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An ancient immunity gene duplication in Daphnia magna: RNA expression and sequence analysis of two nitric oxide synthase genes.

Labbé P, McTaggart SJ, Little TJ - Dev. Comp. Immunol. (2009)

Bottom Line: Both genes bear features commonly found in invertebrate NOS, however, the two genes differ in their rate of evolution, intraspecific polymorphism and expression level.We tested whether the more rapid evolution of NOS2 could be due to positive selection, but found the rate of amino-acid substitutions between Daphnia species to be compatible with a neutral model.A second experiment indicated that NOS transcription does not increase following exposure to Pasteuria.

View Article: PubMed Central - PubMed

Affiliation: University of Edinburgh, Institute of Evolutionary Biology, School of Biological Sciences, Ashworth Laboratory, Kings Buildings, Edinburgh, UK. Pierrick.Labbe@ed.ac.uk

ABSTRACT
NO (nitric oxide) is a highly reactive free radical gas thought to play a major role in the invertebrate immune response by harming pathogens and limiting their growth. Here we report on studies of nitric oxide synthase (NOS) genes in the crustacean Daphnia, one of the few non-insect arthropod models used to study host-pathogen interactions. While the NOS gene is found as a single copy in other invertebrates, we found two copies (NOS1 and NOS2), which a phylogenetic reconstruction showed to be the result of an ancient duplication event. Both genes bear features commonly found in invertebrate NOS, however, the two genes differ in their rate of evolution, intraspecific polymorphism and expression level. We tested whether the more rapid evolution of NOS2 could be due to positive selection, but found the rate of amino-acid substitutions between Daphnia species to be compatible with a neutral model. To associate NOS or NO activity with infection, we performed infection experiments with Daphnia magna and one of its natural pathogens (the bacterium Pasteuria ramosa). In one set of experimental infections, we supplemented D. magna with L-arginine, the NOS substrate, or with L-NAME, a NOS antagonist, and found this to result in lower and higher infection levels, respectively, which is at least compatible with the notion that NO may aid defence against Pasteuria. A second experiment indicated that NOS transcription does not increase following exposure to Pasteuria. Thus, the function of NOS in Daphnia immunity remains uncertain, but the pattern of gene duplication and subsequent divergence suggests evolution via neo- or subfunctionalization.

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Nucleotide (above) and deduced amino-acid (below) sequence of D. magna (A) NOS1 and (B) NOS2. The nucleotide sequences are numbered from the first base at the 5′ end of the transcript. The first methionine (M) is numbered on the first deduced amino-acid of the transcript and the Stop codon is also highlighted (Stop). The hydrophobic residues corresponding to Ca2+-dependent clamodulin-binding 1-5-8-14 Type A motif [43] are boxed. The polyadenylation (AATAAA) signal is black highlighted. As expected, no signal peptide was detected using the online software SignalP 3.0 in any of the protein sequences [http://www.cbs.dtu.dk/services/SignalP/, [56]].
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fig1: Nucleotide (above) and deduced amino-acid (below) sequence of D. magna (A) NOS1 and (B) NOS2. The nucleotide sequences are numbered from the first base at the 5′ end of the transcript. The first methionine (M) is numbered on the first deduced amino-acid of the transcript and the Stop codon is also highlighted (Stop). The hydrophobic residues corresponding to Ca2+-dependent clamodulin-binding 1-5-8-14 Type A motif [43] are boxed. The polyadenylation (AATAAA) signal is black highlighted. As expected, no signal peptide was detected using the online software SignalP 3.0 in any of the protein sequences [http://www.cbs.dtu.dk/services/SignalP/, [56]].

Mentions: NO may be one of the most general immunity effectors [2], and yet, compared to antimicrobial peptides (AMP) or prophenoloxidase (proPO, [39,40–42]), studies of NO are limited. NOS, the enzyme responsible for NO production, usually occurs as a single copy (e.g. [7–11]), with the exception of D. pulex where two gene copies encoding NOS are present [12]. In D. magna, we also found two genes encoding NOS proteins (Dmag-NOS1 and Dmag-NOS2). We assembled a total of 4141 bp and 3438 bp of cDNA for Dmag-NOS1 and Dmag-NOS2, respectively. Dmag-NOS1 contains a 67 bp 5′ untranslated region (UTR), a 528 bp 3′ UTR containing the poly-A tail and the polyadenylation signal, and an open-reading frame (ORF) of 3546 bp corresponding to a deduced protein of 1182 amino-acids (MW = 132.02 kDa, pI = 6.05, Fig. 1A). Dmag-NOS2 contains a 102 bp 5′ UTR, a short 87 bp 3′ UTR containing the poly-A tail and the polyadenylation signal, and an ORF of 3249 bp corresponding to a deduced protein of 1083 amino-acids (MW = 122.97 kDa, p I = 8.67, Fig. 1B). Both Dmag-NOS1 and Dmag-NOS2 possess conserved domains and active sites typical of NOS proteins. In particular the four hydrophobic residues corresponding to the Ca2+-dependent calmodulin-binding motif were found in both sequences (Fig. 1A and B; [43]). The sequences of Dmag-NOS1 and Dmag-NOS2 have been deposited in NCBI GenBank under the accession number FJ593039 and FJ593040, respectively.


