Dogmas and controversies in the handling of nitrogenous wastes: Expression of arginase Type I and II genes in rainbow trout: influence of fasting on liver enzyme activity and mRNA levels in juveniles
Department of Zoology, University of Guelph, Guelph, ON, Canada N1G 2W1
* Author for correspondence (e-mail: patwrigh{at}uoguelph.ca)
Accepted 23 February 2004
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Summary |
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Key words: ornithineurea cycle, arginine, urea, nitrogen metabolism, mitochondrial targeting, Oncorhynchus mykiss
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Introduction |
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Arginase is ubiquitous in fish tissues, with highest activities found in
liver and kidney tissue (Cvancara,
1969; Portugal and Aksnes,
1983
; Singh and Singh,
1988
; Korte et al.,
1997
; Felskie et al.,
1998
). Despite this, only a handful of adult teleost species is
known to have a functional OUC (Randall et
al., 1989
; Saha and Ratha,
1998
; Walsh, 1997
;
reviewed by Anderson, 2001
),
although the OUC is present in the more ancient fish lineages (i.e.
elasmobranchs and the coelacanth;
Griffith, 1991
;
Anderson, 1991
;
Walsh and Mommsen, 2001
). Urea
synthesis occurs early in teleost development
(Kaushik et al., 1982
;
Wright et al., 1995
;
Pilley and Wright, 2000
), and
radiolabeled HCO3, a substrate for the OUC, is
incorporated into urea in rainbow trout (Oncorhynchus mykiss) embryos
(Dépêche et al.,
1979
). The key piscine OUC enzyme, CPSase III, along with OTCase,
arginase and the accessory enzyme glutamine synthetase, is induced early in
the development of rainbow trout and other teleost species, although CPSase
III is absent in adult liver tissue
(Wright et al., 1995
; Terjesen
et al., 1998
,
2001
;
Chadwick and Wright, 1999
;
Steele et al., 2001
;
Todgham et al., 2001
). Thus,
the OUC appears to be functional early in development but, of the OUC enzymes,
only arginase activities remain high in later life stages.
The developmental pattern of OUC enzyme activity raises the question of
whether one or two arginase genes are expressed in rainbow trout. Adult trout
arginase activity is localized to the mitochondria
(Mommsen and Walsh, 1989;
Korte et al., 1997
), as are
other fish arginases (Casey and Anderson,
1985
; Carvajal et al.,
1987
; Mommsen and Walsh,
1989
; Anderson and Walsh,
1995
; Felskie et al.,
1998
). In the present study, we hypothesize that the arginase gene
or genes in trout share more homology with the mammalian mitochondrial
arginase Type II rather than the cytosolic Type I gene. Very little was known
about fish arginase genes at the onset of this project. The only sequences
available were those from the zebrafish EST library, indicating the presence
of one arginase gene with sequence similarity to the mammalian mitochondrial
Type II arginase. The question is further complicated by the fact that
salmonids are tetraploid, and recent studies of trout glutamine synthetase
genes revealed four distinct genes (Murray
et al., 2003
). In the present study, we used PCR cloning
techniques and database searches to determine the complete coding regions of
one arginase Type I gene (Onmy-ARG01) and one arginase Type II gene
(Onmy-ARG02). In addition, the partial coding regions of two similar
genes were also determined (Onmy-ARG02b and Onmy-ARG01b,
respectively). Using northern blot analysis, we determined the pattern of
expression of Onmy-ARG01 and Onmy-ARG02 in several adult
trout tissues.
Our second aim was to investigate whether the two trout arginase genes were
differentially regulated in response to dietary manipulation. In various
teleost fishes, liver arginase activity is induced following several weeks of
fasting (Chiu et al., 1986;
Jürss et al., 1987
;
Singh and Singh, 1988
;
Wright, 1993
). Increased
arginase activity may be important in amino acid catabolism in fasting fish
(Ballantyne, 2001
;
Wood, 2001
). Alternatively,
arginase may be induced during starvation to enhance ornithine synthesis,
modulate nitric oxide production or modulate related pathways (see above).
