Environmental Science and Pollution Research (ESPR)
Supporting Information
Quantification and in situ localisation of abcb1 and abcc9 genes in toxicantexposed sea urchin embryos
Ivana Bošnjaka, Ivana Lepen Pleićb, Marco Borrac, Ivona Mladineob, d,*
a
Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of
Food Technology and Biotechnology, Pierottijeva 6, Zagreb, Croatia
b
Laboratory for Aquaculture, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63,
21000 Split, Croatia
c
Molecular Biology Service, Stazione Zoologica Anton Dohrn, Villa Comunale 80121, Napoli, Italy
d
Assemble Marine Laboratory, Stazione Zoological Anton Dohrn, Villa Comunale, Naples, Italy
*Corresponding author:
Ivona Mladineo
E-mail: mladineo@izor.hr
Phone: + 385 21 408 047
Supporting Information consists of 9 SI pages (S1 – S5), 4 SI tables (Table S1 – Table S4) and 1
SI figures (Figure S1).
S1
SI Materials and Methods
qPCR: The toxicant-exposed and untreated (control) embryos were sampled for qPCR analysis
at the following four developmental stages and corresponding time points: untreated egg cells
(before fertilisation), zygotes (45 min PF), 2-cell (1 h and 30 min PF), and blastula (10 h and 35
min PF). Total RNA was isolated from each sample using the RNAqueous-Micro Kit (Ambion,
Austin, TX). Each RNA sample was immediately DNAse treated using DNase I Treatment and
DNase Inactivation (Ambion, Austin, (TX)) to remove DNA contamination. Purified RNA
samples
were
quantified
by Nanodrop
spectrophotometer
(NanoDrop
Technologies,
Wilmington, Delaware, USA), and 100 – 200 ng of each sample was reverse transcribed to
cDNA in a 20-μl reaction, using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). The
cDNA samples were diluted 1:10 and used as qPCR templates for the measurement of abcb1 and
abcc9 gene expressions throughout the sea urchin embryonic development. The qPCR primers
for the ABC transporter genes and reference gene ubiquitin were designed against partially
obtained mRNA sequences (Table 2). Specific primer sets for each gene amplified products of
100–140 bp that were verified by gel electrophoresis and by sequencing. The amplification
efficiency value (E) of the target and control (ubiquitin) oligo pair was calculated from a cDNA
dilution curve (N=5) in which data were fit using least-squares linear regression analysis (Fig.
S1) [27]. The dilution condition used to generate all the experimental data was within the five
point dilution curves (calibration curve; Fig. S1).
Real-time qPCR measurements were performed in triplicate on a ViiA7 Real-Time PCR
System (Life technologies - Applied Biosystems) for each sample, using the Fast Start SYBR
Green Master (Roche, Mannheim, Germany) with the following thermal cycling parameters:
95°C (10 min) followed by 40 cycles of 95°C (15 sec) and then 60°C (1 min). Data generated by
qPCR were compiled and collected using the ViiA7 RUO software (Applied Biosystems).
Samples that reached the threshold (Ct) in more than 35 cycles were considered below the level
S2
of detection and were not included in the data analysis. The results of the qPCR for abcb1 and
abcc9 gene were calculated relative to the control condition and normalised with the reference
gene ubiquitin, according to the following normalisation procedure of the relative quantification
model with kinetic PCR efficiency correction formula:
(Etarget)ΔCT target (mean control – mean sample)
Q-PCR ratio =
(Eref) ΔCT ref (mean control – mean sample)
where Q-PCR ratio represents the normalised expression; Etarget is the amplification efficiency
for the target gene (e.g., abcb1 or abcc9); Eref is the amplification efficiency for the referent gene
(ubiquitin); ΔCT target (mean control – mean sample) is the difference (Δ) between the mean
sample treatment CT value and the mean control CT value of the target gene; and ΔCT ref (mean
control – mean sample) is the difference (Δ) between the mean referent treatment CT value and
the mean control CT value of the referent gene. Expression data for target genes were presented
relative to the control condition (unexposed embryos). Standard error was calculated according
to the formula for the calculation of the standard error of the mean normalised gene expression
[28].
