ARTICLE pubs.acs.org/est
Differential Display System with Vertebrate-Common Degenerate Oligonucleotide Primers: Uncovering Genes Responsive to Dioxin in Avian Embryonic Liver Hiroki Teraoka,*,† Shino Ito,† Haruki Ikeda,† Akira Kubota,†,‡ Abou Elmagd M. M.,†,^ Takio Kitazawa,† Eun-Young Kim,§,|| Hisato Iwata,|| and Daiji Endoh† †
School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, United States § Department of Life and Nanopharmaceutical Science and Department of Biology, Kyung Hee University, Seoul 130-701, Korea Center for Marine Environmental Studies, Ehime University, Matsuyama 790-8577, Japan
)
‡
bS Supporting Information ABSTRACT: To assess possible impacts of environmental pollutants on gene expression profiles in a variety of organisms, we developed a novel differential display system with primer sets that are common in seven vertebrate species, based on degenerate oligonucleotideprimed PCR (DOP-PCR). An 8-mer inverse repeat motif was found in most transcripts from the seven vertebrates including fish to primates with detailed transcriptome information; more than 10 000 motifs were recognized in common in the transcripts of the seven species. Among them, we selected 275 common motifs that cover about 4070% of transcripts throughout these species, and designed 275 DOP-PCR primers that were common to seven vertebrate species (common DOP-PCR primers). To detect genes responsive to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in developing embryos, differential display with common DOP-PCR primers was applied to embryonic liver of two avian species, the chicken (Gallus gallus) and the common cormorant (Phalacrocorax carbo), which were exposed in ovo to TCDD. The cDNA bands that showed differences between the control and TCDDtreated groups were sequenced and the mRNA expression levels were confirmed by real-time RT-PCR. This approach succeeded in isolating novel dioxin-responsive genes that include 10 coding genes in the chicken, and 1 coding gene and 1 unknown transcript in the cormorant, together with cytochrome P450 1As that have already been well established as dioxin markers. These results highlighted the usefulness of systematically designed novel differential display systems to search genes responsive to chemicals in vertebrates, including wild species, for which transcriptome information is not available.
’ INTRODUCTION Chlorinated dioxins and related compounds (dioxins) including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are persistent and bioaccumulative pollutants that are detected in a variety of animals. Dioxins elicit a broad spectrum of effects in vertebrates through the aryl hydrocarbon receptor (AHR) pathway.1 Studies suggest that the AHR signaling is conserved also in avian species, but with large differences in the sensitivity to dioxins among species.25 Exposure to dioxins has been shown to cause developmental abnormalities, reduced hatchability, and lethality in avian embryos. In avian embryonic liver and avian hepatocyte cultures, the administration of dioxins induces gene expression and/or enzymatic activity of cytochrome P450 1As (CYP1As), ostensibly through the AHR activation.68 However, these and related studies have focused mostly on genes involved in AHR/ARNT-CYP1A pathways and thus little is known about other genes that might be related to some toxicological end points in birds. Gene expression has been used as a sensitive indicator of chemical exposure and their potential effects, and also as a clue to r 2011 American Chemical Society
unveil the mechanism of the chemical effects. Recent advances in microarray technology enabled the evaluation of chemical exposure and downstream toxic effects associated with the gene expression profiles in model animals. However, challenges for applying microarray technology in nonmodel animals include lack of information of genome and/or transcriptome of target species. This limits the widespread application of microarrays to ecotoxicology. During the last two decades, differential display (DD) analysis, which shows the altered expression under some treatments relative to a respective control, has been used to detect unknown genes in a completely randomized fashion.9,10 This method is based upon a Special Issue: Ecogenomics: Environmental Received: April 7, 2011 Accepted: July 22, 2011 Revised: July 14, 2011 Published: July 25, 2011 27