Based on the similarity between Onmy-ARG01 and mammalian Type I
arginase (hepatic OUC-related) and between Onmy-ARG02 and mammalian
Type II arginase (nonhepatic), we hypothesized that only Onmy-ARG02
expression would be enhanced in the livers of fasted juvenile trout where the
OUC is non-functional. We reproduced the protocol used by Chiu et al.
(1986
), where juvenile trout
were either fed or fasted for a 6-week period. Livers were collected at 0 and
6 weeks, and arginase activities, as well as Onmy-ARG01 and
Onmy-ARG02 mRNA levels, were compared between fasted and fed
fish.
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Materials and methods |
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Cloning
The polymerase chain reaction (PCR) was used to amplify the arginase gene
from a primary rainbow trout cDNA library constructed from mRNA isolated from
the combined gill and kidney tissues of 12 trout (1x109 PFU
ml1; S. F. Perry, personal communication). The initial
degenerate primer combinations used, based on known vertebrate arginase
sequences, were ARG*e5f1 (5'-TTGGG CTTAG AGAYG TGGAY C) and ARG*e7r1
(5'-GGGTC CAGNC CATCA ATGTC AAA) or ARG*e7r2 (5'-CCTTC TCTGT ARGTN
AGTCC TCC). To obtain further sequence information of the Onmy-ARG02
transcript, we employed anchored PCR using the vector-specific primers
pBKCMV*r1 and pBKCMV*f1 (Murray et al.,
2003) in combination with either Onmy-ARG02 specific
reverse primers, ARG*e7r1 or ARG-Onmy*e6r2 (5'-TGGTC GAAAG AGACT TCCAT
G), or the forward primer ARG-Onmy*e5f2 (5'-TGAAG GACTT GGGTG TCCAG).
PCR reactions were conducted on a PTC-150 MiniCyclerTM thermal cycler (MJ
Research Inc., Incline Village, NV, USA). The thermal profile consisted of an
initial cycle of 95°C for 5 min, 5560°C for 30 s, and 72°C
for 2 min, followed by 35 cycles of 94°C for 30 s, 5560°C for
30 s, 72°C for 1.5 min, and finished with an elongation of the 72°C
step for 7.5 min. Reaction volumes were 25 µl, consisting of 1.5 mmol
l1 MgCl2, 200 µmol l1 dNTPs,
1x reaction buffer, 0.2 µmol l1 of each primer and
1 unit of HotStar Taq polymerase (Qiagen Inc., Mississauga, ON,
Canada). The PCR products were separated by gel electrophoresis, isolated
using a QIAGEN QIAquick gel extraction kit (Qiagen Inc.) and cloned into
pGEM-T Easy Vector system (Promega Corp., Madison, WI, USA). Plasmid inserts
were sequenced using an ABI Prism 377 DNA Sequencer (Perkin-Elmer, Foster
City, CA, USA).
Analysis of EST and pufferfish genomic databases
The GenBank EST database
(http://ncbi.nlm.nih.gov/)
was searched for all rainbow trout, zebrafish and pufferfish entries with
similarity to the deduced amino acid sequences of the identified trout
arginase genes through tBlastn searches. Resulting EST sequences were then
analysed as described below to establish sets of contiguous sequence
information. In a similar manner, the draft sequence of the pufferfish genome
(release #3;
http://fugu.hgmp.mrc.ac.uk/)
was searched for scaffolds containing arginase genes. Putative coding regions
were extracted from the genomic sequence scaffolds.
Sequence analysis
DNA sequence was edited and contiguous sequences were assembled using the
Sequencher 4.1.2 program (Gene Codes Corp., Ann Arbor, MI, USA). Alignments
were constructed using ClustalW (Thompson
et al., 1994) contained within the MacVector 7.0 program (Oxford
Molecular Group). Gene phylogenies were estimated from a DNA alignment of the
coding sequence using the maximum likelihood method of the computer program
Paup 4.0b10 (Sinauer Associates Inc., Sunderland, MA, USA). Three hundred
bootstrap trees were constructed to estimate confidence in the branch
topology.
