Characterization and mapping to human chromosome 8q24.3 of Ly-6-related gene 9804 encoding an apparent homologue of mouse TSA-1.

The 9804 gene, which encodes a human Ly-6 protein most similar to mouse differentiation Ag TSA-1/Sca-2, has also been called RIG-E. Like mouse TSA-1, it has a broad tissue distribution with varied expression levels in normal human tissues and tumor cell lines. Like some members of the murine Ly-6 family, the 9804 gene is responsive to IFNs, particularly IFN-alpha. Overlapping genomic fragments spanning the 9804 gene (5543 bp) have been isolated and characterized. The gene organization is analogous to that of known mouse Ly-6 genes. The first exon, 2296 bp upstream from exon II, is entirely untranslated. The three coding exons (II, III, and IV) are separated by short introns of 321 and 131 bp, respectively. Primers were developed for specific amplification of 9804 gene fragments. Screening of human-hamster somatic cell hybrids and yeast artificial chromosomes (YACs) indicated that the gene is distal to c-Myc, located in the q arm of human chromosome 8. No positives were detected from the Centre d'Etude du Polymorphisme Humain mega-YAC A or B panels, nor from bacterial artificial chromosome libraries; two positive cosmids (c101F1 and c157F6) were isolated from a human chromosome 8 cosmid library (LA08NC01). Fluorescence in situ hybridization of metaphase spreads of chromosome 8, containing hybrid cell line 706-B6 clone 17 (CL-17) with cosmid c101F1, placed the 9804 gene close to the telomere at 8q24.3. This mapping is significant, since the region shares a homology with a portion of mouse chromosome 15, which extends into band E where Ly-6 genes reside. Moreover, the gene encoding E48, the homologue of mouse Ly-6 molecule ThB, has also been mapped to 8q24.

Increased expression of proopiomelanocortin (POMC) mRNA in adrenal glands of mice undergoing graft-versus-host disease (GVHD): association with persistent elevated plasma corticosterone levels.

GVHD in animal models induces severe thymic atrophy as a result of prolonged secretion of high concentrations of adrenal glucocorticoids. In this study we investigated the mechanism responsible for the persistent stimulation of the adrenal glands to secrete glucocorticoids in mice undergoing GVHD. GVHD was induced across the major and multiple minor histocompatibility antigen difference in unirradiated C57Bl/6 x AF1 hybrid mice by the intravenous injection of A strain parental lymphoid cells. Our results showed plasma corticosterone (CS) levels were elevated in association with high concentrations of corticotropin (ACTH) in both the GVHD and control syngeneic (SYN) groups on day 9. By days 16 and 24, plasma CS and ACTH in the SYN mice returned to basal levels. In contrast, plasma CS levels remained elevated in the GVHD animals on days 16 and 24 despite decreasing concentrations of plasma ACTH. Reverse transcription-polymerase chain reaction (RT-PCR) showed several-fold increase in POMC mRNA in the adrenal glands of GVHD mice compared with SYN animals. In addition, high mRNA levels for murine prohormone convertase 1, the enzyme that cleaves POMC into ACTH, were also detected in GVHD adrenals. Histological analysis of GVHD adrenals failed to show any sign of adrenalitis, and RT-PCR of GVHD adrenals also failed to detect mRNA for interferon-gamma (IFN-gamma), a cytokine expressed by activated T and natural killer (NK) cells. However, mRNA for IL-12, a cytokine produced by activated macrophages, was increased in GVHD adrenals, suggesting that resident adrenal macrophages were activated during GVHD. Our findings suggest that persistent elevated levels of plasma glucocorticoids during GVHD could be mediated by intra-adrenal ACTH produced by resident adrenal macrophages activated as a consequence of GVHD.

Development of a novel type of cloning vector for suicide selection of recombinants.

