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Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes
Fish & Shellfish Immunology (2000) 10, 623–630 doi:10.1006/fsim.2000.0279 Available online at http://www.idealibrary.com on
Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes TAKASHI AOKI1*, IKUO HIRONO1, MIN-GYON KIM1, TAKAYUKI KATAGIRI1, YUKI TOKUDA2, HARUHIKO TOYOHARA2 AND EIICHI YAMAMOTO3 1
Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4-5-7, Minato, Tokyo 108-8477, Japan, 2 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and 3Tottori Prefectural Fisheries Experimental Station, Tottori 689-0602, Japan (Received 9 December 1999, accepted after revision 30 March 2000) Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by di#erential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection. 2000 Academic Press
Key words:
Japanese flounder, Ig+ leucocyte, hirane rhabdovirus, viral induced gene, biodefence gene.
I. Introduction Hirame rhabdovirus (HRV) has been isolated from several types of fish and is considered to be a significant pathogen of cultured fish in Japan (Kimura et al., 1986). HRV is a member of the Rhabdoviridae based upon biochemical and biophysical characteristics, and the similarity of some genes (Nishizawa et al., 1997). *Corresponding author. E-mail: [email protected] 1050–4648/00/070623+08 $35.00/0
623
2000 Academic Press
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T. AOKI ET AL.
In general, viral infections stimulate the host immune system. B lymphocytes (B cells), which express immunoglobulins on their surfaces, are usually produced in response to a microbial infection. When the host immune system recognises a microbial infection, B cells proliferate and di#erentiate into antibody-secreting B cells. Antibody-mediated responses of host cells fight invading pathogens either immediately, at the site of pathogen entry, or later when the infection spreads to the blood and/or organs. Antibodies act directly or indirectly on the invading pathogens. If the host animal has antibodies to a particular invading pathogen, antibodies will bind directly to the pathogens and may prevent further infection. Antibodies may recognise one or more antigens of the pathogen and mediate the removal of the pathogen through complement, cell lysis or phagocytosis. During these responses, B cells express several genes that are specific–responsive stimuli. Some of the genes that are specifically expressed upon human B cell proliferation, di#erentiation or induced by viral infection have been previously characterised (Hong et al., 1993; Yancopoulous et al., 1990). However, there is no information about the specific responsive genes of fish B cells. Genes that are specifically expressed in response to stress often encode proteins that are essential for the function of that tissue or cell type (Bureau et al., 1990; Fujiki et al., 1999; Hong et al., 1993; Yancopoulus et al., 1990). The cDNA subtraction method, which is a method for enriching the clones of cDNAs that are specifically-expressed in response to a particular stimulus, depends on the use of cell types whose mRNA populations are similar, di#ering in only a limited number of their expressed genes (Sive & John, 1988). A subtractive cDNA can be used as a DNA probe for screening a cDNA library, which is called di#erential hybridisation, from cells or tissues that show a stress response. A cloned population of Japanese flounder Paralichthys olivaceus, has been produced and established (Yamamoto, 1999). Such a cloned population is useful for molecular biological and genome analysis of fish. Moreover, Japanese flounder is one of the most important food fish of both the fishing and aquaculture industries in Japan. In this study, cDNAs that are up-regulated in hirame rhabdovirus (HRV)-infected Ig+ leucocyte cells of Japanese flounder were identified by a di#erential hybridisation and cloning strategy. II. Materials and Methods CONSTRUCTION OF CDNA LIBRARY
The immune response of homocloned Japanese flounder Paralichthys olivaceus, was activated in vivo by intramuscular inoculation with 105·8 TCID50 fish 1 of hirame rhabdovirus (HRV). Peripheral blood samples were taken from three fish at 12, 24, and 36 h post-injection. Leucocytes were isolated by centrifugation at 400g for 20 min, with Percoll solution (1·072 g ml 1). mRNA from three individual fish in each time group was isolated using a micro mRNA purification kit (Amersham-Pharmacia, U.S.A.), pooled and used to construct a cDNA library (Aoki et al., 1999; Nam et al., 2000). The cDNA library synthesis was performed using a cDNA synthesis kit
IDENTIFICATION OF VIRAL INDUCED GENES
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(Amersham-Pharmacia, U.S.A.) with an oligo dT primer. The cDNA library was constructed in ZAPII vectors according to the manufacturer’s instructions (Stratagene, U.S.A.). PREPARATION OF IG+ LEUCOCYTES
