Cosmid clones in the HLA-DZ and -DP subregions

Cosmid Clones in the HLA-DZ and -DP Subregions George Blanck and Jack L. Strominger

Proteins intimately involved in antigen presentation and allograft rejection are primarily encoded in the major histocompatibility complex (MHC), which in humans is subdivisible into three nonoverlapping segments on the short arm of chromosome 6: from centromere to telomere, the class II, class III, and class I regions, respectively. Here we report a refinement of the chromosomal organization of the class II region which encodes heavy and light chain proteins constituting heterodimeric surface molecules. These molecules are expressed constitutively on B cells and can be induced on a variety of other cell types. With only rare exception, heavy and light chain genes for a given class II molecule are adjacent [1]. Considerable contiguous portions of each of the three MHC segments have been cloned in sets of overlapping cosmids, yielding two general results [2-9]. First, the cloned regions have afforded a systemic identification and summation of the potential coding regions of the MHC, which is necessary for anticipating the entire repertoire available for antigen presentation and graft rejection. Second, precise molecular maps of the MHC of several species have afforded a better understanding of the evolution of long chromosomal segments [10-12]. For example, a comparison of the IA region of the mouse with its human counterpart, HLA-DQ, shows that relative gene order and orientations have been preserved and DQ is a virtual duplicate of the IA subregion [9]. As part of our continuing effort to link the entire class II segment of the MHC, a number of chromosome walking steps have recently been taken downstream of the DZA gene [13,14] in an attempt to link it with the DPB-DPA cluster, The HLA-DZA gene has been reported to be within approximately 170 kb telomeric of the HLA-DPA gene as a result of pulse-field gel analysis [15,16]. Also the rabbit counterpart of DZA, termed DN, has been linked to the rabbit counterpart of HLA-DPA [17,18]. In the rabbit these genes are arranged 3' to 3' and are 35 kb apart. Since previous chromosome walks have verified a number of evolutionary preservations of relative orientations, such as the TNF-B genes in mouse and man and the A and B genes in the mouse and man class II regions, the HLA-DZA and -DPA genes may be expected to be oriented as shown in Figure 1. The chromosome walk has totaled 120 kb around the DZA gene, primarily in the 3' direction. The only previously published cosmid in this region [ 14] is mainly From the Department of Biochemistry and Molecular Biology. Harvard University, Cambridge, Massachusetts. Address reprint requests to Dr.Jack L. Strominger, Ham,ard University, Department of Biochemistry and Molecular Biology, 7 Divinity Avenue, Cambridge, MA 02138. Received July 17, 1989; accepted October 23. 1989.

Human Immunology 27, 265-268 (1990) © American Society for Histocompatibility and Iramunogenetics, 1990

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HLA-DZ and -DP

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equivalent to 017A. In addition two cosmids extending previously described D P clone segments by about 10 kb have been obtained. K18A is the most telomeric of a previously reported cluster, which includes an additional A and B pseudogene pair and extends an additional 60 kb centromeric of the DPB gene shown in Figure 1 [3]. The D Z and DP regions are unlinked. However, the D Z A and new DPA subregion cosmids reveal that regardless of orientation, D Z A and DPA must be separated by a minimum of 50 kb. If these genes are oriented as in the rabbit and as represented in Figure 1, then they must be separated by a minimum of 110 kb. This would reflect an expansion in the region of the D Z A gene in the human class II region that is common to other class II subregions. Efforts to locate cross-hybridizing heavy and light chain sequences in the D Z A cluster have proved unsuccessful, suggesting there are no additional class II genes neighboring DZA. Attempts to obtain additional clones telomeric of DPA or centromeric of the D Z A cluster have been unsuccessful and are not being continued. The material obtained is therefore being published now to make it available to other, interested investigators.

ACKNOWLEDGMENTS

This research was supported by NIH grants DK-30241 and AI-21163. We thank Roberta Bettano for preparation of the manuscript.

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