Indian Journal of Biotechnology
Vol 13, January 2014, pp 52-56
DNA binding domain of retinoid receptor gene (RXR) from a field crab
inhabiting the Indian peninsula
S N Sarika and Anilkumar Gopinathan*
Molecular Endocrinology Laboratory, School of Biosciences and Technology, VIT University, Vellore 632 014, India
Received 18 January 2013; revised 15 July 2013; accepted 25 August 2013
Retinoid receptors are ubiquitous throughout the animal kingdom both in invertebrates and vertebrates, including the
humans. In vertebrates, the retinoid X receptor (RXR) is suggested to be responsible for a variety of functions including
growth and reproduction. However, reports are very scarce on the exact function of the RXR in crustaceans. In the present
paper, we report the sequence information on the DNA binding domain (DBD) of the retinoid hormone receptor gene (RXR)
of a field crab, Paratelphusa sp. from the Indian peninsula. BLAST analysis of the RXR-DBD shows considerable sequence
homology with other brachyuran crabs, such as, Uca pugilator, Gecarcinus lateralis and Carcinus maenas. Taking cues
from the GenBank database, we could also draw out the identity of this domain of the RXR with major phylogenic groups,
including insects, molluscs and vertebrates. The sequence analysis of the RXR-DBD of the crab revealed remarkable
sequence similarity to crustaceans and vertebrates, as compared with those of the insects.
Keywords: Brachyuran crabs, Decapoda, DNA binding domain (DBD), nuclear receptor (NR), Paratelphusa, retinoid
receptor (RXR)
Introduction
Retinoid X receptor (RXR), a member of the nuclear
receptor (NR) super family, is suggested to play key
roles in the hormonal regulation of both vertebrates and
invertebrates. In humans, for example, several forms of
RXR are known to exist to control various metabolic
functions, including morphogenesis and cellular
differentiation1. Recent studies reveal that RXR (and its
ligands) could involve in anti-inflammatory activity in
humans2. Among invertebrates, especially the insects,
the ultraspiracle gene (USP) (a homolog of the RXR)
encoding the retinoid receptor is suggested to regulate
a wide variety of functions, such as, growth,
reproduction and development3.
In crustaceans, which are phylogenically close to
insects, retinoids―the putative ligands for the RXR
receptor―are known to be endogenous4. However, its
exact role in crustacean system is still enigmatic.
Studies on vertebrates reveal that the RXR share the
typical domain structure characteristic of NRs5.
Accordingly, the receptor molecule has 4 or 5
functional domains, such as, the highly variable A/B
domain, the C-domain or the DNA-binding domain
(DBD) that binds to the hormone-response element
——————
*Author for correspondence:
E-mail: [email protected]
(HRE) situated in the target gene promoter, the linker
D domain, the ligand binding E domain and the
terminal F domain6. The studies conducted on
vertebrates reveal that DBD also plays a pivotal role
in receptor dimerization, an essential step in hormone
action. Despite the previous attempts made on a
number of vertebrates and invertebrates (especially
the insects), nothing was known with respect to the
hormone receptors in crustaceans until 1996. For the
first time, DBD of the gene encoding the retinoid
receptor (RXR) was isolated, cloned and sequenced
from a crustacean, the fiddler crab (Uca pugilator)7,8.
Subsequently, more investigations were made on
crustacean DBDs including those of other brachyuran
crabs (Gecarcinus lateralis & Carcinus maenas),
shrimps (Marsupenaeus japonicus) and the
branchiopod (Daphnia magna)8-13.
Having been the most conserved domain of the NR
and having the ability to induce target gene
expression through interaction with specific NR
elements (NREs) present within the target gene
promoters, DBD is considered to be one of the most
functionally important domains in the hormone
receptor5,6. Further, its involvement in dimerization
with the other partner receptors (for instance, the
ecdysteroid receptor, EcR) has attracted the attention
of molecular endocrinologists. To our knowledge, this
SARIKA & GOPINATHAN: DBD OF RETINOID RECEPTOR GENE (RXR) FROM AN INDIAN FIELD CRAB
domain has been sequenced only in three brachyuran
(true) crabs, viz., U. pugilator (GenBank Acc. No.