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low-specificity polymerase chain reaction (PCR) with approximately 10-mer random primers in a conventional thermal cycler. DD analyses using a small number of primers may not cover entire transcripts with different expression levels. Although 80160 arbitrary primers used in combination with the three one-base anchored dT primers allow coverage of an eukaryotic genome with 73.9 and 93.2% probabilities,11 there has been no method or tool available so far to design additional DD primers. Degenerate oligonucleotide-primed PCR (DOP-PCR) was originally developed to randomly amplify unknown DNA fragments from genomic DNA12 and has been applied to explore diagnostic markers, single nucleotide polymorphisms, and evolutionary relationships 13 The primer used for the original DOPPCR method is a single oligonucleotide, which consists of an approximately 6-mer priming part linked to a 6-mer degenerate part, and a 13-mer specific restriction enzyme part, for a two-step amplification procedure with lower and higher hybridization temperatures. DOP-PCR results in highly reproducible band patterns upon electrophoresis. Priming sequences, which frequently appear in the genome of various vertebrates, can be used to obtain multiple bands in one PCR procedure. In this study, we initially found 7-mer or 8-mer inverse repeats in transcripts of seven vertebrates, from fish to higher primates. Further, large numbers of the inverse repeats were shared in the seven vertebrates. Thus, we attempted to develop a novel DD system based on the DOP-PCR technique, using the inverse repeats on transcripts. We selected 275 DOP-PCR mono primers, of which the priming part (inverse repeat) covers roughly 4070% of transcripts throughout the seven vertebrates. We further applied the DD system to the screening of TCDDresponsive genes in the liver of the chicken treated in ovo with TCDD. A similar investigation was performed also for in ovo TCDD-treated common (great) cormorant, a wild avian species that lacks transcriptome information.
Figure 1. Schematic overview of DOP-PCR. Given that there is a set of 8-mer and its inverse sequences (inverse repeat) in a given mRNA in vertebrates, the cDNAs reverse-transcribed from the mRNA have two sets of inverse repeats for each strand. Thus, this cDNA could be amplified in two-step PCR reactions with DOP-PCR mono primer consisting of 8-mer priming part linked to 4-mer degenerate part (NNNN) and 10-mer anchor part (Anchor).
adult cormorants collected in our previous study16 also were used in this study. Samples in each dosage group consisted of 6 livers for chickens and 5 for cormorants, and mRNA from each liver sample was analyzed for DOP-PCR and real-time RT-PCR to evaluate individual differences. Experiments were conducted according to the Regulation of Animal Experiments of Ehime University and Rakuno Gakuen University. Search for Inverse Repeats and DDs with DOP-PCR Primers. Inverse repeats were sought in transcripts of seven vertebrates from fish to higher primates (Danio rerio, Xenopus tropicalis, Gallus gallus, Mus musculus, Bos taurus, Canis lupus familiariz, Macaca fascicularis) listed in UniGene (as of April 8, 2008), with our developed computer program Mono Search RGU ver.1 mRNA (written with Ruby 1.8.7 and C using MySQL 5.1 as a database). DOP-PCR primers consisted of 8-mer priming part (part of inverse repeat) linked to 4-mer degenerate part (NNNN) and 10-mer anchor part (50 -CCGAGTGGAG-30 ) (Figure 1). Inverse repeats, whose PCR products below 100 bp and above 1000 bp were rejected, considering amplification efficiency and the following cloning procedure. Using the DOP-PCR primers, cDNAs that were reversetranscribed from mRNAs of chicken and cormorant livers and of zebrafish embryos were amplified. The