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J Aruga

Publications and source records attributed to J Aruga.

At least 37 records · Page 2Linked to original sources
PubMed1996

Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell.

There is increasing interest in the role of RNA-binding proteins during neural development. Drosophila Musashi is one of the neural RNA-binding proteins essential for neural development and required for asymmetric cell divisions in the Drosophila adult sensory organ development. Here, a novel mammalian neural RNA-binding protein, mouse-Musashi-1, was identified based on the homology to Drosophila Musashi and Xenopus NRP-1. In the developing CNS, mouse-Musashi-1 protein was highly enriched in the CNS stem cell. Single-cell culture experiments indicated that mouse-Musashi-1 expression is associated with neural precursor cells that are capable of generating neurons and glia. In contrast, in fully differentiated neuronal and glial cells mouse-Musashi-1 expression is lost. This expression pattern of mouse-Musashi-1 is complementary to that of another mammalian neural RNA-binding protein, Hu (a mammalian homologue of a Drosophila neuronal RNA-binding protein Elav), that is expressed in postmitotic neurons within the CNS. In vitro studies indicated that mouse-Musashi-1 possesses binding preferences on poly(G) RNA homopolymer, whereas Hu is known to preferentially bind to short A/U-rich regions in RNA. Based on their differential expression patterns and distinct preferential target RNA sequences, we believe that the mouse-Musashi-1 and Hu proteins may play distinct roles in neurogenesis, either through sequential regulatory mechanisms or differential sorting of mRNA populations during asymmetric division of neural precursor cells.

Amino Acid Sequence↗
PubMed1996

A neural cell-type-specific expression system using recombinant adenovirus vectors.

We demonstrated neural cell-type-specific gene expression using an adenovirus vector, which is useful for delivering foreign genes into quiescent neural cells. We produced eight recombinant replication-defective adenoviruses carrying the lacZ reporter gene driven by various promoters, including those of the L7/PCP2 gene (highly restricted expression in cerebellar Purkinje cells), and the myelin basic protein (in oligodendrocytes) gene. We demonstrated in vitro and in vivo promoter-driven, neural cell-type-specific gene expression by recombinant adenoviruses. The genes were transferred into these recombinant adenoviruses and expressed with high levels of efficiency in vitro and in vivo. In primary culture, the recombinant adenoviruses AdexL7-NL-LacZ and AdexMBP-NL-LacZ appeared to be expressed in a Purkinje cell and oligodendrocyte-specific manner. Introduction of 10(8) pfu of these viruses into the adult rat cerebellum by stereotactic injection yielded neural cell-type-specific expression without apparent toxicity to the animals. Thus, adenovirus vectors are useful for cell-type-specific therapeutic uses and in studies requiring neural cell-type-specific gene expression.

Adenoviruses, Human↗
PubMed1996

Predominant expression of human zic in cerebellar granule cell lineage and medulloblastoma.

Zic is a novel zinc finger protein which displays a highly restricted expression pattern in the adult and developing mouse cerebellum and is highly homologous to the recently cloned Drosophila pair-rule gene Opa. To clarify the mechanism for the development of the human cerebellum and its involvement in human nervous system diseases, we have isolated human Zic cDNA and examined its expression by using monoclonal antibody against recombinant Zic protein. The nucleotide sequence of human Zic cDNA is 85% homologous to that of mouse Zic cDNA. Its putative amino acid sequence is highly conserved (> 99%) except for substitution of only two amino acid residues. In situ chromosome hybridization localized the human Zic gene to chromosome band 3q24. Human Zic protein was immunohistochemically detected in the nuclei of the cerebellar granule cell lineage from the progenitor cells of the external germinal layer to the postmigrated cells of the internal granular layer. Furthermore, Zic protein was detected in medulloblastoma (26/29 cases), whereas no other tumors examined (over 70 cases including primitive neuroectodermal tumors) expressed this protein. These findings suggest that Zic is a potential biomarker for medulloblastoma as well as the human cerebellar granule cell lineage.

Adolescent↗
PubMed1996

The mouse zic gene family. Homologues of the Drosophila pair-rule gene odd-paired.

The mouse Zic gene, which encodes a zinc finger protein, is expressed in the developing or matured central nervous system in a highly restricted manner. We identified two novel Zic-related genes (Zic2, Zic3) through genomic and cDNA cloning. Both genes are highly similar to Zic(1), especially in their zinc finger motif. A comparison of genomic organization among the three Zic genes showed that they share common exon-intron boundaries and belong to the same gene family . Zic1, Zic2, and Zic3 were determined to mouse chromosome 9, 14, and X using an interspecific backcross panel. Northern blotting and ribonuclease protection showed that Zic2 and Zic3 are expressed in a restricted manner in the cerebellum at the adult stage. However, the temporal profile of the mRNA expression in the developing cerebella differ in the three Zic genes. Furthermore, we found that the Drosophila pair-rule gene, odd-paired is highly homologous to the Zic gene family. The similarity was not only the zinc finger motif, but also the exon-intron boundary was the same as those of mouse Zic gene family. These findings suggest that the Zic gene family and Drosophila odd-paired are derived from a common ancestral gene.

