PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 2559446

Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy.

S Narayanswami·K Lundgren·B A Hamkalo
View original source ↗Export metadata ↓

Abstract

Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridization with a biotinylated DNA probe followed by detection with a primary antibiotin antibody and a secondary antibody coupled to colloidal gold. Hybridization probes can also be labelled with alternative ligands such as N-acetoxy-2-acetylaminofluorene (AAF), Dinitrophenyl-dUTP and Digoxigenin-dUTP. Multiple labelling is possible if these differently modified DNA probes are used in conjunction with colloidal gold preparations of varying particle sizes. A substantial signal amplification can be achieved by incubating preparations with successive cycles of primary antibiotin antibody followed by a biotinylated secondary antibody. Detection is with Streptavidin-gold, and in the case of highly and moderately repeated sequences, the signal is visible in the light microscope. Detailed protocols are given for EM in-situ hybridization to whole mount metaphase chromosomes and include instructions necessary to perform multiple sequence localization and signal amplification.

Explore related subjects

AnimalsCells, CulturedChromosome MappingChromosomesDNAGold Colloid, RadioactiveHumansHybridization, GeneticMetaphaseMiceMicroscopy, ElectronRabbitsRepetitive Sequences, Nucleic AcidXenopus laevisJournal ArticleResearch Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

S Narayanswami, K Lundgren, B A Hamkalo. 1989. Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy.. https://pubmed.ncbi.nlm.nih.gov/2559446/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2027

Protocol for Detecting and Sequencing Chikungunya Virus from Field-Collected Mosquitoes.

Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.

Animals↗
PubMed2027

Genomic Profiling of Chromatin State Using CUT&Tag.

Alterations in chromatin state, mediated through histone modifications and the incorporation of histone variants, are fundamental to establishing transcriptional networks and cell identity. Recent advances in low-input epigenome profiling methods, such as CUT&Tag and CUT&RUN, have enabled the study of chromatin states from very limited starting materials. In this chapter, we describe procedures for generating CUT&Tag libraries to profile histone modifications and histone variants in early-developing zebrafish embryos.

Animals↗
PubMed2026

Relaxin-2: Shaping the Proteomic Landscape of Skeletal Muscle Physiology, Glucose Trafficking, and Mitochondrial Function in Rat.

Relaxin-2 is a hormone with robust beneficial effects on the heart and blood vessels and potential as a therapy for cardiovascular (CV) disease. Considering the interorgan communication between skeletal muscle and heart, and the relation between muscle quality/composition and CV events, we hypothesize that relaxin-2 may regulate skeletal muscle physiology and metabolism. We aim to evaluate the impact of relaxin-2 on the proteome of skeletal muscle from healthy Sprague-Dawley rats. Animals were treated with 0.4 mg/kg/day of serelaxin (recombinant form of human relaxin-2) or vehicle (PBS) for 2 weeks employing subcutaneous osmotic minipumps. Skeletal muscle protein identification and quantification were performed by LC-MS/MS using a Data-Independent Acquisition (DIA)-Sequential Window Acquisition of All Theoretical Fragment Ion Spectra (SWATH) method. SWATH/MS quantitative analysis identified that relaxin-2 significantly decreased 95 proteins and significantly increased 32 proteins in rat skeletal muscle when compared to control rats. From these, 34 proteins were associated with muscle function, myogenesis, muscle differentiation and/or regeneration, 20 are mitochondrial proteins (six from the complexes of the electron transport chain), and 10 proteins participate in glucose metabolism. Qualitative data-dependent workflow analysis identified 35 proteins exclusive to the skeletal muscle of the relaxin-2-treated group: eight proteins related to processes of skeletal muscle function (size, ion homeostasis or organization of caveolae structures and cytoskeleton) and myogenesis, and two proteins involved in muscle differentiation. Our work highlighted for the first time the role of relaxin-2 in crucial processes of muscle physiology and energetic metabolism, which could influence several processes involved in myopathy and CV.

Animals↗