Environ. Sci. Technol. 2008, 42, 6997–7002
Application of HiCEP to Screening of Radiation Stress-Responsive Genes in the Soil Microarthropod Folsomia candida (Collembola) T A I Z O N A K A M O R I , * ,† A K I R A F U J I M O R I , ‡ KEIJI KINOSHITA,§ TADAAKI BAN-NAI,† YOSHIHISA KUBOTA,† AND SATOSHI YOSHIDA† Environmental Radiation Effects Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan, Heavy Ion Radiobiology Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan, Nagoya University Avian Bioscience Research Centre, Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Received April 24, 2008. Revised manuscript received June 12, 2008. Accepted July 7, 2008.
The field of ecotoxicogenomics has received increasing attention for its potential to provide insight into pressing ecological issues. However, its applications are limited due to a lack of genetic sequence information for organisms used in ecotoxicological studies. We used high-coverage expression profiling (HiCEP), a method that requires no prior sequence knowledge, to examine stress-responsive genes and their dose dependence in the springtail Folsomia candida using γ radiation as the stressor. Radiation-responsive genes and their dose dependency were detected at effective doses for reproduction, and 16 up-regulated transcript-derived fragments (TDFs) were sequenced. Quantitative PCR analysis also found that most of the TDFs were up-regulated. The sequences of the TDFs showed resemblance to known genes, such as glutathione S-transferase and poly(ADP-ribose) polymerase, but most showed no similarity to any genes in the gene databases. These results suggest that HiCEP is effective for discovering differently expressed genes and their dose dependence, even in organisms for which few sequence data are available. The limited length of the TDFs, however, may impede functional annotation of the genes. In conclusion, HiCEP is useful for ecotoxicogenomic studies in which various organisms with few available genomic resources are involved.
Introduction Recent advances in genomic technologies have had great impact on human toxicology and ecotoxicology research. The application of genomic tools into ecotoxicology, referred to as ecotoxicogenomics (1), has received increasing attention for its potential advantages in applications such as the * Corresponding author phone: (81) 43-206-3158; fax: (81) 43251-4853; e-mail:
[email protected]. † Environmental Radiation Effects Research Group, National Institute of Radiological Sciences. ‡ Heavy Ion Radiobiology Research Group, National Institute of Radiological Sciences. § Nagoya University Avian Bioscience Research Centre. 10.1021/es801128q CCC: $40.75
Published on Web 08/19/2008
2008 American Chemical Society
identification of diagnostic biomarkers, cross-species extrapolation, and ecological impact assessment of contaminant mixtures (2). Although DNA microarrays have become a standard tool in genomics (3), this method relies on the availability of a complete genome sequence or large collections of known transcript sequences. Thus, the lack of extensive sequence information for organisms of ecotoxicological interest has limited the usefulness of DNA microarrays for research in this field. However, for most such organisms, the screening and sequencing of stress-responsive genes is the first priority for ecotoxicogenomics. A number of a gene discovery or gene expression analysis tools do not rely on the availability of prior sequence knowledge (4). For example, cDNA-amplified fragment length polymorphism (AFLP), a polymerase chain reaction (PCR)and electrophoresis-based method, can detect unknown and low-abundance transcripts. However, their coverage is not very high, and there is a substantial rate of false positives caused by misannealing during the PCR step (5). Alternatively, high-coverage expression profiling (HiCEP) offers substantial improvement over cDNA-AFLP, especially with respect to the selective PCR step (6). HiCEP produces an extremely low rate of false positives (less than 4% in mouse) because transcript-derived fragments (TDFs) usually differ in molecular size and can be easily separated by capillary electrophoresis. This enables each peak (the resulting data of capillary electrophoresis) to be unequivocally assigned to a specific gene. HiCEP is useful and efficient for the identification of stress-responsive genes (7, 8) and is applicable to nongenomic model organisms. Thus, HiCEP stands to substantially benefit the field of ecotoxicogenomics, given the need to identify stress-response genes in organisms for which few genomic resources are available; however, the technology has yet to be applied to this type of research. Springtails (Collembola) constitute part of the most abundant soil mesofauna and have been widely used as sentinel model animals for ecological impact assessment (9, 10). The springtail Folsomia candida is commonly used for ecotoxicological testing (11) and is also useful for ecotoxicogenomic applications because a genetically homogeneous population can be easily obtained from a parthenogenic culture. An expressed sequence tag (EST) database for springtails including F. candida was recently constructed for the primary application of diagnostic soil quality testing (12). This database, entitled Collembase, includes stressor-enriched libraries that were constructed on the basis of suppression subtractive hybridization approaches. However, to date, the stressors examined have been limited to cadmium and phenanthrene (12). We used HiCEP to screen for stress-responsive genes in the springtail F. candida. γ Radiation was selected as a stressor because of its increasing relevance as an environmental safety concern (13, 14). Dose-effect relationships of acute γ irradiation on the reproduction, growth, and survival of F. candida are available elsewhere (15). At effective doses for reproduction, radiation-responsive genes in F. candida and their dose dependence were detected using the HiCEP method, and 16 up-regulated transcripts were sequenced. The dose dependence of sequenced transcripts was also analyzed using quantitative PCR.
