Ubiquitin genes in rainbow trout (Oncorhynchus mykiss)

Fish & Shellfish Immunology (2002) 12, 335–351 doi:10.1006/fsim.2001.0375 Available online at http://www.idealibrary.com on

Ubiquitin genes in rainbow trout (Oncorhynchus mykiss) KAZUE OKUBO1, KEISUKE YAMANO2, QIWEI QIN2†, KAZUHIKO AOYAGI2, MITSURU OTOTAKE2*, TERUYUKI NAKANISHI2‡, HIDEO FUKUDA1 AND JOHANNES M. DIJKSTRA2 1

Department of Aquatic Biosciences, Tokyo University of Fisheries, Tokyo 108-8477, Japan and 2National Research Institute of Aquaculture, Tamaki, Mie 519-0423, Japan (Received 6 April 2001, accepted after revision 6 August 2001, published electronically 4 March 2002) Ubiquitin is a small protein involved in intracellular proteolysis. It is highly conserved throughout eukaryotic phyla and has been detected in such diverse species as yeast, barley, Drosophila and man. A previous study showed that chromatin of rainbow trout testis contains free ubiquitin with a sequence similar to that of other phyla. In the present study, which focused on rainbow trout but included eleven other species, it is shown that fish ubiquitin genetic organisation and expression are similar to those of other phylogenetic groups through the following set of observations: (a) Multiple loci were detected, (b) These loci encode repeats of ubiquitin, (c) Although the DNA sequences are not conserved, the encoded amino acid sequences are fully conserved, (d) The expression of ubiquitin was influenced by cell culture conditions and viral infection.  2002 Elsevier Science Ltd. Key words:

ubiquitin, polyubiquitin, sequence, transcription, IHN virus, rainbow trout.

I. Introduction Ubiquitin is a small protein (76 amino acids) abundantly present in all eukaryotes. It is highly conserved, with the amino acid sequences of ubiquitin in yeast and animals di#ering in only three residues [1]. It can be expressed as polyubiquitin, a repeat of identical ubiquitin units that can be cleaved to individual units, or as a fusion with another protein from which it is cleaved [1–4]. Ubiquitin has been found in the nucleus, where it is conjugated with histones [5] preferentially at transcriptionally active sites [6], suggesting a role in transcription [7]. Ubiquitin is also involved in signal transduction and endocytosis [3, 8]. *Corresponding author. E-mail: ototake@a#rc.go.jp †Present address: Tropical Marine Science Institute, National University of Singapore, 10 Kent Ridge Crescent, 119260 Singapore. ‡Present address: Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan. 335 1050–4648/02/040335+17 $35.00/0

 2002 Elsevier Science Ltd.

336

K. OKUBO ET AL.

UBIQUITIN GENES IN RAINBOW TROUT

337

The best-known function of ubiquitin, however, is its cytoplasmic role in the ubiquitin-proteasome pathway [3, 4, 9, 10]. Proteins are first targeted for degradation by covalent attachment of multiple ubiquitin units via a complex system of enzymes (including E1, E2 and E3), after which they are digested by a large structure called the proteasome (Fig. 1). Selective targeting of proteins for degradation is important for numerous processes and allows cells to di#erentiate. The ubiquitin/proteasome pathway is also important for the removal of a large fraction of newly formed proteins; it is estimated that more than 30% of newly synthesized proteins are made incorrectly due to errors during or after translation [11]. In vertebrates, the digestive products of the proteasome include peptides that can be delivered by transporters associated with antigen processing (TAP) to the lumen of the endoplasmic reticulum, where they are bound by MHC class I molecules [12, 13]. Subsequent presentation of these MHC-bound peptides at the cell surface can induce specific cell-mediated immunity. Specific cell-mediated immunity is thought to be the key immune response against many viral diseases [14, 15]. Ubiquitin therefore plays an important role in host defence mechanisms. An outline of this process is depicted in Fig. 1. It is worth noting however that although the process shown in Fig. 1 is the main route of protein digestion and supply of peptides for MHC class I binding, there are other routes not involving ubiquitin [13, 16].

