Project: Research project

Project Details


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.
Effective start/end date12/31/8911/30/96