Percoll-separated leucocytes were allowed to react with mouse anti-Japanese flounder Ig mab (Tokuda et al., 1999) and were then incubated with magnetic beads conjugated to goat anti-mouse IgG mab (Miltenyi Biotec, U.S.A.). Labelled cells were separated from unlabelled cells by magnetic cell sorting using the MiniMACS (Miltenyi Biotec, U.S.A.) according to the manufacturers instructions. The magnetic cells were retained in a MiniMACS column inserted into a MiniMACS magnet while the nonmagnetic cells passed through. Labelled cells were eluted after the column was removed from the magnet. DIFFERENTIAL HYBRIDISATION FOR SCREENING THE CDNA LIBRARY
mRNA was extracted from the Ig+ leucocytes of HRV-infected and apparently healthy Japanese flounder using a micro mRNA purification kit (Amersham-Pharmacia, U.S.A.). The Ig+ leucocytes of HRV-infected Japanese flounder mRNA and apparently healthy Japanese flounder were used for isolation of subtracted and unsubtracted cDNA probes by using a Subtraction kit (CLONTECH, U.S.A.). cDNA obtained by the subtraction method (probe one) and un-subtracted cDNA (probe two) were used for di#erential hybridisation. Plaque hybridisation was performed as described by Sambrook et al. (1989). Clones that were positive for probe one and negative for probe two were selected for further analysis. PLASMID PREPARATION AND DNA SEQUENCING
Conversion of the recombinant ZAPIIs into pBluescript plasmid was carried out by in vivo excision according to the protocol of the manufacturer (Stratagene, U.S.A.). cDNA clones were sequenced using ThermoSequenase (Amersham-Pharmacia, U.S.A.) with M13 forward and/or M13 reverse primers and an automated DNA sequencer LC4000 (Li-Cor, U.S.A.). Each sequence was compared with all sequences available in DDBJ/EMBL/ GenBank using the BLAST version 2.0 (Altschul et al., 1990, 1997) (http:// www.ncbi.nlm.nih.gov). RT-PCR ANALYSIS
From 15 viral infected homocloned fish peripheral blood samples were taken at 0, 12, 24, 48 and 72 h post-injection, three fish in each time group. Total RNA was extracted from leucocytes using TRIZOL (Life Technologies, U.S.A.). The purified total RNA (5 g) was reverse transcribed into cDNA using the AMV Reverse Transcriptase First-strand cDNA Synthesis kit (Life Science, U.S.A.). The final volume of the cDNA synthesis reaction was 25 l. The reversetranscribed sample (1 l) was used in 50 l of PCR reaction mixture. The PCR primers that were used in this study are listed in Table 1. The -actin primer set was used as a positive control of RT-PCR (Katagiri et al., 1997). PCR was
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Table 1. Sequences of used primers for RT-PCR Primer name JFIG19-F JFIG19-R JFIG68-F JFIG68-R JFIG108-F JFIG108-R JFIG106-F JFIG106-R JFIG6-F JFIG6-R -actin-F -actin-R
Sequences 5 -GAACCTGGTCCTCTCCTCCT-3 5 -CTGGACACTCTCTCCGTTCT-3 5 -GACAACTCGGAGCTTCTTCA-3 5 -AGCCAAACCATCCCTAGACC-3 5 -TCCAGCTGGGGTCCATCGAA-3 5 -TCTGGAAGTAATCAGCAGCC-3 5 -GCCTTGAGGGTGCACTGCCC-3 5 -CCCTCCTGGCTGAAAATGCC-3 5 -AAGAACTAGCAGACTGGAAG-3 5 -CACATCACCAGATTCCTTGT-3 5 -ACTACCTCATGAAGATCCTG-3 5 -TTGCTGATCCACATCTGCTG-3
performed with an initial denaturation step of 1 min at 95 C, then 20 cycles were run as follows: 30 s of denaturation at 95 C, 30 s of annealing at 55 C and 30 s of extension at 72 C. The reacted products were electrophoresed on a 2·0% agarose gel. III. Results and Discussion Approximately 25 000 independent clones were initially screened with probes one and two. Most of clones displayed a positive signal with both probes, but some of the clones displayed a positive signal with only probe one. Fifty clones that displayed a positive signal with only probe one were selected. Of these 50 clones (DDBJ/GenBank/EMBL accession No. AU083036AU083141), 30 were found to have significant identities to previously reported genes and cDNA sequences by the BLAST analysis. These 30 clones were comprised of 24 di#erent cDNAs (Table 2). The other 20 clones coded other, unknown sequences (data not shown). Three of the 30 identified clones were clones of genes that encoded IgD and five were of genes that encoded secreted IgM. Recently, an expressed sequence tag (EST) analysis of Japanese flounder leucocyte cells which were infected with HRV was reported (Nam et al., 2000; Aoki et al., 1999). From over 1500 ESTs examined, no IgD cDNA was isolated and only one IgM cDNA was isolated. Magnetic separation using mouse anti-Japanese flounder Ig mab showed that the Ig+ leucocytes were enriched. Interestingly, membrane IgM cDNAs were not isolated in this study although five clones of secreted IgM cDNA were isolated. These results suggest the presence of IgD and secreted IgM cDNAs after the subtraction may be because the Ig+ leucocytes were stimulated by HRV infection, which could have resulted in the activation of these genes. Thus, these cDNAs would have become enriched after the subtraction. The cDNAs for homologues of several biodefence and immune-related genes were isolated (Table 2). This is the first time that each of these genes has been reported in teleostei, except gelatinase-b, collagenase III and Stat3. Several of the cDNAs in Table 2 are known to be stress-inducible genes, including the
AU083136 AU083087 AU083065 AU083059 AU083063 AU083075 AU083102 AU083044 AU083125 AU083116 AU083081 AU083042 AU083110 AU083089 AU083107 AU083038 AU083073 AU083085 AU083092 AU083039 AU083139 AU083071 AU083083 AU083079
Accession no.
