Worm Breeder's Gazette 15(5): 23 (February 1, 1999)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Searching the genome for DNA sites recognized by binding proteins.

Sara Ederle1, John F. Pulitzer1, Adriana La Volpe2

1 Dipartimento di Genetica e Biologia Generale e Molecolare,, Facolta' di Scienze, Universita' di Napoli "Federico II", , 8, Via Mezzocannone, 80134 Naples, Italy
2 International Institute of Genetics and Biophysics -CNR, 10, Via Marconi, 80125 Naples, Italy, lavolpe@iigbna.iigb.na.cnr.it

        In the past two decades much effort in biology has gone into
        cloning and sequencing of genes. The accomplishment of genome
        sequencing projects, such as that of Caenorhabditis elegans, is
        leading to a new scenario, where all genomic information is
        available, and the remaining challenge is to interpret it in
        biological terms. It is becoming essential to develop strategies
        to link non coding sequence to function.  We have  set up  a one
        hybrid system kit, using C. elegans as model, to search the DNA
        for sites of binding of structural and regulatory proteins. We
        have constructed  yeast strains in which C. elegans  non-coding
        DNA sequences (baits) are inserted, in place of the UAS,
        upstream the two test genes HIS-3 and LacZ. The recipient strain
        is a diploid ura- yeast strain carrying a deletion of the HIS-3
        gene on one chromosome and a wild type copy on the other. We
        have chosen to use diploid strains because the most frequent
        cause of HIS spontaneous reversion is the  transposition of a Ty
        element upstream the HIS coding region;  in diploid strains the
        transposable element Ty is quiescent. Our bait sequences are
        cloned upstream the HIS-3 gene in an integrative plasmid and
        transformed in yeast after restriction digestion.  The 
        integrative plasmid had been  constructed by inserting a
        polylinker into the partially deleted HIS-3 promoter for ease of
        manipulation and it contains homology cassettes appropriate for
        gene replacement and to facilitate the screening of the
        integrants. It contains, in fact, HIS-3 flanking regions for
        proper integration of the plasmid, and the selectable marker
        URA3 in such a position to be deleted, after integration, via
        homologous recombination together with the wild type copy of
        HIS-3. Such event can be selected by plating the recombinant
        strains on FOA (5-fluor-orotic acid), only the ura- cells will
        growth in such conditions. Most pop-outs occur by recombination
        in the extensive tracts  of homology between the recombinant
        HIS-3 sequence and the wild type.  The obtained yeast strains
        are auxotrophic for histidin in the presence of only 5mM
        aminotriazol unless a protein binds to the bait sequence and
        activates transcription by mean of an activation domain such as
        that of GAL-4. This is the selection tool  for the screening of
        our cDNA library (described in Ederle et al. European C. elegans
        Meeting 1996). In all cases the second test gene (LacZ) is 
        carried as an episome by the cell.  We have tested our tools by
        cloning the GATA-box as DNA bait and successfully screening our
        library.  We are now proceeding in using this kit for the
        analysis of gene promoter regions and other DNA sequences
        potentially target of binding proteins.

1) Ederle S, De Felice B, Pulitzer JF, La Volpe A (1996)  A C. elegans
        cDNA library for one hybrid/two hybrid system screening in
        yeast. 2nd European C. elegans Meeting Mont Saint-Odile, France.