, 1979). In order to reduce the volume of the hybridization, the spotted filter was cut down to 3 mm squares and the hybridization was carried out in a 1.5 ml eppendorf tube. The selectively bound mKNA molecules were eluted from the filter (Bozzoni et al., 1981) and translated in a reticulocyte ell free system (NEN translation kit, in 25 ~1 assay, 20 @i 5 Translation products were analyzed in 15% H leucine). acrylamide gel electrophoresis (Laemli, 1970). The gel fluorography revealed a single protein band fig. 3, lane c, whose apparent molecular weight is 32.500 daltons. Background radioactivity (essentially two bands), due to endogenous synthesis of the in vitro system, was present also in translation controls (no messenger added), fig. 3, lane b & d). The presence of a single band in the experimental system, selected by this fig. 3, lane c, suggests that mKNA molecules and belong to a single messenger class. method, are integer DISCUSSION We isolated an expressed gene from a genomic library berghei which is transcribed during the bloodstream stage the life cycle of the parasite.
of P. of -
1984
Cell Biology
International
4 Fig.
3:
Reports,
Vol. 8, No. 3, March
1984
261
b)
Fluorography of gel electrophoresis of hybrid-released translation. Products of cell-free translation were analyzed by 15% acrylamide slab gel electrophoresis (Laemli, 1970); Biorad SDS gel electrophoresis standards (low molecular weight range) were used as molecular weight markers. Slab gel was stained, destained and fluorographed. In 1M sodium salicylate solution for 1 h. Dried gel was exposed to preflashed Kodak-X-Omat film AT - 70°C. a) protein pattern of total polyA+ mRNA from P. berghei translated in rabbit reticulocyte lysze -in vitro cell free system. b) rabbit reticulocyte lysate in vitro cell free system translation incubated without exogenous mRNA as control. c) in vitro translation of purified mRNA complementary to 4PL clone. A unique protein band appears after gel electrophoresis of the translation products.
Cell Biology
262
International
Reports,
Vol. 8, No. 3, March
As the corresponding protein, translated in a cell free system, has a molecular weight of about 32.500 daltons, we can approximately calculate that the coding region within the 5Kb cloned fragment, is about 0.9Kb long. Due to the presence of a single, well defined, protein band the hybrid-released translation experiment indicates that, at least at this stage of development: a) the examined gene doesn't cross-hybridize to any other mRNA transcribed; b) the studied recombinant clone doesn't contain any other expressed c) only one strand of the cloned gene is transcribed. portion; This latter observation rules out, at least in this case, the possibility that this eucaryote utilizes a symmetric reading; this possibility could be suggested by the low value of genetic complexity ( 3.8 times that of E. coli), i.e. one of the lowest value found in eucaryotic organisms. Results obtained by Dore et al. (19801, suggest that repetitive DNA may play a role in P. berghei differentiation. Several regulative roles have been attributed to repetitive sequences within genes or in their flanking regions: differential gene transcription (Britten and Davidson, 1964), nuclear processing (Davidson and Britten, 1979), or selection of mRNAs for transport as well as for other forms of translational control. In the light of these observations, it would be of great interest to investigate the presence of in the flanking sequences of our coding repetitive "modules" region. Finally, the clone can be used as a probe to test whether the gene is expressed at other stage of development, e.g. by purifying mRNA from sporozoites or in other Plasmodium species.
ACKNOWLEDGEMENTS We would like to thank dr. C. Frontali and Dr. M. Gennaro the critical reading of the manuscript. Thanks are also due to Mr. E. Chessa for helping in photography. This study was *partially supported by grant M2/181/12(A) from the WHODivision of Malaria and other Parasitic Diseases. for
REFERENCES 1.
2.
Aviv, H. and Leder, P.: (1972) Purification of biologically active globin mRNA by chromatography on oligothymidilic acid cellulose. Proc. Natl. Acad. Sci. USA, 69, 1408-1412. of mouse Auffray, C. and Rougeon, F.: (1980) Purification immunoglobulin heavy chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107, 303-314.
1984
Cell Biology
3.
4.
5. 6. 7.
8.
9.
10.
11.
12.
13. 14.
15.