ATP synthase lipid binding protein P3 Basigin III CEF-10 Collagenase-3 Dihydrolipoamide succinyltransferase Erythropoietin receptor Gelatinase-B (collagenase IV) Herpes virus entry mediator (TNFR family) IgD IgM Inducible polyA binding protein Interferon inducible 56K protein Megacaryocyte stimulating factor Melanoma-associated antigen MUC-18 Nuclear factor Phospholipase C-gamma-2 PKC inhibitor Prolylcarboxylpeptidase Prostaglandintransporter (matrin) Protease-nexin 1 (SERPIN) Rab8-interacting protein RGS5 Stat3 Yrk (tyrosine-protein kinase)
Identification Homo sapiens Gallus gallus Gallus gallus Xenopus laevis Fugu rubripes Homo sapiens Cynops pyrrhogaster Homo sapiens Ictalurus punctatus Oncorhynchus mykiss Homo sapiens Homo sapiens Homo sapiens Homo sapiens Xenopus laevis Rattus norvegicus Bos taurus Homo sapiens Rattus norvegicus Rattus norvegicus Mus musculus Mus musculus Oncorhynchus mykiss Gallus gallus
Species P48201 P17790 P19336 U41824 U40758 P19235 D82052 U70321 U67437 X65261 U33818 P09914 U70136 P43121 S80988 P24135 P29312 P42785 Q00910 P07092 U50595 U67188 U60333 Q02977
Accession no.
Quality of match is given as percent identity of amino acid sequences (%ID) and length in amino acid residues for peptide meatches.
JFIG-M37 JFIG-85-2F JFIG-19F JFIG-141F JFIG-150-3F JFIG-30F JFIG-B280-2F JFIG-117-2F JFIG-IG-59R JFIG-IG-125-2R JFIG-68-3R JFIG-108R JFIG-I184-2R JFIG-92-1R JFIG-B9F JFIG-105-1R JFIG-29R JFIG-73F JFIG-99F JFIG-106R JFIG-M38R JFIG-25-1R JFIG-6F JFIG-58F
Clone
Table 2. Identified clones of Ig+ leucocyte cells from Japanese flounder infected with HRV
74·6 40·8 62·5 45·3 78·8 22·1 64·3 34·2 43·1 42·5 78·1 25·5 21·5 30·1 32·7 54·2 82·9 56·1 52·2 48·8 67·2 40·6 81·7 66·7
%ID
137 262 184 226 146 263 258 184 137 240 113 306 233 173 266 306 246 194 249 258 116 192 231 238
Length
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1. Time course of the expression of subtracted cDNA clones in peripheral blood leucocytes, as detected by RT-PCR. Expression levels at various times after injection of HRV are shown. Lanes 1–3, 0 h; lanes 4–6, 12 h; lanes 7–9, 24 h; lanes 10–12, 48 h; lanes 13–15, 72 h. RT-PCR was carried out with the primer sets of JFIG-108R (a) (IFI56), JFIG-19F (b) (CEF-10), JFIG-6F (c) (Stat3), JFIG-106R (d) (protease-nexin 1), JFIG-68-3R (e) (inducible poly (A) binding protein) and -actin (f).
genes for IFI56 (Wathelet et al., 1986), Stat3 (Johnson et al., 1996), CEF-10 (Simmons et al., 1989), inducible poly (A) binding protein (Yang et al., 1995), prolylcarboxylpeptidase, proteasome-nexin 1 (SERPIN) (Sommer et al., 1987), collagenase III (Brown et al., 1996), gelatinase-b (collagenase IV) (Wilhelm et al., 1989), megakaryocyte stimulating factor (Turner et al., 1991), specific stress inducible protease prolylcarboxylpeptidase (Tan et al., 1993), Herpes virus entry mediator (tumor necrosis factor receptor family) (Montgomery et al., 1996), and Rab8-interacting protein genes (Ren et al., 1996). Recently it was reported that the gelatinase-b (collagenase IV) and collagenase III were highly expressed in Japanese flounder leucocytes which were infected with HRV (Aoki et al., 1999). In mammals, gelatinase and collagenase are expressed in granule cells, and act for defence to infections. It may therefore be proposed that the Japanese flounder gelatinase-b and collagenase III will also act for defence to the HRV infection. Recently, the expression of the Mx gene, which codes for an antiviral protein, of Japanese flounder was shown to be induced by HRV infection (Lee et al., 2000). In this study, RT-PCR was used to analyse the e#ect of HRV on the levels of expression of the genes for homologues of IFI56 (JFIG-108R), CEF-10 (JFIG-19F), Stat3 (JFIG-6F), protease-nexin 1 (JFIG-106R) and inducible poly(A) binding protein (JFIG-68R) genes. The amount of mRNA of each of the examined genes increased after HRV infection although the expression level of house keeping gene -actin were very similar in all samples (Fig. 1). The expression levels of these genes were di#erent but the patterns of increase of the mRNA were similar over time. These expression patterns were also similar to the expression pattern of the antiviral protein Mx (Lee et al., 2000).
