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1.
Development ; 150(4)2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36786333

RESUMO

The first mitotic division of the initial cell is a key event in all multicellular organisms and is associated with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular generations: the sporophyte and the gametophyte. Each generation deploys a distinct developmental programme autonomously from an initial cell, the first cell division of which sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first cell division and subsequent failure to produce basal structures during both generations. BAS encodes a type B″ regulatory subunit of protein phosphatase 2A (PP2A), and transcriptomic analysis identified potential effector genes that may be involved in determining basal cell fate. The bas mutant phenotype is very similar to that observed in distag (dis) mutants, which lack a functional Tubulin-binding co-factor Cd1 (TBCCd1) protein, indicating that TBCCd1 and PP2A are two essential components of the cellular machinery that regulates the first cell division and mediates basal cell fate determination.


Assuntos
Phaeophyceae , Proteína Fosfatase 2 , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Mutação/genética , Perfilação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Phaeophyceae/genética , Phaeophyceae/metabolismo
2.
New Phytol ; 231(5): 2077-2091, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34076889

RESUMO

Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology, but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes. Here, we report that mutations at specific target sites are generated following the introduction of CRISPR-Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method. Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE (APT) gene were isolated following treatment with 2-fluoroadenine, and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism. The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.


Assuntos
Sistemas CRISPR-Cas , Phaeophyceae , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eucariotos , Técnicas de Inativação de Genes , Phaeophyceae/genética
3.
J Phycol ; 57(3): 742-753, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33432598

RESUMO

The haploid-diploid life cycle of the filamentous brown alga Ectocarpus involves alternation between two independent and morphologically distinct multicellular generations, the sporophyte and the gametophyte. Deployment of the sporophyte developmental program requires two TALE homeodomain transcription factors OUROBOROS and SAMSARA. In addition, the sporophyte generation has been shown to secrete a diffusible factor that can induce uni-spores to switch from the gametophyte to the sporophyte developmental program. Here, we determine optimal conditions for production, storage, and detection of this diffusible factor and show that it is a heat-resistant, high molecular weight molecule. Based on a combined approach involving proteomic analysis of sporophyte-conditioned medium and the use of biochemical tools to characterize arabinogalactan proteins, we present evidence that sporophyte-conditioned medium contains AGP epitopes and suggest that the diffusible factor may belong to this family of glycoproteins.


Assuntos
Células Germinativas Vegetais , Phaeophyceae , Haploidia , Plantas , Proteômica
4.
Mol Biol Evol ; 36(12): 2778-2789, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504759

RESUMO

Most eukaryotes inherit their mitochondria from only one of their parents. When there are different sexes, it is almost always the maternal mitochondria that are transmitted. Indeed, maternal uniparental inheritance has been reported for the brown alga Ectocarpus but we show in this study that different strains of Ectocarpus can exhibit different patterns of inheritance: Ectocarpus siliculosus strains showed maternal uniparental inheritance, as expected, but crosses using different Ectocarpus species 7 strains exhibited either paternal uniparental inheritance or an unusual pattern of transmission where progeny inherited either maternal or paternal mitochondria, but not both. A possible correlation between the pattern of mitochondrial inheritance and male gamete parthenogenesis was investigated. Moreover, in contrast to observations in the green lineage, we did not detect any change in the pattern of mitochondrial inheritance in mutant strains affected in life cycle progression. Finally, an analysis of field-isolated strains provided evidence of mitochondrial genome recombination in both Ectocarpus species.


Assuntos
Genes Mitocondriais , Phaeophyceae/genética , Genoma Mitocondrial , Características de História de Vida , Partenogênese/genética , Recombinação Genética
5.
Development ; 144(3): 409-418, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28049657

RESUMO

The sporophyte generation of the brown alga Ectocarpus sp. exhibits an unusual pattern of development compared with the majority of brown algae. The first cell division is symmetrical and the apical-basal axis is established late in development. In the immediate upright (imm) mutant, the initial cell undergoes an asymmetric division to immediately establish the apical-basal axis. We provide evidence which suggests that this phenotype corresponds to the ancestral state of the sporophyte. The IMM gene encodes a protein of unknown function that contains a repeated motif also found in the EsV-1-7 gene of the Ectocarpus virus EsV-1. Brown algae possess large families of EsV-1-7 domain genes but these genes are rare in other stramenopiles, suggesting that the expansion of this family might have been linked with the emergence of multicellular complexity. EsV-1-7 domain genes have a patchy distribution across eukaryotic supergroups and occur in several viral genomes, suggesting possible horizontal transfer during eukaryote evolution.


