Genomic organization and chromosomal location of the human gene encoding the B-lymphocyte activation antigen B7

The human B lymphocyte activation antigen B7 provides regulatory signals for T lymphocytes as a consequence of binding to its ligands CD28 and CTLA-4. The cDNA for B7 has previously been isolated and predicted to encode a type I membrane protein. The predicted polypeptide has a secretory signal peptide followed by two contiguous Ig-like domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Here we report the exon-intron genomic organization of human B7 and the chromosomal location. The gene has six exons that span approximately 32 kilobases of DNA. Exon 1 is not translated and the second exon contains the initiation ATG codon and encodes a predicted signal peptide. This gene structure is characteristic for several eukaryotic genes with tissue-specific expression. The third and fourth exons correspond to two Ig-like domains whereas the fifth and sixth exons encode respectively the trans-membrane portion and the cytoplasmic tail. This close relationship between exons and functional domains is a characteristic feature of genes of the Ig superfamily. Cell surface expression of the B7 gene product has previously been mapped to human chromosome 12 by antibody reactivity with the B7-specific monoclonal antibody BB-1. We here demonstrate that theB7 gene is located to theq21-qter region of chromosome 3 by DNA blot analysis of human × rodent somatic cell hybrids.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83071-M83075, M83077. Address correspondence and offprint requests to: B. Dupont, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue (Room S709), New York, NY 10021, USA.     

Genomic structure and chromosomal mapping of the murine CD40 gene.

The B cell-associated surface molecule, CD40, is likely to play a central role in the expansion of Ag-stimulated B cells, and their interaction with activated Th cells. In our study we have isolated genomic clones of murine CD40 from a mouse liver genomic DNA library. Comparison with the murine CD40 cDNA sequence revealed the presence of nine exons that together contain the entire murine CD40 coding region, and span approximately 16.3 kb of genomic DNA. The intron/exon structure of the CD40 gene resembles that of the low affinity nerve growth factor receptor gene, a close homolog of both human and murine CD40. In both cases the functional domains of the receptor molecules are separated onto different exons throughout the genes. Southern blot analysis demonstrated that murine CD40 is a single copy gene that maps in the distal region of mouse chromosome 2.     

Characterization of the genomic structure of the mouse limbic system-associated membrane protein (Lsamp) gene

The Lsamp gene encodes the limbic system-associated membrane protein (LAMP) an immunoglobulin (Ig) superfamily member with three Ig domains and a glycosylphosphatidylinositol anchor. LAMP is expressed by neurons composing the limbic system, is highly conserved between rodents and human, and has structural and functional properties that substantiate its role in the formation of limbic circuits. We report here the genomic organization of the Lsamp gene. The Lsamp gene is composed of 11 exons distributed over 2.2 megabases (Mb). Two exons 1 are separated by approximately 1.6 Mb and contribute to the unusual large size of the gene. Alternative spliced Lsamp mRNAs are generated from distinct promoter regions associated with the two exons 1 that encode distinct signal peptides and thus generate identical native mature polypetides. Additional diversity is created by the use of two small exons to include an insertion of 23 amino acids within the polypeptide C-terminal region of the mature protein. The genomic features of the Lsamp gene described here indicate an intricate mechanism of gene expression regulation that may be relevant in the context of human neuropsychiatric and neurological disorders, where LAMP expression may be altered.     

A striking organization of a large family of human neural cadherin-like cell adhesion genes.

We have identified 52 novel human cadherin-like genes organized into three closely linked clusters. Comparison of the genomic DNA sequences with those of representative cDNAs reveals a striking genomic organization similar to that of immunoglobulin and T cell receptor gene clusters. The N-terminal extracellular and transmembrane domains of each cadherin protein are encoded by a distinct and unusually large exon. These exons are organized in a tandem array. By contrast, the C-terminal cytoplasmic domain of each protein is identical and is encoded by three small exons located downstream from the cluster of N-terminal exons. This unusual organization has interesting implications regarding the molecular code required to establish complex networks of neuronal connections in the brain and the mechanisms of cell-specific cadherin-like gene expression.     