Based on the primary structure of the rat corticostatin R4 (antimicrobial defensin RatNP-1), a synthetic gene was designed, synthesized, and inserted into the IPTG-inducible prokaryotic expression vector pFLAG, with an additional 13 codons between the FLAG sequence and the synthetic R4 sequence. This construct, N-p1.2, was further developed by inclusion of multiple cloning sites right after the FLAG sequence, forming a new plasmid pSCV-1. Escherichia coli transformants containing pSCV-1 or N-p1.2 could only be propagated on agar plates in the absence of IPTG due to the detrimental expression of R4 fusion peptide to the growth of bacteria upon IPTG induction. A 214-bp bovine IGF-II cDNA and a 700-bp Ly-6C.2 cDNA fragment were subcloned into pSCV-1 and N-p1.2 respectively. Only the E. coli cells transformed with recombinant plasmids grew on IPTG agar plates. This "suicide" selection against nonrecombinants was further tested in cDNA library construction using pSCV-1. Analysis of plasmid DNA prepared from randomly picked colonies growing on ampicillin agar plates containing IPTG showed all plasmids contained cDNA inserts. The lambda Hind III fragments were used for comparing the cloning efficiency of pSCV-1 to pBluescript. Four of the 60 (6.6%) analyzed white colonies transformed with pBluescript were false positives. All of the analyzed pSCV-1-transformed colonies growing on IPTG plates contained recombinant forms of plasmid. The percentage recovery of each ligatable lambda Hind III fragment was similar in both pBluescript and pSCV-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of corticostatins/defensins: higher levels of CS-4 (NP-2) transcripts compared with CS-6 (NP-5) in rabbit lung.

cDNA clones encoding two members of the corticostatin (CS)/defensin family of peptides have been isolated from a rabbit bone marrow cDNA library through cross-hybridization with cDNA encoding the human corticostatin HP-4. They encode the precursors of CS-4 (NP-2, MCP-2) and CS-6 (NP-5). Highly specific probes were generated for Northern blotting of RNA from various normal rabbit tissues. Both detected high levels of message in bone marrow, moderate in spleen, and low in thymus. In lung, however, while moderate levels were detected with the CS-4 probe, CS-6 message was barely detectable. This correlates with the report of CS-4, but not CS-6 peptide in lung macrophages, and implies that differential regulation of CS/defensin expression in rabbit lung is at the level of gene transcript abundance.

The gene encoding the human corticostatin HP-4 precursor contains a recent 86-base duplication and is located on chromosome 8.

The human neutrophil-derived cationic peptide HP-4 exhibits corticostatic activity on adrenal cells and is an L-type calcium channel agonist at nanomolar concentrations. Complementary DNA clones encoding the HP-4 precursor have been isolated from a human bone marrow cDNA library by screening with oligonucleotide probes. The nucleotide sequence shares about 72% identity with the cDNA encoding defensin HP-1, but differs from it, and from other genes of this family characterized to date, by an extra 83-base segment. This extra segment is not adjacent to an intron and is apparently the result of a recent duplication within the coding region corresponding to most of the mature HP-4 peptide. The predicted amino acid sequence shows the HP-4 precursor structure to be typical of this family of molecules. By analysis of DNA from a pannel of hamster/human hybrid cell lines, the HP-4 gene was found to be on chromosome 8, as is the gene for human peptide HP-1. Comparison with the few sequences of other corticostatin/defensin genes available does not indicate distinct lineages of corticostatic and noncorticostatic peptides, since HP-1 and HP-4 cDNA sequences share more identity with each other than either shares with cDNAs encoding rabbit MCP-1 or MCP-2, or guinea pig GNCP-1.

Nucleotide sequence of the central coding region of bovine erythrotropin/insulin-like growth factor II cDNA from fetal intestine and northern analysis of the major IGF II transcripts at the time of hepatic erythropoiesis.

1. cDNA for insulin-like growth factor II (IGF II) was synthesized from poly A(+)-RNA from fetal bovine intestine and amplified. 2. The sequence corresponding to amino acids 6-62 was identical to a published ovine IGF II sequence cDNA with the exception of a single nucleotide change (G to A). 3. Northern blot analysis of intestine, liver, kidney and spleen from bovine fetuses showed multiple IGF II RNA species which are more similar to the human than to the rodent mRNAs. 4. Under the hybridization conditions used, synthesis of antisense strands, as described in embryonic chicken IGF II transcripts, was undetectable.

