Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor
Publication Date
November 10, 2009
Journal
PLOS Neglected Tropical Diseases
Authors
Marcela G. Drummond, Carlos E. Calzavara Silva, Diego S. D'astolfo, Fernanda C. Cardoso, et al
Volume
3
Issue
11
Pages
e547
DOI
https://dx.plos.org/10.1371/journal.pntd.0000547
Publisher URL
http://journals.plos.org/plosntds/article?id=10.1371%2Fjournal.pntd.0000547
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/19901992
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770324
Europe PMC
http://europepmc.org/abstract/MED/19901992
Web of Science
000272032900008
Scopus
73449143459
Mendeley
http://www.mendeley.com/research/molecular-characterization-schistosoma-mansoni-zinc-finger-protein-smzf1-transcription-factor
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Mendeley | Further Information

{"title"=>"Molecular characterization of the Schistosoma mansoni zinc finger protein SmZF1 as a transcription factor", "type"=>"journal", "authors"=>[{"first_name"=>"Marcela G.", "last_name"=>"Drummond", "scopus_author_id"=>"12786226000"}, {"first_name"=>"Carlos E.", "last_name"=>"Calzavara-Silva", "scopus_author_id"=>"6506730075"}, {"first_name"=>"Diego S.", "last_name"=>"D'Astolfo", "scopus_author_id"=>"15218881400"}, {"first_name"=>"Fernanda C.", "last_name"=>"Cardoso", "scopus_author_id"=>"13808351000"}, {"first_name"=>"Matheus A.", "last_name"=>"Rajão", "scopus_author_id"=>"24773928400"}, {"first_name"=>"Marina M.", "last_name"=>"Mourão", "scopus_author_id"=>"36790920400"}, {"first_name"=>"Elisandra", "last_name"=>"Gava", "scopus_author_id"=>"13103590700"}, {"first_name"=>"Sérgio C.", "last_name"=>"Oliveira", "scopus_author_id"=>"35426145900"}, {"first_name"=>"Andréa M.", "last_name"=>"Macedo", "scopus_author_id"=>"7101906771"}, {"first_name"=>"Carlos R.", "last_name"=>"Machado", "scopus_author_id"=>"25959133200"}, {"first_name"=>"Sérgio D J", "last_name"=>"Pena", "scopus_author_id"=>"7102688512"}, {"first_name"=>"Gregory T.", "last_name"=>"Kitten", "scopus_author_id"=>"6701345371"}, {"first_name"=>"Glória R.", "last_name"=>"Franco", "scopus_author_id"=>"7102415382"}], "year"=>2009, "source"=>"PLoS Neglected Tropical Diseases", "identifiers"=>{"scopus"=>"2-s2.0-73449143459", "pmid"=>"19901992", "sgr"=>"73449143459", "doi"=>"10.1371/journal.pntd.0000547", "issn"=>"1935-2735", "pui"=>"358052616"}, "id"=>"60ecb27a-dc01-3635-8c72-0213176cce6c", "abstract"=>"BACKGROUND: During its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite. METHODOLOGY/PRINCIPAL FINDINGS: In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p</=0.003) only when its specific binding site was present. CONCLUSION: Taken together, these results strongly support the hypothesis that SmZF1 acts as a transcription factor in S. mansoni.", "link"=>"http://www.mendeley.com/research/molecular-characterization-schistosoma-mansoni-zinc-finger-protein-smzf1-transcription-factor", "reader_count"=>15, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>1, "Student > Master"=>3, "Student > Bachelor"=>2, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>1, "Student > Master"=>3, "Student > Bachelor"=>2, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>12, "Medicine and Dentistry"=>1, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>12}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}}, "reader_count_by_country"=>{"Brazil"=>2}, "group_count"=>0}

Scopus | Further Information

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  • {"files"=>["https://ndownloader.figshare.com/files/434692", "https://ndownloader.figshare.com/files/434708", "https://ndownloader.figshare.com/files/434719"], "description"=>"<div><h3>Background</h3><p>During its development, the parasite <em>Schistosoma mansoni</em> is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a <em>S. mansoni</em> zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite.</p><h3>Methodology/Principal Findings</h3><p>In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p≤0.003) only when its specific binding site was present.