IDENTIFICATION OF VIRAL INDUCED GENES
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Interestingly, after 24 h of HRV injection, the expression pattern of three fish were di#erent. The CEF-10 expression of a single fish (Fig. 1, fish 8) from the 24 h sample had not increased although the expression levels of the other two fish had. In contrast, the expression levels of other four genes of this fish (fish 8) were higher than those of other two fish in that time sample group (Fig. 1). The reasons for such di#erences in the expression pattern of these five genes, 24 h after HRV injection, is unclear. In this study a clonal line of Japanese flounder was used, the same number of HRV was injected into each fish, and these fish were kept in the same large tank. In future studies, the expression of cloned genes using di#erent species and a number of viruses, bacteria, or chemicals injected into such fish will be analysed to gain a better understanding of the expression mechanism of these genes. Di#erential hybridisation, using subtracted and un-subtracted cDNA probes, has been shown to be useful for the isolation and identification of stressinducible genes of homologues of both known genes and new genes of fish. This research was supported in part by a grant from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF97L00902).
References Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, E. W. (1990). Basic local alignment search tool. Journal of Molecular Biology 215, 403–410. Altschul, S. F., Madden, T. L., Scha#er, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402. Aoki, T., Nam, B.-H., Hirono, I. & Yamamoto, E. (1999). Sequences of 596 cDNA clones (565,977 bp) of Japanese flounder, Paralichthys olivaceus, leucocytes infected with hirame rhabdovirus. Marine Biotechnology 1, 477–488. Brown, D. D., Wang, Z., Furlow, J. D., Kanamori, A., Schwartzman, R. A., Remo, B. F. & Pinder, A. (1996). The thyroid hormone-induced tail resorption program during Xenopus laevis metamorphosis. Proceedings of the National Academy of Science U.S.A. 93, 1924–1929. Bureau, J. F., Chirinian, S., Ozden, S., Aubert, C. & Brahic, M. (1990). Isolation of a specific cellular mRNA by subtractive hybridisation in Theiler’s virus persistent infection. Microbial Pathogenesis 8, 335–341. Fujiki, K., Shin, D. H., Nakao, M. & Yano, T. (1999). Molecular cloning of carp (Cyprinus carpio) CC chemokine, CXC chemokine receptors, allograft inflammatory factor-1, and natural killer cell enhancing factor by use of suppression subtractive hybridization. Immunogenetics 49, 909–914. Hong, J. X., Wilson, G. L., Fox, C. H. & Kehrl, J. H. J. (1993). Isolation and characterization of a novel B cell activation gene. Journal of Immunology 150, 3895–3904. Johnson, M. C., Mourich, D. V. & Leong, J. C. (1996). Stat Genes in Trout—Comparison with Mammalian Sequences. GenBank accession number AAB60926. Katagiri, T., Hirono, I. & Aoki, T. (1997). Identification of a cDNA for medaka cytoskeltal -actin and construction for the reverse transcriptase-polymerase chain reaction (RT-PCR) primers. Fisheries Science 63, 73–76. Kimura, T., Yoshimizu, M. & Gorie, S. (1986). A new rhabdovirus isolated in Japan from cultured hirame (Japanese flounder) Paralichthys olivaceus and ayu Plecoglossus altivelis. Diseases of Aquatic Organisms 1, 209–217. Lee, J.-Y., Hirono, I. & Aoki, T. (2000). Cloning and analysis of expression of Mx cDNA in Japanese flounder, Paralichthys olivaceus. Developmental and Comparative Immunology 24, 407–415.