CD150 is a member of a family of genes that encode glycoproteins on the surface of hematopoietic cells

Human CD150 (SLAM) is a glycoprotein expressed on the surface of T, B, natural killer, and dendritic cells. The extracellular domain of CD150 is the receptor for measles virus and CD150 acts as a co-activator on T and B cells. We characterized the mouse and human CD150 genes, each of which comprises seven exons spanning approximately 32 kb. Mouse CD150 mRNA was detected in T cells and in most thymocyte subsets, except CD4-8- cells. Surprisingly, the CD4-8- thymocytes of CD3gammadeltanull mice, but not of Ragnull or severe combined immunodeficiency mice, expressed CD150. Whereas high levels of CD150 were found in Th1 cells, only small amounts were detectable in Th2 cells. CD150 expression was up-regulated upon in vitro activation of mouse T cells by anti-CD3. The complete mouse CD150 gene is highly homologous to its human orthologue in terms of nucleotide sequences and intron/exon organization. The human genomic sequences indicate that all isoforms detected so far have arisen from alternative splicing events. As judged by fluorescence in situ hybridization, mouse CD150 mapped to Chromosome (Chr) 1, band 1H2.2-2.3, and human CD150 was found on Chr 1q22. Human and mouse CD150 share sequence homologies with six other genes, five of which - CD84, CD229 (Ly-9), CD244 (2B4), CD48, and 19A - are localized in a 250-kb segment in close proximity to the human gene. Their location and their sequence similarities strongly suggest that the CD150 family of cell surface receptors arose via successive duplications of a common ancestral gene.     

Analysis of the genomic structure of the porcine CD1 gene cluster

CD1 is an MHC class I-like protein that presents lipid antigens to T cell receptors. We determined 470,187 bp of the genomic sequence encompassing the region encoding porcine CD1 genes. We identified 16 genes in this region and newly identified CD1A2, CD1B, CD1C, CD1D, and CD1E. Porcine CD1 genes were located in clusters between KIRREL and olfactory receptor (OR) genes, as observed in humans, although they were divided into two regions by a region encoding OR genes. Comparison of the genomic sequences of CD1 gene loci in pigs with other mammals showed that separation of the CD1 gene cluster by ORs was observed only in pigs. CD1A duplication in the porcine genome was estimated to have occurred after the divergence of the human and porcine. This analysis of the genomic sequence of the porcine CD1 family will contribute to our understanding of the evolution of mammalian CD1 genes.     

The human LFA-3 gene is located at the same chromosome band as the gene for its receptor CD2

Lymphocyte function-associated antigen 3 (LFA-3) is a widely distributed cell surface glycoprotein that has been assigned a role in cell-cell adhesion on the basis of its capacity to bind to the T-lymphocyte CD2 antigen. The amino acid sequences of the extracellular domains of these two antigens, predicted from their cDNA sequences, show significant similarities, and both are members of the immunoglobulin supergene family. In this communication, a probe prepared from LFA-3 cDNA has been used in Southern blot analyses of somatic cell hybrids and in in situ hybridization to assign the LFA-3 gene to the human chromosome band 1p13. This is the same location previously assigned to CD2. Thus the LFA-3 and CD2 genes have probably arisen by duplication of a common evolutionary precursor. These genes therefore represent a further instance in which related members of the immunoglobulin superfamily are located in adjacent regions of the genome.     

Genomic and Phylogenetic Analysis of the Human CD1 and HLA Class I Multicopy Genes

The human CD1 proteins belong to a lipid-glycolipid antigen-presenting gene family and are related in structure and function to the MHC class I molecules. Previous mapping and DNA hybridization studies have shown that five linked genes located within a cluster on human chromosome 1q22-23 encode the CD1 protein family. We have analyzed the complete genomic sequence of the human CD1 gene cluster and found that the five active genes are distributed over 175,600 nucleotides and separated by four expanded intervening genomic regions (IGRs) ranging in length between 20 and 68 kb. The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Some L1 sequences have acted as receptors for other subtypes or families of retroelements. Alu molecular clocks that have evolved during primate history are found distributed within the HLA class I duplicated segments (duplicons) but not within the duplicons of CD1. Phylogeny of the alpha3 domain of the class I-like superfamily of proteins shows that the CD1 cluster is well separated from HLA class I by a number of superfamily members including MIC (PERB11), HFE, Zn-alpha2-GP, FcRn, and MR1. Phylogenetically, the human CD1 sequences are interspersed by CD1 sequences from other mammalian species, whereas the human HLA class I sequences cluster together and are separated from the other mammalian sequences. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans. In contrast to the HLA class I genomic structure, the human CD1 duplicons are smaller in size, they lack Alu clocks, and they are interrupted by IGRs at least 4 to 14 times longer than the CD1 genes themselves. The IGRs seem to have been created as "buffer zones" to protect the CD1 genes from disruption by transposable elements.     