program of thermal cycling comprised two steps as follows: 2 min at 94 C, 8 cycles of 1 min at 94 C, 1 min at lower Tm (usually 35 C) and 3 min at 72 C, and then 28 cycles of 1 min at 94 C, 1 min at higher Tm (60 C), and 3 min at 72 C. Lower Tm was calculated as 4 C for C or G and 2 C for A or T for hybridization of oligonucleotidepriming part with the corresponding inverse repeat sequence in transcripts. Higher Tm (60 C) was used for all primers. GoTaq Green Master Mix (Promega, Madison, WI) was used for DOPPCR reactions. Semiquantitative PCR was also carried out with GoTaq Green Master Mix to determine candidates of genes differently expressed by TCDD. All DOP-PCR primers used are listed in the Supporting Information (SI) (Tables S1S7). A detailed description of some conventional molecular experiments for cDNA preparation and DNA sequencing of PCR products is also given in SI. Real-Time RT-PCR. Real-time RT-PCR analysis was carried out to determine fold changes in expression levels of transcripts
’ MATERIALS AND METHODS Samples and TCDD Exposure. In the present study, we used two avian species, the chicken (Gallus gallus) and the common cormorant (Phalacrocorax carbo), to screen TCDD-responsive genes by using our DD system. In addition, the zebrafish (Danio rerio) was used to examine preliminarily whether DOP-PCR primers that were designed based upon inverse repeats for CYP1A, CYP1C1, and CYP1C2 can work specifically in this species. Zebrafish embryos were exposed to waterborne TCDD (1 ppb), from 24 h post fertilization (hpf) to 48 hpf, and harvested at 72 hpf according to the method described in Teraoka et al.14 and details are also given in the Supporting Information (SI). The procedures for handling eggs of chickens and cormorants, including incubation and TCDD injection, were reported elsewhere.8,15 Vehicle (dimethyl sulfoxide/toluene = 9/1) or TCDD (10 and 100 pg/g egg) (10 μL) were injected into the air sac of chicken eggs (White Leghorn) on embryonic day 0. Eggs were incubated by rotating at 37 C and 50% humidity. Embryos were sacrificed and the liver was collected on embryonic day 10. Liver samples from cormorant embryos, which were injected with vehicle or TCDD (1500 and 4500 pg/g egg), were used for the present study. The cormorant eggs were collected at one time from Lake Biwa, Japan. They were at different developmental stages when the eggs were collected, and thus were incubated individually. TCDD was injected after confirming the developmental stage of embryos by candling. The embryos then were sacrificed just before hatching, resulting in 1 to 7 days of TCDD exposure of embryos. Livers of 28
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Figure 2. Abundance of oligomer inverse repeats in 7 vertebrates. Black, white, and gray bars indicate percentages of transcripts with 7-mer, 8-mer, and 9-mer inverse repeats, respectively, for the zebrafish, Xenopus (tropicalis), chicken, mouse, cattle, dog, and Cynomolgus monkey.
Figure 3. Target transcripts for 275 DOP-PCR primers in 7 vertebrates. Bars show percentages of transcripts with 8-mer inverse repeats in each species, which were included in selected 275 motifs that are common to 7 vertebrates. Black, white, and gray bars indicate percentages of transcripts which could be detected with 275 DOP-PCR primers with no, 1-, and 2-mer mismatches in priming part, respectively.
that were suggested to be differentially expressed between control and TCDD-treated groups in our DD analysis. Real-time RT-PCR analysis was performed in Real-Time PCR Detector (Chromo4: Bio-Rad, Hercules, CA) using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA), as described previously.14 Longer PCR products including target sequence of real-time PCR were used as standards. All primer sets used are listed in Table S8. Statistical Analysis. Results are presented as mean ( SEM. Significance of differences in mean values between groups was determined either by one-way ANOVA followed by Dunnett test, or by KruskalWallis test followed by Steel test if variances could not be regarded as equal (p < 0.05).
