ISOLATION OF DIFFERENTIALLY EXPRESSED GENES UPON IMMUNOGLOBULIN CLASSSWITCHING BY A SUBTRACTIVE HYBRIDIZATION METHOD USING URACIL DNA GLYCOSYLASE

Immunoglobulin class switch recombination enables B lymphocytes to seq uentially express antibodies that have identical specificities but tha t differ in class and effector function, Although several cis elements required for class switch recombination have been identified, few tra ns-acting factors which are directly related to class switching have b een found, Previously we have developed an efficient in vitro class sw itching system using a cell line, CH12F3-2. To clarify the molecular m echanism of class switching, we intended to isolate genes induced with interleukin (IL)-4, transforming growth factor (TGF)-beta and CD40L u sing the in vitro class switching system, For that purpose, an improve d method for making subtracted cDNA libraries, using uracil DNA glycos ylase, has been developed, This method can overcome a general problem of subtraction, that rare cDNAs are easily lost, This new subtraction method was applied to the CH12F3-2 switching system to isolate genes i nduced by stimulations with IL-4, TGF-beta and CD40L, and cDNAs encodi ng deiodinase 1 and SS32, an alternatively spliced form of the muscle LIM protein, were obtained, Their expression patterns in response to v arious combinations of stimuli and the time courses of the induction s upported the usefulness of this method.