27 results on '"Tekotte H"'
Search Results
2. Author Correction: Building a global alliance of biofoundries.
- Author
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Hillson N, Caddick M, Cai Y, Carrasco JA, Chang MW, Curach NC, Bell DJ, Feuvre RL, Friedman DC, Fu X, Gold ND, Herrgård MJ, Holowko MB, Johnson JR, Johnson RA, Keasling JD, Kitney RI, Kondo A, Liu C, Martin VJJ, Menolascina F, Ogino C, Patron NJ, Pavan M, Poh CL, Pretorius IS, Rosser SJ, Scrutton NS, Storch M, Tekotte H, Travnik E, Vickers CE, Yew WS, Yuan Y, Zhao H, and Freemont PS
- Abstract
The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as '(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.'. The correct version replaces this with '(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University'. This has been corrected in both the PDF and HTML versions of the Comment.
- Published
- 2019
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3. Building a global alliance of biofoundries.
- Author
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Hillson N, Caddick M, Cai Y, Carrasco JA, Chang MW, Curach NC, Bell DJ, Le Feuvre R, Friedman DC, Fu X, Gold ND, Herrgård MJ, Holowko MB, Johnson JR, Johnson RA, Keasling JD, Kitney RI, Kondo A, Liu C, Martin VJJ, Menolascina F, Ogino C, Patron NJ, Pavan M, Poh CL, Pretorius IS, Rosser SJ, Scrutton NS, Storch M, Tekotte H, Travnik E, Vickers CE, Yew WS, Yuan Y, Zhao H, and Freemont PS
- Subjects
- Biomedical Research methods, Biotechnology instrumentation, Genetic Engineering, International Cooperation, Organisms, Genetically Modified
- Published
- 2019
- Full Text
- View/download PDF
4. Leaf LIMS: A Flexible Laboratory Information Management System with a Synthetic Biology Focus.
- Author
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Craig T, Holland R, D'Amore R, Johnson JR, McCue HV, West A, Zulkower V, Tekotte H, Cai Y, Swan D, Davey RP, Hertz-Fowler C, Hall A, and Caddick M
- Subjects
- Database Management Systems, Databases, Factual, Synthetic Biology instrumentation, Synthetic Biology methods
- Abstract
This paper presents Leaf LIMS, a flexible laboratory information management system (LIMS) designed to address the complexity of synthetic biology workflows. At the project's inception there was a lack of a LIMS designed specifically to address synthetic biology processes, with most systems focused on either next generation sequencing or biobanks and clinical sample handling. Leaf LIMS implements integrated project, item, and laboratory stock tracking, offering complete sample and construct genealogy, materials and lot tracking, and modular assay data capture. Hence, it enables highly configurable task-based workflows and supports data capture from project inception to completion. As such, in addition to it supporting synthetic biology it is ideal for many laboratory environments with multiple projects and users. The system is deployed as a web application through Docker and is provided under a permissive MIT license. It is freely available for download at https://leaflims.github.io .
- Published
- 2017
- Full Text
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5. Genetic Constructor: An Online DNA Design Platform.
- Author
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Bates M, Lachoff J, Meech D, Zulkower V, Moisy A, Luo Y, Tekotte H, Franziska Scheitz CJ, Khilari R, Mazzoldi F, Chandran D, and Groban E
- Subjects
- DNA genetics, Genetic Engineering instrumentation, Software
- Abstract
Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs. A plugin architecture enables contributions from scientists and coders to leverage existing powerful software and connect to DNA foundries. The software is easily accessible and platform agnostic, free for academics, and available in an open-source community edition. Genetic Constructor seeks to democratize DNA design, manufacture, and access to tools and services from the synthetic biology community.
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- 2017
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6. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.
- Author
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Beard PM, Griffiths SJ, Gonzalez O, Haga IR, Pechenick Jowers T, Reynolds DK, Wildenhain J, Tekotte H, Auer M, Tyers M, Ghazal P, Zimmer R, and Haas J
- Subjects
- Gene Expression Regulation, Gene Knockdown Techniques, High-Throughput Screening Assays, Humans, Reproducibility of Results, Signal Transduction, Transcription, Genetic, Vaccinia metabolism, Host-Pathogen Interactions genetics, RNA Interference, RNA, Small Interfering genetics, Vaccinia genetics, Vaccinia virology, Vaccinia virus physiology, Virus Replication
- Abstract
Vaccinia virus (VACV) is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.
