Recovery And Study Of Yeast Artificial Chromosomes In Ci

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Abstract

Previously we have shown that linear Yeast Artificial chromosomes (YACs) which carry hundreds of kilobases of DNA, in some instances complete gene or genes, can be circularized using the vector, pNKG418, and molecules of up to 300 Kb can be transferred successfully into bacteria while maintaining their integrity during the process. These molecules can then be replicated in bacteria and large amounts of DNA can be prepared, which is a limiting factor in studying the YACs directly. The circular molecules can then 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 have now used this method to circularize two YACs, one of which contains PLAC1 gene, which is expressed specifically in placenta and a second gene FOXL2 from chromosome 3, responsible for Premature Ovarian Failure (POF) in women and lies upstream of a translocation breakpoint (see AG000647-05). We are in the process of retrofitting the circular molecules with a mammalian selectable marker, following which we will transfer the circular molecule to bacteria, isolate large quantity of DNA for transfection studies and study their regulation in appropriate cell lines. Following the demonstration that the cell lines can mimic in vivo regulation, we plan to probe the chromatin structure of the artificial chromosome in such cell lines. Further, we plan to study the sequences responsible for such regulation by targeted deletion analysis which will be constructed once again in yeast harboring the circular molecule and transferring the deleting versions into the cell lines to see its effect. The techniques are discussed in two invited reviews.