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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA039068-10
Application #
3177822
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1984-09-01
Project End
1992-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
10
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Collins, John T; Shi, Jian; Burrell, Bryna E et al. (2006) Induced expression of murine gamma2a by CD40 ligation independently of IFN-gamma. J Immunol 177:5414-9
Dunnick, Wesley A; Shi, Jian; Graves, Kevin A et al. (2005) The 3' end of the heavy chain constant region locus enhances germline transcription and switch recombination of the four gamma genes. J Exp Med 201:1459-66
Gao, Ning; Dang, Tam; Dunnick, Wesley A et al. (2005) Receptors and counterreceptors involved in NK-B cell interactions. J Immunol 174:4113-9
Dunnick, Wesley A; Shi, Jian; Graves, Kevin A et al. (2004) Germline transcription and switch recombination of a transgene containing the entire H chain constant region locus: effect of a mutation in a STAT6 binding site in the gamma 1 promoter. J Immunol 173:5531-9
Berton, Michael T; Linehan, Leslie A; Wick, Kerilyn R et al. (2004) NF-kappaB elements associated with the Stat6 site in the germline gamma1 immunoglobulin promoter are not necessary for the transcriptional response to CD40 ligand. Int Immunol 16:1741-9
Williams, Bret R; Mirzoeva, Olga K; Morgan, William F et al. (2002) A murine model of Nijmegen breakage syndrome. Curr Biol 12:648-53
Adams, K; Ackerly, H; Cunningham, K et al. (2000) A DNase I hypersensitive site near the murine gamma1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation. Int Immunol 12:1705-13
Collins, J T; Dunnick, W A (1999) Cutting edge: IFN-gamma regulated germline transcripts are expressed from gamma2a transgenes independently of the heavy chain 3' enhancers. J Immunol 163:5758-62
Cunningham, K; Ackerly, H; Claflin, L et al. (1998) Germline transcription and recombination of a murine VDJmudeltagamma1 transgene. Int Immunol 10:1027-37
Cunningham, K; Ackerly, H; Alt, F et al. (1998) Potential regulatory elements for germline transcription in or near murine Sgamma1. Int Immunol 10:527-36

Showing the most recent 10 out of 30 publications