Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187
Isolation and characterization of cDNA encoding Argonaute,
a component of RNA silencing in shrimp (Penaeus monodon) ☆
Sasimanas Unajak a,b , Vichai Boonsaeng a , Sarawut Jitrapakdee a,b, ⁎
a Center of Excellence for Shrimp Biotechnology, Mahidol University, Bangkok 10400, Thailand
b Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Rd, Bangkok 10400, Thailand
Received 12 February 2006; received in revised form 28 June 2006; accepted 6 July 2006
Available online 13 July 2006
We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila
melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated
molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that
exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D.
melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate
Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference
(RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in
intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2–3-fold increased during the early period of viral infection
but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction
of Pm Ago1 mRNA suggesting that its expression is associated with virus infection.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Argonaute; Differential display RT-PCR; Penaeus monodon; RNA interference; Gene silencing; Yellow head virus; Immune system; RNA virus
Shrimp, like other crustaceans, do not possess an adaptive
immune response, but rely primarily on the innate immune system
including the hemolymph coagulation system (Yeh et al., 1998,
1999), production of anti-bacterial peptides such as penaedins
(Destoumieux et al., 1997), anti-lipopolysaccharide factor
(Somboonwiwat et al., 2005), and the melanization reaction
through the production of prophenol oxidase cascade system
(Perazzolo and Barracco, 1997; Söderhäll, 1999; Sritunyalucksana
et al., 1999) to respond to bacterial or fungal infections.
These mechanisms, in turn, prevent the spread of these pathogens
to the body. However, there are few reports on the defense
molecules produced by shrimp upon infection by viruses. Luo
☆ The nucleotide sequence reported in this paper has been submitted to
GenBank with accession number DQ343133.
⁎ Corresponding author. Tel.: +66 2 201 5460; fax: +66 2 354 7174.
E-mail address: email@example.com (S. Jitrapakdee).
et al. (2003) identified a group of genes that are differentially
expressed in shrimp infected with the white spot syndrome virus
(WSSV). One of them is the PmAV, an antiviral gene. This gene
encodes a 170-amino acid polypeptide with a C-type lectin-like
domain whose function remains to be elucidated. Using a similar
approach, a group of housekeeping genes has been isolated from
WSSV-resistant shrimp, and an interferon-like protein with
antiviral activity has been identified from hemocytes (He et al.,
Here we employed the differential display reverse transcriptase-PCR
technique to identify a group of genes in the lymphoid
organs of Penaeus monodon shrimp that respond to RNA virus
infection. Of particular interest, we identified a cDNA homologue
of human/Drosophila melanogaster Argonaute proteins.
The Argonaute protein family is a group of proteins that are
conserved from fission yeasts to human. Argonaute protein 1
(Ago1) and several other members of this protein family play
crucial role in stem cell differentiation or RNA interference
(RNAi). Members of this family consist of proteins with an N-
1096-4959/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
180 S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187
terminal PAZ and C-terminal PIWI domain. In D. melanogaster
and C .elegans, Ago1 and Ago2 proteins appear to play a key
role in double stranded RNA-induced post-transcriptional gene
silencing (Carmell et al., 2002). Here we also present evidence
that the expression of shrimp putative Argonaute is highly
expressed in lymphoid organ, and that the expression of this
gene is correlated with viral infection.
2. Materials and methods
2.1. Experimental animals and sample preparation
Shrimp (P. monodon)(20–25 g) from local farms in Thailand
were used as a source throughout the experiments. Each white
spot syndrome virus (WSSV)–or yellow head virus (YHV)–free
shrimp was examined by PCR using IQ2000 WSSV Detection
and Prevention System and IQ2000 YHV/GAV Detection and
Typing System (Farming IntelliGene Technology Corporation,
Taiwan). The virus-free shrimp were acclimatized in brackish
water for one day before being injected intramuscularly with
100 μL of 1:100 dilution of YHV suspension in LHM medium
(1.8 M NaCl, 61 mM CaCl 2 , 5 mM KCl, 15 mM MgCl 2 ,0.3mM
Na 2 H 2 PO 4 ).