Fig. 1. Protein destruction by the ubiquitin/proteasome pathway and its supply of antigenic peptides bound by MHC class I as found in mammals. Data shown are reviewed in the studies [3, 9, 10, 12, 13, 16]. (1) The carboxyl terminal glycine residue of free ubiquitin is activated by the ubiquitin-activating enzyme E1. (2) Next, activated ubiquitin is transferred to a family of ubiquitin carrier proteins (ubiquitinconjugating enzymes, E2). Conjugation of ubiquitin to Lys residues of the protein that is to be degraded can occur by direct transfer of ubiquitin from E2 or by a process in which target proteins are first bound to specific sites of ubiquitin protein ligases, E3, before ubiquitin is transferred from E2. (3) Most species have only one E1 enzyme and multiple E2 and E3 enzymes; the E3s play the most crucial role in determining the specificity of the ubiquitination. The ubiquitin attached is further ubiquitinated by a similar process, forming multi-ubiquitin chains. The number of ubiquitin molecules conjugated to each protein is specific for certain proteins and influences the rate by which the proteins are degraded by the 26S proteasome. The proteasome has a$nity for polyubiquitin tags of proteins (4). The proteasome is a huge complex of proteases containing a barrel-shaped core particle consisting of four rings of subunits. In this ‘tunnel’ the target proteins are hydrolysed (5), producing peptides and polyubiquitin. The polyubiquitin is cleaved to monoubiquitin units by members of a family of non-proteasomal ubiquitin-specific proteases (6). The generated peptides can be further digested into amino acids (7) or can be transported to the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP) (8). TAP is a heterodimer with subunits TAP.1 and TAP.2 and is located in the ER membrane. TAP forms a complex together with several MHC class I,
2m (indicated as a grey circle), tapasin (a transmembrane glycoprotein) and calreticulin (a chaperone) molecules. The peptide transported by TAP can bind to the peptide-binding groove of the MHC class I molecule. The MHC class I/b2m/peptide trimer then dissociates from the other components of the complex (9) and is transported through the secretory pathway (10) to the plasma membrane (11), where recognition by the T cell receptors of CD8 positive cytotoxic T cells can occur.

338

K. OKUBO ET AL.

The protective function of the ubiquitin/proteasome pathway is supported by the detection of ubiquitinated forms of several viral proteins [17–19] and findings that interferons [20] or viral infection [21, 22] can up regulate ubiquitin expression. Moreover, proteasome inhibition was found to increase the e$ciency of virus infection [23]. Free ubiquitin has been detected previously in rainbow trout testis chromatin. The protein was isolated and sequenced by Edman degradation and appeared to be similar to calf thymus ubiquitin [24]. Analysis at the protein level also showed that trout ubiquitin can occur conjugated with histones [25]. The present study shows that, at the DNA and RNA levels, fish ubiquitin is organised and regulated in ways similar to those of other phyla. Polyubiquitin units and up regulation of ubiquitin expression after virus infection were observed. II. Materials and Methods FISH

Rainbow trout (Oncorhynchus mykiss), masou salmon (Oncorhynchus masou), amago salmon (Oncorhynchus rhodurus), carp (Cyprinus carpio), goldfish (Carassius auratus), Japanese catfish (Silurus asotus), Japanese eel (Anguilla japonica) and tilapia (Oreochromis niloticus) were raised at the Tokyo University of Fisheries, Tokyo, Japan. Coho salmon (Oncorhynchus kisutch (Walbaum)) were raised at the National Research Institute of Aquaculture, Tamaki, Japan. Brown trout (Salmo trutta), Japanese char (Salvelinus leucomaeris) and brook trout [Salvelinus fontiralis (Mitchill)] were raised at the Hayashi trout farm in Fukushima, Japan. All fish were adults. CELLS AND VIRUSES

Cultured RTG-2 (rainbow trout gonad) cells [26] were maintained in two laboratories. In one laboratory they were grown at 15 C in minimal Eagle’s medium (MEM), supplemented with 10% foetal bovine serum (FBS) and NaHCO3 bu#ered at pH 7·4. These cells were used for the isolation of DNA used for Fig. 3 and of RNA used for Fig. 7 and Fig. 8. In the other laboratory cells were grown at 20 C in MEM medium supplemented with 10% FBS and bu#ered with Tris/HCl at pH 7·4. The RNA isolated from these cells was used for sequence analysis (Fig. 2) and these cells were used for virus infection (Fig. 9). The infectious haematopoietic necrosis virus (IHNV) used in this study was strain G4, isolated in 1992 from rainbow trout in Gifu prefecture, Japan. RTG-2 cells were infected at confluency with IHNV with a multiplicity of infection (M.O.I.) of 0, 0·1 or 5. At the time of infection, 5 ml of the 20 ml of growth medium was replaced with fresh medium in infected and uninfected cell cultures. ISOLATION OF GENOMIC DNA, RNA AND SINGLE STRAND CDNA

DNA was isolated from whole blood. Blood samples of 200 l were mixed with 10 ml lysis bu#er (10 mM Tris-HCl, 125 mM NaCl, 10 mM EDTA, 0·5% SDS,

UBIQUITIN GENES IN RAINBOW TROUT

339

4 M Urea) and incubated overnight at 37 C after addition of 50 g ml
1 proteinase K. After phenolchloroform extraction and ethanol precipitation, the samples were dissolved in 10 mM Tris, 1 mM EDTA. The total RNA samples used for the RT-PCR shown in Fig. 8 were obtained by isolation from kidney or RTG-2 cells using Isogen (Nippon Gene, Tokyo, Japan), following the manufacturer’s instructions. The mRNA used for Northern blot analysis (Fig. 7) was obtained from this total RNA with a Quikprep micro mRNA purification kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The total RNA samples from virus- or mock-infected RTG-2 cells were obtained with TRIzol (GibcoBRL, Grand Island, NY, U.S.A.) following the manufacturer’s recommendations. For the production of cDNA for PCR and sequence analysis, total RNA was isolated from the kidney (including pronephros) using a RNA extraction kit (Amersham Pharmacia Biotech, Uppsala, Sweden), followed by mRNA purification with a mRNA purification kit (Amersham Pharmacia Biotech). Subsequently, cDNA was produced from isolated mRNA using pd(N)6 primers (Amersham Pharmacia Biotech). PRIMERS

