Biomedical subjects

D J Gilbert

Publications and source records attributed to D J Gilbert.

At least 37 records · Page 2Linked to original sources

PubMed2000

Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2).

We report the identification and characterization of a novel CC chemokine designated CCL28 and its receptor CCR10, known previously as orphan G-protein-coupled receptor GPR2. Human and mouse CCL28 share 83% identity at the amino acid and 76% at the nucleic acid levels. We also identified the mouse homologues of CCL28 and of CCR10, which map to mouse chromosomes 13 and 11, respectively. CCL28 is expressed in a variety of human and mouse tissues, and it appears to be predominantly produced by epithelial cells. Both human and mouse CCL28 induce calcium mobilization in human and mouse CCR10-expressing transfectants. CCL28 desensitized the calcium mobilization induced in CCR10 transfectants by CCL27, indicating that these chemokines share this new chemokine receptor. In vitro, recombinant human CCL28 displays chemotactic activity for resting CD4 or CD8 T cells.

Amino Acid Sequence↗

PubMed2000

TROY, a newly identified member of the tumor necrosis factor receptor superfamily, exhibits a homology with Edar and is expressed in embryonic skin and hair follicles.

In a signal sequence trap screening of the murine brain, we identified a new member of the tumor necrosis factor receptor superfamily designated TROY. TROY is a type I membrane protein of 416 amino acids with characteristic cysteine-rich motifs in the extracellular domain and a tumor necrosis factor receptor-associated factor (TRAF) 2 binding sequence in the cytoplasmic domain of 223 amino acids. In fact, activation of nuclear factor kappaB was induced by the overexpression of TROY and inhibited by dominant negative forms of TRAF2, TRAF5, and TRAF6, indicating that TRAFs and nuclear factor kappaB are involved in the signal transduction of TROY. We also cloned a cDNA for a human counterpart, which showed a 75% homology with mouse TROY at the amino acid level. The extracellular domain of TROY exhibits an extensive homology with that of Edar, a receptor that specifies hair follicle fate. TROY mRNA is strongly expressed in brain and embryo and moderately expressed in the heart, lung, and liver but not the spleen. In the embryo, the expression level is particularly strong in the skin. Interestingly, in situ hybridization analysis of the embryo showed that TROY mRNA was exclusively expressed in the epithelium of many tissues. On the other hand, in neonatal mice, TROY is expressed in hair follicles like Edar as well as in the cerebrum, suggesting pleiotropic functions of TROY in development as well as in the adult mice. The Troy gene is located near the waved coat (Wc) locus, a mutant related to abnormalities in skin and hair.

Amino Acid Sequence↗

PubMed2000

Mouse proteasomal ATPases Psmc3 and Psmc4: genomic organization and gene targeting.

PSMC3 and PSMC4, components of the 19S complex of the 26S proteasome, show a significant degree of amino acid similarity, especially in the conserved ATPase domain (CAD). In this study, we characterized the mouse Psmc3 and Psmc4 genes. The genomic structures of both genes showed a significant degree of similarity. The Psmc3 gene was composed of 12 coding exons, whereas the Psmc4 gene had 11 exons. Exons encoding the leucine zipper domain and CAD were identical in number between the Psmc3 and Psmc4 genes. The Psmc3 gene mapped to mouse chromosome 2, whereas Psmc4 mapped to chromosome 7. We further addressed the biological roles of Psmc3 and Psmc4 through the generation of gene targeted mice. Both Psmc3- and Psmc4-deficient mice died before implantation, displaying defective blastocyst development. These findings indicate that Psmc3 and Psmc4 have similar and essential roles in early embryogenesis and further that both ATPases have noncompensatory functions in vivo.

Adenosine Triphosphatases↗

PubMed2000

Molecular cloning and characterisation of GPR74 a novel G-protein coupled receptor closest related to the Y-receptor family.

