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[PMID]:28456689
[Au] Autor:Mandic A; Strebinger D; Regali C; Phillips NE; Suter DM
[Ad] Endereço:The Institute of Bioengineering (IBI), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland.
[Ti] Título:A novel method for quantitative measurements of gene expression in single living cells.
[So] Source:Methods;120:65-75, 2017 May 01.
[Is] ISSN:1095-9130
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.
[Mh] Termos MeSH primário: Imagem Molecular/métodos
Proteínas/genética
Análise de Célula Única/métodos
Coloração e Rotulagem/métodos
Transcrição Genética
[Mh] Termos MeSH secundário: Animais
Linhagem Celular
Genes Reporter/genética
Vetores Genéticos/genética
Meia-Vida
Integrases/genética
Lentivirus/genética
Luminescência
Camundongos
Microscopia de Fluorescência/métodos
Imagem Molecular/instrumentação
Proteínas/química
Proteínas/metabolismo
Proteólise
RNA Mensageiro/genética
RNA Mensageiro/metabolismo
Análise de Célula Única/instrumentação
Coloração e Rotulagem/instrumentação
Imagem com Lapso de Tempo/instrumentação
Imagem com Lapso de Tempo/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Proteins); 0 (RNA, Messenger); EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180309
[Lr] Data última revisão:
180309
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170501
[St] Status:MEDLINE


  2 / 5862 MEDLINE  
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[PMID]:28454901
[Au] Autor:Wu S; Han J; Zhang X; Zhong D; Liu R
[Ad] Endereço:School of Electronic and Information Engineering, Xi'an Jiaotong University, Xi'an 710049, China.
[Ti] Título:A computational model for predicting integrase catalytic domain of retrovirus.
[So] Source:J Theor Biol;423:63-70, 2017 Jun 21.
[Is] ISSN:1095-8541
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Integrase catalytic domain (ICD) is an essential part in the retrovirus for integration reaction, which enables its newly synthesized DNA to be incorporated into the DNA of infected cells. Owing to the crucial role of ICD for the retroviral replication and the absence of an equivalent of integrase in host cells, it is comprehensible that ICD is a promising drug target for therapeutic intervention. However, annotated ICDs in UniProtKB database have still been insufficient for a good understanding of their statistical characteristics so far. Accordingly, it is of great importance to put forward a computational ICD model in this work to annotate these domains in the retroviruses. The proposed model then discovered 11,660 new putative ICDs after scanning sequences without ICD annotations. Subsequently in order to provide much confidence in ICD prediction, it was tested under different cross-validation methods, compared with other database search tools, and verified on independent datasets. Furthermore, an evolutionary analysis performed on the annotated ICDs of retroviruses revealed a tight connection between ICD and retroviral classification. All the datasets involved in this paper and the application software tool of this model can be available for free download at https://sourceforge.net/projects/icdtool/files/?source=navbar.
[Mh] Termos MeSH primário: Domínio Catalítico
Biologia Computacional
Evolução Molecular
Integrases/química
Retroviridae/classificação
Análise de Sequência de Proteína
[Mh] Termos MeSH secundário: Simulação por Computador
Bases de Dados de Proteínas
Anotação de Sequência Molecular
Software
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180305
[Lr] Data última revisão:
180305
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170430
[St] Status:MEDLINE


  3 / 5862 MEDLINE  
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[PMID]:29315333
[Au] Autor:Kang Q; Sun Z; Zou Z; Wang M; Li Q; Hu X; Li N
[Ad] Endereço:State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
[Ti] Título:Cell-penetrating peptide-driven Cre recombination in porcine primary cells and generation of marker-free pigs.
[So] Source:PLoS One;13(1):e0190690, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Cell-penetrating peptides (CPPs) have been increasingly used to deliver various molecules, both in vitro and in vivo. However, there are no reports of CPPs being used in porcine fetal fibroblasts (PFFs). The increased use of transgenic pigs for basic research and biomedical applications depends on the availability of technologies for efficient genetic-modification of PFFs. Here, we report that three CPPs (CPP5, TAT, and R9) can efficiently deliver active Cre recombinase protein into PFFs via an energy-dependent endocytosis pathway. The three CPP-Cre proteins can enter PFFs and subsequently perform recombination with different efficiencies. The recombination efficacy of CPP5-Cre was found to be nearly 90%. The rate-limiting step for CPP-Cre-mediated recombination was the step of endosome escape. HA2 and chloroquine were found to improve the recombination efficiency of TAT-Cre. Furthermore, we successfully obtained marker-free transgenic pigs using TAT-Cre and CPP5-Cre. We provide a framework for the development of CPP-based farm animal transgenic technologies that would be beneficial to agriculture and biomedicine.
[Mh] Termos MeSH primário: Peptídeos Penetradores de Células/metabolismo
Integrases/metabolismo
Recombinação Genética
[Mh] Termos MeSH secundário: Animais
Animais Geneticamente Modificados
Biomarcadores/metabolismo
Células Cultivadas
Transporte Proteico
Suínos
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Biomarkers); 0 (Cell-Penetrating Peptides); EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180215
[Lr] Data última revisão:
180215
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180110
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0190690