The primers pUbi1, 5 -ATGCAGATCTTTGTAAAAACCTTGACC-3 (forward), pUbi2, 5 -TCAGCCACCCCTGAGCCGAAGTACAAG-3 (reverse, including stop codon irrelevant for this study), pUbi3, 5 -CTGGAAGATG GGCGAACCC-3 (forward), and pUbi4, 5 -CTGCTTGCCAGCAAAGATCA ACC-3 (reverse), were derived from the reported Xenopus laevis ubiquitin sequence pXIgC20 [27]. The primers pUbi1 and pUbi2 bind at the beginning and end of the ubiquitin units respectively, and pUbi3 and pUbi4 bind in the middle (Fig. 2). PCR AND RT-PCR

PCR on genomic DNA of rainbow trout as shown in Fig. 5 was performed with primers pUbi3 and pUbi4 and by using Takara Ex Taq polymerase (Takara, Tokyo, Japan), with 0·1 g genomic DNA in 50 l reaction mixtures and following the instructions of the manufacturer. The PCR schedule was: 5 min at 95 C, 30 cycles (30 s at 95 C, 30 s at 58 C, 30 s at 72 C), and finally 5 min at 72 C. PCR with genomic DNA of several fish species (Fig. 6) was performed using primers pUbi3 and pUbi4 and Takara Ex Taq polymerase (Takara, Tokyo, Japan), with 20 ng genomic DNA in 50 l reaction mixtures and following the instructions of the manufacturer. The PCR schedule was: 5 min at 95 C, 30 cycles (30 s at 95 C, 30 s at 56 C, 30 s at 72 C), and finally 5 min at 72 C. RT-PCR with total RNA isolated from RTG-2 cells or rainbow trout head kidney and primers pUbi3 and pUbi4 was performed using 10 ng total RNA and the RT-PCR high-Plus kit (Toyobo, Osaka, Japan) in 20 l reaction mixtures following the manufacturer’s instructions. The RT-PCR schedule was 30 min at 60 C, 2 min at 94 C, and then 30 cycles of 30 s at 94 C, 1·5 min at 55 C.

340

K. OKUBO ET AL.

Fig. 2. Ubiquitin sequences detected in RTG-2 cells. The Ub2-1 and Ub2-2 dimer sequences, and the Ub1-1, Ub1-2, Ub1-3, Ub1-4 and Ub1-5 monomer sequences are shown. Dashes indicate identical nucleotides. The ends of the Ub2-1 sequence depicted in bold letters are derived from the primer sequences pUbi1 and pUbi2 that were used for the amplification of Ub2-1, Ub2-2, Ub1-1, and Ub1-2. The ends of the Ub1-3 sequence depicted in bold letters indicate the primer sequences pUbi3 and pUbi4 that were used for the amplification of Ub1-3, Ub1-4, and Ub1-5. The encoded amino sequence is indicated above the nucleotide sequences and is identical for all clones.

UBIQUITIN GENES IN RAINBOW TROUT

341

Fig. 3. Southern blot analysis of genomic DNA of RTG-2 cells using an ubiquitin probe. DNA was digested with Kpn I (K), Pst I (P) or Hind III (H). Binding of a digoxinin-labelled ubiquitin gene fragment was detected with X-ray film. Size markers are present at the right.

PCR with cDNA of RTG-2 cells and primers pUbi1 and pUbi2, or pUbi3 and pUbi4, was performed using Ampli Taq Gold DNA polymerase (PerkinElmer, Tokyo, Japan), with 200 ng cDNA in 100 l reaction mixtures and following the manufacturer’s instructions. The PCR schedule was 2 min at 94 C, and then 30 cycles of 30 s at 94 C, 30 s at 58 C, 1 min at 72 C. SEQUENCE ANALYSIS

PCR fragments obtained from amplification of RTG-2 cell cDNA were cloned into the pGEM-T Easy Vector (Promega, Madison, U.S.A.) and sequenced using a CEQ Dye terminator cycle sequencing kit (Beckman Coulter, Fullerton, U.S.A.) and vector-based primers. Sequence analysis was performed with an automated sequencer ceqtm2000 DNA analysis system (Beckman Coulter). The computer software GENETYX version 10.1.3 (Software Development Co., Ltd, Tokyo, Japan) was used for translation into proteins and alignment of sequences. SOUTHERN AND NORTHERN BLOT ANALYSIS

For Southern blot analysis of genomic DNA, 10 g DNA was digested with 150 U of the restriction enzymes Apa I, Kpn I, Pst I or Hind III and then electrophoretically separated in 1% agarose gels. For Southern blot analysis of PCR products, 10 ng DNA was electrophoretically separated in 1% agarose gels. For Northern blot analysis 10 g of total RNA or 1 g of mRNA was electrophoretically separated in 1% agarose, 10% formaline gels. The DNA (Southern blots) or RNA (Northern blots) was transferred to Hybond-N + nylon membranes (Amersham Pharmacia Biotech) and incubated with digoxininlabelled PCR fragments obtained from a monomeric ubiquitin clone with primers pUbi3 and pUbi4. Digoxinin labelling was performed with the PCR