- Published
- 2014
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7. Ablation of the regulatory IE1 protein of murine cytomegalovirus alters in vivo pro-inflammatory TNF-alpha production during acute infection.
- Author
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Rodríguez-Martín S, Kropp KA, Wilhelmi V, Lisnic VJ, Hsieh WY, Blanc M, Livingston A, Busche A, Tekotte H, Messerle M, Auer M, Fraser I, Jonjic S, Angulo A, Reddehase MJ, and Ghazal P
- Subjects
- Animals, Cell Line, Cytokines metabolism, DNA Replication, DNA, Viral genetics, Female, Herpesviridae Infections virology, Immediate-Early Proteins metabolism, Liver metabolism, Macrophages metabolism, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Muromegalovirus growth & development, Muromegalovirus physiology, Phenotype, Signal Transduction, Tumor Necrosis Factor-alpha analysis, Tumor Necrosis Factor-alpha genetics, Viral Proteins genetics, Viral Proteins metabolism, Virus Replication, Gene Expression Regulation, Viral genetics, Herpesviridae Infections immunology, Immediate-Early Proteins genetics, Muromegalovirus genetics, Tumor Necrosis Factor-alpha metabolism
- Abstract
Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MΦs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFα production. Intracellular staining for cytokine production and viral early gene expression shows that TNFα production is highly associated with the productively infected MΦ population of cells. The ie1- dependent phenotype of enhanced MΦ TNFα production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFα, and to a lesser extent IFNβ, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1- mediated inhibition of TNFα production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFα inhibition. However, in vivo infection studies show that genetic ablation of TNFα or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFα in vivo.
- Published
- 2012
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8. Sch9 regulates ribosome biogenesis via Stb3, Dot6 and Tod6 and the histone deacetylase complex RPD3L.
- Author
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Huber A, French SL, Tekotte H, Yerlikaya S, Stahl M, Perepelkina MP, Tyers M, Rougemont J, Beyer AL, and Loewith R
- Subjects
- RNA, Transfer biosynthesis, Transcription, Genetic, Gene Expression Regulation, Fungal, RNA, Ribosomal biosynthesis, Ribosomal Proteins biosynthesis, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Signal Transduction
- Abstract
TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription.
- Published
- 2011
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9. SCFCdc4 enables mating type switching in yeast by cyclin-dependent kinase-mediated elimination of the Ash1 transcriptional repressor.
- Author
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Liu Q, Larsen B, Ricicova M, Orlicky S, Tekotte H, Tang X, Craig K, Quiring A, Le Bihan T, Hansen C, Sicheri F, and Tyers M
- Subjects
- Amino Acid Sequence, CDC28 Protein Kinase, S cerevisiae metabolism, Cell Cycle, Gene Silencing, Molecular Sequence Data, Phosphorylation, Phosphothreonine metabolism, Protein Binding, Protein Processing, Post-Translational, Protein Stability, Protein Transport, Repressor Proteins chemistry, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae Proteins chemistry, Ubiquitination, Cell Cycle Proteins metabolism, Cyclin-Dependent Kinases metabolism, F-Box Proteins metabolism, Genes, Mating Type, Fungal, Repressor Proteins metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins metabolism, Transcription, Genetic, Ubiquitin-Protein Ligases metabolism
- Abstract
In the budding yeast Saccharomyces cerevisiae, mother cells switch mating types between a and α forms, whereas daughter cells do not. This developmental asymmetry arises because the expression of the HO endonuclease, which initiates the interconversion of a and α mating type cassettes, is extinguished by the daughter-specific Ash1 transcriptional repressor. When daughters become mothers in the subsequent cell cycle, Ash1 must be eliminated to enable a new developmental state. Here, we report that the ubiquitin ligase SCF(Cdc4) mediates the phosphorylation-dependent elimination of Ash1. The inactivation of SCF(Cdc4) stabilizes Ash1 in vivo, and consistently, Ash1 binds to and is ubiquitinated by SCF(Cdc4) in a phosphorylation-dependent manner in vitro. The mutation of a critical in vivo cyclin-dependent kinase (CDK) phosphorylation site (Thr290) on Ash1 reduces its ubiquitination and rate of degradation in vivo and decreases the frequency of mating type switching. Ash1 associates with active Cdc28 kinase in vivo and is targeted to SCF(Cdc4) in a Cdc28-dependent fashion in vivo and in vitro. Ash1 recognition by Cdc4 appears to be mediated by at least three phosphorylation sites that form two redundant diphosphorylated degrons. The phosphorylation-dependent elimination of Ash1 by the ubiquitin-proteasome system thus underpins developmental asymmetry in budding yeast.
