- INTRODUCTION
- MATERIALS AND METHODS
- Mosquito maintenance and the parasite transmission cycle.
- cDNA libraries.
- Subtractive hybridization protocol.
- Isolation of the Pg93 genomic clone.
- Sporozoite and sporozoite-infected tissue isolation.
- RNA isolation.
- Rapid amplification of cDNA ends.
- Transcriptional expression analysis by RT-PCR.
- DNA isolation and sequence determination of Pg93.
- Degenerate PCR reactions.
- Recombinant protein expression and purification.
- Antibody production and purification.
- Immunoblotting.
- Confocal microscopy.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 24 :3389–3402.
-
2
Angerer L, 1989. In situ hybrization: Methods for detecting DNA and RNA sequences at cellular and subcellular resolution. American Society of Cellular Biology Workshop Manual, 1–21.
-
3
Appel RD, Bairoch A, Hochstrasser DF, 1994. A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. Trends Biochem Sci 19 :258–260.
-
4
Ausbel FM, Breant R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, 2000. Current Protocols in Molecular Biology. Canada: John Wiley & Sons.
-
5
Beerntsen BT, Champagne DE, Coleman JL, Campos YA, James AA, 1999. Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti.Insect Mol Biol 8 :459–467.
-
6
Clemons DJ, Besch-Williford C, Steffen EK, Riley LK, Moore DH, 1992. Evaluation of a subcutaneously implanted chamber for antibody production in rabbits. Lab Anim Sci 42 :307–311.
-
7
Fidock DA, Nguyen TV, Beerntsen BT, James AA, 2002. Production of stage-specific Plasmodium falciparum cDNA libraries using subtractive hybridization. Methods Mol Med 72 :277–289.
-
8
Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ, 2002. A proteomic view of the Plasmodium falciparum life cycle. Nature 419 :520–526.
-
9
Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DM, Fairlamb AH, Fraunholz MJ, Roos DS, Ralph SA, McFadden GI, Cummings LM, Subramanian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C, Davis RW, Fraser CM, Barrell B, 2002. Genome sequence of the human malaria parasite Plasmodium falciparum.Nature 419 :498–511.
-
10
Garnham PCC, 1966. Malaria Parasites and Other Haemosporidia. Oxford: Blackwell Scientific, 593–597.
-
11
Greenwood B, Mutabingwa T, 2002. Malaria in 2002. Nature 415 :670–672.
-
12
Hastings ML, Krainer AR, 2001. Pre-mRNA splicing in the new millennium. Curr Opin Cell Biol 13 :302–309.
-
13
Hayes RO, 1953. Determination of a physiological saline for Aedes aegypti (L.). J Econ Entomol 46 :624–627.
-
14
Hewitt R, 1949. Synopsis of species of avian malaria parasites. Boyd, MF, ed. Malariology. Philadelphia: W.B. Saunders, 148–154.
-
15
Houge G, 1993. Simplified construction of a subtracted cDNA library using asymmetric PCR. PCR Methods Appl 3 :204–209.
-
16
Ibrahim MS, Eisinger SW, Scott AL, 1996. Muscle actin gene from Aedes aegypti (Diptera:Culicidae). J Med Entomol 33 :955–962.
-
17
James AA, Beerntsen BT, Capurro M, Coates CJ, Coleman J, Jasinskiene N, Krettli AU, 1999. Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system. Parassitologia 41 :461–471.
-
18
Kaiser K, Matuschewski K, Camargo N, Ross J, Kappe SH, 2004. Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins. Mol Microbiol 51 :1221–1232.
-
19
Kappe SH, Gardner MJ, Brown SM, Ross J, Matuschewski K, Ribeiro JM, Adams JH, Quackenbush J, Cho J, Carucci DJ, Hoffman SL, Nussenzweig V, 2001. Exploring the transcriptome of the malaria sporozoite stage. Proc Natl Acad Sci USA 98 :9895–9900.
-
20
Kozak M, 1987. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 196 :947–950.
-
21
Krettli AU, Rocha EM, Lopes JD, Carneiro CR, Kamboj KK, Cochrane AH, Nussenzweig RS, 1988. Circumsporozoite protein of Plasmodium gallinaceum characterized by monoclonal antibodies. Parasite Immunol 10 :523–533.
-
22
Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, De La Vega P, Holder AA, Batalov S, Carucci DJ, Winzeler EA, 2003. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301 :1503–1508.
-
23
Matuschewski K, Ross J, Brown SM, Kaiser K, Nussenzweig V, Kappe SH, 2002. Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage. J Biol Chem 44 :41948–41953.
