Document Detail


The use of Tn5 transposable elements in a gene trapping strategy for the protozoan Leishmania.
MedLine Citation:
PMID:  17362967     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.
Authors:
Eliane C Laurentino; Jeronimo C Ruiz; Loislene O Brito; Michael Fiandt; Liliana M Nicoletti; M C Jamur; C Oliver; Luiz R O Tosi; Angela K Cruz
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-02-02
Journal Detail:
Title:  International journal for parasitology     Volume:  37     ISSN:  0020-7519     ISO Abbreviation:  Int. J. Parasitol.     Publication Date:  2007 Jun 
Date Detail:
Created Date:  2007-05-02     Completed Date:  2008-01-08     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0314024     Medline TA:  Int J Parasitol     Country:  England    
Other Details:
Languages:  eng     Pagination:  735-42     Citation Subset:  IM    
Affiliation:
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av Bandeirantes, Ribeirão Preto, São Paulo, Brazil.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Base Sequence
DNA Transposable Elements / genetics*
Gene Library
Genomics / methods
Leishmania braziliensis / genetics*
Microscopy, Confocal
Molecular Sequence Data
Mutagenesis, Insertional
Transfection
Chemical
Reg. No./Substance:
0/DNA Transposable Elements

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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