U31832)7, G. lateralis (DQ067286)9 and C. maenas
(EU683889)10, inhabiting North American shores.
Significantly, no reports are available with respect to
the hormone receptors of the crustaceans inhabiting
the Indian peninsula, despite that it houses a rich
fauna of this class with highly diverse habits and
habitats. In the present paper, we present the first ever
report on the sequence information of the DBD of an
Indian variety of field crab Paratelphusa sp. The
paper also deals with the results of our comparative
study using bioinformatics tools to find homology
with other phylogenic groups.
Materials and Methods
Specimen Collection
Female adult crabs (Paratelphusa sp.), used for the
present study, were collected through conventional
bait technique from the rice farms and adjacent areas
of the Vellore Institute of Technology University
Campus, Vellore (12.56° N; 79.8°E), India. The
specimens were maintained in the laboratory in plastic
cisterns, laid with wet sand. In most of the instances, the
crabs were sacrificed as soon as they were brought to
the laboratory.
Tissue Collection for RNA Isolation
The crabs were dissected in crustacean
physiological saline14. The ovary, being the putative
target for RXR, was chosen (from reproductively
active organism in early vitellogenic stage with yolky
oocytes of 150-200 µ size) as the source. The pair of
ovaries was dissected out by cutting open the dorsal
side of the carapace. Ovarian tissue (100 mg) was
homogenized in TRIzol (Invitrogen, Carlsbad, CA,
USA) reagent and the total RNA was extracted and
precipitated
by
TRIzol-chloroform-isopropanol
method15. The purity of the RNA was assessed
through UV spectrophotometry (at 260 nm) and
agarose gel electrophoresis. The isolated RNA was
stored at −80 °C until further use.
cDNA Synthesis and PCR Amplification
The RNA obtained from the ovarian tissue was
reverse transcribed into cDNA using Verso Enzyme
cDNA Synthesis kit (Abgene-Thermo Fisher
Scientific, USA) as per manufacturer’s instructions.
The primers specific for the RXR gene were designed
using the Primer III software and validated through
Primer-Blast. Previous reports on the RXR sequence
of the fiddler crab, Uca pugilator were also used as
53
the cue for primer design7,8. Accordingly, the forward
primer (5′–AGC CTC CGG TAA GCA CTA TG-3′)
and the reverse primer (5′–CAC TTT TCA TCC CCA
TGG TC–3′) were used for PCR amplification of the
domain. Each 25 µL reaction mixture contained 50 ng
of template DNA, 1 µL reaction buffer (with 1.5 mM
MgCl2), 2.5 mM dNTP, 1 µM each forward (F) and
reverse (R) primers and 1 U of Red Taq
DNA polymerase (Merck-Bangalore GeNeiTM,
Bangalore, India). PCR mixtures were incubated in
Veriti™ Thermal Cycler (ABI, USA) with an
initial denaturation (95°C, 10 min), final denaturation
(95°C, 1 min) and 30 amplification cycles with 60°C
(45 sec) as annealing temperature and 72°C (30 sec)
as extension temperature for each cycle. The last
cycle had a longer extension period (72°C, 10 min). 8
µL of the amplified product was agarose gel (1.5%)
separated at 50 V in Tris-borate-EDTA buffer
(pH 8.3). The band was visualized with ethidium
bromide (Sigma-Aldrich, St. Louis, MO, USA) and
monitored using the Vilber Leumart (France) Gel
Documentation System. The PCR band eluted using
the gel purification kit (Qiagen, Germany) was
sequenced at the facility of Chromous Biotech Ltd,
Bangalore and deposited to GenBank (Acc. No.
JQ239171). The sequence information was further
subjected to Nucleotide BLAST (Blastn) and
CLUSTALW (v5.0) alignments for assessing the
extent of homology with other phylogenic groups.
Phylogenetic Analysis
For phylogenetic analysis, the sequence of the PCR
amplified band was aligned using CLUSTALW in the
MEGA v5.04 software. Further, the sequences were
subjected to DNA maximum likelihood program with
the bootstrap phylogeny test of 1000 replicates so as
to obtain the phylogenic tree.
