Previously we have shown that linear Yeast Artificial chromosomes (YACs) can carry hundreds of kilobases of DNA, and large complete genes, can be circularized using the vector, pNKG418. Molecules of up to 300 Kb can be transferred successfully into bacteria while maintaining their integrity. These molecules can then be replicated in bacteria and large amounts of DNA can be prepared. This eliminates a limiting factor in studying the genes directly. The circular molecules can be used for further studies, such as transfection into mammalian cells to study their regulation, and to specifically dissect the control elements responsible their tissue-specific expression patterns. We are now extending this method to circularize two YACs, one of which contains the PLAC1 gene, which is expressed specifically in placenta, and a second gene, FOXL2, from chromosome 3, responsible for Premature Ovarian Failure (POF) in some women (see AG000647-05). In conjunction with this approach we are exploring the incorporation into bacterial artificial chromosome clones (BACs) of GFP (green fluorescence protein) as a reporter gene and a mammalian selectable drug marker, using recombination-based methods in bacteria. In preliminary studies a BAC containing PLAC1 gene with at least 60 Kb of upstream sequences retrofitted with IRES-GFP cassette in 3'UTR region gives expression, suggesting that the BAC contains all regulatory elements necessary for expression. This is in contrast to construct with 10 Kb uspstream sequence which was not sufficient to drive gene expression. Further experiments are in progress to tag the transfected PLAC1 with a FLAG epitope and resect the promoter region to define the upstream regulatory regions in DNA and chromatin. Additional experiments with rabbit antibodies raised against selected epitopes suggest that the PLAC1 protein exists as a dimer and is modified from in its native state. Experiments are in progress to analyze the protein further and look for interacting partners by immunoprecipitation. Similar studies will be extended to the FOXL2 gene. The techniques are discussed in two invited reviews.
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| Crisponi, Laura; Uda, Manuela; Deiana, Manila et al. (2004) FOXL2 inactivation by a translocation 171 kb away: analysis of 500 kb of chromosome 3 for candidate long-range regulatory sequences. Genomics 83:757-64 |
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