Assuntos
Proteínas de Algas/genética , Phaeophyceae/genética , Proteínas de Algas/antagonistas & inibidores , Proteínas de Algas/química , Sequência de Aminoácidos , Clonagem Molecular , Cisteína/química , Evolução Molecular , Perfilação da Expressão Gênica , Transferência Genética Horizontal , Modelos Genéticos , Família Multigênica , Mutação , Phaeophyceae/crescimento & desenvolvimento , Phaeophyceae/virologia , Filogenia , Interferência de RNA , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética
6.
Plant Cell ; 29(12): 3102-3122, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29208703

RESUMO

Brown algae are one of the most developmentally complex groups within the eukaryotes. As in many land plants and animals, their main body axis is established early in development, when the initial cell gives rise to two daughter cells that have apical and basal identities, equivalent to shoot and root identities in land plants, respectively. We show here that mutations in the Ectocarpus DISTAG (DIS) gene lead to loss of basal structures during both the gametophyte and the sporophyte generations. Several abnormalities were observed in the germinating initial cell in dis mutants, including increased cell size, disorganization of the Golgi apparatus, disruption of the microtubule network, and aberrant positioning of the nucleus. DIS encodes a TBCCd1 protein, which has a role in internal cell organization in animals, Chlamydomonas reinhardtii, and trypanosomes. Our study highlights the key role of subcellular events within the germinating initial cell in the determination of apical/basal cell identities in a brown alga and emphasizes the remarkable functional conservation of TBCCd1 in regulating internal cell organization across extremely distant eukaryotic groups.


Assuntos
Proteínas de Algas/metabolismo , Linhagem da Célula , Phaeophyceae/citologia , Sequência de Bases , Núcleo Celular/metabolismo , Tamanho Celular , Sequência Conservada , Flagelos/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Mutação/genética , Phaeophyceae/genética , Phaeophyceae/ultraestrutura , Filogenia , Transcriptoma/genética
7.
Nature ; 465(7298): 617-21, 2010 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-20520714

RESUMO

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.


Assuntos
Proteínas de Algas/genética , Evolução Biológica , Genoma/genética , Phaeophyceae/citologia , Phaeophyceae/genética , Animais , Eucariotos , Evolução Molecular , Dados de Sequência Molecular , Phaeophyceae/metabolismo , Filogenia , Pigmentos Biológicos/biossíntese , Transdução de Sinais/genética
8.
New Phytol ; 197(2): 503-510, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23106314

RESUMO

The model brown alga Ectocarpus has a haploid-diploid life cycle, involving alternation between two independent multicellular generations, the gametophyte and the sporophyte. Recent work has shown that alternation of generations is not determined by ploidy but is rather under genetic control, involving at least one master regulatory locus, OUROBOROS (ORO). Using cell biology approaches combined with measurements of generation-specific transcript abundance we provide evidence that alternation of generations can also be regulated by non-cell autonomous mechanisms. The Ectocarpus sporophyte produces a diffusible factor that causes major developmental reprogramming in gametophyte cells. Cells become resistant to reprogramming when the cell wall is synthetized, suggesting that the cell wall may play a role in locking an individual into the developmental program that has been engaged. A functional ORO gene is necessary for the induction of the developmental switch. Our results highlight the role of the cell wall in maintaining the differentiated generation stage once the appropriate developmental program has been engaged and also indicate that ORO is a key member of the developmental pathway triggered by the sporophyte factor. Alternation between gametophyte and sporophyte generations in Ectocarpus is surprisingly labile, perhaps reflecting an adaptation to the variable seashore environment inhabited by this alga.


Assuntos
Modelos Biológicos , Phaeophyceae/citologia , Phaeophyceae/crescimento & desenvolvimento , Benzenossulfonatos/metabolismo , Meios de Cultivo Condicionados/farmacologia , Loci Gênicos/genética , Células Germinativas Vegetais/citologia , Células Germinativas Vegetais/efeitos dos fármacos , Células Germinativas Vegetais/crescimento & desenvolvimento , Phaeophyceae/efeitos dos fármacos , Phaeophyceae/genética , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , Regeneração/efeitos dos fármacos
9.
Genome Biol Evol ; 15(7)2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37481260

