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Cold Spring Harbor Protocols

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https://www.readbyqxmd.com/read/28912375/organ-culture-of-the-xenopus-tadpole-intestine
#1
Atsuko Ishizuya-Oka
During Xenopus metamorphosis, most tadpole organs remodel from the larval to adult form to prepare for adaptation to terrestrial life. Organ culture serves as an important tool for studying larval-to-adult organ remodeling independent of the effects of other parts of the body. Here, I introduce a protocol for organ culture in vitro using the Xenopus laevis tadpole intestine before metamorphic climax. During culture in the absence of exogenous 3,3',5-triiodo-l-thyronine (T3), the most potent natural thyroid hormone, the intestine remains in its larval state without any metamorphic changes...
September 14, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28912374/collagen-embedded-tumor-transplantations-in-xenopus-laevis-tadpoles
#2
Maureen Banach, Jacques Robert
The Xenopus laevis tadpole provides a valuable model for studying tumorigenesis and tumor immunity by intravital real-time microscopy. Using well-characterized thymic lymphoid tumor lines (15/0 and ff-2) that are transplantable into their compatible hosts (LG-15 isogenic clones and the F inbred strain, respectively), a system of semisolid tumor engraftment has been designed. Because these lymphoid tumor cell lines are not adherent and grow in suspension, they are first immobilized in a matrix of type I rat tail collagen before transplantation as a semisolid tumor graft under the transparent dorsal skin in the head region of a tadpole...
September 14, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733418/a-simple-method-to-induce-meiosis-and-sporulation-semisynchronously-in-the-fission-yeast-schizosaccharomyces-pombe
#3
Akira Yamashita, Takeshi Sakuno, Yoshinori Watanabe, Masayuki Yamamoto
Schizosaccharomyces pombe cells initiate a sexual differentiation program, which comprises meiosis and spore formation, on nitrogen starvation. This protocol describes a simple procedure to induce meiosis and sporulation semisynchronously in heterozygous diploid S. pombe cells. The procedure is appropriate for a variety of applications, including fluorescence-activated cell sorting (FACS) and northern and western blotting. Zygotic meiosis can also be induced by the same procedure, although less synchronously...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733417/analysis-of-schizosaccharomyces-pombe-meiosis
#4
Akira Yamashita, Takeshi Sakuno, Yoshinori Watanabe, Masayuki Yamamoto
Meiosis is a specialized cell cycle that generates haploid gametes from diploid cells. The fission yeast Schizosaccharomyces pombe is one of the best model organisms for studying the regulatory mechanisms of meiosis. S. pombe cells, which normally grow in the haploid state, diploidize by conjugation and initiate meiosis when starved for nutrients, especially nitrogen. Following two rounds of chromosome segregation, spore formation takes place. The switch from mitosis to meiosis is controlled by a kinase, Pat1, and an RNA-binding protein, Mei2...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733416/genetic-interaction-screens-in-schizosaccharomyces-pombe-using-the-pombe-epistasis-mapper-pem-system-and-a-manual-colony-replicator
#5
Isabelle Colson, Edgar Hartsuiker
In laboratories in which a colony-replicating robot is not available, manual replication provides a good, low-cost alternative for genetic interaction screening using the Pombe Epistasis Mapper (PEM) system. The protocol presented here describes the minimum number of steps required to identify genetic interactions. First, a query deletion is introduced to a library of deletion mutants by mating. Through a series of subsequent selection steps, single and double mutants are isolated and analyzed.
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733415/introduction-to-fission-yeast-as-a-model-system
#6
Jacqueline Hayles, Paul Nurse
Here, we briefly outline the history of fission yeast, its life cycle, and aspects of its biology that make it a useful model organism for studying problems of eukaryotic molecular and cell biology.
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733414/live-cell-imaging-in-fission-yeast
#7
Daniel P Mulvihill
Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast.
