The objective of the work is to identify and characterize special mechanisms for stimulating recombination among the nearly identical repeat units of certain multigene families. Such mechanisms may be important for maintaining sequence homogeneity among the repeat units of the ribosomal RNA genes and the histone genes. In addition, they would play an important role in the evolution of these multigene families. A fragment of the ribosomal DNA of the yeast S. cerevisiae that stimulates recombination in flanking sequences has been identified. The precise DNA sequence within this fragment that is required to stimulate recombination will be determined. Subclones of this fragment will be used initially to localize the important sequences. In vitro mutagenesis of the fragment will be used to precisely identify the sequences responsible for this activity. Preliminary data from subclones suggests that highly efficient transcription from the initiation site for the ribosomal RNA precursor is required to stimulate exchange. Therefore, we will attempt to correlate the stimulation of recombination by this sequence with transcription initiated in the fragment. Trans-acting mutations that specifically affect the recombination-stimulatory activity of this fragment will be isolated. These mutations will be genetically and biochemically characterized to further study the mechanism responsible for the recombination-stimulatory activity. This characterization will include assaying the effect of these mutation on ribosomal DNA transcription. The genes encoding the trans-acting functions will be cloned. These genes and their products will then be characterized using a combination of in vivo and in vitro techniques to determine their mode of action. Parameters that affect the activity of this recombination hotspot will also be rigorously characterized. The parameters to be studied include the distance of DNA over which the hotspot stimulates recombination and whether one or both of the recombining genes must be acted on by the hotspot. Studies to determine whether such recombination-stimulatory sequences occur in the multigene families of other organisms will also be performed. Fragments of the 5S ribosomal DNA from X. laevis and the D. melanogaster histone genes will be studied for their ability to stimulate recombination in yeast. If these sequences stimulate recombination, studies to determine the mechanism by which they function will be initiated.