RESUMO

Macroalgal (seaweed) genomic resources are generally lacking as compared with other eukaryotic taxa, and this is particularly true in the red algae (Rhodophyta). Understanding red algal genomes is critical to understanding eukaryotic evolution given that red algal genes are spread across eukaryotic lineages from secondary endosymbiosis and red algae diverged early in the Archaeplastids. The Gracilariales is a highly diverse and widely distributed order including species that can serve as ecosystem engineers in intertidal habitats and several notorious introduced species. The genus Gracilaria is cultivated worldwide, in part for its production of agar and other bioactive compounds with downstream pharmaceutical and industrial applications. This genus is also emerging as a model for algal evolutionary ecology. Here, we report new whole-genome assemblies for two species (Gracilaria chilensis and Gracilaria gracilis), a draft genome assembly of Gracilaria caudata, and genome annotation of the previously published Gracilaria vermiculophylla genome. To facilitate accessibility and comparative analysis, we integrated these data in a newly created web-based portal dedicated to red algal genomics (https://rhodoexplorer.sb-roscoff.fr). These genomes will provide a resource for understanding algal biology and, more broadly, eukaryotic evolution.


Assuntos
Gracilaria , Rodófitas , Gracilaria/genética , Ecossistema , Rodófitas/genética , Genômica , Genoma
10.
Methods Protoc ; 5(3)2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35645344

RESUMO

The brown algae are an important but understudied group of multicellular marine organisms. A number of genetic and genomic tools have been developed for the model brown alga Ectocarpus; this includes, most recently, chromatin immunoprecipitation methodology, which allows genome-wide detection and analysis of histone post-translational modifications. Post-translational modifications of histone molecules have been shown to play an important role in gene regulation in organisms from other major eukaryotic lineages, and this methodology will therefore be a very useful tool to investigate genome function in the brown algae. This article provides a detailed, step-by-step description of the Ectocarpus ChIP protocol, which effectively addresses the difficult problem of efficiently extracting chromatin from cells protected by a highly resistant cell wall. The protocol described here will be an essential tool for the future application of chromatin analysis methodologies in brown algal research.

11.
Plant Physiol ; 153(1): 128-44, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20200071

RESUMO

Ectocarpus siliculosus is a small brown alga that has recently been developed as a genetic model. Its thallus is filamentous, initially organized as a main primary filament composed of elongated cells and round cells, from which branches differentiate. Modeling of its early development suggests the involvement of very local positional information mediated by cell-cell recognition. However, this model also indicates that an additional mechanism is required to ensure proper organization of the branching pattern. In this paper, we show that auxin indole-3-acetic acid (IAA) is detectable in mature E. siliculosus organisms and that it is present mainly at the apices of the filaments in the early stages of development. An in silico survey of auxin biosynthesis, conjugation, response, and transport genes showed that mainly IAA biosynthesis genes from land plants have homologs in the E. siliculosus genome. In addition, application of exogenous auxins and 2,3,5-triiodobenzoic acid had different effects depending on the developmental stage of the organism, and we propose a model in which auxin is involved in the negative control of progression in the developmental program. Furthermore, we identified an auxin-inducible gene called EsGRP1 from a small-scale microarray experiment and showed that its expression in a series of morphogenetic mutants was positively correlated with both their elongated-to-round cell ratio and their progression in the developmental program. Altogether, these data suggest that IAA is used by the brown alga Ectocarpus to relay cell-cell positional information and induces a signaling pathway different from that known in land plants.


Assuntos
Ácidos Indolacéticos/metabolismo , Morfogênese , Phaeophyceae/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Mutação , Phaeophyceae/genética , Phaeophyceae/crescimento & desenvolvimento , Transdução de Sinais
12.
Genome Biol ; 22(1): 12, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397407

RESUMO

BACKGROUND: Brown algae evolved complex multicellularity independently of the animal and land plant lineages and are the third most developmentally complex phylogenetic group on the planet. An understanding of developmental processes in this group is expected to provide important insights into the evolutionary events necessary for the emergence of complex multicellularity. Here, we focus on mechanisms of epigenetic regulation involving post-translational modifications of histone proteins. RESULTS: A total of 47 histone post-translational modifications are identified, including a novel mark H2AZR38me1, but Ectocarpus lacks both H3K27me3 and the major polycomb complexes. ChIP-seq identifies modifications associated with transcription start sites and gene bodies of active genes and with transposons. H3K79me2 exhibits an unusual pattern, often marking large genomic regions spanning several genes. Transcription start sites of closely spaced, divergently transcribed gene pairs share a common nucleosome-depleted region and exhibit shared histone modification peaks. Overall, patterns of histone modifications are stable through the life cycle. Analysis of histone modifications at generation-biased genes identifies a correlation between the presence of specific chromatin marks and the level of gene expression. CONCLUSIONS: The overview of histone post-translational modifications in the brown alga presented here will provide a foundation for future studies aimed at understanding the role of chromatin modifications in the regulation of brown algal genomes.


