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丁香实验推荐阅读
Defining Pre-mRNA cis Elements that Regulate Cell-Specific Splicing

A large number of genes express multiple mRNAs that encode diverse protein isoforms via alternative pre-mRNA splicing. For many genes, alternative splicing is regulated according to cell-specific patterns (in this chapter, cell-specific is used as a general term to refer to regulation a ...

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A Yeast Spliceosome Assay

The spliceosome is a large ribonucleoprotein that splices precursor messenger RNA (pre-mRNA) in the nuclei of eukaryotic cells (reviewed in refs. 1–3). It is composed of five small nuclear ribonucleoproteins (snRNPs) and numerous non-snRNP proteins. Each snRNP consists of a small nucle ...

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Prespliceosome and Spliceosome Isolation and Analysis

Spliceosomes are multicomponent enzymes that remove introns from premessenger RNAs (pre-mRNAs) (1–3) in the reaction known as pre-mRNA splicing. Spliceosomes are ribonucleoprotein (RNP) machines consisting of both RNA and protein components. SnRNPs, composed of small nuclear ...

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Yeast Pre-mRNA Splicing Extracts

Splicing of eukaryotic precursor messenger RNAs (pre-mRNAs) excises the intron from the precursor and ligates the two exons together to produce the mature mRNA. It occurs via a two-step mechanism (Fig. 1) (reviewed in ref. 1). In the first step the 2′ hydroxyl group of an intronic adenylyl residue init ...

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Mammalian In Vitro Splicing Assays

Splicing reactions are typically carried out using nuclear extracts, S100 extracts complemented with SR proteins, or partially purified fractions derived from the crude extracts. The extract preparation procedures are described in Chapter 24. Extracts derived from HeLa cells are ...

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Preparation of Hela Cell Nuclear and Cytosolic S100 Extracts for In Vitro Splicing

Following the initial discovery of split genes in 1977, it took several years before in vitro systems were successfully developed to study the biochemistry of pre-mRNA splicing. The first systems relied on coupling of transcription and splicing in whole-cell extracts and were fairly inef ...

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In Vivo SELEX in Vertebrate Cells

Iterative selection strategies have been widely used to enrich specific RNA molecules from randomized pools based on binding affinities or an RNA-mediated activity (6,8). The vast majority of these procedures have been performed in cell-free systems. Of particular use would be iterati ...

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Gel Electrophoresis Assays for Analyzing DNA Double-Strand Breaks in Saccharomyces cerevisiae at Various Spatial Resolutions

Meiotic recombination is triggered by programmed DNA double-strand breaks (DSBs), which are catalyzed by Spo11 protein in a type II topoisomerase-like manner. Meiotic DSBs can be detected directly using physical assays (gel electrophoresis, Southern blotting, and indirect end-l ...

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Analysis of Meiotic Recombination Products from Human Sperm

Traditional methods for surveying meiotic recombination in humans are limited to pedigree and linkage disequilibrium analyses. We have developed assays that allow the direct detection of crossover and gene conversion molecules in batches of sperm DNA. To date, we have characterized ...

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Parallel Detection of Crossovers and Noncrossovers in Mouse Germ Cells

The recombination between homologous chromosomes during the prophase of the first meiotic division plays an essential role in the formation of euploid gametes, as well as contributing to genetic diversity through the generation of new allele combinations. Two types of products are for ...

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Genome-Wide High-Resolution Chromatin Immunoprecipitation of Meiotic Chromosomal Proteins in Saccharomyces cerevisiae

Cooperative actions of chromosomal proteins play critical roles in the dynamics, structural transition, segregation, and maintenance of meiotic chromosomes. A high-resolution genome-tiling array combined with a chromatin immunoprecipitation assay (ChIP-chip) is a powe ...

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Analysis of ProteinDNA Interactions During Meiosis by Quantitative Chromatin Immunoprecipitation (qChIP)

During meiotic prophase a number of important events require recombination between maternal and paternal chromosomes, which is initiated through the introduction of DNA double-strand breaks (DSBs). The majority of DSBs, which mostly occur at so-called hotspots, have been located b ...

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Analysis of Chromatin Structure at Meiotic DSB Sites in Yeasts

One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many ...

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Using Schizosaccharomyces pombe Meiosis to Analyze DNA Recombination Intermediates

The fission yeast Schizosaccharomyces pombe has many biological characteristics that make it an ideal model organism for the study of meiosis. A nearly synchronous meiosis is one of the most important. Under certain environmental and genetic conditions, large cultures of S. pombe can be in ...

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Stabilization and Electrophoretic Analysis of Meiotic Recombination Intermediates in Saccharomyces cerevisiae

Joint Molecule (JM) recombination intermediates result from DNA strand-exchange between homologous chromosomes. Physical monitoring of JM formation in budding yeast has provided a wealth of information about the timing and mechanism of meiotic recombination. These assays are ...

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Detection of SPO11-Oligonucleotide Complexes from Mouse Testes

The SPO11 protein generates programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination. Endonucleolytic cleavage 3′ to the DSB sites releases SPO11 from DNA, leaving SPO11 covalently associated with an oligonucleotide. This chapter describes detection of ...

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End-Labeling and Analysis of Spo11-Oligonucleotide Complexes in Saccharomyces cerevisiae

During meiosis Spo11 catalyzes the formation of DNA double-strand breaks, becoming covalently attached to the 5′ ends on both sides of the break during this process. Spo11 is removed from the DSB by single-stranded endonucleolytic cleavage flanking the DSB, liberating a short-lived spec ...

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Detection of Meiotic DNA Breaks in Mouse Testicular Germ Cells

The study of location and intensity of double-strand breaks (DSBs) in mammalian systems is more challenging than in yeast because, unlike yeast, the progression through meiosis is not synchronous and only a small fraction of all testis cells are actually at the stage where DSB formation is initi ...

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Genome-Wide Mapping of Meiotic DNA Double-Strand Breaks in Saccharomyces cerevisiae

DNA double-strand breaks (DSBs) initiate meiotic recombination in eukaryotes. We describe two strategies that use microarrays to determine the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) ...

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Visual Markers for Detecting Gene Conversion Directly in the Gametes of Arabidopsis thaliana

Measuring meiotic gene conversion is important both because of its role in the fundamental mechanisms of meiotic recombination and because of its influence on linkage relationships and allelic diversity in the genome. Historically, gene conversion has been most thoroughly examin ...

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