Evolution of Nitric Oxide Synthase Regulatory Genes by DNA Inversion

Sergei Korneev and Michael O'Shea

Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
DNA inversions are mutations involving major rearrangements of the genome and are often regarded as either deleterious or catastrophic to gene function and can be associated with genomic disorders, such as Hunter syndrome and some forms of hemophilia. Here, we propose that DNA inversions are also an essential and hitherto unrecognized component of gene evolution in eukaryotic cells. Specifically, we provide evidence that an ancestral neuronal nitric oxide synthase (nNOS) gene was duplicated and that one copy retained its original function, whereas an internal DNA inversion occurred in the other. Crucially, the inversion resulted in the creation of new regulatory elements required for the termination and activation of transcription. In consequence, the duplicated gene was split, and two new and independently expressed genes were created. Through its dependence on DNA inversion, this is a fundamentally new scheme for gene evolution, which we show as being of particular relevance to the generation of endogenous antisense-containing RNA molecules. Functionally, such transcripts can operate as natural negative regulators of the expression of the genes to which they are related through a common ancestor.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
The duplication of DNA is an important driving force in the evolution of genomes, leading to the creation of new genes and new gene functions (Ohno 1970Citation ; Graur and Li 2000Citation ). According to a conventional model of gene creation, the duplication of a gene is followed by the gradual accumulation of mutations in one of the two resultant copies. Eventually, the divergent copy becomes a new gene with a related but different role, whereas the other one retains its original function. This process is slow and inefficient, however, principally because the majority of mutations are deleterious, and their accumulation may transform the duplicated gene into a functionless pseudogene. Also hindering gene creation by this process is the removal of duplicated genes from the genome before they acquire the beneficial mutations that enable the evolution of new functions.

Here, we present a different evolutionary scenario, which depends on the inversion of DNA within a duplicated gene. We propose that this can lead more rapidly to the acquisition of new functions for the duplicated gene, perhaps particularly with respect to regulating the expression of the related gene. But how can a DNA inversion create new genes? After all, such major rearrangements of DNA are normally regarded as either deleterious or catastrophic to gene function and are often the cause of the so-called genomic disorders (Lupski 1998Citation ), such as Hunter syndrome (Bondeson et al. 1995Citation ) and some forms of hemophilia (Lakich et al. 1993Citation ). We were led to propose a creative evolutionary role for intragenic DNA inversions through our studies of the nitric oxide–signaling pathway in the nervous system of a pond snail Lymnaea stagnalis (Elphick et al. 1995Citation ; Korneev et al. 1998Citation ; Park, Straub, and O'Shea 1998Citation ). Nitric oxide is now a recognized neurotransmitter in the nervous systems of both vertebrates and invertebrates (Bredt and Snyder 1992Citation ), where it is synthesized by the neuronal isoform of neuronal nitric oxide synthase (nNOS), the product of a constitutively active gene. The Lymnaea version of nNOS (Lym-nNOS) is approximately 40% similar to its mammalian counterpart (Korneev et al. 1998Citation ), indicating the early appearance of this protein in metazoan evolution. Here, we show that the duplication of an ancestor of the Lymnaea nNOS gene was followed by the occurrence of an internal DNA inversion in one of the copies. Remarkably, this produced new regulatory elements required for the termination and the activation of transcription. Consequently, the gene was split, and simultaneously two new genes with entirely new functions were created.

We believe that through its dependence on DNA inversion, this mechanism could be especially important in the creation of genes encoding RNA molecules that are antisense to the mRNA produced from the related gene that did not undergo an inversion. Thus, transcripts from genes created by intragenic DNA inversions may be preadapted to function as natural antisense regulators of the expression of their related genes. Indeed, we have shown directly that one of the two novel genes produced after an inversion in the ancestral nNOS gene does function as a negative regulator of nNOS expression through a natural antisense mechanism (Korneev, Park, and O'Shea 1999Citation ).


