Dr. Jamie McGowan, a postdoctoral scientist at the Earlham Institute, was studying the genome of a protist collected from freshwater. The goal was practical. Researchers wanted to test a DNA sequencing pipeline that could 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 Oligohymenophorea sp. PL0344, turned out to be a previously unknown species with a rare change in how it reads DNA instructions and builds proteins. The PLOS Genetics study reported that two codons normally associated with gene stopping signals had been reassigned to different amino acids, a combination the researchers described as previously unreported.

"It's sheer luck we chose this protist to test our sequencing pipeline, and it just shows what's out there, highlighting just how little we know about the genetics of protists."

A Tiny Organism With a Big Genetic Surprise

Protists are difficult to define neatly because they are so diverse. Many are microscopic, single celled organisms, including amoebas, algae, and diatoms. Others are much larger and multicellular, such as kelp, slime molds, and red algae.

"The definition of a protist is loose -- essentially it is any eukaryotic organism which is not an animal, plant, or fungus," said Dr. McGowan. "This is obviously very general, and that's because protists are an extremely variable group.

"Some are more closely related to animals, some more closely related to plants. There are hunters and prey, parasites and hosts, swimmers and sitters, and there are those with varied diets while others photosynthesize. Basically, we can make very few generalizations."

Oligohymenophorea sp. PL0344 belongs to a group called ciliates. These swimming protists can be seen under a microscope and are found in many watery environments. Ciliates have become especially interesting to geneticists because they are known hotspots for genetic code changes, including changes involving stop codons.

When Genetic Stop Signs Change Meaning

In most living things, three stop codons tell the cell where a gene ends: TAA, TAG, and TGA. These work like punctuation marks in the genetic instructions, signaling that protein construction should stop.

The genetic code is usually described as nearly universal because most organisms use the same basic rules. Variations do occur, but they are rare. In the small number of known genetic code variants, TAA and TAG usually change together and usually end up meaning the same thing. That pattern suggested the two codons were evolutionarily linked.

"In almost every other case we know of, TAA and TAG change in tandem," explained Dr. McGowan. "When they aren't stop codons, they each specify the same amino acid."

This organism did something different. In Oligohymenophorea sp. PL0344, only TGA appears to function as a stop codon. The other two signals have been repurposed. TAA specifies lysine, while TAG specifies glutamic acid. The researchers also found more TGA codons than expected, which may help compensate for the loss of the other two stop signals. The PLOS Genetics paper reported that the remaining UGA stop codon is enriched just after coding regions, suggesting it may help prevent harmful readthrough when translation continues too far.

"This is extremely unusual," Dr. McGowan said. "We're not aware of any other case where these stop codons are linked to two different amino acids. It breaks some of the rules we thought we knew about gene translation -- these two codons were thought to be coupled.

"Scientists attempt to engineer new genetic codes -- but they are also out there in nature. There are fascinating things we can find, if we look for them.

"Or, in this case, when we are not looking for them."

How Cells Read DNA Instructions

DNA can be thought of as a set of instructions, but the instructions must be copied and interpreted before they have an effect. First, a gene is transcribed into RNA. That RNA copy is then translated into amino acids, which are linked together to form proteins and other functional molecules.

Translation begins at the DNA start codon (ATG) and normally ends at a stop codon (normally TAA, TAG, or TGA). In this ciliate, that familiar ending system has been rearranged. The discovery shows that even one of biology's most conserved systems can be more flexible than expected.

The team's genome and transcriptome analysis also identified suppressor tRNA genes that match the reassigned codons, supporting the conclusion that the organism truly 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 Shows Ciliates Are Genetic Rule Breakers

Follow up work has strengthened the idea that ciliates are unusually rich sources of genetic code surprises. In a 2024 PLOS Genetics study, researchers reported multiple independent reassignments of the UAG stop codon in phyllopharyngean ciliates. Some uncultivated ciliates from the TARA Oceans dataset appear to use UAG to encode leucine, while Hartmannula sinica and Trochilia petrani were found to use UAG to encode glutamine.

That later study also found that UAA remains the preferred stop codon in those phyllopharyngean ciliates, while UAG has repeatedly shifted into a protein coding role. The findings point to repeated changes in the genetic code across poorly studied microbial eukaryotes and reinforce the idea that ciliates are among the strongest exceptions to the standard genetic code.

Together, these discoveries suggest that the genetic code is not as fixed as it once seemed. For most organisms, the rules remain remarkably stable. But in overlooked microbial life, especially ciliates, evolution has repeatedly found ways to edit the instructions.

Funding and Publication

The original research was published in PLOS Genetics in 2023. It was funded by the Wellcome Trust as part of the Darwin Tree of Life Project and supported by the Earlham Institute's core funding from the Biotechnology and Biological Sciences Research Council (BBSRC), part of UKRI. The publication reported sequencing data and genome assembly resources deposited in public repositories.