All eukaryotic genomes are packaged into basic units of DNA wrapped around histone proteins called nucleosomes. The ability of histones to specify a variety of epigenetic states at defined chromatin domains is essential for cell survival. The most distinctive type of chromatin is found at centromeres, which are marked by the centromere-specific histone H3 variant CENP-A. Many of the factors that regulate CENP-A chromatin have been identified; however, our understanding of the mechanisms of centromeric nucleosome assembly, maintenance, and reorganization remains limited. This review discusses recent insights into these processes and draws parallels between centromeric and noncentromeric chromatin assembly mechanisms.
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Reconstitution and Purification of Nucleosomes with Recombinant Histones and Purified DNA
Nucleosomes are substrates for a broad range of factors, including those involved in transcription or chromosome maintenance/reorganization and enzymes that covalently modify histones. Given the heterogeneous nature of nucleosomes in vivo (i.e., varying histone composition, post‐translational modifications, DNA sequence register), understanding the specificity and activities of chromatin‐interacting factors has required in vitro studies using well‐defined nucleosome substrates. Here, we provide detailed methods for large‐scale PCR preparation of DNA, assembly of nucleosomes from purified DNA and histones, and purification of DNA and mononucleosomes. Such production of well‐defined nucleosomes for biochemical and biophysical studies is key for studying numerous proteins and protein complexes that bind and/or alter nucleosomes and for revealing inherent characteristics of nucleosomes. © 2020 Wiley Periodicals LLC.
- NSF-PAR ID:
- 10238449
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Molecular Biology
- Volume:
- 133
- Issue:
- 1
- ISSN:
- 1934-3639
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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This article was corrected on 26 June 2021. See the end of the full text for details.
Basic Protocol 1 : Total RNA isolation from camelid leukocytesBasic Protocol 2 : First‐strand cDNA synthesis; VHH and VHrepertoire PCRBasic Protocol 3 : Preparation of the phage display libraryBasic Protocol 4 : Panning of the phage display libraryBasic Protocol 5 : Small‐scale nAb expressionBasic Protocol 6 : Sequence analysis of selected nAb clonesBasic Protocol 7 : Nanobody validation as immunolabelsBasic Protocol 8 : Generation of nAb‐pEGFP mammalian expression constructsBasic Protocol 9 : Nanobody validation as intrabodiesSupport Protocol 1 : ELISA for llama serum testing, phage titer, and screening of selected clonesSupport Protocol 2 : Amplification of helper phage stockSupport Protocol 3 : nAb expression in amber suppressorE. coli bacterial strains -
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Abstract Genome editing technologies have revolutionized genetic studies in the life sciences community in recent years. The application of these technologies allows researchers to conveniently generate mutations in almost any gene of interest. This is very useful for species such as maize that have complex genomes and lack comprehensive mutant collections. With the improvement of genome editing tools and transformation methods, these technologies are also widely used to assist breeding research and implementation in maize. However, the detection and genotyping of genomic edits rely on low‐throughput, high‐cost methods, such as traditional agarose gel electrophoresis and Sanger sequencing. This article describes a method to barcode the target regions of genomic edits from many individuals by low‐cost polymerase chain reaction (PCR) amplification. It also employs next‐generation sequencing (NGS) to genotype the genome‐edited plants at high throughput and low cost. This protocol can be used for initial screening of genomic edits as well as derived population genotyping on a small or large scale, at high efficiency and low cost. © 2021 Wiley Periodicals LLC.
Basic Protocol 1 : A fast genomic DNA preparation method from genome edited plantsBasic Protocol 2 : Barcoding the amplicons of edited regions from each individual by two rounds of PCRBasic Protocol 3 : Bioinformatics analysis