Results and Discussion
Amplification and Sequencing of RXR-DBD of Paratelphusa sp.
The PCR amplification of the cDNA and
subsequent agarose gel (1.5%) separation of the
product gave a distinct band between 200 to 300 bp
(Fig. 1). Upon gel elution (Qiagen) and subsequent
sequencing, the band obtained was shown to contain a
sequence of 225 bp (Fig. 2a). However, a closer
analysis using the coding sequences from other
brachyurans (for instance, U. pugilator) has permitted
us to have a better picture of the sequence, as
represented in Fig. 2b.
54
INDIAN J BIOTECHNOL, JANUARY 2014
Phylogenetic Analysis of Paratelphusa sp.
The phylogenetic analysis of RXR-DBD of
Paratelphusa sp. was performed with RXR
coding sequences (mRNA) of other brachyurans
(U. pugilator, C. maenas & G. lateralis)7,9,10,
non-brachyuran decapods (M. japonicus & Crangon
crangon)5,12, insects (Drosophila melanogaster,
Bombyx mori, Aedes aegyptii & Manduca sexta)16-18,
mollusc
(Biomphalaria
glabrata),
chordates
10
(Danio rerio) and the Homo sapiens (Kaighin
et
al
unpublished,
cited
in
GenBank
acc. no. HQ692843) with MEGA v5.04 software
and the results are shown in Figs 3 & 4. The
analysis showed that Paratelphusa sp. forms a
close cluster with other brachyurans and
non-brachyuran decapods, implicating a closer
Fig. 1—PCR product (DBD) of RXR cDNA of Paratelphusa sp.
on 1.5% agarose gel: Lane 1, 100 bp Mol ladder; Lane 2, RXRDBD.
evolutionary relationship with them. BLAST and
CLUSTAL W analyses revealed that the DBD of
Paratelphusa sp. has about 90% identity with other
brachyuran crabs, such as, U. pugilator and G.
lateralis, while showing 88% identity with
C. maenas.The RXR-DBD of Paratelphusa sp. is
shown to have about 85% homology with the shrimp
M. japonicus (AB295493), but has only 78%
homology with those of the brown shrimp Crangon
crangon (FJ231415)5,12. The extent of homology
seems to decrease as we drew comparisons with
several of the non-crustacean arthropods, especially
the insects. The RXR-DBD of Paratelphusa sp.
has only 76% identity with the USP-DBD of
Fig. 3—Results of the CLUSTALW alignment of the RXR-DBD
of Paratelphusa sp. (RXRPhDBD01) with other brachyurans,
such as, U. pugilator, G. lateralis and C. maenas; insects, such as,
D. ananassae, P. xylostella and A. americanum.
Fig. 2 (a & b)—a. Sequence information on RXR-DBD derived out of cDNA of Paratelphusa sp.; b. Coding region for DNA binding
domain of RXR with the part of the domain underlined indicating the primers used.
SARIKA & GOPINATHAN: DBD OF RETINOID RECEPTOR GENE (RXR) FROM AN INDIAN FIELD CRAB
55
mechanisms involved in the control of major
metabolic events (growth and reproduction, for
instance) in decapods. Pertinently enough, RXR
in crustaceans is being suggested to be a putative
receptor for the gonadotropic hormone, such
as, methyl farnesoate, a terpenoid compound and
homolog of the insect JH III10. In the present study,
we could detect the RXR transcripts from the growing
ovary, implicating that the ovary could be a target for
the receptor gene. Future research involving seasondependent and stage-dependent gene expression of the
hormone receptor genes could provide us with
valuable clues on this subject.
Acknowledgements
The authors wish to thank Dr David S Durica
(University of Oklahoma, USA) for the advice and
guidance. SSN gratefully acknowledges the award of
Associateship from VIT University. Thanks are also
due to Mr A B Aditya Naga Sai Naidu for his help in
collection of the specimens.
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Fig. 4—Phylogenetic tree (MEGA v5.04) created using the
sequence information from RXRPhDBD01 and those from U.
pugilator, G. lateralis, C. maenas, M. japonicus, C. crangon, D.
melanogaster, B. mori, A. aegyptii, M. sexta, B. glabrata, D. rerio
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