Interleuldn 4 (IL4) is a cytokine mainly produced by T lymphocytes, predominantly expressed in T-helper-2 cells (Mosmann et al. 1986;Romagnani et al. 1991). The gene for IL4 is located on chromosome 5 and spans 10 kilobase (kb), comprising four exons and three introns, encoding a 0.9 kb mRNA (Yokota et al. 1988).
RNA from non-stimulated and Phorbol Myristate Acetate (PMA)stimulated human leukemia and lymphoma cell lines was analyzed for the presence of IL4 mRNA. First-strand cDNA synthesis was performed, using oligo dT primers (oligo (dT)ts. The amplification primers used in the polymerase chain reaction (PCR) were /[[-specific and located in exon 1 (upstream primer: 5'ATGGGTCTCACCTCC-CAACTGCTY) and exon 4 (downstream primer: 5'CGAA-CACTTTGAATATTTCTCTCTCATY). The PCR was performed in a volume of 50 gl containing 0.5 gg of 5' and 3' primers, 2 mM MgC12, 2.5 mM of each deoxynucleotide and 1 unit Taq Polymerase. Amplification was established at 94 Β°C for 5 rain, then followed by 35 cycles of 94 Β°C for 1 min, 60 Β°C for 1 min, 72 Β°C for 2 min, and finally 72 Β°C for 5 min. PCR products were transferred to nitrocellulose and hybridized with radioactively labeled internal oligonucleotide (5'GTCCTTCTCATGGTGGCTGTAGAACTGCCGY) specific for IL4 and located in exon 3. Two different I[[ mRNA products were expressed in all human leukemia and lymphoma cell lines after PMA stimulation, even in cells of B-cell origin.
The PCR products were cloned by TA cloning into the Eco RI site of the plasmid pCR TM II (Invitrogen, San Diego, CA). Sequencing of the cloned PCR products was performed in both directions with the 21M13 and M13PR1 primers of a Taq Dye Primer Cycle Sequencing Kit (Applied Biosystems, CA) using an 373A automated sequencer (Applied Biosystems).
In the B-cell-derived line Namalwa (Burkitt lymphoma; Klein et al. 1972) the normal I[[ transcript and a second transcript, which carried a deletion of 48 bp, were identified. The deletion encompasses the sequence of exon 2 of the IL4 gene (Fig. 1). This alternatively spliced RNA could code for a truncated IL4 protein, as the deletion does not result in a frameshift (Fig. 2).