- Published
- 2011
- Full Text
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10. Imaging the migrating border cell cluster in living Drosophila egg chambers.
- Author
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Tekotte H, Tollervey D, and Davis I
- Subjects
- Animals, Drosophila physiology, Female, Green Fluorescent Proteins, Oogenesis, Ovary cytology, Cell Movement, Drosophila cytology, Microscopy, Interference methods
- Abstract
Cell migration is a key process in animal development and central to the spread of cancer. Border cell migration in Drosophila egg chambers is an excellent general model for cell migration, but lacks techniques for studying this process in living cells. Here, we describe a simple and effective method of preparing egg chambers in halocarbon oil. The movement and behavior of the migrating border cells can reproducibly be followed in up to 25 egg chambers simultaneously by time-lapse microscopy using a variety of green fluorescent protein markers on a widefield microscope over a period of 4 hr. Our studies reveal a remarkably linear migration route of the border cell cluster and highly dynamic activity within their cluster. Migrating cells rapidly alter their relative positions and generate transient protrusions. These activities are likely to play key roles in the mechanism of migration and cannot readily be analyzed using fixed samples., (2007 Wiley-Liss, Inc.)
- Published
- 2007
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11. Bruno: a double turn-off for Oskar.
- Author
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Tekotte H and Davis I
- Subjects
- Animals, Drosophila Proteins genetics, Gene Silencing, Protein Biosynthesis, RNA, Messenger metabolism, RNA-Binding Proteins genetics, Drosophila Proteins metabolism, Drosophila melanogaster physiology, RNA-Binding Proteins metabolism
- Abstract
In Drosophila, the posterior localization of oskar mRNA and its translational regulation are essential for axis specification and germline formation. Recently in Cell, demonstrated that Bruno inhibits cap-dependent translation of oskar mRNA and uncovered a novel Bruno-dependent assembly of oskar mRNA into multimeric RNP particles, which are inaccessible to the translational machinery. This work provides a novel link between mRNA localization, particle formation, and translational regulation.
- Published
- 2006
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12. Intracellular mRNA localization: motors move messages.
- Author
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Tekotte H and Davis I
- Subjects
- Animals, Drosophila genetics, Drosophila metabolism, Drosophila Proteins genetics, Dyneins metabolism, Kinesins metabolism, Myosin Type V metabolism, Trans-Activators metabolism, Transcription Factors genetics, Yeasts genetics, Yeasts metabolism, DNA-Binding Proteins, Molecular Motor Proteins metabolism, RNA, Messenger metabolism
- Abstract
Intracellular mRNA localization is a common mechanism of post-transcriptional regulation of gene expression. In a wide range of organisms, mRNA localization coupled with translational regulation target the proteins to their site of function. Here, we describe recent exciting evidence that some mRNAs are transported as particles along the cytoskeleton by the molecular motors dynein, kinesin or myosin. We discuss the key questions of how localized mRNAs might be linked to motors and what determines their cytoplasmic destinations.
- Published
- 2002
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13. Dcas is required for importin-alpha3 nuclear export and mechano-sensory organ cell fate specification in Drosophila.
- Author
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Tekotte H, Berdnik D, Török T, Buszczak M, Jones LM, Cooley L, Knoblich JA, and Davis I
- Subjects
- Active Transport, Cell Nucleus physiology, Animals, Apoptosis, DNA Helicases metabolism, Drosophila melanogaster genetics, Embryo, Nonmammalian physiology, Gene Expression Regulation, Developmental, In Situ Hybridization, Morphogenesis, Phylogeny, RNA, Messenger genetics, Cellular Apoptosis Susceptibility Protein genetics, Drosophila Proteins, Drosophila melanogaster embryology, Mechanoreceptors embryology, Sense Organs embryology, alpha Karyopherins metabolism
- Abstract
We have studied the in vivo function and tissue specificity of Dcas, the Drosophila ortholog of CAS, the importin beta-like export receptor for importin alpha. While dcas mRNA is specifically expressed in the embryonic central nervous system, Dcas protein is maternally supplied to all embryonic cells and its nuclear/cytoplasmic distribution varies in different tissues and times in development. Unexpectedly, hypomorphic alleles of dcas show specific transformations in mechano-sensory organ cell identity, characteristic of mutations that increase Notch signaling. Dcas is essential for efficient importin-alpha3 nuclear export in mechano-sensory cells and the surrounding epidermal cells and is indirectly required for the import of one component of the Notch pathway, but not others tested. We interpret the specificity of the dcas phenotype as indicating that one or more Notch signaling components are particularly sensitive to a disruption in nuclear protein import. We propose that mutations in house keeping genes often cause specific developmental phenotypes, such as those observed in many human genetic disorders., (Copyright 2002 Elsevier Science (USA).)