Isolation and sequence of the granulin precursor cDNA from human bone marrow reveals tandem cysteine-rich granulin domains.

Granulins are candidate growth factors recently discovered in human and rat inflammatory leukocytes and bone marrow. Two granulin homologs, epithelin 1 and 2, occur in the rat kidney. Epithelin 1, which is probably identical to rat leukocyte granulin, exhibits proliferative and antiproliferative effects on epithelial cells in vitro. Here we show by cDNA analysis that the prepropeptide for the human granulins is a 593-residue glycoprotein, containing seven tandem repeats of the 12-cysteine granulin domain. By Northern blot analysis, gene expression was seen in myelogenous leukemic cell lines of promonocytic, promyelocytic, and proerythroid lineage, in fibroblasts and was seen very strongly in epithelial cell lines. Some epithelial cell lines respond to the mature peptide and express the gene. Among tissues examined, the kidney had the highest levels of granulin mRNA.

Role of protein kinase C in IFN-mediated Ly-6E antigen induction.

The YAC T cell lymphoma normally does not express Ly-6E mRNA or Ly-6E surface molecules but can be induced to do so on incubation with either IFN-gamma or IFN-alpha/beta. This system afforded a model to assess the possible role of protein kinase C (PKC) in IFN-mediated Ly-6E induction. First, we used various pharmacologic agents known to interfere with the function of PKC or other kinases. The PKC inhibitors H-7 and phloretin were found to block Ly-6E induction by IFN-gamma or IFN-alpha/beta both at the mRNA and protein levels. In contrast, inhibitors of cyclic nucleotide-dependent kinases (HA1004), of myosin L chain kinase (ML-9, A-3) or of calmodulin (R24157, W-7) failed to suppress this induction. Next, we investigated the effects of the PKC activators PMA and mezerein (MEZ) on Ly-6E expression. Although neither PMA nor MEZ by themselves could induce Ly-6E in YAC cells, both agents enhanced by up to fivefold the induction of Ly-6 mRNA and Ly-6E surface expression triggered by IFN-gamma. However, the induction of Ly-6E expression caused by IFN-alpha/beta was only marginally increased by cotreatment of YAC cells with PMA or MEZ. Altogether, these observations demonstrate that PKC or a related kinase is involved in the transduction mechanisms that lead to Ly-6E induction. However, activation of PKC is not sufficient for this induction and requires other unidentified signal(s) provided by IFN. Our data also indicate that IFN-gamma and IFN-alpha/beta induce Ly-6E through overlapping but distinct intracellular pathways with different sensitivities to PKC activators.

The CD59 antigen is a structural homologue of murine Ly-6 antigens but lacks interferon inducibility.

A cDNA encoding the human leukocyte antigen CD59 has been isolated from the erythroid cell line K-562 and its identity confirmed through expression in COS cells. Northern blotting reveals three message species of approximately 800, 1400 and 2000 bases in size, which are constitutively expressed in all lymphoid, erythroid, myeloid, and neural cell types tested thus far. Southern blotting of human DNA indicates a pattern consistent with the presence of a single gene, which has been mapped to chromosome 11 by somatic cell hybrids. Also, the finding of a transcriptionally active cross-hybridizing gene in monkey cells suggests conservation of CD59 sequences among primates. Comparison of the CD59 protein sequence with those of the Ly-6E and Ly-6C antigens discloses a similarity in overall structure, including the alignment of abundant cysteine residues, hydrophobic carboxy termini and conservation of amino acids surrounding the proposed phosphatidylinositol-glycan modification site for Ly-6 molecules. Unlike Ly-6, however, CD59 expression does not appear to be inducible with interferons. This, along with its limited homology and different tissue distribution, cast doubt upon the functional equivalence of CD59 and either of the well-characterized mouse Ly-6 proteins.

Analysis of three distinct Ly6-A-related cDNA sequences isolated from rat kidney.