Experimental protocol
To determine the tissue distribution of Onmy-ARG01 and
Onmy-ARG02, adult rainbow trout were killed by a sharp blow to the
head. Tissues were removed immediately and frozen in liquid nitrogen, followed
by storage at 80°C until isolation of RNA (<1 month).
To investigate liver arginase expression in response to a dietary manipulation, fish were divided into two groups: fed or fasted. The fed group were distributed equally between two 50-litre tanks and, likewise, the fasted group were distributed equally between two 50-litre tanks. Prior to the start of the experiment, five fish were killed from each tank (control, 0 week). Fish were weighed and the liver was removed. Whole livers were frozen immediately in liquid nitrogen and stored at 80°C for later isolation of RNA and analysis of enzyme activity (<1 month). Subsequently, only the fed group were given a ration of 1.8% daily (fed twice a day). The fasted group did not receive food for the remainder of the experiment. At the end of the 6th week, 10 fish from the fed group (fed control, 6 weeks) and 10 fish from the fasted group (fasted, 6 weeks) were killed, and tissues were collected as described above.
Northern analysis
To determine the tissue distribution of arginase mRNA or whether arginase
mRNA levels changed with fasting, total RNA was isolated from trout liver by
the guanidine isothiocyanate method using Trizol (Gibco-BRL, Burlington, ON,
Canada). RNA (10 µg lane1) was separated on a 1.5%
agarose gel in the presence of 1.0 mol l1 formaldehyde and
transferred to a nylon membrane. Filters were hybridized with 32P-labelled
probes. Probes were generated by PCR using a cloned fragment of cDNA as a
template and primers specific for either Onmy-ARG01 (240 bp),
Onmy-ARG02 (655 bp) or ß-actin (286 bp; control gene).
[-32P]-dCTP (3000 Ci mmol l1) was used to
label probes using a random priming kit (Invitrogen/Gibco-BRL). Following
hybridization with Onmy-ARG01 probe (65°C, overnight),
high-stringency washes were conducted (65°C). Membranes were placed in a
PhosphorImager cassette and relative RNA concentrations were estimated from
the density of bands using the ImageQuant software program (Molecular
Dynamics, Sunnyvale, CA, USA). Membranes were stripped, re-hydridized with the
Onmy-ARG02 probe and analysed as described above. Finally, membranes
were stripped, re-hybridized with the ß-actin probe and analyzed as
before. The ratio of the density of the arginase:ß-actin band was
calculated. Separate membranes containing RNA isolated from different
individuals were analyzed and presented as means ±
S.E.M. (N=34).
Enzyme activity
Liver tissue was homogenized in buffer as described by Felskie et al.
(1998), except tissue was
diluted 200-fold in buffer. Arginase activities were measured after the tissue
homogenate was passed through a Sephadex G25 column, as described previously
(Felskie et al., 1998
). Total
protein measurements were taken before and after the column chromatography
step, and final enzyme activities corrected accordingly. The assay was linear
with time and tissue amount. The assay was performed with 50 µl of
homogenized tissue extract and the reaction was stopped after 10 min.
Activities are presented as µmol min1
g1 wet mass liver tissue or µmol min1
mg1 protein. Protein was measured by the dye-binding method
of Bradford (1976
) using
Bio-Rad Laboratories reagents and bovine serum albumin as a standard.
Statistics
A single factor analysis of variance (ANOVA) was conducted on the arginase
activities between fed control (0 week), fed control (6 week) and fasted (6
week). Where significance was detected, the analysis was followed up with a
Tukey's test. Comparisons between tissues or fed and fasted liver
arginase:ß-actin mRNA levels were assessed with an unpaired Student's
t-test. Significant difference was detected at
P<0.05.