Localisation of gene expression by in situ hybridization: PCR-generated cDNA templates
(abcc9 138 bp, abcb1 137 bp, Ubq 127 bp) were amplified using gene specific primers as they
were for qPCR (Table 2), cloned with the TOPO TA Cloning Kit (Invitrogen) and ligated to a T7
RNA Polymerase promoter adapter. The ligated template cDNAs were directionally amplified
with a T7 promoter adapter primer and a forward/reverse gene-specific primer to obtain
antisense and sense (negative control) cDNA templates. Purified templates were labelled using a
DIG (digoxigenin-UTP) RNA Labeling Kit (SP6/T7) (Roche), following the manufacturer’s
procedure. The template was briefly incubated with 10x NTP labelling mixture, 10x
transcription buffer, protector Rnase inhibitor and RNA Polymerase T7 for 2 h at 37°C. The
S3
template was then removed by Dnase I, by incubation for 15 min at 37°C, and the reaction was
blocked by EDTA. RNA probe purification was performed by means of Mini Quick Spin RNA
columns (Roche). Labelling efficiency was determined by spotting DIG-labelled RNA to a
positively charged nylon membrane, using the DIG Wash and Block Buffer Set (Roche). The
yield of the labelling reaction showed that 1 μg of template had 10 μg of labelled RNA. The
probes obtained were stored at –80°C until they were used.
Two-cell stage embryos and blastulae incubated with FSW (control), HgCl2, TBT and
OXI were fixed overnight at 4°C in EM grade 2.5 % glutaraldehyde in 0.2 M sodium- phosphate
buffer, pH 7.4. The material was washed in a sodium-phosphate buffer with 0.1 % Tween20,
dehydrated in the graded ethanol series for 30 min and stored in 70 % ethanol at 4°C until further
use, according to Ransick [29]. Prior to hybridisation, the material was incubated in proteinase K
(Roche) in TRIS-buffered saline with 0.1 % Tween20 (TBST), whose activity was afterwards
blocked by a glycine TBST solution. For two-cell stage embryos, 5 μg/ ml proteinase K stock
dilution was used for 10 min, and 3 μg/ ml for 5 min for blastula, all at 37°C. Postfixation was
conducted for 30 min at room temperature with a buffered 4 % paraformaldehyde in TBST kept
previously at 4°C. The material was washed consecutively three times in TBST at room
temperature to remove the fixative.
Increasing concentrations (35 %, 65 % and 100 %) of the hybridisation buffer (HB),
composed of 50 % formamide, 6x SSC (saline sodium citrate), 5x Denhardt’s solution, salmon
sperm DNA and 0.1 % Tween20 (all Sigma) in water, were heated to 57°C and allowed to
prehybridise with embryos before adding the probe. The samples were incubated overnight in a
water bath at 50°C with HB and antisense or sense probe (final concentration 0.1 ng/ μl), then
sealed with parafilm. The probe washing steps were performed first by washing two times in
TBST and then three consecutive times with 1x SSC in TBST.
S4
Afterwards, the samples were incubated and shaken with a 5 % sheep serum in TBST for 30 min
at room temperature. The detection of transcripts began with the introduction of Anti DIGalkaline phosphatase (Fab fragments) (Roche) diluted 1/500 in 5 % sheep serum in TBST,
incubating the samples overnight at room temperature in the dark on a shaker. The reaction was
blocked by washing the embryos several times in TBST. The alkaline phosphatase detection
solution in which the material was incubated for 4 h at room temperature consisted of levamisole
(Sigma) and NBT/BCIP (Roche) diluted in alkaline phosphatase staining buffer (APB). The
reaction was stopped by adding EDTA/ TBST. The material was inspected under the microscope
(Zeiss Axio Imager M1), photographed (AxioCam HRc Zeiss) and processed by accompanying
software (AxioVision Rel. 4.7.1). For long-term storage, the material was transitioned through
an increasing concentration to 50% glycerol containing 5 mM sodium-azide.