Mouse Ly6A and Ly6C cDNA probes were hybridized to total RNA of rat tissues and, as in mouse, the highest level of Ly6-related transcripts was detected in kidney. Therefore, Ly6-related cDNA clones were isolated from a commercial rat kidney cDNA library in lambda gt11. Four of these (RK3, RK6, RK10, and RK11) have been fully characterized, and represent transcripts from three distinct genes. Each contains a reading frame encoding an amino acid sequence typical of the known Ly6 molecules: a 26aa leader (except in clone RK6 which has only two of its leader codons), followed by a sequence of 108 or 109aa containing 10 cysteines in excellent alignment with those of Ly6A. The three rat polypeptide sequences were more closely related to Ly6A than Ly6C, and more closely related to each other than to Ly6A. The most striking similarity between all these sequences is in the last 33aa at the C-terminal. Most of this is presumed to be cleaved off during post-translational addition of a phosphatidylinositol-glycan (PI-G) membrane anchor. Southern blot analysis of rat DNA probed with rat-Ly6 cDNA showed multiple band patterns indicative of a multigene family. No restriction fragment length polymorphism (RFLP) was evident amongst the six inbred rat strains tested. Anomalies in two of the rat cDNA clones, resulting from improper splicing of the original transcripts, correlated with Ly6Ca exon boundaries, thus suggesting conserved intron-exon organization.

Kinetic analysis of Ly-6 gene induction in a T lymphoma by interferons and interleukin 1, and demonstration of Ly-6 inducibility in diverse cell types.

The Ly-6 locus contains multiple genes encoding cell surface proteins, two of which, when cross-linked by antibodies, effect antigen-independent activation of T lymphocytes. In this study, cDNA for Ly-6-encoded antigens have been used as probes to examine RNA from various tissues and transformed cell lines for constitutive levels of Ly-6 RNA expression. Analyses of RNA prepared from several different tissues revealed a high level of expression of Ly-6 RNA in kidney, spleen, heart and thymus, with a more moderate level of expression in liver, brain and lung tissue cells. A survey of various cell lines demonstrated the presence of Ly-6 RNA in many, but not all T lymphocytic cell lines, in L cells, the Meth A fibrosarcoma, in the TCMK kidney cell line, and in the Neuro-2a neuroblastoma. We also evaluated the expression of Ly-6 RNA in cells after treatments with interferons (IFN) and interleukin 1 (IL1). Treatment of lymphoid cells with IFN (alpha/beta and gamma), known to increase cell surface Ly-6 antigen expression in normal T cells, was correlated with increases in Ly-6 RNA levels. Increases in levels of RNA correlated with increases in levels of the Ly-6A/E or Ly-6C antigens. Several T lymphoid cell lines exhibiting Ly-6 RNA inducibility by IFN were similarly inducible with IL1. Kinetic experiments using one such line, (YAC-1), showed that the induction of Ly-6 RNA mediated by IFN-alpha/beta occurred rapidly (within 4 h), while the induction by IL1 required relatively more time (approximately 8 h). Although the actions of IFN-alpha/beta were not blocked by cycloheximide, the presence of this protein synthesis inhibitor significantly attenuated the effects of IL1 and IFN-gamma on Ly-6 RNA transcription. Induction by IFN-gamma as well as IL1 could be blocked completely by co-culture with anti-IFN-gamma, implicating IFN-gamma as a mediator of the induction by IL1.

The T-cell activating protein (TAP) is up-regulated by endogenous IFN-gamma in activated T cells.

The surface expression of the T-cell activating protein (TAP) glycoprotein has been shown to be increased following mitogenic stimulation of T cells. Recently, we found that TAP is also augmented by exogenous interferon-gamma (IFN-gamma) in resting T cells. Because T cells are known to secrete IFN-gamma upon activation, we postulated that TAP enhancement in activated T cells may reflect an autocrine action of endogenous IFN-gamma. This possibility was examined using a potent neutralizing anti-IFN-gamma mAb (H-22.10). Supernatants from Concanavalin A (Con A)-stimulated T cells were found to induce TAP enhancement in resting T cells, and this effect was blocked by the anti-IFN-gamma mAb. Stimulation of T cells with Con A or with the combination of ionomycin plus phorbol myristate acetate produced a marked increase of TAP expression. Addition of the H-22.10 mAb at the initiation of such stimulated T-cell cultures was found to prevent the augmentation of TAP but not to affect the emergence of IL-2 receptors or the increase of Pgp-1 expression. Taken together, these data demonstrate that IFN-gamma is the principal TAP-enhancing mediator released by stimulated T cells. Endogenously produced IFN-gamma, rather than cell activation per se, is thus responsible for the augmentation of TAP expression in stimulated T cells.

