©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Warm Temperature Acclimation-related 65-kDa Protein, Wap65, in Goldfish and Its Gene Expression (*)

(Received for publication, April 3, 1995)

Kiyoshi Kikuchi (1)(§), Michiaki Yamashita (3), Shugo Watabe (2)(¶), Katsumi Aida (1)

From the  (1)Laboratory of Fish Physiology and (2)Laboratory of Marine Biochemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo, Tokyo 113, Japan and (3)National Research Institute of Fisheries Science, Kanazawa, Yokohama, Kanagawa 236, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

cDNAs encoding a warm temperature acclimation-related protein (Wap65) were cloned from the muscle and hepatopancreas cDNA libraries of the warm temperature-acclimated goldfish Carassius auratus, and their nucleotide sequences containing 5`- and 3`-noncoding regions together with their polyadenylation signal were determined. The deduced amino acid sequence of Wap65 was 31% homologous to rat hemopexin. However, goldfish Wap65 lacked a few possible glycosylation sites and presumed functional histidine residues, implying that it may have different functions from hemopexin. Wap65 contained a leader peptide of 30 amino acids and a mature protein region of 415 amino acids. Southern blot analysis demonstrated that the protein is expressed by a single copy gene in the goldfish haploid genome. In RNA blot analysis using isolated cDNA clones, a single transcript of about 2.0 kilobases was detected in the hepatopancreas but not in brain, muscle, or hemocytes. The abundancy of this transcript markedly increased in the hepatopancreas as a result of warm temperature acclimation. Electrophoretic analysis of plasma proteins revealed a good correlation of plasma Wap65 levels to those of the corresponding transcript in the hepatopancreas, suggesting that serum Wap65 concentrations are regulated mainly by transcript levels in the hepatopancreas via the secretion process.


INTRODUCTION

Water temperature is one of the most notable factors that bears a spatial and temporal influence on poikilotherms such as fish and aquatic invertebrates. While seasonal temperature changes take place over weeks or months, physiological reorganization compensating for such changes is often referred to as an acclimatory response (Hazel and Prosser, 1974). This temperature acclimation is distinguished from short-term adjustment such as heat shock responses in terms of time scale and the range of temperature fluctuation. Exposure to an acute increase reaching extremely high temperatures triggers heat shock responses involving the transient enhancement of the protein synthesis of heat shock proteins (HSPs)()in cells, regardless of whether the organism is a poikilotherm or homoiotherm. The mechanisms underlying such responses including expressions of these HSPs have been extensively studied (Morimoto et al., 1990).

In contrast to heat shock responses, the acclimatory process has been much less understood at the cellular and molecular levels despite its marked significance for eurythermal fish such as goldfish and carp, which inhabit environments in which temperature varies widely and fluctuates seasonally from near zero to over 30 °C. However, some information on acclimatory processes at the level of biochemistry has been obtained; for example, goldfish and carp are known to increase their myofibrillar ATPase activity after cold acclimation within 4 weeks (Johnston et al., 1975; Heap et al., 1985). In accordance with these changes, myosin isoforms having different primary structures are expressed (Hwang et al., 1990; Watabe et al., 1992; Guo et al., 1994); this is possibly regulated by different genes (Gerlach et al., 1990; Watabe et al., 1995).

On two-dimensional electrophoretic analysis, we have recently found a 65-kDa protein that shows an increased abundance in various tissues in association with warm temperature acclimation of goldfish and carp (Watabe et al., 1993; Kikuchi et al., 1993). No such protein was detected in muscle tissues from 10 and 20 °C-acclimated goldfish on immunoblots using specific antibody raised against the 65-kDa protein (Kikuchi et al., 1993). When water temperature was increased from 20 to 30 °C over a 20-h period, this protein appeared in muscle tissues within 5 days and maintained high concentrations for at least 9 days of additional rearing. In addition, the 10-N-terminal amino acid of the protein was clearly different from the sequences of HSP70 so far reported for other vertebrates and cultured cells (Kikuchi et al., 1993). These results suggest that the 65-kDa protein is expressed in response to raising water temperature in a different way from HSP.