</p><h3>Conclusion</h3><p>Taken together, these results strongly support the hypothesis that SmZF1 acts as a transcription factor in <em>S. mansoni</em>.</p></div>", "links"=>[], "tags"=>["molecular", "characterization", "zinc", "smzf1", "transcription"], "article_id"=>145743, "categories"=>["Cell Biology", "Genetics", "Molecular Biology"], "users"=>["Marcela G. Drummond", "Carlos E. Calzavara-Silva", "Diego S. D'Astolfo", "Fernanda C. Cardoso", "Matheus A. Rajão", "Marina M. Mourão", "Elisandra Gava", "Sergio C. Oliveira", "Andréa M. Macedo", "Carlos R. Machado", "Sérgio D. J. Pena", "Gregory T. Kitten", "Glória R. Franco"], "doi"=>["https://dx.doi.org/10.1371/journal.pntd.0000547.s001", "https://dx.doi.org/10.1371/journal.pntd.0000547.s002", "https://dx.doi.org/10.1371/journal.pntd.0000547.s003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Molecular_Characterization_of_the_Schistosoma_mansoni_Zinc_Finger_Protein_SmZF1_as_a_Transcription_Factor/145743", "title"=>"Molecular Characterization of the <em>Schistosoma mansoni</em> Zinc Finger Protein SmZF1 as a Transcription Factor", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2009-11-10 01:35:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/876608"], "description"=>"<p>Fixed parasites were incubated with a rabbit anti-SmZF1 antibody, and then with a Cy-5 conjugated anti-rabbit IgG in a solution containing Alexa Fluor 488 phalloidin to stain actin microfilaments (phalloidin staining was not used with adult male worms). Samples were incubated with propidium iodide to visualize cells nuclei. Fluorescent images were obtained using a 63x oil-immersion objective lens and confocal microscopy (Carl Zeiss LSM 510 META). Images were analyzed with the Zeiss LSM Image Browser software and edited with Adobe Photoshop CS. To help distinguish the individual fluorescence signals, the original fluorescence colors were digitally modified. In the figure, fluorescence from Cy-5 is shown in red, propidium iodide in blue and phalloidin-Alexa fluor 488 in green. The merged blue and red produces a pink color. For adult male worms a phase contrast image is also presented. The following developmental stages were assayed: adult male (A–D) and female (F–I) worms, cercaria (K–N) and schistossomulum (P–S). Rabbit pre-immune serum was used as a control and was negative in all samples (E, J, O and T). Bars = 20 µm.</p>", "links"=>[], "tags"=>["smzf1", "displays", "localization", "stages"], "article_id"=>547047, "categories"=>["Cell Biology", "Genetics", "Molecular Biology"], "users"=>["Marcela G. Drummond", "Carlos E. Calzavara-Silva", "Diego S. D'Astolfo", "Fernanda C. Cardoso", "Matheus A. Rajão", "Marina M. Mourão", "Elisandra Gava", "Sergio C. Oliveira", "Andréa M. Macedo", "Carlos R. Machado", "Sérgio D. J. Pena", "Gregory T. Kitten", "Glória R. Franco"], "doi"=>["https://dx.doi.org/10.1371/journal.pntd.0000547.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Native_SmZF1_displays_a_nuclear_localization_at_diverse_stages_of_S_mansoni_development_/547047", "title"=>"Native SmZF1 displays a nuclear localization at diverse stages of <i>S. mansoni</i> development.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-10 01:57:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/876750"], "description"=>"<p>(A) COS-7 cells were transfected either with pEYFP-c1 (control) or pEYFP-SmZF1 plasmids for transient expression of the proteins YFP or YFP-SmZF1. After 48 h the cells were fixed with paraformaldehyde, quenched, permeabilized and the cells nuclei stained using Hoechst 33342 dye. Images were directly captured at 200x magnification using Carl Zeiss LSM 510 META. In order to obtain a clearer visualization of fluorescent signals in merged images, the original yellow fluorescence of YFP and blue fluorescence of Hoechst were modified to green and red, respectively, using Adobe Photoshop CS2. (B) SmZF1-transfected and control COS-7 cells were used to generate the total (TE), cytoplasmic (CE) and nuclear (NE) extracts. The extracts were submitted to 10% SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were blocked overnight and samples were reacted with anti-myc, anti-GFP or anti-c-erbB-2 peroxidase conjugated antibodies (1∶1000). Subsequently, blots were washed and developed with ECL enhanced chemiluminescence reagents and exposed to X-ray films. The exclusively cytoplasmic human oncoprotein c-erbB-2 was used as a quality control for extracts. YFP can be visualized as an approximately 30 kDa protein and the recombinant protein YFP-SmZF1 as an approximately 50 kDa protein.