FIGURE 7.

Selective HLA-A2 Ag loss caused by a single cytosine deletion in the HLA-A2 gene exon 4 in 1259MEL melanoma cells. A, Diagram of HLA-A*0201 mRNA structure marked with the location of the primers (arrows) and of the premature stop codon (1259stop) identified in 1259MEL cells. B, HLA-A*0201 mRNA expression by 1259MEL cells. Total RNA was isolated from cells, reversely transcribed to first-strand cDNA, and subjected to PCR analysis with different combinations of HLA-A- or HLA*0201 gene-specific primers. PCR products were run on an agarose gel and visualized by ethidium bromide staining. C, Sequencing histogram of the single cytosine deletion found in the HLA-A*0201 cDNA isolated from 1259MEL cells. RT-PCR-amplified HLA-A2 exon1-exon8 (E1-E8) cDNA fragments from 1259MEL and 1074MEL cells were gel purified and subjected to nucleotide sequencing analysis with sense primers 5pE1A2, 5pE2A, and A2E3–5′ or antisense primers AP2, 3pE3A2, A2E4–3′, and 3pE8A. The histograms obtained with primer A2E3–5′ are shown. Nucleotides are numbered according to Arnett and Parham (46) and indicated above the 1074MEL histogram. The cytosine stretch starting from nt 699 is underlined. The premature stop codon TGA generated as a result of a shifted reading frame is boxed. D, Nucleotide and deduced amino acid sequence of the partial HLA-A2 exon 4 in 1074MEL and 1259MEL cells. The regions corresponding to codon 232–245 are shown. The cytosine stretches are underlined. The missense codon-deduced amino acids are indicated in bold letters.