An ancient immunity gene duplication in Daphnia magna: RNA expression and sequence analysis of two nitric oxide synthase genes.

Labbé P, McTaggart SJ, Little TJ - Dev. Comp. Immunol. (2009)

Nucleotide (above) and deduced amino-acid (below) sequence of D. magna (A) NOS1 and (B) NOS2. The nucleotide sequences are numbered from the first base at the 5′ end of the transcript. The first methionine (M) is numbered on the first deduced amino-acid of the transcript and the Stop codon is also highlighted (Stop). The hydrophobic residues corresponding to Ca2+-dependent clamodulin-binding 1-5-8-14 Type A motif [43] are boxed. The polyadenylation (AATAAA) signal is black highlighted. As expected, no signal peptide was detected using the online software SignalP 3.0 in any of the protein sequences [http://www.cbs.dtu.dk/services/SignalP/, [56]].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724039&req=5

fig1: Nucleotide (above) and deduced amino-acid (below) sequence of D. magna (A) NOS1 and (B) NOS2. The nucleotide sequences are numbered from the first base at the 5′ end of the transcript. The first methionine (M) is numbered on the first deduced amino-acid of the transcript and the Stop codon is also highlighted (Stop). The hydrophobic residues corresponding to Ca2+-dependent clamodulin-binding 1-5-8-14 Type A motif [43] are boxed. The polyadenylation (AATAAA) signal is black highlighted. As expected, no signal peptide was detected using the online software SignalP 3.0 in any of the protein sequences [http://www.cbs.dtu.dk/services/SignalP/, [56]].
Mentions: NO may be one of the most general immunity effectors [2], and yet, compared to antimicrobial peptides (AMP) or prophenoloxidase (proPO, [39,40–42]), studies of NO are limited. NOS, the enzyme responsible for NO production, usually occurs as a single copy (e.g. [7–11]), with the exception of D. pulex where two gene copies encoding NOS are present [12]. In D. magna, we also found two genes encoding NOS proteins (Dmag-NOS1 and Dmag-NOS2). We assembled a total of 4141 bp and 3438 bp of cDNA for Dmag-NOS1 and Dmag-NOS2, respectively. Dmag-NOS1 contains a 67 bp 5′ untranslated region (UTR), a 528 bp 3′ UTR containing the poly-A tail and the polyadenylation signal, and an open-reading frame (ORF) of 3546 bp corresponding to a deduced protein of 1182 amino-acids (MW = 132.02 kDa, pI = 6.05, Fig. 1A). Dmag-NOS2 contains a 102 bp 5′ UTR, a short 87 bp 3′ UTR containing the poly-A tail and the polyadenylation signal, and an ORF of 3249 bp corresponding to a deduced protein of 1083 amino-acids (MW = 122.97 kDa, p I = 8.67, Fig. 1B). Both Dmag-NOS1 and Dmag-NOS2 possess conserved domains and active sites typical of NOS proteins. In particular the four hydrophobic residues corresponding to the Ca2+-dependent calmodulin-binding motif were found in both sequences (Fig. 1A and B; [43]). The sequences of Dmag-NOS1 and Dmag-NOS2 have been deposited in NCBI GenBank under the accession number FJ593039 and FJ593040, respectively.

Bottom Line: Both genes bear features commonly found in invertebrate NOS, however, the two genes differ in their rate of evolution, intraspecific polymorphism and expression level.We tested whether the more rapid evolution of NOS2 could be due to positive selection, but found the rate of amino-acid substitutions between Daphnia species to be compatible with a neutral model.A second experiment indicated that NOS transcription does not increase following exposure to Pasteuria.

View Article: PubMed Central - PubMed

Affiliation: University of Edinburgh, Institute of Evolutionary Biology, School of Biological Sciences, Ashworth Laboratory, Kings Buildings, Edinburgh, UK. Pierrick.Labbe@ed.ac.uk

ABSTRACT
NO (nitric oxide) is a highly reactive free radical gas thought to play a major role in the invertebrate immune response by harming pathogens and limiting their growth. Here we report on studies of nitric oxide synthase (NOS) genes in the crustacean Daphnia, one of the few non-insect arthropod models used to study host-pathogen interactions. While the NOS gene is found as a single copy in other invertebrates, we found two copies (NOS1 and NOS2), which a phylogenetic reconstruction showed to be the result of an ancient duplication event. Both genes bear features commonly found in invertebrate NOS, however, the two genes differ in their rate of evolution, intraspecific polymorphism and expression level. We tested whether the more rapid evolution of NOS2 could be due to positive selection, but found the rate of amino-acid substitutions between Daphnia species to be compatible with a neutral model. To associate NOS or NO activity with infection, we performed infection experiments with Daphnia magna and one of its natural pathogens (the bacterium Pasteuria ramosa). In one set of experimental infections, we supplemented D. magna with L-arginine, the NOS substrate, or with L-NAME, a NOS antagonist, and found this to result in lower and higher infection levels, respectively, which is at least compatible with the notion that NO may aid defence against Pasteuria. A second experiment indicated that NOS transcription does not increase following exposure to Pasteuria. Thus, the function of NOS in Daphnia immunity remains uncertain, but the pattern of gene duplication and subsequent divergence suggests evolution via neo- or subfunctionalization.

Show MeSH
Related in: MedlinePlus