targeting 100500 transcripts seemed constant, while the efficiency tended to decrease as the number of target transcripts decreased between 50 and 100. As for CYP1Cs, primers with 7-mer inverse repeat had lower detection rates than those of 8-mer (Table S9). Thus, we decided to select 8-mer inverse repeats in the following study to construct DOP-PCR primers. Selection of Vertebrate-Common DOP-PCR Primer Sets. We then examined the UniGene database for inverse repeats that are conserved in the seven vertebrates from zebrafish to Cynomolgus monkey, excluding palindromes in rRNA, as these are immensely expressed. As the result, 11 143 transcripts containing 8-mer inverse repeats were identified, while 8932 and 1517 transcripts were found for 7-mer and 9-mer, respectively. Among these 11 143 transcripts, we finally selected 275 different 8-mer inverse repeats, which were present in 50500 transcripts and had 5 or 6 CGs in sequences. These 275 chosen inverse repeats were present in 41.870.7% transcripts in the chosen 7 species (average 54.2%) (Figure 3). We connected 4 degenerate sequences (NNNN) plus anchor sequences with these 8-mer repeats to prepare 275 vertebratecommon DOP-PCR primers for DDs (Table S7). TCDD-Responsive Gene Transcripts Identified with DDs in Embryonic Chicken Livers. Four independent biological sample pairs of livers of chickens treated with vehicle and TCDD were set for each DOP-PCR primer to consider individual differences. As shown in Figure 4A, DOP-PCR primers designed in the previous section produced several clear bands upon agarose-gel electrophoresis. When the same cDNA was used, practically the same band pattern was obtained (data not shown). Thus, different band patterns between replications should be derived from individual differences. The experiments suggest a high reproducibility of the DDs with DOP-PCR primers. Among the 275 primers examined, 28 positive bands were obtained with 26 primers; the intensity either increased or decreased upon TCDD exposure (100 pg/g egg). When sequencing transcripts in these positive bands, plural sequences (up to 6) were recognized from each single band in most cases. An average of 2.07 unique genes was identified from a single band (58 genes in 28 bands). The number of bands that contained plural genes was 17 (60.71%). We were not able to determine unique gene in 12 bands that had differential expression. A combination of semiquantitative RT-PCR and real-time
’ RESULTS AND DISCUSSION Distributions of Inverse Repeat in 7 Vertebrates. We selected seven vertebrates, from fish (zebrafish) to higher primates (Cynomolgus monkey), for which the UniGene database is available on GenBank. Inverse repeats of 7-mer and 8-mer were identified in 92.199.3% (average 97.2%) and 79.697.0% (average 90.3%) of transcripts, respectively, while inverse repeats of 9-mer were found in smaller numbers of transcripts (49.790.3%; average 68.4%) (Figure 2). Basic Properties of DDs with DOP-PCR Primers. Developing zebrafish is one of the most suitable organisms to characterize the effects of TCDD.17 It is well-known that CYP1As are robustly induced by TCDD in many species including developing zebrafish.18 CYP1C1 and CYP1C2 (CYP1Cs) are also induced by TCDD in larval fish including zebrafish.19 To obtain basic information on the application of these numerous inverse repeats as priming parts of DOP-PCR primers for DDs, we searched inverse repeats in zebrafish CYP1A and CYP1Cs to design DOPPCR primers, and tested them with the larvae exposed to vehicle or TCDD. DOP-PCR primers with 6-mer inverse repeat for CYP1A and CYP1Cs failed to produce any positive bands of these CYPs induced by TCDD. In contrast, many primers with 7or 8-mer inverse repeats succeeded in producing positive bands of CYP1A and CYP1Cs (Table S9). Primers with 9-mer inverse repeats also produced positive bands of CYP1s: however, only a few inverse 9-mer repeats could be found in zebrafish as well as other organisms. Detection efficiency of DOP-PCR primers 29
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Figure 4. Representative images of agarose gel electrophoresis of differential displays with DOP-PCR primers. A: Chicken embryos were treated with vehicle (0.1% mixture of DMSO and minimal toluene) (control, C) or TCDD (100 pg/g egg) (T), on incubation day 0 and were euthanized to collect their livers on embryonic day 10. B: Common cormorant embryos were treated with vehicle (C), lower dose of TCDD (1500 pg/g egg) (T1), or higher dose of TCDD (4500 pg/g egg) (T2) at 17 days prior to hatch and their livers were collected just before hatching. Results of four different pairs of control (C) and TCDD-treated livers (T for A, and T1 and T2 for B) are indicated (No. 1No. 4) in A and B. M: λ DNA marker. Arrow head: The bands emerged in A and B in which TCDD notably augmented the intensity was found to be CYP1A4 by following sequence analysis.