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Results |
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Additional sequence information was obtained from an analysis of the growing rainbow trout EST database (May 2003). From the 15 trout arginase ESTs found in the database, four sets of contiguous sequences were determined, Onmy-EST contig1 (eight clones) and Onmy-EST contig24 (two clones each). One clone, CA358388, presumably an incompletely spliced mRNA, was identical to contig2 but was found to contain additional intron sequence. The EST contigs were combined with the previous sequences, confirming and extending the sequence information and revealing the presence of a second arginase Type I gene (Fig. 1). The four resulting sequence contigs, two complete Onmy-ARG01 and Onmy-ARG02 and two partial Onmy-ARG01b and Onmy-ARG02b, were reported to the Third Party Annotation database (TPA accession nos. BK001403, BK001400, BK001401 and BK001404, respectively).
In a similar fashion, the zebrafish EST database was searched for arginase sequences (May 2003). Twenty-two zebrafish ESTs were found, 20 of which form a single arginase Type II contig (TPA accession nos. BK001402). The two remaining ESTs contain unique arginase-like sequences. One (BG738104) contains stop codons in the putative reading frame, while the other (BI474173) is more similar to the arginase Type I sequences but also appears to contain frameshift mutations. In either case, this sequence information is not supported by additional ESTs, is incomplete and, therefore, was not included in further analyses.
Putative arginase Type I and Type II genes were also identified from pufferfish, Taru-ARG01 and Taru-ARG02, from the genomic scaffolds FM:M000333 and FM:M002889 (release version 3), respectively. Both genes contain all the expected exons as determined through comparison with available Genscans and with the bony fish sequences reported here. Two pufferfish ESTs were identified in the database (May 2003). One is identical to the reported Taru-ARG01 gene (CA330269) while the other is 92% similar to the Taru-ARG02 gene.
Sequence comparison
Previous comparisons of 21 arginase enzymes in both eukaryotes and
prokaryotes (Perozich et al.,
1998) revealed 20 residues that are strictly conserved, six that
display an invariant similarity (i.e. D/E, S/T or V/I/L/M) and 10 that are
highly conserved (at least 80% conserved). An alignment of the putative amino
acid sequences of all known vertebrate arginase genes shows that these
residues are also conserved in the seven bony fish arginases reported (20/20
strictly conserved, 6/6 invariant similarity and 9/10 highly conserved;
Fig. 2).
|
Interestingly, the three bony fish Type I arginases (identified through
phylogenetic analysis below) contain a much longer N-terminal domain than do
the amphibian or mammalian Type I arginases. As a presumptive mitochondrial
protein, Type II arginase requires an N-terminal mitochondrial targeting
peptide (mTP) in order to be transported into the matrix of the mitochondria.
These mTPs can be diverse in both length and sequence but are characteristic
in the over-representation of positively charged residues (i.e. Arg, Ala,
Ser), whereas negatively charged residues (Asp and Glu) are rare
(Grivell, 1988;
Emanuelsson et al., 2000
). Two
computer programs were used to detect the presence of mTPs in the six bony
fish arginase genes for which N-terminal information is available;
TargetP predicts the subcellular location of newly identified
proteins (Emanuelsson et al.,
2000
), and MitoProt predicts the probability that a
nuclear gene is exported to the mitochondria and identifies probable mTP
cleavage sites (Claros and Vincens,
1996
). These computer analyses indicate that it is highly probable
that all six of the bony fish arginases, whether Type I or Type II, are
targeted to the mitochondria. In addition, no physical restraints for the
import of the proteins were found, and putative cleavage sites of the mTP were
identified at position 2325 and/or 43
(Fig. 2). Further analysis of
all other vertebrate arginases (Fig.
2) reveals that only the amphibian and mammalian Type I arginases
are not predicted to be imported into the mitochondria.
Phylogenetic analysis
A maximum likelihood tree (Fig.