In the present study, we isolated cDNA clones encoding the 65-kDa protein from warm temperature-acclimated goldfish and named it Wap65. Transcription levels of Wap65 were clearly regulated by acclimation to warm water temperature. It showed about 30% homology in the primary structure to mammalian hemopexins, but our protein was significantly different from them in some presumed functional regions.


MATERIALS AND METHODS

Fish

Goldfish Carassius auratus (15-24 g) were acclimated in laboratory aquariums to either 10 or 30 °C for a minimum of 5 weeks. All fish were fed commercial pellets daily ad libitum. The acclimation period was determined in reference to the data of Watabe et al.(1993) and Kikuchi et al.(1993).

RNA Preparation and cDNA Synthesis

Total RNA was extracted from various tissues of temperature-acclimated goldfish according to the guanidium isothiocyanate procedure (Sambrook et al., 1989) or the manufacturer's protocol with RNA extraction solution (Isogen, Nippon Gene). Poly(A) RNAs were isolated with oligo(dT)-cellulose spin columns (Takara), and their corresponding cDNAs were synthesized using Amersham cDNA synthesis kits.

Polymerase Chain Reaction Conditions

The conditions for polymerase chain reaction (PCR) were as follows. 10 µg each of 5`- and 3`-primers and 100 ng of a given template DNA were combined with 10 µl of 10 Tth DNA polymerase buffer (67 mM Tris-HCl, pH 8.8, 16.6 mM (NH)SO, 6.7 mM MgCl, 10 mM 2-mercaptoethanol) and 2 µl of 20 mM dNTP solution. The volume was brought to 100 µl with HO, and the mixture was overlaid with 50 µl of mineral oil to prevent evaporation. 2 units of Tth DNA polymerase was added to the reaction mixture, and the cycle reaction was initiated. Denaturation was at 94 °C for 1 min, annealing at 5565 °C for 2 min, and polymerization at 72 °C for 1 min. The cycle was repeated 30 times.

Construction and Screening of a cDNA Library

A hepatopancreas cDNA library was constructed in ZAP II vectors (Stratagene) using cDNAs prepared from the hepatopancreas of the warm temperature-acclimated goldfish. cDNAs synthesized were blunt-ended with T4 DNA polymerase, tailed with EcoRI (NotI) adapters (Life Technologies, Inc.), size-fractionated in agarose gel, and ligated into the EcoRI sites of the ZAP II vector. Following packaging and amplification, the resultant library was screened, employing the plaque hybridization method with randomly labeled [P]DNA probes. Positives were plaque purified, and the inserts were excised in the form of pBluescript SK plasmid vectors according to the manufacturer's protocol. The plasmid DNAs were purified utilizing an alkaline lysis method (Sambrook et al., 1989) and used for further analysis.

Sequencing Analysis

Sequencing was performed for both strands on subclones deleted by exonuclease III and mung bean nuclease (Barnes et al., 1983), with a DNA sequencer model 373A using dye deoxy terminator cycle sequencing kits (Applied Biosystems). The protein homology search was performed by using the SWISS-PROT data base coordinated with the Inherit program (Applied Biosystems).

Northern Blot Analysis

10 µg of total RNA isolated from the goldfish tissues were denatured at 65 °C for 15 min in 50% formamide and subjected to electrophoresis on a 0.9% agarose gel in 0.2 M MOPS, pH 7.0, containing 2.2 M formamide, 0.05 M sodium acetate, and 5 mM EDTA, then transferred to Hybond N nylon membranes (Amersham Corp.). Total hepatopancreatic RNA, primarily composed of 18 and 28 S rRNAs, was treated in the same manner as above and used as size markers. The membranes were air-dried and baked at 80 °C for 15 min prior to hybridization with randomly labeled [P]DNA probes. Membrane filters were washed at 65 °C with several buffer changes of decreasing SSC concentrations from 5 to 0.1 and autoradiographed on x-ray films with intensifying screens at -80 °C. The hybridized membranes were scanned by a Fujix BAS 1000 computerized densitometer scanner and quantified using a recommended scanning program. The quantified mRNA levels of Wap65 were statistically analyzed using the Student's t test.