342

K. OKUBO ET AL.

Fig. 4. Southern blot analysis of genomic DNA from several fish species using an ubiquitin probe. The species were coho salmon (Oncorhynchus kisutch) (1), rainbow trout (Oncorhynchus mykiss) (2), masou salmon (Oncorhynchus masou) (3), amago salmon (Oncorhynchus rhodurus) (4), brown trout (Salmo trutta) (5), Japanese char (Salvelinus leucomaeris) (6), brook trout (Salvelinus fontiralis) (7), carp (Cyprinus carpio) (8), goldfish (Carassius auratus) (9), Japanese catfish (Silurus asotus) (10), Japanese eel (Anguilla japonica) (11) and tilapia (Oreochromis niloticus) (12). DNA was digested with Pst I and binding of a digoxinin-labelled ubiquitin gene fragment was detected with X-ray film. Size markers are present at the right.

DIG probe synthesis kit (Boehringer, Mannheim, Germany), and analysis of hybridisation signals was performed according to the manufacturer’s recommendations. For all blots the hybridisation temperatures were 37 C, but the washing conditions di#ered. The washing steps for the Southern blots in Figs 3, 5 and 8 and the Northern blot in Fig. 7 consisted of two 15 min washes in 0·1SSC/0·1% SDS at 68 C. The washing steps for the Southern blot in Fig. 4 consisted of two 15 min washes in 1SSC/0·1% SDS at room temperature. The washing step for the Northern blot in Fig. 9 consisted of two 15 min washes in 2SSC/0·1% SDS at 56 C. The hybridisation patterns were detected using X-ray film. III. Results SEQUENCE ANALYSIS OF UBIQUITIN IN RTG-2 CELLS

Ubiquitin-specific PCR was performed on cDNA from RTG-2 cells. Sequences were determined for monomeric and dimeric ubiquitin fragments obtained with primers pUbi1 and pUbi2, and for monomeric ubiquitin fragments obtained with primers pUbi3 and pUbi4. It was not possible to clone the larger repeats (see below) presumably due to illegitimate recombination

UBIQUITIN GENES IN RAINBOW TROUT

343

Fig. 5. Southern blot of ubiquitin repeats amplified from the genome of a rainbow trout. Genomic DNA was used as target for PCR amplification with primers pUbi3 and pUbi4. Binding of a digoxinin-labelled ubiquitin gene fragment was detected with X-ray film. Size markers are present at the right. The hybridising PCR fragments likely represent di#erent repeats of the 228 bp ubiquitin unit (1–6).

Fig. 6. Agarose gel electrophoresis of ubiquitin-specific PCR products from genomic DNA of several fish species. Genomic DNA was used as target for PCR amplification with primers pUbi3 and pUbi4. The numbers 1–12 represent fish species as shown in Fig. 4. The 228 bp monomer (1) and the 456 bp dimer (2) appear to be present in all samples. The outer lanes were loaded with a 100 bp ladder.

events. Fig. 2 shows a comparison of two dimeric repeats and four monomeric ubiquitin fragments. The clones contain di#erent nucleotide sequences, but the encoded proteins are identical to each other and to those in other species such as man (GenBank P02248), Xenopus laevis (GenBank AAA49978) and Drosophila melanogaster (GenBank AAA28997).

344

K. OKUBO ET AL.

SOUTHERN BLOT ANALYSIS OF RTG-2 CELLS

A Southern blot was performed using RTG-2 cell genomic DNA digested with several restriction enzymes and labelled ubiquitin as a probe (Fig. 3). The labelling pattern indicates that there may be four loci containing ubiquitin sequences. SOUTHERN BLOT ANALYSIS OF SEVERAL FISH SPECIES

A Southern blot was performed using Pst I-digested genomic DNA of salmonids, common carp, goldfish, Japanese catfish, Japanese eel and tilapia (Fig. 4). The data indicate that all fish have several loci containing ubiquitin sequences. The Pst I fragments hybridising with the ubiquitin probe are di#erent for the rainbow trout individual and the RTG-2 cells (compare Fig. 4 with Fig. 3). PCR OF GENOMIC DNA OF RAINBOW TROUT

PCR was performed on genomic DNA of a rainbow trout individual with the primers pUbi3 and pUbi4. This resulted in a ladder of amplified fragments that likely consist of multiple repeats of the 228 bp ubiquitin unit. All of the fragments of this ladder reacted with an ubiquitin probe in Southern blot analysis (Fig. 5). The size of the largest hybridising fragment was in agreement with the size expected for ubiquitin hexamers. UBIQUITIN-SPECIFIC PCR IN SEVERAL FISH SPECIES

PCR was performed using ubiquitin-specific primers and genomic DNA of several salmonids, common carp, goldfish, catfish, eel and tilapia (Fig. 6). Although sequence analysis was not performed, probable 228 and 456 bp products were amplified in all species. This indicates that the presence of ubiquitin repeats is common in fish. NORTHERN BLOT ANALYSIS OF RAINBOW TROUT

