Genes and Immunity (2000) 1, 280–287 2000 Macmillan Publishers Ltd All rights reserved 1466-4879/00 $15.00 www.nature.com/gene The centromeric part of the human NK gene complex: linkage of LOX-1 and LY49L with the CD94/NKG2 region C Bull1, Y Sobanov2,BRo¨hrdanz1, J O’Brien1, H Lehrach1 and E Hofer2 1Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany; 2Department of Vascular Biology and Thrombosis Research, Vienna International Research Cooperation Center, University of Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria The natural killer (NK) gene complex is a genomic region containing lectin-type receptor genes. We have established a contig of PAC and BAC clones comprising about 1 Mb of the centromeric part of the NK gene complex. This region extends from the LOX-1 gene, which encodes a receptor for oxidized LDL and was found within 100 kb telomeric of the STS marker D12S77, contains the CD94 and NKG2 NK receptor genes and reaches beyond D12S852 on the proximal side. In this part we have mapped the human LY49L gene, a homologue of the rodent Ly49 genes, which encode important MHC class I receptors for the regulation of NK cell activity in rodents. The LY49L gene is localized 100 to 200 kb centromeric of the NKG2 gene cluster and 300 to 400 kb telomeric of the STS marker D12S841. Genomic sequencing of the complete gene including promoter and intron sequences confirmed that the structure is similar to the mouse Ly49 genes. Screening of several cDNA libraries did not detect any transcripts of putative additional human LY49 genes. In addition, in the course of these studies several EST sequences were localized in the region, one immediately upstream of the LY49L gene. Genes and Immunity (2000) 1, 280–287. Keywords: natural killer cells; NK gene complex; chromosome 12; LOX, CD94, NKG2 and LY-49 genes Introduction NKR-P1 families. Whereas members of the CD94/NKG2 and the NKR-P1 families have been found to be Cytotoxicity of natural killer (NK) cells towards tumor or expressed in mice,17–19 rats20 and humans,5,21–24 1 virally infected target cells is regulated by inhibitory and expression of functional Ly49 proteins has so far only activating signals from cell surface receptors. NK cell been detected in rodents.15,19 receptors that recognize MHC class I or related molecules Recently, an aberrantly spliced cDNA coding for a 2–6 on target cells can be grouped into two structurally dis- human LY49 molecule (named Ly49L) has been tinct classes, namely the immunoglobulin-(Ig)-like and detected25 in the expressed sequence tags (EST) database the C-type lectin receptors. The killer immunoglobulin- and in cDNA libraries derived from human NK cells. like receptors (KIR) have been found to be expressed on Therefore genes related to all different NK cell receptor human NK cells and different variants display either classes have now been found in rodents and in humans. repressive or triggering functions. They are encoded by However, it remains unclear whether intact gene families a cluster of genes in a region on chromosome 19 named related to the human KIR receptors and the rodent Ly49 7,8 the leukocyte receptor complex (LRC). A gene distantly receptors coexist and fulfill similar functions in human as related to the Ig-like receptor gene family has recently well as in rodent NK cells or whether these two receptor also been detected in mice, but its relevance for rodent classes, which bind classical MHC class I molecules, have 9 NK cell function has not been firmly established. evolved differently from common ancestor genes in C-type lectin NK receptors, which occur in both inhibi- humans and rodents. tory and activating forms and are encoded within a syn- Furthermore, there are additional lectin-type receptors 10–13 tenic region on human chromosome 12, mouse chro- encoded in the NK gene complex which possess different 14,15 16 mosome 6 and rat chromosome 4, called the NK functions in various cell types. The CD69 or AICL recep- gene complex, can be sub-grouped into different families. tors26,27 are widely expressed in the hematopoietic lineage Among these are the CD94/NKG2, the Ly49 and the and their functions and ligands are not clearly defined. Recently, the lectin-type LLT1 receptor was reported to be broadly expressed in lymphocytes.28 Furthermore, a Correspondence: Erhard Hofer, Department of Vascular Biology and member of another lectin-type receptor class, LOX-1, has Thrombosis Research, Vienna International Research Cooperation Center, been implicated as a receptor for oxidized low-density University of Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria lipoprotein on endothelial cells and monocytes.29 This work was