viernes, 6 de octubre de 2017

Expandiendo y reprogramando el código genético

Nature uses a limited, conservative set of amino acids to synthesize proteins. The ability to genetically encode an expanded set of building blocks with new chemical and physical properties is transforming the study, manipulation and evolution of proteins, and is enabling diverse applications, including approaches to probe, image and control protein function, and to precisely engineer therapeutics. Underpinning this transformation are strategies to engineer and rewire translation. Emerging strategies aim to reprogram the genetic code so that noncanonical biopolymers can be synthesized and evolved, and to test the limits of our ability to engineer the translational machinery and systematically recode genomes.

Protein translation uses transfer RNAs (tRNAs), which are aminoacylated with their cognate amino acids by aminoacyl-tRNA synthetase enzymes, to read triplet codons in messenger RNAs (mRNAs) via base pairing interactions between the mRNA codon and the anticodon of the tRNA. The ribosome facilitates both the sequential decoding of triplet codons on mRNAs by cognate tRNAs, and the polymerization of the corresponding amino acids into a polypeptide (Fig. 1). The near-universal genetic code defines how 64 triplet codons are decoded in a template-directed manner on the ribosome to synthesize proteins with complex three-dimensional structures. The code is read by an evolutionarily conserved translational machinery and, with the notable exceptions of the atypical amino acids selenocysteine and pyrrolysine, all proteins are synthesized from a limited set of building blocks: the 20 canonical amino acids.

Figure 1: The natural translational machinery performs encoded amino acid polymerization.
The natural translational machinery performs encoded amino acid polymerization.
An aminoacyl-tRNA synthetase (aaRS) aminoacylates its cognate tRNA with a specific amino acid (blue circle). The aminoacylated tRNA is decoded on the ribosome in response to a cognate codon in the mRNA during translational elongation, leading to the addition of an amino acid to the growing polymer. Iterative decoding of aminoacylated tRNAs leads to the synthesis of an amino acid polymer with a defined sequence.
Over the past two decades, approaches have been developed to expand the genetic code, enabling the co-translational and site-specific incorporation of diverse noncanonical amino acids (ncAAs) into proteins synthesized in cells. These approaches extend strategies for the stoichiometric, chemical aminoacylation of tRNAs—for use in in vitro studies or (via injection) in oocytes—to diverse in vivo settings, using catalytically and specifically aminoacylated tRNAs, and complement other strategies for the synthesis of modified proteins.

Figure 2: Genetic code expansion for ncAA incorporation into proteins in vivo and its use for creating attenuated viruses.

Genetic code expansion for ncAA incorporation into proteins in vivo and its use for creating attenuated viruses.
ncAAs and viral genes containing amber (TAG) codons are introduced into cells containing the orthogonal aminoacyl-tRNA synthetase (O-aaRS) and orthogonal tRNA. The orthogonal aaRS transfers the ncAA onto the tRNA, and the aminoacylated tRNA is used to decode UAG codons in the viral mRNA. Virus particles containing UAG codons in their packaged genomes are produced from these cells. Immunization with this ‘replication-incompetent virus’ leads to protection in animal models.
The site-specific incorporation of diverse chemistries into proteins has led to a wave of powerful, transformative new ways to control, evolve and understand biological functions and to provide new strategies for medicines. Ongoing efforts are testing our ability to radically engineer the translational machinery, and to recode entire genomes, with the goal of reprogramming organisms to synthesize noncanonical biopolymers. Here I focus on key developments in in vivo code expansion over the past three years and on emerging strategies that may contribute to in vivocode reprogramming in the future; more details on earlier in vivo work can be found in previous reviews.

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lunes, 2 de octubre de 2017

Premio Nobel Medicina 2017

Premio Nobel Medicina 2017

Press Release

The Nobel Assembly at Karolinska Institutet has today decided to award
the 2017 Nobel Prize in Physiology or Medicine
jointly to
Jeffrey C. Hall, Michael Rosbash and Michael W. Young
for their discoveries of molecular mechanisms controlling the circadian rhythm


Life on Earth is adapted to the rotation of our planet. For many years we have known that living organisms, including humans, have an internal, biological clock that helps them anticipate and adapt to the regular rhythm of the day. But how does this clock actually work? Jeffrey C. Hall, Michael Rosbash and Michael W. Young were able to peek inside our biological clock and elucidate its inner workings. Their discoveries explain how plants, animals and humans adapt their biological rhythm so that it is synchronized with the Earth's revolutions.
Using fruit flies as a model organism, this year's Nobel laureates isolated a gene that controls the normal daily biological rhythm. They showed that this gene encodes a protein that accumulates in the cell during the night, and is then degraded during the day. Subsequently, they identified additional protein components of this machinery, exposing the mechanism governing the self-sustaining clockwork inside the cell. We now recognize that biological clocks function by the same principles in cells of other multicellular organisms, including humans.
With exquisite precision, our inner clock adapts our physiology to the dramatically different phases of the day. The clock regulates critical functions such as behavior, hormone levels, sleep, body temperature and metabolism. Our wellbeing is affected when there is a temporary mismatch between our external environment and this internal biological clock, for example when we travel across several time zones and experience "jet lag". There are also indications that chronic misalignment between our lifestyle and the rhythm dictated by our inner timekeeper is associated with increased risk for various diseases.

