Transcriptional activation of genes within the nucleus of eukaryotic cells occurs by a variety of mechanisms. Typically, these mechanisms rely on the interaction of regulatory proteins (transcriptional activators or repressors) with specific DNA sequences that control gene expression. Upon DNA binding, regulatory proteins also interact with other proteins that are part of the RNA polymerase II transcriptional complex.
One type of transcriptional activation relies on inducing a conformational change in chromatin, the DNA-protein complex that makes up each chromosome within a cell. In a broad sense, “extended” or loosely wound chromatin is more accessible to transcription factors and can signify an actively transcribed gene. In contrast, “condensed” chromatin hinders access to transcription factors and is characteristic of a transcriptionally inactive state. Acetylation of lysine residues in histones—the primary constituents of the chromatin backbone—results in opening up the chromatin and consequent gene activation. Disruption of histone acetylation pathways is implicated in many types of cancer (1).
They existed 3.5 billion years before humans evolved on Earth. They’re neither dead nor alive. Their genetic material is embedded in our own DNA, constituting close to 10% of the human genome. They can attack most forms of life on our planet, from bacteria to plants to animals. And yet, if it wasn’t for them, humans might never have existed.
No, that’s not the blurb for a new Hollywood blockbuster, although recent developments have proven, once again, that truth is decidedly more bizarre than fiction. Now that “coronavirus” has become a household word, the level of interest in all things virus-related is growing at an unprecedented rate. At the time of writing, coronavirus and COVID-19 topics dominated search traffic on Google, as well as trends on social media. A recent FAQ on this blog addresses many of the questions we hear on these topics.
Our cells have evolved multiple mechanisms for “taking out
the trash”—breaking down and disposing of cellular components that are defective,
damaged or no longer required. Within a cell, these processes are balanced by
the synthesis of new components, so that DNA, RNA and proteins are constantly
Proteins are degraded by two major components of the cellular
machinery. The discovery of the lysosome in the mid-1950s
provided considerable insight into the first of these degradation mechanisms
for extracellular and cytosolic proteins. Over the next several decades,
details of a second protein degradation mechanism emerged: the ubiquitin-proteasome system
(UPS). Ubiquitin is a small, highly conserved polypeptide that is used to
selectively tag proteins for degradation within the cell. Multiple ubiquitin
tags are generally attached to a single targeted protein. This ill-fated, ubiquitinated
protein is then recognized by the proteasome, a large protein complex with
proteolytic activity. Ubiquitination is a multistep process, involving several
specialized enzymes. The final step in the process is mediated by a family of ubiquitin
ligases, known as E3.
Understanding the expression, function and dynamics of
proteins in their native environment is a fundamental goal that’s common to
diverse aspects of molecular and cell biology. To study a protein, it must
first be labeled—either directly or indirectly—with a “tag” that allows
specific and sensitive detection.
Using a labeled antibody to the protein of interest is a
common method to study native proteins. However, antibody-based assays, such as
ELISAs and Western blots, are not suitable for use in live cells. These
techniques are also limited by throughput and sensitivity. Further, suitable
antibodies may not be available for the target protein of interest.
In the fall of 1989, a small group of forensic scientists, law enforcement officials and representatives from Promega Corporation gathered in Madison, Wisconsin, for the very first International Symposium on Human Identification (ISHI). At the time, DNA typing was in its infancy and had not yet been validated as a forensic method. The available technology consisted of two methods: detection of restriction fragment length polymorphisms (RFLPs) and variable number of tandem repeats (VNTRs). Promega had developed products based on both analytical methods, which essentially provide a DNA “fingerprint” or profile for each individual tested.
Among the attendees at that first symposium was Tom Callaghan, then a graduate student. That experience made a significant impact on his career path. Last week, at ISHI 30, he presented a session on rapid DNA testing. Dr. Callaghan currently serves as a Senior Biometric Scientist for the FBI. In 1999, he was instrumental in launching the FBI’s Combined DNA Index System (CODIS) and in 2003, he became the first CODIS Unit Chief.
G protein-coupled receptors (GPCRs) are a large family of receptors that traverse the cell membrane seven times. Functionally, GPCRs are extremely diverse, yet they contain highly conserved structural regions. GPCRs respond to a variety of signals, from small molecules to peptides and large proteins. Many GPCRs are involved in disease pathways and, not surprisingly, they present attractive targets for both small-molecule and biologic drugs.
In response to a signal, GPCRs undergo a conformational change, triggering an interaction with a G protein—a specialized protein that binds GDP in its inactive state or GTP when activated. Typically, the GPCR exchanges the G protein-bound GDP molecule for a GTP molecule, causing the activated G protein to dissociate into two subunits that remain anchored to the cell membrane. These subunits relay the signal to various other proteins that interact with or produce second-messenger molecules. Activation of a single G protein can result, ultimately, in the generation of thousands of second messengers.
For over a decade, obesity has been called an “epidemic”, both in the popular and scientific literature. Traditionally, the term “epidemic” is associated with a highly contagious disease that carries with it a significant risk of mortality. A comprehensive review of observational studies (1) suggested that obesity did not fit this definition, despite the use of the term in a widely disseminated report by the World Health Organization in 2002.
Regardless of the etymological fine points, the worldwide prevalence of obesity and its associated health risks are clear. These risks include type 2 diabetes, hypertension, several cancers, gall bladder disease, coronary artery disease and stroke (2). Yet, the debate over obesity and options for reducing its risks has become increasingly polarized. As a result, some health researchers are advocating a “health at every size” (HAES) approach to address the social, cultural and lifestyle implications of obesity (2).
No protein is an island. Within a cell, protein-protein interactions (PPIs) are involved in highly regulated and specific pathways that control gene expression and cell signaling. The disruption of PPIs can lead to a variety of disease states, including cancer.
Two general approaches are commonly used to study PPIs. Real-time assays measure PPI activity in live cells using fluorescent or luminescent tags. A second approach includes methods that measure a specific PPI “after the fact”; popular examples include a reporter system, such as the classic yeast two-hybrid system.
Redundancy equips us to survive. We have more than one lung or one kidney for a reason—if one organ in a pair gets damaged, we can still manage if the other is functional. At the cellular level, we have two copies of each chromosome in every non-germline cell. Each copy was inherited originally from a single sperm and ovum, which are “haploid” cells. Consequently, there are two copies of any given gene in non-germline “diploid” cells. In many cases, should one copy of a gene be damaged, the cell can still survive with the other, functional copy of a gene. In plants, this redundancy is common, and many plants exhibit polyploidy. In an extreme example of polyploidy, the large (by bacterial standards) but otherwise unassuming species Epulopiscium contains tens of thousands of copies of its genome.
The rapid advancement of next-generation sequencing technology, also known as massively parallel sequencing (MPS), has revolutionized many areas of applied research. One such area, the analysis of mitochondrial DNA (mtDNA) in forensic applications, has traditionally used another method—Sanger sequencing followed by capillary electrophoresis (CE).
Although MPS can provide a wealth of information, its initial adoption in forensic workflows continues to be slow. However, the barriers to adoption of the technology have been lowered in recent years, as exemplified by the number of abstracts discussing the use of MPS presented at the 29th International Symposium for Human Identification (ISHI 29), held in September 2018. Compared to Sanger sequencing, MPS can provide more data on minute variations in the human genome, particularly for the analysis of mtDNA and single-nucleotide polymorphisms (SNPs). It is especially powerful for analyzing mixture samples or those where the DNA is highly degraded, such as in human remains. Continue reading “Harnessing the Power of Massively Parallel Sequencing in Forensic Analysis”