International
Reports,
Vol. 8, No. 3, March
1984
Benton, W.D. and Davies, R.W.: (1977) Screening Agt recombinant clones by hybridization to single plaques -in situ. Science 196, 180-182. Bozzoni, I., Beccari, E., Luo, X.Z., Amaldi, F., Pierandrei-Amaldi, P. and Campioni, N.: (1981) Xenopus laevis ribosomal protein genes: isolation of recombinant cDNA clones and study of the genomic organization. Nucl. Acids Res. 9(5), 1069-1085. Britten, R.J. and Davidson, E.H.: (1969) Gene regulation for higher cells: a theory. Science 165, 349-357. Davidson, E.H. and Britten, R.J.: (1979) Regulation of possible role of repetitive sequences. gene expression: Science 204, 1052-1059. C. and Battaglia, P.A.: Dore, E., Birago, C., Frontali, (1980) Kinetic complexity and repetitivity of Plasmodium berghei DNA. Mol. Biochem. Parasitol. 1, 199-208. Efstradiatis, A., Maniatis, T., Kafatos, F.C., Jeffrey, A. and Vournakis, J.N.: (1975) Full length and discrete partial reverse transcripts of globin and chorion mRNAs. Cell 4, 367-378. Ellis, J., Ozaki, L.S., Gwadz, R.W., Cochrane, A.H., Nussenzweig, V., Nussenzweig, R.S. and Godson, G.N.: (1983) Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi. Nature 302, 7 April, 536-538. Goman, M., Langsley, G., Hyde, J.E., Yankovsky, N.K., Werner Zolg, J. and Scaife J.G.: (1982) The establishment of genomic DNA libraries for human malaria parasite P. falciparum and identification of individual clones by hybridization. Mol. Biochem. Parasitol. 5, 391-400. Gross-Bellard, M., Oudet, P. and Chambon, P.: (1973) Isolation of high molecular weight DNA from mammalian cells. Eur. J. Biochem. 36, 32-38. A.: (1979) Kafatos, C.F., Jones, C.W. and Efstradiatis, Determination of nucleic acid sequence homologies and relative concentrations by a dot-hybridization procedure. Nucl. Acids Res. 7, 1541. Laemli, V.K.: (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature New Biology London 227, 680-685. Loenen, W.A.M. and Brammar, W.J.: (1980) A bacteriophage Lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene 10, 249-259. Maniatis, T., Hardison, R.C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, D.K. and Efstradiatis, A.:
263
264
16. 17.
Cell Biology
Jnternational
Reports,
Vol. 8, No. 3, March
(1978) The isolation of structural genes from libraries eucaryotic DNA. Cell 15, 687. Maniatis, T., Fritsch, E.F. and Sambrook, J.: (1982) Molecular cloning - a laboratory manual - page 271 C.S.H.L. Miller, J.H.: (1972) Experiments in Molecular Genetics. Page 331-337. Cold Spring Harbor Laboratory.
Received:
21st
February
1984.
Accepted:
2nd March
1984.
of
1984
International
Reports,
Vol. 8, No. 3, March
1984
257
ISOLATION OF A SINGLE MESSENGERRNA AND OF THE CORRESPONDING GENE IN Plasmodium berghei C. Birago, M. Ponzi, P.A. Battaglia Laboratorio di Biologia Cellulare Istituto Superiore di Sanita, Rome, Italy
SUMMARY A genomic library of Plasmodium berghei DNA was constructed using 147.1 as a vector. It represents 90% of Plasmodium genome. Genes expressed during the intraerythrocytic stage of P. berghei were isolated among the recombinant clones of +he library using labelled cDNA complementary to the polyA Plasmodium mRNA extracted during this stage. The purified coding strand of an expressed clone was utilized to catch the corresponding mRNA(s). The hybridized mRNA fraction was eluted and in vitro translated. Translation products were analyzed by gel electrophoresis; the gel fluorography revealed a single protein band of 32.500 daltons of molecular weight, corresponding to a 900bp coding region in the examined clone. INTRODUCTION Malaria parasites undergo several differentiative stages during their life cycle, some of which have a fundamental role in parasite's transmission. For instance, Dore et al, (1980) evidenced genome rearrangements in Plasmodium berghei strains which had lost their ability to infect the vector as they did gametocytes any longer. The understanding of not produce viable the mechanisms involved in differential gene expression, which are at the basis of cellular differentiation, is therefore required to study the parasite's biology as well as to carry out an effective disease control. The isolation of expressed genes constitutes the first step in the study of their structure and expression; molecular cloning of the parasite DNA into procaryotic vectors makes the isolation of single genes feasible. Genomic libraries of Plasmodium falciparum have first been constructed at the Department of Molecular Biology of the University of Edinburgh (Goman et al., 1982). These libraries were screened for expression of different cloned genes without success (Goman et al., 1982). 0309-1651W4/030257-08/$03.0010
@ 1984 Academic
Press Inc. (London)
Ltd.
