Saccharomyces cerevisiae (commonly known as baker’s yeast) is a single-celled eukaryote that is frequently used in scientific research. S. cerevisiae is an attractive model organism due to the fact that its genome has been sequenced, its genetics are easily manipulated, and it is very easy to maintain in the lab. Because many yeast proteins are similar in sequence and function to those found in other organisms, studies performed in yeast can help us to determine how a particular gene or protein functions in higher eukaryotes (including humans). This video provides an introduction to the biology of this model organism, how it was discovered, and why labs all over the world have selected it as their model of choice. Previous studies performed in S. cerevisiae that have contributed to our understanding of important cellular processes such as the cell cycle, aging, and cell death are also discussed. Finally, the video describes some of the many ways in which yeast cells are put to work in modern scientific research, including protein purification and the study of DNA repair mechanisms and other cellular processes related to Alzheimer’s and Parkinson’s diseases.…
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification. Plasmid purification includes three basic steps: growth of the bacterial culture, harvesting and lysis of the bacteria, and purification of the plasmid DNA. The video contains an explanation where the plasmid can be found in each step of the protocol and to quantitatively and qualitatively analyze plasmid DNA with a spectrophotometer and/or gel electrophoresis. There are different types of plasmid purification methods available, which are geared toward desired yield, plasmid copy number, and bacterial culture volume.…
The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a radiolabeled nucleic acid and test protein are mixed. Binding is determined via gel electrophoresis which separates components based on mass, charge, and conformation.
This video shows the concepts of EMSA and a general procedure, including gel and protein preparation, binding, electrophoresis, and detection. Applications covered in this video include the analysis of chromatin-remodeling enzymes, a modified EMSA that incorporates biontinylation, and the study of binding sites of bacterial response regulators. EMSA, the electrophoretic mobility shift assay, also known as the gel shift assay, is a versatile and sensitive biochemical procedure. EMSA elucidates binding between proteins and nucleic acids by detecting a shift in bands in gel electrophoresis. This video describes the principles of EMSA, provides a general procedure, and discusses some applications. DNA replication, transcription, and repair, as well as RNA processing are all critical biochemical processes. They all involve binding between proteins and nucleic acids. Many serious diseases and disorders are associated with modifications in this …
Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Alex Wassimi
Viruses are a unique group of biological entities that infect both eukaryotic and prokaryotic organisms. They are obligate parasites that have no metabolic capacity, and in order to replicate, rely on host metabolism to produce viral parts that self-assemble inside host cells.
Viruses are ultramicroscopic—too small to be viewed with the light microscope, visible only with the greater resolution of the electron microscope. A viral particle consists of a nucleic acid genome, either DNA or RNA, surrounded by a protein coat, known as a capsid, composed of protein subunits or capsomers. In some more complex viruses, the capsid is surrounded by an additional lipid envelope, and some have spike-like surface appendages or tails.
Viruses that infect the intestinal tract of humans and animals are known as enteric viruses. They are excreted in feces and can be isolated from domestic wastewater. Viruses which infect bacteria are known as bacteriophages, and those which infect coliform bacteria are called coliphages (Figure 1). The phages of coliform bacteria are found anywhere coliform bacteria are found.
Since the early days of genetics research, scientists have noted certain heritable phenotypic differences that are not due to differences in the nucleotide sequence of DNA. Current evidence suggests that these “epigenetic” phenomena might be controlled by a number of mechanisms, including the modification of DNA cytosine bases with methyl groups, the addition of various chemical groups to histone proteins, and the recruitment of protein factors to specific DNA sites via interactions with non-protein-coding RNAs.In this video, JoVE presents the history of important discoveries in epigenetics, such as X-chromosome inactivation (XCI), the phenomenon where an entire X-chromosome is silenced in the cells of female mammals. Key questions and methods in the field are reviewed, including techniques to identify DNA sequences associated with different epigenetic modifications. Finally, we discuss how researchers are currently using these techniques to better understand the epigenetic regulation of gene function.…
Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple stages, and any misregulation could lead to diseases such as cancer.
This video highlights important historical discoveries relating to gene expression, including the understanding of how distinct combinations of DNA bases encode the amino acids that make up proteins. Key questions in the field of gene expression research are explored, followed by a discussion of several techniques used to measure gene expression and investigate its regulation. Finally, we look at how scientists are currently using these techniques to study gene expression.…
Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells
1Philips Institute for Oral Health Research, Virginia Commonwealth University, 2Department of Microbiology and Immunology, Virginia Commonwealth University, 3Department of Biochemistry, Virginia Commonwealth University
Genetic engineering – the process of purposefully altering an organism’s DNA – has been used to create powerful research tools and model organisms, and has also seen many agricultural applications. However, in order to engineer traits to tackle complex agricultural problems such as stress tolerance, or to realize the promise of gene therapy for treating human diseases, further advances in the field are still needed. Important considerations include the safe and efficient delivery of genetic constructs into cells or organisms, and the establishment of the desired modification in an organism’s genome with the least “off-target” effects. JoVE’s Overview of Genetic Engineering will present a history of the field, highlighting the discoveries that confirmed DNA as the genetic material and led to the development of tools to modify DNA. Key questions that must be answered in order to improve the process of genetic engineering will then be introduced, along with various tools used by genetic engineers. Finally, we will survey several applications demonstrating the types of experimental questions and strategies in the field today.…
Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells
Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions
1Department of Rheumatology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 2Department of Nephrology and Rheumatology, Yongchuan Hospital of Chongqing Medical University, 3Department of Medical Oncology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University
An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness
1Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, 2Department of Pharmacology, College of Medicine, Chungnam National University, 3Department of Nursing, Gwangju Women's University
Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis
1Centre for Biospectroscopy, Monash University, 2Department of Microbiology, Faculty of Medicine, Nursing and Health Sciences, Monash University, 3Centre for Biomedical Research, Burnet Institute, 4Department of Medicine, University of Melbourne
1Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, 2Facultad de Medicina, Universidad Nacional Autónoma de México, 3Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro
Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)
1The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 2Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 3RCSI Molecular Medicine, Royal College of Surgeons in Ireland, 4Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, 5School of Medical Sciences, University of New South Wales, 6St Vincent's Hospital Clinical School, University of New South Wales, 7School of Medicine and Medical Science, University College Dublin
Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells
1Department of Oncohematology and Genetics. Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del Rocío, 2Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Member of the German Center for Lung Research, Justus-Liebig-University
A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast
1Instituto de Neurociencias de Castilla y León (INCYL), Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, 2Centre for Cancer Research & Cell Biology (CCRCB), School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast
Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes
High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials
1The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 2Department of Neurology and neurosurgery, Johns Hopkins University School of Medicine, 3Departments of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 4Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine
Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages
1Institute for Lung Research, Universities of Giessen and Marburg Lung Center, Philipps-University Marburg, 2German Center for Lung Research, 3Department of Medicine, Pulmonary and Critical Care Medicine, University Medical Center Giessen and Marburg
System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis
Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions
Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum
Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity