Genome

How Low Can You Go? Designing a Minimal Genome

By Elizabeth Ohneck, PhD

How many genes are necessary for life? We humans have 19,000 – 20,000 genes, while the water flea Daphnia pulex has over 30,000 and the microbe Mycoplasma genitalium has only 525. But how many of these genes are absolutely required for life? Is there a minimum number of genes needed for a cell to survive independently? What are the functions of these essential genes? Researchers from the J. Craig Venter Institute and Synthetic Genomics, Inc., set out to explore these questions by designing the smallest cellular genome that can maintain an independently replicating cell. Their findings were published in the March 25th version of Science.

The researchers started with a modified version of the Mycoplasma mycoides genome, which contains over 900 genes. Mycoplasmas are simplest cells capable of autonomous growth, and their small genome size provides a good starting point for building minimal cells. To identify genes unnecessary for cell growth, the team used Tn5 transposon mutagenesis, in which a piece of mobile DNA is introduced to the cells and randomly “jumps” into the bacterial chromosome, thereby disrupting gene function. If many cells were found to have the transposon inserted into the same gene at any position in the gene sequence, and these cells were able to grow normally, the gene was considered non-essential, since its function was not required for growth; such genes were candidates for deletion in a minimal genome. In some genes, the transposon was only found to insert at the ends of the genes, and cells with these insertions grew slowly; such genes were considered quasi-essential, since they were needed for robust growth but were not necessary for cell survival. Genes which were never found to contain the transposon in any cells were considered essential, since cells that had transposon insertions in these genes did not survive; these essential genes were required in the minimal genome.

The researchers then constructed genomes with various combinations of non-essential and quasi-essential gene deletions using in vitro DNA synthesis and yeast cells. The synthetic chromosomes were transplanted into Mycoplasma capricolum, replacing its normal chromosome with the minimized genome. If the M. capricolum survived and grew in culture, the genome was considered viable. Some viable genomes, however, caused the cells to grow too slowly to be practical for further experiments. The team therefore had to find a compromise between small genome size and workable growth rate.

The final bacterial strain containing the optimized minimal genome, JCVI-syn3.0, had 473 genes, a genome smaller than any autonomously replicating cell found in nature. Its doubling time was 3 hours, which, while slower than the 1 hour doubling time of the M. mycoides parent strain, was not prohibitive of further experiments.

What genes were indispensable for an independently replicating cell? The 473 genes in the minimal genome could be categorized into 5 functional groups: cytosolic metabolism (17%), cell membrane structure and function (18%), preservation of genomic information (7%), expression of genomic information (41%), and unassigned or unknown function (17%). Because the cells were grown in rich medium, with almost all necessary nutrients provided, many metabolic genes were dispensable, aside from those necessary to effectively use the provided nutrients (cytosolic metabolism) or transport nutrients into the cell (cell membrane function). In contrast, a large proportion of genes involved in reading, expressing, replicating, and repairing DNA were maintained (after all, the presence of genes is of little use if there is no way to accurately read and maintain them). As the cell membrane is critical for a defined, intact cell, it’s unsurprising that the minimal genome also required many genes for cell membrane structure.

Of the 79 genes that could not be assigned to a functional category, 19 were essential and 36 were quasi-essential (necessary for rapid growth). Thirteen of the essential genes had completely unknown functions. Some were similar to genes of unknown function in other bacteria or even eukaryotes, suggesting these genes may encode proteins of novel but universal function. Those essential genes that were not similar to genes in any other organisms might encode novel, unique proteins or unusual sequences of genes with known function. Studying and identifying these genes could provide important insight into the core molecular functions of life.

One of the major advancements resulting from this study was the optimization of a semi-automated method for rapidly generating large, error-free DNA constructs. The technique used to generate the genome of JCVI-syn3.0 allows any small genome to be designed and built in yeast and then tested for viability under standard laboratory conditions in a process that takes about 3 weeks. This technique could be used in research to study the function of single genes or gene sets in a well-defined background. Additionally, genomes could be built to include pathways for the production of drugs or chemicals, or to enable cells to carry out industrially or environmentally important processes. The small, well-defined genome of a minimal cell that can be easily grown in laboratory culture would allow accurate modeling of the consequences of adding genes to the genome and lead to greater efficiency in the development of bacteria useful for research and industry.