Amino Acid Sequence↗
PubMed1996

Calcium-dependent phospholipid binding to the C2A domain of a ubiquitous form of double C2 protein (Doc2 beta).

Rabphilin 3A and Doc2 alpha are synaptic vesicle-associated proteins, and are thought to function as Ca2+ sensors in neurotransmitter release. If either rabphilin 3A or Doc2 alpha plays a role in membrane trafficking, like the synaptotagmins, then non-neural forms should be present. Here we describe the isolation of a mouse cDNA which encodes a novel Doc2 homologue (Doc2 beta) that is present in all tissues. The encoded protein, which is highly homologous to human Doc2 alpha (70% identity), is composed of 412 amino acids with a calculated relative molecular mass (M(r)) of 45,837. The sequence identity is especially high in two C2 domains (74% in C2A and 84% in C2B). Northern and Western blot analyses have shown that Doc2 beta is expressed in all cell lines and tissues tested. Ca(2+)-dependent phospholipid binding assaying of recombinant fusion proteins revealed that the single C2A domain, but not the C2B domain, of Doc2 beta binds phosphatidycholine and phosphatidylserine (2.5:1, w/w) liposomes. The binding is Ca(2+)-dependent, with an EC50 value of approximately 1 microM and a Hill coefficient of approximately 3, which are comparable to those of synaptotagmins, rabphilin 3A and Doc2 alpha. Our results suggest that Doc2 beta is involved in constitutive membrane trafficking.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Functional diversity of C2 domains of synaptotagmin family. Mutational analysis of inositol high polyphosphate binding domain.

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca(2+)-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.

Amino Acid Sequence↗
PubMed1994

Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II.

IP4BP/Synaptotagmin II is an inositol-1,3,4,5-tetrakisphosphate (IP4) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals. Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca(2+)-dependent binding to phospholipid membranes. A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/synaptotagmin II (315 IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF 346) are essential for inositol polyphosphate binding. This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release. Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP4, indicating that the IP4 binding property is specific to the C2B domain of synaptotagmin. Phospholipid and IP4 binding experiments clearly indicated that the C2A and C2B domains have different functions. The C2A domain binds phospholipid in a Ca(2+)-dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca2+. Our data suggest that the C2B domain of synaptotogamin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.

Amino Acid Sequence↗
PubMed1994

Glial cell degeneration and hypomyelination caused by overexpression of myelin proteolipid protein gene.

Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Plp gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Plp genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plp gene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.

Animals↗
PubMed1994

A novel zinc finger protein, zic, is involved in neurogenesis, especially in the cell lineage of cerebellar granule cells.

To clarify the mechanism of cerebellar development, we have cloned a gene, named zic, encoding a zinc finger protein that is expressed abundantly in granule cells throughout development of the cerebellum. zic has a significant homology to the zinc finger domain of the Caenorhabditis elegans tra1 gene, the Drosophila cubitus interruptus Dominant gene, and the human GLI oncogene. An in situ hybridization study revealed that zic showed a restricted expression pattern in the granule cells and their putative precursor cells. It is also expressed at an early embryonic stage in the dorsal half of the neural tube. The expression pattern and nuclear localization were confirmed by immunohistochemical study. Furthermore, the bacterially expressed zic protein containing the zinc finger domains bound to the GLI-binding sequence. These findings suggest that zic is one of a number of nuclear factors involved in both differentiation in early development and maintenance of properties of the cerebellar granule cells.

Amino Acid Sequence↗
PubMed1993

The Xenopus IP3 receptor: structure, function, and localization in oocytes and eggs.

To study the role of the IP3 receptor (IP3R) upon egg activation, cDNA clones encoding IP3R expressed in the Xenopus oocytes were isolated. By analyses of the primary structure and functional expression of the cDNA, Xenopus IP3R (XIP3R) was shown to have an IP3-binding domain and a putative Ca2+ channel region. Immunocytochemical studies revealed polarized distribution of XIP3R in the cytoplasm of the animal hemisphere in a well-organized endoplasmic reticulum-like structure and intensive localization in the perinuclear region of stage VI immature oocytes. In ovulated unfertilized eggs, XIP3R was densely enriched in the cortical region of both hemispheres in addition to its polarized localization. After fertilization, XIP3R showed a drastic change in its distribution in the cortical region. These results imply the predominant role of the XIP3R in both the formation and propagation of Ca2+ waves at fertilization.

Amino Acid Sequence↗
PubMed1993

Novel isoforms of mouse myelin basic protein predominantly expressed in embryonic stage.

Myelin basic protein (MBP), a major protein of myelin, is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well-known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages. These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of 18 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here.

Animals↗
PubMed1993

Isolation of a Drosophila gene encoding a head-specific guanylyl cyclase.

