Circular Chromosomes Straightened

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A newly described method linearizes circular chromosomes in yeast and caps them with telomeres to mimic natural chromosomes.

By Kerry Grens | November 6, 2014

 

FLICKR, AJ CANN

Synthetic biologists often work with circular chromosomes to engineer genetic material because they’re stable and easy to manipulate, but they don’t resemble the natural shape of chromosomes in eukaryotes. Reporting in PNAS this week (November 5), Jef Boeke of NYU Langone Medical Center and postdoc Leslie Mitchell designed a tool, which they dubbed the telomerator, that straightens circular yeast chromosomes and adds telomeres to either end.

“To convert circular DNA to something more akin to a natural chromosome is appealing,” said Timothy Lu, a synthetic biologist at MIT who was not involved in the study. Lu said the telomerator could help advance a number of goals, from designing artificial chromosomes that encode complex pathways to testing the significance of telomere location in the genome. “It’s really a platform technology for downstream applications.”

 

The telomerator includes an endonuclease target—the site where the DNA loop will be severed—flanked by telomere seed sequences that form the basis of telomere construction. The telomerator is inserted into a gene of interest in the circular chromosomes, and when an endonuclease cuts the sequence at the recognition site, each exposed end carries a seed sequence on which to build telomeres. “The reason you can actually linearize the molecule and produce a stable molecule is because this telomere seed sequence gets exposed,” explained Mitchell. She and Boeke engineered the telomerator so that it would be induced only in the presence of galactose, giving scientists an easy way to turn it on by simply changing the growth medium.

 

The researchers tested the telomerator on a synthetic chromosome designed a few years earlier, cutting it at 54 different genes. For 51 of the permutations, “we got pretty happy yeast at the end of the experiment,” said Boeke. For another three, it appeared that the proximity of essential genes to the new telomere interfered with their expression, a phenomenon known as telomeric silencing. Blocking telomeric silencing rescued the cells’ growth. “I would say we’ve worked out most of the kinks for yeast,” said Boeke. “All you need to decide right now is where you want to put it.”

 

Yo Suzuki, a synthetic biologist at the J. Craig Venter Institute, said that linearizing DNA is important to avoiding problems in meiotic recombination that can emerge with circular chromosomes. In particular, crossover events between two circular chromosomes can result in a chromosome with two centromeres, which would break during chromosome segregation. “If you have a linear molecule in yeast, you don’t have that problem,” Suzuki said.

 

Suzuki envisions that the telomerator will open up more opportunities for chromosome engineering, such as splitting chromosomes into customized fragments, fitting them back together, and then delivering the genetic material to recipient cells such as bacteria. Suzuki and colleagues have developed an approach of moving genomes into new cells via cell-to-cell transfer, and being able to do all the chromosome engineering within the yeast cell before transferring the material to a recipient will accelerate progress in the field, he said. “Boeke’s approach of linearizing is the first step,” he told The Scientist.

 

Alina Chan, a postdoc in Pam Silver’s lab at Harvard University, said it will be informative to explore the characteristics of the telomeres on the linearized chromosomes. “That’s something that would be interesting to look at in an artificial context: how long telomere seeds get extended and whether they get truncated over time,” Chan told The Scientist. Additionally, she said, the telomerator will be useful to study why eukaryotes have linear chromosomes. “What are the evolutionary advantages of the linear format?” she asked.

 

Boeke said his next goal is to apply the telomerator to mammalian cells. “We’d like to be able to build large molecules in the form of circles because they’re relatively easy to move around, sequence, et cetera,” he said. “But when we deliver them to mammalian cells we’d like to deliver a linear chromosome that looks like a native chromosome. And we think the telomerator allows us to do that.”

 

Chan pointed out that some of the technology the telomerator calls upon was developed two decades ago. And since then, others have induced chromosome splitting. Boeke’s advance was to achieve it in a circular chromosome. “It’s important to point out that various bits and widgets of this thing we pulled together are not unique to us, but it’s a combination of all these pieces that uniquely defines the telomerator,” Boeke said.

 

The name, too, was inspired by past successes in bioengineering, he noted. The repressilator is a genetic circuit developed more than a decade ago that cycles the expression of a fluorescent signal. The term telomerator “is a nod to the first synthetic biology device,” Boeke said.

 

L.A. Mitchell and J.D. Boeke, “Circular permutation of a synthetic eukaryotic chromosome with the telomerator,” PNAS, doi:10.1073/pnas.1414399111, 2014.

Tags

yeast, telomeres, telomerase, synthetic biology, genome, genetics & genomics and chromosomes

 

 

The RNA World: molecular cooperation at the origins of life.