A novel gene product, GPR74, with homology to the seven transmembrane-domain receptor superfamily, has been cloned. GPR74 has been identified from the expressed sequence tags (EST) database. Subsequent PCR amplification of that sequence and screening of a human heart cDNA library led to the isolation of a 1.7-kb cDNA clone encoding a protein of 408 amino acids. GPR74 shows highest amino acid identity (33%) to the human neuropeptide Y-receptor subtype Y2. The human and mouse genes for GPR74 have been isolated and their exon-intron structures determined. In both species the gene consists of four exons spanning around 20 kb with the exon-intron borders being 100% conserved. Northern analysis of various human tissues reveals highest levels of mRNA expression in brain and heart. In situ hybridisation analysis of rat brain tissue confirms this result and identifies the hippocampus and amygdala nuclei as the brain areas with particular high expression of GPR74 mRNA. Fluorescence in situ hybridisation, PCR analysis on a radiation hybrid panel and interspecific mouse backcross mapping have localised the genes to human chromosome 4q21 and mouse chromosome 5. Expression of the human GPR74 cDNA as a GFP-fusion protein in various cell lines reveals the inability of the recombinant receptor protein to reach the cell surface. This is consistent with the lack of NPY specific binding in these cells and suggests that unknown factors are required for a full functional receptor complex.

Amino Acid Sequence↗

PubMed2000

Y-receptor-like genes GPR72 and GPR73: molecular cloning, genomic organisation and assignment to human chromosome 11q21.1 and 2p14 and mouse chromosome 9 and 6.

Two novel G-protein-coupled receptors, one from human, GPR72, and one from mouse, GPR73 have been isolated, sequenced and their genomic organisation determined. Non-isotopic in situ hybridisation and radiation hybrid mapping have identified GPR72 to be localised on human chromosome 11q21.1, and GPR73 on human chromosome 2p14. Interspecific mouse backcross mapping has localised the genes to mouse chromosomes 9 and 6, respectively. Northern analysis reveals GPR72 mRNA expression only in brain tissue. However, GPR73 mRNA can be found in heart, skeletal muscle and pancreas. Both receptors are closely related with 36 and 33% overall amino acid identity, respectively, to the Y-receptor family. However, although successful cell surface expression in a heterologous expression system can be achieved no specific binding to this ligand family can be detected, indicating that perhaps additional factors are required for binding.

Animals↗

PubMed2000

Molecular cloning of a novel type 1 cytokine receptor similar to the common gamma chain.

In a complementary DNA (cDNA) screening of murine Th2-skewed lymphocytes with our recently developed signal sequence trap method termed SST-REX, a novel type 1 cytokine receptor, Delta1 (delta1), was identified. Although delta1 is ubiquitously expressed in multiple tissues, the expression level is higher in Th2-skewed lymphocytes than in Th1-skewed ones. The delta1 cDNA encodes a 359-amino acid type 1 membrane protein. The extracellular domain of 206 amino acids showed 24% identity with the murine common gamma receptor that is shared among the receptors for interleukin(IL)-2, IL-4, IL-7, IL-9, and IL-15. The membrane-proximal region of delta1 includes a box1 motif, which is important for association with Janus kinases (JAKs), and showed a significant homology with that of the mouse erythropoietin receptor (EPOR). A box2 motif was also found in close proximity to the box1 region. Dimerization of the cytoplasmic region of delta1 alone did not transduce proliferative signals in IL-3-dependent cell lines. However, the membrane-proximal region of delta1 could substitute for that of human EPOR in transmitting proliferative signals and activating JAK2. These results suggest that delta1 is a subunit of cytokine receptor that may be involved in multiple receptor systems and play a regulatory role in the immune system and hematopoiesis.

Amino Acid Sequence↗

PubMed2000

The mouse N-acetylgalactosamine-6-sulfate sulfatase (Galns) gene: cDNA isolation, genomic characterization, chromosomal assignment and analysis of the 5'-flanking region.

Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.

Amino Acid Sequence↗

PubMed2000

Characterization of the mouse JAB1 cDNA and protein.

JAB1 was originally described as a transcriptional coactivator of c-Jun and Jun D. Recent data suggests that JAB1 is a component of a large protein complex, the JAB1 signalosome in mammals and the COP9 complex in plants. The JAB1 signalosome is implicated in the phosphorylation of selected transcription factors, while the COP9 complex is involved in repression of photomorphogenesis in Arabidopsis. In this study, we describe the partial characterization of mouse JAB1 (mJAB1). The murine JAB1 protein is encoded by a gene located on mouse chromosome 1. mJAB1 mRNA is abundantly expressed in a variety of adult tissues as well as in mouse embryos. The JAB1 protein was readily detectable in many cell types and localized to both the nucleus and cytoplasm. Endogenous JAB1 protein is relatively stable and its degradation is not perturbed by blocking 26S proteasome activity, suggesting that this protein is not degraded by the ubiquitin-mediated proteolytic pathway.