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
cDNA Library Construction and Screening
Approximately 3 µg of poly(A)+ RNA isolated from total Lymnaea CNS RNA by means of Dynabeads oligo-dT25 (Dynal) was used to construct a cDNA library according to the manufacturer's protocol for SuperScript Choice System (Life Technologies). The cDNA library was subjected to hybridization with 32P-labeled Lymnaea NOS cDNA. Positive clones were selected and sequenced. For further details see Korneev, Park, and O'Shea (1999)Citation .

Northern Blot Hybridization
A blot containing poly(A)+ RNA extracted from Lymnaea CNS was prepared as described previously (Korneev, Park, and O'Shea 1999Citation ). Hybridization with the 32P-labeled probe containing a fragment of the 3' untranslated region of antiNOS-2 cDNA (positions 1426–2100) was performed in the ULTRAhyb buffer according to the manufacturer's protocol (Ambion).

In Vitro Translation
Approximately 1 µg of a plasmid-containing antiNOS-2 cDNA was used for the in vitro translation reaction performed according to the manufacturer's protocol for the TNT T7 Coupled Reticulocyte Lysate System (Promega). Labeled products were resolved on SDS-PAGE. Control reaction had no added RNA template.

RT-PCR on Isolated Identified Neurons
The cell bodies of six cerebral giant cells (CGCs) were identified and individually dissected from the CNS. Total RNA was extracted from pooled cells and used as a template in a reverse transcription reaction in the presence of random primers and Sensiscript reverse transcriptase (QIAGEN). Synthesized cDNA was then subjected to 35 cycles of PCR using the following parameters: denaturation, 94°C, 20 s; annealing, 55°, 30 s; extension, 68°C, 60 s. For detection of antiNOS-1 RNA the primers were as follows: 5'-ATCTTCCTGTCTCCGAGGC-3' and 5'-TGTGGAAATGTGTTGCCCTT-3'. For the detection of antiNOS-2 RNA the primers were 5'-TGTAGCTGGGATCTTTCACTC-3' and 5'-ATCCTCGTCAATCGATTGCAC-3'. Nested PCRs were then performed under the same cycling parameters. The primers used for the nested PCR were as follows: 5'-GCTAGTAGCCCAAGTCTCTT-3' and 5'-CACTATGGCATCTAAATGTTAAG-3' for detection of antiNOS-1, and 5'-TGAAGGGCTCTACTTTCTTCC-3' and 5'-CTCGATCACTCAACATTGTCC-3' for detection of antiNOS-2. PCRs were performed using Taq Supreme and a reaction buffer supplied by Helena BioSciences.

Long-Distance PCR
Approximately 200 ng of genomic DNA was used as a template in a long-distance PCR (LD-PCR) to amplify the intergenic region in the anti-NOS locus. The reaction was performed using the Extensor system (ABgene) according to the manufacturer's protocol. PCR primers were as follows: 5'-CGCCTGTGCAATATTCAACC-3' and 5'-ATAGTCTGATGACTAGCAAAGC-3'. Nested amplification was then performed in the presence of 5'-GTAAGCATTAGATCCCAGTG-3' and 5'-TTGACCTTTGAACTACTGATAG-3' primers. The products of the reaction were purified from an agarose gel, cloned into pCRII-TOPO (Invitrogen), and sequenced.

Computational Analysis
The Promoter 2.0 software designed to predict transcription start sites was used (Knudsen 1999Citation ) to analyze the intergenic region in the anti-NOS locus. On average, the software picks up about 80% of all PolII promoters. Typically, a sequence scoring 0.5–0.8 (marginal prediction) contains about 65% of the true promoter sequence. For a sequence scoring 0.8–1.0 (medium likely prediction) this figure is about 80%, and for a region scoring above 1.0 (highly likely prediction) it reaches 95%.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
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 Lym-nNOS mRNA (Korneev et al. 1998Citation ) 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 1999Citation ). 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.