-
24
McCutchan TF, Kissinger JC, Touray MG, Rogers MJ, Li J, Sullivan M, Braga EM, Krettli AU, Miller LH, 1996. Comparison of circumsporozoite proteins from avian and mammalian malarias: Biological and phylogenetic implications. Proc Natl Acad Sci USA 93 :11889–11894.
-
25
Nakai K, Horton P, 1999. PSORT: a program for detecting the sorting signals of proteins and predicting their subcellular localization. Trends Biochem Sci 24 :34–35.
-
26
Nielsen H, Engelbrecht J, Brunak S, von Heijne G, 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10 :1–6.
-
27
Putnam CD, Copenhaver GP, Denton ML, Pikaard CS, 1994. The RNA polymerase I transactivator upstream binding factor requires its dimerisation domain and high-mobility-group (HMG) box 1 to bend, wrap and positively supercoil enhancer DNA. Mol Cell Biol 14 :6476–6488.
-
28
Robson KJ, Naitza S, Barker G, Sinden RE, Crisanti A, 1997. Cloning and expression of the thrombospondin related adhesive protein gene of Plasmodium berghei.Mol Biochem Parasitol 84 :1–12.
-
29
Sambrook J, Russell DW, 2001. Molecular Cloning: A Laboratory Manual. 3rd edition. New York: Cold Spring Harbor Laboratories Press.
-
30
Schultz RM, Liebman MN, 1997. Proteins II: structure-function and relationships in protein families. Devlin, TM, ed. Textbook of Biochemistry with Clinical Correlations. Sixteenth edition. New York: John Wiley & Sons, 108–114.
-
31
Stratmann T, Schmid SR, Harper JF, Kang AS, 1997. Bacterial expression and purification of recombinant Plasmodium yoelii circumsporozoite protein. Protein Expr Purif 11 :72–78.
-
32
Struhl K, 1989. Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eukaryotic transcriptional regulatory proteins. Trends Biochem Sci 14 :137–140.
-
33
Su XZ, Wu Y, Sifri CD, Wellems TE, 1996. Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res 24 :1574–1575.
-
34
The Plasmodium Genome Database Collaborative, 2001. PlasmoDB: An integrative database of the Plasmodium falciparum genome. Tools for analyzing finished and unfinished sequence data. Nucleic Acids Res 29 :66–69.
-
35
Touray M, Warburg A, Laughinghouse A, Krettli AU, Miller LH, 1992. Developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host. J Exp Med 175 :1607–1612.
-
36
Waters AP, Higgins DG, McCutchan TF, 1994. Plasmodium falciparum appears to have arisen as a result of lateral transfer between avian and human hosts. Proc Natl Acad Sci USA 88 :3140–3144.
-
37
Wattam AR, Christensen BM, 1992. Induced polypeptides associated with filarial worm refractoriness in Aedes aegypti.Proc Natl Acad Sci USA 89 :6502–6505.
-
39
Yoshihara CM, Lee JD, Dodgson JB, 1997. The chicken carbonic anhydrase II gene: evidence for a recent shift in intron position. Nucleic Acids Res 15 :753–770.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 125 | 109 | 14 |
| Full Text Views | 186 | 2 | 0 |
| PDF Downloads | 24 | 2 | 0 |
THE NOVEL PLASMODIUM GALLINACEUM SPOROZOITE PROTEIN, PG93, IS PREFERENTIALLY EXPRESSED IN THE NUCLEUS OF OOCYST SPOROZOITES
ALEXIS N. LACRUE
ALEXIS N. LACRUE
Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri; Microbiology & Molecular Genetics, Molecular Biology & Biochemistry, University of California, Irvine, California
Search for other papers by ALEXIS N. LACRUE in
Current site
Google Scholar
PubMed
Search for other papers by ALEXIS N. LACRUE in
Current site
Google Scholar
PubMed
ANTHONY A. JAMES
ANTHONY A. JAMES
Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri; Microbiology & Molecular Genetics, Molecular Biology & Biochemistry, University of California, Irvine, California
Search for other papers by ANTHONY A. JAMES in
Current site
Google Scholar
PubMed
Search for other papers by ANTHONY A. JAMES in
Current site
Google Scholar
PubMed
BRENDA T. BEERNTSEN
BRENDA T. BEERNTSEN
Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri; Microbiology & Molecular Genetics, Molecular Biology & Biochemistry, University of California, Irvine, California
Search for other papers by BRENDA T. BEERNTSEN in
Current site
Google Scholar
PubMed
Search for other papers by BRENDA T. BEERNTSEN in
Current site
Google Scholar
PubMed
To study gene expression differences between oocyst and salivary gland sporozoites, cDNA libraries previously constructed from the two sporozoite populations of the avian malaria parasite, Plasmodium gallinaceum, were used in a subtractive hybridization protocol to isolate Pg93, a novel oocyst sporozoite gene. Pg93 encodes a putative ~76 kDa translated protein that was predicted to localize to the nucleus. Transcriptional analysis indicates that Pg93 is preferentially expressed in oocyst sporozoites versus salivary gland sporozoites. Immunolocalization assays confirm both the nuclear prediction and transcriptional analysis, suggesting that Pg93 is a nuclear protein. BLAST sequence analysis indicates that Pg93 represents a novel gene that has significant homology with a Plasmodium falciparum hypothetical protein and translated Plasmodium knowlesi and Plasmodium vivax nucleotide sequences. This is the first characterization of a Plasmodium nuclear protein that shows preferential expression in one sporozoite population as compared with the other population.