Isolation of Genomic DNA and Southern Blot Analysis

Genomic DNAs were isolated by homogenizing the male goldfish hepatopancreas and subsequently treating with proteinase K (Gross-Bellard et al., 1972). For Southern blot analysis, 20 µg of genomic DNAs were digested with a series of restriction endonucleases and electrophoresed in 0.7% agarose gels. The gels were processed with slight modifications after Sambrook et al. (1989), denatured with 0.5 M NaOH containing 1.5 M NaCl, transferred to nylon membranes omitting renaturing steps, and baked at 80 °C for 15 min. Membranes were hybridized with randomly labeled [P]DNA probes and washed under stringent conditions in the Northern blots.

Blood Sampling

Approximately 0.05 ml of blood was drawn from the caudal vasculature with a heparinized syringe fitted with a 23-gauge needle after anesthetizing fish with 600 ppm of 2-phenoxyethanol (Wako). Blood samples were centrifuged at 3000 rpm, and plasma was stored at -20 °C until electrophoretic analysis. Precipitated hemocytes were subjected to RNA isolation.

Electrophoretic Analysis

Two-dimensional electrophoresis was performed by the method of O'Farrell(1975), using 4% polyacrylamide gels in the presence of 8 M urea and 1% Ampholine (composed of 0.8% pH range 5-8 and 0.2% pH range 3.5-10) for isoelectric focusing and 12.5% slab gels for SDS-polyacrylamide gel electrophoresis. Gels were stained with 0.1% Coomassie Brilliant Blue R250 after electrophoresis. Sample volumes used were 3 µl of plasma for analysis.

N-terminal Amino Acid Sequencing

The N-terminal amino acid sequence was determined by the method of Matsudaira(1987) as follows. Plasma proteins separated on SDS-polyacrylamide gel electrophoresis were electrically transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore) and stained with Coomassie Brilliant Blue R250. Parts of the membrane carrying the blotted Wap65 were cut out with a clean razor and subjected to an Applied Biosystems model 477A protein sequencer with an on-line system model 120A.

cDNA Cloning of Goldfish -Actin

cDNAs coding for -actin were isolated from a goldfish hepatopancreas cDNA library with a DNA probe coding for -actin of medaka, Oryzias latipes, provided by Dr. Takashi Aoki (Tokyo University of Fisheries) (details will be described elsewhere).


RESULTS

PCR Amplification of a DNA Fragment Encoding an N-terminal Region of Wap65

We isolated Wap65 from muscle tissues of warm temperature-acclimated goldfish using a sequential series of column chromatography, TSKgel DEAE 5PW, TSKgel G3000 SWG, and TSKgel phenyl 5PW columns (details will be described elsewhere). In total, 44 amino acids were determined from the N terminus, which was sufficient for developing PCR primers (Fig. 1). We synthesized three sets of oligonucleotides encoding three peptides of Wap65 (Fig. 1). Either a BamHI or EcoRI linker was added to each primer to facilitate subsequent analysis.


Figure 1: N-terminal amino acid sequence of Wap65 and nucleotide sequences of synthesized DNA primers for PCR. The N-terminal amino acid sequence was determined for Wap65 purified from warm temperature-acclimated goldfish muscle (details will be reported elsewhere). An X at position 41 from the N terminus indicates an unidentified amino acid. The primers contain all possible combinations of nucleotides that encode respective amino acids except for primer 1, where one of the triplets for leucine, TT(AG), was omitted.



We used cDNAs synthesized from poly(A) RNA preparations of the warm temperature-acclimated goldfish muscle as templates. The first PCR was carried out at an annealing temperature of 55 °C with primers 1 and 2, yielding products of 50300 bp with contamination due to nonspecific amplification. PCR products with sizes from 80 to 150 bp were eluted from agarose gels. The second PCR was performed with primers 1 and 3 using the eluted PCR products as templates, yielding a DNA fragment of 102 bp, which was consistent with the size expected from the amino acid sequence between the two primers. Subsequently, the 102-bp product was blunted and digested with BamHI and EcoRI and then subcloned into pUC118 vectors. The amino acid sequence deduced from a clone, pw65-N, concurred with that directly determined for the isolated protein, GANLDRCGGMEFDAIAV.