  4 / 5862 MEDLINE  
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[PMID]:29232693
[Au] Autor:Rai SK; Sangesland M; Lee M; Esnault C; Cui Y; Chatterjee AG; Levin HL
[Ad] Endereço:Section on Eukaryotic Transposable Elements, Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (NIH), Bethesda, Maryland, United States of America.
[Ti] Título:Host factors that promote retrotransposon integration are similar in distantly related eukaryotes.
[So] Source:PLoS Genet;13(12):e1006775, 2017 12.
[Is] ISSN:1553-7404
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.
[Mh] Termos MeSH primário: Fatores Hospedeiros de Integração/genética
Recombinação Genética
Retroelementos/genética
Sequências Repetidas Terminais/genética
Ubiquitina-Proteína Ligases/genética
[Mh] Termos MeSH secundário: Reparo do DNA/genética
Eucariotos/genética
Regulação Fúngica da Expressão Gênica
Integrases/genética
Regiões Promotoras Genéticas
DNA Polimerase Dirigida por RNA/genética
Retroviridae/genética
Saccharomyces cerevisiae/genética
Proteínas de Saccharomyces cerevisiae/genética
Schizosaccharomyces/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL
[Nm] Nome de substância:
0 (Integration Host Factors); 0 (Retroelements); 0 (Saccharomyces cerevisiae Proteins); 0 (Spp382 protein, S cerevisiae); EC 2.3.2.27 (Rhp18 protein, S pombe); EC 2.3.2.27 (Ubiquitin-Protein Ligases); EC 2.7.7.- (Integrases); EC 2.7.7.49 (RNA-Directed DNA Polymerase); EC 2.7.7.49 (reverse transcriptase Ty3, S cerevisiae)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180111
[Lr] Data última revisão:
180111
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171213
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pgen.1006775


  5 / 5862 MEDLINE  
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[PMID]:28455376
[Au] Autor:Tavares ALP; Cox TC; Maxson RM; Ford HL; Clouthier DE
[Ad] Endereço:Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
[Ti] Título:Negative regulation of endothelin signaling by SIX1 is required for proper maxillary development.
[So] Source:Development;144(11):2021-2031, 2017 06 01.
[Is] ISSN:1477-9129
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Jaw morphogenesis is a complex event mediated by inductive signals that establish and maintain the distinct developmental domains required for formation of hinged jaws, the defining feature of gnathostomes. The mandibular portion of pharyngeal arch 1 is patterned dorsally by Jagged-Notch signaling and ventrally by endothelin receptor A (EDNRA) signaling. Loss of EDNRA signaling disrupts normal ventral gene expression, the result of which is homeotic transformation of the mandible into a maxilla-like structure. However, loss of Jagged-Notch signaling does not result in significant changes in maxillary development. Here we show in mouse that the transcription factor SIX1 regulates dorsal arch development not only by inducing dorsal expression but also by inhibiting endothelin 1 ( ) expression in the pharyngeal endoderm of the dorsal arch, thus preventing dorsal EDNRA signaling. In the absence of SIX1, but not JAG1, aberrant EDNRA signaling in the dorsal domain results in partial duplication of the mandible. Together, our results illustrate that SIX1 is the central mediator of dorsal mandibular arch identity, thus ensuring separation of bone development between the upper and lower jaws.
[Mh] Termos MeSH primário: Endotelina-1/metabolismo
Proteínas de Homeodomínio/metabolismo
Maxila/embriologia
Maxila/metabolismo
Transdução de Sinais
[Mh] Termos MeSH secundário: Animais
Padronização Corporal/genética
Região Branquial/metabolismo
Anormalidades Craniofaciais/embriologia
Anormalidades Craniofaciais/genética
Anormalidades Craniofaciais/patologia
Embrião de Mamíferos/metabolismo
Regulação da Expressão Gênica no Desenvolvimento
Integrases/metabolismo
Camundongos
Modelos Biológicos
Crista Neural/metabolismo
Receptor de Endotelina A/metabolismo
Receptores Notch/metabolismo
Proteínas Serrate-Jagged/metabolismo
Fator de Transcrição Sp7
Fatores de Transcrição/genética
Fatores de Transcrição/metabolismo
Regulação para Cima/genética
Zigoma/embriologia
Zigoma/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Endothelin-1); 0 (Homeodomain Proteins); 0 (Receptor, Endothelin A); 0 (Receptors, Notch); 0 (Serrate-Jagged Proteins); 0 (Six1 protein, mouse); 0 (Sp7 Transcription Factor); 0 (Sp7 protein, mouse); 0 (Transcription Factors); EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:171126
[Lr] Data última revisão:
171126
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170430
[St] Status:MEDLINE
[do] DOI:10.1242/dev.145144


