Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton
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Abstract |
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Introduction |
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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 oxidesignaling 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 (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
).
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Materials and Methods |
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Northern Blot Hybridization
A blot containing poly(A)+ RNA extracted from Lymnaea CNS was prepared as described previously (Korneev, Park, and O'Shea 1999
). Hybridization with the 32P-labeled probe containing a fragment of the 3' untranslated region of antiNOS-2 cDNA (positions 14262100) 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 1999
) 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 0.50.8 (marginal prediction) contains about 65% of the true promoter sequence. For a sequence scoring 0.81.0 (medium likely prediction) this figure is about 80%, and for a region scoring above 1.0 (highly likely prediction) it reaches 95%.
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Results |
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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) in the anti-NOS transcripts appear at this locus in the reversed order to that of the corresponding sense regions in the nNOS gene (
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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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 1999
) 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.
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Discussion |
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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 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 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
; 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 vitrogenerated 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
).
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Keywords: 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. s.korneev{at}sussex.ac.uk
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