Northern blot analysis was performed with mRNA isolated from RTG-2 cells and from the head kidney of a rainbow trout individual (Fig. 7). Surprisingly, the transcripts that hybridised with an ubiquitin probe were di#erent. In the head kidney, mRNA transcripts 1·2, 1·6, 2·2 and 3·0 kb in size were detected. In RTG-2 cells the 1·2 and 2·2 kb transcripts were detected, while the other transcripts found in head kidney were absent and additional transcripts of 2·5 and 3·6 kb were present. Although not indicated clearly, transcripts somewhat larger than the 1·2 kb transcript appear to be present in the RTG-2 cells. RT-PCR FOR RAINBOW TROUT

RT-PCR using primers pUbi3 and pUbi4 and total RNA isolated from the head kidney of a rainbow trout individual or from RTG-2 cells resulted in the amplification of several fragments that appeared to include a ladder of repeats of the 228 bp ubiquitin unit. A Southern blot with an ubiquitin probe

UBIQUITIN GENES IN RAINBOW TROUT

345

Fig. 7. Ubiquitin-specific Northern blot analysis of messenger RNA from RTG-2 cells (1) and from the head kidney of a rainbow trout (2). Binding of a digoxinin-labelled ubiquitin gene fragment was detected with X-ray film. Band sizes are indicated at the left and right.

Fig. 8. Southern blot analysis of ubiquitin repeats amplified by RT-PCR using total RNA from a rainbow trout individual (1) and RTG-2 cells (2). RT-PCR amplification was performed with primers pUbi3 and pUbi4. Binding of a digoxinin-labelled ubiquitin gene fragment was detected with X-ray film. Size markers are present at the right. The hybridising PCR fragments likely represent di#erent repeats of the 228 bp ubiquitin unit (1–4).

confirmed that this ladder was indeed present (Fig. 8). The largest hybridising fragment appeared to represent a four-fold repeat. UBIQUITIN TRANSCRIPTS ARE DIFFERENTIALLY REGULATED

RTG-2 cells were infected with IHN virus at low multiplicity of infection (M.O.I.) (0·1) or high M.O.I. (5) and total RNA was isolated at 12 and 24 h

346

K. OKUBO ET AL.

Fig. 9. Ubiquitin transcription after virus infection. Total RNA was isolated from RTG-2 cell monolayers at 12 h (12) or 24 h (24) after mock infection (U) or infection with a low dose (L) or high dose (H) of IHN virus. Total RNA of the RTG-2 cells was electrophoretically separated, A, and binding of a digoxinin-labelled ubiquitin gene fragment probe was analysed by Northern blot analysis. A short exposure of the blot to X-ray film is shown in B, and a long exposure in C. The sizes of the ribosomal RNA are shown on the left.

post-infection. Northern blot analysis using an ubiquitin probe revealed at least six transcripts of approximately 1·2, 1·4, 1·6, 2·2, 2·5 and 3·4 kb in length (Fig. 9). In uninfected controls the major transcript was 1·2 kb in length, whereas in infected cells the 2·2 and 2·5 kb transcripts were also abundant. The 1·2 kb transcript appeared to be more abundant in uninfected cells at 24 h after mock infection than at 12 h after mock infection. IHN virus infection appeared to stimulate the expression of all six transcripts. Longer exposure of the Northern blot [Fig. 9(C)] showed that all six transcripts could be detected in uninfected cells as well. IV. Discussion Ubiquitin tagging is a major determinant for the destruction of proteins by the proteasome, a process that supplies antigenic peptides that can bind to MHC class I molecules. Therefore, to understand this process is of fundamental immunological interest. Dysfunction of the ubiquitin/proteasome pathway has been shown to increase the e$ciency of virus infection [23] and has also been suggested to be a possible cause of diseases characterised by protein aggregations such as Parkinson’s and Huntington’s diseases [28].

