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Resources » Paper

Abad P et al. (1991) International C. elegans Meeting "DNA binding activity of the putative transposase of Tcl element (TcA) overexpressed in a baculovirus system."

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  • Comments on Abad P et al. (1991) International C. elegans Meeting "DNA binding activity of the putative transposase of Tcl element (TcA) overexpressed in a baculovirus system." (0)

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    Status:
    Publication type:
    Meeting_abstract
    WormBase ID:
    WBPaper00020967

    Abad P, Cerutti M, & Devauchelle G (1991). DNA binding activity of the putative transposase of Tcl element (TcA) overexpressed in a baculovirus system presented in International C. elegans Meeting. Unpublished information; cite only with author permission.

    The aim of our work is to analyse the mechanism of Tcl transposition in Caenorhabditis elegans by carrying out the reaction in vitro . To do so, we choosed at first to overproduce the transposase of Tcl (TcA) in a baculovirus vector. C.elegans TcA protein obtained from the pIM55 plasmid DNA has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedosis virus (Ac NPV ). Recombinant Ac NPV s have been isolated and their genomes have been partly characterized upon the location of the foreign DNA insert. Upon infection of S.frugiperda cells with the recombinant Ac NPV, C.elegans Tcl element specific messenger RNAs, as well as newly synthesized polypeptide with an apparent molecular weight of about 35 kD have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. TcA is detected by antisera which have been raised against fusion proteins containing TcA sequences synthesized in pAR vector of E.coli in Western blotting experiments. The TcA specific protein is nuclear and exhibits a strong DNA binding activity for both single stranded and double stranded DNA. We have purified the overproduced TcA and shown that this protein is partially soluble from 0.7M NaCl upwards. In the next step we intend to make DNA binding experiments with different parts of the Tcl sequence in a gel retardation assays. In soluble extract proteins of C.elegans bergerac RW7000 strain and using antisera made with TcA fusion protein in pAR vector, we detect a protein wich has the same apparent molecular weight as the polypeptide overproduced in baculovirus. This protein seems to be absent in C. elegans bristol N2 strain. We have obtained the same result with antisera which have been raised against TcA overproduced in baculovirus vector.


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