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
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
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 1970;
Graur and Li 2000). 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
1998), such as Hunter syndrome (Bondeson et al. 1995)
and some forms of hemophilia (Lakich et al. 1993). 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. 1995;
Korneev et al. 1998; Park, Straub, and O’Shea 1998).
Nitric oxide is now a recognized neurotransmitter in the
nervous systems of both vertebrates and invertebrates
Key words: DNA inversion, gene evolution, NOS, antisense
RNA, mollusc.
Address for correspondence and reprints: Sergei Korneev, Sussex
Centre for Neuroscience, School of Biological Sciences, University of
Sussex, Falmer, Brighton BN1 9QG, U.K.
E-mail: [email protected].
Mol. Biol. Evol. 19(8):1228–1233. 2002
q 2002 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
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(Bredt and Snyder 1992), 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. 1998), 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 1999).
Materials and Methods
cDNA Library Construction and Screening
Approximately 3 mg of poly(A)1 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 32Plabeled Lymnaea NOS cDNA. Positive clones were selected and sequenced. For further details see Korneev,
Park, and O’Shea (1999).
Northern Blot Hybridization
A blot containing poly(A)1 RNA extracted from
Lymnaea CNS was prepared as described previously
Evolution by DNA Inversion
1229
(Korneev, Park, and O’Shea 1999). Hybridization with
the 32P-labeled probe containing a fragment of the 39
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 mg 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, 948C, 20 s; annealing, 558, 30 s; extension, 688C, 60 s. For detection of antiNOS-1 RNA
the primers were as follows: 59-ATCTTCCTGTCTCC
GAGGC-39 and 59-TGTGGAAATGTGTTGCCCTT-39.
For the detection of antiNOS-2 RNA the primers were
59-TGTAGCTGGGATCTTTCACTC-39 and 59-ATCC
TCGTCAATCGATTGCAC-39. Nested PCRs were then
performed under the same cycling parameters. The
primers used for the nested PCR were as follows: 59GCTAGTAGCCCAAGTCTCTT-39 and 59-CACTAT
GGCATCTAAATGTTAAG-39 for detection of antiNOS-1, and 59-TGAAGGGCTCTACTTTCTTCC-39
and 59-CTCGATCACTCAACATTGTCC-39 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: 59-CGCCTGTGCAAT
ATTCAACC-39 and 59-ATAGTCTGATGACTAGCA
AAGC-39. Nested amplification was then performed in
the presence of 59-GTAAGCATTAGATCCCAGTG-39
and 59-TTGACCTTTGAACTACTGATAG-39 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 1999) to analyze
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 LymnNOS mRNA and antiNOS-1 RNA. Antisense regions in the anti-NOS
transcripts (antia and antib) and their complementary counterparts in
the Lym-nNOS mRNA (a and b) are hatched. The antisense similarity
of antia to a and of antib to b is approximately 80%.
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
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 59 end of the molecule,
whereas in antiNOS-2 it is located at the 39 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.
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 tran-
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Korneev and O’Shea
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).
FIG. 2.—AntiNOS-2 RNA is expressed in the CNS and encodes
a truncated NOS homolog. In A, Northern blot hybridization of
poly(A)1 RNA from Lymnaea CNS with a probe specific for antiNOS2 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.
script 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 calmodulinbinding site (for a review see Alderton, Cooper, and
Knowles 2001). 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).
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 1989; Kemenes, Hiripi, and Benjamin 1994). The CGCs were
known to contain Lym-nNOS mRNA and antiNOS-1
(Korneev, Park, and O’Shea 1999), 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.
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 39 end of antiNOS-2 and a reverse primer specific
for the 59 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 (antib and antia) in the anti-NOS transcripts
appear at this locus in the reversed order to that of the
corresponding sense regions in the nNOS gene (a and
b) 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.
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 ra-
Evolution by DNA Inversion
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 antia and
antib in the anti-NOS locus and a and b 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 exonintron 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 1 6, ex 1 7 and ex 1 8) and two introns (ix 1 6 and ix 1 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 1 2 and intron
ix 1 1 of the ancestral gene.
tionale 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 antiNOS 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 in-
1231
verted 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 antiNOS1 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 1999) designed to identify the regulatory elements critical for
transcription. Crucially, the intergenic region in the antiNOS 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
Generally, DNA inversions are known for their
devastating effects on the structural and functional integrity of genes in eukaryotic cells (Lupski 1998). 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
1232
Korneev and O’Shea
VIII (Lakich et al. 1993). Another example is Hunter
syndrome, which results from an inversion of the gene
encoding iduronate-2-sulfatase (Bondeson et al. 1995).
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. 1990; Muller et al. 2000; Seoighe et
al. 2000; Podemski, Ferrer, and Locke 2001). 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. 2000). 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 2001). 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 1999); 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 presentday 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 antisensecontaining 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 1999) 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. 1996;
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 1999). 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.
Crane et al. 1998), 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 1995). 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. 2001).
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
Evolution by DNA Inversion
inversions, and perhaps other types of major DNA rearrangements, occurring within duplicated sequences
soon after they are created.
Supplementary Material
GenBank accession number for the antiNOS-2
cDNA is AF373019. GenBank accession number for an
inverted region of the anti-NOS locus is AF373020.
Acknowledgments
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.
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KEN WOLFE, reviewing editor
Accepted February 25, 2002