UBIQUITIN GENES IN RAINBOW TROUT

347

Watson et al. [24] previously reported the rainbow trout ubiquitin sequence obtained by protein sequencing; that sequence di#ers by one amino acid from the one presented in this study, with a phenylalanine at position 30 instead of isoleucine. It is unclear if this is due to experimental error, but the predicted sequence in the present study is identical to that in such divergent species as man, frogs and insects. In this study Southern blot and Northern blot data indicated that ubiquitin in rainbow trout and other fish is encoded by several di#erent loci. Genomic PCR and RT-PCR analysis indicated that at least one of these loci expresses ubiquitin repeats. The genomic PCR for rainbow trout with primers pUbi3 and pUbi4 amplified what appeared to be ubiquitin hexamers. Because these primers bind in the middle of the ubiquitin unit, heptamers are expected to be present. Sequence analysis of the ubiquitin units showed variation at the nucleotide level, but no variation in the encoded amino acid sequence. The expression of ubiquitin as polyubiquitin, with silent variation at the nucleotide level, has also been described in other species [1, 27, 29, 30]. Polyubiquitin has been found in diverse species such as yeast (5 ubiquitin repeats), barley (d3), Drosophila (15), Xenopus (>12), chicken (4) and humans (9) [1]. There is more than one ubiquitin locus in each of these species [1], which agrees with the probable two to four loci detected in the fish species investigated in this study (Fig. 4). Polyubiquitin is cleaved between the glycine at position 76 and the methionine at position 1 of the following unit, resulting in single unit ubiquitin molecules that can label proteins for destruction (reviews [1, 3, 4]). Ubiquitin is abundant in many cells, e.g. human fibroblasts contain approximately 108 molecules per cell [31], and the multiple locus and the polyubiquitin organisation allow for high expression levels. Under certain conditions the ubiquitin expression is upregulated. For example, increased ubiquitin transcripts were found during sepsis in rat muscle concomitant with increased proteolysis [9]. Many kinds of stress appear to stimulate ubiquitin expression, including heat shock (hamster: [32]; chicken: [33]; yeast: [34]), starvation (yeast: [34]), respiratory stress (yeast: [34]), incubation with DNA damaging agents (hamster [32]; yeast: [34, 35]) and virus infection (hamster: [21]; humans: [22]). Furthermore, up regulation of ubiquitin genes has been observed during meiosis (yeast: [35]) and after induction by interferons (pig: [20]), and over expression of ubiquitin has been found in cells with a sustained state of proliferation (mouse: [36]). The green alga Chlamydomonas reinhardtii expressed more ubiquitin during the dark [37] and exponentially growing yeast expressed more ubiquitin than yeast in the stationary phase [38]. Clear proof of the function of these up regulations seems absent, but in some studies it may relate to attempts of the cell to deal with abnormal proteins formed during stresses or to viral proteins which are abnormal to the cell [21, 23, 32]. In summary, a large number of factors can influence ubiquitin expression, which is not surprising since a large number of cellular processes require selective protein degradation. In RTG-2 cells all of the di#erent ubiquitin transcripts were stimulated by IHN virus infection (Fig. 9). Figure 9 shows that expression of the 1·2 kb transcript was also stimulated by other (unknown) factors related to the state

348

K. OKUBO ET AL.

of the cells. That factors other than virus infection can influence the expression of the di#erent transcripts is also shown in the present study by comparison of the Northern blots for uninfected RTG-2 cells in Fig. 7 and Fig. 9. Comparison between Fig. 7 and Fig. 9 shows that the 2·2 and 2·5 kb transcripts are not solely regulated by virus infection. Presumably di#erences in cell cycle or cell metabolism may explain the di#erences in the ubiquitin gene transcription between the di#erent uninfected RTG-2 cultures, but more research remains to be done. The transcripts detected by Northern blot analysis using RNA from RTG-2 cells and RNA from the head kidney of an individual rainbow trout were clearly di#erent (Fig. 7). The Southern blot data for the RTG-2 cells and the rainbow trout individual were di#erent as well (Figs 3 and 4). Di#erences in ubiquitin genes between individuals were similarly detected at both the genomic and the transcription levels in Xenopus laevis [27]. Unequal crossover has been suggested to generate variation in ubiquitin coding unit numbers in human polyubiquitin genes [39]. Such recombination events may also have created the di#erences observed in genome fragments or transcript sizes between rainbow trout individuals. It is unclear whether the rainbow trout genome fragments or mRNAs detected in this study encode polyubiquitin or fusions between ubiquitin gene and a di#erent protein. These fusions have been found in a number of species. Human cells contain three size classes of ubiquitin mRNAs 0·65, 1·1 and 2·5 kb in length [30, 40, 41], and it has been shown that the 1·1 kb and 2·5 kb transcripts encode polyubiquitin whereas the smaller transcripts encode a fusion protein. In yeast, three gene hybrids between ubiquitin and other protein fragments have been found in addition to polyubiquitin [38]; these fusion proteins are cleaved into ubiquitin and a protein incorporated in the ribosome [42]. Gene fusions of ubiquitin with ribosomal proteins have also been described for other species (humans: [43]; the amoeba Acanthamoeba castellanii: [44]; rat: [2]). Presumably, the ubiquitin in these fusions functions as a chaperone to promote correct formation of intracellular structures [42]. Why Southern blot analysis of RTG-2 cells detected only four ubiquitin loci, whereas Northern blot analysis indicated six transcripts, is unclear. Some loci may have polymorphism not detected in the Southern blot analysis, or perhaps some loci give rise to more than one transcript due to variable RNA processing. Another possible explanation is that some genes with high sequence homology to ubiquitin are more easily recognised by Northern blot analysis than by Southern blot analysis. Co-migration of loci could also explain the discrepancy, although these loci would have to be very similar since three di#erent restriction enzymes (Fig. 3) did not separate them. The di#erences in intensity of the bands detected in Southern blot analysis may be related to di#erences in the number of ubiquitin units. This study adds to the rapidly increasing knowledge regarding genes related to the MHC class I pathway in fish [45, 46]. The data presented here for ubiquitin agree completely with findings for other species. Presently it is unclear which gene transcripts express polyubiquitin and which express fusions between ubiquitin and another protein; this should be clarified in the future, along with a more detailed analysis of gene regulation.

