Identifying the Ancestor of a Domesticated Animal Using Whole-Genome Sequencing

What animal can be found around the globe that outnumbers humans three to one? Gallus gallus domesticus, the humble chicken. The human appetite for eggs and lean meat drive demand for this feathered bird, resulting in a poultry population of over 20 billion.

Controversy over the origin of the domestic chicken (when, where and which species) have lead some researchers to look for that information in the genomes of contemporary chicken breeds and wild jungle fowl, the candidates from which chickens were derived. By sequencing over 600 genomes from Asian domestic poultry as well as 160 genomes from all four wild jungle fowl species and the five red jungle fowl subspecies, Wang et al. wanted to understand and identify the relationships and relatedness among these species and derive where domesticated chickens first arose.

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Using the Power of Technology for Viral Outbreaks

Artist’s rendition of a virus particle.

When the world is experiencing a viral pandemic, scientists and health officials quickly want data-driven answers to understand the situation and better formulate a public health response. Technology provides tools that researchers can use to develop a rapid sequencing protocol. With such a protocol, the data generated can help answer questions about disease epidemiology and understand the interaction between host and virus. Even better: If the protocol is freely available and based on cheap, mobile sequencing systems.

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Small Changes With Large Consequences: The Role of Genetic Variance in Disease Development

Structure of Human Ferrochelatase
Human Ferrochelatase 2 angstrom crystal structure. Generated from 1HRK (RCSB PDB) using Pymol. Copyright: Sarah Wilson / CC BY-SA

Understanding how disease states arise from genetic variants is important for understanding disease resistance and progression. What can complicate our understanding of disease development is when two people have the same genetic variant, but only one has the disease. To investigate what might be happening with ferrochelatase (FECH) variant alleles that result in erythropoietic protoporphyria (EPP), scientists used next-generation sequencing (NGS) along with RNA analysis and DNA methylation testing to assess the FECH locus in 72 individuals from 24 unrelated families with EPP.

What is FECH and its relationship to EPP?

FECH is the gene for ferrochelatase, the last enzyme in the pathway that synthesizes heme. The inherited metabolic disorder, EPP, is caused when the activity of FECH is reduced to less than a third of normal levels thus, increasing the levels of protoporphyrin (PPIX) without metal in erythrocytes. The consequences of the low-metal PPIX include severe phototoxic skin reactions and hepatic injury due to PPIX accumulation in the liver.

How does FECH expression affect EPP?

The EPP disease state is not simply the lack of two functional FECH genes. Disease occurs with a hypomorphic allele, mutations in FECH that reduce its function, in trans to a null FECH allele. Researchers focused on three common variants called the GTC haplotype that are associated with expression quantitative trait loci (eQTL) that reduce FECH activity. Interestingly, these three variants have been found in trans, but researchers wanted to learn if there were individuals who were homozygous for the GTC allele and how EPP manifested for them.

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Uncovering the Origins of the Commensal House Mouse

Figure of house mouse. Copyright George Shuklin.
📷: George Shuklin

When I encounter my cat fixated on specific locations in my kitchen, her behavior shows me that she has heard some mice in those areas. In fact, mice have been attributed as a reason that cats became companions to humans. Mice start gathering and reproducing so cats followed the food source and hunted the rodents, thus endearing themselves to humans, who were storing food for their own use. However, new evidence described in Scientific Reports has shown that mice have been associated with humans even before grain storage was widespread. In fact, by making our dwellings comfortable, we also created an inviting place for mice to live.

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Celebrating the 100th Cartoon with a Few Words from the Promega Cartoonist

Heading into 2020, we realized that our Cartoon Lab was reaching a milestone: the 100th cartoon! We asked the “official” Promega Cartoonist Ed Himelblau to list his Top Five Cartoons and what inspired them. See what he has chosen in his own words:

This was the first of my cartoons that Promega published and it’s still one of my favorites. The file on my computer is dated February, 1999. I have been an undergraduate in a lab. I’ve mentored undergraduates in lab. Today I have lots of undergraduates working in my plant genetics lab at Cal Poly in San Luis Obispo. For the record, I enjoy having undergraduates in the lab and I never make them dress like robots. In this cartoon, I particularly like the centrifuge and stir plate on the right. I’ve always tried to put something in each cartoon (a tube rack, an enzyme shipping box, a desiccator) that make molecular biologists say, “I know that!”