Abstract

The RNA World concept posits that there was a period of time in primitive Earth's history — about 4 billion years ago — when the primary living substance was RNA or something chemically similar. In the past 50 years, this idea has gone from speculation to a prevailing idea. In this Review, we summarize the key logic behind the RNA World and describe some of the most important recent advances that have been made to support and expand this logic. We also discuss the ways in which molecular cooperation involving RNAs would facilitate the emergence and early evolution of life. The immediate future of RNA World research should be a very dynamic one.

Key points

• Research into the RNA World paradigm is active, and new discoveries in synthetic organic chemistry and biochemistry routinely provide new insights.
• The field of prebiotic chemistry is increasingly discovering phenomena that provide solutions to multiple (as opposed to single) problems simultaneously.
• A key issue in RNA World research is how RNAs might have made copies of themselves (that is, how they replicated). There are now several possible mechanisms of this process, and increasing focus is being placed on those that display autocatalytic feedback.
• Cooperation among various molecules was probably a key aspect of the RNA World, and at least three types of molecular cooperation could have been at play during the origins of life.
• Chemical alternatives to RNA per se may have existed at some point in the Earth's earliest history, and many efforts are underway to find and evaluate such structures.
• Network establishment was another process that had a large impact on the organization of the living state, from small molecules to large molecules and cell-like structures.

Introduction

 

The RNA World is the conceptual idea that there was a period in the early history of life on Earth when RNA, or something chemically very similar, carried out most of the information processing and metabolic transformations needed for biology to emerge from chemistry. This scenario, if it indeed existed, took place some 4 billion years ago. By contrast, the realization that RNA is a good candidate for the emergence of life is an idea that is only ~50 years old. It was recognized early on by Crick¹, Orgel² and others that RNA has both a genotype and a phenotype, and that a system based on RNA would be a plausible precursor to the much more complex system of DNA–RNA–proteins on which current life is based. It was also realized that the ribonucleotide coenzymes now used by many proteins may be molecular 'fossils' from an RNA-based metabolism³. Discoveries of naturally occurring ribozyme catalysts, such as self-splicing introns⁴ and the ribonuclease P catalyst⁵, were made in the 1980s and, with the demonstration that ribosomal RNA catalyses peptide bond formation in the ribosome⁶, the credentials of RNA as a catalyst became firmly established.

 

Spiegelman's classic experiments with the bacteriophage Qβ showed how viral RNA could evolve over time in response to selection⁷. This study gave rise to the field of in vitro evolution⁸ by demonstrating that RNA could behave in a Darwinian manner in the absence of cells. Once this realization has been made, pioneers such as Orgel⁹, Eigen¹⁰, Joyce^{11, 12}, Gold¹³ and Szostak¹⁴ fully demonstrated the evolutionary capabilities of RNA and made it difficult to ignore the possibility that life started with RNA. However, additional pieces of data from both new and old angles then became available. The catalytic repertoire of RNA was shown to be diverse¹⁵, RNA riboswitches were detected in bacteria and shown to be widespread in biology¹⁶, and an autocatalytic cycle based on RNA ligases was found¹⁷. Furthermore, polymerase ribozymes that can use another sequence as a template were selected and improved^{18, 19, 20} (see below).

 

Different research questions are gradually being brought together to assemble a complete picture of the emergence of life via the RNA World scenario (Fig. 1). New routes of chemical synthesis of ribonucleotides that could operate prebiotically are being studied, and there are also further experiments to isolate ribozymes from random RNA sequences. These areas are reviewed below. Theoretical models of RNA action are being developed to describe the origin and evolution of replicating systems. A point that emerges both from theoretical work and from laboratory experiments is that cooperation at the molecular level is essential for the survival of replicating sequences. A key aim of this Review is to describe the different senses in which cooperation is relevant in the RNA World. We argue that RNA replication must also fit into a broader thermodynamic and biological context if this were to form the basis of life (Fig. 1). What was the energy source that drove the synthesis of large macromolecules on early Earth? What were the environmental conditions at the location where these molecules were forming? How did RNA replication become associated with growth and division of protocells? Current ideas on some of these questions are considered later in this Review.

 

Figure 1: Research in different fields is coming together to assemble a more complete picture of the way the RNA World began and operated.

a | Progress in organic chemistry helps to show how nucleotides and RNA oligomers could have been synthesized before life. b | In vitro evolution studies discover functional ribozymes in the very large RNA sequence space. c | Theoretic…

 

Figure 2: RNA polymerases as altruistic cooperators.