For the bacterial challenge, a single colony of Vibrio harveyi
(114GL) grown on tryptic soy agar (supplemented with 1.5%
NaCl) (Difco) was inoculated in Müller–Hinton broth (Difco)
and incubated overnight at 30 °C with shaking. The next day, the
bacteria were washed 3 times with sterile phosphate buffer and
diluted to an optical density at 600 nm (OD 600 ) of 0.5. 100 μLof
bacterial suspension were injected intramuscularly into the live
shrimp and their lymphoid organs were collected at 6, 10 and
11 h post infection. The presence of V. harveyi in the infected
lymphoid organs after infection was examined by smearing the
lymphoid organ to the TCBS (thiosulphate, citrate, bile salts,
sucrose agar) agar (Difco). Plates were incubated for 18–24 h at
30 °C to examine the blue or blue-green colonies of V. harveyi.
2.2. RNA isolation
Pooled lymphoid organs from shrimp infected with YHV for
6, 12, 18, 24, 30, 48, 60 h or those infected with V. harveyi for 6,
10, 11 h and from the moribund shrimp were homogenized in
TRI reagent (Pacific science) and snap-frozen in liquid
nitrogen. Chloroform (0.2 vol) was added to the frozen samples.
The RNA was precipitated from an aqueous phase by adding
0.5 vol of chilled isopropanol, before being washed with 75%
(v/v) ethanol, dried and resuspended in 20 μLdiethylpyrocarbonate
(DEPC)-treated water. The RNA concentrations were
determined by spectrophotometry, and the integrity was verified
by 1% formaldehyde agarose gel electrophoresis. Contaminating
DNA in RNA samples was eliminated by digestion with the 10 U/
μL DNaseI (Amersham Bioscience) at 37 °C for 1 h.
2.3. Differential display RT-PCR (ddRT-PCR)
The Delta® Differential Display kit (BD Bioscience) was
used to identify the differentially expressed RNA transcripts.
Briefly, first stranded cDNAs were synthesized with Moloney
murine leukemia virus reverse transcriptase (MMLV) from 2 μg
of total RNA extracted from mock- or YHV-infected shrimp
using random hexamer primers. The PCR was performed in a
20-μL final volume containing 1 μM of each forward and
reverse primer, 50 μM of each dNTP, and 50 nM [α- 32 P]dATP
(specific activity of 3000 Ci/mmol; Amersham Bioscience) with
the supplied buffer. The thermal cycle consisted of an initial
cycle of 94 °C for 5 min, 40 °C for 5 min, 68 °C for 5 min,
followed by 5 cycles of 94 °C for 2 min, 40 °C for 5 min, 68 °C
for 5 min; 25 cycles of 94 °C for 1 min, 60 °C for 1 min, 68 °C
for 2 min; and 68 °C for 7 min. The reactions were terminated
by mixing with a denaturing loading dye (95% formamide/0.2%
bromophenol blue/0.2% xylene cyanol), and heated at 95 °C for
10 min and chilled. The samples were then subjected to 6%
(w/v) polyacrylamide −6 M urea gel electrophoresis for 6 h at
70 W. The gel was transferred to Whatman paper, dried and
autoradiographed for 48 h.
2.4. Identification of differential display products
To recover the differentially expressed cDNA bands, the
autoradiogram was aligned with the dried gel and the marked
fragments were excised and eluted in 50 μL sterile water before
heating at 100 °C for 5 min. The eluted DNA was re-amplified
in a 50 μL reaction mixture using the same set of primer used in
the ddRT-PCR. The reaction was performed with 8 μL of eluted
DNA, 1× PCR buffer (Advantage cDNA polymerase mix, BD
Bioscience), 50 μM of each dNTP, 1 μM each P and T primers
(supplied in the kit) and 1 unit of Advantage polymerase mix.
Cycling parameters were 94 °C, 2 min, 25 cycles of 94 °C,
1 min, 60 °C, 1 min, 68 °C, 2 min and 68 °C, 7 min. The PCR
products were purified using a QiaQuick gel extraction kit
(Qiagen) before cloning into pGEM®-T Easy vector (Promega).
