Genomic organization of a human killer cell inhibitory receptor gene

We have cloned a region of human chromosome 19q13.4 which contains multiple killer cell inhibitory receptor (KIR) loci. By random and directed sequence analysis of these KIR-specific clones, we deduced the genomic structure of KIR genes. A locus encoding a member of the NKAT-2 family of KIRs is presented here. The structure of the gene is reminiscent of loci of the Fc receptor gene family, and the two sets of genes may derive from a common ancestor. The KIR gene contains potentially nine exons. The first two exons encode the leader sequence, as in Fc receptor genes. The third exon encodes an untranslated pseudo exon specifying an immunoglobulin domain with an in-frame stop codon. Expressed cDNAs do not contain this exon. This finding is consistent with the hypothesis that certain KIR genes may have been derived from the duplication of a primordial three immunoglobulin domain structure with subsequent skipping of one exon to derive genes with two expressed immunoglobulin domains. Variation in numbers of immunoglobulin domains in different KIR genes is facilitated by conservation of splicing frame in respect to the codon triplet for each immunoglobulin domain.     

p49, a putative HLA class I-specific inhibitory NK receptor belonging to the immunoglobulin superfamily

NK cells display several killer inhibitory receptors (KIR) specific for different alleles of MHC class I molecules. A family of KIR are represented by type I transmembrane proteins belonging to the immunoglobulin superfamily (Ig-SF). Besides cDNA encoding for these KIR, additional cDNA have been identified which encode for Ig-SF receptors with still undefined specificity. Here we analyze one of these cDNA, termed cl.15.212, which encodes a type I transmembrane protein characterized by two extracellular Ig-like domains and a 115-amino acid cytoplasmic tail containing a single immuno-receptor tyrosine-based inhibitory motif (ITIM) which is typical of KIR. cl.15.212 cDNA displays approximately 50 % sequence homology with other Ig-SF members. Different from the other KIR, cl.15.212 mRNA is expressed by all NK cells and by a fraction of KIR+ T cell clones. cl.15.212 cDNA codes for a membrane-bound receptor displaying an apparent molecular mass of 49 kDa, thus termed p49. To determine the specificity of the cl.15.212-encoded receptor, we generated soluble fusion proteins consisting of the ectodomain of p49 and the Fc portion of human IgG1. Soluble molecules bound efficiently to 221 cells transfected with HLA-G1, -A3, -B46 alleles and weakly to -B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA-A2, -B51, -Cw3 or -Cw4. The binding specificity of soluble p49-Fc was confirmed by competition experiments using an anti-HLA class I-specific monoclonal antibody. Finally, different cDNA encoding for molecules homologous to cl.15.212 cDNA have been isolated, two of which lack the sequence encoding the transmembrane portion, thus suggesting they may encode soluble molecules.     

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal ("K") region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid "spacer" domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of approximately 1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-alpha (interferon-alpha/beta-stimulated response elements), but two motifs resembling interferon-gamma activation sites were located. IFNs alpha and gamma both induce IFI16 mRNA expression in myeloid cells. Interferon-alpha inducibility of IFI16 may be regulated by an interferon-alpha/beta-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural organization of the mouse and human GALR1 galanin receptor genes (Galnr and GALNR) and chromosomal localization of the mouse gene

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15-20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouse Galnr gene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the human GALNR gene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.     

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The genes encoding the murine paired immunoglobulin-like receptors PIR-A and PIR-B are members of a novel gene family which encode cell-surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and their non-inhibitory/activatory counterparts. PIR-A and PIR-B have highly homologous extracellular domains but distinct transmembrane and cytoplasmic regions. A charged arginine in the transmembrane region of PIR-A suggests its potential association with other transmembrane proteins to form a signal transducing unit. PIR-B, in contrast, has an uncharged transmembrane region and several ITIMs in its cytoplasmic tail. These characteristics suggest that PIR-A and PIR-B which are coordinately expressed by B cells and myeloid cells, serve counter-regulatory roles in humoral and inflammatory responses. In the present study we have determined the genomic structure of the single copy PIR-B gene. The gene consists of 15 exons and spans approximately 8 kilobases. The first exon contains the 5' untranslated region, the ATG translation start site, and approximately half of the leader peptide sequence. The remainder of the leader peptide sequence is encoded by exon 2. Exons 3-8 encode the six extracellular immunoglobulin-like domains and exons 9 and 10 code for the extracellular membrane proximal and transmembrane regions. The final five exons (exons 11-15) encode for the ITIM-bearing cytoplasmic tail and the 3' untranslated region. The intron/exon boundaries of PIR-B obey the GT-AG rule and are in phase I, with the notable exception of the three boundaries determined for ITIM-containing exons. A microsatellite composed of the trinucleotide repeat AAG in the intron between exons 9 and 10 provides a useful marker for studying population genetics.     