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733413/live-cell-imaging-of-the-schizosaccharomyces-pombe-sexual-life-cycle
#8
Laura Merlini, Aleksandar Vjestica, Omaya Dudin, Felipe Bendezú, Sophie G Martin
The fission yeast Schizosaccharomyces pombe is an invaluable model system for studying the principles that drive sexual differentiation and the meiotic cell division cycle. We describe a simple protocol for microscopic observation of the entire sexual life cycle that can be adapted to focus on specific stages of sexual differentiation. After growth to exponential phase in a nitrogen-rich medium, cell cultures are switched to a nitrogen-deprived medium until the population is enriched for the specific stage of the sexual lifecycle to be studied...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733412/schizosaccharomyces-japonicus-a-distinct-dimorphic-yeast-among-the-fission-yeasts
#9
Keita Aoki, Kanji Furuya, Hironori Niki
Genomic sequencing data and morphological properties demonstrate evolutionary relationships among groups of the fission yeast, Schizosaccharomyces Phylogenetically, S. japonicus is the furthest removed from other species of fission yeast. The basic characteristics of cell proliferation are shared among all fission yeast, including the process of binary fission during vegetative growth, conjugation and karyogamy with horsetail movement, mating-type switching, and sporulation. However, S. japonicus also exhibits characteristics that are unique to filamentous fungi...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733411/dna-preparation-from-schizosaccharomyces-pombe
#10
Assen Roguev, Jiewei Xu, Nevan Krogan
This protocol includes two methods for genomic DNA preparation from Schizosaccharomyces pombe The first is a quick method for preparation of DNA suitable for polymerase chain reaction (PCR) genotyping. The second, longer method yields high-quality DNA that can be used for amplification of targeting cassettes.
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733410/radioactive-labeling-and-fractionation-of-fission-yeast-walls
#11
Pilar Pérez, Juan C Ribas
Fungal cells contain an essential structure external to the cell, made of polysaccharides and proteins, termed the cell wall. Polysaccharides represent ∼96% of the cell wall on a dry-weight basis. They are complex insoluble polymers connected to each other by covalent linkages and hydrogen bonds with specific localizations in the cell wall and septum. Fission yeast contains three β-glucans (a major branched β(1,3)-glucan, a minor linear β(1,3)-glucan, and a minor branched β(1,6)-glucan), two α-glucans (a major α(1,3)-glucan and a minor α(1,4)-glucan), and a minor amount of galactomannan-linked glycoproteins...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733409/using-pulsed-field-gel-electrophoresis-to-analyze-schizosaccharomyces-pombe-chromosomes-and-chromosomal-elements
#12
Chen-Chun Pai, Carol Walker, Timothy Humphrey
Pulsed field gel electrophoresis (PFGE) uses alternatively oriented pulsed electrical fields to separate large DNA molecules. Here, we describe PFGE protocols and conditions for separating and visualizing chromosomes between 0.5 and 6 Mb (optimal for analyzing the endogenous fission yeast chromosomes of 5.7, 4.6, and 3.5 Mb), and for shorter chromosomal elements of between 50 and 600 kb, such as the 530 kb Ch(16) minichromosome. In addition to determining chromosome size, this technique has a wide range of applications, including determining whether DNA replication or repair is complete, defining the molecular karyotype of cells, analyzing chromosomal rearrangements, assigning genes or constructs to particular chromosomes, and isolating DNA from specific chromosomes...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733408/dna-double-strand-break-repair-assay
#13
Chen-Chun Pai, Elizabeth Blaikley, Timothy C Humphrey
DNA double-strand breaks (DSBs), arising during normal DNA metabolism or following exposure to mutagenic agents such as ionizing radiation can lead to chromosomal rearrangements and genome instability, and are potentially lethal if unrepaired. Therefore, understanding the mechanisms of DSB repair and misrepair, and identifying the factors involved in these processes is of biological as well as medical interest. Here we describe a DSB assay in Schizosaccharomyces pombe that can be used to identify and quantify different repair, misrepair, and failed repair events resulting from a site-specific DSB within the context of a nonessential minichromosome, Ch(16) This assay can be used to determine the contribution of most genes or genetic backgrounds to DSB repair and genome stability, and can also provide mechanistic insights into their function...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733407/fission-yeast-cell-wall-analysis
#14
Pilar Pérez, Juan C Ribas