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Montgomery, R. I., Warner, M. S., Lum, B. J. & Spear, P. G. (1996). Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427–436. Nam, B-H., Katagiri, T., Yamamoto, E., Hirono, I. & Aoki, T. (2000). A survey of expressed genes in the Leucocytes of Japanese flounder, Paralichthys olivaceus, infected with hirame rhabdovirus. Developmental and Comparative Immunology 24, 13–24. Nishizawa, T., Kurath, G. & Winton, J. R. (1997). Sequence analysis and expression of the M1 and M2 matrix protein genes of hirame rhabdovirus (HIRRV). Diseases of Aquatic Organisms 31, 9–17. Ren, M., Zeng, J., De Lemos-Chiarandini, C., Rosenfeld, M., Adesnik, M. & Sabatini, D. D. (1996). In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase. Proceedings of the National Academy of Science U.S.A. 93, 5151–5155. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular cloning: A laboratory manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press. Simmons, D. L., Levy, D. B., Yannoni, Y. & Erikson, R. L. (1989). Identification of a phorbol ester-repressible v-src-inducible gene. Proceedings of the National Academy of Science U.S.A. 86, 1178–1182. Sive, H. L. & St John, T. (1988). A simple subtractive hybridisation technique employing photoactivatable biotin and phenol extraction. Nucleic Acids Research 16, 10937. Sommer, J., Gloor, S. M., Rovelli, G. F., Hofsteenge, J., Nick, H., Meier, R. & Monard, D. (1987). cDNA sequence coding for a rat glia-derived nexin and its homology to members of the serpin superfamily. Biochemistry 26, 6407–6410. Tan, F., Morris, P. W., Skidgel, R. A. & Erdos, E. G. (1993). Sequencing and cloning of human prolylcarboxylpeptidase (angiotensinase C). Similarity to both serine carboxypeptidase and prolylendopeptidase families. Journal of Biological Chemistry 268, 16631–16638. Tokuda, Y., Toyohara, H., Kina, T. & Sakaguchi, M. (1999). Characterization of distinct subpopulations of Japanese flounder lymphocytes with monoclonal antibody against serum immunoglobulin. Fisheries Science 65, 347–352. Turner, K. J., Fitz, L. J., Temple, P., Jacobs, K., Larson, D., Leary, A. C., Kelleher, K., Giannotti, J., Calvetti, J., Fitzgerald, M., Kriz, M.-J., Ferenz, C., Grobholz, J., Fraser, H., Bean, K., Norton, C. R., Gesner, T., Bhatia, S., Kriz, R., Hewick, R. & Clark, S. C. (1991). Purification, biochemical characterization, and cloning of a novel megakaryocyte stimulating factor that has megakaryocyte colony stimulating activity. Blood 78, 279. Wathelet, M., Moutschen, S., Defilippi, P., Cravador, A., Collet, M., Huez, G. & Content, J. (1986). Molecular cloning, full-length sequence and preliminary characterization of a 56-kDa protein induced by human interferons. European Journal of Biochemistry 155, 11–17. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A. & Goldberg, G. I. (1989). SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secretal by normal human macrophages. Journal of Biological Chemistry 264, 17213–17221. Yamamoto, E. (1999). Studies on sex-manipulation and production of cloned populations in hirame flounder, Paralichthys olivaceus (Temminck et Schlegel). Aquaculture 173, 235–246. Yancopoulos, G. D., Oltz, E. M., Rathbun, G., Berman, J. E., Smith, R. K., Lansford, R. D., Rothman, P., Okada, A., Lee, G., Morrow, M., Kaplan, K., Prockop, S. & Alt, F. W. (1990). Isolation of coordinately regulated genes that are expressed in discrete stages of B-cell development. Proceedings of the National Academy of Science U.S.A. 87, 5759–5763. Yang, H., Duckett, C. S. & Lindsten, T. (1995). iPABP, an inducible poly(A)-binding protein detected in activated human T cells. Molecular and Cellular Biology 15, 6770–6776.
Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes TAKASHI AOKI1*, IKUO HIRONO1, MIN-GYON KIM1, TAKAYUKI KATAGIRI1, YUKI TOKUDA2, HARUHIKO TOYOHARA2 AND EIICHI YAMAMOTO3 1
Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4-5-7, Minato, Tokyo 108-8477, Japan, 2 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and 3Tottori Prefectural Fisheries Experimental Station, Tottori 689-0602, Japan (Received 9 December 1999, accepted after revision 30 March 2000) Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by di#erential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection. 2000 Academic Press
Key words:
Japanese flounder, Ig+ leucocyte, hirane rhabdovirus, viral induced gene, biodefence gene.
I. Introduction Hirame rhabdovirus (HRV) has been isolated from several types of fish and is considered to be a significant pathogen of cultured fish in Japan (Kimura et al., 1986). HRV is a member of the Rhabdoviridae based upon biochemical and biophysical characteristics, and the similarity of some genes (Nishizawa et al., 1997). *Corresponding author. E-mail: [email protected] 1050–4648/00/070623+08 $35.00/0