- Published
- 2002
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14. Perturbing nuclear transport in Drosophila eye imaginal discs causes specific cell adhesion and axon guidance defects.
- Author
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Kumar JP, Wilkie GS, Tekotte H, Moses K, and Davis I
- Subjects
- Animals, Animals, Genetically Modified, Axons ultrastructure, Blastoderm metabolism, Cell Adhesion, Drosophila genetics, Drosophila Proteins genetics, Drosophila Proteins metabolism, Eye cytology, Humans, Photoreceptor Cells, Invertebrate embryology, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Deletion, beta Karyopherins genetics, beta Karyopherins metabolism, Active Transport, Cell Nucleus, Drosophila embryology, Drosophila metabolism, Eye embryology
- Abstract
To study nucleocytoplasmic transport during multicellular development, we developed a sensitive nuclear protein import assay in living blastoderm embryos. We show that dominant negative truncations of the human nuclear transport receptor karyopherinbeta/Importinbeta (DNImpbeta) disrupt mRNA export and protein import in Drosophila. To test the sensitivity of different developmental processes to nuclear trafficking perturbations, we expressed DNImpbeta behind the morphogenetic furrow of the eye disc, at a time when photoreceptors are patterned and project their axons to the brain. DNImpbeta expression does not disrupt the correct specification of different photoreceptors, but causes a defect in cell adhesion that leads to some photoreceptors descending below the layer of ommatidia. The photoreceptors initially project their axons correctly to the posterior, but later their axons are unable to enter the optic stalk en route to the brain and continue to project an extensive network of misguided axons. The axon guidance and cell adhesion defects are both due to a disruption in the function of Ketel, the Drosophila ortholog of Importinbeta. We conclude that cell adhesion and axon guidance in the eye have specific requirements for nucleocytoplasmic transport, despite involving processes that occur primarily at the cell surface.
- Published
- 2001
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15. The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program.
- Author
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Pijnappel WW, Schaft D, Roguev A, Shevchenko A, Tekotte H, Wilm M, Rigaut G, Séraphin B, Aasland R, and Stewart AF
- Subjects
- Amino Acid Sequence, Haploidy, Mass Spectrometry, Molecular Sequence Data, Phenotype, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Sirtuin 2, Time Factors, Fungal Proteins chemistry, Histone Deacetylases metabolism, Meiosis, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces pombe Proteins, Sirtuins
- Abstract
Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.
- Published
- 2001
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16. Cooperative interaction of ets-1 with USF-1 required for HIV-1 enhancer activity in T cells.
- Author
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Sieweke MH, Tekotte H, Jarosch U, and Graf T
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Cell Line, Cloning, Molecular, DNA, Viral metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Genes, Dominant, HIV-1 physiology, Helix-Loop-Helix Motifs, Humans, Jurkat Cells, Leucine Zippers, Molecular Sequence Data, Mutation, Proto-Oncogene Protein c-ets-1, Proto-Oncogene Proteins c-ets, Quail, Recombinant Fusion Proteins, Transcription Factors genetics, Transcriptional Activation physiology, Transfection, Upstream Stimulatory Factors, HIV Enhancer genetics, HIV-1 genetics, Proto-Oncogene Proteins metabolism, T-Lymphocytes virology, Transcription Factors metabolism
- Abstract
The distal enhancer region of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) is known to be essential for HIV replication and to contain immediately adjacent E-box and Ets binding sites. Based on a yeast one-hybrid screen we have identified the E-box binding protein USF-1 as a direct interaction partner of Ets-1 and found that the complex acts on this enhancer element. The binding surfaces of USF-1 and Ets-1 map to their DNA-binding domains and although these domains are highly conserved, the interaction is very selective within the respective protein family. USF-1 and Ets-1 synergize in specific DNA binding as well as in the transactivation of reporter constructs containing the enhancer element, and mutations of the individual binding sites dramatically reduce reporter activity in T cells. In addition, a dominant negative Ets-1 mutant inhibits both USF-1-mediated transactivation and the activity of the HIV-1 LTR in T cells. The inhibition is independent of Ets DNA-binding sites but requires the Ets binding surface on USF-1, highlighting the importance of the direct protein-protein interaction. Together these results indicate that the interaction between Ets-1 and USF-1 is required for full transcriptional activity of the HIV-1 LTR in T cells.