  6 / 5862 MEDLINE  
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[PMID]:29054407
[Au] Autor:Wang Z; Hou X; Wang Y; Xu A; Cao W; Liao M; Zhang R; Tang J
[Ad] Endereço:College of Veterinary Medicine, China Agricultural University, Beijing 100193, China; State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
[Ti] Título:Ubiquitination of non-lysine residues in the retroviral integrase.
[So] Source:Biochem Biophys Res Commun;494(1-2):57-62, 2017 Dec 09.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Retroviral integrase catalyzes the integration of retroviral genome into host chromosomal DNA, which is a prerequisite of effective viral replication and infection. The human immunodeficiency virus type 1 (HIV-1) integrase has previously been reported to be regulated by the ubiquitination, but the molecular characterization of integrase ubiquitination is still unclear. In this study, we analyzed the ubiquitination of avian leukosis virus (ALV) integrase in detail. The ubiquitination assay showed that, like HIV-1, ALV integrase could also be modified by ubiquitination when expressed in 293 T and DF-1 cells. Domain mapping analysis revealed that the ubiquitination of ALV integrase might mainly occurred in the catalytic core and the N-terminal zinc-binding domains. Both lysine and non-lysine residues within integrase of ALV and HIV-1 were responsible for the ubiquitin conjugation, and the N-terminal HHCC zinc-binding motif might play an important role in mediating integrase ubiquitination. Interestingly, mass spectrometry analysis identified the Thr10 and Cys37 residues in the HHCC zinc-binding motif as the ubiquitination sites, indicating that ubiquitin may be conjugated to ALV integrase through direct interaction with the non-lysine residues. These findings revealed the detailed features of retroviral integrase ubiquitination and found a novel mechanism of ubiquitination mediated by the non-lysine residues within the N-terminal zinc-binding domain of integrase.
[Mh] Termos MeSH primário: Vírus da Leucose Aviária/enzimologia
Integrase de HIV/química
Integrase de HIV/metabolismo
Integrases/química
Integrases/metabolismo
Proteínas dos Retroviridae/química
Proteínas dos Retroviridae/metabolismo
Retroviridae/enzimologia
[Mh] Termos MeSH secundário: Motivos de Aminoácidos
Sequência de Aminoácidos
Animais
Vírus da Leucose Aviária/genética
Vírus da Leucose Aviária/fisiologia
Linhagem Celular
Galinhas
Células HEK293
Integrase de HIV/genética
HIV-1/enzimologia
HIV-1/genética
HIV-1/fisiologia
Seres Humanos
Integrases/genética
Lisina/química
Mutagênese Sítio-Dirigida
Retroviridae/genética
Retroviridae/fisiologia
Proteínas dos Retroviridae/genética
Ubiquitinação
Zinco/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Retroviridae Proteins); 0 (p31 integrase protein, Human immunodeficiency virus 1); EC 2.7.7.- (HIV Integrase); EC 2.7.7.- (Integrases); J41CSQ7QDS (Zinc); K3Z4F929H6 (Lysine)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171113
[Lr] Data última revisão:
171113
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171022
[St] Status:MEDLINE


