22(3)-383-389-2009] An EST-based approach for identifying genes expressed in the gills of olive flounder Paralichthys olivaceus

J. Fish Pathol., 22(3) ： 383 ~ 389 (2009)

383 383

�Corresponding Author :

The olive flounder Paralichthys olivaceus is one of the most significant fish species in Korea due to human interests in aquaculture and fisheries. The olive flounder stocks currently used in aquaculture have become appreciably different to their wild counterparts and it seems likely that genetics will play an increasingly more important role in achiev- ing further improvements in the performance of the brood stocks. Traits that may be amenable to genet- ic improvement include growth, disease resistance and sex determination. Despite this interest, rela- tively little information is available about olive flounder genes.

Recently, we have conducted expressed sequence

tags (ESTs) analyses of liver, kidney and testis libraries in olive flounder (Lee et al., 2003, 2006, 2007). ESTs are widely used for identification of genes and for studying expression profiles (Adams et al., 1991; Liew et al., 1994). Several of these studies were specifically designed to identify genes involved in innate and acquired immunity. These studies include the EST researches on olive floun- der leucocytes infected with Hirame rhabdovirus (Aoki et al., 1999; Nam et al., 2000), olive flounder leucocytes stimulated with Con A/PMA (Nam et al., 2003) and kidney tissues from peptidoglycan- stimulated olive flounder (Kono et al., 2001). In addition, ESTs have been obtained from kidney and

An EST-based approach for identifying genes expressed in the gills of olive flounder Paralichthys olivaceus

Jeong-Ho Lee, Jae Koo Noh, Hyun Chul Kim, Choul-Ji Park, Byung Hwa Min, Young-Ok Kim

, Jong-Hyun Kim

, Kyung-Kil Kim

, Woo-Jin Kim

and Jeong-In Myeong

Genetics and Breeding Research Center, NFRDI, Geoje, 656-842, Korea

�

Biotechnology Research Division, NFRDI, Busan, 619-902, Korea

��

Aquaculture Management Division, NFRDI, Busan, 619-902, Korea

Analysis of expressed sequence tags (ESTs) is an efficient approach for gene discovery, expression pro- filing, and development of resources useful for functional genomics studies. As part of studies on the immune system of olive flounder, a total of 251 EST sequences from gill cDNA library were generated to identify and characterize important genes in the immune machanisms of olive flounder. Of the 251 clones, 126 clones (50.2%) were identified as orthologues of known genes from olive flounder and other organ- isms. Among the 126 EST clones, 16 clones (12.7%) were representing 9 unique genes identified as homol- ogous to the previously reported olive flounder ESTs, 100 clones (79.4%) representing 103unique genes were identified as orthologs of known genes from other organisms. We also identified several kinds of immune associated proteins, indicating EST as a powerful method for identifying immune related genes of fish as well as identifying novel genes. Further studies using cDNA microarrays are needed to identify the differentially expressed transcripts after disease infection.

Key words: Olive flounder, Gill, Expressed sequence tags (ESTs), Expression profile, Immune related

genes

�Corresponding Author : Jeong-In Myeong, Tel : 055-633-1272 Fax : 055-633-0891, E-mail : [email protected]

20...

gill tissues of rainbow trout infected with infectious hematopoietic necrosis virus (IHNV) and from head kidney cells of common carp after stimulation with lipopolysaccharide and concanavalin A (Kono et al., 2001; Savan and Sakai, 2002).

Gene cataloguing and profiling of the gill is an essential part of our effort in EST analysis of immune organs because gills directly contact with the external environment; therefore, the cells cru- cially respond to external biotic and abiotic factors.

For example, gills are the siteof formation of hemo- cyte nodules during foreign particle injection (Mar- tin et al., 2000) and accumulation of viable bacteria during infection (Burgents et al., 2005).

As a part of studies on the immune system of olive flounder, partial cDNA sequences or ESTs were generated to identify and characterize impor- tant genes in the immune machanisms. Here, we report a total of 251 EST sequences from olive flounder gill cDNA library on the purpose of identi- fying genes and their expression profiles in the gill, and improving our understanding of the immune system of olive flounder and the availability as a genomic tool.