Characterization and identification of Tage4 as the murine orthologue of human poliovirus receptor/CD155

CD155 is a member of the immunoglobulin superfamily also known as the human receptor for poliovirus (PVR). Transmembrane glycoproteins related to CD155, the nectins, are well-characterized cell adhesion receptors displaying a high degree of sequence conservation across species. In contrast, CD155 belongs to the category of rapidly evolving genes wherefore a mouse CD155 gene distinguished by an affirmative extent of amino acid conservation as observed for nectins is absent. Consequently, the existing genetic evidence by itself is an inferior indicator to consider whether Tage4, a mouse orphan receptor, represents the murine orthologue of CD155. In the present study Tage4 cDNA was cloned from mouse lung and further characterized genetically. CD155 and Tage4 possess an identical genomic organization and reside in syntenic chromosomal regions. The Tage4 expression pattern was explored applying a newly generated antibody. Both receptors, CD155 in human and Tage4 in mouse, are expressed by intestinal epithelia as well as by follicle associated epithelium and follicular dendritic cells inside Peyer's patches of the gut associated lymphoid tissue. Furthermore, Tage4 lacks self-adhesion capacity but binds to vitronectin, two known features of CD155. These data indicate that Tage4 represents the functional orthologue of CD155 in mouse. Therefore, we suggest to rename Tage4 into rodent CD155.     

Mouse novel Ly9: a new member of the expanding CD150 (SLAM) family of leukocyte cell-surface receptors

Human CS1, also known as novel Ly9, 19A24, or CRACC, is a member of the immunoglobulin gene superfamily (IgSF) expressed on natural killer cells and other leukocytes. Here we describe the cloning of the mouse homologue of this gene. The mouse novel Ly9 gene is shown to encode a transmembrane protein composed of two extracellular immunoglobulin-like domains, a transmembrane region and an 88-amino acid cytoplasmic domain. Mouse novel Ly9 is structurally similar to the extracellular domains of CD84 and CD229 (Ly9). Both mouse and human novel Ly9 genes mapped close to the CD229gene in a region where other members of the CD150 family have also been mapped, and analysis of their genomic sequences showed that they have an identical intron/exon organization. Northern blot analysis revealed that the expression of mouse and human novel Ly9 was predominantly restricted to hematopoietic tissues, with the exception of testis. Here we show that SAP (SH2D1A), an adapter protein responsible for the X-linked lymphoproliferative disease, binds to the phosphorylated cytoplasmic tail of human but not mouse novel Ly9. Taken together, these data indicate that mouse novel Ly9 is a new member of the expanding CD150 family of cell surface receptors.     

Direct human T helper cell-induced B cell activation is not mediated by inositol lipid hydrolysis

The Ag-specific interaction between cloned allospecific human Th cells and class II MHC determinants on the surface of allogeneic B cells induces a significant fraction of resting B cells to express a B cell specific activation Ag BLAST-2 (CD23). On the other hand, cross-linking of B cell surface Ig R by Ag analogues does not lead to BLAST-2 expression. By utilizing the BLAST-2 induction assay as a positive control for efficient Th-B cell interaction, we have investigated the biochemical basis of human B cell activation mediated by Ag and Th cells. Our data demonstrate that ligands for sIg R, including F(ab')2 goat anti-human IgM and Staphylococcus aureus protein A, stimulate the metabolism of B cell membrane inositol lipids as assessed by: 1) increased [3H]inositol phosphates formation in myo-[3H]inositol-labeled B cells; 2) selective incorporation of [32P]orthophosphate into phosphatidic acid and phosphatidylinositol, but not into phosphatidylethanolamine or phosphatidylcholine; and 3) rapid increase in B cell cytoplasmic ionized Ca2+ concentration ([Ca2+]i). In contrast, direct Th-B cell interaction leads to high intensity BLAST-2 expression on the B cell surface but this response is not mediated by changes in inositol lipid metabolism or [Ca2+]i. Further, Th-B cell interaction does not affect the changes in B cell inositol lipid metabolism or [Ca2+]i triggered by sIg cross-linking. Taken together, our results suggest that Ag and Th cells induce different functional B cell responses by activating distinct second messenger systems within the B cell.     

Physical and genetic linkage of the genes encoding Ly-9 and CD48 on mouse and human chromosomes 1

By virtue of sequence similarity, the genes encoding CD2, CD48, CD58, and Ly-9 have been assigned to a distinct subset within the immunoglobulin superfamily. Previous gene mapping studies in human and mouse have suggested that CD2, CD48, and CD58 arose by gene duplication. Here we show the gene encoding Ly-9 to be located adjacent to CD48 and the Na,K-ATPase 2 subunit gene on human and mouse chromosome 1. The proximity in human and mouse genomes of the genes encoding CD2, CD58, and the Na,K-ATPase 1 subunit, and of the Ly-9, CD48, and the Na,K-ATPase 2 subunit genes may be explained by the occurence of two, successive duplication events during vertebrate evolution, and suggest that Ly-9 may also participate in adhesion reactions between T lymphocytes and accessory cells by homophilic interaction.