Assuntos
Código das Histonas , Histonas , Estágios do Ciclo de Vida , Phaeophyceae/genética , Processamento de Proteína Pós-Traducional , Cromatina/metabolismo , Epigênese Genética , Genoma , Células Germinativas Vegetais , Phaeophyceae/fisiologia , Filogenia , Plantas/genética
13.
New Phytol ; 188(1): 82-97, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20618907

RESUMO

• Brown algal cell walls share some components with plants (cellulose) and animals (sulfated fucans), but they also contain some unique polysaccharides (alginates). Analysis of the Ectocarpus genome provides a unique opportunity to decipher the molecular bases of these crucial metabolisms. • An extensive bioinformatic census of the enzymes potentially involved in the biogenesis and remodeling of cellulose, alginate and fucans was performed, and completed by phylogenetic analyses of key enzymes. • The routes for the biosynthesis of cellulose, alginates and sulfated fucans were reconstructed. Surprisingly, known families of cellulases, expansins and alginate lyases are absent in Ectocarpus, suggesting the existence of novel mechanisms and/or proteins for cell wall expansion in brown algae. • Altogether, our data depict a complex evolutionary history for the main components of brown algal cell walls. Cellulose synthesis was inherited from the ancestral red algal endosymbiont, whereas the terminal steps for alginate biosynthesis were acquired by horizontal gene transfer from an Actinobacterium. This horizontal gene transfer event also contributed genes for hemicellulose biosynthesis. By contrast, the biosynthetic route for sulfated fucans is an ancestral pathway, conserved with animals. These findings shine a new light on the origin and evolution of cell wall polysaccharides in other Eukaryotes.


Assuntos
Parede Celular/metabolismo , Evolução Molecular , Matriz Extracelular/metabolismo , Phaeophyceae/genética , Phaeophyceae/metabolismo , Polissacarídeos/metabolismo , Alginatos/metabolismo , Vias Biossintéticas , Celulose/biossíntese , Phaeophyceae/enzimologia , Filogenia , Polissacarídeos/biossíntese
14.
New Phytol ; 188(1): 67-81, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20618908

RESUMO

• Brown algae exhibit a unique carbon (C) storage metabolism. The photoassimilate D-fructose 6-phosphate is not used to produce sucrose but is converted into D-mannitol. These seaweeds also store C as ß-1,3-glucan (laminarin), thus markedly departing from most living organisms, which use α-1,4-glucans (glycogen or starch). • Using a combination of bioinformatic and phylogenetic approaches, we identified the candidate genes for the enzymes involved in C storage in the genome of the brown alga Ectocarpus siliculosus and traced their evolutionary origins. • Ectocarpus possesses a complete set of enzymes for synthesis of mannitol, laminarin and trehalose. By contrast, the pathways for sucrose, starch and glycogen are completely absent. • The synthesis of ß-1,3-glucans appears to be a very ancient eukaryotic pathway. Brown algae inherited the trehalose pathway from the red algal progenitor of phaeoplasts, while the mannitol pathway was acquired by lateral gene transfer from Actinobacteria. The starch metabolism of the red algal endosymbiont was entirely lost in the ancestor of Stramenopiles. In light of these novel findings we question the validity of the 'Chromalveolate hypothesis'.


Assuntos
Metabolismo dos Carboidratos/genética , Carbono/metabolismo , Evolução Molecular , Phaeophyceae/metabolismo , Ciclo do Carbono/genética , Genoma/genética , Phaeophyceae/enzimologia , Filogenia , Amido/metabolismo , Simbiose
15.
Bio Protoc ; 10(18): e3753, 2020 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-33659412

RESUMO

The brown alga Ectocarpus has a haploid-diploid life cycle that involves alternation between two multicellular generations, the sporophyte and the gametophyte. Life cycle generation is not determined by ploidy but by a genetic system that includes two different three amino acid loop extension homeodomain transcription factors called OUROBOROS and SAMSARA. In addition, sporophytes have been shown to secrete a diffusible factor into the medium that can induce gametophyte initial cells to switch from the gametophyte to the sporophyte developmental program. The protocol presented here describes how to produce sporophyte-conditioned medium containing the diffusible sporophyte-inducing factor and how to assay for activity of the factor using a meio-spore-based bioassay. The protocol, which describes how several steps of these procedures can be optimised, will represent a useful tool for future work aimed at characterising the diffusible factor and investigating its mode of action.