Materials and Methods

Tissue preparation and RNA isolation

All experimental fish were raised at Genetics and Breeding Research Center, National Fisheries Research and Development Institute (NFRDI) and maintained in 10 tons flow-through tank at 15 ±1

℃ under a natural photoperiod. Gill samples of 10 randomly selected fish were collected and frozen in liquid nitrogen until isolation of RNA. Pooled gill tissues were ground with a mortar/pestle and then homogenized with a hand-held tissue tearor in RNA extraction buffer followed by the guanidium thio-

cyanate method (Chomczynski and Sacchi, 1987).

Poly (A)+ RNA was purified from total cellular RNA using the PolyA Tract mRNA isolation kit (Promega) according to the manufacturer's instruc- tions.

cDNA library construction

A directional cDNA library of the gills was con- structed using the Uni-ZAP XR cDNA synthesis/Gigapack cloning kit (Stratagene Cloning Systems). cDNA synthesis was carried out using an oligo-(dT)

primer for the reverse transcription of approximately 5 ㎍ of mRNA and the libraries were constructed by directional cloning based on the man- ufacturer ’ s instruction manual. All primary libraries were amplified and aliquots of each amplified library were stored at both 4 and -70℃. Mass exci- sion was performed and the cDNA inserts from the amplified Uni-ZAP XR libraries were rescued as pBluescript phagemids in SOLR Escherichia coli.

Plasmid preparation and sequencing analysis The plasmid cDNA library was plated to a density appropriate for picking individual colonies. Random clones were grown in 1.5-ml LB medium overnight in 12 × 75 mm culture tubes. Plasmid DNA was prepared by alkaline lysis method (Sambrook et al., 1989) using the Qiagen Spin Column Mini-plasmid kits. Three microlitres of plasmid DNA (about 0.5- 1.0 ㎍) were used in sequencing reactions. Single- pass sequencing of the 5'-termini of selected cDNA clones in phagemid form was performed using the ABI 3100 automatic DNA sequencer (PE Applied Biosystems) and the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems).

Bioinformatic analysis

Bioinformatic analysis was conducted to deter-

mine gene identities using GeneMaster software (Ensoltek). ESTs were assembled in clusters of con- tigous sequences (contig) using ICAtools program (Parsons, 1995). Gene annotation procedures and homology searches of the sequenced ESTs have been locally done by BLASTX for amino acid simi- larity comparisons (Altschul et al., 1997). Matches with the Expect value (E) less than 1.0 × e

were considered to be significant. After the BLAST searches, a visual inspection was made to determine if the significant similarity was caused by simple sequences. ESTs with significant similarities in searches were considered orthologs of known genes only when the similarities were not caused by sim- ple sequences. All ESTs that were not identified as orthologs of known genes were designated as unknown EST clones.

Results and Discussion

Gene and orthologue identification

To identify genes expressed in the olive flounder gill and establish their expression profiles, we

sequenced 251 ESTs from a non-normalized cDNA library of the olive flounder gill. The number of clones sequenced from the cDNA library, the aver- age size of inserts, and the redundancy of the obtained sequences, are given in Table 1. The aver- age insert size was estimated to be 1.6 ± 0.6 kb by PCR amplification of inserts from 20 randomly selected clones. Clustering of the sequences using the assembly program ICAtools software (Parsons, 1995) showed that the 251 gill ESTs were com- posed of 15 clusters and 203 singletons, suggesting that the overall redundancy of the library was 13.1%.

Of the 251clones, 126 (50.2%) were identified as orthologues of known genes from olive flounder and other organisms. The remaining 125 clones (49.8%) could not be identified by similarity com- parisons (E≥1×e

). Among the 126 EST clones, 16 clones (12.7%) were representing 9 unique genes identified as homologous to the previously reported olive flounder ESTs, 100 clones (79.4%) representing 103 unique genes were identified as orthologs of known genes from other organisms.

Table 1. General characteristics of olive flounder gill ESTs.

