Tag Archive | "DNA"

All change as researchers find new meanings in our genetic code


 In the Lewis Carroll classic, Through the Looking Glass, Humpty Dumpty states, “When I use a word, it means just what I choose it to mean — neither more nor less.” In turn, Alice (of Wonderland fame) says, “The question is, whether you can make words mean so many different things.” All organisms on Earth use a genetic code, which is the language in which the building plans for proteins are specified in their DNA. It has long been assumed that there is only one such “canonical” code, so each word means the same thing to every organism. While a few examples of organisms deviating from this canonical code had been serendipitously discovered before, these were widely thought of as very rare evolutionary oddities, absent from most places on Earth and representing a tiny fraction of species. Now, this paradigm has been challenged by the discovery of large numbers of exceptions from the canonical genetic code, published by a team of researchers from the U.S. Department of Energy Joint Genome Institute (DOE JGI) in the May 23, 2014 edition of the journal Science.

“All along, we presumed that the code or vocabulary used by organisms was universal, applying to all branches of the tree of life, with vanishingly few exceptions,” said DOE JGI Director Eddy Rubin, and senior author on the Science paper. “We have now confirmed that this just isn’t so. There is a significant portion of life that uses different vocabularies where the same word means different things in different organisms.”

This research was conducted under the DOE JGI’s continuing effort to explore the biological frontier known as “microbial dark matter.” These are the vast number of microbes that are difficult-to-impossible to grow and study in the laboratory but populate nearly all environments from the human gut to the hot vents at the bottom of the ocean. Approximately 99% of all microbial species on Earth fall in this category, defying culture in the laboratory but profoundly influencing the most significant environmental processes from plant growth and health, to the carbon and other nutrient cycles on land and sea, and even climate processes.

“The tools of metagenomics and single-cell genomics, with which we determine the genetic blueprints of microbes without the need to grow them in the laboratory, provide us a window into the unexplored, uncultured microbial world,” Rubin said. “The metaphor we use is that up until very recently we have just been looking under the lamppost for new life, studying organisms that we can grow in the laboratory while we know most microbial life is very resistant to being grown in the lab. In this project, using metagenomics and single-cell genomics to explore uncultured microbes, we really had the opportunity to see how the genetic code operates in the wild. It is helping us get an unbiased view of how nature operates and how microbes manage our planet.”

It has been 60 years since the discovery of the structure of DNA and the emergence of the central dogma of molecular biology, wherein DNA serves as a template for RNA and these nucleotides form triplets of letters called codons. There are 64 codons, and all but three of these triplets encode actual amino acids — the building blocks of protein. The remaining three are “stop codons,” that bring the molecular machinery to a halt, terminating the translation of RNA into protein. Each has a given name: Amber, Opal and Ochre. When an organism’s machinery reads the instructions in the DNA, builds a protein composed of amino acids, and reaches Amber, Opal or Ochre, this triplet would signal that they have arrived at the end of a protein.

“This is sort of a ‘stop sign,'” Rubin said. “But what we saw in the study was that in certain organisms, the stop sign was not interpreted as stop, rather it signaled to continue adding amino acids and expand the protein.”

The particular observation that caught the team’s interest in looking for breakdowns in the canonical genetic code was when the study’s lead investigator, DOE JGI’s Natalia Ivanova, came across an anomaly: bacteria with extraordinarily short genes of only 200 base pairs in length. Typically, genes from microbes are about 800-900 base pairs long.

“When trying to interpret the sequence of these bacteria using the canonical codon table, Opal, normally interpreted as a stop sign, resulted in the bacteria having unbelievably short genes. When Natalia applied a different vocabulary where Opal, instead of be interpreted as a stop, was assumed to encode the amino acid glycine, the genes in the bacteria suddenly appeared to be of normal length,” Rubin said. Their interpretation of the finding was that “Opal-recoded” organisms, instead of stopping, incorporated an amino acid into the polypeptide, which kept growing and eventually produced normal-sized proteins.

Following this finding they wanted to see how frequently this occurs in nature and looked for similar occurrences in enormous amounts of sequence data from uncultured microbes. Computationally they sifted through a massive “haystack” of sequence data, 5.6 trillion letters of genetic code (the equivalent of nearly 2,000 human genomes). These came from over 1,700 samples sourced from far-flung and esoteric locations that span the globe — marine, fresh water, and terrestrial environments — to those much closer to home and more prosaic — from the human mouth and gut.

“We were surprised to find that an unprecedented number of bacteria in the wild possess these codon reassignments, from “stop” to amino-acid encoding “sense,” up to 10 percent of the time in some environments,” said Rubin.

Another observation the researchers made was that beyond bacteria, these reassignments were also happening in phage, viruses that attack bacterial cells. Phage infect bacteria, injecting their DNA into the cell and exploiting the translational machinery of the cell to create more of themselves, to the point when the bacterial cell explodes, releasing more progeny phage particles to spread to neighboring bacteria and run amok.

“To make this all happen, the established dogma was that phage needed to employ the exact genetic code that the host cell uses, otherwise, whatever DNA they inject wouldn’t be properly translated,” Rubin said. “But we observed phage with codon vocabularies that did not match any we found in their bacterial hosts. We scratched our heads at this result, because we wondered about what was up with the host. The dogma tells us that the phage to need to share the same code as the host, but we saw no Amber in bacteria. So what were these phage doing?”

The punch line, Rubin said, is that the dogma is wrong.