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[PMID]:28985409
[Au] Autor:Grieb MS; Nivina A; Cheeseman BL; Hartmann A; Mazel D; Schlierf M
[Ad] Endereço:B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstraße 18, 01307 Dresden, Germany.
[Ti] Título:Dynamic stepwise opening of integron attC DNA hairpins by SSB prevents toxicity and ensures functionality.
[So] Source:Nucleic Acids Res;45(18):10555-10563, 2017 Oct 13.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Biologically functional DNA hairpins are found in archaea, prokaryotes and eukaryotes, playing essential roles in various DNA transactions. However, during DNA replication, hairpin formation can stall the polymerase and is therefore prevented by the single-stranded DNA binding protein (SSB). Here, we address the question how hairpins maintain their functional secondary structure despite SSB's presence. As a model hairpin, we used the recombinogenic form of the attC site, essential for capturing antibiotic-resistance genes in the integrons of bacteria. We found that attC hairpins have a conserved high GC-content near their apical loop that creates a dynamic equilibrium between attC fully opened by SSB and a partially structured attC-6-SSB complex. This complex is recognized by the recombinase IntI, which extrudes the hairpin upon binding while displacing SSB. We anticipate that this intriguing regulation mechanism using a base pair distribution to balance hairpin structure formation and genetic stability is key to the dissemination of antibiotic resistance genes among bacteria and might be conserved among other functional hairpins.
[Mh] Termos MeSH primário: Sítios de Ligação Microbiológicos
DNA Bacteriano/química
DNA de Cadeia Simples
Proteínas de Ligação a DNA/metabolismo
Proteínas de Escherichia coli/metabolismo
Integrons
[Mh] Termos MeSH secundário: DNA Bacteriano/metabolismo
Integrases/metabolismo
Conformação de Ácido Nucleico
Ligação Proteica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Bacterial); 0 (DNA, Single-Stranded); 0 (DNA-Binding Proteins); 0 (Escherichia coli Proteins); 0 (SSB protein, E coli); EC 2.7.7.- (Integrases); EC 2.7.7.- (integron integrase IntI1)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171107
[Lr] Data última revisão:
171107
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171007
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx670


  8 / 5862 MEDLINE  
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[PMID]:28950225
[Au] Autor:Flynn CT; Kimura T; Frimpong-Boateng K; Harkins S; Whitton JL
[Ad] Endereço:Dept. of Immunology and Microbiology, SP30-2110, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
[Ti] Título:Immunological and pathological consequences of coxsackievirus RNA persistence in the heart.
[So] Source:Virology;512:104-112, 2017 Dec.
[Is] ISSN:1096-0341
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Type B coxsackieviruses (CVB) can cause myocarditis and dilated cardiomyopathy (DCM), a potentially-fatal sequela that has been correlated to the persistence of viral RNA. Herein, we demonstrate that cardiac RNA persistence can be established even after an inapparent primary infection. Using an inducible Cre/lox mouse model, we ask: (i) Does persistent CVB3 RNA cause ongoing immune activation? (ii) If T1IFN signaling into cardiomyocytes is ablated after RNA persistence is established, is there any change in the abundance of persistent CVB3 RNA and/or does cytopathic infectious virus re-emerge? (iii) Does this loss of T1IFN responsiveness by cardiomyocytes lead to the recurrence/exacerbation of myocarditis? Our findings suggest that persistent enteroviral RNAs probably do not contribute to ongoing myocardial disease, and are more likely to be the fading remnants of a recent, possibly sub-clinical, primary infection which may have set in motion the process that ultimately ends in DCM.
[Mh] Termos MeSH primário: Enterovirus/fisiologia
Miócitos Cardíacos/virologia
RNA Viral/fisiologia
[Mh] Termos MeSH secundário: Animais
Cardiomiopatia Dilatada/virologia
Infecções por Coxsackievirus/virologia
Ativação Enzimática/efeitos dos fármacos
Deleção de Genes
Regulação da Expressão Gênica
Integrases/metabolismo
Interferon Tipo I/genética
Interferon Tipo I/metabolismo
Camundongos
Camundongos Endogâmicos C57BL
Miocardite/virologia
Miócitos Cardíacos/metabolismo
RNA Mensageiro/genética
RNA Mensageiro/metabolismo
Tamoxifeno/farmacologia
Carga Viral
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Interferon Type I); 0 (RNA, Messenger); 0 (RNA, Viral); 094ZI81Y45 (Tamoxifen); EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171109
[Lr] Data última revisão:
171109
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170927
[St] Status:MEDLINE