Northern blots with the pw65-N clone were performed to examine expression levels of Wap65 mRNA in several tissues including hemocytes, hepatopancreas, muscle, and brain. The highest amount of Wap mRNA was observed in the warm temperature-acclimated hepatopancreas among tissues examined. Unexpectedly, mRNA levels were markedly low in the muscle, which was used for isolation of Wap65 and subsequent analysis of the N-terminal amino acid sequence (Fig. 2).


Figure 2: Northern blot analysis of Wap65 mRNA in hemocytes, hepatopancreas, muscle, and brain from warm temperature-acclimated goldfish. Lanes1-4 contain 10 µg of total RNAs from every tissue. The blots were probed with pw65-N DNA.



cDNA Cloning of Wap65

To isolate the clones that cover the entire coding region of Wap65, we constructed a new cDNA library from the warm temperature-acclimated goldfish hepatopancreas. Screening of 1.0 10 plaques probed with the aforementioned pw65-N DNA of 102 kbp yielded four clones, pw65-14. The longest clone, pw65-1, had 1749 bp with initiator and terminator codons. It contained two EcoRI sites and one each of the PstI and BamHI sites. These restriction endonuclease sites, together with a series of ad hoc deletion mutants, facilitated determination of the DNA nucleotide sequence. However, no clones containing a putative polyadenylation site were found (Fig. 3). To obtain such sites, the library was rescreened with pw65-1, yielding an additional five positive clones. The longest clone, pw65-5, from the second screening contained one polyadenylation signal together with a poly(A) tail but lacked a part of 5`-coding region (Fig. 3).


Figure 3: A partial restriction endonuclease map and two cDNA clones coding for Wap65. The arrow indicates a site of the codon coding for aspartic acid, which occurs at the N terminus of Wap65 isolated from the warm temperature-acclimated goldfish (Kikuchi et al., 1993).



DNA nucleotide and deduced amino acid sequences from the two clones are shown in Fig. 4. In total, 1761 bases were determined where 1335 bases encoded 445 amino acid residues. The coding region was followed by a 3`-noncoding region of 341 bp that contained a polyadenylation signal, AATAAA, in 17 bp upstream from the poly(A) tail. The first methionine was followed by a short polypeptide rich in hydrophobic amino acids that may serve as a signal peptide for secreting Wap65 across cell membranes. This peptide, consisting of 30 amino acids, was followed by aspartic acid, which was identified as the N-terminal amino acid of Wap65 isolated from the warm-acclimated goldfish muscle (Kikuchi et al., 1993).


Figure 4: DNA nucleotide and deduced amino acid sequences of Wap65. The partial amino acid sequence directly determined for Wap65 purified from the muscle is underlined. Potential N-linked glycosylation sites are double-underlined. A putative polyadenylation signal is boxed.



The molecular mass from deduced amino acids was 47.5 kDa, which is smaller than that determined by SDS-polyacrylamide gel electrophoresis (65 kDa). Three possible glycosylation sites of Wap65 may explain these differences (Fig. 4).

Homology Search for Protein Sequence

A homology search for the Wap65-deduced amino acid sequence was conducted using the SWISS-PROT data base, revealing that some parts of Wap65 contained the structures similar to those of hemopexin from rat and human (Takahashi et al., 1985; Nikkila et al., 1991). Hemopexin is a serum glycoprotein that is mainly synthesized in the liver and plays an important role in scavenging hemes from the blood (Muller-Eberhard, 1983). It is highly conserved in its primary structure and shows 78% homology between human and rat (Nikkila et al., 1991). 10 of 12 cysteine residues were conserved between Wap65 and hemopexin, indicating that the disulfide bridges may be similarly arranged in the two proteins (Fig. 5) (Takahashi et al., 1985). A comparison of the N- and C-terminal halves of goldfish Wap65 resulted in 22% homology between these two, suggesting that an internal duplication event had occurred in goldfish as has been described for human and rat hemopexins (Altruda et al., 1985; Nikkila et al., 1991). Seven of eight internal repeats characteristic of the pexin gene family reported by Jenne and Stanley(1987) were also observed in Wap65 (Fig. 5).