3), including all vertebrate and three nonvertebrate eukaryotic
arginase sequences for reference, was constructed from a nucleotide sequence
alignment corresponding to the codons for positions 29335
(Fig. 2). The tree topology is
robust, with bootstrap values over 75% for most branching points. Two lineages
of arginase genes are suggested in vertebrates, labelled Arginase Type I and
Type II; however, bootstrap for the Arginase I lineage is not as well
supported. Interestingly, both lineages contain the predicted branching order
of vertebrate evolution; an initial split of bony fish and tetrapod genes
followed by the divergence of tetrapod genes into amphibian and mammalian
lineages.
|
Expression of arginase genes in various tissues
Onmy-ARG01 and Onmy-ARG02 mRNA were detected in liver,
kidney, gill, intestine, red muscle and heart tissues
(Fig. 4). Onmy-ARG01
was expressed at a significantly higher level relative to Onmy-ARG02
in liver and red muscle tissue (Fig.
4). Although a similar trend was observed in kidney, this
difference was not significant (Fig.
4). When comparing between tissues, expression relative to
ß-actin is not appropriate because ß-actin mRNA levels vary in
different tissues (e.g. Foss et al.,
1998; Murray et al.,
2003
). The amount of total RNA loaded appeared to be consistent
between lanes, as observed from the ethidium bromide-stained gel. For each
transcript, the order of the highest to the lowest level of mRNA was:
Onmy-ARG01: liver > kidney > red muscle
gill > heart
intestine; Onmy-ARG02: liver > kidney
gill >
intestine
red muscle
heart.
|
Expression of arginase genes during fasting
Following the 6-week experiment, there was a significant difference between
the body mass of fasted (23±2 g; N=6) and fed fish
(56±1 g; N=6). Body mass at the start of the experiment (0
week) was 26±2 g (N=6) and was not significantly different
from the fasted fish at 6 weeks.
Hepatic arginase activity and specific activity were significantly higher
in fasted relative to fed control fish, with a 2- to 3-fold higher level
in fasted compared with fed fish at 0 and 6 weeks, respectively
(Table 1).
|
The levels of Onmy-ARG01 mRNA (relative to ß-actin mRNA levels) were not significantly different between fasted and fed fish at 6 weeks (Fig. 5A). By contrast, there was a 2-fold elevation of Onmy-ARG02 mRNA levels in fasted relative to fed fish (Fig. 5B).
|
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Discussion |
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Recent studies of glutamine synthetase genes have revealed four separate
genes in rainbow trout, also found in two evolutionarily distinct lineages
(Murray et al., 2003). It was
speculated that glutamine synthetase gene number rose from two to four
following the tetraploidisation event that proceeded the evolution of
salmonids. Given the current phylogeny of arginase genes
(Fig. 3), it is tempting to
invoke a similar argument here for the evolution of two Type I or Type II
genes; however, preliminary sequencing evidence suggests the existence of
additional Type II sequences in trout. It is interesting to note that three
mRNA species hybridized to the Onmy-ARG02 probe (data not shown),
suggesting the possibility that multiple ARG02 mRNAs arise from differential
processing, the presence of alternative promoters or three distinct Type II
genes. In Xenopus, three closely related nonhepatic arginase genes
(Type II) have been isolated with distinct tissue, hormone-dependent and
ontogeny-related regulation (Patterton and
Shi, 1994
). Interestingly, five separate bands were detected when
Human Type II was used as a probe on a northern blot of human RNA, but only a
single band was observed in the mouse
(Morris et al., 1997
).
Clearly, further studies are necessary to explore the number, role and nature
of multiple Type I or Type II arginase genes in rainbow trout.
Tissue distribution of arginase genes
Our results indicate that Onmy-ARG01 is expressed at a higher
level than Onmy-ARG02 in trout liver tissue. This finding is in
agreement with studies of Type I genes in Xenopus tadpole
(Patterton and Shi, 1994) and
mammalian liver tissue (Morris et al.,
1997
). Our results also show that Onmy-ARG01 is expressed
at a higher level than Onmy-ARG02 in trout red muscle tissue. In
other trout tissues (kidney, gill, intestine, heart), both transcripts were
expressed, but there was no significant difference between Onmy-ARG01
and ARG02 expression (Fig. 4).