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Fig. 1.—Schematic organization of Lym-nNOS mRNA and nNOS-related transcripts. All three transcripts were isolated from the L. stagnalis CNS cDNA library. Dark gray boxes indicate regions of similarity (76%) shared by Lym-nNOS mRNA and antiNOS-2 RNA. Light gray boxes show regions of similarity (80%) shared by Lym-nNOS mRNA and antiNOS-1 RNA. Antisense regions in the anti-NOS transcripts (anti{alpha} and antiß) and their complementary counterparts in the Lym-nNOS mRNA ({alpha} and ß) are hatched. The antisense similarity of anti{alpha} to {alpha} and of antiß to ß is approximately 80%

 
To verify that antiNOS-2 is transcribed in vivo, we have performed a Northern blot analysis of RNA extracted from the CNS of Lymnaea. The result shown in figure 2A demonstrates clearly the presence of a transcript of the expected size. A protein-encoding function of the antiNOS-2 RNA was confirmed by in vitro translation experiments, in which a single protein band of exactly the expected size (45 kDa) was detected (fig. 2B ). The functional nNOS protein is a homodimer of subunits consisting of two major functional domains (oxygenase and reductase) separated by a calmodulin-binding site (for a review see Alderton, Cooper, and Knowles 2001Citation ). The antiNOS-2 protein shares about 70% identity with the oxygenase domain, which includes the regions required for the formation of the functional homodimer. It does not, however, possess other important functional regions essential for enzymatic activity of the NOS, such as calmodulin-binding sites and the cofactor- and cosubstrate-binding sites of the reductase domain (fig. 2C ).



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Fig. 2.—AntiNOS-2 RNA is expressed in the CNS and encodes a truncated NOS homolog. In A, Northern blot hybridization of poly(A)+ RNA from Lymnaea CNS with a probe specific for antiNOS-2 identifies a transcript of about 3,000 nt (arrow). In B the result of in vitro translation of antiNOS-2 cRNA (lane 2) shows that the main labeled product (arrow) is the protein of the expected size (approximately 45 kDa). For comparison, no product can be observed in the control experiment (lane 1) in which an RNA template was not added. In C the schematic representation of Lymnaea nNOS and antiNOS-2 proteins is shown. The upper bar represents the nNOS protein, which is composed of two major functional domains (oxygenase domain and reductase domain), connected by the calmodulin-binding site (hatched). Importantly, it was shown that the oxygenase domain contains regions essential for NOS dimerization. Note that the antiNOS-2 protein (lower bar) contains the oxygenase domain only. Oxygenase domains in the nNOS and antiNOS-2 proteins share 70% identity

 
In order to investigate the expression of the two antisense transcripts at the cellular level, we performed RT-PCR on RNA extracted from individually identified neurons called CGCs (Benjamin and Elliott 1989Citation ; Kemenes, Hiripi, and Benjamin 1994Citation ). The CGCs were known to contain Lym-nNOS mRNA and antiNOS-1 (Korneev, Park, and O'Shea 1999Citation ), but the results of RT-PCR show that antiNOS-2 is not expressed in these neurons (fig. 3 ). This demonstrates that although both antiNOS-1 and antiNOS-2 transcripts are present in the CNS, their expression is independently regulated at the cellular level.



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Fig. 3.—AntiNOS-1 and antiNOS-2 RNAs are differentially expressed. The results of RT-PCR experiments on RNA extracted either from Lymnaea CNS (lanes 1 and 2) or identified CGCs (lanes 3 and 4) show that both antiNOS-1 (lane 1, expected size of PCR product is 431 bp) and antiNOS-2 (lane 2, expected size of PCR product is 652 bp) are expressed in the CNS. Note, however, that although the antiNOS-1 transcript is present in the CGCs (lane 3), the antiNOS-2 is not detectable in these neurons (lane 4)

 
The structural organization and independent expression of the antiNOS-1 and antiNOS-2 RNAs, as described previously, suggest that they might be transcribed from two different but adjacent and related genes. Moreover, considering the positions of the antisense regions and the localization of their counterparts in the nNOS mRNA (see fig. 1 ), we hypothesize that these two genes are the result of a single internal DNA inversion that occurred in an ancestral gene. According to this hypothesis the inversion must have resulted in the generation of new instructions for the termination and initiation of transcription within the body of the mutated gene. Importantly, not only can this scenario explain the unusual structural features of the anti-NOS transcripts and their independent expression but it also represents a novel mechanism for the generation of new genes with new functions.