“Phage apparently don’t really ‘care’ about the codon usage of the host. They have ways to get around that, and in fact they use differences to attack the host.” The phage use certain molecular tricks, just those slight changes in the codon table, to suppress the host cell’s protective mechanisms to conduct a ‘hostile takeover’ of the cell. “We call this strategy ‘codon warfare’,” Rubin said. “We need to keep this in mind when characterizing environments and how their resident microbes contribute to biochemical and biogeochemical processes. Now that our assumptions about the canonical nature of the codon table are shaken up, we will be able to devise new analysis methods that take this phenomenon of unexpected complexity into consideration so we can obtain a better understanding of how these environments function.”

Additional food for thought, Rubin noted, is whether adequate controls can effectively be established for those emergent organisms developed through synthetic biology. Some of these organisms have been engineered with an intentionally altered genetic code, designed as a “firewall” to prevent the exchange of genetic information between laboratory-engineered microbes and their cousins in the wild.

Alice in Wonderland certainly captured the vexations of nature’s complexity: “If I had a world of my own, everything would be nonsense.”

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Scientists create first living organism that transmits added letters in DNA ‘alphabet’


Scientists at The Scripps Research Institute (TSRI) have engineered a bacterium whose genetic material includes an added pair of DNA “letters,” or bases, not found in nature. The cells of this unique bacterium can replicate the unnatural DNA bases more or less normally, for as long as the molecular building blocks are supplied.

“Life on Earth in all its diversity is encoded by only two pairs of DNA bases, A-T and C-G, and what we’ve made is an organism that stably contains those two plus a third, unnatural pair of bases,” said TSRI Associate Professor Floyd E. Romesberg, who led the research team. “This shows that other solutions to storing information are possible and, of course, takes us closer to an expanded-DNA biology that will have many exciting applications — from new medicines to new kinds of nanotechnology.”

The report on the achievement appears May 7, 2014, in an advance online publication of the journal Nature.

Romesberg and his laboratory have been working since the late 1990s to find pairs of molecules that could serve as new, functional DNA bases — and, in principle, could code for proteins and organisms that have never existed before.

The task hasn’t been a simple one. Any functional new pair of DNA bases would have to bind with an affinity comparable to that of the natural nucleoside base-pairs adenine-thymine and cytosine-guanine. Such new bases also would have to line up stably alongside the natural bases in a zipper-like stretch of DNA. They would be required to unzip and re-zip smoothly when worked on by natural polymerase enzymes during DNA replication and transcription into RNA. And somehow these nucleoside interlopers would have to avoid being attacked and removed by natural DNA-repair mechanisms.

Despite these challenges, by 2008 Romesberg and his colleagues had taken a big step towards this goal; in a study published that year, they identified sets of nucleoside molecules that can hook up across a double-strand of DNA almost as snugly as natural base pairs and showed that DNA containing these unnatural base pairs can replicate in the presence of the right enzymes. In a study that came out the following year, the researchers were able to find enzymes that transcribe this semi-synthetic DNA into RNA.

But this work was conducted in the simplified milieu of a test tube. “These unnatural base pairs have worked beautifully in vitro, but the big challenge has been to get them working in the much more complex environment of a living cell,” said Denis A. Malyshev, a member of the Romesberg laboratory who was lead author of the new report.

In the new study, the team synthesized a stretch of circular DNA known as a plasmid and inserted it into cells of the common bacterium E. coli. The plasmid DNA contained natural T-A and C-G base pairs along with the best-performing unnatural base pair Romesberg’s laboratory had discovered, two molecules known as d5SICS and dNaM. The goal was to get the E. coli cells to replicate this semi-synthetic DNA as normally as possible.

The greatest hurdle may be reassuring to those who fear the uncontrolled release of a new life form: the molecular building blocks for d5SICS and dNaM are not naturally in cells. Thus, to get the E. coli to replicate the DNA containing these unnatural bases, the researchers had to supply the molecular building blocks artificially, by adding them to the fluid solution outside the cell. Then, to get the building blocks, known as nucleoside triphosphates, into the cells, they had to find special triphosphate transporter molecules that would do the job.

The researchers eventually were able to find a triphosphate transporter, made by a species of microalgae, that was good enough at importing the unnatural triphosphates. “That was a big breakthrough for us — an enabling breakthrough,” said Malyshev.

Though the completion of the project took another year, no hurdles that large arose again. The team found, somewhat to their surprise, that the semi-synthetic plasmid replicated with reasonable speed and accuracy, did not greatly hamper the growth of the E. coli cells, and showed no sign of losing its unnatural base pairs to DNA repair mechanisms.

“When we stopped the flow of the unnatural triphosphate building blocks into the cells, the replacement of d5SICS-dNaM with natural base pairs was very nicely correlated with the cell replication itself — there didn’t seem to be other factors excising the unnatural base pairs from the DNA,” Malyshev said. “An important thing to note is that these two breakthroughs also provide control over the system. Our new bases can only get into the cell if we turn on the ‘base transporter’ protein. Without this transporter or when new bases are not provided, the cell will revert back to A, T, G, C, and the d5SICS and dNaM will disappear from the genome.”

The next step will be to demonstrate the in-cell transcription of the new, expanded-alphabet DNA into the RNA that feeds the protein-making machinery of cells. “In principle, we could encode new proteins made from new, unnatural amino acids — which would give us greater power than ever to tailor protein therapeutics and diagnostics and laboratory reagents to have desired functions,” Romesberg said. “Other applications, such as nanomaterials, are also possible.”

Other contributors to the paper, “A semi-synthetic organism with an expanded genetic alphabet,” were Kirandeep Dhami, Thomas Lavergne and Tingjian Chen of TSRI, and Nan Dai, Jeremy M. Foster and Ivan R. Corrêa Jr. of New England Biolabs, Inc.

The research was funded in part by the U.S. National Institutes of Health

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