Ly-6 A/E antigen of murine T cells is associated with a distinct pathway of activation. Requirements for interferon and exogenous interleukin 2.

The Ly-6 pathway of T cell activation was analyzed to identify its essential requirements. Using a monospecific chicken antiserum to Ly-6E, fully cross-reactive to its allelic counterpart, Ly-6A, but unreactive with other members of the Ly-6 family, we have found that interferon (IFN)-alpha/beta or gamma, Ly-6 antibody and interleukin 2 (IL 2) act synergistically in inducing T cell proliferation. The action of IFN can be attributed to induction of Ly-6A/E antigen on T cells, as described previously, and this induction is transcriptionally controlled. Exposure of T cells with elevated Ly-6 concentrations to chicken anti-Ly-6 antibody leads to expression of IL 2 receptors. Consequently, the addition of IL 2 drives T cell proliferation. Thus, in BALB/c mice the minimum requirements for activation by the Ly-6 pathway are IFN (as a means of inducing Ly-6). Ly-6 antibody (as inducer of IL 2 receptors) and IL 2. In mice of the Ly-6.2 haplotype, IFN is not an absolute requirement. This may be related to the fact that these animals, in contrast to those of Ly-6.1 haplotype, express Ly-6 constitutively on a substantial proportion of resting T cells. Thus, T cells of C57BL/6 or DBA/2 mice can be induced to proliferate with Ly-6 antibody and IL 2 alone, although IFN pretreatment enhances this response. In BALB/c mice the IL 2-driven proliferative response induced through the Ly-6 pathway occurs selectively in the L3T4- population.

Mls and the helper T-cell repertoire. I. Mls as a regulator of T-helper cell activation.

We provide evidence that the Mls reaction involves a broad cross-section of the helper cell population. In addition to those cells reacting overtly to Mls stimulatory spleen cells, there is a second large population of helper cells that are affected by an Mls difference. This latent Mls effect is manifested by either synergy or antagonism in mitogen-mediated, Ia-dependent T-cell activation, depending on Mlsa or Mlsb phenotypes of stimulator cells, respectively. Our data can be explained by attributing to Mls alleles the function of differential regulation of autoreactivity of T cells. The results show that the concept of the Mls exerting a negative signal deserves serious consideration.

N-terminal and cDNA characterization of murine lymphocyte antigen Ly-6C.2.

The Ly-6C.2 molecule was purified from K36 tumor cells by affinity chromatography and gel filtration. The electrophoretically homogeneous preparation, with m.w. 15,000, was tested with a panel of antibodies that confirmed the presence of the LY-6C.2 epitope. An N-terminal sequence of 39 amino acids was obtained showing 59% homology with the corresponding portion of the Ly-6A.2 polypeptide. Based on the least homologous (29%) 14 amino acid segment, an oligonucleotide probe was constructed, and Ly-6C.2 cDNA was cloned from a BW5147 cDNA library. A 794-base pair cDNA containing the entire coding region had 82% homology with Ly-6A.2 cDNA. The encoded polypeptide sequence of 131 amino acids containing a perfect correlation with the N-terminal sequence data was 63% homologous with that of Ly-6A.2. The greatest homology was in the leader, first 16 N-terminal and last 39 C-terminal amino acids. The latter are likely to be important in determining the attachment of glycophosphatidylinositol. Despite results indicating fewer disulfide constraints in the Ly-6C molecule, the predicted sequence contains 10 cysteine residues nearly perfectly matched with those predicted in Ly-6A.