 

a | A trans-acting RNA polymerase (or replicase) such as the R18 polymerase¹⁸ is a likely mechanism for supporting replication in the RNA World. b | Such a polymerase can use a template that is either another copy of itself (red) or an unrelated sequence (grey). Well-mixed systems of altruistic replicators are destroyed by parasites. There are two ways in which cooperators can resist parasites. c | First, survival of replicases is possible in two-dimensional models on surfaces. A simulation of altruistic replicators (red) diffusing on a surface in the presence of parasites (grey) demonstrates survival of the replicators as a result of spatial clustering⁸². d | Second, when small groups of molecules are packaged in compartments, group selection can occur. Functional RNAs (red and blue) are shown replicating in a protocell compartment in the presence of parasites (grey). Random segregation creates cells of varying composition. Cells with an excess of parasites are unviable, and this prevents parasites from over-running the system, even if the parasites multiply at a higher rate within a cell. Part a reproduced from Ref. 50, Nature Publishing Group. Part c reprinted from J. Theor. Biol., Vol. 364, Shay, J. A., Huynh, C. & Higgs, P. G., The origin and spread of a cooperative replicase in a prebiotic chemical system, 249–259, Copyright (2014), with permission from Elsevier.

 

Figure 3: Different senses of molecular cooperation.

 

a | Cooperation in networks of mutually dependent strands with different functions is shown. An autocatalytic set involves precursor molecules present in the environment (blue circles) and molecules synthesized by the cycle (green circles). Each reaction (red triangles) is catalysed by a molecule that is part of the set (dashed arrows). b | An autocatalytic set composed of two cross-catalytic ligases¹⁷ is shown. RNA A and RNA B are ligated together by ribozyme E′ to create ribozyme E, which can reciprocate and ligate RNA A′ and RNA B′ to create ribozyme E′. c |Cooperation between multiple strands that assemble to perform a single function is shown. Ribozymes, such as the Azoarcus recombinase⁵⁷, can be made from several short strands that assemble as a result of RNA secondary structure formation and information contained in internal guide sequences (IGSs) and complementary targets (grey). d | While strands can cooperate to form ribozymes, these ribozymes can then potentially cooperate at an even higher level to construct an autocatalytic set, such as a three-membered cycle⁶⁰. In this scheme, the Azoarcus ribozyme is fragmented into two pieces in three different ways (that is, at three different junctions). The IGS of the ribozyme in E₁ is adjusted in such a way that it can only covalently assemble the E₂ ribozyme and so on, such that for assembly of all the ribozymes an obligatory cooperative process must (and does) occur. Part b from Lincoln, T. A. & Joyce, G. F. Self-sustained replication of an RNA enzyme. Science 323, 1229–1232 (2009). Reprinted with permission from AAAS. Parts c and d adapted from Ref. 60, Nature Publishing Group.

Conclusions and perspectives

 

Contemporary studies both in the laboratory and by simulation are beginning to reveal the cooperative nature of the RNA World, as well as how various types of cooperation and conflict probably guided the earliest evolutionary processes. The basics of the RNA World concept are well established, but the details continue to evolve. A powerful theme has begun to emerge from many of these new approaches and their results. One way to describe this is systems chemistry¹⁰⁰. By focusing not on single reactions in isolation but on the collective set of processes that must all occur contemporaneously, systems chemistry can lead to the discovery of 'multi-fers' in which the system as a whole can establish itself through the shared use of common conditions, reactants or products. Another way to look at this process, one that we highlighted in this Review, is that of chemical cooperation (Box 1). This newer view, embedded in the broader perspective of intermolecular cooperation and conflict, is pervading other aspects of study of the RNA World, as we explored above.

 

Key questions for the next decade of research include the following. How long were the first ribozymes? In other words, what is the extent of the error threshold problem? How specific were the first ribozymes? Was life sparked by a single polymerase or a random autocatalytic set? How far can the RNA World go without being encapsulated in a cell, or were there cells already at the earliest stage? What was the energy source for the RNA World? Thus, how was it possible to link a stable energy supply to a metabolic synthesis of RNA? The RNA World idea emphasizes replication, but thermodynamic driving force is still needed for synthesis. We anticipate that the answers to many of these questions will not only be within our conceptual reach in the next decade or two, but will also invoke the insights gained from a more systematic appreciation of how conflict and cooperation can influence molecular processes. 

Nature Reviews Genetics | Review
Article series: Non-coding RNA
Paul G. Higgs^1, & Niles Lehman^2,

Year published (2014)

 

 

DNA Extraction Kits Contaminated

The Scientist » News & Opinion » Daily News

Sequencing study reveals low levels of microbes in lab reagents that can create big problems for some microbiome studies.

By Kerry Grens | November 11, 2014

 

FLICKR, CIMMYT

Researchers studying microbiomes can do their best to prevent contamination, but a new study reveals widespread, low-level contamination in DNA extraction kits. Reporting in BMC Biology today (November 11), Alan Walker of the University of Aberdeen in the U.K. and colleagues list dozens of contaminating taxa that can swamp out a sample’s true microbial signal, if starting concentrations are low.