258
Cell Biology
International
Reports,
Vol. 8, No. 3, March
R.S. Nussenzweig and ~011. have recently constructed a cDNA library of Plasmodium knowlesi and succeeded in isolating a recombinant clone expressing a sporozoite surface antigen under the control of a procaryotic promoter (Ellis et al., 1983). The different strategies adopted indicate that expression in procaryotes has, so far, been successful only when cDNA libraries have been used. This, in turn, suggests that the Plasmodium gene structure is probably complex and not directly translatable in a procaryotic system. To overcome this problem, we used a cloned gene from P. berghei total DNA to catch the complementary mRNA fractionrthe mRNA which hybridized with the gene was eluted and translated in vitro. This approach allows direct investigation of both eucaryotic gene structure and gene product. RESULTS -~ Construction of Plasmodium berghei library The P. berghei strain used NK65 obtained from Wellcome Research Laboratories was serially transmitted by syringe infection in our Institute. We purified Plasmodia from contaminating leucocytes as described by Dore et al. (1980) and extracted high molecular weight DNA ( -3OKb as determined by agarose gel electrophoresis) (Gross-Bellard et al. 1973). The absence of contaminating host DNA was verified by analytical CsCl density gradient. Parasite DNA was digested with BamHI restriction enzyme. Restriction fragments distribution ranged from 5-15Kb as determined by gel electrophoresis of the digest. These fragments were used to construct a genomic library 1978) in BamBI digested A47.1 (Loenen and (Maniatis et al., Brammar, 1980). Following the method of Maniatis (Maniatis et 1982), we calculated that our genomic library was al., representative of about 90% of Plasmodium genome, assuming 1OKb as the average molecular weight of the inserted fragments. Screening of transcribed genes To isolate and characterize expressed genes of P. berghei cloned DNA in the genomic library, we extracted mRNA polyAt from intraerythrocytic stages of the parasites (Auffray and Rougeon, 1980; Aviv and Leder, 1972). As a first step we thoroughly purified parasites from 1980), before lysis in order to leucocytes (Dore et al., eliminate the presence of contaminating mRNA and to avoid nuclease activity. Total RNA was then passed twice through oligo (dT)-column (Aviv and Leder, 1972) to isolate polyadenylate mRNA fraction, which represents about 1% of total RNA. Labelled single stranded DNA, complementary to the mRNA fraction, was synthesized with AMV reverse transcriptase using oligo (dT) as a primer (Efstradiatis et al., 1975). Only
1984
Cell Biology
Fig.
1:
International
Reports,
Vol. 8, No. 3, March
1984
Agarose gel electrophoresis of .il recombinant phage DNA (4PL), digested with BamHI (a). The arrow indicates the 5Kb inserted fragment; the other bands correspond to 1 DNA fragments. The molecular weight of the inserted fragment was calculated by comparison double digested (EcoRI-HindIII), as marker (b).
polyadenylate mRNA will be primed by oligo (dT) and will function as a template, thus eliminating any contamination of residual r-RNA. This cDNA was used as a radioactive probe to screen the genomic library for the presence of expressed genes as described by Benton and Davies (Benton and Davies, 1977). Twelve hybridization positive plaques were found among 1000 recombinant phages tested; hence, approximatively, 1% of cloned DNA fragments were transcribed into mRNAs. Isolation of a single mRNA and of its translation product A first step towards the study of gene expression is the identification of the protein product of a single cloned gene in the pool of parasite proteins, in a cell free system, see fig. 3, lane a. To this aim, we purified DNA from one of the hybridization-positive clones, which was named 4PL. The BamHI digestion pattern showed a 5Kb insert, as shown in fig. 1. The identification of the mRNA-complementary DNA strand was performed by separation of the heavy and light strands of 4PL clone through CsCl gradients after hybridization of denatured DNA with polyUG, which binds only to one DNA strand, making it heavier than the untreated single stranded DNA (Miller, 1972).
