Structural features of a close homologue of L1 (CHL1) in the mouse: a new member of the L1 family of neural recognition molecules

We have identified a close homologue of L1 (CHL1) in the mouse. CHL1 comprises an N-terminal signal sequence, six immunoglobulin (Ig)-like domains, 4.5 fibronectin type III (FN)-like repeats, a transmembrane domain and a C-terminal, most likely intracellular domain of approximately 100 amino acids. CHL1 is most similar in its extracellular domain to chicken Ng-CAM (approximately 40% amino acid identity), followed by mouse L1, chicken neurofascin, chicken Nr-CAM, Drosophila neuroglian and zebrafish L1.1 (37-28% amino acid identity), and mouse F3, rat TAG-1 and rat BIG-1 (approximately 27% amino acid identity). The similarity with other members of the Ig superfamily [e.g. neural cell adhesion molecule (N-CAM), DCC, HLAR, rse] is 16-11%. The intracellular domain is most similar to mouse and chicken Nr-CAM, mouse and rat neurofascin (approximately 60% amino acid identity) followed by chicken neurofascin and Ng-CAM, Drosophila neuroglian and zebrafish L1.1 and L1.2 (approximately 40% amino acid identity). Besides the high overall homology and conserved modular structure among previously recognized members of the L1 family (mouse/human L1/rat NILE; chicken Ng-CAM; chicken/mouse Nr-CAM; Drosophila neuroglian; zebrafish L1.1 and L1.2; chicken/mouse neurofascin/rat ankyrin-binding glycoprotein), criteria characteristic of L1 were identified with regard to the number of amino acids between positions of conserved amino acid residues defining distances within and between two adjacent Ig-like domains and FN-like repeats. These show a collinearity in the six Ig-like domains and four adjacent FN-like repeats that is remarkably conserved between L1 and molecules containing these modules (designated the L1 family cassette), including the GPI-linked forms of the F3 subgroup (mouse F3/chicken F11/human CNTN1; rat BIG-1/mouse PANG; rat TAG-1/mouse TAX-1/chicken axonin-1). The colorectal cancer molecule (DCC), previously introduced as an N-CAM-like molecule, conforms to the L1 family cassette. Other structural features of CHL 1 shared between members of the L1 family are a high degree of N-glycosidically linked carbohydrates (approximately 20% of its molecular mass), which include the HNK-1 carbohydrate structure, and a pattern of protein fragments comprising a major 185 kDa band and smaller fragments of 165 and 125 kDa. As for the other L1 family members, predominant expression of CHL1 is observed in the nervous system and at later developmental stages. In the central nervous system CHL1 is expressed by neurons, but, in contrast to L1, also by glial cells. Our findings suggest a common ancestral L1-like molecule which evolved via gene duplication to generate a diversity of structurally and functionally distinct yet similar molecules.     

Cloning of zebrafish cDNA for 3beta-hydroxysteroid dehydrogenase and P450scc

P450scc and 3beta-HSD cDNA were isolated from a zebrafish lambda gt10 cDNA library using trout SCC and 3beta-HSD cDNA as the probes. The zebrafish SCC cDNA encodes a protein of 509 amino acids, which shares a 78% similarities with the trout SCC and 58% with the human SCC. As for 3beta-HSD, two forms of cDNA were isolated, termed HSD 5 and HSD 17, which may have resulted from alternative splicing. HSD 5 and HSD 17 encode proteins of 374 and 341 amino acids respectively. Both share 77% amino acid similarities with trout 3beta-HSD and 53% similarities with the mouse 3beta-HSD. Zebrafish has been increasingly used as a genetic model system to study organ development and to investigate human diseases. The cloning and the characterization of zebrafish P450scc and 3beta-HSD should facilitate study of steroidogenesis and human disease associated with steroid imbalance.     

