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Walter J. Gehring - One of the best experts on this subject based on the ideXlab platform.
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functional synthetic Antennapedia genes and the dual roles of ypwm motif and linker size in transcriptional activation and repression
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Dimitrios K Papadopoulos, Diana Resendezperez, Diana L Cardenaschavez, Karina Villanuevasegura, Ricardo Canalesdelcastillo, Daniel A Felix, Raphael Funfschilling, Walter J. GehringAbstract:Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. Here we derive synthetic Drosophila Antennapedia genes, consisting only of the YPWM motif and homeodomain, and investigate their functional role throughout development. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-a-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. Our results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary—whereas the entire N terminus of the protein is dispensable—for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors.
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molecular basis for the inhibition of drosophila eye development by Antennapedia
The EMBO Journal, 2001Co-Authors: Serge Plaza, Susanne Flister, Frederic Prince, Johannes Jaeger, Urs Kloter, Corinne Benassayag, David L Cribbs, Walter J. GehringAbstract:Hox genes encoding homeodomain transcriptional regulators are known to specify the body plan of multicellular organisms and are able to induce body plan transformations when misexpressed. These findings led to the hypothesis that duplication events and misexpression of Hox genes during evolution have been necessary for generating the observed morphological diversity found in metazoans. It is known that overexpressing Antennapedia (Antp) in the head induces antenna-to-leg as well as head-to-thorax transformation and eye reduction. At present, little is known about the exact molecular mechanism causing these phenotypes. The aim of this study is to understand the basis of inhibition of eye development. We demonstrate that Antp represses the activity of the eye regulatory cascade. By ectopic expression, we show that Antp antagonizes the activity of the eye selector gene eyeless. Using both in vitro and in vivo experiments, we demonstrate that this inhibitory mechanism involves direct protein-protein interactions between the DNA-binding domains of EY and ANTP, resulting in mutual inhibition.
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a sequence conserved in vertebrate hox gene introns functions as an enhancer regulated by posterior homeotic genes in drosophila imaginal discs
Mechanisms of Development, 1997Co-Authors: Liam P Keegan, Theodor E Haerry, David Crotty, Alan I Packer, Debra J Wolgemuth, Walter J. GehringAbstract:The intron of the mouse Hoxa-4 gene acts as a strong homeotic response element in Drosophila melanogaster leg imaginal discs. This activity depends on homeodomain binding sites present within a 30 bp conserved element, HB1, in the intron. A similar arrangement of homeodomain binding sites is found in many other potential homeotic target genes. HB1 activity in Drosophila imaginal discs is activated by Antennapedia and more posterior homeotic genes, but is not activated by more anterior genes. Testing a reporter gene construct with mutated binding sites in mouse embryos shows that HB1 is also active in the expression domains of posterior Hox genes in the mouse neural tube.
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the des 1 6 Antennapedia homeodomain comparison of the nmr solution structure and the dna binding affinity with the intact Antennapedia homeodomain
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Yan Qiu Qian, Diana Resendezperez, Walter J. Gehring, Kurt WuthrichAbstract:The nuclear magnetic resonance (NMR) solution structure of an N-terminally truncated mutant Antennapedia homeodomain, des(1-6)Antp(C39S), has been determined from 935 nuclear Overhauser effect upper distance constraints and 148 dihedral angle constraints by using the programs DIANA and OPAL. Twenty conformers representing the solution structure of des(1-6)Antp(C39S) have an average root-mean-square distance relative to the mean coordinates of 0.56 A for the backbone atoms of residues 8-59. Comparison with the intact Antp(C39S) homeodomain shows that the two proteins have identical molecular architectures. The removal of the N-terminal residues 1-6, which are flexibly disordered in the intact homeodomain, causes only strictly localized structure variations and does not noticeably affect the adjoining helix I from residues 10-21. The DNA-binding constant of des(1-6)Antp(C39S) is approximately 10-fold reduced relative to the intact Antp(C39S) homeodomain, which can now be attributed to the absence of the previously reported contacts of the N-terminal polypeptide segment of the intact Antp(C39S) homeodomain with the minor groove of the DNA duplex.
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functional specificity of the Antennapedia homeodomain
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Katsuo Furukubotokunaga, Susanne Flister, Walter J. GehringAbstract:The segmental identity in animal development is determined by a set of homeotic selector genes clustered in the invertebrate HOM or vertebrate Hox homeo box complexes. These genes encode proteins with very similar homeodomains and highly diverged N- and C-terminal sequences. The Antennapedia (Antp) homeodomain, for instance, differs at only five amino acid positions from that of Sex combs reduced (Scr) protein. Using a heat shock assay in which chimeric Antp-Scr proteins are expressed ectopically in Drosophila, we have shown that the functional specificity of the Antp protein is determined by the four specific amino acids located in the flexible N-terminal arm of the homeodomain. The three-dimensional structure of the Antp homeodomain-DNA complex shows that this N-terminal arm is located in the minor groove of the DNA, suggesting that the functional specificity is determined either by slight differences in DNA binding and/or by selective interactions with other transcription factor(s).
Eldon E Ball - One of the best experts on this subject based on the ideXlab platform.