Table 1. Profiles of Transcripts Differentially Expressed by TCDD in the Liver of Chicken Embryosa mRNA expression (low TCDD/control) gene
P value
ratio
mRNA expression (high TCDD/control) ratio
P value
cytochrome P450 1A4
15.47 ( 2.93
0.004
62.14 ( 16.15
0.004
cytochrome P450 1A5
2.65 ( 0.65
0.015
15.92 ( 5.51
0.004
12.94 ( 3.69
0.004
34.23 ( 11.14
0.004
8.78 ( 2.42 1.40 ( 0.14
0.004 0.063
5.49 ( 1.38 1.62 ( 0.16
0.004 0.013
3-hydroxymethyl-3-methylglutaryl-coenzyme A lyase
2.49 ( 0.72
0.010
6.75 ( 1.34
0.004
N-acetylneuraminic acid synthase
1.56 ( 0.26
0.072
2.60 ( 0.44
0.000
ovocalyxin-36 precursor
5.40 ( 2.32
0.015
19.15 ( 10.58
0.006
ovoinhibitor
4.60 ( 0.62
0.006
5.78 ( 1.66
0.004
transmembrane protein 183B
2.99 ( 0.89
0.010
2.15 ( 0.38
0.006
L-gulono-gamma-lactone
1.89 ( 0.56
0.329
0.15 ( 0.08
0.004
0.86 ( 0.22
0.462
0.41 ( 0.09
0.004
aminolevulinate acid synthase 1 antigen p97 CCR4-NOT transcription complex, subunit 1
unknown gene
oxidase
a
Chicken embryos were treated with TCDD (10 and 100 pg/g egg indicated as low and high TCDD, respectively) or vehicle, on embryonic day 0. At embryonic day 10, chickens were euthanized and livers (n = 6) in each dosage group were collected for cDNA synthesis. Ratio of transcript levels in TCDD treated group to those in vehicle (Control) was indicated for two separate doses of TCDD. Transcript levels were estimated with real-time quantitative RT-PCR
in vivo was suppressed.22 This suggests that ALAS1 is a promising dioxin marker at least in the liver of chicken embryos. CCR4-NOT transcription complex subunit 1 (CNOT1), 3-hydroxymethyl-3-methylglutaryl-coenzyme A lyase (HMGCoA lyase), and N-acetylneuraminic acid synthase (NANS) also were identified as genes induced by TCDD (Table 1). The roles of these three genes have not been studied in the chicken and other avian species, but in rodents or humans they were found to function as components of transcriptional repressor,23 the synthesizing enzyme for the final step of ketogenesis,24 and the enzyme for biosynthetic pathways of sialic acids,25 respectively. Thus, TCDD could exert profound effects on some facets of metabolic processes related to these genes identified in the chicken. Conversely, a higher concentration of TCDD decreased L-gulono-gamma-lactone oxidase (GULO), an enzyme involved in vitamin C synthesis.26 Deficiency in vitamin C could cause systematic oxidative stress, which is a well-known toxicological effect induced by TCDD in vertebrates. Only limited information is available on the other two genes (Table 1). Antigen p97 was induced and Transmembrane protein 183B was decreased by TCDD in this study. Antigen p97 was
quantitative RT-PCR was conducted to confirm transcripts responsible for changes in band pattern. The results of real-time quantitative RT-PCR showed that induction of marker enzymes of TCDD exposure, CYP1A4 and CYP1A5, was detected in TCDD-treated chicken livers (Table 1). This indicates again that our experimental system is able to detect TCDD-responsive genes. In addition, 8 and 2 genes in chicken liver were identified as transcripts that were increased and decreased by TCDD treatment, respectively. The nucleotide sequences of most of the genes identified were found in the chicken sequence database, but the responses of these genes to exposure to TCDD or other AHR agonists have not been so far reported for avian species. The transcripts identified did not overlap with the results of a proteomics study, in which liver from chickens at the day of hatch were used for the analysis.20 Interestingly, delta-aminolevulinate synthase 1 (ALAS1) transcript level was increased by TCDD approximately 12-fold for low dose and 30-fold for high dose, reaching levels of induction comparable to or greater than the CYP1As (Table 1). This result agrees with a previous study indicating significant induction of hepatic ALAS1 by TCDD in TCDD-sensitive SpragueDawley rats,21 while hepatic ALAS1 mRNA in TCDD-resistant adult rats 30
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Table 2. Profiles of Transcripts Differentially Expressed by TCDD in the Liver of Common Cormorant Embryosa mRNA expression (low TCDD/control)
mRNA expression (high TCDD/control)
ratio
P value
ratio
cytochrome P450 1A4
10.96 ( 2.41
0.009
121.32 ( 30.07
0.009
cytochrome P450 1A5
12.27 ( 5.93
0.015
152.33 ( 67.62
0.009
gene
P value
cytochrome P450 2AF1 (long)
3.03 ( 0.21
0.009
11.41 ( 4.52
0.009
cytochrome P450 2AF1 (short)
4.82 ( 2.32
0.015
13.52 ( 7.19
0.015
unknown ncRNA
2.70 ( 0.71
0.043
2.59 ( 1.25
0.276
a
Cormorant embryos were treated with TCDD (1500 or 4500 pg/g egg indicated as low and high TCDD, respectively) or vehicle, 17 days prior to hatch. Just before hatch, embryonic cormorants were euthanized and livers (n = 5) in each dosage group were collected. Other conditions are the same as given in the footnote of Table 1.
