Cloning, expression analysis, and chromosomal localization of BH-protocadherin (PCDH7), a novel member of the cadherin superfamily

A method is described that allows simultaneous measurement of two spectrally distinguishable green fluorescent protein (GFP) mutants with a confocal microscope. In contrast to previously described methods, neither UV excitation nor repetition of scans is required. Therefore the method is well-suited to the long-time observation of living cells in three-dimensional microscopy and time series recording, as demonstrated with GFP-expressing Dictyostelium discoideum cells.     

Cloning and characterization of a novel serine/threonine protein kinase expressed in early Xenopus embryos

We have cloned from a Xenopus ovary cDNA library a novel protein kinase gene whose expression peaks in the oocyte and unfertilized egg, begins to decrease gradually after fertilization, and disappears during the gastrulation stage of embryogenesis. The cloned gene, termed XEEK1 (for Xenopus egg and embryo kinase), encodes a protein with a predicted molecular mass of 49 kDa. Bacterially expressed XEEK1 migrates at 57 kDa upon polyacrylamide gel electrophoresis analysis, and a XEEK1-specific antibody recognizes a protein of 57 kDa in Xenopus oocyte and egg extracts. The XEEK1 kinase domain shares 35% identity (approximately 65% similarity) with the yeast SNF1 kinase and related kinases. However, expression of XEEK1 does not complement a snf1 deletion mutation in yeast, which suggests that it is probably not a Xenopus homolog of SNF1. Recombinant XEEK1 protein autophosphorylates on threonine residues in vitro in a reaction that prefers Mn2+ to Mg2+ ions. Site-directed mutagenesis of the conserved lysine residue (Lys-81) within the kinase domain to isoleucine totally abolishes kinase activity, and threonine 192 has been identified as the autophosphorylation site. This site is distinct from the conserved threonine (Thr-215 in XEEK1) present in the protein kinase activation loop that is the site of autophosphorylation for many protein kinases. XEEK1 is a substrate for the cyclic AMP-dependent protein kinase both in vitro and in vivo, suggesting a possible mode of regulation of XEEK1. An immunoprecipitate of oocyte/egg extracts with anti-XEEK1 serum contains a protein of approximately 155 kDa that may be a substrate and/or a regulatory component of the kinase.     

Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic beta-cells, brain, heart and skeletal muscle

A cDNA clone encoding an inwardly-rectifying potassium channel subunit (Kir6.2) was isolated from an insulinoma cDNA library. The mRNA is strongly expressed in brain, skeletal muscle, cardiac muscle and in insulinoma cells, weakly expressed in lung and kidney and not detectable in spleen, liver or testis. Heterologous expression of Kir6.2 in HEK293 cells was only observed when the cDNA was cotransfected with that of the sulphonylurea receptor (SUR). Whole-cell Kir6.2/SUR currents were K(+)-selective, time-independent and showed weak inward rectification. They were blocked by external barium (5 mM), tolbutamide (Kd = 4.5 microM) or quinine (20 microM) and by 5 mM intracellular ATP. The single-channel conductance was 73 pS. Single-channel activity was voltage-independent and was blocked by 1 mM intracellular ATP or 0.5 mM tolbutamide. We conclude that the Kir6.2/SUR channel complex comprises the ATP-sensitive K-channel.     

NF-protocadherin, a novel member of the cadherin superfamily, is required for Xenopus ectodermal differentiation

BACKGROUND: The assembly of complex tissues during embryonic development is thought to depend on differential cell adhesion, mediated in part by the cadherin family of cell-adhesion molecules. The protocadherins are a new subfamily of cadherins; their extracellular domains comprise cadherin-like repeats but their intracellular domains differ significantly from those of classical cadherins. Little is known about the ability of protocadherins to mediate the adhesion of embryonic cells, or whether they play a role in the formation of embryonic tissues. RESULTS: We report the isolation and characterization of a novel protocadherin, termed NF-protocadherin (NFPC), that is expressed in Xenopus embryos. NFPC showed a striking pattern of expression in early embryos, displaying predominant expression within the deep, sensorial layer of the embryonic ectoderm and in a restricted group of cells in the neural folds, but was largely absent from the neural plate and surrounding placodal regions. Ectopic expression in embryos demonstrated that NFPC could mediate cell adhesion within the embryonic ectoderm. In addition, expression of a dominant-negative form of NFPC disrupted the integrity of embryonic ectoderm, causing cells in the deep layer to dissociate, though leaving the outer layer relatively intact. CONCLUSIONS: Our results indicate that NFPC is required as a cell-adhesion molecule during embryonic development, and its function is distinct from that of classical cadherins in governing the formation of a two-layer ectoderm. These results suggest that NFPC, and protocadherins in general, are involved in novel cell-cell adhesion mechanisms that play important roles in tissue histogenesis.     

Cloning of human cDNA encoding a novel heptahelix receptor expressed in Burkitt's lymphoma and widely distributed in brain and peripheral tissues

Using PCR with degenerate primers and screening of a human B-cell lymphoblast cDNA library, a full-length cDNA encoding a 375-amino-acid protein was isolated. It contains seven regions of hydrophobic amino acids probably representing membrane-spanning domains of a novel heptahelix receptor, tentatively named CMKRL2. It shows nearly 30% overall identity with the high-affinity IL8 receptor and similar degree of homology with other chemoattractant receptors, including the "fusin" coreceptors for HIV1. Measurements of various transduction pathways following application of a panel of chemokines to transfected cells failed to evoke any reproducible response. Although the natural ligand for CMKRL2 could, thus, not be identified, receptor expression in spleen and lymph nodes as well as in Burkitt's lymphoma (irrespective of EBV status) supports a functional role in activated B-cells. Receptor message was ubiquitously distributed in normal peripheral tissues and CNS, suggesting that CMKRL2 is expressed in widespread cell populations, such as macrophages and neuroglia.     