259
Cell Biology
260
Fig.
2.
International
Reports,
Vol. 8, No. 3, March
Dot-blot of purified heavy and light strand of 4PL recombinant phage DNA hybridized with labelled cDNA complementary to mKNA as a probe; 1) Millipore filter spotted with light strand of 4PL recombinant phage DNA, in which no hybridization signal appears; 2) Millipore filter spotted with heavy strand of 4PL a recombinant phage DNA: the positive hybridization appears as a dark spot.
0.5 pg of either purified DNA strands were bound to Millipore filters and incybated overnight with 1 !hg of labelled single stranded cDNA ( 10 cpmlyg) complementary of the Plasmodium mBNA. Only the heavy strand gave a positive signal, namely a dark radioactive spot, fig. 2: therefore the light strand was subsequently used to catch the complementary mBNA fraction. 5 pg of the light strand were spotted onto a HAWP Millipore filter and incubated with 5 pg of mKNA for 18 hs (Kafatos, 1979). In order to reduce the volume of the hybridization, the spotted filter was cut down to 3 mm squares and the hybridization was carried out in a 1.5 ml eppendorf tube. The selectively bound mKNA molecules were eluted from the filter (Bozzoni et al., 1981) and translated in a reticulocyte ell free system (NEN translation kit, in 25 ~1 assay, 20 @i 5 Translation products were analyzed in 15% H leucine). acrylamide gel electrophoresis (Laemli, 1970). The gel fluorography revealed a single protein band fig. 3, lane c, whose apparent molecular weight is 32.500 daltons. Background radioactivity (essentially two bands), due to endogenous synthesis of the in vitro system, was present also in translation controls (no messenger added), fig. 3, lane b & d). The presence of a single band in the experimental system, selected by this fig. 3, lane c, suggests that mKNA molecules and belong to a single messenger class. method, are integer DISCUSSION We isolated an expressed gene from a genomic library berghei which is transcribed during the bloodstream stage the life cycle of the parasite.
of P. of -
1984
Cell Biology
International
4 Fig.
3:
Reports,
Vol. 8, No. 3, March
1984
261
b)
Fluorography of gel electrophoresis of hybrid-released translation. Products of cell-free translation were analyzed by 15% acrylamide slab gel electrophoresis (Laemli, 1970); Biorad SDS gel electrophoresis standards (low molecular weight range) were used as molecular weight markers. Slab gel was stained, destained and fluorographed. In 1M sodium salicylate solution for 1 h. Dried gel was exposed to preflashed Kodak-X-Omat film AT - 70°C. a) protein pattern of total polyA+ mRNA from P. berghei translated in rabbit reticulocyte lysze -in vitro cell free system. b) rabbit reticulocyte lysate in vitro cell free system translation incubated without exogenous mRNA as control. c) in vitro translation of purified mRNA complementary to 4PL clone. A unique protein band appears after gel electrophoresis of the translation products.
Cell Biology
262
International
Reports,
Vol. 8, No. 3, March
As the corresponding protein, translated in a cell free system, has a molecular weight of about 32.500 daltons, we can approximately calculate that the coding region within the 5Kb cloned fragment, is about 0.9Kb long. Due to the presence of a single, well defined, protein band the hybrid-released translation experiment indicates that, at least at this stage of development: a) the examined gene doesn't cross-hybridize to any other mRNA transcribed; b) the studied recombinant clone doesn't contain any other expressed c) only one strand of the cloned gene is transcribed. portion; This latter observation rules out, at least in this case, the possibility that this eucaryote utilizes a symmetric reading; this possibility could be suggested by the low value of genetic complexity ( 3.8 times that of E. coli), i.e. one of the lowest value found in eucaryotic organisms. Results obtained by Dore et al. (19801, suggest that repetitive DNA may play a role in P. berghei differentiation. Several regulative roles have been attributed to repetitive sequences within genes or in their flanking regions: differential gene transcription (Britten and Davidson, 1964), nuclear processing (Davidson and Britten, 1979), or selection of mRNAs for transport as well as for other forms of translational control. In the light of these observations, it would be of great interest to investigate the presence of in the flanking sequences of our coding repetitive "modules" region. Finally, the clone can be used as a probe to test whether the gene is expressed at other stage of development, e.g. by purifying mRNA from sporozoites or in other Plasmodium species.