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NanoLuc® Luciferase Powers More than Reporter Assays

Bright NanoLuc® Luciferase

NanoLuc® luciferase has been discussed many times on this blog and our web site because the enzyme is integral to studying genetic responses and protein dynamics. While NanoLuc® luciferase was first introduced as a reporter enzyme to assess promoter activity, its capabilities have expanded far beyond a genetic reporter, creating tools used to study endogeneous protein interactions, target engagement, protein degradation and more. So where did the NanoLuc® luciferase come from and how does a one enzyme power several technologies?

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Working in the Lab to Save Animals in the Wild

Asian elephants with babies in Planckendael zoo, Muizen near Mechelen, Flanders, Belgium. Image copyright: Ad Meskens [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)] via Wikimedia Commons

Wildlife conservation is a major focus around the world. With habitat loss and climate change, Asian elephant populations are under severe pressure. Add in an infectious disease that is fatal to the young and you have a recipe for disaster. Even with efforts to breed the endangered Asian elephants in zoos to build the population, elephant endotheliotropic herpesvirus (EEHV) thwarts conservation efforts. EEHV causes hemorrhagic disease in Asian elephants younger than 10 years old, a disease with rapid onset and high mortality. In fact, some numbers indicate EEHV is the cause of death for at least 25% of Asian elephants born in zoos and the wild globally.

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Choosing a Tag for Your Protein

Flow diagram for purifying HaloTag fusion proteins
Overview of the HaloTag® Mammalian Protein Purification System.

You have identified and cloned your protein of interest, but you want to explore its function. A protein fusion tag might help with your investigation. However, choosing a tag for your protein depends on what experiments you are planning. Do you want to purify the protein? Would you like to identify interacting proteins by performing pull-down assays? Are you interested in examining the endogenous biology of the protein? Here we cover the advantages and disadvantages of some protein tags to help you select the one that best suits your needs.

Affinity Tags

The most commonly used protein tags fall under the category of affinity tags. This means that the tag binds to another molecule or metal ion, making it easy to purify or pull down your protein of interest. In all cases, the tag will be fused to your protein of interest at either the amino (N) or carboxy (C) terminus by cloning into an expression vector. This protein fusion can then be expressed in cells or cell-free systems, depending on the promoter the vector contains. Continue reading “Choosing a Tag for Your Protein”

All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing

Ribonucleoprotein complex with Cas9, guide RNA and donor ssDNA. Copyright Promega Corporation.

With the advent of genome editing using CRISPR-Cas9, researchers have been excited by the possibilities of precisely placed edits in cellular DNA. Any double-stranded break in DNA, like that induced by CRISPR-Cas9, is repaired by one of two pathways: Non-homologous end joining (NHEJ) or homology-directed repair (HDR). Using the NHEJ pathway results in short insertions or deletions (indels) at the break site, so the HDR pathway is preferred. However, the low efficiency of HDR recombination to insert exogenous sequences into the genome hampers its use. There have been many attempts at boosting HDR frequency, but the methods compromise cell growth and behave differently when used with various cell types and gene targets. The strategy employed by the authors of an article in Communications Biology tethered the DNA donor template to Cas9 complexed with the ribonucleoprotein and guide RNA, increasing the local concentration of the donor template at the break site and enhancing homology-directed repair. Continue reading “All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing”

Delving into the Diversity of The Plague of Justinian

Wayson stain of Yersinia pestis showing the characteristic
Yersinia pestis. U.S. Center for Disease Control [Public domain], via Wikimedia Commons.
Human teeth play a key role in our understanding of how organisms evolve. Whenever a possible new member of the hominid family is uncovered, the shape and number of teeth are used to place that individual in the family tree. Teeth also harbor information about pathogens that have plagued humans for millennia. Because bacteria use our bloodstream as a transport system, protected places that can preserve DNA—like the pulp of teeth—are a rich medium for uncovering information about humans and the microbes that infected them.

Teeth have been the choice for identifying the infectious agent behind the Plague of Justinian in the sixth century and the Black Plague in the 14th century. In fact, Yersinia pestis, the bacterium responsible for these plagues, has infected humans as far back as the Neolithic. But what can we learn about the pandemic strain or strains of Y. pestis described in historical records? A team of researchers from Europe and the US, many of whom have been delving into the history of Y. pestis for the last decade, wanted to further investigate the Plague of Justinian. They studied bacterial DNA extracted from human remains found in Western European communal graves that were dated to around 541–750, the period of the historically documented Plague of Justinian. Their investigation examined the bacteria’s diversity and how far it spread during this “First Pandemic” of plague. Continue reading “Delving into the Diversity of The Plague of Justinian”