We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.

1. Preparação Chromosome <ol><li> A germinação das sementes e fixação de pontas de raiz <ol><li> 10-20 germinar sementes de cevada em duas camadas de papel de filtro húmido numa placa de Petri em condições escuras durante 2 dias a 22-24 ° C. Cortar raízes vigorosos com o comprimento de 1-2 cm a partir da semente, utilizando uma lâmina de barbear. </li><li> Prepare água gelada, colocando uma garrafa de vidro de 500 ml contendo água fria da torneira em esmagado gelo-água. Arejar a água gelada e mergulhe dicas de raiz durante 20 horas para aumentar a freqüência de células em metafase. </li><li> Transferir raízes a partir de água a 50 ml de etanol: ácido acético (3: 1) fixador para fixá-los à temperatura ambiente durante 2 dias. Armazenar em raízes recentemente preparada etanol: ácido acético (3: 1) fixador, a 4 ° C até à sua utilização até um ano. </li></ol></li><li> Lavar e tratamento enzima <ol><li> Lavam-se as raízes 10-20 com 30 ml de gelo-água fria da torneira durante 5 minutos duas vezes, utilizando uma proveta de vidro de 50 ml. Use microscópio binocular. Transferir raízes, um por um em30 ml de tampão 0,01 M de citrato (0,01 M de ácido cítrico + citrato de sódio 0,01 M, pH 4,8) usando uma pinça e lavar por agitação da proveta de vidro durante 5 min, duas vezes. Coloque raízes em papel de filtro para remover completamente o líquido e de corte do tecido meristemático não indesejada utilizando uma lâmina de barbear. </li><li> Incubar até 20 pontas de raízes em 1 ml de mistura de enzima a 37 ° C durante cerca de 50 minutos para amaciar o tecido da planta (Tabela 1) em um vidro de relógio. Mistura enzimática contém 0.7% celulase R10, 0.7% celulase, 1% e 1% pectolyase cytohelicase diluída em tampão de citrato 0,01 M. Armazenar a mistura da enzima, a -20 ° C e reutilizado até cinco vezes. </li><li> Remover a enzima por pipetagem e lavar as pontas de raiz em gelo com 5 ml de tampão de citrato 0,01 M, duas vezes para substituir a enzima residual. </li></ol></li><li> Maceração Root <ol><li> Pontas de raiz de lavagem com 1 ml de etanol a 96%, duas vezes cuidadosamente no mesmo vidro de relógio. Substituir etanol com fixador preparado de fresco (75% de ácido acético: etanol a 25%). Use 10-15fixador ul por ponta da raiz. </li><li> Pontas de raiz de transferência juntamente com o fixador para um tubo de 2 mL e se desintegram meristemas da raiz com uma agulha de dissecação ou fórceps. Toque o tubo 20 vezes para as células re-suspensão para se obter uma suspensão de células. Armazenar a suspensão de células a -20 ° C até dois meses. </li></ol></li><li> Deixando cair da suspensão de células <ol><li> Coloque 2-3 camadas de tecido de papel embebido em água em uma placa quente a 50 ° C. Imergir as lâminas microscópicas na água gelada da torneira na geladeira por 30 minutos. E lugar desliza em cima do lenço de papel úmido. </li><li> Pipeta 7-10 ul de suspensão celular e solte-o a uma distância de 20 cm para o slide arrefecida colocada sobre a placa quente. Pipeta 10 ml de mistura de ácido acético-etanol no mesmo lugar como suspensão de células no slide e manter a lâmina sobre a placa quente durante mais 2 min. Coloque o slide no prato quente sem o tecido molhado e deixe secar por 1 min. </li></ol></li><li> Controle de qualidade e de armazE de lâminas <ol><li> Verificar lâminas através de um microscópio de contraste de fase para controlar a qualidade da propagação cromossoma. Utilizar lâminas, quer no mesmo dia ou da loja por imersão em etanol a 96% em um frasco Coplin a -20 ° C. </li></ol></li><li> O pré-tratamento das lâminas antes FISH; todos os passos são realizados à TA <ol><li> Colocar as lâminas em um frasco Coplin contendo 50 ml de 2x SSC (20x SSC contém 3 M NaCl e 300 mM de citrato trissódico) por 5 min. Utilizando uma pinça, slides de transferência para uma jarra de Coplin contendo 50 ml de ácido acético 45% para 3-10 min. </li><li> Transferir as lâminas para um frasco Coplin contendo 50 ml de SSC 2x durante 10 min. Lâminas de transferência para uma jarra de Coplin contendo 50 ml de formaldeído 4% (em 2x SSC) e mergulhe as lâminas por 10 min para corrigir cromossomos. </li><li> Remover o formaldeído por lavagem das lâminas 3 vezes durante 4 min cada, num frasco Coplin contendo 50 ml de SSC 2X. Desidratar slides em uma jarra de Coplin por 2 min em séries de 70%, 90% e 100% de etanol, respectivamente, e lâminas secas no plano verticalposição. </li></ol></li></ol> 2. fluorescente hibridação in situ (FISH) <ol><li> Para cada lâmina, preparar uma solução de hibridação de 20 ul, no total, utilizando 10 ul de formamida desionizada, 5 uL de tampão de 4x hibridação (ul de tampão 200 contém 80 ul de SSC 20x, 8 ul de 1 M de Tris-HCl pH 8,0, 1,6 mL de 0,5 M EDTA, 11,2 ul 10 mg / mL de esperma de salmão e 99,2 ul DNase-livre água), 3 ul da sonda e 2 mL de água livre de DNase. </li><li> Adicionar 20 ml de solução de hibridação por lâmina e cobrir com uma lamela de 24 x 32 mm e prender a lamínula com cimento de borracha. Desnaturar lâminas com sondas simultaneamente a 80 ° C durante 2 minutos numa placa de aquecimento. </li><li> Transferir as lâminas para uma câmara húmida e incubar a 37 ° CO / N evitando a luz. Remover lamelas enxaguando os slides em uma jarra de Coplin com 2x SSC. Colocar as lâminas em um frasco Coplin contendo 55-60 ° C 2x SSC e incubar por 20 min. </li><li> Colocar as lâminas 2x SSC em um frasco Coplin durante 2 min à temperatura ambiente. Desidratar lâminas em um frasco Coplin durante 2 min em série de 70%, 90% e etanol a 100%, respectivamente. </li><li> Secar as lâminas e contrastante com 1 mg / ml 4 &#39;, 6-diamidino-2-phenylindoline (DAPI) em antifade meio de montagem, evitar a luz intensa. </li></ol> 3. A análise microscópica e armazenamento <ol><li> Analisar os slides usando um microscópio de epifluorescência. A seleção do filtro depende do fluorocromo utilizado para a rotulagem sonda. Se necessário, armazenar as lâminas a 4 ° C, no escuro, até um ano. </li></ol>