To test this hypothesis, we have performed a PCR on genomic DNA using a forward primer specific for the 3' end of antiNOS-2 and a reverse primer specific for the 5' end of antiNOS-1. The identification of a relatively short PCR product would confirm that the two transcripts are indeed encoded in proximity to each other in the genome. Remarkably, a clear band of about 3 kb was produced, which when sequenced revealed the organization presented in the upper part of figure 4 (the sequence is deposited in GenBank, accession number AF373020). Crucially, it shows that the antiNOS-1 and antiNOS-2 genes are separated by an intergenic region of about 2 kb and are positioned in a tail-to-head manner. Hereafter, we refer to this region of the genome, which includes the two anti-NOS genes and the intergenic region as the anti-NOS locus. Note that the two regions responsible for the generation of the antisense sequences (antiß and anti{alpha}) in the anti-NOS transcripts appear at this locus in the reversed order to that of the corresponding sense regions in the nNOS gene ({alpha} and ß) shown in the lower part of figure 4 . This is consistent with their having been created by a single internal DNA inversion in an ancestral gene.



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Fig. 4.—Structural organization of the anti-NOS locus and the nNOS gene. The upper bar represents a segment of the anti-NOS locus, and the lower bar shows an internal region of the nNOS gene. Introns are labeled by "i" and shown by black boxes. Exons are marked by "e" and indicated by gray boxes. Dashed lines indicate the limits of the inverted region in the anti-NOS locus and the corresponding region in the nNOS gene. The inverted part of the anti-NOS locus contains the last intron and exon of the antiNOS-2 gene, an intergenic region, and a part of the first exon of the antiNOS-1 gene. The corresponding region in the nNOS gene is composed of eight exons and eight introns as indicated. Below the upper bar the positions and sequences of inverted repeats which flank the inverted segment are shown. Their significance is discussed in the text. The hatched boxes labeled anti{alpha} and antiß in the anti-NOS locus and {alpha} and ß in the nNOS gene correspond to the similarly labeled regions in their associated RNA molecules shown in figure 1 . Note that because of the DNA inversion the exon-intron organization of the locus has been considerably altered. Consequently, both the antiNOS-1 and the antiNOS-2 RNAs have acquired some sequences which are not present in the nNOS mRNA. Specifically, the last exon (exon e1367) in the antiNOS-2 gene is composed of three exons (ex + 6, ex + 7 and ex + 8) and two introns (ix + 6 and ix + 7) from the ancestral gene. Another example of this amazing transformation of introns into exons is the first exon (e542) of the antiNOS-1 gene, which has strong sequence similarity with exon ex + 2 and intron ix + 1 of the ancestral gene

 
To identify the break points of the inversion, we have performed a detailed sequence comparison between the anti-NOS locus and the nNOS gene. Our rationale for this is that the similarity between the two antisense transcripts and the nNOS mRNA indicates that an ancestral gene underwent duplication and that subsequently an inversion occurred in one of the copies. The other copy became the nNOS gene, which must retain similarity to the ancestral gene. Remarkably, this comparison has revealed a clear continuous antisense homology between the central region of the anti-NOS locus (about 3 kb in length) and a corresponding region in the nNOS gene. Outside these regions the similarity between the anti-NOS locus and the nNOS gene reverts to the normal sense homology. In addition, we have identified a pair of short inverted repeats flanking the inverted segment of DNA within the anti-NOS locus (fig. 4 ). Together, this evidence suggests that the anti-NOS locus was generated by an internal DNA inversion in its ancestor, occurring as a result of a recombination event between the inverted repeats.