Lethal Weapon: How Many Lethal Mutations Do We Carry?

 

By John McLaughlin

Many human genetic disorders, such as cystic fibrosis and sickle cell anemia, are caused by recessive mutations with a predictable pattern of inheritance. Tracking hereditary disorders such as these is an important part of genetic counseling, for example when planning a family. In fact, there exists an online database dedicated to medical genetics, Mendelian Inheritance in Man, which contains information on most human genetic disorders and their associated phenotypes.

 

The authors of a new paper in Genetics set out to estimate the number of recessive lethal mutations carried in the average human’s genome. The researchers’ rationale for specifically focusing on recessive mutations is their higher potential impact on human health; because deleterious mutations that are recessive are less likely to be purged by selection, they can be maintained in heterozygotes with little impact on fitness, and therefore occur in greater frequency. For the purposes of their analysis, recessive lethal disorders (i.e. caused by a recessive lethal mutation) were defined by two main criteria: first, when homozygous for its causative mutation, the disease leads to the death or effective sterility of its carrier before reproductive age, and second, mutant heterozygotes do not display any disease symptoms.

 

For this study, the researchers had access to an excellent sample population, a religious community known as the Hutterian Brethren. This South Dakotan community of ~1600 individuals is one of three closely related groups that migrated from Europe to North America in the 19th century. Importantly, the community has maintained a detailed genealogical record tracing back to the original 64 founders, which also contains information on individuals affected by genetic disorders since 1950. An additional bonus is that the Hutterites practice a communal lifestyle in which there is no private property; this helps to reduce the impact of confounding socioeconomic factors on the analysis.

 

Four recessive lethal genetic disorders have been identified in the Hutterite pedigree since their more detailed records began: cystic fibrosis, nonsyndromic mental retardation, restrictive dermopathy, and myopathy. To estimate the number of recessive lethal mutations carried by the original founders, the team used both the Hutterite pedigree and a type of computational simulation known as “gene dropping”. In a typical gene dropping simulation, alleles are assigned to a founder population, the Mendelian segregation and inheritance of these alleles across generations is simulated, and the output is compared with the known pedigree. One simplifying assumption made during the analysis is that no de novo lethal mutations had arisen in the population since its founding; therefore, any disorders arising in the pedigree are attributed to mutations carried by the original founder population.

 

After combining the results from many thousands of such simulations with the Hutterite pedigree, the authors make a final estimate of roughly one or two recessive lethal mutations carried per human genome (the exact figure is ~0.58). What are the implications of this estimate for human health? Although mating between more closely related individuals has been long known to increase the probability of recessive mutations homozygosing in offspring, a more precise risk factor was generated from this study’s mutation estimate. In the discussion section it is noted that mating between first cousins, although fairly rare today in the United States, is expected to increase the chance of a recessive lethal disorder in offspring by ~1.8%.

 

Perhaps the most interesting finding from this paper was the consistency of the predicted lethal mutation load across the genomes of different animal species. The authors compared their estimates for human recessive lethal mutation number to those from previous studies examining this same question in fruit fly and zebrafish genomes, and observed a similar value of one or two mutations per genome. Of course, the many simplifying assumptions made during their analyses should be kept in mind; the estimates are considered tentative and will most likely be followed up with similar future work in other human populations. It will certainly be interesting to see how large-scale studies such as this one will impact human medical genetics in the future.

 


DNA gel

Biotech Breakthrough: The CRISPR/Cas System

 

By John McLaughlin

In the last few years, a huge amount of excitement has grown over the CRISPR/Cas system and its use in targeted genome editing; this acronym derives from Clustered Regularly Interspaced Short Palindromic Repeats and their CRISPR-associated genes (Cas). CRISPR loci, which are found in many species of bacteria and most archae, have been collectively described as an RNA-based “immune system,” because of their ability to recognize and destroy foreign phage and plasmid DNA.