Materials and Methods Animals. Stock cultures of F. candida originating from a population in central Japan were reared on baker’s yeast as described by Nakamori et al. (15). Age-synchronized springtail VOL. 42, NO. 18, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Example of HiCEP analysis peak profiles and detection of a differentially expressed peak induced by γ irradiation: (a) nonirradiated control; (b) 4 Gy-irradiated; (c) 26 Gy-irradiated; (d) superposition of a-c. Arrowheads indicate a differentially expressed peak among treatments.
TABLE 1. Intensity of HiCEP Peaks Up-Regulated in γ-Irradiated F. candida intensitya peak no. 0 Gy 1 2b 3 4 5b 6 7 8 9 10b 11 12 13 14 15 16
69 -c 53 232 -c 200 685 1876 78 -c 283 3002 1528 4204 3702 5216
relative intensity to blank control
4 Gy
26 Gy
0 Gy
4 Gy
26 Gy
13823 1764 1535 2360 543 2502 3428 11060 219 179 642 10323 4531 12014 10067 9433
15274 5257 3533 9151 1268 5039 8170 20473 624 380 1896 15798 7592 16029 12628 11252
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
201.8 35.3 29.2 10.2 10.9 12.5 5.0 5.9 2.8 3.6 2.3 3.4 3.0 2.9 2.7 1.8
223.0 105.1 67.3 39.4 25.4 25.2 11.9 10.9 8.0 7.6 6.7 5.3 5.0 3.8 3.4 2.2
a Means of technical duplicates of a pooled sample (n ) 1). b Peaks with intensity below measurable limits were given a minimum detectable quantity of 50 to calculate relative intensity. c -, below measurable limits.
specimens used in experiments were prepared according to the International Organization for Standardization (11). Experimental Design. For HiCEP analyses, newly hatched springtail specimens from a synchronized egg culture were reared on autoclaved baker’s yeast in several test vessels. The vessels and food were replaced every other day to minimize contamination by fungi. When the specimens were 10-12 days old, they were exposed to 4 or 26 Gy of 137Cs γ radiation in the test vessels at a constant dose rate of 8.3 Gy/min. Two hours after the irradiation, all springtail specimens within a treatment group were pooled, soaked in liquid nitrogen, and stored at -80 °C until RNA extraction. Each treatment consisted of one replicate of 2500 pooled 6998
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specimens. Further details can be found in the Supporting Information. Irradiation experiments were carried out in the same manner for quantitative PCR analysis, except that 90 specimens were used per sample, four replicates were conducted per treatment, nonautoclaved baker’s yeast was used for food, and irradiation doses at 8, 20, and 48 Gy were administered, which correspond to approximately 10, 50, and 80% effective doses for reproduction, respectively (15). Preparation of Total RNA from Springtails. A group of whole springtail specimens in each sample was ground in liquid nitrogen in a 1.5-mL tube. Total RNA was extracted using an RNeasy mini kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). RNase-free DNase I treatment (Qiagen) was also performed. Analysis of Gene Expression Using HiCEP. The HiCEP method (6), developed at the National Institute of Radiological Sciences (NIRS) of Japan, was applied to determine differentially expressed genes between irradiated and nonirradiated springtail populations. HiCEP analyses were carried out as described by Fukumura et al. (6). Briefly, 1 µg of total RNA treated with DNase I was converted to cDNA using the SuperScriptIII First Strand Synthesis system (Invitrogen) with 5′-biotinylated oligo(dT) primers. Double-strand cDNA was prepared, digested with MspI, and trapped by avidin bound to magnetic beads. After the fragments digested by MspI [except for most of the 3′-region bearing oligo(dT)-biotin] were washed off, a synthetic adaptor was ligated, and the trapped templates were digested by MseI. The resulting solution was used as a template for 256 runs of selective PCR. The products were denatured and loaded on an ABI PRISM 3100 electrophoresis system (Applied Biosystems, Foster City, CA) for separation, and the fluorescent intensity of each peak height was recorded. Electrophoretic pattern data were normalized using a global normalization program developed by Maze Inc. (http://www.maze.co.jp/). Technical duplicates were created by independently generating selected peak profiles by applying HiCEP to mRNA from an identical springtail sample, and the means of the technical duplicates were used for analysis. For peaks with intensities below the measurable limits, the minimum detectable quantity was used to calculate the quantity relative to other treatments. Fractionation and Sequencing of HiCEP Peaks. Peaks of interest were physically detected using polyacrylamide gel electrophoresis and sequenced directly (see the Supporting Information for details). Sequence Analysis of TDFs. Functional annotation of TDFs was carried out on the basis of sequence similarity using the blastx and blastn algorithms of the basic local alignment search tool (BLAST) against a nonredundant protein database and an F. candida EST database, respectively. Sequences of restriction enzyme sites and 2-bp 3′ends of selective PCR primers (5′-CCGGNN-NNTTAA-3′; N ) A, G, T, or C) were included for database searching. An E value of