Animals↗

PubMed2000

Collapsin response mediator protein-3/unc-33-like protein-4 gene: organization, chromosomal mapping and expression in the developing mouse brain.

CRMPs (collapsin response mediator proteins)/ULIPs (unc-33-like proteins) are a family of intracytoplasmic proteins that are expressed mainly in the brain. The involvement of CRMP/ULIP members in neuronal differentiation, growth cone motility and axonal collapse has been suggested. We recently found that a member of this family, CRMP3/ULIP4, corresponds to POP66 (paraneoplastic oligodendrocyte protein of 66 kDa), a protein which may be associated with auto-immune induced-neuronal degeneration in paraneoplastic neurological syndromes. However, the physiological functions of these proteins remain to be elucidated. Further studies, including the generation of cell lines and of animals with modified/disrupted CRMP/ULIP gene expression, are necessary to explore the functions of this protein. We have cloned and determined the organization and chromosomal localization of the mouse gene encoding CRMP3/ULIP4. The gene is composed of 14 exons and spans more than 20 kb. We assigned the mouse CRMP3/ULIP4 gene to the distal end of chromosome 7. In mouse brain, in situ hybridization showed that CRMP3/ULIP4 mRNA is expressed mainly in the dentate gyrus of hippocampus, in the granular layers of cerebellum and in the inferior olive of the pons, the nucleus which controls movement and posture, and adjusts the major output of descending motor system.

Amino Acid Sequence↗

PubMed2000

Structure, expression, and chromosome mapping of LATS2, a mammalian homologue of the Drosophila tumor suppressor gene lats/warts.

We have cloned and characterized LATS2, a novel mammalian homologue of the Drosophila tumor suppressor gene lats/warts. Northern blot analysis showed ubiquitous expression of mouse LATS2 (MmLATS2) mRNA, whereas expression of human LATS2 (HsLATS2) mRNA was enhanced in skeletal muscle and heart. Immunoblotting analysis of fractionated cell lysates showed HsLats2 to be a nuclear protein. We mapped the MmLATS2 gene to mouse chromosome 14 by interspecific backcross analysis. We also mapped the HsLATS2 gene (by fluorescence in situ hybridization) to the 13q11-q12 region, in which a loss of heterozygosity has been frequently observed in many primary cancers and to which the tumor suppressor genes RB and BRCA2 have also been mapped.

Amino Acid Sequence↗

PubMed2000

Cloning, characterization, and chromosomal localization of Pnck, a Ca(2+)/calmodulin-dependent protein kinase.

Calcium is an important second messenger in eukaryotic cells. Many of the effects of calcium are mediated via its interaction with calmodulin and the subsequent activation of Ca(2+)/calmodulin-dependent (CaM) kinases. CaM kinases are involved in a wide variety of cellular processes including muscle contraction, neurotransmitter release, cell cycle control, and transcriptional regulation. While CaMKII has been implicated in learning and memory, the biological role of the other multifunctional CaM kinases, CaMKI and CaMKIV, is largely unknown. In the course of a degenerate RT-PCR protein kinase screen, we identified a novel serine/threonine kinase, Pnck. In this report, we describe the cloning, chromosomal localization, and expression of Pnck, which encodes a 38-kDa protein kinase whose catalytic domain shares 45-70% identity with members of the CaM kinase family. The gene for Pnck localizes to mouse chromosome X, in a region of conserved synteny with human chromosome Xq28 that is associated with multiple distinct mental retardation syndromes. Pnck is upregulated during intermediate and late stages of murine fetal development with highest levels of expression in developing brain, bone, and gut. Pnck is also expressed in a tissue-specific manner in adult mice with highest levels of expression detected in brain, uterus, ovary, and testis. Interestingly, Pnck expression in these tissues is restricted to particular compartments and appears to be further restricted to subsets of cells within those compartments. The chromosomal localization of Pnck, along with its tissue-specific and restricted pattern of spatial expression during development, suggests that Pnck may be involved in a variety of developmental processes including development of the central nervous system.