originally identified as a surface antigen in melanoma and belongs to the transferrin superfamily.27 In the chicken, however, antigen p97 was reported as a marker antigen for newly formed eosinophils.28 No information was available on Transmembrane protein 183B (also known as c1orf37-dup) except that it is one of the transmembrane proteins in humans.29 Two avian specific genes also were identified (Table 1). It is known that coding products of ovocalyxin-36 precursor and ovoinhibitor are present mainly in egg white and may participate in natural immunity to block bacterial infection of chicken embryos. Ovocalyxin-36 was recently identified as a lipopolysaccharide-binding protein that has bactericidal permeability-increasing property.30 Ovoinhibitor is a major protease inhibitor synthesized in liver and oviduct and could play essential role as an antimicrobial defense by inhibiting microbial protease.31 These two genes were highly induced in embryonic chicken liver by TCDD exposure (Table 1). Both proteins could contribute to embryonic defense mechanisms against bacteria and other pathogens, although opposite effects of TCDD might also be anticipated as a possible toxicity. The results suggest that the natural defense mechanisms could respond to environmental chemicals such as dioxins in avian embryos. We found a partial sequence of an unknown coding gene (AB614396) induced by TCDD (Table 1). This sequence contains 1285 bp with a stop codon, giving a deduced protein of 279 amino acids. A Blastx search revealed that this unknown gene shared 4455% identity with mug-1 protein of Xenopus or alpha-2-macroglobulin of some vertebrates including the chicken. TCDD-Responsive Transcripts Identified with DDs in Embryonic Cormorant Livers. In embryonic cormorant livers, we found several distinct bands, similar to those observed in chicken liver, in agarose-gel electrophoresis by using the designed DOPPCR primers. This indicates again the high reproducibility of this system in species other than the seven vertebrates studied (Figure 4B). Among the 275 primers examined in the cormorant liver, 41 positive bands were detected with 37 primers. Nearly half of the bands (19 of 41) contained CYP1A4 or CYP1A5. This result is consistent with our previous study, which showed the hepatic induction of CYP1A4 and CYP1A5 in TCDD-treated cormorant embryos.8 An average of 2.20 unique genes per band was identified (90 genes in 41 bands). The number of bands that contained plural genes was 29 (70.73%) for cormorant liver. Moreover, in semiquantitative RT-PCR and real-time quantitative RT-PCR experiments, expression levels of CYP2AF1 and an unknown noncoding transcript were found to be increased (Table 2). This noncoding transcript (AB614395) (774 bp)
showed no homology with any transcripts or fragments of known genomic sequences of vertebrates or invertebrates. In addition, the quantitative PCR assay revealed a 2-fold increase in transcripts of vascular endothelial growth factor receptor 3 (VEGFR3) by TCDD, although this fold change was not statistically significant (results not shown). We were not able to determine unique gene in 12 bands that had differential expression. We have already reported CYP2AF as a new CYP subfamily in the cormorant, with members found also in the genomes of the finch and the anole.32 In the present study we identified a CYP2AF1 variant gene that has a much longer 30 -UTR (long CYP2AF1; 1190 bp) (AB614394), together with the original CYP2AF1 reported previously (short CYP2AF1; 382 bp). Both of the two CYP2AF1s were dose-dependently induced by TCDD in a similar manner (Table 2). The Short CYP2AF1 was predominant in cormorant livers and its expression level in vehicle-treated birds was about 40 times higher than that of the long CYP2AF1. Recent studies have shown that alternative polyadenylation contributes to the complexity of the transcriptome by producing mRNAs with different 30 -UTRs and/or encoding variable protein isoforms,33 as 30 -UTRs contain various regulatory elements. An alternative polyadenylation of CYP2AF1 in embryonic cormorant livers is unlikely to be affected by TCDD. We