 

Although the acronym was first coined in a 2002 paper, CRISPR has only recently been exploited as a research tool. How does the system work and what is its use in the lab? There are at least three distinct types of CRISPR system. A typical “type II” CRISPR locus consists of several protein-coding Cas genes adjacent to an array of direct repeat and spacer sequences. The direct repeats are usually palindromic and conserved, in contrast to the much more variable spacers; these repeat-spacer sequences are transcribed as one unit and then processed into short CRISPR-RNAs (crRNAs).  A 2007 Science article demonstrated that a bacterial population could acquire resistance to phage infection by incorporating DNA fragments from the invading phage genome into a CRISPR locus, in the form of new spacer sequences. The newly acquired spacers are then transcribed and processed into crRNAs, associate with a trans-activating RNA (tracRNA) and Cas protein, and are eventually guided to a homologous DNA sequence to catalyze a double-stranded break.

 

The CRISPR system can be flexibly “reprogrammed” by designing custom chimeric RNAs (chiRNA), which serve the function of both crRNA and tracRNA in one molecule. By co-expressing a “designer” chiRNA with a Cas protein, a targeted and specific DNA break can be created in the genome; after providing an exogenous DNA template to help repair the break, customized knock-ins or knock-outs can be generated. Judging from the rapid technical advances made in the last few years, the system promises to be an efficient and high-throughput format for genome editing. To date, knock-outs have been created in a variety of organisms including rats, flies, and human cells.

 

CRISPR/Cas technology has attracted scientific attention as well as commercial interests. In November 2014, biologists Jennifer Doudna and Emmanuelle Charpentier were honored as co-recipients of the 2015 Breakthrough Prize in the Life Sciences, for their work in dissecting the mechanism of CRISPR’s sequence-specific DNA cleavage. According to its proponents, the possible applications of the CRISPR system seem almost limitless. CRISPR Therapeutics, a recently formed company dedicated to translating the technology into genetic disease therapies, has raised 25 million dollars from new investors. And just last month, the pharmaceutical company Novartis began collaborations with Intellia Therapeutics and Caribou Biosciences in order to pursue new therapeutics using CRISPR/Cas.

 

A technology as potentially lucrative as this one does not develop without controversy. MIT Technology Review recently reported on the competing startup companies aiming to exploit CRISPR technology, and the ensuing battles over intellectual property rights in different organisms. In fact, last year the Broad Institute and MIT were awarded a patent which covers the use of CRISPR genome-editing technology in eukaryotes. Feng Zhang, who is listed as Inventor on the patent, and his lab at MIT were the first to publish on CRISPR’s functionality in human cells.

 

In a few years, this exciting technology may be a commonplace fixture of the biology lab. Only time will tell if the CRISPR craze produces the amazing breakthroughs that scientists, and the general public, are eagerly awaiting.


Sizzling Papers of the Week - Dec 6

 

The Scizzle Team

 

Genetics don't lie

A cave in Spain - Sima de los huesos - had he largest collections of hominin bones. Now mitochondrial DNA from a femur bone collected from the cave in the 1990s was sequenced. It was believed that the bones found in Sima de los huesos were of Neanderthals but the DNA sequence suggests otherwise and left the researchers bewildered: the phylogenetic analysis showed that the DNA is closer to Denisovans than to Neanderthals - a population believed to live in southwestern Siberia. So the mystery of where our ancestors came from still remains and only more sequencing of ancient DNA will help solve it.

A mitochondrial genome sequence of a hominin from Sima de los huesos. Meyer at al., Nature. 2013.

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Future of genome therapy is looking CRISPR

Two studies published in Cell Stem Cell using the CRISPR-Cas9 system to cure diseases in mice and human stem cells.  They CRISPR/Cas9 system was originally discovered as the "immune system" of archaea and bacteria.  In the first study  the system was used in mice to correct the Crygc gene that causes cataracts; in the second study the CRSPR-Cas9 system was used to correct the CFTR locus in cultured intestinal stem cells of CF patients. These findings serve as a proof-of-concept that diseases caused by a single mutation can be "fixed" with genome editing using the CRISPR-Cas9 system.

Correction of genetic disease in mouse via use of CRISPR-Cas9. Wu et al. Cell Stem Cell. 2013.

Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of Cystic Fibrosis patients. Schwank et al., Cell Stem Cell. 2013.