Total cDNA sequenced

251

Average insert size

1.6 ± 0.6 kb

Average EST length 621 bp

EST clusters

15

Singletons

203

Redundancy

13.1 %

ESTs with E value<1 ×e

(matched) 126 (50.2%)

ESTs with E value ≥1×e

(unknown) 125 (49.8%)

a

Length of sequence used for comparison after editing (inserts<100 base pairs were excluded).

b

The average insert size was calculated for 20 randomly selected cDNA clones.

c

ESTs with 90% or greater identity over a 100 bp region were clustered together forming 15 EST clusters.

d

203 sequences did not sufficiently match any sequence in the data set to allow assembly.

e

Redundancy = The number of genes / total ESTs.

Ten clones (7.9%) representing 10 unique genes showed significant similarities to known sequences of unknown functions from other organisms.

Functional categorization of the identified ESTs The 126 known genes were categorized into 6 functional groups. Eight (3.2%) of the total ESTs were found to be homologous to genes involved in cell/organism defence (Table 2). The most abundant functional group consisted of genes involved in gene/protein expression for which there were 61 ESTs (24.3% of the total ESTs). Other ESTs were assigned to the following categories: metabolism with 30 ESTs (12.0% of the total ESTs); cell signal- ing/cell communication with 4 ESTs (1.6% of the total ESTs) and cell structure/motility with 13 ESTs (5.2% of the total ESTs).

Expression profile and gene identification Expression profiles of the EST clones identified from the olive flounder gill are shown in Fig. 1. Of the 251 gill ESTs, 203 genes (80.9%) were sequenced only once; 30 genes (11.9%) were sequenced 2-5 times; 18 genes (7.2%) were sequenced over five times. These results suggest that the gill library has a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST pro- jects; thus, the gill library can supply more unique

and novel cDNA sequences in an olive flounder EST project.

The putative amino acid sequence deduced from one cDNA clone, gill1-E05 was identified as Beclin-1, a Bcl-2-interacting protein. The alignment showed that the putative sequence is 82.2%, 80.0%

and 77.8% identical to fugu, salmon and human beclin, respectively (Fig. 2). Beclin-1 is originally identified as a Bcl-2 binding protein and it is known to be a promoter of autophagy (Aita et al., 1999;

Liang et al., 1999, 2001). Beclin 1-dependent autophagy may function in antiviral host defense by several different mechanisms. There is an increas- ing evidence that cytoplasmic bacteria are targeted for lysosomal degradation by an autophagy gene- dependent pathway (Levine, 2005; Amano et al., 2006), and it seems likely that a similar pathway is involved in the degradation of intracytoplasmic viri- ons. Autophagy may also play a direct role in pro- moting neuronal survival during viral infection. In support of this hypothesis, autophagy genes protect against programmed cell death in plant cells infected with tobacco mosaic virus (Liu et al., 2005), in mouse neurons infected with Sindbis virus (Liang et al., 1999), and in neurons subjected to noninfectious forms of cellular stress (Levine and Yuan, 2005).

Another clone, gill1-C05 showed homology to interleukin-17 receptor of salmon, mouse and human, with identities of 70.7%, 44.4% and 42.7%,

Table 2. Functional classification of identified ESTs in the gill library.

Classification Number of clones Clones(%) Number of genes Redundancy factor

Cell signaling/Cell communicaion 4 1.6 4 1.0

Cell structure/Motility 13 5.2 12 1.1

Cell/ Organism defence 8 3.2 8 1.0

Gene/Protein expression 61 24.3 54 1.1

Metabolism 30 12.0 24 1.3

Unknown functions 10 4.0 10 1.0

respectively. IL-17 is a proinflammatory cytokine that plays an important role in the clearance of extracellular bacteria and contributes to the patholo- gy of many autoimmune and allergic conditions (Nakae et al., 2003; Kolls and Linde´n, 2004;

Stark et al., 2005). The receptor for IL-17A (IL- 17R) is a single-pass transmembrane protein of approximately 130 kDa. While the IL-17A cytokine is expressed only by T-cells, its receptor is expressed in all tissues examined to date. The acti- vation of the receptor by IL-17A generally results in the induction of other pro-inflammatory cytokines, through the activation of NF-κ B (Shi et al., 2000;

Lee et al., 2001). Much work on IL-17 and its receptor has been done in humans and higher verte- brates while little work has been conducted in lower vertebrates including fish. As a first step to eluci- date the role of IL-17R in fish, we identified IL-17R in olive flounder for the first time.