previously found no significant relationship between TEQ and expression levels of CYP2AF1s (including both short and long types) in the liver of adult cormorants,32 while a significant positive relationship was found between TEQ and both CYP1A4 and CYP1A5.34 These observations suggest that responsiveness of CYP2AF1 to TCDD could change during the development from embryos to adults. Other than CYP1A4 and CYP1A5, only CYP2AF1 and an unknown noncoding transcript were detected as TCDD-responsive transcripts in the embryonic cormorant liver by our DD system. The number of TCDD-responsive genes found in the cormorant was much smaller than that in the chicken. The difference in the results between the two species may be explained by the following reasons. First, TCDD doses needed to obtain similar induction levels of CYP1A4 and CYP1A5, in the case of embryonic cormorant liver, were much greater than those of the chicken (Tables 1 and 2). We previously reported that the cormorant AHR1 had TCDD-EC50 approximately 10-fold greater than the chicken AHR1 in terms of in vitro transactivation potency of CYP1A5.5 The less sensitivity in the cormorant might have resulted in the smaller number of TCDD-responsive genes identified in our DD system. Second, data for cormorant embryos that were exposed to TCDD for different periods (17 days before hatching) were pooled for the statistical 31
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analysis, while data for chicken embryos were obtained from individuals at the same stage of development. Third, the stage difference in embryos also should be taken into account, as chicken liver samples were collected at relatively early stage, while cormorant livers were collected at the perihatch period. Analysis of Nucleotide Sequences of Priming Part of Transcripts Identified with DDs. We analyzed 8-mer priming part of nucleotide sequences from a variety of PCR products identified with DDs in both chicken and cormorants (Table S10). Primer sequences were found only in both ends of DOPPCR products. Transcripts with a complete priming part occurred in 54.5% of transcripts in embryonic chicken liver and 19.6% in embryonic cormorant liver, and the rest was from transcripts with mismatches in the priming part. Nucleotide insertions were very rare. In both birds, the most common cases were one-nucleotide mismatches, which accounted for major percentages together with two-nucleotide mismatches (94.5% for chickens and 89.2% for cormorants). Also in both birds, the rate of mismatch of the priming part of the PCR products tended to decrease as one moved from 50 - to 30 - along the sequences. Most mismatches were the result of conversion of G or C to A or T, respectively. Assuming that DOP primer could also detect genes with 1 or 2 nucleotide mismatches consistently, we estimated the number of target transcripts possibly detected by DOP-PCR primers common to the selected 7 vertebrate species. The results indicated that 83.597.7% of total UniGenes could be covered with 275 common DOP-PCR primers in the 7 species (Figure 3). The results of gene detection by mismatch priming support the findings of Yang and Liang.11 Predicted palindomes with 1 or 2 nucleotide mismatches are presented on the site http://dop.ver2.asia/login. Taken together, mismatches in the priming part of the DOP-PCR primers with our DD system result in the detection of many more transcripts than the original target detected in both avian species. In the present study we proposed a theoretically based systematic approach for primer design for DD, using 8-mer inverse repeats present in many transcripts of vertebrates as priming part of DOPPCR primer. The theory of primer design for DD has not been clear in previous studies. As an extended application of this theory, smaller numbers of primer sets also could be designed, if one focuses on particular genes. For example, new information on DOP-PCR primer sets targeting CYP1s or other CYP members will be very useful for future studies on environmental toxicology on dioxins and other chemicals. We used purified mRNA to prepare cDNA in this study. Many more noncoding