The nucleotide sequences were determined in both directions by
automated sequencing. The nucleotide sequences were analyzed
against the GenBank database using the Basic Local
Alignment Search Tool (BLAST X) (Thompson et al., 1997).
2.5. RACE PCR
Rapid amplification of cDNA ends (RACE) was performed
using SMART RACE cDNA amplification Kit, (BD Bioscience).
The sequences of specific primer were designed for the 5′
or 3′ ends of cDNAs (Table 1). The PCR profile consisted of
5 cycles of 94 °C for 30 s, 72 °C for 1 min and 5 cycles of 94 °C for
30 s, 70 °C for 30 s, 72 °C for 1 min, followed by 25 cycles of
94 °C for 30 s, 68 °C for 30 s, 72 °C for 1 min and 72 °C for 7 min.
All RACE PCR products were cloned into pGEM® T-Easy vector
(Promega) and subjected to automated sequencing in both
2.6. Semi-quantitative reverse transcriptase polymerase chain
The expression of shrimp Argonaute (Ago1) and β-actin
genes in lymphoid organs was compared between the mock-
S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187
Primers used for the PCR
and the YHV-infected shrimps using semi-quantitative RT-PCR
with specific primer set of 162F and 162R for Argonaute gene
or with primers actinF and actinR for β-actin gene (Table 1).
The RNAs were subjected to a one-step RT-PCR using
SuperScript III reverse transcriptase (Invitrogen) with
100 ng of RNA. The PCR amplified in a 25 μL-reaction
volume containing 1× PCR buffer, 1.6 mM MgSO 4 , 200 μM of
each dNTP, 0.2 μM of each forward and reverse primers and
1 μL of SuperScript III RT/Platinum® Taq Mix. Reverse
transcription was carried out at 50 °C for 30 min followed by
94 °C for 2 min. The cycling parameters were 25 cycles of 94 °C
for 30 s, 55 °C for 30 s, 72 °C for 30 s and 72 °C for 7 min. PCR
products were separated on 1.4% NuSieve 3:1 agarose (FMC
from this technique. One of the differentially expressed bands
(PCR1) when sequenced showed that it encoded a protein with
71.4% and 76.9% respective similarity to the Argonaute protein
from human (GenBank accession no. Q9UL78) and D.
melanogaster (GenBank accession no. BAA88078.1). This
band was down-regulated upon YHV-infection.
3.2. Cloning of putative Pm Ago1 cDNA
To further characterize this cDNA, we performed 5′- and 3′-
RACE PCR to obtain its 5′- and 3′-ends using cDNAs prepared
from YHV-infected shrimp. Upon amplification 502 bp (named
162_5_AS6), 762 bp (162_5_AS3) and 740 bp (162_5_AS1)
2.7. Phylogenetic tree construction
ClustalX (Thompson et al., 1997) was used to align amino
acid sequences and generate bootstrapping trees. GenBank
accession numbers are: Dme, D. melanogaster; DmeAgo1
(BAA88078), DmeAgo2 (Q9VUQ5), Gga, Gallus gallus;
GgaAgo2 (XP_418421), GgaAgo3 (NP_001026071),
GgaAgo4 (XP_417776), XlaAgo, Xenopus laevis
(AAH77863), Homo sapiens; HsaAgo1 (Q9UL78), HsaAgo2
(Q9UKV8), HsaAgo3 (Q9H9G7), HsaAgo4 (NP_060099)
AgaAgo, Anopheles gambiae; (Eaa00062), Dre, Danio rerio;
DreAgo1 (XP_699226), DreAgo3 (XP_696563), DreAgo4
(XP_691861), Ath, Arabidopsis thaliana, AthAgo1
(NP_175274), AthAgo4 (NP_565633), Mmu, Mus musculus;
MmuAgo1 (Q8CJG1), MmuAgo2 (Q8UKV8), MmuAgo3
(Q8CJF9), MmuAgo4 (Q8CJF8), Cel, Caenorhabditis elegans;
CelAlg1 (NP_510322.2), CelAlg2 (NP_871992.1).