- Published
- 1998
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17. MafB represses erythroid genes and differentiation through direct interaction with c-Ets-1.
- Author
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Sieweke MH, Tekotte H, Frampton J, and Graf T
- Subjects
- Animals, Cell Differentiation, Erythroblasts physiology, Erythrocytes physiology, Hematopoietic Stem Cells physiology, Heme biosynthesis, Leucine Zippers, Proto-Oncogene Protein c-ets-1, Proto-Oncogene Proteins c-ets, Receptors, Transferrin biosynthesis, Recombinant Proteins metabolism, Repressor Proteins metabolism, Saccharomyces cerevisiae, Transcriptional Activation, Transfection, Avian Proteins, DNA-Binding Proteins, Erythroblasts cytology, Erythrocytes cytology, Erythropoiesis physiology, Hematopoietic Stem Cells cytology, Oncogene Proteins metabolism, Proto-Oncogene Proteins metabolism, Trans-Activators metabolism, Transcription Factors metabolism
- Abstract
Using a yeast interaction screen with a DNA-bound Ets-1 protein we have identified MafB as a direct interaction partner. This distant AP-1 relative, which is specifically expressed in myelomonocytic cells, binds to the DNA binding domain of Ets-1 via its basic region/leucine zipper domain. MafB represses Ets-1 transactivation of synthetic promoters containing Ets binding sites in yeast as well as avian cells. Both Ets-1 and Maf family proteins have been implicated in erythroid specific gene expression. Here we show that MafB inhibits Ets-1-mediated transactivation of the transferrin receptor, which is essential for heme synthesis and erythroid differentiation. Consequently, overexpression of MafB in an erythroblast cell line down-regulates the endogenous transferrin receptor gene and inhibits the cells' potential to differentiate into erythrocytes, without affecting cellular proliferation.
- Published
- 1997
18. Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p.
- Author
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Zabel U, Doye V, Tekotte H, Wepf R, Grandi P, and Hurt EC
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Fungal Proteins genetics, Fungal Proteins isolation & purification, Fungal Proteins metabolism, Genes, Fungal genetics, Genes, Lethal, Genetic Complementation Test, Molecular Sequence Data, Molecular Weight, Mutation, Nuclear Envelope ultrastructure, Nuclear Proteins analysis, Nuclear Proteins genetics, Nuclear Proteins isolation & purification, Protein Binding, Recombinant Fusion Proteins isolation & purification, Temperature, Yeasts cytology, Yeasts genetics, Fungal Proteins chemistry, Membrane Proteins, Nuclear Envelope chemistry, Nuclear Pore Complex Proteins, Nuclear Proteins chemistry, Nuclear Proteins metabolism, Saccharomyces cerevisiae Proteins
- Abstract
The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport. Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature. Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction. Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology. We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.
- Published
- 1996
- Full Text
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19. Nuclear pore proteins are involved in the biogenesis of functional tRNA.
- Author
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Simos G, Tekotte H, Grosjean H, Segref A, Sharma K, Tollervey D, and Hurt EC
- Subjects
- Amino Acid Sequence, Molecular Sequence Data, Nuclear Proteins chemistry, Nucleic Acid Conformation, RNA Splicing, RNA, Transfer chemistry, Sequence Homology, Amino Acid, Nuclear Envelope metabolism, Nuclear Proteins metabolism, RNA, Transfer biosynthesis
- Abstract
Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.
