New instrumentation will be developed for pulse field gel electrophoresis, using a contour clamped homogeneous electric field, for the analytical and preparative separation of very large DNA molecules (up to 10 million base pairs). A two dimensional- apparatus for the unique separation of large DNA molecules with a topological constraint is also planned. This is accomplished by tagging a specific DNA molecule by a D-loop promoted by the E. coli RecA reaction. The topological constraint caused by the D- loop will cause a unique mobility in the proposed two dimensional pulse field gel apparatus. This will allow genes for specific human diseases to be isolated, including cystic fibrosis. Techniques for the unique cleavage of high molecular weight DNA will be developed. This makes use of tethering EDTA molecules to a small single-stranded DNA. Complexed with ferrous ion and in the presence of oxygen, duplex DNA can be cleaved when the DNA probe is bound to a large DNA molecule through a paranemic or plectonemic joint catalyzed by the E. coli RecA reaction. It will allow a correlation to be drawn between the human physical and genetic RFLP map. A new yeast vector will be developed for the specific cloning of these very large DNA molecules. This vector will allow a foreign DNA sequence, including human, to be maintained in a yeast cell as an artificial chromosome. It is anticipated that molecules as large as 1 million base pairs can be directly isolated in yeast. A method will be developed for rapidly mapping single base pair changes in large stretches of DNA. This technique relies on the thermodynamics of branch migration allowing a branched DNA molecule to reside at a mismatched location in DNA and cleavage at the branch point by single strand nucleases. Also, a new vector will be developed for the direct cloning of a gene coding for a DNA protein. It will be used to isolate the DNA binding proteins that bind to a yeast centromere and the sequences involved in the cell cycle and DNA damage regulation of yeast ribonucleotide reductase. DNA sequences have been isolated that protect those sequences involved in the maintenance of yeast chromosomes, including centromeres and ARS from transcription. A sensitive, quantitative colony color assay has been developed for the direct measurement of the effectiveness of this protective sequence, thus allowing its detailed investigation.
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