</p>", "links"=>[], "tags"=>["localization", "cos-7"], "article_id"=>547196, "categories"=>["Cell Biology", "Genetics", "Molecular Biology"], "users"=>["Marcela G. Drummond", "Carlos E. Calzavara-Silva", "Diego S. D'Astolfo", "Fernanda C. Cardoso", "Matheus A. Rajão", "Marina M. Mourão", "Elisandra Gava", "Sergio C. Oliveira", "Andréa M. Macedo", "Carlos R. Machado", "Sérgio D. J. Pena", "Gregory T. Kitten", "Glória R. Franco"], "doi"=>["https://dx.doi.org/10.1371/journal.pntd.0000547.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SmZF1_has_a_nuclear_localization_in_COS_7_cells_/547196", "title"=>"SmZF1 has a nuclear localization in COS-7 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-10 01:59:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/876882"], "description"=>"<p>For EMSA experiments, SmZF1 target oligonucleotide D1-3DNA was incubated with no extract (lane 1) or total extracts of COS-7 cells expressing the proteins YFP (lanes 2–4) or YFP-SmZF1 (lanes 5–7). For supershift reactions, the mixture DNA/extracts was pre-incubated with anti-GFP (lanes 3 and 6) or anti-SmZF1 antibodies (lanes 4 and 7). As negative controls, the anti-GFP and anti-SmZF1 antibodies were also incubated with the oligonucleotide in the absence of cell extracts (lanes 8 and 9, respectively). The resulting 4% PAGE was stained with VISTRA Green DNA specific dye for visualization of bands.</p>", "links"=>[], "tags"=>["heterologously", "cos-7", "cells"], "article_id"=>547322, "categories"=>["Cell Biology", "Genetics", "Molecular Biology"], "users"=>["Marcela G. Drummond", "Carlos E. Calzavara-Silva", "Diego S. D'Astolfo", "Fernanda C. Cardoso", "Matheus A. Rajão", "Marina M. Mourão", "Elisandra Gava", "Sergio C. Oliveira", "Andréa M. Macedo", "Carlos R. Machado", "Sérgio D. J. Pena", "Gregory T. Kitten", "Glória R. Franco"], "doi"=>["https://dx.doi.org/10.1371/journal.pntd.0000547.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SmZF1_heterologously_expressed_in_COS_7_cells_is_able_to_bind_to_its_target_oligonucleotide_/547322", "title"=>"SmZF1 heterologously expressed in COS-7 cells is able to bind to its target oligonucleotide.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-10 02:02:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/876994"], "description"=>"<p>The plasmids of interest plus the TK-Renilla reporter plasmid were transfected into COS-7 cells using the LipofectamineTM 2000 Transfection Reagent. Each transfection was performed in triplicate, and transfection efficiency was normalized to TK-Renilla luciferase reporter plasmid. Luciferase activity was assayed in cell lysates using a TD20/20 luminometer (Promega) and is indicated as Relative Light Units (RLU). Statistical analysis of the data was carried out with Minitab Version 1.4 using Student's t test, and p values<0.05 were considered as significant. Data is representative of three independent experiments.</p>", "links"=>[], "tags"=>["luciferase", "cos-7"], "article_id"=>547444, "categories"=>["Cell Biology", "Genetics", "Molecular Biology"], "users"=>["Marcela G. Drummond", "Carlos E. Calzavara-Silva", "Diego S. D'Astolfo", "Fernanda C. Cardoso", "Matheus A. Rajão", "Marina M. Mourão", "Elisandra Gava", "Sergio C. Oliveira", "Andréa M. Macedo", "Carlos R. Machado", "Sérgio D. J. Pena", "Gregory T. Kitten", "Glória R. Franco"], "doi"=>["https://dx.doi.org/10.1371/journal.pntd.0000547.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SmZF1_is_able_to_increase_luciferase_gene_expression_in_COS_7_cells_/547444", "title"=>"SmZF1 is able to increase luciferase gene expression in COS-7 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-10 02:04:04"}

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