A test designed to push the bounds of single-cell DNA sequencing revealed something way more surprising: a microscopic organism from a pond in Oxford University Parks appears to be manipulating the genetic code in a way scientists have never seen before.
Dr. Jamie McGowan, a postdoctoral scientist on the Earlham Institute, was studying the genomes of protists collected from freshwater. The goal was practical. The researchers desired to test a DNA sequencing pipeline that might work with extremely small amounts of DNA, including DNA from a single cell.
Instead, the team found an unexpected genetic outlier. The organism, identified as sp. PL0344, a previously unknown species with an unusual mutation in the way in which it reads DNA instructions and makes proteins. The study showed that two codons normally related to gene inhibition signals were reassigned to different amino acids, a mix the researchers had previously unreported.
“It’s sheer luck that we chose this protist to test our sequencing pipeline, and it just goes to show what’s out there, highlighting how little we know about protist genetics.”
A tiny creature with a giant genetic surprise.
Protesters are difficult to define neatly because they’re so diverse. Many are microscopic, single-celled organisms, including amoeba, algae, and diatoms. Others are very large and multicellular, similar to kelp, slime molds, and red algae.
“The definition of a protist is loose — basically any eukaryotic organism that is not an animal, plant or fungus,” Dr. McGowan said. “This is clearly quite common, and that is because protists are a highly variable group.
“Some are more closely related to animals, some more closely related to plants. There are predators and prey, parasites and hosts, swimmers and sessiles, and individuals with different diets while others photosynthesize. Basically, we can generalize very little.”
SP PL0344 belongs to the group called ciliates. These swimming protists could be seen under a microscope and are present in many aquatic environments. Ciliates have develop into particularly interesting to geneticists because they’re known sites for genetic code changes, including changes in stop codons.
When Genitive Stop Signs Change Meaning
In most organisms, three stop codons tell the cell where a gene ends: TAA, TAG, and TGA. They act like punctuation marks in genetic instructions, signaling that protein constructing must stop.
The genetic code is normally described as nearly universal because most organisms use the identical basic principles. Variations do occur, but they’re rare. In the small variety of known genetic codes, TAA and TAG normally change together and frequently mean the identical thing. This pattern suggested that the 2 codons were evolutionarily linked.
“In almost every other case that we know of, TAA and TAG mutate in tandem,” explained Dr. McGowan. “When they are not stop codons, they each specify the same amino acid.”
This creature did something different. PL0344 in SP, appears to operate only as a TGA stop codon. The other two signals are reused. TAA specifies lysine, while TAG specifies glutamic acid. The researchers also found more TGA codons than expected, which can help compensate for the lack of the opposite two stop signals. The paper reports that the remaining UGA stop codons are enriched only after coding regions, suggesting that this will help prevent harmful read-through when translation proceeds an excessive amount of.
“It’s extremely unusual,” Dr. McGowan said. “We’re not aware of some other case where these stop codons are linked to 2 different amino acids. This breaks a few of the rules we thought we knew about gene translation — these two codons were considered linked.
“Scientists attempt to engineer latest genetic codes — but additionally they exist in nature. If we search for them, we are able to find interesting things.
“Or, for that matter, when we’re not looking for them.”
How cells read DNA instructions.
DNA could be regarded as a set of instructions, however the instructions should be copied and interpreted before they will take effect. First, a gene is transcribed into RNA. This RNA copy is then translated into amino acids, that are linked together to form proteins and other functional molecules.
Translation starts at a DNA start codon (ATG) and frequently ends at a stop codon (normally TAA, TAG, or TGA). In this ciliate, this familiar end system has been rearranged. The discovery suggests that even one of the crucial conserved systems in life could also be more resilient than expected.
The team’s genome and transcriptome evaluation also identified suppressor tRNA genes that corresponded to the reassigned codons, supporting the conclusion that the organism indeed reads these former stop signals as amino acids. In the study, UAA was found to code for lysine and UAG for glutamic acid.
Later work showed that ciliates are genetic rule breakers.
Follow-up work has reinforced the concept that ciliates are extraordinarily wealthy sources of genetic code surprises. In a 2024 study, researchers reported multiple independent assignments of the UAG stop codon in phyllopharyngean ciliates. Some non-cultivated ciliates from the TARA Oceans dataset appear to make use of UAG to encode leucine, while Hart Manola seneca and Trochelia petrani were found to make use of UAG to encode glutamine.
This latter study also found that UAA stays the popular stop codon in these phyllopharyngean ciliates, while UAG has repeatedly shifted to a protein-coding role. The results point to frequent changes within the genetic code in poorly studied microbial eukaryotes and reinforce the concept that ciliates are among the many strongest exceptions to the usual genetic code.
Together, these findings suggest that the genetic code is just not as fixed because it once seemed. For most organisms, the principles remain remarkably stable. But in neglected microbial life, particularly ciliates, evolution has repeatedly found ways to switch instructions.
Funding and publication
The original research was published in 2023. It was funded by the Wellcome Trust as a part of the Darwin Tree of Life project and supported by core funding from the Earlham Institute from the Biotechnology and Biological Sciences Research Council (BBSRC), a part of UKRI. The publication reported sequencing data and genome assembly resources deposited in public repositories.