Gregor Siebenkotten

Nucleotide sequence and restriction fragment length polymorphisms of the equine Cε gene

IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Ce gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Ce alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cea and Ceb. Sequence analysis of both, Cea and Ceb cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences have only 95.9% homology of the coding region sequence with the published equine Ce sequence, which could represent a third haplotype. Polymorphism of the IgE heavy chain constant region gene, as described here, might well impose genetic variability on the effector functions of equine IgE predisposition to allergic diseases in horses.

Sensitive analysis of recombination activity using integrated cell surface reporter substrates.

BACKGROUND Recombination processes play a crucial role in the functioning of the immune system and are also involved in mutation events that result in various malignancies. So far the study of recombination activity has frequently relied on the use of reporter substrates that are limited by low sensitivity as well as tedious and distorting readout procedures. METHODS Immunoglobulin class switch recombination substrates were generated which, upon recombination, resulted in the surface expression of human CD4 or murine MHC class I H-2K(k) and thus allowed for cytometric evaluation. RESULTS Recombining cells harboring integrated reporter substrates were analyzed by immunofluorescence and flow cytometry and could easily be isolated by high-gradient magnetic cell sorting (MACS). The analysis was not influenced by cloning efficiencies, as would be the case after drug selection, or prokaryotic recombination that might occur after analysis of recovered substrates in bacteria. In addition, cytometric readout is much faster, as it can be performed immediately after recombination. The substrate exhibited properties compatible with the detection of immunoglobulin class switch recombination and permitted the detection of recombination events down to 10(-5) per cell and generation. CONCLUSIONS The high sensitivity of this system allows precise detection of very rare recombination events and thus permits the study of cell types with extremely low recombination activities.