<ol>
	<li>Geber, G., Schweizer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+heterochromatin+differentiation+in+Sinapis+alba+(Cruciferae)+using+a+simple+air-drying+technique+for+producing+chromosome+spreads.">Cytochemical heterochromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads.</a> Pl Syst Evol. 158 (2-4), 97-106 (1988).</li><li>Andras, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drop-spreading+technique+to+produce+cytoplasm-free+mitotic+preparations+from+plants+with+small+chromosomes.">A drop-spreading technique to produce cytoplasm-free mitotic preparations from plants with small chromosomes.</a> Chromosome Res. 7 (8), 641-647 (1999).</li><li>Rothfels, K. H., Siminovitch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+air-drying+technique+for+flattening+chromosomes+in+mammalian+cells+grown+in+vitro.">An air-drying technique for flattening chromosomes in mammalian cells grown in vitro.</a> Stain Technology. 33 (2), 73-77 (1958).</li><li>Martin, R., Busch, W., Herrmann, R. G., Wanner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+preparation+of+plant+chromosomes+for+high-resolution+scanning+electron+microscopy.">Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy.</a> Chromosome Res. 2 (5), 411-415 (1994).</li><li>Kato, A., Albert, P. S., Vega, J. M., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+fluorescence+in+situ+hybridization+signal+detection+in+maize+using+directly+labeled+probes+produced+by+high+concentration+DNA+polymerase+nick+translation.">Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation.</a> Biotech. Histochem. 81 (2-3), 71-78 (2006).</li><li>Kirov, I., Divashuk, M., Van Laere, K., Soloviev, A., Khrustaleva, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy+'SteamDrop'+method+for+high+quality+plant+chromosome+preparation.">An easy 'SteamDrop' method for high quality plant chromosome preparation.</a> Mol. Cytogenet. 7, 21 (2014).</li><li>Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., Jalal, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+chromosome+spreading.">Dynamics of chromosome spreading.</a> Am J Med Genet. 61 (4), 387-393 (1996).</li><li>Ma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synteny+between+Brachypodium+distachyon+and+Hordeum+vulgare+as+revealed+by+FISH.">Synteny between Brachypodium distachyon and Hordeum vulgare as revealed by FISH.</a> Chromosome Res. 18 (7), 841-850 (2010).</li><li>Kato, A., Lamb, J. C., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+painting+using+repetitive+DNA+sequences+as+probes+for+somatic+chromosome+identification+in+maize.">Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize.</a> Proc. Natl. Acad. Sci. U S A. 101 (37), 13554-13559 (2004).</li><li>Pan, W. H., Houben, A., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+effective+cell+synchronization+in+plant-roots+by+hydroxyurea+and+amiprophos-methyl+or+colchicine.">Highly effective cell synchronization in plant-roots by hydroxyurea and amiprophos-methyl or colchicine.</a> Genome. 36 (2), 387-390 (1993).</li><li>Kim, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+karyotyping+of+sorghum+by+in+situ+hybridization+of+landed+BACs.">Integrated karyotyping of sorghum by in situ hybridization of landed BACs.</a> Genome. 45 (2), 402-412 (2002).</li><li>Lapitan, N. L. V., Brown, S. E., Kennard, W., Stephens, J. L., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+physical+mapping+with+barley+BAC+clones.">FISH physical mapping with barley BAC clones.</a> Plant J. 11 (1), 149-156 (1997).</li><li>Aliyeva-Schnorr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+mapping+with+centromeric+BAC+contigs+shows+that+this+recombination-poor+region+comprises+more+than+half+of+barley+chromosome+3H.">Cytogenetic mapping with centromeric BAC contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H.</a> Plant J. 84, 385-394 (2015).</li></ol>

The authors have nothing to disclose.

O experimento cromossoma preparação foi levada a cabo usando raízes novas de cereais pertencentes à família das gramíneas (Poaceae). Todas as espécies analisadas têm 14 relativamente longo mitóticas cromossomos metafásicos (11-15 um) no conjunto de genoma diplóide e pertencem a espécies de grande genoma (5,1-7,9 GBP). Comprimento de raízes germinadas não era mais do que 2 cm, para se obter um máximo de tecido meristemático. Sincronização de células em divisão foi atingido...

Hibridização fluorescente in situ (FISH) é uma ferramenta eficaz para o mapeamento físico de sequências de cópia única e de alta no nível cromossômico. Pré-requisito é a preparação dos spreads de cromossomos de alta qualidade. Não existe um protocolo de preparação geral descrito cromossoma que seriam igualmente adequados para células animais e vegetais. Preparação dos cromossomas de plantas é particularmente difícil devido à rígida parede celular e vários consistência citoplasma dentro de espécies diferentes. Um dos métodos mais favoráveis ​​para a preparação dos cromossomas de plantas é uma assim chamada técnica de gota também conhecida como técnica de espalhamento gota e a técnica de secagem ao ar 1,2. Este método foi introduzido pela primeira vez em 1958 por Rothfels e Siminovitch in vitro para células de mamíferos cultivadas 3. Mais tarde, Martin et al. 4 e Kato et al. 5 este método adaptado para plantas. Mais recentemente, um método chamado &#39;&amp; SteamDrop# 39; foi desenvolvido o qual utilizado vapor de água para a preparação de cromossomas que não se sobrepõem 6. Embora, a influência positiva da alta umidade foi observado anteriormente 7, &#39;SteamDrop&#39; oferece um fluxo de trabalho controlado de preparações cromossômicas de alta qualidade 6. O tratamento de vapor faz com que o alongamento de cromossomas, provavelmente ligados a algumas modificações de proteínas cromossómicas. A qualidade dos resultando metáfase é muito elevado, apesar de retenção de número suficiente de metaphase completa espalha para experimentos FISH subsequentes exige conhecimentos técnicos. Aqui é apresentado um protocolo para a preparação dos cromossomas de cereais mitóticas adequados para a detecção de sondas FISH individuais e elevado número de cópias 5,8. Este método é uma variante melhorada do método de gotejamento, seca ao ar descrito por Kato 9 realizada sob uma humidade relativa de 50% -55% (Figura 1). Este protocolo compreende um número reduzidode etapas de lavagem fazendo sua fácil aplicação, eficiente e reprodutível. Usando este protocolo obtivemos os spreads de cromossomos de alta qualidade e os resultados peixe para Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum e Secale cereale.