Want to stay on top of  the CRISPR/Cas9 genome editing and curing diseases? Create a feed for CRISPR, Cas9 and diseases.

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You smell so good!

After being traumatized by what really happens inside you when a mosquito bites we thought there's no hope that we'll ever be mosquito-bite free. But a new study published in Cell opens the door to new, safe and pleasantly smelling way to lure mosquitoes away. The female mosquito detects CO2 using a class of olfactory receptor neurons, but the neurons and receptors that detects skin odor are a mystery. The researchers found  one neuron important for attraction to skin odor and then screened half a million compounds to find those who lured mosquitoes to traps effectively as CO2 does. Joking aside, finding safe and affordable ways to control mosquitoes is a key way to preventing them from transferring deadly diseases.

Targeting a dual detector of skin and CO2 to modify mosquito host seeking. Tauxe et al. Cell. 2013.

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It Runs in the family

Scientists showed that behavioral experiences can shape mice epigenetically in a way that is transmittable to offspring.  Male mice conditioned to fear an odor showed hypomethylation for the respective odor receptor in their sperm; offspring of these mice showed both increased expression of this receptor, and increased sensitivity to the odor that their fathers had been conditioned on.  Does this suggest that memories can be inherited?

Parental olfactory experience influences behavior and neural structure in subsequent generations, Dias, B.G. and Ressler, K.J., Nature Neuroscience. Published online December 1st 2013

Fascinated by the possibility of inheriting memories? Create a feed for epigenetics and memory and don't miss our post about this article and all the buzz -  family fear?

A pathway for the worst sides of addiction?

Opioid drugs, such as heroin, appear to have a specific pathway mediating some of the worst aspects of addiction; the κ opioid receptor is involved the dysphoria of withdrawal and the need to constantly increase dosage.  Inhibiting the κ receptor blocked dosage escalation in rats and reduced their motivation to administer the drug.  This receptor pathway may be crucial to the urge to avoid withdrawal, which itself is a powerful component of addiction.

Long-Term Antagonism of κ Opioid Receptors Prevents Escalation of and Increased Motivation for Heroin Intake, Schlosburg, J.E, et al., The Journal of Neuroscience. December 4 2013

From bacteria to behavior

Is there anything gut bacteria can't do?  A new study this week shows that the little critters may have a role in ameliorating autism.  It's been known for some time that autistic individuals are more likely to suffer various gastrointestinal problems.  Researchers found that a mouse model for autism suffers from gut inflammation similar to that seen in colitis.  The bacteria B. fragilis, which has been show to help repair these symptoms in illnesses such as Chrons disease. help repair the autistic mice's intestines as well.  More amazingly, treatment with the bacteria also improved behavioral symptoms of autism.

Microbiota Modulate Behavioral and Physiological Abnormalities Associated with Neurodevelopment Disorders, Hsiao, E.Y., et al., Cell. 2013.

Stay on top of the growing list of things we can thank or blame gut microbiota for? Create a feed for gut microbiota.

It wasn't my fault...line

Japan's Tohoku-Oki earthquake in 2011 was not only devastating, but in many ways surprising even to scientists.  Investigators are striking back in a full throttle attempt to glen information about the fault zone implicated in the 2011 quake.  A collection of papers published this week in Science characterize the fault zone's structure and composition, examined the physics underlying slippage during the quake, and tracked the physical conditions and stresses that the fault zone is exposed to, allowing an unprecedented understanding of the underlying causes of this natural disaster.

Chester et al, Ujiie et al, and Fulton et al   Science. December 6 2013.


Division Doppelgangers

Alisa Moskaleva

 

Cyclin A is a confounded nuisance for cell biologists. Noticed serendipitously in 1982 in sea urchins and clams in an experiment that earned a share of the 2001 Nobel Prize in Physiology or Medicine, cyclin A and its doppelganger protein, cyclin B, help cells of all animals grow and divide properly. Cells stockpile both proteins before dividing, use them to control division, and then degrade them after they have served their purpose. If cells are deprived of cyclin A or cyclin B, they can’t divide. If cells have too much of these proteins they start dividing early and get stuck, unable to separate into two new cells. But whereas cyclin B sticks around until the step before the two new cells separate, when the two copies of the cell genome are all set to separate, cyclin A disappears several minutes earlier when those two copies of the genome are nowhere near ready to split. Why does a responsible regulator like cyclin A leave its post so scandalously early? And why does a cell need cyclin A to regulate division when it has cyclin B there willing and present?