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sequence and expression of grasshopper Antennapedia comparison to drosophila
Developmental Biology, 1995Co-Authors: David C Hayward, Nipam H Patel, Jay E Rehm, Corey S Goodman, Eldon E BallAbstract:We have cloned and characterized the Antennapedia (Antp) gene from the grasshopper Schistocerca americana. The Antennapedia protein contains seven blocks of sequence, including the homeodomain, that are conserved in the homologous proteins of other insects, interspersed with (usually repetitive) sequences unique to each species. There is no similarity between 1.8 kb of 3 * untranslated sequence in grasshopper and Drosophila. We examined Antennapedia protein expression in grasshopper using an antibody raised against a grasshopper fusion protein and reexamined its expression in Drosophila using several different antibodies. Early patterns of expression in the two insects are quite different, reflecting differing modes of early development. However, by the germband stage, expression patterns are quite similar, with relatively uniform epithelial expression throughout the thoracic and abdominal segments which later retracts to the thorax. Expression is observed in muscle pioneers, the peripheral nervous system, and the central nervous system (CNS). In the CNS expression is initially limited to a few neurons, but eventually becomes widespread. Both insects show strong expression in certain homologous identified neurons and similar temporal modulation of expression. q 1995 Academic Press, Inc.
Kurt Wuthrich - One of the best experts on this subject based on the ideXlab platform.
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Determination of h2J(NN) and h1J(HN) coupling constants across Watson-Crick base pairs in the Antennapedia homeodomain-DNA complex using TROSY.
Journal of Biomolecular NMR, 2000Co-Authors: Konstantin Pervushin, Masatsune Kainosho, César Fernández, Roland Riek, Kurt WuthrichAbstract:This paper describes NMR measurements of 15N–15N and 1H–15N scalar couplings across hydrogen bonds in Watson–Crick base pairs, h2JNN and h1JHN, in a 17 kDa Antennapedia homeodomain–DNA complex. A new NMR experiment is introduced which relies on zero-quantum coherence-based transverse relaxation-optimized spectroscopy (ZQ-TROSY) and enables measurements of h1JHN couplings in larger molecules. The h2JNN and h1JHN couplings open a new avenue for comparative studies of DNA duplexes and other forms of nucleic acids free in solution and in complexes with proteins, drugs or possibly other classes of compounds.
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nmr with 13 c 15 n doubly labeled dna the shape Antennapedia homeodomain complex with a 14 mer dna duplex
Journal of Biomolecular NMR, 1998Co-Authors: César Fernández, Masatsune Kainosho, Thomas Szyperski, Akira Ono, Hideo Iwai, Shinichi Tate, Kurt WuthrichAbstract:Nearly complete 1H, 13C and15 N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly 15N-labeled Antennapedia homeodomain. The DNA was either fully 13C,15N-labeled or contained uniformly 13C, 15N-labeled nucleotides only at those positions which form the protein–DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain–DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via1 H–1H NOEs observed in 3D13 C-resolved NOESY; and (iii) assignment of the imino and amino groups via 1H–1H NOEs and15 N–1H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein–DNA complex was greatly facilitated by the fact that 1H signals of the protein were filtered out in 13C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding 13C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.
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the des 1 6 Antennapedia homeodomain comparison of the nmr solution structure and the dna binding affinity with the intact Antennapedia homeodomain
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Yan Qiu Qian, Diana Resendezperez, Walter J. Gehring, Kurt WuthrichAbstract:The nuclear magnetic resonance (NMR) solution structure of an N-terminally truncated mutant Antennapedia homeodomain, des(1-6)Antp(C39S), has been determined from 935 nuclear Overhauser effect upper distance constraints and 148 dihedral angle constraints by using the programs DIANA and OPAL. Twenty conformers representing the solution structure of des(1-6)Antp(C39S) have an average root-mean-square distance relative to the mean coordinates of 0.56 A for the backbone atoms of residues 8-59. Comparison with the intact Antp(C39S) homeodomain shows that the two proteins have identical molecular architectures. The removal of the N-terminal residues 1-6, which are flexibly disordered in the intact homeodomain, causes only strictly localized structure variations and does not noticeably affect the adjoining helix I from residues 10-21. The DNA-binding constant of des(1-6)Antp(C39S) is approximately 10-fold reduced relative to the intact Antp(C39S) homeodomain, which can now be attributed to the absence of the previously reported contacts of the N-terminal polypeptide segment of the intact Antp(C39S) homeodomain with the minor groove of the DNA duplex.
Brenda J Brizuela - One of the best experts on this subject based on the ideXlab platform.
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the drosophila homeotic gene moira regulates expression of engrailed and hom genes in imaginal tissues
Mechanisms of Development, 1997Co-Authors: Brenda J Brizuela, James A KennisonAbstract:moira is a member of the trithorax group of homeotic gene regulators in Drosophila melanogaster. We show that moira is required for the function of multiple homeotic genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal tissues and that the requirement for moira function is at the level of transcription. moira is also required for transcription of the engrailed segmentation gene in the imaginal wing disc. The abnormalities caused by the loss of moira function in germ cells suggests that at least one other target gene requires moira for normal oogenesis.
Roland Brock - One of the best experts on this subject based on the ideXlab platform.
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A comprehensive model for the cellular uptake of cationic cell-penetrating peptides
Traffic, 2007Co-Authors: Falk Duchardt, Mariola Fotin-mleczek, Heinz Schwarz, Rainer Fischer, Roland BrockAbstract:The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so-called cell-penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis-independent cellular entry. In this study, we provide evidence that the Antennapedia-homeodomain-derived Antennapedia (Antp) peptide, nona-arginine and the HIV-1 Tat-protein-derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis-independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant-negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.