Microtubule-associated protein-2 in the rat testis: a novel site of expression

The testis is one of the most abundant sources of microtubule networks. These networks include mitotic and meiotic spindles, the spermatid manchette and axoneme, and the Sertoli cell cytoskeleton. Microtubules are composed of alpha- and beta-tubulin subunits that are polymerized and stabilized by a variety of microtubule-associated proteins (MAPs). One of these, MAP2, has been extensively characterized as a brain-specific protein with the capacity to bind tubulin, cAMP-dependent kinase, and calmodulin. MAP2 mRNA is processed into at least two variants encoding proteins designated MAP2a, MAP2b, and MAP2c. Of the 5.7 kb of coding sequence in the 9-kb mRNA that encodes MAP2a and MAP2b, a deletion of approximately 4 kb produces mRNA encoding MAP2c, which consists of only the N- and C- terminal regions of MAP2b. To determine whether MAP2 was present in the rat testis, microtubule preparations were isolated from adult rat testis and brain by means of taxol-mediated polymerization and analyzed by gel filtration, ELISA, and Western blotting using polyclonal and monoclonal antibodies reactive with MAP2. A 74-kDa protein corresponding to MAP2c was detected in the testis. These results were confirmed by Northern blot analysis of total RNA from adult rat brain and testis with cDNA probes that distinguish between the known MAP2 splice variants. The predominant mRNAs in testis of 6 kb and 2.5-3.5 kb corresponded to MAP2c. A single 6-kb mRNA with the potential to encode MAP2c was detected in enriched preparations of immature Sertoli cells and adult Leydig cells. Round spermatids contained at least two MAP2 mRNAs between approximately 2.5 and 3.5 kb in size that displayed a stage-specific pattern of expression. Immunohistochemistry showed a MAP2-like protein in both somatic and germ cells, with a particularly distinct localization within the cytoplasm of primary and secondary spermatocytes at stage XIV of the seminiferous cycle during meiotic metaphase. In addition to cytoplasmic staining, a novel localization of this protein was observed in the nucleus of many testicular cells.     

Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.     

Factors associated with marijuana use among American Indian adolescents

The BCMA gene is a new gene discovered by the molecular analysis of a t(4;16) translocation, characteristic of a human T cell lymphoma. It has no significant similarity with any known protein or motif, so that its function was unknown. This report describes the cloning of murine BCMA cDNA and its genomic counterpart. The mouse gene is organized into three exons, like the human gene, and lies in murine chromosome 16, in the 16B3 band, the counterpart of the human chromosome 16p13 band, where the human gene lies. Murine BCMA cDNA encodes a 185 amino acids protein (184 residues for the human), has a potential central transmembrane segment like the human protein and is 62% identical to it. The murine BCMA mRNA is found mainly in lymphoid tissues, as is human BCMA mRNA. Alignment of the murine and human BCMA protein sequences revealed a conserved motif of six cysteines in the N-terminal part, which strongly suggests that the BCMA protein belongs to the tumor necrosis factor receptor (TNFR) superfamily. Human BCMA is the first member of the TNFR family to be implicated in a chromosomal translocation.     

The cDNA cloning and ontogeny of mouse alpha-synuclein

Alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease. To investigate the role of alpha-synuclein in the brain, the cDNA clone encoding the mouse cognate of the human alpha-synuclein was isolated from a mouse brain cDNA library. The open reading frame coded for 140 amino acids that share 95% identity with human alpha-synuclein. Northern blot analysis showed that alpha-synuclein mRNA was primarily expressed in brain and spleen of adult mouse. In situ hybridization histochemistry revealed the highest expression of alpha-synuclein mRNA in the hippocampal formation and neocortex of the adult mouse. alpha-Synuclein mRNA expression in the brain was first observed in the hippocampus and neocortex on postnatal day 1. Levels of alpha-synuclein mRNA in these forebrain areas were nearly maximal at postnatal day 7 and remained relatively high until the adult stage. alpha-Synuclein mRNA was expressed in the liver transiently during embryogenesis.     