Lilian Kabeche and Duane A. Compton begin to answer both of these questions in their October 3 Nature paper. They took a close, microscope-assisted look at what goes on during cell division. The general process of cell division has been known for over a hundred years. Before starting to divide, the cell replicates its contents, including its DNA, so it can pass on a copy to both cells of the new generation. Then, during the prometaphase stage, the cell packs up its DNA really tightly and simultaneously builds up lots of microtubules, which are long fibers of protein that act as miniature ropes and sprout from two opposite sides of the dividing cell. The microtubules attempt to lasso the DNA, so that half of the DNA is attached to microtubules from one end of the cell and the other half is attached to microtubules from the other end of the cell. At this time cyclin A disappears. Then, at a stage called metaphase when the DNA is all lined up in the middle of the cell and properly attached to microtubules, cyclin B disappears. What follows is separation of the two copies of DNA to the two sides of the cell, pulled by microtubules; this is called anaphase. Finally, in telophase the two cells pinch off from each other and resume growing.

Kabeche and Compton focused on how cyclin A may be regulating the way microtubules attach to DNA. The big blob of DNA inside a cell is quite easy to see under a microscope, but it’s much harder to see the thin individual microtubules. Thus, Kabeche and Compton labeled microtubules with a photoactivatable fluorescent protein, a protein that can be made to glow by shining a certain wavelength of light on it. Then they looked for microtubules that approached DNA, shone light on them to make them glow, and assessed whether the glowing microtubules would stay in place or wander off. They observed that in prometaphase microtubules were much more likely to wander off than in metaphase. This makes sense. In metaphase, the DNA is organized and aligned, so it should be easy for microtubules to grab it. In prometaphase, by contrast, the DNA is still unorganized and in the process of aligning, so mistakes in attaching microtubules are likely. Microtubules from both sides of the cell may grab the same copy of DNA. Or microtubules from only one side of the cell may grab both DNA copies. These attachment mistakes, if not corrected, would distribute DNA unevenly or even tear it up, leading to deleterious mutations. So, it’s good that microtubules in prometaphase do not attach stably. When Kabeche and Compton gave cells extra cyclin A, they saw that microtubules would wander much longer than normally even in cells that were in metaphase and had their DNA aligned properly. And when Kabeche and Compton deprived cells of cyclin A, they noticed that the DNA separated unevenly, suggesting that microtubules attached at the wrong place.

All of these observations suggest that cyclin A somehow makes microtubules restless, whereas cyclin B, still present when microtubules make stable attachments, does not. The cell uses cyclin A to control the attachment of microtubules to DNA, and then disposes of it, while relying on cyclin B to control the separation of DNA copies. Given its distinct function, cyclin A disappears not early, but at precisely the right time. If it were to stick around, microtubules would never attach to DNA and division would never proceed. On the other hand, if it were not present at all, microtubules would attach too early and in all the wrong places, leading to mistakes in partitioning the genome to the new generation. Of course, there are many vexing questions that remain to be answered, the most obvious of which is how does cyclin A cause microtubules to no longer attach to DNA? It looks like cyclin A has many more mysteries to reveal.

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What’s Chromatin Got to Do With It?

Alisa Moskaleva

 

We know that cells lead intricate lives of growth, change, and division. We also know that DNA has not only the four letters A, T, C, and G, but also an intricate grammar of modifications on DNA-associated proteins, termed chromatin, that changes over time. We can surmise that there is a connection between the life cycle of a cell, called the cell cycle, and its chromatin. But how does the cell cycle influence chromatin? Yang Xu and colleagues shed new light on this question in a paper in the latest issue of the Cell Cycle.

 

Before a cell can divide, it must first condense its chromatin into packages called mitotic chromosomes, so that its genome may be evenly divided between its two daughters. One of the chromatin modifications that promotes this condensation is the deubiquitination of histone H2A. It’s been known for six years that a protein called Ubp-M can deubiquitinate histone H2A. Now Xu and colleagues explain what causes Ubp-M to deubiquitinate histone H2A before mitosis and not at other times in the cell cycle.