The results of sequence analysis also explain why the antisense homology, which is widespread at the genomic level, was actually restricted to a couple of relatively short regions in the RNA transcripts. The first and most obvious reason is that a large part of the inverted DNA has become an intergenic region and is not transcribed. The second reason is that because of the inversion, the exon-intron organization of the locus has been dramatically altered. As a result, both the antiNOS-1 and antiNOS-2 RNAs have acquired some sequences which are not present in the Lym-nNOS mRNA. Specifically, the last exon in the antiNOS-2 gene is actually composed of three exons and two introns from the ancestral gene. This extraordinary transition of introns into exons is also evident in case of the antiNOS-1 gene. Here, the first exon has clear similarity with an exon and intron of the ancestral gene.

No doubt, the DNA inversion has drastically changed the organization of one duplicate of the ancestral nNOS gene. An important consequence of this is an interruption of the original open reading frame. Sequencing analysis suggested that a new stop codon, which was introduced by the DNA inversion and is therefore not present in Lym-nNOS mRNA, is used as a signal for the termination of translation in the antiNOS-2 RNA. This suggestion was confirmed by our in vitro translation experiments (see fig. 2B ). Furthermore, we have also identified in the antiNOS-2 transcript a classical polyadenylation signal AAUAAA located in the expected position, 16 nt upstream of the poly(A) tail, which was generated as a consequence of the DNA inversion.

The independent expression pattern of the antiNOS-1 and antiNOS-2 genes indicates that they must have separate promoters. Although the promoter for antiNOS-2 was not necessarily affected by the DNA rearrangement because it resides outside the inverted region, the promoter for the antiNOS-1 gene must have been created as a consequence of the inversion and is expected to be located in the intergenic region. We have, therefore, analyzed the DNA sequence of this region using the Promoter 2.0 software (Knudsen 1999Citation ) designed to identify the regulatory elements critical for transcription. Crucially, the intergenic region in the anti-NOS locus has scored 1.087, confirming the very high likelihood of the presence of a functional promoter for the antiNOS-1 gene. Furthermore, the predicted transcription start is just 40 bp upstream of the start site indicated from our cDNA clone of the antiNOS-1 transcript. For comparison, no promoter sequences were identified when the intergenic region was analyzed in the opposite direction. Thus, these data strongly support our hypothesis that a new promoter has been created by the inversion. Together with new instructions for the termination of translation and transcription, this served to split the ancestral gene, creating two new and independently expressed genes.


    Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Generally, DNA inversions are known for their devastating effects on the structural and functional integrity of genes in eukaryotic cells (Lupski 1998Citation ). They can lead to the so-called genomic disorders such as hemophilia A, 47% of the cases of which are caused by an inversion of a portion of the gene encoding factor VIII (Lakich et al. 1993Citation ). Another example is Hunter syndrome, which results from an inversion of the gene encoding iduronate-2-sulfatase (Bondeson et al. 1995Citation ). Despite this rather negative image of their effects, DNA inversions are also now recognized as contributing significantly to genome evolution in eukaryotes. Specific examples involve inversions ranging in size from 10s of thousands up to several million base pairs which cause dramatic changes in the order and relative transcriptional orientation of a number of genes in a chromosome (Weichhold et al. 1990Citation ; Muller et al. 2000Citation ; Seoighe et al. 2000Citation ; Podemski, Ferrer, and Locke 2001Citation ). These large DNA inversions leading to global changes in genome organization, however, are not directly associated with the generation of new genes or new gene functions. This is in direct contrast to the much smaller intragenic DNA inversion described here, which has drastically altered the structure and function of an individual gene and resulted in gene splitting and the simultaneous creation of two novel and independently expressed genes.

Although the mechanism of gene splitting that we describe is unprecedented, the phenomenon of gene splitting itself is well recognized. For example, during the evolution of immunoglobulin L chain genes in sharks, a mobile DNA element was inserted in the V exon of the precursor and split it into V and J segments (Lee et al. 2000Citation ). In this and other examples of gene splitting, in contrast to our findings, the resultant new genes have functions that are highly related to that of the original gene (Krem and Di Cera 2001Citation ). The novel feature of the type of gene splitting that we describe here is that it results from an internal DNA inversion within a duplicated gene. Thus, as a direct consequence of the molecular mechanism of their evolution, two genes producing trans-encoded antisense RNA molecules are created. Such genes would, therefore, appear to be preadapted to regulate the expression of the related gene through an antisense mechanism (see Korneev, Park, and O'Shea 1999Citation ); this is a function that is entirely unlike that of the original gene.