3.1. Differential display of normal and infected shrimp gene
We employed differential display PCR from the cDNAs
prepared from normal and infected shrimp with 90 pairs of
arbitrary primers supplied with the differential display PCR kit.
Approximately, 161 bands showed differential expression
between the normal and the YHV-infected shrimp. Fig. 1
shows an example of the differentially displayed bands obtained
Fig. 1. Representative ddRT-PCR generated from PCR with different sets of
primers shown in Table 1. Total RNAs were isolated from the lymphoid organs
of mock- or YHV-infected shrimp collected at 6 h post infection. Arrows
indicate differentially expressed transcripts. M1 is a 100 bp DNA marker; C,
mock-infected shrimp; V, YHV-infected shrimp. T and P indicate the name of
arbitrary and anchor primers used in each reaction.
182 S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187
PCR products were obtained from 5′-RACE while a 776 bp
PCR product (163_3_S1) was obtained from 3′-RACE. These 4
RACE clones together with PCR1 clone were overlapped and
spanned the entire coding region of the putative cDNA. The full
length of the cDNA comprised 3178 nucleotides including the
5′-untranslated region, coding sequence, 3′-untranslated region
and poly(A) tail (Fig. 2). The longest open reading frame
commenced at nucleotide 115 and ended at nucleotide 2931,
encoding a putative 939-amino acid polypeptide with calculated
molecular weight (Mr) of 104 kDa and pI of 9.49. Using a
potential initiation codon at position 115, 62 amino acid
residues at the N-terminus showed relatively low homology to
argonaute proteins in the database, while a second potential
initiation codon located at nucleotide 309 gave much higher
similarity. Therefore, it is unclear whether the authentic
initiation codon is located at nucleotide position 115 or 309.
The amino acid sequence inferred from nucleotide position 309
to the stop codon showed 73.8% identity with D. melanogaster
Fig. 2. Nucleotide and deduced amino acid sequences of shrimp Ago1 cDNA. (GenBank accession no. DQ343133). A, schematic diagram to show the strategy for isolating
the full length shrimp Ago1 cDNA. PCR1 fragment was initially isolated by the differential display PCR while the other four overlapping fragments, 162_5_AS6,
162_5_AS3, 162_5_AS1 and 162_3_S1, spanning an entire coding region of Pm Ago1 cDNAwere isolated by 5′-and3′-RACE PCR. Each fragment was named after the
primers used in the PCR amplification. B, nucleotide and inferred amino acid sequences of Pm Ago1. Nucleotide sequences are numbered while the amino acid number is in
bold type. Amino acids are shown in a single capital letter.
S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187
Ago1 protein (GenBank accession no. BAA88078.1), and
68.4% with human translation initiation factor 2C1 (eIF2C1),
also called Argonaute-1 (GenBank accession no. Q9UL78). The
highest homologies were observed between residues 280–401
and 569–897, that correspond to the PAZ and the PIWI domains
of the mammalian and Drosophila proteins, as shown in Fig. 3.
Fig. 3. Alignment of the amino acid sequences of shrimp Ago1 and other species' Ago. The amino acid sequence of Pm Ago1 was aligned with sequences as follows: Aga,
A. gambiae (GenBank accession no. EAA00062), Dme, D. melanogaster (GenBank accession no. BAA88078), Hsa, Human; (GenBank accession no. Q9UL78), Mmu, M.
musculus (GenBank accession no. Q8CJG1), Xla, X. laevis (GenBank accession no. AAH77863), using ClustalW. Gaps were introduced to maximize the alignment. The
highly conserved residues are shown as white text against shaded background. PAZ and PIWI domains are shown by open boxes.
184 S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187
Interestingly, P. monodon Argonaute contains an extra 27
amino acid (NGSTTQGQSASDGSRPRQLTFARTAHD) in the
PIWI domain. This insertion is not present in Argonaute
proteins of other species.