 

Xu and colleagues focused on a phosphorylation on the 552nd amino acid, a serine, of Ubp-M. This serine is in a motif that a kinase called CDK1 likes to phosphorylate. CDK1 is to the cell cycle what a conductor is to the symphony orchestra: it coordinates all the events, so that they happen in the right sequence and at the appropriate time. By knocking down CDK1 and using chemical inhibitors, Xu and colleagues established that CDK1 indeed phosphorylates Ubp-M on its serine 552.

 

Phosphorylation changes interactions between proteins. To find the function of the phosphorylation of serine 552, Xu and colleagues looked at the interaction between Ubp-M and a nuclear exporter called CRM1. This is a particularly interesting interaction because Ubp-M spends most of the cell cycle in the cytoplasm, even though it must go to the nucleus to deubiquitinate histone H2A. Therefore, Ubp-M is actively exported from the nucleus, and Xu and colleagues used an inhibitor of CRM1 to show that CRM1 participates in this export. Interestingly, a mutant version of Ubp-M that cannot be phosphorylated on the 552nd amino acid does not get exported as much. This mutant version also decreases cell proliferation and reduces the number of cells that enter mitosis. However, the mutation has no effect on the ability of Ubp-M to deubiquitinate histone H2A. Since CDK1 becomes more active before mitosis, Xu and colleagues propose that it phosphorylates Ubp-M on serine 552 and increases the fraction of Ubp-M in the nucleus, thus promoting chromatin condensation and mitosis.

Serine 552 of Ubp-M is present in primates but is not conserved in the mouse or rat homolog of Ubp-M. Though this particular example of temporal control using phosphorylation and localization occurs in only a few animal species, the principle is likely more general. Moreover, Ubp-M may contain other more conserved phosphorylation sites that function in the same way. And it is intriguing to speculate what special function this phosphorylation may serve in primates. Regardless, Xu and colleagues flesh out a direct connection between the cell cycle and chromatin modification to a rare level of detail.


Leafing Through The Literature

Thalyana Smith-Vikos

Highlighting recently published articles in molecular biology, genetics, and other hot topics

 

Aging is inherited maternally

 

Credit: Bob AuBuchon (Flickr)
Credit: Bob AuBuchon (Flickr)

Ross and colleagues investigated how mitochondrial DNA (mtDNA) mutations, which are exclusively maternally inherited, can contribute to aging. The researchers found that these mutations result in mild aging in otherwise wild-type mice, while decreasing fertility and accelerating premature aging in respectively heterozygous and homozygous PolgA mutants with increased mtDNA mutations. Additionally, maternal and somatic mtDNA mutations also resulted in brain developmental disorders. The authors posit that aging tissues may arise from the rapid expansion of mutated respiratory chain factors as mutated mtDNA replicates.

 

MicroRNAs regulate micro food portions

Vora et al. have identified a conserved microRNA (miRNA), miR-80, which regulates dietary restriction in C. elegans. Similar to dietary restriction-mediated effects, these mir-80 mutant worms are long-lived and maintain a healthy state for a prolonged period, regardless of the presence of food. Transcription factors DAF-16 and HSF-1 and transcription co-factor CBP-1 are required for these mir-80 mutant phenotypes. Expression of this miRNA is decreased when worms are subjected to a restricted diet, resulting in increased levels of CBP-1.

 

A fatty reward

Credit: Quinn Dombrowski (Flickr)
Credit: Quinn Dombrowski (Flickr)

Researchers have proposed that lowered dopaminergic function from a high-fat diet leads to obesity by promoting excessive food intake to restore this food-reward relationship. Tellez et al. further investigated how a high-fat diet can affect dopamine levels. The authors identified an intestinal lipid messenger, oleoylethanolamine, which is normally suppressed under a high-fat diet but can restore dopamine release upon administration. Additionally, administration of oleoylethanolamine increased consumption of low-fat foods, indicating that this signaling molecule may be responsible for promoting reward of low-fat foods.