623
2000 Academic Press
624
T. AOKI ET AL.
In general, viral infections stimulate the host immune system. B lymphocytes (B cells), which express immunoglobulins on their surfaces, are usually produced in response to a microbial infection. When the host immune system recognises a microbial infection, B cells proliferate and di#erentiate into antibody-secreting B cells. Antibody-mediated responses of host cells fight invading pathogens either immediately, at the site of pathogen entry, or later when the infection spreads to the blood and/or organs. Antibodies act directly or indirectly on the invading pathogens. If the host animal has antibodies to a particular invading pathogen, antibodies will bind directly to the pathogens and may prevent further infection. Antibodies may recognise one or more antigens of the pathogen and mediate the removal of the pathogen through complement, cell lysis or phagocytosis. During these responses, B cells express several genes that are specific–responsive stimuli. Some of the genes that are specifically expressed upon human B cell proliferation, di#erentiation or induced by viral infection have been previously characterised (Hong et al., 1993; Yancopoulous et al., 1990). However, there is no information about the specific responsive genes of fish B cells. Genes that are specifically expressed in response to stress often encode proteins that are essential for the function of that tissue or cell type (Bureau et al., 1990; Fujiki et al., 1999; Hong et al., 1993; Yancopoulus et al., 1990). The cDNA subtraction method, which is a method for enriching the clones of cDNAs that are specifically-expressed in response to a particular stimulus, depends on the use of cell types whose mRNA populations are similar, di#ering in only a limited number of their expressed genes (Sive & John, 1988). A subtractive cDNA can be used as a DNA probe for screening a cDNA library, which is called di#erential hybridisation, from cells or tissues that show a stress response. A cloned population of Japanese flounder Paralichthys olivaceus, has been produced and established (Yamamoto, 1999). Such a cloned population is useful for molecular biological and genome analysis of fish. Moreover, Japanese flounder is one of the most important food fish of both the fishing and aquaculture industries in Japan. In this study, cDNAs that are up-regulated in hirame rhabdovirus (HRV)-infected Ig+ leucocyte cells of Japanese flounder were identified by a di#erential hybridisation and cloning strategy. II. Materials and Methods CONSTRUCTION OF CDNA LIBRARY
The immune response of homocloned Japanese flounder Paralichthys olivaceus, was activated in vivo by intramuscular inoculation with 105·8 TCID50 fish 1 of hirame rhabdovirus (HRV). Peripheral blood samples were taken from three fish at 12, 24, and 36 h post-injection. Leucocytes were isolated by centrifugation at 400g for 20 min, with Percoll solution (1·072 g ml 1). mRNA from three individual fish in each time group was isolated using a micro mRNA purification kit (Amersham-Pharmacia, U.S.A.), pooled and used to construct a cDNA library (Aoki et al., 1999; Nam et al., 2000). The cDNA library synthesis was performed using a cDNA synthesis kit
IDENTIFICATION OF VIRAL INDUCED GENES
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(Amersham-Pharmacia, U.S.A.) with an oligo dT primer. The cDNA library was constructed in ZAPII vectors according to the manufacturer’s instructions (Stratagene, U.S.A.). PREPARATION OF IG+ LEUCOCYTES
Percoll-separated leucocytes were allowed to react with mouse anti-Japanese flounder Ig mab (Tokuda et al., 1999) and were then incubated with magnetic beads conjugated to goat anti-mouse IgG mab (Miltenyi Biotec, U.S.A.). Labelled cells were separated from unlabelled cells by magnetic cell sorting using the MiniMACS (Miltenyi Biotec, U.S.A.) according to the manufacturers instructions. The magnetic cells were retained in a MiniMACS column inserted into a MiniMACS magnet while the nonmagnetic cells passed through. Labelled cells were eluted after the column was removed from the magnet. DIFFERENTIAL HYBRIDISATION FOR SCREENING THE CDNA LIBRARY
mRNA was extracted from the Ig+ leucocytes of HRV-infected and apparently healthy Japanese flounder using a micro mRNA purification kit (Amersham-Pharmacia, U.S.A.). The Ig+ leucocytes of HRV-infected Japanese flounder mRNA and apparently healthy Japanese flounder were used for isolation of subtracted and unsubtracted cDNA probes by using a Subtraction kit (CLONTECH, U.S.A.). cDNA obtained by the subtraction method (probe one) and un-subtracted cDNA (probe two) were used for di#erential hybridisation. Plaque hybridisation was performed as described by Sambrook et al. (1989). Clones that were positive for probe one and negative for probe two were selected for further analysis. PLASMID PREPARATION AND DNA SEQUENCING
