Insight into the mechanism of involvement of HLA-B27 in ankylosing spondylitis (AS) (Brewerton et al. 1973, Schlosstein et al. 1973) has previously been hampered by the inability to directly examine the HLA-B27 gene and/or contiguous genetic sequences. Accordingly, we investigated the HLA-B27 gene by generating restriction fragments of genomic DNA derived from healthy and diseased HLA-B27 ÷ subjects. Using an HLA class I-specific cDNA probe, we have previously reported the assignment of a restriction fragment length polymorphism (RFLP) which segregates strongly with the HLA-B27 allele (Trapani et al. 1985). We now describe the molecular cloning and partial nucleotide sequence of this fragment of genomic DNA derived from a patient with AS. It is anticipated that further sequencing of this cloned fragment, which represents the first isolation of a segment of the HLA-B27 gene, may resolve the question of whether susceptibility to AS is related to a particular polymorphism of the B27 gene.
High molecular weight genomic DNA derived from HLA-B27 + patients with AS, healthy HLA-B27 ÷ relatives, and B27-control subjects was purified from peripheral blood leukocytes according to standard techniques (Wyman and White 1980), restricted with Taq I (New England Biolabs, Beverley, Massachusetts) according to the manufacturer's recommended assay conditions and electrophoresed in 0.8% agarose gels. Following transfer to nitrocellulose (Southern 1975), DNA sequences were probed with nick-translated pDP001 (Biro et al. i983), whose insert represents 85% of the mRNA sequence for HLA-B7. Examination of the resultant autoradiograph revealed multiple polymorphic bands (Fig. 1). The smaller of two bands which formed a doublet at approximately 3.6 and 3.5 kb was associated with the presence of the HLA-B27 allele as the 3.5 kb band segregated with the B27 status of nine of the ten individuals tested in this experiment. Of a total of 33 HLA=B27 + and B27-individuals examined thus far, all 17 HLA-B27 ÷ subjects have exhibited this band. Of the remaining 16 negative control subjects, 2 exhibited the 3.5 kb fragment (Z 2 > 20; P < 0.0001). These studies have included two families, and in both, the polymorphic fragment was shown to segregate with the