Identification of cortexin: a novel, neuron-specific, 82-residue membrane protein enriched in rodent cerebral cortex

Nucleotide sequence analysis of a cDNA clone of a rat cortex-enriched mRNA identifies a novel integral membrane protein of 82 amino acids. The encoded protein is named cortexin to reflect its enriched expression in cortex. The amino acid sequence of rat cortexin and its mouse homologue are nearly identical (98% similarity), and both contain a conserved single membrane-spanning region in the middle of each sequence. Northern blot analysis shows that cortexin mRNA is brain-specific, cortex-enriched, and present at significant levels in fetal brain, with peak expression in postnatal rodent brain. In situ hybridization studies detect cortexin mRNA primarily in neurons of rodent cerebral cortex, but not in cells of the hindbrain or white matter regions. The function of cortexin may be particularly important to neurons of both the developing and adult cerebral cortex.     

Identification and characterisation of a human calmodulin-stimulated phosphodiesterase PDE1B1

A cDNA encoding a calmodulin-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE) was isolated from a human brain cDNA library. The cDNA, designated HSPDE1B1, encoded a protein of 536 amino acids that shared 96% sequence identity with the bovine "63 kDa" calmodulin-stimulated PDE. The recombinant protein had cyclic nucleotide phosphodiesterase activity that was stimulated approximately 2-fold by Ca2+/calmodulin and preferred cGMP as substrate. In addition, the enzymatic activity of HSPDE1B1 was inhibited by phosphodiesterase inhibitors with potencies similar to that displayed toward the bovine PDE1 enzymes: IBMX approximately equal to 8-methoxymethyl-IBMX > vinpocetine approximately equal to zaprinast > cilostamide > rolipram. HSPDE1B1 mRNA was found predominantly in the brain. Lower mRNA levels were found in heart and skeletal muscle. In situ hybridisation of brain revealed expression of HSPDE1B1 predominately in neuronal cells of the cerebellum, hippocampus and caudate. The HSPDE1B1 gene was mapped to human chromosome 12. A partial genomic sequence of HSPDE1B1 was isolated and shown to contain two splice junctions that are conserved in the rat PDE4 and the Drosophila dunce genes.     

Molecular cloning and expression of the KIF3A gene in the frog brain and testis

KIF3A is a member of the kinesin superfamily proteins (KIFs), but its gene has been cloned only in mouse and sea urchin. We have cloned a homolog of KIF3A from the frog, Rana rugosa (rrKIF3A). The sequence encoded a 699 amino acid protein that shares 93% similarity with mouse KIF3A (mKIF3A) and 69% with sea urchin kinesin-related protein (SpKRP85). The putative ATP-binding domain was completely identical to that of mKIF3A and SpKRP85. The level of rrKIF3A mRNA appeared to be high in the brain and testis of adult frogs, but low in the heart, lung and kidney. The results suggest that the rrKIF3A gene is expressed in the brain and testis more than other tissues of adult frogs examined, and that KIF3A is widely distributed in eukaryotic organisms.     

Westernized food habits and concentrations of serum lipids in the Japanese

It has recently been reported that there exists a new 'RING-finger' protein family among the zinc-finger (Zf) proteins. Previously, we had isolated the mouse Mel-18 cDNA (mMel-18) encoding the nuclear RING-finger protein that exhibits an ability to bind to a nonspecific DNA column. Here, we have isolated and characterized the human Mel-18 cDNA (hMel-18) using the mMel-18 cDNA as a probe. The deduced hMel-18 protein contains 344 amino acids (38 kDa) with a RING-finger motif, a helix-loop-helix (HLH)-like structure and a Pro/Ser-rich region. The hMel-18 gene is conserved among vertebrates. Its mRNA is highly expressed in placenta, lung and kidney, but the level is low in liver, pancreas and skeletal muscle. Using in situ hybridization, we mapped hMel-18 to band q22 of chromosome 12. It is possible that the Mel-18/bmi-1 gene family represents a mammalian homologue of the Drosophila polycomb gene group.     

