Organization of the gene encoding the human macrophage mannose receptor (MRC1).

The gene for the human macrophage mannose receptor (MRC1) has been characterized by isolation of clones covering the entire coding region. Sequence analysis reveals that the gene is divided into 30 exons. The first three exons encode the signal sequence, the NH2-terminal cysteine-rich domain, and the fibronectin type II repeat, while the final exon encodes the transmembrane anchor and the cytoplasmic tail. The intervening 26 exons encode the eight carbohydrate-recognition domains and intervening spacer elements. However, no simple correlation between intron boundaries and functional carbohydrate-recognition domains is apparent. The pattern of intron positions as well as comparison of the sequences of the carbohydrate-recognition domains suggests that the duplication of these domains was an evolutionarily ancient event.     

The mouse homologue of the leukocyte-associated Ig-like receptor-1 is an inhibitory receptor that recruits Src homology region 2-containing protein tyrosine phosphatase (SHP)-2, but not SHP-1

We report the molecular cloning and characterization of the first leukocyte-associated Ig-like receptor 1 (LAIR-1) homologue in mice that we have named mouse LAIR-1 (mLAIR-1). The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region syntenic with human chromosome 19q13.4 where the leukocyte receptor cluster is located. The protein shares 40% sequence identity with human LAIR-1, has a single Ig-like domain, and contains two immunoreceptor tyrosine-based inhibitory motif-like structures in its cytoplasmic tail. Mouse LAIR-1 is broadly expressed on various immune cells, and cross-linking of the molecule on stably transfected RBL-2H3 and YT.2C2 cells results in strong inhibition of their degranulation and cytotoxic activities, respectively. Upon pervanadate stimulation, the mLAIR-1 cytoplasmic tail becomes phosphorylated, thereby recruiting Src homology region 2-containing tyrosine phosphatase-2. Interestingly, unlike human LAIR-1, Src homology region 2-containing tyrosine phosphatase-1 is not recruited to the mLAIR-1 cytoplasmic tail. Screening human and mouse cell lines for mLAIR-1 and human LAIR-1 binding partners identified several lines expressing putative ligand(s) for both receptors.     

Cloning and molecular characterization of two splice variants of a new putative member of the Siglec-3-like subgroup of Siglecs.

The sialic acid binding immunoglobulin-like lectin (Siglec) family is a recently described member of the immunoglobulin superfamily. Within this Siglec family there exists a subgroup of molecules which bear a very high degree of homology with the molecule Siglec-3 (CD33), and has thus been designated the Siglec-3-like subgroup of Siglecs. The members of this subgroup have been localized to chromosome 19q13.4, through both in situ hybridization and precise genomic mapping at the nucleotide level. Through the positional cloning approach we have identified and characterized a Siglec-like gene (SLG), a putative novel member of the Siglec-3-like subgroup of Siglecs. We have characterized the complete genomic structure of SLG, as well as two alternative splice variants, and determined its chromosomal localization. The short isoform, SLG-S, consists of seven exons, with six intervening introns, while the longer isoform, SLG-L, consists of eight exons and seven intervening introns. The SLG gene is localized 32.9 kb downstream of Siglec-8 on chromosome 19q13.4. The putative SLG-S and SLG-L proteins, of 477 and 595 amino acid residues, respectively, show extensive homology to many members of the Siglec-3-like subgroup. This high degree of homology is conserved in the extracellular Ig-like domains, as well as in the cytoplasmic tyrosine-based motifs. Interestingly, the SLG-L protein contains two N-terminal V-set Ig-like domains, as opposed to SLG-S and other Siglec-3-like subgroup members which contain only one such domain. Through RT-PCR we have examined the expression profile of both SLG splice variants in a panel of human tissues and have found that SLG-S is highly expressed in spleen, small intestine and adrenal gland, while SLG-L exhibits high levels of expression in spleen, small intestine, and bone marrow. This gene is quite likely the latest novel member of the CD33-like subgroup of Siglecs, and given its high degree of homology, it may also serve a regulatory role in the proliferation and survival of a particular hematopoietic stem cell lineage, as has been found for CD33 and Siglec7.     

Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.     