Author Notes
- INTRODUCTION
- MATERIALS AND METHODS
- Mosquito maintenance and the parasite transmission cycle.
- cDNA libraries.
- Subtractive hybridization protocol.
- Isolation of the Pg93 genomic clone.
- Sporozoite and sporozoite-infected tissue isolation.
- RNA isolation.
- Rapid amplification of cDNA ends.
- Transcriptional expression analysis by RT-PCR.
- DNA isolation and sequence determination of Pg93.
- Degenerate PCR reactions.
- Recombinant protein expression and purification.
- Antibody production and purification.
- Immunoblotting.
- Confocal microscopy.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 24 :3389–3402.
-
2
Angerer L, 1989. In situ hybrization: Methods for detecting DNA and RNA sequences at cellular and subcellular resolution. American Society of Cellular Biology Workshop Manual, 1–21.
-
3
Appel RD, Bairoch A, Hochstrasser DF, 1994. A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. Trends Biochem Sci 19 :258–260.
-
4
Ausbel FM, Breant R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, 2000. Current Protocols in Molecular Biology. Canada: John Wiley & Sons.
-
5
Beerntsen BT, Champagne DE, Coleman JL, Campos YA, James AA, 1999. Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti.Insect Mol Biol 8 :459–467.
-
6
Clemons DJ, Besch-Williford C, Steffen EK, Riley LK, Moore DH, 1992. Evaluation of a subcutaneously implanted chamber for antibody production in rabbits. Lab Anim Sci 42 :307–311.
-
7
Fidock DA, Nguyen TV, Beerntsen BT, James AA, 2002. Production of stage-specific Plasmodium falciparum cDNA libraries using subtractive hybridization. Methods Mol Med 72 :277–289.
-
8
Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ, 2002. A proteomic view of the Plasmodium falciparum life cycle. Nature 419 :520–526.
-
9
Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DM, Fairlamb AH, Fraunholz MJ, Roos DS, Ralph SA, McFadden GI, Cummings LM, Subramanian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C, Davis RW, Fraser CM, Barrell B, 2002. Genome sequence of the human malaria parasite Plasmodium falciparum.Nature 419 :498–511.
-
10
Garnham PCC, 1966. Malaria Parasites and Other Haemosporidia. Oxford: Blackwell Scientific, 593–597.
-
11
Greenwood B, Mutabingwa T, 2002. Malaria in 2002. Nature 415 :670–672.
-
12
Hastings ML, Krainer AR, 2001. Pre-mRNA splicing in the new millennium. Curr Opin Cell Biol 13 :302–309.
-
13
Hayes RO, 1953. Determination of a physiological saline for Aedes aegypti (L.). J Econ Entomol 46 :624–627.
-
14
Hewitt R, 1949. Synopsis of species of avian malaria parasites. Boyd, MF, ed. Malariology. Philadelphia: W.B. Saunders, 148–154.
-
15
Houge G, 1993. Simplified construction of a subtracted cDNA library using asymmetric PCR. PCR Methods Appl 3 :204–209.
-
16
Ibrahim MS, Eisinger SW, Scott AL, 1996. Muscle actin gene from Aedes aegypti (Diptera:Culicidae). J Med Entomol 33 :955–962.
-
17
James AA, Beerntsen BT, Capurro M, Coates CJ, Coleman J, Jasinskiene N, Krettli AU, 1999. Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system. Parassitologia 41 :461–471.
