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 alternative splicing mechanism in which exon 5 of HLA class I heavy chain transcripts is deleted. Reverse transcription-polymerase chain reaction (RT-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 RPMI1788 cells can be selectively or preferentially amplified by RT-PCR with three different DNA polymerases. Cloning and sequencing of the resulting cDNA confirmed that the spliced transcript lacks exon 5. The targeted amplification method may be useful and important for studies with respect to the regulation of class I sHLA expression and the mechanism by which alternative splicing of HLA class I heavy chain mRNA is induced.
Yang
D
D
Department of Microbiology, New York University Medical Center, NY 10016.
Le
J
J
D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005784:Gene Amplification / Q000235:genetics*; D006680:HLA Antigens / Q000235:genetics*; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics
D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I