transcripts might be detected with this DD system if total RNA were used instead. Even with mRNA, one noncoding transcript was detected in the cormorant livers. This study provides information on 12 coding genes including 1 unknown gene, and 1 unknown noncoding transcript as novel TCDD-responsive genes in embryonic chicken and cormorant. As far as we know, relationships of these transcripts with TCDD exposure or AHR signaling pathways have not been reported so far in avian embryonic liver. Whether these genes are involved directly or indirectly in TCDD-induced toxicity should be determined. A variety of custom-made DNA microarray systems have been developed for wild species to obtain information on the effects of toxic substances on gene expression profiles.35,36 These approaches require a cDNA database for each species for which arrays are not commercially available. Therefore, target genes need to be selected based on the available information on cDNAs. DDs we developed here could also be useful to identify
transcripts of interest for screening strategies. Studies on the impacts of chemicals on gene expression profiles in various wild species should reveal novel pollution markers, which will help to evaluate toxicological effects and risk and to understand mechanism(s) underlying the basis of species difference in the sensitivity and response to chemicals.
’ ASSOCIATED CONTENT
bS
Supporting Information. Detailed descriptions of experimental procedures, including TCDD exposure to developing zebrafish and molecular protocols, lists of primer sequences including 275 DOP-PCR primer and quantitative RT-PCR (Table S1S8), relationship between number of target transcripts and detection efficiency with CYP1s-targeted DOP-PCR primers in zebrafish larvae (Table S9), mismatches of priming part of PCR products with vertebrate-common DOP-PCR primer sets in livers of chicken and cormorant (Table S10) are available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Telephone and fax: +81-11-388-4791; e-mail: hteraoka@rakuno. ac.jp. Present Addresses ^
Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Mansoura University, Mansoura City 35516, Dakahlia Governorate, Egypt.
’ ACKNOWLEDGMENT We thank Dr. John J. Stegeman, Woods Hole Oceanographic Institution and Dr. Annamalai Subramanian, Ehime University for critical reading of this manuscript. We also thank Dr. Akiko Sudo and Dr. Michio X. Watanabe for the collection of cormorant eggs and Ms. Naoko Kounosu, Ms. Kaori Igarashi, Ms. Miki Tamura, and Mr. Noriyoshi Ukai for excellent experimental techniques. This study was supported by Grants-in-Aid for Scientific Research (C) (18580298) and (B) (21310044) to H. T. from the Japan Society for the Promotion of Science (JSPS), Grant-in-Aid for JSPS Fellows to A.K. (4313), Akiyama Foundation to H.T., the Promotion and Mutual Aid Corporation for Private Schools of Japan to H.T., Grant-in-Aid for the High Technological Research Center (Rakuno Gakuen University) from the Ministry of Education to H.T., and by Grant-in-Aid for Scientific Research (S) (21221004) to H.I. ’ REFERENCES (1) Peterson, R. E.; Theobald, H. M.; Kimmel, G. L. Developmental and reproductive toxicity of dioxins and related compounds: Crossspecies comparisons. Crit. Rev. Toxicol. 1993, 23 (3), 283–335. (2) Karchner, S. I.; Franks, D. G.; Kennedy, S. W.; Hahn, M. E. The molecular basis for differential dioxin sensitivity in birds: Role of the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (16), 6252–6257. (3) Yasui, T.; Kim, E. Y.; Iwata, H.; Franks, D. G.; Karchner, S. I.; Hahn, M. E.; Tanabe, S. Functional characterization and evolutionary history of two aryl hydrocarbon receptor isoforms (AhR1 and AhR2) from avian species. Toxicol. Sci. 2007, 99 (1), 101–117. 32
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’ NOTE ADDED AFTER ASAP PUBLICATION This paper published August 3, 2011 with errors. There were corrections to text in paragraphs 1 and 4 of the Materials and Methods section, and typographical corrections to Tables 1 and 2. The correct version published August 11, 2011. 33
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