Our inner clock

Most living organisms anticipate and adapt to daily changes in the environment. During the 18th century, the astronomer Jean Jacques d'Ortous de Mairan studied mimosa plants, and found that the leaves opened towards the sun during daytime and closed at dusk. He wondered what would happen if the plant was placed in constant darkness. He found that independent of daily sunlight the leaves continued to follow their normal daily oscillation (Figure 1). Plants seemed to have their own biological clock.
Other researchers found that not only plants, but also animals and humans, have a biological clock that helps to prepare our physiology for the fluctuations of the day. This regular adaptation is referred to as the circadianrhythm, originating from the Latin words circa meaning "around" and diesmeaning "day". But just how our internal circadian biological clock worked remained a mystery.
Mimosa plants in window
Figure 1. An internal biological clock. The leaves of the mimosa plant open towards the sun during day but close at dusk (upper part). Jean Jacques d'Ortous de Mairan placed the plant in constant darkness (lower part) and found that the leaves continue to follow their normal daily rhythm, even without any fluctuations in daily light.

Identification of a clock gene

During the 1970's, Seymour Benzer and his student Ronald Konopka asked whether it would be possible to identify genes that control the circadian rhythm in fruit flies. They demonstrated that mutations in an unknown gene disrupted the circadian clock of flies. They named this gene period. But how could this gene influence the circadian rhythm?
This year's Nobel Laureates, who were also studying fruit flies, aimed to discover how the clock actually works. In 1984, Jeffrey Hall and Michael Rosbash, working in close collaboration at Brandeis University in Boston, and Michael Young at the Rockefeller University in New York, succeeded in isolating the period gene. Jeffrey Hall and Michael Rosbash then went on to discover that PER, the protein encoded by period, accumulated during the night and was degraded during the day. Thus, PER protein levels oscillate over a 24-hour cycle, in synchrony with the circadian rhythm.

A self-regulating clockwork mechanism

The next key goal was to understand how such circadian oscillations could be generated and sustained. Jeffrey Hall and Michael Rosbash hypothesized that the PER protein blocked the activity of the period gene. They reasoned that by an inhibitory feedback loop, PER protein could prevent its own synthesis and thereby regulate its own level in a continuous, cyclic rhythm (Figure 2A).
Simplified illustration of the feedback regulation of the period gene
Figure 2A. A simplified illustration of the feedback regulation of the periodgene. The figure shows the sequence of events during a 24h oscillation. When the period gene is active, period mRNA is made. The mRNA is transported to the cell's cytoplasm and serves as template for the production of PER protein. The PER protein accumulates in the cell's nucleus, where the period gene activity is blocked. This gives rise to the inhibitory feedback mechanism that underlies a circadian rhythm.
The model was tantalizing, but a few pieces of the puzzle were missing. To block the activity of the period gene, PER protein, which is produced in the cytoplasm, would have to reach the cell nucleus, where the genetic material is located. Jeffrey Hall and Michael Rosbash had shown that PER protein builds up in the nucleus during night, but how did it get there? In 1994 Michael Young discovered a second clock gene, timeless, encoding the TIM protein that was required for a normal circadian rhythm. In elegant work, he showed that when TIM bound to PER, the two proteins were able to enter the cell nucleus where they blocked period gene activity to close the inhibitory feedback loop (Figure 2B).
The molecular components of the circadian clock.
Figure 2B. A simplified illustration of the molecular components of the circadian clock.
Such a regulatory feedback mechanism explained how this oscillation of cellular protein levels emerged, but questions lingered. What controlled the frequency of the oscillations? Michael Young identified yet another gene, doubletime, encoding the DBT protein that delayed the accumulation of the PER protein. This provided insight into how an oscillation is adjusted to more closely match a 24-hour cycle.
The paradigm-shifting discoveries by the laureates established key mechanistic principles for the biological clock. During the following years other molecular components of the clockwork mechanism were elucidated, explaining its stability and function. For example, this year's laureates identified additional proteins required for the activation of the period gene, as well as for the mechanism by which light can synchronize the clock.