UBIQUITIN GENES IN RAINBOW TROUT

349

Hopefully, increasing knowledge of fish ubiquitin will contribute to research on the MHC class I pathway. For example, e$cient targeting of protein into this pathway might be achieved by linking them to a noncleavable ubiquitin unit [15, 47]. In mammals some of these fusions abrogated the humoral response and enhanced the cell-mediated cytotoxic response [15]. If this is found in fish as well, such constructs could contribute to basic research on cell-mediated cytotoxicity and to the development of better vaccines. This study was supported by ‘the promotion of basic research activities for innovative biosciences’ funded by Bio-oriented Technology Research Advancement Institution (BRAIN) and supported by a post-doctoral fellowship for Qiwei Qin from the Science and Technology Agency of Japan. We thank Mr Soichiro Hayashi from the Hayashi trout farm in Fukushima, Japan, for kindly giving us the fish used in this study. We thank Dr James D. Moore, Bodega Marine Laboratory, U.S.A., for his help in editing this article.

References 1 Sharp, P. M. & Li, W. H. (1987). Ubiquitin genes as a paradigm of concerted evolution of tandem repeats. Journal of Molecular Evolution 25, 58–64. 2 Chan, Y. L., Suzuki, K. & Wool, I. G. (1995). The carboxyl extensions of two rat ubiquitin fusion proteins are ribosomal proteins S27a and L40. Biochemical and Biophysical Research Communications 215, 682–690. 3 Weissman, A. M. (1997). Regulating protein degradation by ubiquitination. Immunology Today 18, 189–198. 4 Ciechanover, A. (1998). The ubiquitin-proteasome pathway: on protein death and cell life. EMBO Journal 17, 7151–7160. 5 Goldknopf, I. L. & Busch, H. (1977). Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24. Proceedings of the National Academy of Sciences of the United States of America 74, 864–868. 6 Levinger, L. & Varshavsky, A. (1982). Selective arrangement of ubiquitinated and D1 protein-containing nucleosomes within the Drosophila genome. Cell 28, 375–385. 7 Thomas, D. & Tyers, M. (2000). Transcriptional regulation: Kamikaze activators. Current Biology 10, 341–343. 8 Rotin, D., Staub, O. & Haguenauer-Tsapis, R. (2000). Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. Journal of Membrane Biology 176, 1–17. 9 Hasselgren, P. O. & Fischer, J. E. (1997). The ubiquitin-proteasome pathway: review of a novel intracellular mechanism of muscle protein breakdown during sepsis and other catabolic conditions. Annals of Surgery 225, 307–316. 10 Yamao, F. (1999). Ubiquitin system: selectivity and timing of protein destruction. Journal of Biochemistry 125, 223–229. 11 Schubert, U., Anton, L. C., Gibbs, J., Norbury, C. C., Yewdell, J. W. & Bennink, J. R. (2000). Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404, 770–774. 12 Howard, J. C. (1995). Supply and transport of peptides presented by class I MHC molecules. Current Opinion in Immunology 7, 69–76. 13 Pamer, E. & Cresswell, P. (1998). Mechanisms of MHC class I—restricted antigen processing. Annual Review of Immunology 16, 323–358. 14 Oldstone, M. B. (1997). How viruses escape from cytotoxic T lymphocytes: molecular parameters and players. Virology 234, 179–185. 15 Rodriguez, F., Zhang, J. & Whitton, J. L. (1997). DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. Journal of Virology 71, 8497–8503.