The exon/intron organization and chromosomal mapping of the mouse ADAMTS-1 gene encoding an ADAM family protein with TSP motifs

ADAM is a recently discovered gene family that encodes proteins with a disintegrin and metalloproteinase. ADAMTS-1 is a gene encoding a new member protein of the ADAM family with the thrombospondin (TSP) type I motif, the expression of which is associated with inflammatory processes. In the present study, we have characterized the exon/intron organization of the mouse ADAMTS-1 gene. The ADAMTS-1 gene is composed of nine exons, all of which are present within the 9.2-kb genomic region. Among the nine exons, exons 1, 5, and 6 encode a proprotein domain, a disintegrin-like domain, and a TSP type I motif, respectively, of the ADAMTS-1 protein, suggesting that there is a correlation between exon/intron organization and functional domains. In addition, the exon/ intron organization of the ADAMTS-1 gene is very different from that of the metalloproteinase-like/disintegrin-like/ cysteine-rich protein gene (MDC) (ADAM11), suggesting that the genomic structure of ADAM family genes is not necessarily conserved. Furthermore, fluorescence in situ hybridization revealed that the ADAMTS-1 gene is located in region C3-C5 of chromosome 16, to which none of the previously identified ADAM genes have been mapped.     

Complete cDNA sequence, genomic structure, and chromosomal localization of the LPA receptor gene, lpA1/vzg-1/Gpcr26

The lpA1/Gpcr26 locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3' rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5' sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor, Edg1, but is identical to another more similar orphan receptor (lpA2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 in Mus spretus, and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for the vzg-1/lpA1 subfamily of receptors compared to those of homologous orphan receptor genes.     

Validation of the indicators of abuse (IOA) screen

The ephrins are a family of ligands that bind to Eph family receptor tyrosine kinases, and have been implicated in axon guidance and other patterning processes during vertebrate development. We describe here the identification and characterization of murine ephrin-B3. The cDNA encodes a 340 amino acid transmembrane molecule, most closely related to the two other known transmembrane ligands, ephrin-B1 and ephrin-B2. In addition to homology in their extracellular receptor binding domains, these transmembrane ligands share striking homology between their cytoplasmic domains, with 31 of the last 34 amino acids of ephrin-B3 being identical to ephrin-B2, suggesting functional interactions of the cytoplasmic tail. While most Eph family ligands are promiscuous in their interactions with Eph receptors, binding studies with the five receptors known to bind other transmembrane ligands only revealed a high affinity interaction of ephrin-B3 with EphB3, with a dissociation constant of approximately 1 nM. In situ hybridization of mouse embryos showed ephrin-B3 is expressed prominently at the dorsal and ventral midline of the neural tube, particularly in the floor plate, a structure with key functions in patterning the nervous system. The isolation of this ligand may help to elucidate the molecular basis of patterning activities at the neural tube midline.     

CTX, a Xenopus thymocyte receptor, defines a molecular family conserved throughout vertebrates

CTX, a cortical thymocyte marker in Xenopus, is an immunoglobulin superfamily (Igsf) member comprising one variable and one constant C2-type Igsf domain, a transmembrane segment and a cytoplasmic tail. Although resembling that of the TCR and immunoglobulins, the variable domain is not encoded by somatic rearrangement of the gene but by splicing of two half-domain exons. The C2 domain, also encoded by two exons, has an extra pair of cysteines. The transmembrane segment is free of charged residues, and the cytoplasmic tail (70 amino acids) contains one tyrosine and many glutamic acid residues. ChT1, a chicken homologue of CTX, has the same structural and genetic features, and both molecules are expressed on the thymocyte surface. We cloned new mouse (CTM) and human (CTH) cDNA and genes which are highly homologous to CTX/ChT1 but not lymphocyte specific. Similarity with recently described human cell surface molecules, A33 antigen and CAR (coxsackie and adenovirus 5 receptor), and a number of expressed sequence tags leads us to propose that CTX defines a novel subset of the Igsf, conserved throughout vertebrates and extending beyond the immune system. Strong homologies within vertebrate sequences suggest that the V and C2 CTX domains are scions of a very ancient lineage.     

Structure of the murine CD156 gene, characterization of its promoter, and chromosomal location

The murine cell surface antigen mCD156 is a glycoprotein that is expressed in monocytic cell lines and consists of a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, and an epidermal growth factor-like domain in the extracellular region. The mCD156 gene is composed of 24 exons and 23 introns and spans approximately 14 kilobases. The first exon encodes most of the signal peptide sequence, and the transmembrane region is encoded by a single exon (19). In contrast, the other regions are composed of multiple exons. Of these, exons 7-12 and 12-15 encode a metalloprotease domain and a disintegrin domain, respectively. Sequence analysis of the 5'-flanking DNA revealed many potential regulatory motifs. Chloramphenicol acetyltransferase analysis demonstrated that nucleotides at positions -183, -334, and -623 contained cis-acting enhancing elements in a mouse monocytic cell line, aHINS-B3. Nucleotides at positions -183 and -390 contained elements responsible for lipopolysaccharide (LPS) inducibility, although several other 5'-flanking regions were also involved in LPS responsiveness. Regions -202, -507, and -659 play a role in interferon-gamma inducibility. Some of the potential regulatory motifs and other unknown cis elements may be involved in the constitutive expression, and LPS and interferon-gamma inducibilities. The mCD156 gene was mapped to chromosome 7, region F3-F4.     

Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.