3.3. Evolution of Argonaute protein of P. monodon
Phylogenetic analysis was performed to characterize the
evolutionary relationships of the Pm Ago and other invertebrate
and invertebrate Argonaute protein subtypes using the CLUS-
TAL W and PHYLIP programs. As shown in Fig. 4, three
clusters in the phylogenetic tree were identified which included
group 1 (X. laevis Ago1, D. rerio Ago1, Pm Ago1, D.
melanogaster Ago1 and 2, A. gambiae Ago, C. elegans Ago1
and 2, A. thaliana Ago1 and 4), group 2 (G. gallus Ago2, M.
musculus Ago2 and H. sapiens Ago2) and group 3 (G. gallus
Ago3 and 4, M. musculus Ago 1, 3, D. rerio Ago3 and 4, H.
sapiens Ago 1, 3 and 4). As Pm Ago1 appears to be clustered into
group 1 and is more closely related to the insect rather than
vertebrate Argonautes, we therefore suggest that Pm Ago belongs
to Ago1 subfamily.
3.4. Expression of Pm Ago1 in various tissues
To determine whether Pm Ago1 mRNA was expressed in a
tissue-specific manner, we performed a semi-quantitative RT-
PCR of mRNA in various tissues including gill, heart,
hemocytes, hepatopancreas, intestine, pleopod, lymphoid
organ and muscle of normal shrimp. As shown in Fig. 5A, the
expression of Pm Ago1 mRNA showed tissue-specific expression.
It is highly expressed in lymphoid organ, expressed at
a low level in hemocytes, intestine, pleopod and muscle, but
was barely detectable in gill, heart and hepatopancreas.
3.5. Expression of Pm Ago1 in YHV infected lymphoid organ
We next examined whether the expression of Pm Ago1 is
affected by an RNA virus. We used semi-quantitative RT-PCR
to detect the expression level of Pm Ago1 mRNA in lymphoid
organ of shrimp infected with YHV at various time points. As
shown in Fig. 5B and C, the expression level of Pm Ago1 was
increased to 3-fold at 24 h post infection. However, the level of
Pm Ago1 mRNA rapidly declined at 30 h and onward,
becoming undetectable in the moribund shrimp (60 h). In
contrast the expression of Pm Ago1 in mock-infected shrimp
was unchanged at various time points. Infection of shrimp with
the white spot syndrome virus (WSSV) yielded similar results
i.e. the level of Pm Ago1 mRNA was decreased at the late stage
of infection, becoming undetectable in moribund shrimp (data
We next investigated whether the alteration in the level of Pm
Ago1 mRNA expression is specific to the viral infection. We
Fig. 4. Phylogenetic analysis of Argonaute proteins. Multiple alignment was constructed by ClustalX (version 1.83) (Thompson et al., 1997). Treeview was used in tree
construction. GenBank accession numbers are: Dme, D. melanogaster; DmeAgo1 (GenBank accession no. BAA88078), DmeAgo2 (GenBank accession no.
Q9VUQ5), Gga, G. gallus; GgaAgo2 (GenBank accession no. XP_418421), GgaAgo3 (GenBank accession no. NP_001026071), GgaAgo4 (GenBank accession no.
XP_417776), XlaAgo, X. laevis (GenBank accession no. AAH77863), Has, H. sapiens; HsaAgo1 (GenBank accession no. Q9UL78), HsaAgo2 (GenBank accession
no. Q9UKV8), HsaAgo3 (GenBank accession no. Q9H9G7), HsaAgo4 (GenBank accession no. NP_060099) AgaAgo, A. gambiae; (GenBank accession no.
Eaa00062), Dre, Danio rerio; DreAgo1 (GenBank accession no. XP_699226), DreAgo3 (GenBank accession no. XP_696563), DreAgo4 (GenBank accession no.