ACKNOWLEDGEMENTS We would like to thank dr. C. Frontali and Dr. M. Gennaro the critical reading of the manuscript. Thanks are also due to Mr. E. Chessa for helping in photography. This study was *partially supported by grant M2/181/12(A) from the WHODivision of Malaria and other Parasitic Diseases. for
REFERENCES 1.
2.
Aviv, H. and Leder, P.: (1972) Purification of biologically active globin mRNA by chromatography on oligothymidilic acid cellulose. Proc. Natl. Acad. Sci. USA, 69, 1408-1412. of mouse Auffray, C. and Rougeon, F.: (1980) Purification immunoglobulin heavy chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107, 303-314.
1984
Cell Biology
3.
4.
5. 6. 7.
8.
9.
10.
11.
12.
13. 14.
15.
International
Reports,
Vol. 8, No. 3, March
1984
Benton, W.D. and Davies, R.W.: (1977) Screening Agt recombinant clones by hybridization to single plaques -in situ. Science 196, 180-182. Bozzoni, I., Beccari, E., Luo, X.Z., Amaldi, F., Pierandrei-Amaldi, P. and Campioni, N.: (1981) Xenopus laevis ribosomal protein genes: isolation of recombinant cDNA clones and study of the genomic organization. Nucl. Acids Res. 9(5), 1069-1085. Britten, R.J. and Davidson, E.H.: (1969) Gene regulation for higher cells: a theory. Science 165, 349-357. Davidson, E.H. and Britten, R.J.: (1979) Regulation of possible role of repetitive sequences. gene expression: Science 204, 1052-1059. C. and Battaglia, P.A.: Dore, E., Birago, C., Frontali, (1980) Kinetic complexity and repetitivity of Plasmodium berghei DNA. Mol. Biochem. Parasitol. 1, 199-208. Efstradiatis, A., Maniatis, T., Kafatos, F.C., Jeffrey, A. and Vournakis, J.N.: (1975) Full length and discrete partial reverse transcripts of globin and chorion mRNAs. Cell 4, 367-378. Ellis, J., Ozaki, L.S., Gwadz, R.W., Cochrane, A.H., Nussenzweig, V., Nussenzweig, R.S. and Godson, G.N.: (1983) Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi. Nature 302, 7 April, 536-538. Goman, M., Langsley, G., Hyde, J.E., Yankovsky, N.K., Werner Zolg, J. and Scaife J.G.: (1982) The establishment of genomic DNA libraries for human malaria parasite P. falciparum and identification of individual clones by hybridization. Mol. Biochem. Parasitol. 5, 391-400. Gross-Bellard, M., Oudet, P. and Chambon, P.: (1973) Isolation of high molecular weight DNA from mammalian cells. Eur. J. Biochem. 36, 32-38. A.: (1979) Kafatos, C.F., Jones, C.W. and Efstradiatis, Determination of nucleic acid sequence homologies and relative concentrations by a dot-hybridization procedure. Nucl. Acids Res. 7, 1541. Laemli, V.K.: (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature New Biology London 227, 680-685. Loenen, W.A.M. and Brammar, W.J.: (1980) A bacteriophage Lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene 10, 249-259. Maniatis, T., Hardison, R.C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, D.K. and Efstradiatis, A.:
263
264
16. 17.
Cell Biology
Jnternational
Reports,
Vol. 8, No. 3, March
(1978) The isolation of structural genes from libraries eucaryotic DNA. Cell 15, 687. Maniatis, T., Fritsch, E.F. and Sambrook, J.: (1982) Molecular cloning - a laboratory manual - page 271 C.S.H.L. Miller, J.H.: (1972) Experiments in Molecular Genetics. Page 331-337. Cold Spring Harbor Laboratory.
Received:
21st
February
1984.
Accepted:
2nd March
1984.
of
1984