Polymerase chain reaction analysis of transcriptional patterns of expression of class I HLA genes.
In order to study transcriptional patterns of expression of individual class I HLA genes we have constructed a series of cDNA libraries from human cell lines including normal lymphoblastoid cell lines MANN and HOM2, two colorectal carcinoma cell lines, WiDr and SW480, and a fetal lung fibroblast cell line, MRC-5. Between 0.5 and 1 x 10(6) independent clones were screened in each library using a class I HLA-specific DNA probe and the frequency of class I HLA cDNA clones was found to vary between 0.23% (WiDr) and 0.76% (HOM2). Polymerase chain reaction (PCR)-based analyses of possible alternative splicing events showed that each of 161 class I HLA cDNA clones which had insert sizes exceeding 0.6 kb exhibited normal splicing patterns for exons 5 and 6. Similar PCR-based analyses in clones with appropriately large inserts revealed no exceptions to the normal splicing patterns for each of exons 2, 3, 4, and 7. Sixty of the class I HLA cDNA clones selected from the WiDr, MRC-5, and MANN cDNA libraries were assigned to individual loci following identification of locus-specific DNA sequences by PCR sequencing across exon 5. The sequences obtained from the 60 clones were each interpreted to correspond to one of the classical loci, HLA-A, HLA-B, and HLA-C. While representatives of the HLA-A locus predominated in the MANN library, HLA-B-specific clones were the most abundant in the WiDr and MRC-5 libraries.
Summers
C W
CW
University Department of Medical Genetics, St. Mary's Hospital, Manchester, United Kingdom.
Woodcock
V
V
Pook
M A
MA
Strachan
T
T
D001483:Base Sequence; D002460:Cell Line; D016384:Consensus Sequence; D004247:DNA / Q000032:analysis; D015342:DNA Probes; D004587:Electrophoresis, Agar Gel; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D015723:Gene Library; D006680:HLA Antigens / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D006680:HLA Antigens; D004247:DNA