Figure 5: Comparison of the amino acid sequence of Wap65 with those of rat and human hemopexins. The number starts from the N-terminal amino acid of matured proteins. Identical amino acids between Wap65 and rat hemopexin (Hx) or between rat and human hemopexin are indicated by asterisks. Dashes denote gaps introduced to maximize homology. The conserved cysteine residues are meshed, whereas the conserved histidine residues, which are assumed to serve as heme axial ligands in human hemopexin, are boxed. Potential N-linked glycosylation sites are underlined. Internal repeats characteristic of pexin gene family (Jenne and Stanley, 1987) are boxed. Amino acid sequences of human and rat hemopexin are cited from Takahashi et al.(1985) and Nikkila et al.(1991), respectively.



Despite such similarity, there were significant differences between Wap65 and mammalian hemopexins. Wap65 contained no tryptophan, which is unusually abundant in mammalian hemopexins (Fig. 5) (Muller-Eberhard and Liem, 1974). Furthermore, Wap65 had few of the possible glycosylation sites, which seem to involve heme binding in human hemopexin (Satoh et al., 1994) (Fig. 5).

Genomic Organization

To analyze the genomic organization of Wap65, we carried out Southern blot experiments probing with an EcoRI restriction fragment of the pw65-1 clone harboring 217 bases from the 5` terminus of the DNA (Fig. 3). Probing hepatopancreas genomic DNA with the above DNA fragment showed one band each of 2.1, 5.4, and 2.0 kbp after digestion with EcoRI, BamHI, and PstI, respectively (Fig. 6). These results suggest that Wap65 is encoded by a single copy gene. The occurrence of two bands observed after HindIII digestion suggests a possible presence of an intron with a HindIII site inserted into the pw65-N DNA region of the Wap65 gene, since no corresponding site was found in the Wap65 cDNA.


Figure 6: Southern blot analysis of the Wap65 gene. Goldfish hepatopancreas genomic DNAs (10 µg/lane) were digested with a series of restriction endonucleases, electrophoresed in a 0.7% agarose gel, and transferred to a nylon membrane. A probe consisting of 217 bp was obtained after digestion of the Wap65 cDNA clone, pw65-1, with EcoRI. Molecular weight markers are EcoT14I digests of phage DNA.



Effects of Acclimation Temperature on Wap65 Transcription Levels

The pw65-1 DNA clone was used as a probe for investigating changes in mRNA levels of Wap65 in the hepatopancreas after warm and cold acclimation. As seen in Fig. 7A, a single transcript was observed by RNA blot analysis. Levels of these hybridized transcripts were determined with a BAS 1000 densitometer, revealing that temperature acclimation of goldfish from 10 to 30 °C resulted in a 10-fold increase at the mRNA level of Wap65 without appreciable changes in molecular weight. These changes were significant at p < 0.01, even after transcriptional levels of Wap65 were standardized with those of -actin (Fig. 7B).


Figure 7: Wap65 mRNA levels in hepatopancreas from goldfish acclimated to warm and cold water temperature. A, Northern blot analysis for three individuals each from goldfish acclimated to 30 and 10 °C. Lanes1-3 contain 10 µg of total RNAs from fish acclimated to 30 °C, whereas lanes4-6 contain those from fish acclimated to 10 °C. RNAs were electrophoresed in 0.9% agarose gels, transferred to nylon membranes, and hybridized with randomly labeled pw65-1 [P]DNA or with that coding for goldfish -actin. B, relative transcriptional levels of Wap65 and -actin in hepatopancreas. RNA blots were quantified using a computerized densitometer.



The Occurrence of Wap65 in the Serum

The tissue-specific transcription of Wap65 revealed by the Northern analysis in this study, together with a nonspecific distribution of the translated product (Watabe et al., 1993; Kikuchi et al., 1993), led us to postulate that Wap65 may be circulated as a plasma protein after being synthesized in the hepatocytes. Two-dimensional electrophoretic analysis demonstrated that the 30 °C acclimated goldfish showed an increased abundance of a 65-kDa protein, possibly corresponding to Wap65 (Fig. 8). N-terminal amino acid sequencing confirmed that this plasma protein was identical to Wap65 from muscle extracts (data not shown). A diffused band observed on the acrylamide gel may be due to multiple glycosylation levels of this protein as expected from its amino acid sequence.


