Characterization of the human homolog of the rat MRC OX-2 membrane glycoprotein

The MRC OX-2 antigen is a membrane glycoprotein present on rat thymocytes, neurons, follicular dendritic cells, endothelium, and some smooth muscle. The sequence of 248 amino acids has similarities to Ig domains organized with one V-like domain, one C-like domain, and transmembrane and cytoplasmic regions. Thus it resembles a T-cell receptor chain but shows no sequence divergence. We report the characterization of the human gene for this molecule. Its exon organization is similar to that found for immunoglobulins although the region with similarities to Ig J regions is found within the same exon as the V-like domain. Human MRC OX-2 is expressed at the mRNA level in brain and B-cell lines but not detected in liver or T-cell lines. It does not obviously correspond to any previously defined leukocyte antigen. The sequence homology for the human and rat MRC OX-2 molecules is higher for the Ig-related region (75 %) than for many other Ig-related molecules and very high in the transmembrane region (96 %), implying a functional role other than simply its anchoring into the membrane.     

The human leucocyte surface antigen CD53 is a protein structurally similar to the CD37 and MRC OX-44 antigens

CD53 is an N-glycosylated pan-leucocyte antigen of 35–42 000 M _r. The sequence of the CD53 polypeptide deduced from a cDNA clone is 219 amino acids in length. It appears to lack a conventional leader sequence because the deduced NH₂-terminal amino acid sequence is very similar to the rat MRC OX-44 and human CD37 antigens. The CD53 molecule is likely to consist of four transmembrane regions and a major extracellular hydrophilic loop containing two potential N-glycosylation sites. It is suggested that the CD53 glycoprotein is the true human homologue of the rat OX-44 antigen, rather than the CD37 antigen of more restricted expression and lower NH₂-terminal sequence similarity to OX-44.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M37033. Offprint requests to: P. Angelisová.     

MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain.

The MRC OX-2 antigen is a rat cell surface glycoprotein of mol. wt. 41 000-47 000 found on neurones, thymocytes, B cells, follicular dendritic cells and endothelium. We now report the amino sequence for this antigen as deduced from the nucleotide sequence of cDNA clones detected by use of an oligonucleotide probe. The sequence contains 248 amino acid residues of which 202 residues are likely to be outside the cell with two domains that show homology with immunoglobulins. The N-terminal domain fits best with Ig V domains and Thy-1 antigen while the C-terminal part is like an Ig C domain. Thus the structure overall is similar to an Ig light chain or the T cell receptor beta chain. Three glycosylation sites are identified on each of the MRC OX-2 antigen domains.     

Purification, chain separation and sequence of the MRC OX-8 antigen, a marker of rat cytotoxic T lymphocytes.

The MRC OX-8 antigen is a marker of the rat cytotoxic T lymphocytes that consists of disulphide-linked chains of mol. wts. 37 and 32 kd. It is thought to be equivalent to the human T8 and mouse Lyt2,3 antigens (all now called CD8 antigens). MRC OX-8 antigen was purified from thymocytes using a monoclonal antibody column and because antigenicity was retained after reduction and alkylation the two polypeptide chains could be separated by a subsequent affinity chromatography step. Peptides were isolated from each chain and their sequences determined. A cDNA probe coding for the mouse CD8 antigen (pLY2C-1 provided by Dr L. A. Herzenberg) was used to obtain rat cDNA clones from which the sequence of the equivalent rat molecule was determined. Peptides from the 32-kd chain were identified in this translated sequence whereas peptides from the 37-kd chain were not. The 32-kd polypeptide sequence consisted of 210 amino acids and had one possible N-linked glycosylation site. The N-terminal part of the sequence was surprisingly different from both its mouse and human counterparts but, as in the other two species, it showed a clear relationship to Ig V domains.     

Two coding regions closely linked to the rat apolipoprotein E gene: nucleotide sequences of rat apolipoprotein C-I and ECL cDNA.

Restriction fragments isolated from a 17-kb rat genomic DNA clone containing the gene for apolipoprotein (apo) E were radiolabeled and used to screen a rat liver cDNA library. A cDNA clone hybridizing to a 6-kb genomic DNA fragment was isolated and the nucleotide sequence of the cDNA insert determined. The sequence was homologous to the sequence for human apo C-I and was used to derive the corresponding amino acid sequence. Unlike human apo C-I, mature rat apo C-I contains histidine, lacks valine, and has alanine at the C terminus and aspartate as the N terminus. Screening the rat liver cDNA library with a radiolabeled 1.9-kb restriction fragment from the genomic DNA clone containing the rat apo E gene identified another cDNA clone (ECL cDNA). Nucleotide sequencing yielded a derived 75-amino-acid sequence for the ECL protein with a hydrophobicity profile similar to that of rat apo C-I. Northern analysis demonstrated a 0.50-kb band for ECL mRNA. The tissue-specific expression of the gene is similar to that of rat apo C-I. This study indicates that the rat apo C-I and ECL genes are closely linked, about 4.5 and 12 kb downstream of the apo E gene, respectively.     