  9 / 5862 MEDLINE  
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[PMID]:28941984
[Au] Autor:Schock EN; Brugmann SA
[Ad] Endereço:Division of Plastic Surgery, Department of Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA; Division of Developmental Biology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
[Ti] Título:Neural crest cells utilize primary cilia to regulate ventral forebrain morphogenesis via Hedgehog-dependent regulation of oriented cell division.
[So] Source:Dev Biol;431(2):168-178, 2017 11 15.
[Is] ISSN:1095-564X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Development of the brain directly influences the development of the face via both physical growth and Sonic hedgehog (SHH) activity; however, little is known about how neural crest cells (NCCs), the mesenchymal population that comprise the developing facial prominences, influence the development of the brain. We utilized the conditional ciliary mutant Wnt1-Cre;Kif3a to demonstrate that loss of primary cilia on NCCs resulted in a widened ventral forebrain. We found that neuroectodermal Shh expression, dorsal/ventral patterning, and amount of proliferation in the ventral neuroectoderm was not changed in Wnt1-Cre;Kif3a mutants; however, tissue polarity and directional cell division were disrupted. Furthermore, NCCs of Wnt1-Cre;Kif3a mutants failed to respond to a SHH signal emanating from the ventral forebrain. We were able to recapitulate the ventral forebrain phenotype by removing Smoothened from NCCs (Wnt1-Cre;Smo ) indicating that changes in the ventral forebrain were mediated through a Hedgehog-dependent mechanism. Together, these data suggest a novel, cilia-dependent mechanism for NCCs during forebrain development.
[Mh] Termos MeSH primário: Divisão Celular
Cílios/metabolismo
Proteínas Hedgehog/metabolismo
Morfogênese
Crista Neural/citologia
Prosencéfalo/citologia
Prosencéfalo/embriologia
[Mh] Termos MeSH secundário: Animais
Padronização Corporal/genética
Polaridade Celular
Face/embriologia
Regulação da Expressão Gênica no Desenvolvimento
Integrases/metabolismo
Cinesina/metabolismo
Camundongos
Modelos Biológicos
Morfogênese/genética
Mutação/genética
Crista Neural/metabolismo
Placa Neural/citologia
Placa Neural/embriologia
Placa Neural/metabolismo
Fenótipo
Prosencéfalo/metabolismo
Recombinação Genética/genética
Fatores de Transcrição SOXE/metabolismo
Telencéfalo/embriologia
Telencéfalo/metabolismo
Proteínas Wnt/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Hedgehog Proteins); 0 (Kif3a protein, mouse); 0 (SOXE Transcription Factors); 0 (Shh protein, mouse); 0 (Wnt Proteins); EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (Integrases); EC 3.6.4.4 (Kinesin)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171111
[Lr] Data última revisão:
171111
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170925
[St] Status:MEDLINE


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[PMID]:28934476
[Au] Autor:Meinke G; Karpinski J; Buchholz F; Bohm A
[Ad] Endereço:Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA.
[Ti] Título:Crystal structure of an engineered, HIV-specific recombinase for removal of integrated proviral DNA.
[So] Source:Nucleic Acids Res;45(16):9726-9740, 2017 Sep 19.
[Is] ISSN:1362-4962
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:As part of the HIV infection cycle, viral DNA inserts into the genome of host cells such that the integrated DNA encoding the viral proteins is flanked by long terminal repeat (LTR) regions from the retrovirus. In an effort to develop novel genome editing techniques that safely excise HIV provirus from cells, Tre, an engineered version of Cre recombinase, was designed to target a 34-bp sequence within the HIV-1 LTR (loxLTR). The sequence targeted by Tre lacks the symmetry present in loxP, the natural DNA substrate for Cre. We report here the crystal structure of a catalytically inactive (Y324F) mutant of this engineered Tre recombinase in complex with the loxLTR DNA substrate. We also report that 17 of the 19 amino acid changes relative to Cre contribute to the altered specificity, even though many of these residues do not contact the DNA directly. We hypothesize that some mutations increase the flexibility of the Cre tetramer and that this, along with flexibility in the DNA, enable the engineered enzyme and DNA substrate to adopt complementary conformations.
[Mh] Termos MeSH primário: Repetição Terminal Longa de HIV
HIV-1/genética
Integrases/metabolismo
Proteínas Recombinantes/química
Proteínas Recombinantes/metabolismo
[Mh] Termos MeSH secundário: Domínio Catalítico
Cristalografia por Raios X
DNA Viral/química
DNA Viral/metabolismo
Repetição Terminal Longa de HIV/genética
Integrases/química
Integrases/genética
Modelos Moleculares
Mutação
Conformação de Ácido Nucleico
Conformação Proteica
Engenharia de Proteínas/métodos
Proteínas Recombinantes/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (DNA, Viral); 0 (Recombinant Proteins); EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171024
[Lr] Data última revisão:
171024
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170922
[St] Status:MEDLINE
[do] DOI:10.1093/nar/gkx603



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