The chain of evolutionary events suggested by our studies and leading to the rapid creation of novel genes is shown in figure 5 . The first event was the duplication of the ancestral nNOS gene. One copy of the gene retained its original function and evolved into the present-day nNOS gene. The function of the other copy was disrupted by an internal DNA inversion. Critically, it also introduced new instructions for the termination and initiation of transcription. This resulted in the creation of two novel and independently expressed genes, antiNOS-1 and antiNOS-2. We know that the antisense-containing RNA transcribed from the antiNOS-1 gene functions as a negative regulator of Lym-nNOS translation. This has been demonstrated in the uniquely identified neurons (Korneev, Park, and O'Shea 1999Citation ) in which the nNOS gene and antiNOS-1 gene are coexpressed. Concerning the second antisense-containing transcript (antiNOS-2), unlike antiNOS-1 it has an open reading frame encoding a protein with 70% identity to the oxygenase domain of nNOS. Because the nNOS enzyme is active only as a homodimer (Klatt et al. 1996Citation ; Crane et al. 1998Citation ), it is of considerable interest that the truncated NOS homolog produced by the antiNOS-2 gene includes the domain required for dimerization but lacks the other functional regions essential for NO synthesis. Importantly, when heterodimers are formed between similarly organized, in vitro–generated truncated variants of the NOS protein and the normal nNOS momomer, a strong suppressive effect on enzyme activity is observed (Lee, Robinson, and Michel 1995Citation ). One intriguing possibility, therefore, is that the antiNOS-2 protein functions as a natural dominant negative regulator of nNOS activity through binding to the normal nNOS monomer, forming a nonfunctional heterodimer (fig. 5 ). Interestingly, a similar dominant negative regulatory role for a truncated nNOS protein encoded by a spliced variant of the NOS gene in Drosophila has recently been suggested (Stasiv et al. 2001Citation ).



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Fig. 5.—Evolution of new genes by DNA inversion. An ancestral nNOS gene underwent a duplication, producing nNOS gene I and nNOS gene II. The internal DNA inversion in nNOS gene II resulted in the generation of a new terminator and new start site, which in effect split the gene resulting in the generation of two new and independently expressed genes, antiNOS-1 and antiNOS-2. The original functions of the nNOS gene I were unaffected, but its expression can now be regulated by a natural antisense-containing RNA transcribed from the antiNOS-1 gene (Korneev, Park, and O'Shea 1999Citation ). The antiNOS-2 gene produces a message encoding a truncated nNOS homolog. This protein can form a nonfunctional heterodimer and might therefore have a natural dominant negative effect on nNOS enzyme activity

 
Our observations indicate that intragenic DNA inversions could be an important and hitherto unrecognized component of genome evolution in eukaryotic cells. They suggest an alternative to the conventional scheme in which additional genes are derived from copies of duplicated genes by the slow and gradual accumulation of point mutations. Certainly, DNA duplication provides evolutionary opportunities for the acquisition of new genes, and our results demonstrate that these can be exploited quite rapidly as a consequence of DNA inversions, and perhaps other types of major DNA rearrangements, occurring within duplicated sequences soon after they are created.


    Supplementary Material
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
GenBank accession number for the antiNOS-2 cDNA is AF373019. GenBank accession number for an inverted region of the anti-NOS locus is AF373020.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
We are grateful to Elena Korneeva for technical assistance. This work was funded by the Biotechnology and Biological Sciences Research Council of the U.K.


    Footnotes
 
Ken Wolfe, Reviewing Editor

Keywords: DNA inversion gene evolution NOS antisense RNA mollusc Back

Address for correspondence and reprints: Sergei Korneev, Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K. s.korneev{at}sussex.ac.uk Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 

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Accepted for publication February 25, 2002.