S5
SI Tables
Table S1. Percent identity table with ABCB1 from P. lividus compared with representative
orthologues from different species. See Table S3 for NCBI codes
Table S2. Percent identity table with ABCC9 from P. lividus compared with representative
orthologues from different species. See Table S4 for NCBI codes
S6
Table S3. List of ABCB transporters from NCBI and Sea Urchin Genome Database (SUGD)
used for phylogenetic tree analysis (Fig. 3)
I: ABCB transporters
Species name
ABC transporter name
NCBI accession number
Ciona intestinalis
Gallus gallus
Homo sapiens
"P-gp"
ABCB1
ABCB1
XM_002122107.1
Mytilus californianus
"ABCB-like"
EF521414.1
Mytilus galloprovincialis
"ABCB-like"
FM999809.2
Mus musculus
ABCB1A
NM_011076.2
Mus musculus
ABCB1B
NM_011075.2
Oncorhynchus. mykiss
"P-gp"
AY863423.3
Oreochromis niloticus
"Mdr"
XM_003450949.1
Platichthys flesus
"mdr gene"
AJ344049.1
Paracentrotus lividus
ABCB1-like
JQ793791
Strongylocentrotus purpuratus
ABCB1
NM_001033950.1
Xiphophorus hellerii
"P-gp"
HQ829295.1
Xenopus laevis
ABCB1
NM_001087925.1
NM_204894.1
NM_000927.4
Table S4. List of ABCC transporters from NCBI and Sea Urchin Genome Database (SUGD)
used for phylogenetic tree analysis (Fig. 3).
S7
II: ABCC transporters
Species name
ABC transporter name
NCBI accession number /
SUGD accession number
Cenorhabditis elegans
Ciona intestinalis
"MRP"
ABCC1
AB199793.1
XM_002121587.1
Danio rerio
ABCC1
XM_001341859.3
Danio rerio
ABCC2
NM_200589.1
Danio rerio
ABCC4
NM_001007038.1
Danio rerio
ABCC5
HQ161064.1
Danio rerio
ABCC7
NM_001044883.1
Danio rerio
ABCC8
NM_001172647.2
Danio rerio
ABCC9
BC163584.1
Gallus gallus
ABCC1
NM_001012522.1
Homo sapiens
Homo sapiens
ABCC1
ABCC2
EF419769.1
AJ132244.1
Homo sapiens
ABCC3
NM_003786.3
Homo sapiens
ABCC4
NM_005845.2
Homo sapiens
ABCC5
NM_005688.1
Homo sapiens
ABCC6
BC131732.1
Homo sapiens
ABCC7
NM_000492.3
Homo sapiens
ABCC8
NM_000352.3
Homo sapiens
ABCC9
NM_005691.1
Homo sapiens
ABCC10
NM_001198934.1
Homo sapiens
ABCC11
NM_032583.3
Homo sapiens
ABCC2
NM_033226.2
Mytilus californianus
ABCC1
EF521415.1
Mus musculus
Mus musculus
ABCC1
ABCC2
NM_008576.2
NM_013806.2
Mus musculus
ABCC3
BC150788.1
Mus musculus
ABCC5
BC090629.1
Mus musculus
ABCC7
NM_021050.2
Mus musculus
ABCC8
NM_011510.3
Mus musculus
ABCC9
NM_011511.2
Oncorhynchus mykiss
ABCC1
NM_001168330.1
Oncorhynchus mykiss
ABCC3
GQ888533.1
Paracentrotus lividus
ABCC9-like
JQ867397
Saccoglossus kowalevkii
ABCC9
XM_002738322.1
Strongylocentrotus purpuratus
ABCC4
XM_001190458.1
Strongylocentrotus purpuratus
ABCC5
XM_789939.2
Strongylocentrotus purpuratus
ABCC9a
SPU_025903
Strongylocentrotus purpuratus
ABCC9b
SPU_028797
Strongylocentrotus purpuratus
ABCC9d
SPU_007764
S8
SI Figures
Figure S1: Dilution curves for RT-qPCR primer pair efficiency (E) calculation.
S9