The mouse kappa immunoglobulin gene contains a DNA sequence that binds tightly to the nuclear matrix, to topoisomerase II, and to other proteins known to specify higher order chromatin structures. This sequence has been termed a matrix association region (MAR). The mouse kappa gene MAR is adjacent to the intronic transcriptional enhancer. This juxtaposition has been evolutionarily conserved in the rabbit and human kappa genes. In previous studies we found that deletion of the MAR resulted in lower and erratic expression of a rearranged mouse kappa gene, when it was stably integrated into ectopic sites in either mouse plasmacytoma cells or into the mouse germline for the production of transgenic mice. Here we propose to take advantage of the powerful technique of site directed integration in embryonic stem cells to determine the functional significance of this MAR in its natural chromosomal location by addressing the following specific aims: 1. The possible function of the MAR in recombination will be explored in germline alleles containing a MAR deletion introduced by site-directed integration into: a. mouse pre-B cells capable of undergoing V-J joining in culture b. mouse embryonic stem cells for the generation of knockout mice 2. The possible function of the MAR in transcription will be explored in the systems described under specific aim 1. Levels of germline transcripts arising from unrearranged C-kappa-O and V-kappa-O alleles and spliced transcripts arising from rearranged alleles will be assayed in pre-B and B cells, respectively. If its is found in specific aim 1 that the MAR is absolutely required for recombination, then a rearranged kappa allele containing a MAR deletion will be site-directed-integrated into plasmacytoma cells for transcription studies. 3. The possible function of the MAR in somatic hypermutation will be explored in the system described under specific aim 1b, if it is found that the MAR is not absolutely required for recombination. The antiarsonate response will be assessed specifically. 4. The sequence requirements of the MAR to perform functions uncovered from the above studies will be evaluated by mutagenesis and heterologous MAR substitution experiments.
| Li, S; Hammer, R E; George-Raizen, J B et al. (2000) High-level rearrangement and transcription of yeast artificial chromosome-based mouse Ig kappa transgenes containing distal regions of the contig. J Immunol 164:812-24 |
| Liu, X; Zou, H; Widlak, P et al. (1999) Activation of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease). Oligomerization and direct interaction with histone H1. J Biol Chem 274:13836-40 |
| Yi, M; Wu, P; Trevorrow, K W et al. (1999) Evidence that the Igkappa gene MAR regulates the probability of premature V-J joining and somatic hypermutation. J Immunol 162:6029-39 |
| Scheuermann, R H; Garrard, W T (1999) MARs of antigen receptor and co-receptor genes. Crit Rev Eukaryot Gene Expr 9:295-310 |
| Hale, M A; Garrard, W T (1998) A targeted kappa immunoglobulin gene containing a deletion of the nuclear matrix association region exhibits spontaneous hyper-recombination in pre-B cells. Mol Immunol 35:609-20 |