The Schizosaccharomyces pombe cell wall is a rigid exoskeletal structure mainly composed of interlinked glucose polysaccharides and galactomannoproteins. It is essential for survival of the fission yeast, as it prevents cells from bursting from internal turgor pressure and protects them from mechanical injuries. Additionally, the cell wall determines the cell shape and, therefore, a better knowledge of cell wall structure and composition could provide valuable data in S. pombe morphogenetic studies. Here, we provide information about this structure and the current reliable methods for rapid analysis of the cell wall polymers by specific enzymatic and chemical degradations of purified cell walls...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733406/genetic-interaction-score-s-score-calculation-clustering-and-visualization-of-genetic-interaction-profiles-for-yeast
#15
Assen Roguev, Colm J Ryan, Jiewei Xu, Isabelle Colson, Edgar Hartsuiker, Nevan Krogan
This protocol describes computational analysis of genetic interaction screens, ranging from data capture (plate imaging) to downstream analyses. Plate imaging approaches using both digital camera and office flatbed scanners are included, along with a protocol for the extraction of colony size measurements from the resulting images. A commonly used genetic interaction scoring method, calculation of the S-score, is discussed. These methods require minimal computer skills, but some familiarity with MATLAB and Linux/Unix is a plus...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733405/analysis-of-dna-replication-in-fission-yeast-by-combing
#16
Divya R Iyer, Shankar Das, Nick Rhind
DNA replication studies based on population experiments give an average estimate of replication kinetics from many cells. This average replication profile masks the stochastic nature of origin firing in eukaryotes, which is revealed by using single-molecule techniques, such as DNA combing. The analysis of replication kinetics by DNA combing involves isolating DNA from cells that have been pulse-labeled with thymidine analogs and stretching it on a silanized coverslip. The analog-labeled patches on the stretched DNA fibers can then be detected using fluorescent antibodies against the analog...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733404/high-throughput-quantitative-genetic-interaction-mapping-in-the-fission-yeast-schizosaccharomyces-pombe
#17
Assen Roguev, Colm J Ryan, Edgar Hartsuiker, Nevan J Krogan
Epistasis mapping, in which the phenotype that emerges from combining pairs of mutations is measured quantitatively, is a powerful tool for unbiased study of gene function. When performed at a large scale, this approach has been used to assign function to previously uncharacterized genes, define functional modules and pathways, and study their cross talk. These experiments rely heavily on methods for rapid sampling of binary combinations of mutant alleles by systematic generation of a series of double mutants...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733403/transformation-of-schizosaccharomyces-japonicus
#18
Keita Aoki, Hironori Niki
This protocol describes the use of electroporation to transform Schizosaccharomyces japonicus with plasmids or linear DNA. Plasmids are helpful for the complementation testing of mutations and for the expression of specific genes. Linear DNA fragments integrated into chromosomal DNA by homologous recombination are useful for gene deletion or to fuse a gene with a tag sequence (e.g., encoding a fluorescent protein). To introduce DNA into S. japonicus, electroporation methods are recommended because S. japonicus is sensitive to lithium acetate (LiOAc)...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733402/analysis-of-dna-metabolism-in-fission-yeast
#19
Francisco Antequera, Timothy C Humphrey
The fission yeast Schizosaccharomyces pombe is an excellent model organism to study DNA metabolism, in which the DNA replication and repair mechanisms are evolutionarily conserved. In this introduction we describe a range of methods commonly used to study aspects of DNA metabolism in fission yeast, focusing on approaches used for the analysis of genome stability, DNA replication, and DNA repair. We describe the use of a minichromosome, Ch(16), for monitoring different aspects of genome stability. We introduce two-dimensional gel electrophoresis and immunofluorescent visualization of combed DNA molecules for the analysis of DNA replication...
July 21, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28733401/mating-spore-dissection-and-selection-of-diploid-cells-in-schizosaccharomyces-japonicus
#20
Kanji Furuya, Hironori Niki
Haploid yeast cells mate to form heterozygotes and subsequently undergo meiosis to form spores. This process can be used to produce gene combinations and variants that are useful for genetic analysis. For example, these spores can be used to generate double mutants or to measure genetic distances in a mutational analysis. Here, we describe mating and spore dissection procedures for Schizosaccharomyces japonicus cells. Although the overall procedures resemble those used in Schizosaccharomyces pombe, some differences exist, including the use of EMM2 medium without nitrogen (EMM-N) for mating and the shorter incubation time of 16-20 h for S...
July 21, 2017: Cold Spring Harbor Protocols
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