Keeping time on our human physiology

The biological clock is involved in many aspects of our complex physiology. We now know that all multicellular organisms, including humans, utilize a similar mechanism to control circadian rhythms. A large proportion of our genes are regulated by the biological clock and, consequently, a carefully calibrated circadian rhythm adapts our physiology to the different phases of the day (Figure 3). Since the seminal discoveries by the three laureates, circadian biology has developed into a vast and highly dynamic research field, with implications for our health and wellbeing.
The circadian clock
Figure 3. The circadian clock anticipates and adapts our physiology to the different phases of the day. Our biological clock helps to regulate sleep patterns, feeding behavior, hormone release, blood pressure, and body temperature.

Key publications

Zehring, W.A., Wheeler, D.A., Reddy, P., Konopka, R.J., Kyriacou, C.P., Rosbash, M., and Hall, J.C. (1984). P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic Drosophila melanogaster. Cell 39, 369–376.
Bargiello, T.A., Jackson, F.R., and Young, M.W. (1984). Restoration of circadian behavioural rhythms by gene transfer in Drosophila. Nature 312, 752–754.
Siwicki, K.K., Eastman, C., Petersen, G., Rosbash, M., and Hall, J.C. (1988). Antibodies to the period gene product of Drosophila reveal diverse tissue distribution and rhythmic changes in the visual system. Neuron 1, 141–150.
Hardin, P.E., Hall, J.C., and Rosbash, M. (1990). Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels. Nature 343, 536–540.
Liu, X., Zwiebel, L.J., Hinton, D., Benzer, S., Hall, J.C., and Rosbash, M. (1992). The period gene encodes a predominantly nuclear protein in adult Drosophila. J Neurosci 12, 2735–2744.
Vosshall, L.B., Price, J.L., Sehgal, A., Saez, L., and Young, M.W. (1994). Block in nuclear localization of period protein by a second clock mutation, timeless. Science 263, 1606–1609.
Price, J.L., Blau, J., Rothenfluh, A., Abodeely, M., Kloss, B., and Young, M.W. (1998). double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation. Cell 94, 83–95.

Jeffrey C. Hall was born 1945 in New York, USA. He received his doctoral degree in 1971 at the University of Washington in Seattle and was a postdoctoral fellow at the California Institute of Technology in Pasadena from 1971 to 1973. He joined the faculty at Brandeis University in Waltham in 1974. In 2002, he became associated with University of Maine.
Michael Rosbash was born in 1944 in Kansas City, USA. He received his doctoral degree in 1970 at the Massachusetts Institute of Technology in Cambridge. During the following three years, he was a postdoctoral fellow at the University of Edinburgh in Scotland. Since 1974, he has been on faculty at Brandeis University in Waltham, USA.
Michael W. Young was born in 1949 in Miami, USA. He received his doctoral degree at the University of Texas in Austin in 1975. Between 1975 and 1977, he was a postdoctoral fellow at Stanford University in Palo Alto. From 1978, he has been on faculty at the Rockefeller University in New York.

The Nobel Assembly, consisting of 50 professors at Karolinska Institutet, awards the Nobel Prize in Physiology or Medicine. Its Nobel Committee evaluates the nominations. Since 1901 the Nobel Prize has been awarded to scientists who have made the most important discoveries for the benefit of mankind.

Nobel Prize® is the registered trademark of the Nobel Foundation

jueves, 22 de junio de 2017

Una cucharada de azucar puede acabar con las infecciones urigenitales

The mammalian intestine is a natural habitat for trillions of bacteria, including harmless species called commensals and potentially pathogenic species. However, long-term persistence is a challenge for these resident bacteria, which must resist expulsion by waves of peristalsis — the process that pushes the intestinal contents downstream. The ways in which pathogenic bacteria adhere to host cells at sites of infection have been extensively investigated, but little is known about how commensal bacteria cling to mucosal surfaces in the gut. Spaulding et al (2017) report that uropathogenic Escherichia coli (UPEC), a bacterium that is a commensal in the gut but pathogenic in the bladder, persists in the intestine thanks to filamentous protein complexes called pili that project from the bacterium and promote its adhesion to the gut wall. The authors also identify a sugar derivative that can combat pilus-mediated adhesion.

A small molecule clears pathogens from the gut. 
Fig. 1. Inhibición de la adherencia de  Escherichia coli (UPEC) en el intestino mediado por el manósido M4284.

Uropathogenic Escherichia coli (UPEC) strains colonize the gut and are shed in faeces. If, from faeces, they gain access to the area around the urethra, the bacteria can work their way up the urethra, causing urinary-tract infections (UTIs). Preventing the colonization of the gut by these bacteria could therefore be a way to stop UTIs (particularly recurrent infections) from arising. 

UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. 

Using a mouse model, authors show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, they show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.


