Evolutionary origins of fish antifreeze.

Nature has a surefire way of keeping cold-blooded fish alive in ice water: antifreeze. All fish in polar regions produce special blood proteins that bind to ice crystals and keep them from growing. The fish, it turns out, have taken different evolutionary routes to the same end point.

For the perchlike notothenioids, the dominant group of fish in Antarctica, the starting point was the gene for a digestive protein. In the April 15 Proceedings of the National Academy of Sciences, researchers from the University of Illinois at Urbana-Cham paign describe the evolutionary transformation of this gene and report that convergent evolution in other fish has produced the same antifreeze from a different starting point.

Biologist Arthur L. DeVries discovered antifreeze proteins in the 1960s and continues to study how they have allowed fish to colonize the freezing Antarctic waters (SN: 11/22/86, p. 330).

The notothenioid adaptation seems to have come about by an unusual reworking of the gene for trypsinogen. DeVries and his Illinois colleagues Liangbiao Chen and Chi-Hing C. Cheng had first searched a database of sequenced genes for any similarity to the D NA sequence of the gene for the antifreeze protein. When they found a close match to the end of a fish gene for trypsinogen, they did a detailed comparison of the antifreeze DNA to the trypsinogen DNA of a notothenioid and found an even better match.

"The signatures of trypsinogen in the antifreeze are quite clear at the two ends," says DeVries. More than 90 percent of the nucleotide bases making up the DNA there are identical, indicating a close evolutionary relationship.

In the middle of the gene, they found another match-nine DNA bases coding for the three amino acids that do the ice-binding work of the antifreeze protein. This sequence of bases from trypsinogen seems to have been copied and strung together many times in the antifreeze gene.

This evolutionary scenario is different from the previously known methods of crafting new genes, says molecular evolutionist John M. Logsdon Jr. of Dalhousie University in Halifax, Nova Scotia. New genes can arise, for example, when a whole gene sequence is duplicated, then gradually altered over time to perform a new function.

In this case, says Logsdon, part of a working protein arose from a bit of the trypsinogen gene that does not code for protein. "[The new gene] evolved from a gene duplication at some point, but then it did something special. It created this new functional segment out of the middle of the trypsinogen gene."

The use of a digestive protein makes sense from a design standpoint, the researchers write. The reorganized protein could go to work on any freezing seawater ingested with food in the intestine-at least in Antarctic fish.

In the Arctic cod, in contrast, the researchers' analysis of the antifreeze protein doesn't show the same sequences of bases from the trypsinogen gene, even though the protein is made up of the same three ice-binding amino acids. "It's clearly convergence ," says Logsdon.

The researchers found another convergence-between the estimated age of the notothenioid antifreeze gene and the geophysical estimate of the time of cooling in the Antarctic Ocean, about 14 million years ago. Even though it's based on the rate of genetic c hange in salmon mitochondrial DNA, the estimated antifreeze age of 5 million to 14 million years "is right in the ballpark," says Chi-Hing Cheng.

"Demonstrations of this sort . . . are rare and noteworthy," say Logsdon and Dalhousie colleague W. Ford Doolittle in an accompanying commentary. This story may become "a textbook example of molecular evolution."