Association with beta 2-microglobulin controls the expression of transfected human class I genes.

The genes encoding HLA-B27K and HLA-B27W were transfected into murine recipient cells. A monoclonal antibody HC-10, directed against free B-locus heavy chain, was the only reagent capable of efficiently detecting the HLA-B27 heavy chains in detergent lysates. These heavy chains were devoid of sialic acid. Trace amounts of HLA-B27 could be isolated with the anti-HLA-A,-B antibody W6/32, which reacts with the heavy chain beta 2-microglobulin complex. In marked contrast, HLA-A2 and -B7 genes, when transfected, yielded easily detectable amounts of antigen precipitable with W6/32, which carried the usual complement of sialic acids. Because the alpha 3 domains of HLA-B27 and HLA-B7 and the more COOH-terminal portions are identical in amino acid sequence, structural elements in the polymorphic alpha 1 and alpha 2 domains must control association of heavy chain with beta 2-microglobulin. Introduction of a human beta 2-microglobulin gene into L cells transfected with the HLA-B27 gene rescued the expression of HLA-B27 at the cell surface, as evidenced by reactivity with W6/32, surface staining, and the presence of sialic acid on the heavy chain.

Isolation, expression, and the primary structure of HLA-Cw1 and HLA-Cw2 genes: evolutionary aspects.

The HLA-Cw1 and -Cw2 genes were identified in a genomic library and their products characterized by biochemical methods. The HLA-Cw1 and -Cw2 genes, upon transfection in mouse L cells, give rise to class I antigen heavy chains that associate with neither mouse nor human beta-2 microglobulin. They are indistinguishable in isoelectric point from polypeptides identified as HLA-Cw1 and -Cw2 in human cells. The nucleotide sequence of HLA-Cw1 and -Cw2 and their comparison with HLA-Cw3, the only other known HLA-C sequence, reveal a characteristic pattern of locus-specific amino acids. A comparison of 13 different human class I primary structures leads us to speculate that the most variable region in HLA class I antigens, positions 61-83, could assume an alpha helical structure of critical importance for class I antigen function. The locus specificity and the higher degree of intralocus conservation in the COOH-terminal region, especially in the transmembrane and cytoplasmic domains, must reflect evolutionary ancestry rather than positive selection. In view of the pattern and types of substitutions observed for HLA-C locus products, their function as immune response gene products is questioned.

Gene conversion-like mechanisms may generate polymorphism in human class I genes.

The nucleotide sequences of the human class I major histocompatibility complex genes HLA-B27k and HLA-B27w have been determined. They differ by only four nucleotides over a stretch of 14 bp in exon 2, resulting in three amino acid exchanges at positions 77 (Asp to Asn), 80 (Thr to Ile) and 81 (Leu to Ala). The distribution of these nucleotide substitutions suggests a gene conversion-like event responsible for the generation of these HLA-B27 subtypes. The mechanisms underlying the generation of new polymorphic variants in man are therefore probably identical to the gene conversion-like events postulated in the generation of H-2Kbm class I mutants in the mouse.

DNA sequence of the coding region of the HLA-B44 gene.

An HLA-B44 cDNA clone was identified in a cDNA library constructed from an HLA-B44 homozygous cell line. The DNA sequence was determined and was found to contain the complete coding sequence but for (probably) the three N-terminal codons. Comparisons of the derived amino acid sequence with other HLA-A and -B locus amino acid sequences revealed four HLA-B44-specific substitutions including a new polymorphic site. Regions of strong sequence conservation for HLA-B-locus products were found at the nucleotide and amino acid levels.