- Published
- 1996
20. MafB is an interaction partner and repressor of Ets-1 that inhibits erythroid differentiation.
- Author
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Sieweke MH, Tekotte H, Frampton J, and Graf T
- Subjects
- Animals, Base Sequence, Binding Sites physiology, Cell Differentiation drug effects, Cell Differentiation physiology, Chick Embryo, Clone Cells physiology, DNA, Complementary physiology, DNA-Binding Proteins metabolism, DNA-Binding Proteins physiology, Erythroblasts physiology, G-Box Binding Factors, Gene Expression Regulation physiology, Genetic Testing, Hematopoiesis genetics, Macrophages physiology, MafB Transcription Factor, Molecular Sequence Data, Oncogene Proteins metabolism, Proto-Oncogene Protein c-ets-1, Proto-Oncogene Proteins antagonists & inhibitors, Proto-Oncogene Proteins c-ets, Receptors, Transferrin genetics, Repressor Proteins genetics, Trans-Activators metabolism, Transcription Factors antagonists & inhibitors, Vertebrates, Yeasts genetics, Avian Proteins, DNA-Binding Proteins genetics, Erythroblasts cytology, Oncogene Proteins genetics, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins metabolism, Trans-Activators genetics, Transcription Factors metabolism
- Abstract
Using a yeast one-hybrid screen with a DNA-bound Ets-1 protein, we have identified MafB, an AP-1 like protein, as a direct interaction partner. MafB is specifically expressed in myelomonocytic cells and binds to the DNA-binding domain of Ets-1 via its basic region or leucine-zipper domain. Furthermore, it represses Ets-1 transactivation of synthetic promoters containing Ets binding sites and inhibits Ets-1-mediated transactivation of the transferrin receptor, which is known to be essential for erythroid differentiation. Accordingly, overexpression of MafB in an erythroblast cell line down-regulates the endogenous transferrin receptor gene and inhibits differentiation without affecting cell proliferation. These results highlight the importance of inhibitory interactions between transcription factors in regulating lineage-specific gene expression.
- Published
- 1996
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21. A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores.
- Author
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Siniossoglou S, Wimmer C, Rieger M, Doye V, Tekotte H, Weise C, Emig S, Segref A, and Hurt EC
- Subjects
- Amino Acid Sequence, Biological Transport, Carboxypeptidases metabolism, Cathepsin A, Epitopes, Fungal Proteins isolation & purification, Membrane Proteins isolation & purification, Molecular Sequence Data, Molecular Weight, Nuclear Envelope ultrastructure, Nuclear Proteins analysis, Nuclear Proteins chemistry, Nuclear Proteins isolation & purification, Poly A metabolism, Proto-Oncogene Proteins c-myc genetics, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins isolation & purification, Sequence Homology, Amino Acid, Staphylococcal Protein A genetics, Yeasts metabolism, Fungal Proteins metabolism, Membrane Proteins metabolism, Nuclear Envelope chemistry, Nuclear Pore Complex Proteins, Nuclear Proteins metabolism, Nuclear Proteins physiology, Saccharomyces cerevisiae Proteins
- Abstract
In a genetic screen for nucleoporin-interacting components, a novel nuclear pore protein Nup84p, which exhibits homology to mammalian Nup107p, was isolated. Nup84p forms a complex with five proteins, of which Nup120p, Nup85p, Sec13p, and a Sec13p homolog were identified. Upon isolation of Sec13p-ProtA, nucleoporins were still associated, but the major copurifying band was a 150 kDa protein, showing that Sec13p occurs in two complexes. Disruption of any of the genes encoding Nup84p, Nup85p, or Nup120p caused defects in nuclear membrane and nuclear pore complex organization, as well as in poly(A)+ RNA transport. Thus, the Nup84p complex in conjunction with Sec13-type proteins is required for correct nuclear pore biogenesis.
- Published
- 1996
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22. Yeast nucleoporin mutants are defective in pre-tRNA splicing.
