During antigen-driven differentiation of immunoglobulin- producing cells, a switch from mu to gamma, epsilon, or a heavy chain synthesis can occur. This protein switch is the result of a DNA deletion that moves a V region from a donor C mu to a C gamma, C epsilon, or C alpha gene. This deletion begins and ends in switch segments, DNA regions composed of tandemly repeated sequences. We propose to molecularly clone and sequence the switch recombination sites in six to ten rearranged gamma 3 and gamma 1 genes from hybridoma DNA. The gamma 3 and gamma 1 switch regions each include a characterisitic 49 base pair tandemly repeated sequence. Our previous data indicate that the gamma 3 switches are uniformly distributed throughout its 49mer consensus sequence, but that the S gamma 1 switches are localized to a part of its 49mers consensus sequence. Furthermore, sequences near gamma switch sites suggest that a single enzyme may mediate the cutting/religation for all four gamma genes in the mouse. We will use our new sequence data from gamma switch sites to test these tentative conclusions. We will determine if switch recombination occurs in the tandemly repeated sequences only, or if it can occur in non-49mer sequences in the S gamma 1 region. We will also use data derived from nucleotide sequences of cloned switch regions to understand any correlative relationships in switch sites on the two chromosomes from a single immunoglobulin expressing cell. Under the same group of experiments, we will examine the structure of rearranged switch regions from cells with simple switching patterns (typical switches) and complex switching patterns (atypical switches). We will also examine the switch region content of murine hybridomas generated using an alpha- expressing myeloma partner. We will ask if the two spleen cell- derived Igh loci tend to rearrange to the same switch region, and indication of isotype specificity in the heavy chain switch recombinational event. So that we might understand the DNA sequence recognition, specificity, and genetics of switch recombinases we propose to select a cell line that constitutively produces these enzymes.
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