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed lambdaSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.     

Cloning, expression and chromosome mapping of adducin-like 70 (ADDL), a human cDNA highly homologous to human erythrocyte adducin

From a human fetal-brain cDNA library we isolated a novel human cDNA, termed human adducin-like 70 (gene symbol ADDL), whose predicted amino acid sequence showed a high degree of homology to adducins. This cDNA clone (ADDL), which contained an open reading frame of 2,022 nucleotides encoding 674 amino acids, revealed 54%, 53%, and 59% identity in predicted amino acid sequence with alpha and beta components of human adducin and rat adducin 63, respectively. Human adducin-like 70 is likely to play an important role in the skeletal organization of the cell membrane. Northern blot analysis indicated ubiquitous expression of this gene in adult human tissues. We localized the gene to chromosome bands 10q24.2-->q24.3 by fluorescence in situ hybridization (FISH).     

Cloning and characterization of a cDNA encoding a novel heterogeneous nuclear ribonucleoprotein-like protein and its expression in myeloid leukemia cells

Breast cancer is the second leading cause of cancer-related deaths among women in the United States. Approximately 180,000 new cases of breast cancer are diagnosed each year and a quarter of these are fatal. Early detection is a key to survival of these patients. Unfortunately, no definitive markers are available to diagnose breast cancer at early stages. Identification of such early markers, therefore, is an important priority in breast cancer research. In order to identify early markers, we have focussed on understanding the molecular mechanisms that can lead to conversion of the normal mammary epithelial cells into precancerous immortal cells. Over last several years, we have developed in vitro models of human mammary epithelial cell immortalization which have allowed us to invoke the critical roles of the known tumor suppressor pathways in the maintenance of the untransformed state of mammary epithelial cells. These models are now being used to identify novel genes whose expression is important for normal mammary epithelial cell growth and whose altered expression contributes to breast cell transformation. Characterization of the molecular machinery whose alterations result in early preneoplastic transformation should help identify candidate genes for evaluation as potential early diagnostic markers.     

Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family

We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controlling O-linked glycosylation.     

Cloning and expression of human deoxyguanosine kinase cDNA

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.     

Cloning, sequencing and expression of a cDNA encoding mammalian valyl-tRNA synthetase

The incus of the right ear from 4 growing mongrel dogs was surgically disarticulated and left in the middle ear space. The external auditory canal was then filled with teflon paste and sutured. After 6 weeks (D-6 group) and 13 weeks (D-13 group) the animals were sacrificed. The right experimental incus and the left control one were embedded in methyl methacrylate and sectioned in single 50-microns-thick sections according to the principal axis of the two processes. On the microradiographs of each section we evaluated the thickness of the body and of both processes and the percentage area of the primary channels of the secondary osteons and that of the appositional bone tissue. The thickness of the body and of the two processes was more pronounced in all the experimental incuses, in which 6% (in D-6) and 8% (in D-13) of the total area were occupied by new appositional woven bone. In the body of the D-13 group, 9% of the pre-existing bone was substituted by secondary osteons. The results indicate that the incus react to the variations of mechanical stimuli.     

Isolation of a cDNA for a novel 120-kDa GTP-binding protein expressed in motor neurons in the salmon brain

A six-year-old boy with homozygous beta-thalassemia in the favorable class 1 risk group received a bone marrow transplant, from his histocompatible sister. He developed grade IV skin and eye graft-versus-host disease (GVHD) following varicella zoster reactivation. Despite the appropriate prophylactic use of cyclosporin A (CsA), methotrexate (MTX), and prompt treatment with high-dose steroids, GVHD progressed resulting in total body epidermal necrolysis. Anti-IL-2 receptor monoclonal antibodies (anti-IL-2R moAb) in combination with steroids were administered to selectively block the activated T cells. After 27 days of daily administration, followed by 17 doses of alternate-day therapy with anti-IL-2R moAb, the severe skin and eye GVHD resolved. The patient, at two years posttransplant, has full engraftment and immune reconstitution without chronic GVHD (cGVHD). In conclusion, we suggest that in the HLA-genoidentical bone marrow transplantation setting, very severe and steroid-resistant GVHD can be controlled through the use of anti-IL-2 receptor antibodies which specifically block the activated IL-2 receptor expressing T cells.     

Cloning, expression and mapping of a novel RING-finger gene (RNF5), a human homologue of a putative zinc-finger gene from Caenorhabditis elegans

The RING-finger is a unique zinc-chelating domain involved in mediating protein-protein interactions. The extensive sequence homology within the RING-finger domain allowed us to clone a novel member of the RING-finger family of genes. This cDNA clone, designated RNF5 (Ring-finger protein 5), contained an open reading frame of 540 nucleotides. Its predicted amino acid sequence revealed significant homology to a hypothetical protein encoded by Caenorhabditis elegans cosmid C16C10.7. The expression of RNF5 was detected in a variety of human tissues. The RNF5 gene was mapped by fluorescence in situ hybridization to chromosome 6p21.31. Radiation hybrid mapping further assigned RNF5 to a region proximal to the major histocompatibility complex (MHC) on chromosome 6. RNF5 is the third RING-finger gene identified in the region proximal to MHC raising the possibility that the RING-finger family of genes may exist as a cluster in this region.     

Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.     

cDNA cloning and expression of a novel serine protease, TLSP

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.