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.
|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, firstname.lastname@example.org|
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. p.23.