 

Pathogen-host relationship therapy

C. albicans can exist as part of the non-pathogenic gastrointestinal microbiota or can be pathogenic to mammals. Pande and colleagues report that, while this pathogenic switch is due to the host’s suppressed immune system, a microbial genetic program is also at play. The researchers found that passage of C. albicans through the gut results in a switch to commensalism, driven by the transcription factor Wor1. These C. albicans cells that have transitioned into a commensal state are phenotypically different and express a unique transcriptome. The findings suggest that disrupting this genetic program results in reversion to a pathogenic state.

 

Breakthrough in wheat stem rust resistance

A highly resistant race of wheat stem rust, Ug99, has been plaguing wheat production areas all over the world for a number of years. Saintenac et al. report that the Sr35 gene cloned from T. monococcum provides near resistance to Ug99 and similar races, and the gene can be successfully transferred to polyploidy wheat. Periyannan et al. similarly identified a resistance gene, Sr33, which was cloned from another wild relative, A. tauschii. Both Sr33 and Sr35 encode coiled-coil, nucleotide-binding, leucine-rich repeat proteins that resemble other pathogen resistance proteins.

 

Transcribing autism genes

King and colleagues have provided a link to a recent correlation between mutated topoisomerases in individuals with autism and other autism spectrum disorders (ASDs). The researchers showed that a topoisomerase inhibitor, topotecan, reduces the expression of ASD-associated genes in a dose-dependent manner. Intriguingly, these ASD candidate genes are substantially longer than other genes on average. Topectan specifically prevents transcriptional elongation of extremely long genes (>200 kb), which was also achieved by knocking down topoisomerase 1 or 2b in neurons.


Paratransgenesis: Going the Long Way Round to Beat Sleeping Sickness

Chris Spencer

You may be familiar with transgenesis, but are you familiar with paratransgenesis? If not, read on because it’s awesome.

Transgenesis is the genetic modification of an organism such that it produces a particular phenotype. Despite controversies when it comes to crops and animals, it’s a pretty common place technique in molecular biology. Paratransgenesis is pretty much exactly the same thing; the only difference is that you genetically modify Organism A in order to yield a change in Organism B. This might seem like pretty radical, outside the box thinking, and it is, so well done to the scientists responsible.

Read more


BROMODOMAIN-IA!

Tara Burke

Bromodomain inhibitors show potential as a treatment for heart failure

Heart failure is the leading cause of hospitalizations, healthcare expenditures and death in America today. Heart failure occurs when the heart can no longer pump efficiently to accommodate the body’s needs. To date, most heart failure medications target hormonal signaling pathways, a process which initiates at the cell’s outer surface and eventually converges on specific transcription factors that control heart failure pathogenesis. These current treatments have improved patient survival but said treatments are far from optimal or ideal.  Although it is well established that chromatin and transcriptional changes drive heart failure pathogenesis in cardiomyocytes there is currently no treatment to directly block these detrimental nuclear changes and target damaging changes at their source. There are a number of transcription factors and epigenetic changes known to be important in heart failure pathogenesis but, as is the case for numerous other diseases, drug design for transcription factors and specific epigenetic marks proves difficult.Read more


Leafing through the Literature

Thalyana Smith-Vikos

Highlighting recently published articles in molecular biology, genetics, and other hot topics

Small Molecules Achieve Pluripotency

Hou et al. have reached uncharted territory in stem cell research: rather than achieving pluripotency using the well-established transcription factor cocktail or recent advances in somatic cell nuclear transfer, mouse somatic cells were reprogrammed to generate pluripotent stem cells with a frequency of 0.2% using a cocktail of seven small molecules. These reprogrammed cells, termed chemically induced pluripotent stem cells (CiPSCs), were shown to resemble embryonic stem cells (ESCs) based on gene expression and epigenetic profiling, which is not case for other types of iPSCs.

