Somatic hypermutation contributes to affinity maturation and the additional diversity of secondary antibodies. An understanding of its mechanism will make it possible to manipulate the mutation process in clinical application, for example, autoimmune diseases and tumors. The goal of this research is to identify cis-elements in the 5'-region of the Vkappa locus that are associated with a functional mutator.
Two specific aims are proposed: (1) To determine whether the 5'-region of a rearranged Vkappa gene, in concert with the VJ region, has a regulatory function in the control of somatic hypermutation, and (2) To determine the specific segment in the 5'-region of a rearranged Vkappa gene that is responsible for the inhibition of the somatic hypermutation. Transgenic mice carrying modified Vkappa8Jkappa5-Ckappa constructs will be made. Germinal center B cells will be sorted from Peyer's patches of these mice. The Vkappa and its flanking regions will be cloned and sequenced. The frequency and distribution of mutation will then be accessed.
In Aim 1, a DNA construct (K+5'oct) with a 3 kb deletion 5' of the Vkappa promoter will be tested to verify our hypothesis that an inhibitory element resides in that region. Then, exon 6 of the murine carbonic anhydrase II gene will replace the VJ exon in the K+5'oct construct to determine if a VJ exon itself is involved in the control of somatic hypermutation, or serves as a specific target for the mutator.
In Aim 2, a series of DNA constructs (K+5't series) with progressive deletions in their 5' region will be tested to localize the specific segment within the 3 kb fragment 5' of Vkappa promoter that is responsible for the inhibition of somatic hypermutation.
