The hypotrichous ciliated protozoans are multinuclear cells. The micronucleus (MI) is generative and is not needed for vegetative growth, which is directed by the macronucleus (MA). The MA is derived from the MI by a complex process in which approximately 90% of the MI sequences are lost, the surviving sequences of the MA existing on short linear DNA duplexes. We are seeking to learn the structure of junctions between MA and non MA sequences in MI DNA, such sites necessarily being acted upon in the sequence diminution process. Three approaches have been initiated. 1) Random cloned MI restriction fragments are being screened by their patterns of hybridization to blots of MA DNA; certain patterns indicate the cloned fragmett carries a MA/non MA junction. 2) These same clones will also be screened for the neck sequence, which we predict to be present at all junctions, because it is found at the ends of all MA DNA duplexes. 3) Cloned MA duplexes will be used as probes to screen a to-be-constructed lambda Charon 4 library of large MI DNA fragments. Any or all of these approaches should allow us to isolate and sequence across several MA/non MA junctions to determine common features. These junction fragments eventually are to be used as substates for the isolation and characterization of proteins involved in the sequence diminution process.
NIH ExPorter
NIH ExPorter