I MENTIONED BEFORE the IAD model for gene synthesis–innovation in a gene producing a new secondary function, amplification of that gene by duplication, and divergence of the gene copies. However, sometimes things can happen more suddenly. In 2002 studies of a pond snail reveal the generation of two new genes by duplication of an ancestral gene, and then a intragenic inversion converting one duplicate into two new genes!

The simple compound nitric oxide is used as a neurotransmitter in both vertebrates and invertebrates, and nitric oxide in the brain is made by neuronal nitric oxide synthase (nNOS). In studying the nitric oxide signalling pathway in pond snails Korneev and O’Shea found nNOS as expected (40% sequence similarity to mammalian nNOS) and two unexpected gene transcripts.

In our investigation of the molecular biology and function of the NO-signaling pathway in the CNS of the snail Lymnaea we have cloned and sequenced the LymnNOS mRNA (Korneev et al. 1998) and two smaller nNOS-related transcripts (fig. 1). All three have a polyadenylation signal and a poly(A) tail, characteristic features of messenger RNAs. Although both smaller RNA molecules are homologous to the Lym-nNOS mRNA, they do not show sequence similarity to each other. This is because the regions of similarity are localized in different parts of the Lym-nNOS mRNA and do not overlap. Surprisingly, these NOS-related RNA molecules also contain regions of significant antisense homology to the Lym-nNOS message, and we will therefore refer to them here as antiNOS-1 and antiNOS-2. The cloning, sequencing, and expression of antiNOS-1 was reported by us previously (Korneev, Park, and O’Shea 1999). AntiNOS-2 is a novel transcript of about 3,000 nt in length (its sequence is deposited in GenBank, accession number AF373019). Note that in antiNOS-1 the antisense region is located at the 5′ end of the molecule, whereas in antiNOS-2 it is located at the 3′ end. Another important difference is that although antiNOS-1 cannot be translated into a protein because all three reading frames contain multiple stop codons, the antiNOS-2 transcript contains an open reading frame encoding a truncated nNOS-homologous protein of 397 amino acids.

Neither antiNOS-1 nor antiNOS-2 can function in nitric oxide synthesis. The first, antiNOS-1, does not produce a protein at all, but does produce an RNA transcript. The second, antiNOS-2, produces a truncated version of nNOS that is not functional for nitric oxide synthesis. The curious sequence similarity of both to nNOS and dissimilarity to each other suggested to the researchers that these two genes were the produce of a nNOS gene that had been split in two by an intragenic inversion. Indeed, when they examined the positioning of these genes, they are adjacent to each other. The antisense sequences at the 3′ end of antiNOS-1 and 5′ end of antiNOS-2 are remnants of the inverted portion of the gene. The middle portion of the inversion is now noncoding and places a 2 kb sequence between the two genes. The inversion introduced a stop codon to truncate antiNOS-2 and a novel promoter for new gene antiNOS-1.

Another strange result of the inversion was to convert some sequences that were introns, sequences spliced out before translating RNA to protein (thus not incorporated in the protein sequence), into exons, which are not spliced out. The last exon of antiNOS-2 is made of three introns and two exons from nNOS, while the first exon of antiNOS-1 is from an exon and an intron of nNOS.

The authors determined that both genes are transcribed into RNA and antiNOS-2 is translated into protein. They suggest that these two genes now have a function in helping to regulate nNOS. The truncated protein from antiNOS-2 can form nonfunctional heterodimers with nNOS, which is active only in dimer form. The antisense portion of antiNOS-1’s RNA transcript could be active in post-transcriptional regulation of nNOS, lowering the rate of synthesis of nNOS.

New genes by intragenic inversion of nNOS.

This is an exciting twist on the more modestly paced IAD model. In most cases new genes are the result of duplication and gradual divergence, but sometimes due to a fortuitous accident something more innovative will happen.



Korneev, S.; O’Shea, M. “Evolution of Nitric Oxide Synthase Regulatory Genes by DNA Inversion.” Molecular Biology and Evolution 2002, 19, 1228-1233.

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