We have isolated and characterized a new guanylyl cyclase gene (dgc1) in Drosophila. The deduced amino acid sequence (683 amino acids) most closely resembled the mammalian soluble-type guanylyl cyclase alpha subunit. The cyclase catalytic domain was highly conserved between the mammalian and Drosophila guanylyl cyclases. The dgc1 mRNA was detected in wild-type heads but not in bodies, and its level was reduced in the mutant eyes absent (eya), indicating that dgc1 is preferentially expressed in the CNS and in the eye. The enriched distribution in the eye suggests that dgc1 may have a role in phototransduction.

Amino Acid Sequence↗
PubMed1992

The inositol 1,4,5-trisphosphate receptor.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger that releases Ca2+ from its intracellular stores. The InsP3 receptor has been purified and its cDNA has been cloned. We have found that the InsP3 receptor is identical to P400 protein, first identified as a protein enriched in cerebellar Purkinje cells. We have generated an L-fibroblast cell transfectant that produces cDNA-derived InsP3 receptors. The protein displays high affinity and specificity for InsP3. InsP3 induces greater Ca2+ release from membrane vesicles from transfected cells than from those from control L-fibroblasts. After incorporation of the purified InsP3 receptor into lipid bilayers InsP3-induced Ca2+ currents were demonstrated. These results suggest that the InsP3 receptor is involved in physiological Ca2+ release. Immunogold labelling using monoclonal antibodies against the receptor showed that it is highly concentrated on the smooth-surfaced endoplasmic reticulum and slightly on the outer nuclear membrane and rough endoplasmic reticulum; no labelling of Golgi apparatus, mitochondria and plasmalemma was seen. Cross-linking experiments showed that the receptor forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila, and this region contains the critical sequences for InsP3 binding. We have investigated the heterogeneity of the InsP3 receptor using the polymerase chain reaction and have found novel subtypes of the mouse InsP3 receptor that are expressed in a tissue-specific and developmentally specific manner.

Amino Acid Sequence↗
PubMed1991

The subtypes of the mouse inositol 1,4,5-trisphosphate receptor are expressed in a tissue-specific and developmentally specific manner.

Additional subtypes of the inositol 1,4,5-trisphosphate (InsP3) receptor are expressed in a tissue-specific and developmentally specific manner. They differ from the InsP3 receptor structure previously reported in two small variably spliced segments. One segment (SI) is located within the InsP3 binding site, whereas another segment (SII) is located near putative sites for phosphorylation and ATP binding to modulate InsP3 action on Ca2+ flux. Therefore, we speculate that selective use of InsP3 receptor subtypes permits a tissue-specific and developmentally specific expression of functionally distinct channels.

Aging↗
PubMed1991

Myelin basic protein gene and the function of antisense RNA in its repression in myelin-deficient mutant mouse.

The myelin-deficient (mld) mouse is an autosomal recessive mutant characterized by hypomyelination of the CNS due to reduced expression of the myelin basic protein (MBP) gene. In the mld mutant, the MBP gene is duplicated in tandem. One gene is intact, but a large portion is inverted upstream of the other copy, and its transcription yields the antisense RNA. This antisense RNA was shown to be localized in the nucleus and to form an RNA:RNA duplex with sense RNA. These findings suggested that inhibition of transport from the nucleus or selective degradation of the duplex is responsible for the reduced expression of the MBP gene in the mld mutant. The mechanism of gene rearrangement at the MBP locus was also characterized. Cosmid clones encompassing whole MBP gene loci from control and mld genomic DNA libraries were isolated. The recombination points indicated that the duplication and inversion observed in mld occurred due to nonhomologous recombination.

Animals↗
PubMed1991

Identification of the new isoforms of mouse myelin basic protein: the existence of exon 5a.

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.

Animals↗
PubMed1991

Molecular biology of myelin basic protein: gene rearrangement and expression of anti-sense RNA in myelin-deficient mutants.

1. Myelin is an important structure for facilitating the conduction of impulses along the axons both in the central nervous system (CNS) and peripheral nervous system (PNS). 2. Myelin basic protein (MBP) is a major protein in CNS myelin. 3. MBP is expressed specifically in the nervous system. 4. The MBP gene has been cloned and characterized. 5. Two mutant mice, Shiverer (shi) and myelin-deficient (mld. shimid), are autosomal recessive mutants that show severe symptoms such as intentional tremor. They have been found to have a mutation in the MBP gene that results in poor myelination in the central nervous system. 6. It was found that rearrangement within the MBP gene results in low expression of the gene. 7. In Shiverer, the MBP gene is partially deleted (from exons 3 to 7), and in mld, the gene is duplicated tandemly and a large portion of the duplication is inverted upstream of the intact copy. 8. In mld, anti-sense RNA complementary to exons 3-7, which correspond to the inverted segment, was detected by RNase protection studies, and presumed to be responsible for the reduced expressions of MBP. 9. The mechanism of gene rearrangement in MBP was also characterized. 10. This article reviews the recent progress in the study of the MBP gene, especially the rearrangement of the gene and its expression in mutant mice.

Animals↗