XP_691861), Ath, A. thaliana, AthAgo1 (GenBank accession no. NP_175274), AthAgo4 (GenBank accession no. NP_565633), Mmu, M. musculus; MmuAgo1
(GenBank accession no. Q8CJG1), MmuAgo2 (GenBank accession no. Q8UKV8), MmuAgo3 (GenBank accession no. Q8CJF9), MmuAgo4 (GenBank accession
no. Q8CJF8), Cel, C. elegans; CelAlg1 (GenBank accession no. NP_510322.2), CelAlg2 GenBank accession no. (NP_871992.1).
S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187
Fig. 5. Expression of Pm Ago1 in different tissues of shrimp. (A) Total RNA
from various tissues of normal shrimp were subjected to semi-quantitative RT-
PCR. These samples were subjected to RT-PCR using specific primers for Pm
Ago1 or β-actin genes. 1, gill; 2, heart; 3, hemocytes; 4, hepatopancreas; 5,
intestine; 6, pleopod; 7, lymphoid organ; 8, muscle; 9, negative control. (B)
Time course expression of Pm Ago1 mRNA at 0, 6, 12, 18, 24, 30, 48 and 60 h
(moribund) upon infection by YHV (upper panel) or mock-infected (lower
panel). (C) The abundance of Pm Ago1 mRNA of YHV-infected shrimp at
above time points is normalized with β-actin and is shown as relative gene
expression+standard deviations of three independent experiments. The relative
gene expression detected at 0 h was arbitrarily set as 1. In each experiment 3
shrimp were infected at each time point and the lympoid organs were combined
for RNA extraction. (D) Expression of Pm Ago1 in lymphoid organs of shrimp
infected with V. harveyi. Normal shrimp were infected with 0.5 OD of V. harveyi
and the lymphoid organs were collected at 6 and 19 h post infection. Total RNA
samples were extracted from lymphoid organs and subjected to RT-PCR using
specific primers for Pm Ago1 or β-actin genes.
infected shrimp with bacterial pathogen, V. harveyi, and
followed the expression of Pm Ago1 mRNA at different time
points. As shown in Fig. 5D, bacterial infection did not cause
the reduction of Pm Ago1 mRNA seen throughout the infection
as in the case of viral infection. These data suggest that the rapid
fall of Pm Ago1 mRNA towards the late phase of infection is
specific to the virus but not the bacteria.
It is known that crustaceans, including shrimp, lack the humoral
immune response against invading pathogen(s), but contain the
innate immune system which is an ancient defense mechanism.
This includes phagocytic activity, encapsulation and the release of
the prophenoloxidase system, production of antibacterial peptides
and a protease inhibitor. All of these mechanisms are mediated by
circulating hemocytes in the hemolymph. Recent studies showed
that the lymphoid organ is a major organ which contains
exocytosed, granular cells that have phagocytosed foreign
materials, particularly viruses, suggesting that the lymphoid
organ constitutes a major site for penaeid antiviral defense (Hasson
et al., 1999; Anggraeni and Owens, 2000). Using subtraction
hybridization and differential hybridization, He et al. (2005) were
able to isolate a group of housekeeping genes including cofilin,
translational control tumor protein (TCTP), hepatic lectin and
interferon-like protein that are up-regulated in the WSSV resistant
shrimp. These gene products were suggested to play important roles
in the antiviral process. However, the biological activities of these
gene products with respect to an antivirus mechanism in shrimp
remain unknown. Luo et al. (2003) isolated an antiviral gene using
differential display PCR, similar to our approach. They isolated
PmAV as an important defense molecule with antiviral activity.
Taken together, it appears that shrimp utilize an innate immune
system via the network of various defense molecules to protect
itself from viral infection. We have undertaken a differential display
approach to identify a cDNA encoding the Argonaute protein from
P. monodon shrimp as one of various genes that are differentially
expressed during viral infection.