16.
Elife ; 82019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30644818

RESUMO

Three amino acid loop extension homeodomain transcription factors (TALE HD TFs) act as life cycle regulators in green algae and land plants. In mosses these regulators are required for the deployment of the sporophyte developmental program. We demonstrate that mutations in either of two TALE HD TF genes, OUROBOROS or SAMSARA, in the brown alga Ectocarpus result in conversion of the sporophyte generation into a gametophyte. The OUROBOROS and SAMSARA proteins heterodimerise in a similar manner to TALE HD TF life cycle regulators in the green lineage. These observations demonstrate that TALE-HD-TF-based life cycle regulation systems have an extremely ancient origin, and that these systems have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata.


Assuntos
Embriófitas/crescimento & desenvolvimento , Embriófitas/metabolismo , Proteínas de Homeodomínio/metabolismo , Phaeophyceae/crescimento & desenvolvimento , Phaeophyceae/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Embriófitas/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Mutação/genética , Phaeophyceae/genética , Fenótipo , Ligação Proteica , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
17.
Front Genet ; 5: 241, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25120558

RESUMO

Rhizobiales and related orders of Alphaproteobacteria comprise several genera of nodule-inducing symbiotic bacteria associated with plant roots. Here we describe the genome and the metabolic network of "Candidatus Phaeomarinobacter ectocarpi" Ec32, a member of a new candidate genus closely related to Rhizobiales and found in association with cultures of the filamentous brown algal model Ectocarpus. The "Ca. P. ectocarpi" genome encodes numerous metabolic pathways that may be relevant for this bacterium to interact with algae. Notably, it possesses a large set of glycoside hydrolases and transporters, which may serve to process and assimilate algal metabolites. It also harbors several proteins likely to be involved in the synthesis of algal hormones such as auxins and cytokinins, as well as the vitamins pyridoxine, biotin, and thiamine. As of today, "Ca. P. ectocarpi" has not been successfully cultured, and identical 16S rDNA sequences have been found exclusively associated with Ectocarpus. However, related sequences (≥97% identity) have also been detected free-living and in a Fucus vesiculosus microbiome barcoding project, indicating that the candidate genus "Phaeomarinobacter" may comprise several species, which may colonize different niches.

18.
Cold Spring Harb Protoc ; 2012(3): 361-4, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22383637

RESUMO

This article describes how to obtain isolated cells with no surrounding cell wall by enzymatic digestion of Ectocarpus filaments. The resultant protoplasts are totipotent and regenerate to produce individual algae under appropriate culture conditions. The yield of protoplasts and their capacity to regenerate are highly dependent on the Ectocarpus strain used, the stage of the life cycle, and the culture conditions. The highest yields are obtained with young gametophyte filaments cultivated at low density. The naked, wall-less cells produced by this protocol can be used for several applications, including studies of cell wall regeneration, investigation of the role of the cell wall in determining cell fate, and as a source of naked cells for the development of methods for introducing diverse molecules into the cell.


Assuntos
Técnicas Citológicas/métodos , Phaeophyceae/citologia , Protoplastos/fisiologia
19.
Cold Spring Harb Protoc ; 2012(3): 365-8, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22383638

RESUMO

For some applications, such as genome sequencing and high-throughput genotyping with multiple markers, it is necessary to use high-quality genomic DNA. This article describes how to obtain several micrograms of high-quality, cesium chloride-purified DNA from 1 g of Ectocarpus filaments. We also recommend using DNA of this quality for quantitative RT-PCR control reactions. However, simpler, more rapid, kit-based methods are preferable for experiments that involve the treatment of large numbers of individuals, such as genotyping large populations with a small number of markers or PCR screening of large populations.


Assuntos
DNA/isolamento & purificação , Biologia Molecular/métodos , Phaeophyceae/genética , Tipagem Molecular , Análise de Sequência de DNA
20.
Cold Spring Harb Protoc ; 2012(3): 369-72, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22383639

RESUMO

This article describes an immunostaining protocol for Ectocarpus that was optimized for the detection of tubulin but could be used with any suitable antibody. Ectocarpus has small but relatively transparent cells and the uniseriate filaments can be grown directly attached to the surface of microscope slides. These features make Ectocarpus particularly suitable for high resolution imaging approaches, both in vivo or after fixation. All incubations described below are carried out on a platform shaker at room temperature. Use high-quality microscope slides to avoid imperfections in the glass that can be a problem for confocal laserscan microscopy analysis.


Assuntos
Imuno-Histoquímica/métodos , Phaeophyceae/citologia , Coloração e Rotulagem/métodos , Actinas/imunologia , Anticorpos/imunologia
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