Figure 8: Two-dimensional electrophoretic patterns of plasma proteins from goldfish acclimated to either 30 or 10 °C. The arrow indicates Wap65 dominating in the 30 °C acclimated fish.




DISCUSSION

The purpose of this study was to clone cDNAs encoding the warm temperature acclimation-related protein Wap65 to reveal its possible functions, which may be defined by its DNA nucleotide and deduced amino acid sequences.

A homology search of protein data bases revealed that Wap65 had overall 31% homology to and shared several homologous regions with rat hemopexin. Hemopexin is a mammalian serum glycoprotein that transports heme to liver parenchymal cells via a receptor-mediated process (Muller-Eberhard and Liem, 1974). A high conservation of cysteine residues was seen between Wap65 and mammalian hemopexin, suggesting similar disulfide bridges in the two proteins (see Fig. 5) (Nikkila et al., 1991). Several other regions showed very high homology between Wap65 and mammalian hemopexin, implying that Wap65 may have physiological functions similar to those of hemopexin. It seems that mammalian hemopexin and Wap65 belong to the same gene family. Despite the above similarities, distinct structural differences between Wap65 and hemopexin aroused the question whether they can share the same function. The 31% homology at the amino acid level between the two proteins was not as high as the 78% homology between rat and human hemopexins. Furthermore, no tryptophan was found in Wap65. It has been reported that the content of tryptophan is unusually high in the hemopexins and several heme binding proteins, suggesting that certain tryptophan residues are essential for their interaction with heme (Muller-Eberhard and Liem, 1974; Takahashi et al., 1984).

Satoh et al.(1994) expressed a recombinant human hemopexin in baculovirus-infected insect cells with site-directed mutagenesis and demonstrated that N-linked glycosylation and His-127 are essential to bestow a high affinity of hemopexin for blood-circulating hemes. Wap65 contained no histidine residue in the region of His-126 in rat or His-127 in human hemopexin and was poor in possible glycosylation sites as compared with rat and human hemopexins (see Fig. 5). Therefore, Wap65 is not expected to have the same heme binding properties as conventional hemopexins. Alternatively, histidine residues in Wap65 at the sites different from those of mammalian hemopexins may serve as heme axial ligands.

Northern blot analysis suggested that the increased abundance of Wap65 translation levels in the hepatopancreas in response to warm environmental temperature (Watabe et al., 1993) appears to be regulated for the most part by its increased mRNA levels. Wap65 in plasma as well as in muscle and brain (Watabe et al., 1993) seems to be transported from the hepatopancreas as in the cases of many serum proteins including mammalian hemopexins (Kushner, 1988).

It is well established that the concentration of a subset of acute phase proteins (e.g. -acid glycoprotein, -antitrypsin, ceruloplasmin, C-reactive protein, fibrinogen, and haptoglobin) is increased in a pathological state following infection in mammals (Koj, 1974). These acute proteins include hemopexin, and their levels are considered to be mediated under cytokine regulations during inflammation (Poli and Cortese, 1989). Two-dimensional electrophoretic analysis accounts for our tentative conclusion that no possible goldfish acute phase proteins except for Wap65 increase their quantities in the blood during warm acclimation (see Fig. 8). Thus, the regulation of Wap65 expression under warm temperature acclimation may be different from that of mammalian hemopexins during inflammation. Though an upstream regulation in the gene expression of Wap65 is still ambiguous, such regulatory systems for temperature acclimation may exist widely in poikilotherms, whose body temperatures are closely related to those of environment.


FOOTNOTES

*
This study was funded in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GSDB/DDBJ/EMBL/NCBI Data Bases with accession number(s) D50437.

§
Supported by a fellowship from the Japan Society for the Promotion of Science for Japanese Junior Scientists.

To whom correspondence should be addressed. Tel.: 81-3-3812-2111 (ext. 5297); Fax: 81-3-5684-0622.

The abbreviations used are: HSPs, heat shock proteins; bp, base pairs; kbp, kilobase pairs; PCR, polymerase chain reaction; Wap65, warm acclimation-related 65-kDa protein; MOPS, 3-(N-morpholino)propanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. M. N. Wilder of Japan International Research Center for Agricultural Sciences for reading the manuscript.