Sint1, a common integration site in SL3-3-induced T-cell lymphomas, harbors a putative proto-oncogene with homology to the septin gene family

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma, Pad3. Two overlapping genomic lambda clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.     

Human carboxypeptidase A identifies a BglII RFLP and maps to 7q31-qter.

A genomic clone for human carboxypeptidase has been isolated with a probe for rat CPA1 cDNA. A 1.7-kb HindIII/EcoRI fragment from the 3' flanking region of human carboxypeptidase detects a DNA polymorphism with BglIII. Multipoint linkage analysis with an established map of chromosome 7 markers shows that the most likely location of carboxypeptidase is at 7q31-qter, between D7S87 and D7S93. All other placements can be excluded with odds greater than 100:1. These and other markers confirm that carboxypeptidase lies distal to the locus for cystic fibrosis, at a distance of approximately 12 centimorgans. The regions containing identity to the rat gene were sequenced and shown to be 82% identical to exons 9 and 10 of the rat gene. The presence of a codon for isoleucine at the residues corresponding to codon 255 of rat CPA1 cDNA strongly suggests that the A form of human carboxypeptidase has been isolated.     

Isolation of four HSP70 genes in the pig and localization on Chromosomes 7 and 14

For insight into the general organization of the swine leukocyte antigen (SLA) complex, the swine major histocompatibility complex (MHC), four sequences related to the heat-shock proteins HSP70 were characterized by screening of a pig genomic cosmid library with a swine cDNA HSP70 2.6-kb probe. This yielded three positive clones: HC2.2, HC3.2, and HC4.2. Restriction site maps revealed a large overlap of HC2.2 with HC3.2, whereas HC4.2 was independent. Southern blot hybridization with the 5 section, the central section, and the 3 section of the 2.6-kb probe and also with a swine 4.5-kb HSP70 genomic probe suggested the existence, within the overlapping clones, of three distinct HSP70 sequences encompassing a segment no longer than 22 kb. The HC4.2 clone, which hybridized with the same probes, displayed a single band of 7.3 kb, probably corresponding to one gene only. Fluorescent in situ hybridization on swine chromosome metaphases with the whole HC2.2 or HC4.2 cosmids allowed the assignment of HC2.2 to MHC region on Chromosome (Chr) 7 (Cen-p1.1), and of HC4.2 to Chr 14 (q2.4–2.5). Thus, as in humans, the swine MHC comprises three closely linked HSP70 loci. The presence of additional genes belonging to the same inducible HSP70 gene family can be expected from what is known in humans. The HSP70 gene found here on the pig Chr 14 may be one of these putative unidentified genes.     

Membrane antigen phenotype of sensitized T lymphocytes mediating tuberculin-delayed hypersensitivity in rats

The membrane antigen phenotype of immune lymph node cells (LNC) which mediate tuberculin-delayed hypersensitivity (DH) in Lewis rats was examined. The results show that the T-cell population which expresses the RT7.1 alloantigen defined by the BC 84A monoclonal antibody contained cells capable of transferring DH. Separation of the RT7.1-positive T-cell population with the monoclonal antibodies W3/25, MRC OX-8, or DS 4.23 (which defines the RT6.1 alloantigen) revealed that either the W3/25-positive or the RT6.1-negative T-cell subpopulations contained DH effector cells, whereas the corresponding MRC OX-8-positive or RT6.1-positive T-cell subpopulations did not. Moreover, when the W3/25-positive T-cell subpopulation was divided into either RT6.1-positive or RT6.1-negative T-cell subsets, only the W3/25-positive, RT6.1-negative subset transferred DH. These results indicate that the effector cells that mediate tuberculin DH are contained within the immune T-cell subset which bears both the RT7.1 and the W3/25 markers, but lacks both the MRC OX-8 and the RT6.1 markers.     

Surface phenotype of rat bone marrow-derived mononuclear phagocytes

Utilizing a panel of currently available monoclonal antibodies, the surface phenotype of a pure population of resting rat bone marrow-derived mononuclear phagocytes (BMM phi) was analyzed by means of flow cytometry. The present work provides an extensive list of surface markers expressed by BMM phi and also outlines advantages and limitations of flow cytometric analysis of this cell type. The results show that the majority of surface markers considered to be expressed selectively by T lymphocytes, such as Thy-1, CD2 and CD5 antigens, leukosialin (W3/13), or an alloantigen of peripheral T cells, are not expressed by BMM phi. On the other hand, the CD8 antigen and the leukocyte common antigen recognized by MRC OX-33, considered to represent specific markers of cytotoxic T cells and/or peripheral B cells, are expressed on a variable, often considerable proportion of BMM phi. Monoclonal antibodies W3/25, MRC OX-35, and MRC OX-38, directed against epitopes on the CD4 molecule, labeled a variable proportion of BMM phi. Among the 39 monoclonal antibodies examined, none appeared to recognize an epitope which is expressed selectively by mononuclear phagocytes.     