<table><tbody><tr><td>Hot Plate</td><td>MEDAX GmbH</td><td>12603</td><td></td></tr><tr><td>Cellulase R10</td><td>Duchefa</td><td>C8001</td><td></td></tr><tr><td>Cellulase&amp;nbsp;</td><td>CalBioChem</td><td>219466</td><td></td></tr><tr><td>Pectolyase</td><td>Sigma</td><td>P3026</td><td></td></tr><tr><td>Cytohelicase</td><td>Sigma</td><td>C8274</td><td></td></tr><tr><td>Texas Red-12-dUTP</td><td>Invitrogen</td><td>C3176</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluor488-5-dUTP</td><td>Invitrogen</td><td>C11397</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluorecsence microscope</td><td>Olympus BX61</td><td>BX61</td><td></td></tr><tr><td>CCD camera</td><td>Orca ER, Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1200</td><td>fluorecsent dye</td></tr></tbody></table>

a fast air-dry dropping chromosome preparation method suitable for fish in plants

Preparação dos spreads de cromossomos é um pré-requisito para o bom desempenho de hibridização fluorescente in situ (FISH). Preparação de alta qualidade para barrar planta cromossômicas é desafiador devido à parede celular rígida. Um dos métodos aprovados para a preparação dos cromossomas de plantas é uma preparação gota chamada, também conhecido como espalhando-gota ou técnica de secagem ao ar. Aqui, apresentamos um protocolo para a preparação rápida do cromossomo mitótico espalha adequado para a detecção de sondas de DNA FISH individuais e elevado número de cópias. Este método é uma variante melhorada do método de gota, seca ao ar realizada sob uma humidade relativa de 50% -55%. Este protocolo compreende um número reduzido de etapas de lavagem fazendo sua fácil aplicação, eficiente e reprodutível. Óbvios benefícios desta abordagem são bem-propagação, cromossomos metafásicos não danificadas e numerosos que servem como pré-requisito perfeito para análise FISH bem sucedido. Usando este protocolo obtivemos chrom de alta qualidadeosome spreads e resultados PEIXES reprodutíveis para Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum e Secale cereale.

Lâminas microscópicas com os diferenciais de mitose em metafase foram preparados pelo método de preparação rápida, seca ao ar de gotejamento cromossoma descrito acima (Supl. Figura 1). Análise dos peixes foi realizada utilizando ambos, seqüências repetitivas e de cópia única. As imagens foram obtidas através de um microscópio de epifluorescência, com um conjunto de filtros que permitem excitação de fluoróforos correspondentes e captada por uma câmara CCD monocromática de alta sensibil...

Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Um cromossomo Dropping Ar seco rápido Método de Preparação Adequado para FISH em plantas

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

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Universidad de Cartagena UDEC

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Biology

13.1K visualizações.  Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Vídeo: A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Sincronismo de replicação de DNA usando Zebrafish como um sistema modelo In Vivo de criação de perfil

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genética

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Preparação do Cromossomo Meioético Se espalha de espermatocitocitos de zebrafish

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Co de expressão de várias proteínas da fusão fluorescente quimérico de forma eficiente em plantas

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Química

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Um método de alta taxa de transferência para Zebrafish criopreservação de espermatozóides e In Vitro Fertilização

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Biologia

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Preparação de Abóboras Drosophila cromossomos politênicos para Rotulagem de anticorpos

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Desvendando a função de uma bacteriana Effector de um patógeno não-cultiváveis ​​meio de uma levedura Screen Two-hybrid

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Imunologia e Infecção

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Tempo cromossomo Replicando Combinado com Fluorescente<em&gt; Em situ</em&gt; Hibridização

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Método simples para DNA de Fluorescência<em&gt; In Situ</em&gt; A hibridação com Cromossomos Squashed

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Preparação de organismos procariotas e eucariotas, utilizando secagem química para análise morfológica em microscopia eletrônica (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

Um cromossomo Dropping Ar seco rápido Método de Preparação Adequado para FISH em plantas

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Desvendando a função de uma bacteriana Effector de um patógeno não-cultiváveis ​​meio de uma levedura Screen Two-hybrid

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Método simples para DNA de Fluorescência

Preparação de organismos procariotas e eucariotas, utilizando secagem química para análise morfológica em microscopia eletrônica (SEM)

Hibridização in situ fluorescente em preparações de halo de DNA para revelar cromossomos inteiros, telômeros e loci gênicos

Desvendando a função de uma bacteriana Effector de um patógeno não-cultiváveis meio de uma levedura Screen Two-hybrid