Immunogenetics (1995) 41:246-250 © Springer-Verlag 1995 BRIEF COMMUNICATION Suminder M. S. Sawhney • Noor N. Hasima Elizabeth J. Glass • Samer W. K. AI-Murrani Anil K. Nichani • Roger L. Spooner • John L. Williams George C. Russell Transfection, expression, and DNA sequence of a gene encoding a BolaS-All antigen Received: 3 May 1994 / Revised: 25 November 1994 Cattle major histocompatibility complex [(MHC) (BoLA)] class I molecules are heterodimeric glycoproteins which present endogenous antigenic peptides to CD8 ÷ T lympho- cytes, initiating a cellular immune response. The MHC- encoded heavy chains are highly polymorphic and, in cattle, have been characterized mainly by using allo-antisera raised by reciprocal calf/dam immunisations (Spooner et al. 1978). This has enabled the identification of about 50 serological specificities, most of which behave as alleles of a single highly polymorphic class I locus (Davies et al. 1994). However, evidence from biochemical (Joosten et al. 1992; A1-Murrani et al. 1993, 1994) and molecular biolo- gical studies (Ennis et al. 1988; Brown et al. 1989; Toye et al. 1990; Bensaid et al. 1991; Ellis et al. 1992; Garber et al. 1993, 1994) suggest that more than one BoLA class I locus is expressed. These loci are apparently in linkage disequili- brium (A1-Murrani et al. 1993), making them difficult to distinguish by conventional methods. In order to investigate the expression and function of individual class I locus products, we are correlating BoLA class I gene sequences with the expressed products by the transfection and char- acterization of genomic class I clones. Shotgun transfection and expression of BoLA class I molecules has been described previously, but the genes involved were not isolated (Toye et al. 1990). In this paper we report the isolation, DNA sequencing, transfection, and expression of a genomic clone encoding a BoLA-All determinant from an animal expressing A10 and A 11 serological specificities. The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence databases, and have been assigned the accession numbers X82671 -X82675 S. M. S. Sawhney - N. N. Hasima I • E. J. Glass • S. W_ K. A1-Murrani A. K. Nichani - R. L. Spooner - J. L. Williams • G. C. Russell (~) Division of Molecular Biology, Roslin Institute, Roslin, Midlothian, EH25 9PS, UK Present address: 1 Department of Genetics and Cellular Biology, University of Malaya, 59100 Kuala Lumpur, Malaysia A genomic library was prepared in the lambda vector EMBL3, using peripheral blood mononuclear cell (PBM) DNA from an animal with A10 and All serotypes. The library was screened by hybridization with a 32P-labeled cattle MHC class I cDNA clone, pBoLA-1 (Brown et al. 1989). A total of 15 independent clones were selected from the library, which hybridized with both 5' and 3' ends of pBoLA-1 and were considered candidates for full-length genes. These clones were tested for expression by calcium phosphate mediated transfection into murine L cells, as described by Wigler and co-workers (1979), with the thymidine kinase (tk) gene as a selectable marker (Hasima 1992). Expression was examined by flow cytometry (FACS), using monoclonal antibody (mAb) IL-A88, which recognizes a monomorphic, non-conformational epitope on BoLA class I heavy chains (Toye et al. 1990). Only one clone, 19.1, showed significant levels of expres- sion of a BoLA class I product. Three rounds of FACS sorting produced a population in which all of the cells expressed a 19.1 product. This purified population of transfected cells was characterized by serological, cellu- lar, and biochemical methods. The identity of the expressed antigen was initially investigated by using a BoLA class I micro-lymphocyto- toxicity test (Spooner et al. 1978) with allo-antisera routi- nely used to define the All specificity (Davies et al. 1994). The transfected L cells expressing the product from clone 19.1 were lysed by three All-specific allo-antisera: Ed73, Edl02, and Edll0, but were not killed with a fourth All- specific allo-antiserum, Ed76. Control L cells were not killed with any of the All sera, and neither the control nor the transfected L cells were killed with sera defining other BoLA specificities. The killing of the transfected cells required the use of higher concentrations of the allo- antisera than for normal cattle PBM. This may reflect the degree of expression of the transfected gene, or may indicate that L cells are more robust than cattle PBM. Functional analysis of the product expressed by the clone 19.1-transfected cells was done using anti-All and anti-A10 allo-reactive cytotoxic T lymphocytes (CTL). The CTL lines were generated in vitro as described previously