In conclusion, 251 ESTs were identified from a non-normalized cDNA library of the olive flounder gill in this study. Expression profiles of these genes were revealed by their frequency in a cDNA library.

We also identified several kinds of immune associ- ated proteins, indicating EST as a powerful method for identifying immune-related genes of fish as well as identifying novel genes. Further studies using cDNA microarrays are needed to identify the differ- entially expressed transcripts after disease infection.

Acknowledgement

This research was supported by National Fish- eries Research and Development Institute (RP- 2009-BT-027) grant.

References

Adams, M.D., Kelley, J.M., Gocayne, J.D., Dub- nick, M., Polymeropoulos, M.H., Xiao, H., Merril, C.R., Wu, A., Olde, B., Moreno, R.F., Kerlavage, A.R., McCombie, W.R. and Venter, J.C.: Complementary DNA sequenc- ing: expressed sequence tags and human genome project. Science, 252:1651-1656, 1991.

Aita, V.M., Liang, X.H., Murty, V.V., Pincus, D.L., Fig. 2. Expression profiles and sequencing redundancy in the analysis of ESTs from the olive flounder gill. Of the 251 gill ESTs, 203 genes (80.9%) were sequenced only once; 30 genes (11.9%) were sequenced 2-5 times; 18 genes (7.2%) were sequenced over five times.

Fig. 1. Alignment of deduced amino acid sequence of clone gill1-E05 with fugu, salmon and human beclin. Identical amino

acid residues with clone gill1-E05are indicated by dots(.). Asterisk(*) indicates the invariant and conserved residues in

beclin. The percentages in parentheses indicate the overall amino acid identities.

Yu, W., Cayanis, E., Kalachikov, S., Gilliam, T.C. and Levine, B.: Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21.

Genomics, 59:59-65, 1999.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J.: Basic local alignment search tool. J. Mol. Biol., 215:403-410, 1990.

Amano, A., Nakagawa, I. and Yoshimori, T.:

Autophagy in innate immunity against intra- cellular bacteria. J. Biochem. (Tokyo), 140:161-166, 2006.

Aoki, T., Nam, B-H., Hirono, I. and Yamamoto, E.:

Sequences of 596 cDNA (565,977 bp) clones of Japanese flounder (Paralichthys olivaceus) leukocytes infected with hirame rhabdovirus.

Mar. Biotechnol., 1:477-488, 1999.

Burgents, J.E., Burnett, L.E., Stabb, E.V. and Bur- nett, K.G.: Localization and bacteriostasis of Vibrio introduced into the Pacific white shrimp, Litopenaeus vannamei. Dev. Comp.

Immunol., 29:681-691, 2005.

Kolls, J.K. and Linde´n, A.: Interleukin-17 family members and inflammation. Immunity, 21:467-476, 2004.

Kono, T. and Sakai, M.: The analysis of expressed genes in the kidney of Japanese flounder, Paralichthys olivaceus, injected with the immunostimulant peptidoglycan. Fish Shell- fish Immunol., 1:357-366, 2001.

Kono, T., Sakai, M. and LaPatra, S.E.: Expressed sequence tag analysis of kidney and gill tis- sues from rainbow trout (Oncorhynchus mykiss) infected with infectious hematopoi- etic necrosis virus. Mar. Biotechnol., 2:493- 498, 2000.

Lee, J.H., Kim, Y.O., Kim, K.K., Kim, W.J., Park, D.W. and Kim, K.W.: Expressed sequence tag

analysis of olive flounder (Paralichthys oli- vaceus): genes and expression profiles from the liver. J. Fish. Sci. Tech., 6:110-115, 2003.

Lee, J.H., Kim, Y.O., Kim, J.H, Noh, J.K., Kim, H.C., Kim, K.K. and Kim, K.W.: Immune gene discovery by expressed sequence tags generated from olive flounder (Paralichthys olivaceus) kidney. Korean J. Ichthyol., 18:283-292, 2006.

Lee, J.H., Kim, J.H., Noh, J.K., Kim, H.C., Kim, Y.O., Kim, W.J., Kim, K.W. and Kim, K.K.:

Expressed sequence tags of expression pro- files of olive Flounder (Paralichthys oli- vaceus) testis. Korean J. Ichthyol., 19:257- 265, 2007.