The Argonaute protein family is composed of highly conserved
proteins whose functions are implicated in the mechanism of RNA
interference, and the development and maintenance of stem cell
fate determination. Here, we report the cloning and characterization
of cDNA encoding a putative Argonaute protein from a
crustacean for the first time. The inferred amino acid sequence of
to Ago1/eukaryotic initiation factor 2C1 (eIF2C1), a member of
the Argonaute protein family, and provided the first evidence that
the cDNA clone we isolated is indeed a member of this protein
family. Secondly, the putative Pm Ago1 possesses a molecular
weight and pI similar to the eIF2C1 of higher eukaryote species,
i.e. they form a conserved family of ∼100 kDa highly basic
proteins, characterized by an N-terminal PAZ domain and a C-
terminal PIWI domain (Carmell et al., 2002). Structural
determinations by nuclear magnetic resonance and X-ray
crystallography demonstrate that the PAZ domain of Ago1 and
Ago2 of D. melanogaster contains a nucleic acid binding domain
while the PIWI domain is less well characterized. Recently, the
structure of the PIWI domain of the archaebacterium, Pyrococcus
furiosis, has been shown to adopt an RNase H fold, similar to the
RNase H from E. coli (Lingel and Izaurralde, 2004). In fission
yeasts, only one isoform of Ago has been identified and this
protein plays a crucial role in meiotic gene silencing (Volpe et al.,
2002). However, in multicellular organisms, this gene family has
increased up to 24 members as in the case of C. elegans (Carmell
et al., 2002). In D. melanogaster, Ago1 and Ago2 are required for
186 S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187
an efficient RNAi system (Williams and Rubin, 2002; Pal-Bhadra
et al., 2002). There is only one member of the Argonaute family,
Ago1 present in the protozoan parasite, Trypanosoma brucei and
this protein is indeed essential for RNAi (Shi et al., 2004).
However, in A. gambiae, five members of Argonaute proteins
have been identified but only Ago2 and Ago3 are involved in
RNAi (Hoa et al., 2003). In human, up to 8 Argonaute proteins
have been reported. Although the function of each isoform of
human Agonaute protein is not yet clear, some family members
have been shown to be associated with the DICER protein of the
RNAi system (Sasaki et al., 2003; Liu et al., 2004).
As the lymphoid organ is one of the important antiviral tissues,
our finding that Pm Ago1 mRNA is highly expressed in this tissue
suggests that Pm Ago1 may play a defense role against viral
infection. An RNAi-like mechanism against viral infection in two
species of shrimp i.e. Liptopenaeus vannamei and P. monodon
has recently been reported (Robalino et al., 2004; Tirasophon
et al., 2005; Yodmuang et al., 2006). More recently, sequencespecific
antiviral protection or silencing of an endogenous shrimp
gene can be achieved after administration of the cognate double
stranded RNAs, suggesting that an RNAi-like mechanism exists
in shrimp (Robalino et al., 2005). In this present study, an
increased level of Pm Ago1 mRNA at 24 h post infection suggests
that an RNAi mechanism may operate efficiently during the early
stages of infection. However, down-regulation of Pm Ago1
mRNA during the late or towards the final stage (moribund) of
infection may reflect the failure of an RNA silencing system of the
host to inhibit viral replication. Robalino et al. (2004) have shown
that the efficiency of an RNA silencing system to suppress viral
infection in the shrimp L. vannamei can be overwhelmed by viral
load. The production of silencing suppressors from viruses via
transcriptional repression of key components and/or effector
molecules of RNA silencing during the late stage of infection
(Lecellier and Voinnet, 2004) may contribute to this observation.
Our report on the cloning of a cDNA encoding the putative Pm
Ago1 and determination of its expression during viral infection
provides supporting evidence to suggest that the RNA silencing
does occur in shrimp. Whether our reported Pm Ago1 is the only
member of this family that plays a crucial role in RNA interference
in shrimp, remains to be elucidated by a knock-down experiment.
The authors thank Mr. K. Manopwisedjaroen for technical
assistance with V. harveyi detection. We thank Professors T.W.
Flegel and J.C. Wallace, for critically reading the manuscript.
This work was partly supported by Mahidol University Grant.
S.U. was supported by the Royal Golden Jubilee Ph.D. program
(Grant No.PHD/0098/2546) from the Thailand Research Fund.
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cells in the penaeid prawn Penaeus monodon.Dis.Aquat.Org.40,85–92.
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