TARGETED AMPLIFICATION OF ALTERNATIVELY SPLICED TRANSCRIPTS OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I HEAVY-CHAIN

The RPMI1788 cell line was found to produce soluble form of HLA class I molecules (sHLA) constitutively, due at least in part to an alternat ive splicing mechanism in which exon 5 of HLA class I heavy chain tran scripts is deleted. Reverse transcription-polymerase chain reaction (R T-PCR) of cytoplasmic RNA of RPMI1788 cells using a pair of primers (A ,B) complementary to the conserved sequences of HLA class I exon 4 and 6 yielded almost exclusively the full-length class I heavy chain cDNA . In order to amplify the alternatively spliced transcripts, primer C corresponding to the 5' boundary conserved region of exon 6 juxtaposed with three conserved nucleotides in 3' boundary region of exon 4 was synthesized. Using the primers A and C the spliced transcripts of RPMI 1788 cells can be selectively or preferentially amplified by RT-PCR wi th three different DNA polymerases. Cloning and sequencing of the resu lting cDNA confirmed that the spliced transcript lacks exon 5. The tar geted amplification method may be useful and important for studies wit h respect to the regulation of class I sHLA expression and the mechani sm by which alternative splicing of HLA class I heavy chain mRNA is in duced.