Lee, J., Ho, W.H., Maruoka, M., Corpuz, R.T., Baldwin, D.T., Foster, J.S., Goddard, A.D., Yansura, D.G., Vandlen, R.L., Wood, W.I.

and Gurney, A.L.: IL-17E, a novel proin- flammatory ligand for the IL-17 receptor homolog IL-17Rh1. J. Biol. Chem., 276:1660-1664, 2001.

Levine, B.: Eating oneself and uninvited guests:

Autophagyrelated pathways in cellular defense. Cell, 120:159-162, 2005.

Levine, B. and Yuan, J.: Autophagy in cell death:

An innocent convict? J. Clin. Invest., 115:2679-2688, 2005.

Liew, C.C., Hwang, D.M., Fung, Y.W., Laurenssen, C., Cukerman, E., Tsui, S. amd Lee, C.Y.: A catalogue of genes in the cardiovascular sys- tem as identified by expressed sequence tags (ESTs). Proc. Natl. Acad. Sci. USA, 91:10645-10649, 1994.

Liang, X.H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H. and Levine, B.:

Induction of autophagy and inhibition of

tumorigenesis by beclin 1. Nature, 402:672-

676, 1999.

Liang, X.H., Yu, J., Brown, K. and Levine, B.:

Beclin 1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function.

Cancer Res., 61:3443-3449, 2001.

Liu, Y., Schiff, M., Czymmek, K., Talloczy, Z., Levine, B. and Dinesh-Kumar, S.P.:

Autophagy regulates programmed cell death during the plant innate immune response.

Cell, 121:567-577, 2005.

Martin, G.G., Quintero, M., Quigley, M. and Khos- rovian, H.: Elimination of sequestered mate- rial from the gills of decapod crustaceans. J.

Crustac. Biol. 20:209-217, 2000.

Nakae, S., Saijo, S., Horai, R., Sudo, K., Mori, S.

and Iwakura, Y.: IL-17 production from acti- vated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist. Proc.

Natl. Acad. Sci. USA, 100:5986-5990, 2003.

Nam, B-H., Yamamoto, E., Hirono, I. and Aoki, T.:

A survey of expressed genes in the leuko- cytes of Japanese flounder, Paralichthys oli- vaceus, infected with hirame rhabdovirus.

Dev. Comp. Immunol., 24:13-24, 2000.

Nam B-H., Hirono, I.and Aoki, T.: Bulk isolation of immune response-related genes by expressed sequenced tags of Japanese floun- der Paralichthys olivaceus leucocytes stimu- lated with Con A/PMA. Fish Shellfish

Immunol., 14:467-476, 2003.

Parsons, J.D.: Improved tools for DNA comparison and clustering. Comp. Appl. Biosci., 11:603- 613, 1995.

Savan, R. and Sakai, M.: Analysis of expressed sequence tags (EST) obtained from common carp, Cyprinus carpio L., head kidney cells after stimulation by two mitogens, lipopolysaccharide and concanavalin-A.

Comp. Biochem. Physiol., 131:71-82, 2002.

Sambrook, J., Frisch, E.F. and Maniatis, T.: Molec- ular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.

Shi, Y., Ullrich, S.J., Zhang, J., Connolly, K., Grze- gorzewski, K.J., Barber, M.C.,Wang, W., Wathen, K., Hodge, V., Fisher, C.L., Olsen, H., Ruben, S.M., Knyazev, I., Cho, Y.H., Kao, V., Wilkinson, K.A., Carrell, J.A. and Ebner, R.:A novel cytokine receptor-ligand pair. Identification, molecular characteriza- tion, and in vivo immunomodulatory activi- ty. J. Biol. Chem., 275:19167-19176, 2000.

Stark, M.A., Huo, Y., Burcin, T.L., Morris, M.A., Olson, T.S. and Ley, K.: Phagocytosis of apoptotic neutrophils regulates granu- lopoiesis via IL-23 and IL-17. Immunity, 22:285-294, 2005.

Manuscript Received : November 19, 2009

Revised : December 15, 2009

Accepted : December 21, 2009