-
18
Kaiser K, Matuschewski K, Camargo N, Ross J, Kappe SH, 2004. Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins. Mol Microbiol 51 :1221–1232.
-
19
Kappe SH, Gardner MJ, Brown SM, Ross J, Matuschewski K, Ribeiro JM, Adams JH, Quackenbush J, Cho J, Carucci DJ, Hoffman SL, Nussenzweig V, 2001. Exploring the transcriptome of the malaria sporozoite stage. Proc Natl Acad Sci USA 98 :9895–9900.
-
20
Kozak M, 1987. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 196 :947–950.
-
21
Krettli AU, Rocha EM, Lopes JD, Carneiro CR, Kamboj KK, Cochrane AH, Nussenzweig RS, 1988. Circumsporozoite protein of Plasmodium gallinaceum characterized by monoclonal antibodies. Parasite Immunol 10 :523–533.
-
22
Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, De La Vega P, Holder AA, Batalov S, Carucci DJ, Winzeler EA, 2003. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301 :1503–1508.
-
23
Matuschewski K, Ross J, Brown SM, Kaiser K, Nussenzweig V, Kappe SH, 2002. Infectivity-associated changes in the transcriptional repertoire of the malaria parasite sporozoite stage. J Biol Chem 44 :41948–41953.
-
24
McCutchan TF, Kissinger JC, Touray MG, Rogers MJ, Li J, Sullivan M, Braga EM, Krettli AU, Miller LH, 1996. Comparison of circumsporozoite proteins from avian and mammalian malarias: Biological and phylogenetic implications. Proc Natl Acad Sci USA 93 :11889–11894.
-
25
Nakai K, Horton P, 1999. PSORT: a program for detecting the sorting signals of proteins and predicting their subcellular localization. Trends Biochem Sci 24 :34–35.
-
26
Nielsen H, Engelbrecht J, Brunak S, von Heijne G, 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10 :1–6.
-
27
Putnam CD, Copenhaver GP, Denton ML, Pikaard CS, 1994. The RNA polymerase I transactivator upstream binding factor requires its dimerisation domain and high-mobility-group (HMG) box 1 to bend, wrap and positively supercoil enhancer DNA. Mol Cell Biol 14 :6476–6488.
-
28
Robson KJ, Naitza S, Barker G, Sinden RE, Crisanti A, 1997. Cloning and expression of the thrombospondin related adhesive protein gene of Plasmodium berghei.Mol Biochem Parasitol 84 :1–12.
-
29
Sambrook J, Russell DW, 2001. Molecular Cloning: A Laboratory Manual. 3rd edition. New York: Cold Spring Harbor Laboratories Press.
-
30
Schultz RM, Liebman MN, 1997. Proteins II: structure-function and relationships in protein families. Devlin, TM, ed. Textbook of Biochemistry with Clinical Correlations. Sixteenth edition. New York: John Wiley & Sons, 108–114.
-
31
Stratmann T, Schmid SR, Harper JF, Kang AS, 1997. Bacterial expression and purification of recombinant Plasmodium yoelii circumsporozoite protein. Protein Expr Purif 11 :72–78.
-
32
Struhl K, 1989. Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eukaryotic transcriptional regulatory proteins. Trends Biochem Sci 14 :137–140.
-
33
Su XZ, Wu Y, Sifri CD, Wellems TE, 1996. Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res 24 :1574–1575.
-
34
The Plasmodium Genome Database Collaborative, 2001. PlasmoDB: An integrative database of the Plasmodium falciparum genome. Tools for analyzing finished and unfinished sequence data. Nucleic Acids Res 29 :66–69.
-
35
Touray M, Warburg A, Laughinghouse A, Krettli AU, Miller LH, 1992. Developmentally regulated infectivity of malaria sporozoites for mosquito salivary glands and the vertebrate host. J Exp Med 175 :1607–1612.
-
36
Waters AP, Higgins DG, McCutchan TF, 1994. Plasmodium falciparum appears to have arisen as a result of lateral transfer between avian and human hosts. Proc Natl Acad Sci USA 88 :3140–3144.
-
37
Wattam AR, Christensen BM, 1992. Induced polypeptides associated with filarial worm refractoriness in Aedes aegypti.Proc Natl Acad Sci USA 89 :6502–6505.
-
39
Yoshihara CM, Lee JD, Dodgson JB, 1997. The chicken carbonic anhydrase II gene: evidence for a recent shift in intron position. Nucleic Acids Res 15 :753–770.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 125 | 109 | 14 |
| Full Text Views | 186 | 2 | 0 |
| PDF Downloads | 24 | 2 | 0 |