Conversion of the recombinant ZAPIIs into pBluescript plasmid was carried out by in vivo excision according to the protocol of the manufacturer (Stratagene, U.S.A.). cDNA clones were sequenced using ThermoSequenase (Amersham-Pharmacia, U.S.A.) with M13 forward and/or M13 reverse primers and an automated DNA sequencer LC4000 (Li-Cor, U.S.A.). Each sequence was compared with all sequences available in DDBJ/EMBL/ GenBank using the BLAST version 2.0 (Altschul et al., 1990, 1997) (http:// www.ncbi.nlm.nih.gov). RT-PCR ANALYSIS
From 15 viral infected homocloned fish peripheral blood samples were taken at 0, 12, 24, 48 and 72 h post-injection, three fish in each time group. Total RNA was extracted from leucocytes using TRIZOL (Life Technologies, U.S.A.). The purified total RNA (5 g) was reverse transcribed into cDNA using the AMV Reverse Transcriptase First-strand cDNA Synthesis kit (Life Science, U.S.A.). The final volume of the cDNA synthesis reaction was 25 l. The reversetranscribed sample (1 l) was used in 50 l of PCR reaction mixture. The PCR primers that were used in this study are listed in Table 1. The -actin primer set was used as a positive control of RT-PCR (Katagiri et al., 1997). PCR was
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Table 1. Sequences of used primers for RT-PCR Primer name JFIG19-F JFIG19-R JFIG68-F JFIG68-R JFIG108-F JFIG108-R JFIG106-F JFIG106-R JFIG6-F JFIG6-R -actin-F -actin-R
Sequences 5 -GAACCTGGTCCTCTCCTCCT-3 5 -CTGGACACTCTCTCCGTTCT-3 5 -GACAACTCGGAGCTTCTTCA-3 5 -AGCCAAACCATCCCTAGACC-3 5 -TCCAGCTGGGGTCCATCGAA-3 5 -TCTGGAAGTAATCAGCAGCC-3 5 -GCCTTGAGGGTGCACTGCCC-3 5 -CCCTCCTGGCTGAAAATGCC-3 5 -AAGAACTAGCAGACTGGAAG-3 5 -CACATCACCAGATTCCTTGT-3 5 -ACTACCTCATGAAGATCCTG-3 5 -TTGCTGATCCACATCTGCTG-3
performed with an initial denaturation step of 1 min at 95 C, then 20 cycles were run as follows: 30 s of denaturation at 95 C, 30 s of annealing at 55 C and 30 s of extension at 72 C. The reacted products were electrophoresed on a 2·0% agarose gel. III. Results and Discussion Approximately 25 000 independent clones were initially screened with probes one and two. Most of clones displayed a positive signal with both probes, but some of the clones displayed a positive signal with only probe one. Fifty clones that displayed a positive signal with only probe one were selected. Of these 50 clones (DDBJ/GenBank/EMBL accession No. AU083036AU083141), 30 were found to have significant identities to previously reported genes and cDNA sequences by the BLAST analysis. These 30 clones were comprised of 24 di#erent cDNAs (Table 2). The other 20 clones coded other, unknown sequences (data not shown). Three of the 30 identified clones were clones of genes that encoded IgD and five were of genes that encoded secreted IgM. Recently, an expressed sequence tag (EST) analysis of Japanese flounder leucocyte cells which were infected with HRV was reported (Nam et al., 2000; Aoki et al., 1999). From over 1500 ESTs examined, no IgD cDNA was isolated and only one IgM cDNA was isolated. Magnetic separation using mouse anti-Japanese flounder Ig mab showed that the Ig+ leucocytes were enriched. Interestingly, membrane IgM cDNAs were not isolated in this study although five clones of secreted IgM cDNA were isolated. These results suggest the presence of IgD and secreted IgM cDNAs after the subtraction may be because the Ig+ leucocytes were stimulated by HRV infection, which could have resulted in the activation of these genes. Thus, these cDNAs would have become enriched after the subtraction. The cDNAs for homologues of several biodefence and immune-related genes were isolated (Table 2). This is the first time that each of these genes has been reported in teleostei, except gelatinase-b, collagenase III and Stat3. Several of the cDNAs in Table 2 are known to be stress-inducible genes, including the
AU083136 AU083087 AU083065 AU083059 AU083063 AU083075 AU083102 AU083044 AU083125 AU083116 AU083081 AU083042 AU083110 AU083089 AU083107 AU083038 AU083073 AU083085 AU083092 AU083039 AU083139 AU083071 AU083083 AU083079
Accession no.
ATP synthase lipid binding protein P3 Basigin III CEF-10 Collagenase-3 Dihydrolipoamide succinyltransferase Erythropoietin receptor Gelatinase-B (collagenase IV) Herpes virus entry mediator (TNFR family) IgD IgM Inducible polyA binding protein Interferon inducible 56K protein Megacaryocyte stimulating factor Melanoma-associated antigen MUC-18 Nuclear factor Phospholipase C-gamma-2 PKC inhibitor Prolylcarboxylpeptidase Prostaglandintransporter (matrin) Protease-nexin 1 (SERPIN) Rab8-interacting protein RGS5 Stat3 Yrk (tyrosine-protein kinase)
Identification Homo sapiens Gallus gallus Gallus gallus Xenopus laevis Fugu rubripes Homo sapiens Cynops pyrrhogaster Homo sapiens Ictalurus punctatus Oncorhynchus mykiss Homo sapiens Homo sapiens Homo sapiens Homo sapiens Xenopus laevis Rattus norvegicus Bos taurus Homo sapiens Rattus norvegicus Rattus norvegicus Mus musculus Mus musculus Oncorhynchus mykiss Gallus gallus
Species P48201 P17790 P19336 U41824 U40758 P19235 D82052 U70321 U67437 X65261 U33818 P09914 U70136 P43121 S80988 P24135 P29312 P42785 Q00910 P07092 U50595 U67188 U60333 Q02977
Accession no.