supported in part by a grant from the Austrian National Bank (Jubila¨umsfond No. 7567) to E.H. In order to establish the basis to search for novel forms Supplementary material to this paper is available from the author. of lectin-type receptor genes, to link the NKG2 and CD94 Received 28 January 2000; revised and accepted 22 February 2000 NK cell receptor genes13,23 with the region containing the Human NK gene complex C Bull et al 281 LY49L and other potentially related genes and to get bp] and 2729 bp) was confirmed as has been previously some clues to the evolution of the locus, we have now published.30 The first exon, however, containing only constructed a physical map based on PAC and BAC untranslated sequence extends at least 285 bp beyond the clones of the centromeric part of the NK complex region. 5Ј-end described by others.25,30 We also noticed that the We have focused here on the centromeric part of the NK exon-intron boundaries of the human LY49L gene (Figure gene complex since the available data indicate that the 2a) differ for all exons from the sequences that have been syntenic region of rodents contains more than nine Ly49 found most frequently at mammalian splice sites.31,32 The genes and resistance loci for cytomegalovirus and pox cDNAs isolated by us and others always contained a part virus.15 In addition we find the LOX-1 gene to be located of intron 5. This is due to the use of a cryptic splice site 200 kb telomeric of CD94. Sequencing of the complete in intron 5. An alignment of all known Ly49 genes from LY49L gene confirmed previous results concerning the mouse, rat and human (Figure 2b) shows that the very exon/intron structure.30 Furthermore, we have identified 3Ј base of exon 5 is a G in all rodent genes, whereas it is several genes represented as EST sequences in the ana- a C in the human LY49L gene. The G is most frequently lyzed region, one EST is encoded within a very short dis- found at the 3Ј end of mammalian exons. tance to the 5Ј-end of the LY49L gene. The potential promoter region of the LY49L gene The approximate position of the potential transcription Results start site was inferred from a cDNA clone that contained a 381 bp sequence stretch upstream from the predicted A contig of PAC and BAC clones comprising the translation initiation codon. This region was not func- centromeric part of the NK gene complex tionally tested but as another gene (EST AA005053) was 13,23 Starting from the NKG2/CD94 gene region and the already located only 559 bp upstream of this site, it seems LY49L gene we have established contigs of PAC and BAC unlikely that transcription initiates much further clones that extend over about 1 Mb and contain the cen- upstream. The 559 bp sequence was analyzed in order to tromeric part of the NK gene complex. Most parts of the find potential promoter binding sites by the SIGSCAN region are covered by several clones (see Figure 1). The algorithm (Version 4.05, http://bimas.dcrt.nih.gov:80/ 29 LOX-1 gene was detected in clone L21228, the first clone molbio/signal/) developed by Dan S Prestridge. This on the telomeric side of this contig, and this gene is there- analysis suggested some potential TATA boxes and tran- fore located about 100 kb telomeric of the STS marker scription factor consensus binding sites based on the D12S77, which flanks the NKG2/CD94 region. The CD94 TRANSFAC database33 (see Figure 3). gene and all five NKG2 genes are placed between 100 to 200 kb centromeric of D12S77 and their relative orien- Screening for additional LY49L genes 13 tation and linkage has been described previously. The After screening two natural killer cell cDNA libraries as LY49L gene is 100 to 200 kb proximal of the NKG2A gene well as human fetal liver, fetal lung, fetal spleen and and has been linked to the STS marker D12S841 which spleen cDNA libraries we detected another incompletely is found 200 to 300 kb centromeric of LY49L. The contig spliced cDNA in a human fetal lung library in addition region extends further beyond the STS markers D12S112 to the one described previously.25 This cDNA lacked exon and D12S852 and therefore covers about 600 kb of the 4 and contained part of intron 5 due to the use of a cryp- region proximal of LY49L. tic splice site in the intron. However, we did not identify any clone that would encode an LY49L molecule with a Additional genes and ESTs in the region complete carbohydrate recognition domain. Taking into By end-sequencing of individual PAC clones several account the possibility that other human LY49 genes sequences identical or closely similar to database EST might have diverged quite far from the rodent genes, we entries were detected in the region.