Cloning, expression and chromosomal localization of Wnt-13, a novel member of the Wnt gene family

The Wnt genes, encoding structurally-related secreted glycoproteins, are implicated in mammary carcinogenesis induced by mouse mammary tumor virus. In search of the Wnt gene(s) expressed in human gastric cancer, a WTGC1 cDNA fragment sharing 66.9% amino-acid homology with human and mouse Wnt-2 was isolated by degenerate polymerase chain reaction. The human gene corresponding to WTGC1 was designated as Wnt-13 and overlapping Wnt-13 cDNAs were cloned. Nucleotide sequence analysis indicated that the Wnt-13 gene encodes the protein of 372 amino acids, including a signal peptide, two potential N-glycosylation sites and 24 cystein residues highly conserved among members of the Wnt gene family. The Wnt-13 mRNA of 2.5 kb in size was detected in heart, brain, placenta, lung, prostate, testis, ovary, small intestine and colon of adult human and also in brain, lung and kidney of fetal human. Among various cancer cell lines, the Wnt-13 mRNA was detected in HeLa (cervical cancer), MKN28 and MKN74 (gastric cancer). The Wnt-13 gene has been mapped to human chromosome 1p13. These results suggest that the Wnt-13 gene may be involved in normal human development or differentiation as well as in human carcinogenesis.     

cDNA cloning and characterization of a new human microsomal NAD+-dependent dehydrogenase that oxidizes all-trans-retinol and 3alpha-hydroxysteroids

We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (相似文献   

Stability of vancomycin-resistant enterococcal genotypes isolated from long-term-colonized patients

The Eph-related receptor tyrosine kinases constitute a large family of receptors with most members displaying specific expression patterns in the developing embryo. Ligands for Eph receptor tyrosine kinases, recently renamed ephrins, comprise a family of at least 8 membrane-bound members that display promiscuous binding to Eph receptors. Here we report the characterization of a human cDNA clone with high homology to the gene encoding the murine ephrin-A2 ligand. The human gene encodes a single 2.4-kb mRNA with a restricted and developmentally-regulated tissue distribution pattern. In the fetus, ephrin-A2 mRNA is expressed in brain and intestine, whereas in the adult, high levels of ephrin-A2 mRNA are detectable in lung and intestine. Using PCR-based screening of genomic DNA from human x rodent hybrid cell lines, the gene encoding ephrin-A2 (EFNA2) was assigned to chromosome 19. Fluorescence in situ hybridization to metaphase chromosome preparations refined this localization to band p13.3.     

Decreased release of glutathione into the systemic circulation of patients with HIV infection

The human DDX6 gene (alias RCK) at chromosome 11 band q23 was identified through the study of the breakpoint of t(11;14)(q23;q32) translocation in a B-cell lymphoma cell line, RC-K8. DDX6 encodes a DEAD box protein/RNA helicase. Positive mouse genomic and cDNA recombinant clones were obtained by screening mouse B-cell genomic and cDNA libraries with a human DDX6 cDNA probe. The deduced amino acid sequence of an open reading frame from a cDNA clone revealed a protein with 92.5% identity to human ddx6/p54. All positive mouse genomic recombinant clones, and cDNA clones containing mouse Ddx6 (previous gene symbol: Rck), were localized by fluorescent in situ hybridization to band B of mouse Chromosome 9, a region showing conserved linkage homology to human chromosome 11 band q23. Mouse Ddx6 was localized to the region between Ncam and D9Mit45 by molecular linkage analysis. A 7.5-kb mRNA and a 54-kDa protein were identified as mouse Ddx6 gene products which are similar in size to products of the human DDX6 gene, as shown by Northern and Western blot analyses.