Expressed genes in interleukin-4 treated B cells identified by cDNA representational difference analysis.
Interleukin-4 (IL-4) stimulates B cell growth and differentiation, such as inducing mature B cells to switch to IgG1 and IgE production. To further characterize IL-4 effects on B cells, we used a sensitive PCR-based subtraction approach to isolate genes expressed in IL-4 treated cells. Our approach combined an adaptation of the genomic representational difference analysis (RDA) method to cDNA analysis with a physical separation method (magnetic bead depletion). This cDNA RDA technique allowed us to perform subtraction on the relatively small number of highly, characterized, purified B cells that can be conveniently prepared. In the hopes of removing genes responsible for general cell growth, we subtracted cDNA made from lipopolysaccharide (LPS)-stimulated B cells from cDNA from LPS+IL-4 stimulated B cells. Two rounds of subtraction resulted in greater than 100-fold enhancement of expected IL-4-induced Cgamma1 cDNA. At that point, we cloned this subtraction library and analysed 154 randomly picked clones for sequence similarities. From these clones, 37 individual genes were obtained. Most of these genes (30) could be functionally identified by sequence similarity. These included genes encoding Cgamma1 (1), cytoskeletal components (4) and products involved in DNA replication (3), metabolism (5), signal transduction (4), transcription (4), translation (6) and transport (3). Only 7 genes had no similarity to known sequences in the GenBank, EMBL or Swiss Prot databases. One unknown gene (designated Fig1 for IL-Four Induced Gene 1) and one gene with homology to the human transcription factor E4BP4 were confirmed by Northern blot analysis to be induced 10-20-fold by IL-4 treatment. This list of expressed genes in LPS + IL-4 treated B cells may shed further insight on the action and mechanism of IL-4 stimulation of cells.
Chu
C C
CC
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1892, USA. cchu@nshs.edu
Paul
W E
WE
D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D005260:Female; D005784:Gene Amplification / Q000235:genetics; D015870:Gene Expression; D018189:Immunomagnetic Separation / Q000379:methods; D015847:Interleukin-4 / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis
D018076:DNA, Complementary; D012333:RNA, Messenger; D015847:Interleukin-4