Demonstration of phosphatidylinositol anchors on Ly-6 molecules by specific phospholipase C digestion and gel electrophoresis in octylglucoside.

Release by phosphatidylinositol-specific phospholipase C is frequently provided as evidence for membrane anchorage of a protein through phosphatidylinositol. Demonstration that the enzyme removes a lipophilic moiety from the protein is stronger evidence, and is presented here for members of the Ly-6 family of lymphocyte antigens: Ly-6A, C and E. Treatment of these molecules with the enzyme greatly increased their electrophoretic mobilities on polyacrylamide gels containing nonionic detergent. Furthermore, the mobilities of the digested, but not native Ly-6 molecules, were independent of detergent. This analytical method was applied to pure antigen, radiolabelled immunoprecipitate, or immunochemically detected Ly-6 antigens on blots.

Selective up-regulation by interferon-gamma of surface molecules of the Ly-6 complex in resting T cells: the Ly-6A/E and TAP antigens are preferentially enhanced.

Surface molecules encoded by the murine Ly-6 locus can transduce triggering signals in T cells and thus may play important roles in T cell function. Previously, we found that Ly-6 molecules are up-regulated by interferon (IFN)-alpha/beta in resting T cells. Here, we examined the possible influence of IFN-gamma on these molecules. Purified T cells from C57BL/6 (Ly-6.2) and BALB/c (Ly-6.1) mice were incubated in vitro with recombinant murine IFN-gamma and the expression of Ly-6 antigens was measured by flow cytofluorometry. It was found that both Ly-6A/E and T cell-activating protein (TAP) molecules are markedly enhanced while Ly-6C is less affected. Under the same conditions, other T cell surface molecules showed no or marginal changes. The effect of IFN-gamma on Ly-6A/E and TAP expression reached a maximum with as little as 10 U/ml and required only 18-24 h of incubation. Moreover, the enhancement of Ly-6A expression induced by IFN-gamma was stable for at least 5 days. Analysis of T cell subsets further revealed that IFN-gamma-induced augmentation of Ly-6A (C57BL/6 mice) involves both Lyt-2+ and L3T4+ cells while the increase of Ly-6E (BALB/c mice) is more pronounced in Lyt-2+ cells. The functional consequence of these phenotypic alterations was evaluated by studying the mitogenic responses of T cells to antibody-mediated Ly-6 cross-linking in the presence of phorbol myristate acetate. Pretreatment of resting T cells with IFN-gamma dramatically increased the responses to anti-Ly-6A and anti-Ly-6E monoclonal antibodies. IFN-gamma treatment also boosted the stimulation induced by anti-TAP monoclonal antibody when this stimulation was performed under suboptimal conditions. Therefore, IFN-gamma selectively up-regulates the Ly-6A/E and TAP activation pathways in resting T cells. We speculate that this effect may contribute to the immunoregulatory activities of IFN-gamma.

Murine Ly-6 multigene family is located on chromosome 15.

Murine Ly-6-encoded molecules play an important role in the antigen-independent activation of lymphocytes. We have described the cloning of a cDNA encoding the protein component of an Ly-6 molecule. Hybridization studies indicated that this cDNA identified multiple DNA fragments on Southern blots. The banding pattern exhibits a restriction fragment length polymorphism from mice bearing either the Ly-6a or the Ly-6b allele. We have employed three independent chromosomal mapping techniques, somatic cell hybrids, in situ hybridization, and strain distribution pattern analysis of the restriction fragment length polymorphism of DNA from recombinant inbred lines, to ascertain the chromosomal origins of these bands. We report that all members of the Ly-6 multigene family are tightly linked on chromosome 15 and have been regionalized by in situ hybridization analysis to band 15E on the distal portion of this chromosome. Linkage analysis has indicated that the Ly-6 genes are located within 1 map unit of Env-54 (a retroviral envelope restriction fragment length polymorphism probe), 3 map units from ins-1, (insulin-related gene), and 4 map units from the protooncogene c-sis. The possible involvement of the Ly-6 lymphocyte activation and differentiation antigen genes in chromosome 15-related lymphoid malignancies is discussed.