- Author
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Sharma K, Fabre E, Tekotte H, Hurt EC, and Tollervey D
- Subjects
- Base Sequence, DNA, Fungal genetics, Fungal Proteins genetics, Fungal Proteins metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Molecular Sequence Data, Mutation, Nuclear Envelope metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, RNA Precursors genetics, RNA, Fungal genetics, RNA Precursors metabolism, RNA Splicing genetics, RNA, Fungal metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
We have screened nucleoporin mutants for the inhibition of tRNA splicing, which has previously been proposed to be coupled to transport. Strains mutant for Nup49p or Nup116p, or genetically depleted of Nup145p, strongly accumulated unspliced pre-tRNAs. Splicing was inhibited for all 10 families of intron-containing pre-tRNA, but no effects on 5' or 3' end processing were detected. Strains mutant for Nup133p or Nsp1p accumulated lower levels of several unspliced pre-tRNAs. In contrast, no accumulation of any pre-tRNA was observed in strains mutant for Nup1p, Nup85p, or Nup100p. Other RNA processing reactions tested, pre-rRNA processing, pre-mRNA splicing, and small nucleolar and small nuclear RNA synthesis, were not clearly affected for any nucleoporin mutant. These data provide evidence for a coupling between pre-tRNA splicing and nuclear-cytoplasmic transport. Mutation of NUP49, NUP116, or NUP145 has previously been shown to lead to nuclear poly(A)+ RNA accumulation, indicating that these nucleoporins play roles in the transport of more than one class of RNA.
- Published
- 1996
- Full Text
- View/download PDF
23. Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p.
- Author
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Grandi P, Schlaich N, Tekotte H, and Hurt EC
- Subjects
- Amino Acid Sequence, Base Sequence, Biological Transport genetics, Cell Compartmentation, Cell Nucleus metabolism, Cytoplasm metabolism, DNA Mutational Analysis, Fungal Proteins genetics, Fungal Proteins metabolism, Genes, Fungal genetics, Genes, Lethal genetics, Genetic Complementation Test, Macromolecular Substances, Membrane Proteins genetics, Membrane Proteins metabolism, Molecular Sequence Data, Nuclear Envelope genetics, Nuclear Proteins genetics, Protein Binding, Repetitive Sequences, Nucleic Acid, Selection, Genetic, Structure-Activity Relationship, Yeasts, Calcium-Binding Proteins, Nuclear Envelope metabolism, Nuclear Pore Complex Proteins, Nuclear Proteins metabolism, Saccharomyces cerevisiae Proteins
- Abstract
Nic96p has been isolated previously in a complex together with the nuclear pore proteins Nsp1p, Nup49p and a p54 polypeptide. In a genetic screen for Nsp1p-interacting components, we now find NIC96, as well as a novel gene NUP57 which encodes the p54 protein (called Nup57p). Nup57p which is essential for cell growth contains GLFG repeats in the N-terminal half and heptad repeats in the C-terminal half. The domain organization of Nic96p is more complex: N-terminally located heptad repeats mediate binding to a trimeric Nsp1p-Nup49p-Nup57p complex, but are not required for the formation of this core complex; single amino acid substitutions in the central domain yield thermosensitive mutants, which do not impair interaction with the Nsp1 complex; the C-terminal domain is neither essential nor required for binding to the nucleoporin complex, but strikingly mutations in this part cause synthetic lethality with nsp1 and nup57 mutant alleles. Since a strain in which the Nic96p heptad repeats were deleted shows, similar to nsp1 and nup49 mutants, cytoplasmic mislocalization of a nuclear reporter protein, we propose that the interaction of the heterotrimeric Nsp1p-Nup49p-Nup57p core complex with Nic96p is required for protein transport into the nucleus.
- Published
- 1995
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24. Disaccharide composition of heparan sulfates: brain, nervous tissue storage organelles, kidney, and lung.
- Author
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Tekotte H, Engel M, Margolis RU, and Margolis RK
- Subjects
- Aging metabolism, Animals, Animals, Newborn, Brain metabolism, Brain Chemistry, Cattle, Chromatography, High Pressure Liquid, Disaccharides metabolism, Heparitin Sulfate metabolism, Kidney chemistry, Kidney metabolism, Lung chemistry, Lung metabolism, Nerve Tissue chemistry, Nerve Tissue metabolism, Organelles metabolism, Rabbits, Rats, Disaccharides chemistry, Heparitin Sulfate chemistry
- Abstract
We have characterized the structural properties of heparan sulfates from brain and other tissues after depolymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46-69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17-21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10-14%, together with smaller and approximately equal proportions (5-9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues. In chromaffin granules, the catecholamine- and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5-10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71%) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large dense cored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for approximately 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21% unsulfated disaccharides, 10% mono- and disulfated disaccharides, and 69% trisulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
- Full Text
- View/download PDF
25. Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies.