Quality of match is given as percent identity of amino acid sequences (%ID) and length in amino acid residues for peptide meatches.
JFIG-M37 JFIG-85-2F JFIG-19F JFIG-141F JFIG-150-3F JFIG-30F JFIG-B280-2F JFIG-117-2F JFIG-IG-59R JFIG-IG-125-2R JFIG-68-3R JFIG-108R JFIG-I184-2R JFIG-92-1R JFIG-B9F JFIG-105-1R JFIG-29R JFIG-73F JFIG-99F JFIG-106R JFIG-M38R JFIG-25-1R JFIG-6F JFIG-58F
Clone
Table 2. Identified clones of Ig+ leucocyte cells from Japanese flounder infected with HRV
74·6 40·8 62·5 45·3 78·8 22·1 64·3 34·2 43·1 42·5 78·1 25·5 21·5 30·1 32·7 54·2 82·9 56·1 52·2 48·8 67·2 40·6 81·7 66·7
%ID
137 262 184 226 146 263 258 184 137 240 113 306 233 173 266 306 246 194 249 258 116 192 231 238
Length
IDENTIFICATION OF VIRAL INDUCED GENES
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1. Time course of the expression of subtracted cDNA clones in peripheral blood leucocytes, as detected by RT-PCR. Expression levels at various times after injection of HRV are shown. Lanes 1–3, 0 h; lanes 4–6, 12 h; lanes 7–9, 24 h; lanes 10–12, 48 h; lanes 13–15, 72 h. RT-PCR was carried out with the primer sets of JFIG-108R (a) (IFI56), JFIG-19F (b) (CEF-10), JFIG-6F (c) (Stat3), JFIG-106R (d) (protease-nexin 1), JFIG-68-3R (e) (inducible poly (A) binding protein) and -actin (f).
genes for IFI56 (Wathelet et al., 1986), Stat3 (Johnson et al., 1996), CEF-10 (Simmons et al., 1989), inducible poly (A) binding protein (Yang et al., 1995), prolylcarboxylpeptidase, proteasome-nexin 1 (SERPIN) (Sommer et al., 1987), collagenase III (Brown et al., 1996), gelatinase-b (collagenase IV) (Wilhelm et al., 1989), megakaryocyte stimulating factor (Turner et al., 1991), specific stress inducible protease prolylcarboxylpeptidase (Tan et al., 1993), Herpes virus entry mediator (tumor necrosis factor receptor family) (Montgomery et al., 1996), and Rab8-interacting protein genes (Ren et al., 1996). Recently it was reported that the gelatinase-b (collagenase IV) and collagenase III were highly expressed in Japanese flounder leucocytes which were infected with HRV (Aoki et al., 1999). In mammals, gelatinase and collagenase are expressed in granule cells, and act for defence to infections. It may therefore be proposed that the Japanese flounder gelatinase-b and collagenase III will also act for defence to the HRV infection. Recently, the expression of the Mx gene, which codes for an antiviral protein, of Japanese flounder was shown to be induced by HRV infection (Lee et al., 2000). In this study, RT-PCR was used to analyse the e#ect of HRV on the levels of expression of the genes for homologues of IFI56 (JFIG-108R), CEF-10 (JFIG-19F), Stat3 (JFIG-6F), protease-nexin 1 (JFIG-106R) and inducible poly(A) binding protein (JFIG-68R) genes. The amount of mRNA of each of the examined genes increased after HRV infection although the expression level of house keeping gene -actin were very similar in all samples (Fig. 1). The expression levels of these genes were di#erent but the patterns of increase of the mRNA were similar over time. These expression patterns were also similar to the expression pattern of the antiviral protein Mx (Lee et al., 2000).
IDENTIFICATION OF VIRAL INDUCED GENES
629
Interestingly, after 24 h of HRV injection, the expression pattern of three fish were di#erent. The CEF-10 expression of a single fish (Fig. 1, fish 8) from the 24 h sample had not increased although the expression levels of the other two fish had. In contrast, the expression levels of other four genes of this fish (fish 8) were higher than those of other two fish in that time sample group (Fig. 1). The reasons for such di#erences in the expression pattern of these five genes, 24 h after HRV injection, is unclear. In this study a clonal line of Japanese flounder was used, the same number of HRV was injected into each fish, and these fish were kept in the same large tank. In future studies, the expression of cloned genes using di#erent species and a number of viruses, bacteria, or chemicals injected into such fish will be analysed to gain a better understanding of the expression mechanism of these genes. Di#erential hybridisation, using subtracted and un-subtracted cDNA probes, has been shown to be useful for the isolation and identification of stressinducible genes of homologues of both known genes and new genes of fish. This research was supported in part by a grant from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF97L00902).
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