 

“It’s really important to sequence a negative extraction control,” said Patrick Schloss, a microbiome researcher at the University of Michigan who did not participate in this study. “That’s something people should be doing and are not doing.”

 

Contamination or signal?

The presence of microbial DNA in laboratory reagents is nothing new. Studies have even found bacterial DNA in ultrapure water, and just a few weeks ago researchers pointed out ubiquitous contamination in next generation sequencing runs. In many cases such extraneous DNA may not be an issue, but for highly sensitive deep sequencing of amplified samples, contaminants can start to compete with signal, as Walker’s team found.

 

Walker’s group made serial dilutions of Salmonella bongori, beginning with 100 million cells and reducing the sample down to 1,000 cells. When the sample of S. bongori was 10,000 cells or fewer, the abundance of DNA from other microbial taxa exceeded 50 percent of the sequences. This happened using four different commercial extraction kits. Bradyrhizobiaceae, Burkholderiaceae, Pseudomonadaceae, and dozens of other bacterial groups were present, although each kit had a different profile.

 

“The assumption has been they are sterile,” said Walker, adding that, to be fair, DNA extraction kits aren’t marketed as such. “What [researchers] need to do is go back and do some negative controls and with a bit of confidence say, ‘they really are in the samples,’ in cases where maybe there are potentially suspicious results,” he said. Schloss made the point that investigators should not rely on the list Walker’s team produced as a comprehensive catalog of potential contaminants, but be sure to do their own controls given that contamination can vary from batch to batch.

Safeguarding samples

 

Walker’s paper pointed to a couple of studies in which human diseases have been linked with unexpected microbes. In one, for example, Delphine Lee of the John Wayne Cancer Institute in Santa Monica, California, and colleagues found different microbial profiles in breast tissue from cancer patients and normal controls, namely, different abundances of Methylobacterium radiotolerans and Sphingomonas yanoikuyae. Although Methylobacteriaceae and Shingomonadaceae popped up in some of the DNA extraction kits Walker’s group tested, Lee said that she and her team are very aware of contamination concerns—especially given that her work involves low biomass samples—and that they performed the right controls.

 

“With low biomass the tiniest little change can be detected,” Lee told The Scientist. In contrast, a fecal sample would have many more microbial cells. “You could sneeze all over it, you’re not going to affect the findings.”

 

Another study, by Matthew Meyerson of Harvard Medical School and colleagues, identified a novel bacterial species, Bradyrhizobium enterica, in patients with a colitis syndrome. Bradyrhizobium were also detected in Walker’s study, although Meyerson said his team did not detect any DNA from the organism in the reagents used. Additionally, his group performed in situ hybridization on the samples, from which they could see a signal from B. enterica in tissue from patients with colitis but not in controls. Meyerson said contamination is always a possibility, and performing additional cellular assays adds a very strong layer of confidence in the sequencing results.

 

Schloss has experienced his own problems with reagent contamination. In a study on cystic fibrosis, his team found Pseudomonas DNA present in a commercial kit. Such contamination is particularly problematic given that these bacteria are an important pathogen in the lung, especially among people with cystic fibrosis. “We need to be thoughtful in how we design our experiments [and determine] what types of controls are critical.”

 

Clean kits

Martin Laurence, the founder of Montreal-based ShipShaw Labs, which develops bioinformatics tools, said that sequencing studies would greatly benefit from kits that are free of any microbial DNA. “That would simplify studies a lot,” said Laurence, who reported with colleagues earlier this year the presence of bacterial DNA in human genome sequencing reads.

 

In an e-mail to The Scientist, Qiagen spokesperson Przemek Jedrysik pointed out that the QIAamp DNA Stool Mini Kit, one of the four Walker tested, “was not designed to be DNA-free or to be used in low-biomass applications.” However, he added, Qiagen recognizes that certain analyses require cleaner reagents and the company has been developing products with ultra clean product (UCP) spin columns. “It has been confirmed by independent laboratories that the new UCP kits have a significantly lower background and thus meet requirements for high sensitivity analyses.”

 

It’s not just researchers and kit manufacturers with the responsibility of managing contamination issues, but peer reviewers as well, said Laurence. “Going forward, this article should make the peer-review process more strict for articles that find novel bugs and associate them with human disease.” In particular, that means ensuring studies include the appropriate controls. “Many, many studies have used these DNA extraction kits,” he said. “I’m very surprised that it took so long” for someone to report on the contamination.

 

S.J. Salter et al., “Reagent and laboratory contamination can critically impact sequence-based microbiome analyses,” BMC Biology, 12:87, 2014.

Tags

next-gen sequencing, microbiome, microbiology, genetics & genomics, DNA and contamination