REFERENCES
  1. Altruda, F., Poli, V., Restagno, G., Argos, P., Cortese, R., and Silengo, L.(1985) Nucleic Acids Res. 13, 3841-3859 [Abstract]
  2. Barnes, W. M., Bevan, M., and Son, P. H.(1983)Methods Enzymol. 101, 98-122 [Medline] [Order article via Infotrieve]
  3. Gerlach, G., Turay, L., Malik, K. T. A., Lida, J., Scutt, A., and Goldspink, G.(1990) Am. J. Physiol.259,R237-R244
  4. Gross-Bellard, M., Oudet, P., and Chambon, P.(1973)Eur. J. Biochem. 36, 32-38 [Medline] [Order article via Infotrieve]
  5. Guo, X.-F., Nakaya, M., and Watabe, S.(1994)J. Biochem. (Tokyo)116,728-735 [Abstract]
  6. Hazel, J. R., and Prosser, C. L.(1974)Physiol. Rev. 54, 620-677 [Free Full Text]
  7. Heap, S. P., and Goldspink, G.(1985)J. Fish Biol. 26, 733-738
  8. Hwang, G-C., Watabe, S., and Hashimoto, K.(1990)J. Comp. Physiol. B 160, 233-239
  9. Jenne, D., and Stanley, K. K.(1987)Biochemistry 26, 6735-6742 [Medline] [Order article via Infotrieve]
  10. Johnston, I. A., Davison, W., and Goldspink, G.(1975)FEBS Lett. 50, 293-295 [CrossRef][Medline] [Order article via Infotrieve]
  11. Kikuchi, K., Watabe, S., Suzuki, Y., Aida, K., and Nakajima, H.(1993)J. Comp. Physiol. B 163, 349-354
  12. Koj, A. (1974) in Structure and Function of Plasma Protein (Allison, A. C., ed) Vol. 1, pp. 73-121, Plenum, London
  13. Kushner, I.(1988) Methods Enzymol. 163, 373-383 [Medline] [Order article via Infotrieve]
  14. Matsudaira, P. (1987)J. Biol. Chem. 262, 10035-10038 [Abstract/Free Full Text]
  15. Morimoto, R. I., Tissieres, A., and Georgopoulos, C. (1990) in Stress Proteins in Biology and Medicine, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  16. Muller-Eberhard, M. (1983)Methods Enzymol. 101, 536-565
  17. Muller-Eberhard, M., and Liem, H. H. (1974) in Structure and Function of Plasma Protein (Allison, A. C., ed) Vol. 1, pp. 35-53, Plenum, London
  18. Nikkila, H., Gitlin, J. D., and Muller-Eberhard, U.(1991)Biochemistry 30, 823-829 [Medline] [Order article via Infotrieve]
  19. O'Farrell, P. H. (1975)J. Biol. Chem. 250, 4007-4021 [Abstract]
  20. Poli, V., and Cortese, R.(1989)Proc. Natl. Acad. Sci. U. S. A. 86, 8202-8206 [Abstract]
  21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  22. Satoh, I., Satoh, H., Iwahara, S., Harkal, Z., Peyton, D. H., and Muller-Eberhard, U. (1994)Proc. Natl. Acad. Sci. U. S. A. 91, 8423-8427 [Abstract]
  23. Takahashi, N., Takahashi, Y., and Putnam, F. W.(1984)Proc. Natl. Acad. Sci. U. S. A. 81, 2021-2025 [Abstract]
  24. Takahashi, N., Takahashi. Y., and Putnam, F. W.(1985)Proc. Natl. Acad. Sci. U. S. A. 82, 73-77 [Abstract]
  25. Watabe, S, Hwang, G.-C., Nakaya, M., Guo, X.-F., and Okamoto, Y.(1992)J. Biochem. (Tokyo)111,113-122 [Abstract]
  26. Watabe, S., Kikuchi, K., and Aida, K.(1993)Nippon Suisan Gakkaishi 59, 151-156
  27. Watabe, S., Imai, J., Nakaya, M., Hirayama, H., Okamoto, Y., Masaki, H., Uozumi, T., Hirono, I., and Aoki, T.(1995)Biochem. Biophys. Res. Commun.,208,118-125 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.