- Author
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Rauch U, Gao P, Janetzko A, Flaccus A, Hilgenberg L, Tekotte H, Margolis RK, and Margolis RU
- Subjects
- Animals, Antibodies, Monoclonal, Autoradiography, Axons metabolism, Axons ultrastructure, Blotting, Western, Carbohydrate Sequence, Chondroitin Sulfate Proteoglycans chemistry, Chondroitin Sulfate Proteoglycans immunology, Chondroitin Sulfates chemistry, Chondroitin Sulfates immunology, Chromatography, Gel, Chromatography, Thin Layer, Electrophoresis, Polyacrylamide Gel, Immunohistochemistry, Keratan Sulfate chemistry, Keratan Sulfate immunology, Lumican, Molecular Sequence Data, Proteoglycans chemistry, Proteoglycans isolation & purification, Rats, beta-Galactosidase chemistry, Brain Chemistry immunology, Chondroitin Sulfate Proteoglycans isolation & purification, Chondroitin Sulfates isolation & purification, Glycoside Hydrolases, Keratan Sulfate isolation & purification
- Abstract
A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1991
26. Effects of beta-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum.
- Author
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Margolis RK, Goossen B, Tekotte H, Hilgenberg L, and Margolis RU
- Subjects
- Adrenal Gland Neoplasms, Animals, Cell Division, Cells, Cultured, Cerebellum cytology, Chondroitin Sulfates metabolism, Chromatography, Gel, Nerve Growth Factors pharmacology, Pheochromocytoma, Proteoglycans metabolism, Rats, Tumor Cells, Cultured, Cerebellum metabolism, Glycosides pharmacology, Proteoglycans biosynthesis
- Abstract
We have examined the effects of beta-xylosides, which act as exogenous acceptors for glycosaminoglycan chain initiation, on the morphology and proteoglycan biosynthesis of PC12 pheochromocytoma cells, and on monolayer, aggregate and explant cultures of early postnatal rat cerebellum. PC12 cells cultured for 13 days in the presence of nerve growth factor (NGF) and beta-xyloside, and labeled during days 11-13 with sodium [35S]sulfate, showed an 8- to 11-fold increase in [35S]sulfate-labeled macromolecules released into the culture medium. Most of the increase was accounted for by chondroitin sulfate, which was in the form of free glycosaminoglycan chains, which were not acid-precipitable. The presence of beta-xyloside also led to a 65-115% increase in [35S]sulfate incorporation into cell-associated glycosaminoglycans and glycoproteins of untreated and NGF-treated PC12 cells, respectively. beta-Xyloside treatment reduced the size of the chondroitin sulfate chains in both the cells and medium from approximately 34,000 to 10,000 Mr, but had much less effect on heparan sulfate, which decreased in size from 16,000 to 13,000-14,500 Mr (in the medium and cells, respectively). beta-Xyloside inhibition of proteoglycan biosynthesis was accompanied by significant morphological effects in NGF-treated PC12 cells, consisting of an increase in length and decrease in the branching, diameter and adhesion to the collagen substratum of the PC12 cell processes. p-Nitrophenyl- and 4-methylumbelliferyl-beta-D-xylosides produced similar effects, which were not seen with p-nitrophenyl-beta-D-galactoside. beta-Xylosides also produced distinct alterations in the adhesion and morphology of monolayer, aggregate, and explant cultures of early postnatal rat cerebellum, which occurred together with inhibition of chondroitin sulfate proteoglycan biosynthesis and a decrease in glycosaminoglycan chain size. These studies indicate that chondroitin sulfate (and probably also heparan sulfate) proteoglycans play a significant role in modulating cell-cell and cell-matrix interactions in nervous tissue development and differentiation.
- Published
- 1991
- Full Text
- View/download PDF
27. Immunocytochemical localization of chondroitin and chondroitin 4- and 6-sulfates in developing rat cerebellum.
- Author
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Flaccus A, Janetzko A, Tekotte H, Margolis RK, and Margolis RU
- Subjects
- Animals, Cerebellum growth & development, Cerebellum ultrastructure, Chromatography, High Pressure Liquid, Immunohistochemistry methods, Isomerism, Rats, Rats, Inbred Strains, Staining and Labeling, Tissue Distribution, Cerebellum metabolism, Chondroitin metabolism, Chondroitin Sulfates metabolism
- Abstract
Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1991
- Full Text
- View/download PDF
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