Structure of a Moloney murine leukemia virus-virus-like 30 recombinant: implications for transduction of the c-Ha-ras proto-oncogene.

Tumor progression locus 2 (Tpl-2) encodes a novel serine-threonine protein kinase which is activated by provirus integration in the late stages of oncogenesis in Moloney leukemia virus (MoMuLV) induced rat T-cell lymphomas. In this report, we present evidence that the provirus integrated in the Tpl-2 locus in 1 of 10 T-cell lymphomas harboring a Tpl-2 rearrangement (2779) is a recombinant between MoMuLV and virus-like 30 (VL30) sequences (Mo-VL30). Recombination between MoMuLV and VL30 may contribute to the transduction of ras, as suggested by the finding that VL30 flanks the ras oncogene in all of the ras transducing viruses isolated from rats to date. The Mo-VL30 recombinant described here represents evidence that recombination between MoMuLV and VL30 can be uncoupled from the transduction of ras, and it may precede the transduction. Sequence comparison between clones of Mo-VL30, Harvey sarcoma virus (Ha-MSV), and genomic c-Ha-ras revealed that all three share a 124-bp region of 87.3% homology. This region was detected at nucleotide positions -1845 to -1720 of c-Ha-ras and 20 bp 5' of the recombination breakpoint between VL30 and ras in Ha-MSV. On the basis of the sequence comparison between VL30, Ha-MSV, and c-Ha-ras, we are proposing a model which explains how VL30 may have facilitated the transduction of c-Ha-ras and perhaps the other ras proto-oncogenes. According to this model, the sequence homology between VL30 and c-Ha-ras targets this gene for transduction by promoting the integration of the provirus in this locus through homologous recombination.     

Expression of leucocyte antigens by cells from the metrial gland of the pregnant rat

Summary The function of the metrial gland of the rat, and particularly of its characteristic population of granulated cells, remains unknown. However, several lines of evidence suggest that the granulated cells may derive from lymphocytes, and play a role in the immunology of pregnancy. In this study, antigen expression by granulated and other cell populations from the metrial glands of rats at Days 13 and 14 of pregnancy was studied by an indirect immunoperoxidase method. Acetone-fixed frozen sections, and cytocentrifuge preparations of collagenase-dispersed metrial gland tissue in which numbers of granulated cells had been increased by density-gradient centrifugation, were used. The primary antibodies used recognised, inter alia, B lymphocytes (MRC OX-3, MRC OX-6, MRC OX-12), T lymphocytes (MRC OX-8, W3/25, MRC OX-19), neutrophils (MRC OX-42) and cells of the monocyte/macrophage series (MRC OX-3, MRC OX-6, MRC OX-42, MRC OX43). The majority of the granulated cells, including smaller, immature forms, were unlabelled by any of these antibodies. Some lymphocytes, and varying numbers of larger, non-granulated cells, were labelled by OX-6, OX-12, W3/ 25, OX-42 and OX-43. In addition to lymphocytes, labelled cells included neutrophils (OX-42), endothelial cells (OX-43), and probably some macrophages (OX-6, OX-43). OX-12, which recognises the kappa chain of rat IgG, labelled some large cells which may have been stromal cells. These findings do not support the concept that the granulated cells are derived from lymphocytes.     

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.     

Identification of an H-2Kd gene using a specific cDNA probe.

A cDNA clone known to code for a mouse histocompatibility (class I) antigen was found to contain a sequence specific for a subpopulation of H-2 genes. This unique sequence is located in the 3'' non-coding region close to the stretch of poly(A) nucleotides. A subclone containing this fragment (pH-2d-5) has been used to select hybridizing mRNA. Translation of the mRNA in vitro shows that H-2Kd mRNA is selected. Southern blot analysis of DNA from congenic recombinant mice show that at least one gene containing this sequence is located at the K locus (region) of the major histocompatibility complex. This gene contains a 3.7-kb BglII and a 13-kb EcoRI restriction endonuclease fragment. This gene has been isolated from a genomic DNA library.     

Localisation of the MRC OX-2 Glycoprotein on the Surfaces of Neurones

The MRC OX-2 monoclonal antibody recognises membrane glycoproteins of Mr 41,000 in rat brain and 47,000 on thymocytes. It also reacts with follicular dendritic cells in lymphoid organs, endothelium, smooth muscle, and B-lymphocytes. Indirect immunoperoxidase staining of cryostat sections showed that OX-2 antigen was present throughout the cerebellum, with staining of both grey and white matter. Blood vessels were also stained. The Purkinje cell layer appeared to be unlabelled. Double-immunofluorescence staining of cerebellar interneurone cultures with MRC OX-2 antibody and tetanus toxin showed that all tetanus-positive cells (neurones) were MRC OX-2-positive. Glial fibrillary acidic protein-positive astrocytes were not labelled by MRC OX-2 antibody. Thus OX-2 antigen is one of the few biochemically characterised components of neuronal membranes and its properties are compared with those of the neuronal membrane glycoprotein Thy-1.