Amino Acid Sequence↗

PubMed2000

Cloning, expression, and chromosomal localization of a mouse gene homologous to the germ cell migration regulator wunen and to type 2 phosphatidic acid phosphatases.

The wunen gene of Drosophila melanogaster encodes a multipass membrane-spanning protein that negatively regulates primordial germ cell migration. Here we describe the cloning of a mouse gene that encodes a protein homologous to wunen and to the Type 2 phosphatidic acid phosphatases. This gene encodes a 251-amino-acid protein that most closely resembles the human Type 2 phosphatidic acid phosphatase PAP-2c. Northern blot analysis revealed the presence of a single 1.9-kb Ppap2c transcript. The Ppap2c gene was localized to the central portion of mouse Chromosome 10 by interspecific backcross analysis.

Amino Acid Sequence↗

PubMed2000

Cloning and characterization of Hunk, a novel mammalian SNF1-related protein kinase.

We previously identified a novel protein kinase, Hunk, by means of a degenerate PCR screen designed to isolate kinases expressed in the murine mammary gland. We now describe the molecular cloning, chromosomal localization, and activity of this kinase and characterize its spatial and temporal pattern of expression in the mouse. We have isolated a 5.0-kb full-length cDNA clone that contains the 714-amino-acid open reading frame encoding Hunk. Analysis of this cDNA reveals that Hunk is most closely related to the SNF1 family of serine/threonine kinases and contains a newly described SNF1 homology domain. Accordingly, antisera specific for Hunk detect an 80-kDa polypeptide with associated phosphotransferase activity. Hunk is located on distal mouse chromosome 16 in a region of conserved synteny with human chromosome 21q22. During fetal development and in the adult mouse, Hunk mRNA expression is developmentally regulated and tissue-specific. Moreover, in situ hybridization analysis reveals that Hunk expression is restricted to subsets of cells within a variety of organs in the adult mouse. These findings suggest a role for Hunk in murine development.

Amino Acid Sequence↗

PubMed2000

Cloning, expression analysis, and chromosomal localization of murine and human homologues of a Xenopus mix gene.

We report the cloning and chromosomal localization of murine and human Mix genes, members of a subclass of paired-like homeobox genes of which the Xenopus laevis Mix.1 gene is the founding member. The murine Mix gene was mapped to the distal region of chromosome 1 and the human region to the syntenic region 1q41-42. Northern analysis and RT-PCR of murine adult and embryonic tissues demonstrated that Mix expression was restricted to the early embryo. Whole-mount in situ hybridization revealed patchy but symmetrical Mix expression in visceral endoderm of embryonic day (E)5.5 embryos. In slightly older embryos, the expression was skewed to one side of the embryo and by E6.5, at the onset of gastrulation, expression was seen in the epiblast, visceral endoderm, nascent mesoderm, and the primitive streak. This expression pattern was maintained in mid- and late-streak embryos. In early bud-stage embryos, expression was strongest in the proximal two thirds of the streak, extending to the base of the allantois. By the headfold-stage, expression was confined to the remnant of the primitive streak in the caudal region of the embryo and, after E8.0, in the caudal notochord and tail bud mesoderm. Mix transcripts were no longer detectable after embryonic day 9.5.

Amino Acid Sequence↗

PubMed2000

A molecular analysis of NKT cells: identification of a class-I restricted T cell-associated molecule (CRTAM).

cDNA library subtraction techniques were used to identify transcripts expressed by activated mouse alphabetaTCR+ CD4-CD8- (double-negative; DN) T cells, a subset of natural killer T (NKT) cells. The most frequent cDNAs identified included the chemokines TCA3, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and lymphotactin (LPTN), the cytokines interleukin-4 (IL-4) and interferon-gamma (IFN-gamma), and a granzyme. We also identified a new member of the immunoglobulin superfamily (Ig-SF). This molecule was designated class I-restricted T cell-associated molecule (CRTAM) as a result of its restricted expression pattern in T cells. Human CRTAM was also identified, and shares the same expression pattern as the mouse molecule. LPTN and CRTAM exhibit the same expression pattern in T cells, suggesting the existence of a gene expression program common to class I-MHC-restricted T cells.

Amino Acid Sequence↗

PubMed2000

Characterization of the mouse interleukin-13 receptor alpha1 gene.