Tissue and Organ Generation from Pluripotency

Takebe et al. report the first case of successful generation of a three-dimensional vascularized organ Read more


Stick a PIN in it

Nicole Crown

Post-translational modification (PTM) of proteins is an essential cellular process used to regulate protein function and stability.  PTMs are assumed to have an impact on protein structure and conformation, but the effects of specific protein conformations on biological processes are difficult to understand and test in vivo.  The importance of protein conformation is of particular interest in DNA repair as many of the same proteins are involved in context specific repair processes.  For example, the Mre11-Rad50-Nbs1 complex is an essential DNA double-strand break sensor that is found at every type of double-strand break.  It has been proposed that this complex can, in theory, adopt up to 216 conformational states and acts as a “molecular computer” to detect damage and regulate repair pathway choice (i.e. should the break be repaired via nonhomologous end joining or homologous recombination)1In vivo studies of the roles that particular protein conformations play are rare, yet critical for understanding the regulation of DSB repair.

New work from Steger and colleagues2 shows that Pin1, a prolyl isomerase that catalyzes cis/trans isomerization, binds multiple DNA repair proteins, including CtIP, a key player in double-strand break end resection.  The authors show that the interaction between Pin1 and CtIP is induced by phosphorylation of CtIP at two S/T-Proline sites, and that Pin1 binding does indeed cause a conformational change in CtIP.  This conformational change leads to polyubiquitylation of CtIP and subsequent degradation. Overexpression of Pin1 causes hyporesection and a decrease in homologous recombination; similarly, depleting Pin1 causes hyperresection and decreased NHEJ.  These data and others presented in the paper lead to a model in which CtIP is phosphorylated, causing Pin1 to bind and isomerize CtIP.  This isomerization leads to ubiquitin-mediated CtIP degradation and appropriate end resection.  Pin1 overexpression leads to reduced CtIP activity and therefore hyporesection and decreased homologous recombination, whereas Pin1 depletion leads to increased CtIP activity, hyperresection and decreased NHEJ.

In this particular case, the researchers were lucky to find a specific protein, Pin1, that induces a known conformational change, cis/trans isomerization, by acting directly on the substrate protein. However, most conformational changes may be caused indirectly by the effects of post-translational modifications.  While the identification of Pin1 as a critical regulator of DNA repair is a large step forward in understanding the role protein conformation plays in function, this same understanding for other proteins will require more cross-disciplinary studies that are able to modify protein conformation in vivo and determine the biological outcomes.

1. Williams, G. J., et al. (2010). "Mre11-Rad50-Nbs1 conformations and the control of sensing, signaling, and effector responses at DNA double-strand breaks." DNA Repair 9(12): 1299-1306.

2. Steger, M., et al. (2013). "Prolyl Isomerase PIN1 Regulates DNA Double-Strand Break Repair by Counteracting DNA End Resection." Molecular Cell 50(3): 333-343.

 


Leafing through the Literature

Thalyana Smith-Vikos

Avian Influenza Transmission in Mammals

Avian influenza viruses can reassort their genomes to infect mammals. To investigate how this is done, Zhang et al. generated all possible 127 reassorted viruses by combining the hemagglutinin gene of an avian H5N1 influenza virus with an H1N1 virus capable of infecting humans. The researchers examined the virulence of these viruses in mice, as well as their ability to transmit in guinea pigs, which, like certain livestock, have both avian and mammalian airway receptors. Certain H1N1 genes allowed the H5N1 virus to transmit between guinea pigs. The virus was transferred by respiration between guinea pigs without killing them, indicating that livestock could be carriers of this virus without the farmer even knowing. Read more


First Fluorescent Protein Identified in Vertebrates

Sophia David

A novel fluorescent protein discovered in Japanese eels may offer superior experimental advantages and clinical applications

In the early 1960s, researchers investigating the bioluminescent properties of the Aequorea victoria jellyfish discovered a protein that has since revolutionized experimental biology. The protein is, of course, green fluorescent protein (GFP).Read more


Leafing through the Literature

Thalyana Smith-Vikos

Highlighting recently published articles in molecular biology, genetics, and other hot topics

Trusting Your Gut Microbiota

Changes in human gut microbiota have been linked to an increasing likelihood of developing metabolic diseases. Karlsson et al. sequenced the fecal metagenome of 145 European women with normal, impaired or diabetic glucose control. From these profiles, the researchers developed a mathematical model to identify cases of type 2 diabetes, and predicted an individual’s diabetes-like metabolism by applying the model to women with impaired glucose control. They also discriminated between metagenomic markers for type 2 diabetes in their European cohort compared to a recently published Chinese cohort. Read more