350

K. OKUBO ET AL.

16 Germain, R. N. (1998). Antigen processing and presentation. In Fundamental Immunology (W. E. Paul, eds) pp. 287–340. Phladelphia: Lippincott-Raven Publishers. 17 Aviel, S., Winberg, G., Massucci, M. & Ciechanover, A. (2000). Degradation of the epstein-barr virus latent membrane protein 1 (LMP1) by the ubiquitin-proteasome pathway. Targeting via ubiquitination of the N-terminal residue. Journal of Biological Chemistry 275, 23491–23499. 18 Bültmann, A., Eberle, J. & Haas, J. (2000). Ubiquitination of the human immunodeficiency virus type 1 env glycoprotein. Journal of Virology 74, 5373–5376. 19 Suzuki, R., Tamura, K., Li, J., Ishii, K., Matsuura, Y., Miyamura, T. & Suzuki, T. (2001). Ubiquitin-mediated degradation of hepatitis C virus core protein is regulated by processing at its carboxyl terminus. Virology 280, 301–309. 20 Chwetzo#, S. & d’Andrea, S. (1997). Ubiquitin is physiologicall induced by interferons in luminal epithelium of porcine uterine endometrium in early pregnancy: global RT-PCR cDNA in place of RNA for di#erential display screening. FEBS Letters 405, 148–152. 21 Latchman, D. S., Estridge, J. K. & Kemp, L. M. (1987). Transcriptional induction of the ubiquitin gene during herpes simplex virus infection is dependent upon the viral immediate-early protein ICP4. Nucleic Acids Research 15, 7283–7293. 22 Kemp, L. M. & Latchman, D. S. (1988). The herpes simplex virus type 1 immediateearly protein ICP4 specifically induces increased transcription of the human ubiquitin B gene without a#ecting the ubiquitin A and C genes. Virology 166, 258–261. 23 Schwartz, O., Marechal, V., Friguet, B., Arenzana-Seisdedos, F. & Heard, J. M. (1998). Antiviral activity of the proteasome on incoming human immunodeficiency virus type 1. Journal of Virology 72, 3845–3850. 24 Watson, D. C., Levy, W. B. & Dixon, G. H. (1978). Free ubiquitin is a non-histone protein of trout testis chromatin. Nature 276, 196–198. 25 Nickel, B. E. & Davie, J. R. (1989). Structure of polyubiquitinated histone H2A. Biochemistry 28, 964–968. 26 Wolf, K. & Quimby, M. C. (1962). Established eurythermic line of fish cells in vitro. Science 135, 1065–1066. 27 Dworkin-Rastl, E., Shrutkowski, A. & Dworkin, M. B. (1984). Multiple ubiquitin mRNAs during Xenopus laevis development contain tandem repeats of the 76 amino acid coding sequence. Cell 39, 321–325. 28 Bence, N. F., Sampat, R. M. & Kopito, R. R. (2001). Impairment of the ubiquitinproteasome system by protein aggregation. Science 292, 1552–1555. 29 Özkaynak, E., Finley, D. & Varshavsky, A. (1984). The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein. Nature 312, 663–666. 30 Lund, P. K., Moats-Staats, B. M., Simmons, J. G., Hoyt, E., D’Ercole, A. J., Martin, F. & Van Wyk, J. J. (1985). Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor. Journal of Biological Chemistry 260, 7609–7613. 31 Haas, A. L. & Bright, P. M. (1985). The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. Journal of Biological Chemistry 260, 12464–12473. 32 Fornace, A. J. Jr, Alamo, I. Jr, Hollander, M. C. & Lamoreaux, E. (1989). Ubiquitin mRNA is a major stress-induced transcript in mammalian cells. Nucleic Acids Research 17, 1215–1230. 33 Bond, U. & Schlesinger, M. J. (1985). Ubiquitin is a heat shock protein in chicken embryo fibroblasts. Molecular and Cellular Biology 5, 949–956. 34 Cheng, L., Watt, R. & Piper, P. W. (1994). Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast (Saccharomyces cerevisiae). Molecular and General Genetics 243, 358–362. 35 Treger, J. M., Heichman, K. A. & McEntee, K. (1988). Expression of the yeast UB14 gene increases in response to DNA-damaging agents and in meiosis. Molecular and Cellular Biology 8, 1132–1136.

UBIQUITIN GENES IN RAINBOW TROUT

351

36 Finch, J. S., St John, T., Krieg, P., Bonham, K., Smith, H. T., Fried, V. A. & Bowden, G. T. (1992). Overexpression of three ubiquitin genes in mouse epidermal tumors is associated with enhanced cellular proliferation and stress. Cell Growth & Differentiation 3, 269–278. 37 von Kampen, J. & Wettern, M. (1991). Ubiquitin-encoding mRNA and mRNA recognized by genes encoding ubiquitin-conjugating enzymes are di#erentially expressed in division-synchronized cultures of Chlamydomonas reinhardtii. European Journal of Cell Biology 55, 312–317. 38 Özkaynak, E., Finley, D., Solomon, M. J. & Varshavsky, A. (1987). The yeast ubiquitin genes: a family of natural gene fusions. EMBO Journal 6, 1429–1439. 39 Baker, R. T. & Board, P. G. (1989). Unequal crossover generates variation in ubiquitin coding unit number at the human Ubc polyubiquitin locus. American Journal of Human Genetics 44, 534–542. 40 Wiborg, O., Pedersen, M. S., Wind, A., Berglund, L. E., Marcker, K. A. & Vuust, J. (1985). The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences. EMBO Journal 4, 755–759. 41 Baker, R. T. & Board, P. G. (1987). The human ubiquitin gene family: structure of a gene and pseudogenes from the Ub B subfamily. Nucleic Acids Research 15, 443–463. 42 Finley, D., Bartel, B. & Varshavsky, A. (1989). The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338, 394–401. 43 Baker, R. T. & Board, P. G. (1991). The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes. Nucleic Acids Research 19, 1035–1040. 44 Ahn, K. S. & Henney, H. R. Jr (1994). An Acanthamoeba ubiquitin-fusion protein; cDNA and deduced protein sequence. Biochimica et Biophysica Acta 1218, 109–111. 45 Hansen, J. D., Strassburger, P., Thorgaard, G. H., Young, W. P. & Du Pasquier, L. (1999). Expression, linkage, and polymorphism of MHC-related genes in rainbow trout, Oncorhynchus mykiss. Journal of Immunology 163, 774–786. 46 Hashimoto, K., Okamura, K., Yamaguchi, H., Ototake, M., Nakanishi, T. & Kurosawa, Y. (1999). Conservation and diversification of MHC class I and its related molecules in vertebrates. Immunological Reviews 167, 81–100. 47 Fu, T. M., Guan, L., Friedman, A., Ulmer, J. B., Liu, M. A. & Donnelly, J. J. (1998). Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens. Vaccine 16, 1711–1717.