Interleukin (IL)-13 is a pleiotropic immune regulatory cytokine that shares structural and biological characteristics with IL-4. The receptor for IL-13 is comprised of the IL-4 receptor alpha (IL-4Ralpha) subunit and a low-affinity IL-13-binding subunit, IL-13Ralpha1. An additional receptor, IL-13Ralpha2, binds to IL-13 with high affinity, but lacks the cytoplasmic domain for signaling. In this study, we isolated the mouse IL-13Ralpha1 gene (Il13ra1) of approximately 56 kb that spans the entire coding region. The mouse Il13ra1 gene is composed of 11 exons, and shows striking similarity in genomic structure to the previously reported class I cytokine receptor genes. Motifs characteristic of the cytokine receptor family are similarly organized on the genome, including conserved cysteines, a WSxWS motif, and Box1, indicating closely related genetic evolution of the cytokine receptor superfamily. Alternative mRNA splicings were demonstrated to generate variant transcripts that encode soluble IL-13Ralpha1. The mouse Ill13ra1 gene was mapped to the proximal region of the mouse X chromosome, and was closely linked to the DXPas3 locus by interspecific backcross analysis. Il13ra1 mRNA was co-expressed with I14ra mRNA in mouse myeloid and natural killer cells on which IL-13 has been known to act, whereas the Il13ra2 mRNA was not detected in these cells, indicating that IL-13Ralpha1 is the major component of the IL-13 receptor complex in lymphohematopoietic cells.

Alternative Splicing↗

PubMed2000

Organization of the mouse microfibril-associated glycoprotein-2 (MAGP-2) gene.

A 1.4-kb EST clone encoding mouse microfibril-associated glycoprotein-2 (MAGP-2), identified by its similarity with the reported human cDNA, was used to screen a mouse 129 genomic bacterial artificial chromosome (BAC) library. The mouse gene contains 10 exons spanning 16 kb, located on the distal region of Chromosome (Chr) 6. The exons range in size from 24 to 963 bp, with the ATG located in exon 2. The tenth and largest exon contains 817 bp of 3' untranslated sequence, including a B2 repetitive element. Northern analysis demonstrates abundant expression of MAGP-2 mRNA in skeletal muscle, lung, and heart. Sequence analysis of additional cDNA clones suggests that the two mRNA forms of MAGP-2 in the mouse arise from alternative polyadenylation site usage. The promoter does not contain an obvious TATA box, and the sequence surrounding the start site does not conform to the consensus for an initiator promoter element. Additionally, the mouse promoter contains 22 copies of a CT dinucleotide repeat sequence located approximately 155 bp 5' to exon 1. MAGP-2 gene and compared it with that of the human gene (Hatzinikolas and Gibson 1998). While the mouse and human MAGP-2 proteins are similar in sequence, the promoters for the two genes share little in common. The presence of two mRNA species for MAGP-2 in the mouse raised the possibility that more than one isoform of the protein might be synthesized. We have characterized both mRNA species and determined that they do not code for different variants of the protein.

Alternative Splicing↗

PubMed2000

Molecular cloning and functional characterization of murine transmembrane activator and CAML interactor (TACI) with chromosomal localization in human and mouse.

The human Taci gene (Transmembrane Activator and CAML Interactor) encodes a recently discovered member of the Tumor Necrosis Factor Receptor family. TACI is expressed in B-lymphocytes and may act to regulate humoral immunity. To identify functionally important regions of the protein, we have isolated and characterized the murine homolog of the human Taci cDNA. The proteins display 61.5% similarity and 54.6% identity. Mouse TACI is a type III transmembrane protein, as judged by the lack of a cleaved signal sequence and its N-terminal extracellular exposure. The intracellular domains of the mouse and human proteins share a single, defined region of high sequence conservation (19 of 23 residues identical). This constitutes a novel domain that may play a part in the initiation of signal transduction through TACI. In support of this notion, mouse TACI was found to activate NFAT, NFkB, and AP1 transcription factors in a transient transfection assay. The Taci gene was localized to human Chromosome (Chr) 17p11 by fluorescence in situ hybridization. The murine homolog was localized by intraspecific backcross analysis to the middle of Chr 11, a region that is syntenic to human Chr 17p. This work identifies conserved domains within TACI that may mediate the cellular distribution and signal transduction function of the protein and extend the details of homology between mouse Chr 11 and human 17p.

Amino Acid Sequence↗