Here we propose to continue our studies on elucidating cis-acting regulatory elements in the mouse kappa immunoglobulin (Ig) gene locus, namely, the elements that target locus accessibility to the recombinational and transcriptional machinery in B lymphocytes. We plan on focusing on the three following regions that are both conceptually attractive as well as experimentally proven to contain accessible chromatin structures and in some cases novel regulatory elements: (1) The upstream sequence (US) 5' of the most distal variable (V) region, which is always preserved after any type of V gene rearrangement; (2) The intervening sequence (IS) between the joining (J) region and the most proximal V region, which upon V-J joining is either deleted or inverted and far removed from the rearranged V gene destined to be expressed; and (3) Germline V region promoter and recombination signal sequences (RSSs). During the previous period of support, our previous results established that these regions in chromatin contain pre-B specific nuclease hypersensitive sites. In addition, we have evidence that both the US and IS contain recombination enhancers and that the IS possesses a developmentally regulated transcriptional silencer. This proposal presents the following three major aims: 1. To functionally elucidate the regulatory DNA sequences within the US. 2. To functionally elucidate the regulatory DNA sequences within the IS. And 3. To investigate possible mechanisms responsible for generating chromatin accessibility within germline V region promoters and recombination signal sequences (RSSs).