Molecular cloning, characterization, and chromosomal localization of the mouse homologue of CD84, a member of the CD2 family of cell surface molecules

 CD84 is a member of the immunoglobulin gene superfamily (IgSF) with two Ig-like domains expressed primarily on B lymphocytes and macrophages. Here we describe the cloning of the mouse homologue of human CD84. Mouse CD84 cDNA clones were isolated from a macrophage library. The nucleotide sequence of mouse CD84 was shown to include an open reading frame encoding a putative 329 amino acid protein composed of a 21 amino acid leader peptide, two extracellular immunoglobulin (Ig)-like domains, a hydrophobic transmembrane region, and an 87 amino acid cytoplasmic domain. Mouse CD84 shares 57.3% amino acid sequence identity (88.7%, considering conservative amino acid substitutions) with the human homologue. Chromosome localization studies mapped the mouse CD84 gene to distal chromosome 1 adjacent to the gene for Ly-9, placing it close to the region where other members of the CD2 IgSF (CD48 and 2B4) have been mapped. Northern blot analysis revealed that the expression of mouse CD84 was predominantly restricted to hematopoietic tissues. Two species of mRNA of 3.6 kilobases (kb) and 1.5 kb were observed. The finding that the pattern of expression was restricted to the hematopoietic system and the conserved sequence of the mouse CD84 homologue suggests that the function of the CD84 glycoprotein may be similar in humans and mice. Received: 1 July 1998 / Revised: 31 August 1998     

Cloning,expression, and characterization of fugu CD4, the first ectothermic animal CD4

We have cloned and sequenced the first ectothermic animal CD4 gene from fugu, Takifugu rubripes, using a public database of the third draft sequence of the fugu genome. The fugu CD4 gene encodes a predicted protein of 463 amino acids containing four extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail. Fugu CD4 shares low identity of about 15–20% with avian and mammalian CD4 proteins. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair of the first Ig-like domain, but has a unique possible disulfide bond in the third domain. These differences suggest that fugu CD4 may have a different structure that could affect binding of major histocompatibility complex class II molecules and subsequent T-cell activation. In the putative fugu cytoplasmic region, the protein tyrosine kinase p56^lck binding motif is conserved. The predicted fugu CD4 gene is composed of 12 exons, differing from other CD4 genes, but showing conserved synteny and many conserved sequence motifs in the promoter region. RT-PCR analysis demonstrated that the fugu CD4 gene is expressed predominantly in lymphoid tissues. We also show that fugu CD4 can be expressed on the surface of cells via transfection. Molecular characterization of CD4 in fish provides insights into the evolution of both the CD4 molecule and the immune system.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain.

The CD19 molecule is a 95,000 Mr cell-surface protein of human B lymphocytes with two extracellular Ig-like domains and a 240 amino acid cytoplasmic tail. cDNA encoding human CD19 and the cytoplasmic domain of the mouse CD19 Ag were previously isolated. In this report, those cDNA were used to isolate cDNA or genomic DNA encoding the complete mCD19 protein and a portion of CD19 from the guinea pig. Mouse pre-B and B cell lines expressed two CD19 mRNA species of 2.7 and 2.2 kb, whereas myeloma cell lines were negative as were T cell lines. Similarly, among mouse organs, only spleen contained detectable CD19 mRNA. These results suggest that only B cells express CD19 in mouse, as in man. Sequence determination revealed substantial conservation, with hCD19 and mCD19 being 66% and hCD19 and gpCD19 being 73% identical in amino acid sequence. The cytoplasmic region of CD19 was most highly conserved with human/mouse being 73% identical and human/guinea pig being 83% identical in amino acid sequence. Isolation of the hCD19 and mCD19 genes and determination of exon/intron boundaries revealed that both genes were structurally similar and were composed of at least 15 exons, 4 encoded extracellular domains, and 9 encoded cytoplasmic domains. Six of the exons that encoded cytoplasmic domains were essentially identical in sequence in all three species indicating that these regions have undergone considerable selective pressure to conserve sequences. Thus, CD19 appears to be well conserved in structure and expression through recent mammalian evolution and the highly conserved cytoplasmic domains may play a critical role in the transduction of CD19-mediated signals.     

The HumanHNRPDLocus Maps to 4q21 and Encodes a Highly Conserved Protein

The hnRNP D protein interacts with nucleic acids bothin vivoandin vitro.Like many other proteins that interact with RNA, it contains RBD (or “RRM”) domains and arg-gly-gly (RGG) motifs. We have examined the organization and localization of the human and murine genes that encode the hnRNP D protein. Comparison of the predicted sequences of the hnRNP D proteins in human and mouse shows that they are 96.9% identical (98.9% similar). This very high level of conservation suggests a critical function for hnRNP D. Sequence analysis of the humanHNRPDgene shows that the protein is encoded by eight exons and that two additional exons specify sequences in the 3′ UTR. Use of two of the coding exons is determined by alternative splicing of theHNRPDmRNA. The humanHNRPDgene maps to 4q21. The mouseHnrpdgene maps to the F region of chromosome 3, which is syntenic with the human 4q21 region.     

Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene

The mouse pre-T-cell receptor alpha (pT) chain is a 33 000 M _r glycoprotein expressed on the surface of immature thymocytes as a disulfide-linked heterodimer with the T-cell receptor beta (TCR) chain, and in association with proteins of the CD3 complex. The cDNA for pT, isolated previously, encodes a type I transmembrane protein that is a member of the immunoglobulin (Ig) superfamily. Here we report the complete nucleotide sequence, the exon/intron structure, and the chromosomal location of the pTa gene. The gene spans about 8.4 kilobases (kb) and consists of four exons. Exon 1 encodes the 5 untranslated region, the leader peptide, and the first three amino acids of the mature protein. This exon is followed by a relatively long intron of 4.9 kb that contains many short interspersed repeats (SINEs) of the B1 and B2 family. The second exon encodes the extracellular Ig-like domain and exon 3 with just 45 base pairs the connecting peptide (CP), including the cysteine required for heterodimer formation. A similar exon/intron structure encoding corresponding parts of the mature polypeptide is found both in the Tcra and Tcrd constant region genes. The last exon encodes the transmembrane portion, the cytoplasmic tail, and about 540 nucleotides of 3 untranslated sequence, including a B2 repetitive element. In situ hybridization maps the pTa gene to the D/E1 region of mouse chromosome 17.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U27268     

The Human Lamin B Receptor/Sterol Reductase Multigene Family

LBR (lamin B receptor) is an integral protein of the inner nuclear membrane encoded by a gene on human chromosome 1q42.1. LBR has a nucleoplasmic, amino-terminal domain of approximately 200 amino acids followed by a carboxyl-terminal domain similar in sequence to yeast and plant sterol reductases. We have determined the primary structures of two human proteins with strong sequence similarity to the carboxyl-terminal domain of LBR and sterol reductases. Their genes have recently been assigned the symbols TM7SF2 and DHCR7. TM7SF2 mRNA is most predominantly expressed in heart and DHCR7 mRNA mostly in liver and brain. Whereas LBR is localized to the inner nuclear membrane, these two related proteins are in the endoplasmic reticulum. TheTM7SF2gene contains 10 coding exons, and its intron positions are exactly conserved in the part of theLBRgene encoding its carboxyl-terminal domain. Intron positions in theDHCR7gene are also similar. Both of these new LBR-like genes are on chromosome 11q13. These results describe a human gene family encoding proteins of the inner nuclear membrane and endoplasmic reticulum that function in nuclear organization and/or sterol metabolism.     

Isolation and characterization of cDNA clones for Humly9: the human homologue of mouse Ly9

Ly9 is a mouse cell membrane antigen found on all lymphocytes and coded for by a gene that maps to chromosome 1. We previously described the isolation and characterization of a full-lenght cDNA clone for mouse Ly9. Using cross-species hybridization we isolated cDNA clones encoding the human homologue Humly9. Analysis of the predicted protein sequence suggests that the extra-cellular portion of the Humly9 molecules is composed of four Ig-like domains: a V domain (V) without disulphide bonds and a truncated C2 domain (tC2) with two disulphide bonds, a second V domain without disulphide bonds and a second tC2 with two disulphide bonds, i.e., as V-tC2-V-tC2. The gene encoding Humly9 was mapped to chromosome 1 by analysis of human/hamster hybrids, and more specifically to the 1q22 region by in situ hybridization. The protein sequence data support the view that Humly9 belongs to the immunoglobulin-superfamily subgroup which includes CD48, CD2, and LFA-3.The nucleotide sequence data reported in this paper has been submitted to the GenBank nucleotide sequence database has been assigned the accession number L42621     

Lineage-specific diversification of killer cell Ig-like receptors in the owl monkey,a New World primate

Killer cell Ig-like receptors (KIRs) modulate the cytotoxic effects of natural killer cells. In primates, the KIRs are highly diverse as a consequence of variation in gene content, alternative domain composition, and loci polymorphism. We analyzed a bacterial artificial chromosome (BAC) clone draft sequence spanning the owl monkey KIR cluster. The draft sequence had seven ordered yet unconnected contigs containing six full-length and two partial gene models, flanked by the LILRB and FcAR framework genes. Gene models were predicted to encode KIRs with inhibitory, activating, or dual functionality. Four gene models encoded three Ig domain receptors, while three others encoded molecules with four Ig domains. The additional domain resulted from an insertion in tandem of a 2,101 bp fragment containing the last 289 bp of intron 2, exon 3, and intron 3, resulting in molecules with two D0 domains. Re-screening of the owl monkey BAC library and sequencing of partial cDNAs from an owl monkey yielded five additional KIRs, four of which encoded receptors with short cytoplasmic domains with premature stop codons due to either a single nucleotide substitution or deletion or the absence of exon 8. Phylogenetic analysis by domains showed that owl monkey KIRs were monophyletic, clustering independently from other primate KIR lineages. Retroelements found in introns, however, were shared by KIRs from different primate lineages. This suggests that the owl monkey inherited a KIR cluster with a rich history of exon shuffling upon which positive selection for ligand binding operated to diversify the receptors in a lineage-specific fashion. Nucleotide sequence data reported are available in the GenBank database under accession numbers FJ154791–5.     

Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.