Para más información:

Fig. 2. Estructura del manósido M4284

miércoles, 22 de marzo de 2017

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers


Most DNA polymerases initiate DNA synthesis by extending a preexisting primer. Exceptions to this dogma are recently characterized bifunctional primase–polymerases (prim–pols) that resemble archaeal primases in their structure and initiate DNA synthesis de novo using only NTPs or dNTPs. We report here a DNA polymerase encoded by a phage NrS-1 from deep-sea vents. NrS-1 has a genome organization unlike any other known phage. Although this polymerase does not contain a zinc-binding motif typical for primases, it is nonetheless able to initiate DNA synthesis from a specific DNA sequence exclusively using dNTPs. Thus, it represents a unique de novo replicative DNA polymerase that possesses features found in DNA polymerases, primases, and RNA polymerases.


A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase–polymerases (prim–pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

jueves, 23 de junio de 2016

Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing

In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity1. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing23, may be skewed by mutational ‘hot’ or ‘cold’ spots34. Both approaches are affected by numerous caveats567. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription–replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.

De novo mutations in bacteria remain a notoriously difficult target for high-throughput sequencing. Whereas E. coli mutate fewer than 1 in 109 bases per generation, high-fidelity polymerases used for library preparation polymerase chain reaction (PCR) cause errors in ~4 out of 106 bases8. Illumina machines misread ~1 in 103 bases9. Recent methods, such as barcoding of reads from the same original DNA molecule8, have lowered the error rate of sequencing. However, such methods can have low yields10 and do not address errors introduced by PCR. PCR errors can be overcome using duplex barcoding, which forms a consensus from both strands of a DNA template molecule11. However, even when a small region is targeted12, duplexing lowers yield even further. The mutational landscape of an RNA virus with mutation rate 104-fold greater than E. coli was recently mapped using ‘circle sequencing’. However, this technique is not designed for targeted coverage of a single locus, and its accuracy is limited by sequence read length1013.

jueves, 26 de mayo de 2016

Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation

Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and wellbeing1, 2. Intestinal colonization begins at birth and climaxes with the acquisition of two dominant groups of strict anaerobic bacteria belonging to the Firmicutes and Bacteroidetes phyla2. Culture-independent, genomic approaches have transformed our understanding of the role of the human microbiome in health and many diseases1. However, owing to the prevailing perception that our indigenous bacteria are largely recalcitrant to culture, many of their functions and phenotypes remain unknown3

Here we describe a novel workflow based on targeted phenotypic culturing linked to large-scale whole-genome sequencing, phylogenetic analysis and computational modelling that demonstrates that a substantial proportion of the intestinal bacteria are culturable. 

Applying this approach to healthy individuals, we isolated 137 bacterial species from characterized and candidate novel families, genera and species that were archived as pure cultures. Whole-genome and metagenomic sequencing, combined with computational and phenotypic analysis, suggests that at least 50–60% of the bacterial genera from the intestinal microbiota of a healthy individual produce resilient spores, specialized for host-to-host transmission. Our approach unlocks the human intestinal microbiota for phenotypic analysis and reveals how a marked proportion of oxygen-sensitive intestinal bacteria can be transmitted between individuals, affecting microbiota heritability.

 a, Relative abundance of bacteria in faecal samples (x axis) compared with relative abundance of bacteria growing on YCFA agar plates (y axis) as determined by metagenomic sequencing. Bacteria grown on YCFA agar are representative of the complete faecal samples as indicated by Spearman ρ = 0.75 (n = 6). b, Principal component analysis plot of 16S rRNA gene sequences detected from six donor faecal samples (n = 6), representing bacteria in complete faecal samples (green), faecal bacterial colonies recovered from YCFA agar plates without ethanol pre-treatment (black) or with ethanol pre-treatment to select for ethanol-resistant spore-forming bacteria (red). Culturing without ethanol selection is representative of the complete faecal sample, ethanol treatment shifts the profile, enriching for ethanol-resistant spore-forming bacteria and allowing their subsequent isolation. c, Phylogenetic tree of bacteria cultured from the six donors constructed from full-length 16S rRNA gene sequences. Novel candidate species (red), genera (blue) and families (green) are shown by dot colours. Major phyla and family names are indicated. Proteobacteria were not cultured, but are included for context.

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miércoles, 25 de mayo de 2016

Making a decision: to stay or move

Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole
Making a right decision to stay or move is critical for survival in a changing environment. Here we identify a novel mechanism for glucose-dependent on-off switching of flagellar synthesis in Vibrio vulnificus: When enzyme IIAGlc of the bacterial PEP:carbohydrate phosphotransferase system is dephosphorylated in the presence glucose, it delocalizes a protein required for flagellar biosynthesis from the flagellated pole. This leads to a loss of motility and enables bacteria to stay in a favorable habitat.

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