This pattern of expression in nonhepatic trout tissues, regardless of the
unknown expression of other possible arginase genes, is clearly different from
that reported in ureotelic vertebrates. Although there are small discrepancies
between the tissue distribution of arginase genes among different mammalian
species, Type I arginase is generally found in liver whereas Type II arginase
has a wide distribution in nonhepatic tissues
(Grody et al., 1987
;
Gotoh et al., 1996
;
Morris et al., 1997
;
Yu et al., 2001
). Thus, the
wide tissue distribution of Onmy-ARG01 and Onmy-ARG02 in
rainbow trout tissues suggests that the prerequisites were in place for the
evolution in terrestrial ureotelic vertebrates of hepatic Type I arginase
linked to the production of urea via the OUC and the more widely
distributed Type II arginase playing other cellular roles. The evolution of a
cytosolic form of arginase in ureotelic vertebrates appears to be a derived
trait that occurred in the common ancestor of amphibians and mammals
(Fig. 3). Indeed, Mommsen and
Walsh (1989
) determined the
subcellular location of arginase activity in a variety of fish species and
proposed that cytosolic arginase first appeared in the lungfish. They
postulate that this shift in the intracellular location of liver arginase,
along with the evolution of a mitochondrial ornithine transporter and
ammonia-dependent CPSase I, were key events in the evolution of the OUC found
in terrestrial ureotelic vertebrates. If a functioning OUC exists in bony
fishes (e.g. early life stages; see Introduction), then it represents the
ancestral state or has undergone a parallel (or separate) evolution.
Upregulation of arginase with fasting
Our results show that food deprivation in rainbow trout results in the
induction of liver arginase activity (23-fold), similar to other
reports (e.g. Chiu et al.,
1986; Jürss et al.,
1987
). We hypothesized that the two trout arginase genes would be
differentially regulated in response to dietary manipulation. Indeed,
Onmy-ARG02 mRNA levels, but not Onmy-ARG01, were modestly
elevated (2-fold) in liver tissue collected from fasted fish. These results
suggest that the increase in hepatic arginase activity is mainly due to the
accumulation of mRNA because the magnitude of the changes in each were very
similar. Gene expression may be turned on early and sustained during the
6-week fasting trial or the response may be delayed. Regardless, the results
suggest that Onmy-ARG01 and Onmy-ARG02 play different
physiological roles in trout liver. Exactly what those roles are will be an
important question for future studies.
Many fish undergo significant episodes of food deprivation in nature
without pronounced changes in their physical capabilities, although there are
numerous metabolic adjustments. In the laboratory, metabolic rate decreased in
rainbow trout fasted for 17 days (Alsop and
Wood, 1997). Protein use during fasting accounts for only
1430% of oxidative metabolic rate (for review, see
Wood, 2001
), although total
body protein content declined 66% after a 15-day fast in juvenile (4 g)
rainbow trout (Lauff and Wood,
1996
). The mismatch between protein use as fuel and the fall in
body protein content may be due to a redistribution of proteins into
carbohydrates (i.e. gluconeogenesis) and lipids (i.e. lipogensis). Not
surprisingly, intracellular amino acid levels have also been reported to
decline considerably (50%) during a long-term fast (140 days;
Timoshina and Shabalina,
1970
). Arginolysis, catalyzed by arginase, results in the
formation of ornithine and urea. Ornithine can be transaminated to proline or
oxidized to glutamate, both of which may enter the citric acid cycle after
conversion to
-ketoglutarate
(Nelson and Cox, 2000
),
providing fuel for the fasted fish. At this point, we can only speculate that
Onmy-ARG02, but not Onmy-ARG01, is regulated with dietary
manipulations in trout to supply ornithine, similar to one of the putative
functional roles of nonhepatic Type II arginase in mammals.
In the present study, rainbow trout lost only 12% of their body mass
after a 6-week fast (not significant), which is within the range reported by
others (e.g. Chiu et al.,
1986
; Jürss et al.,
1987
). Although arginase activity per gram of liver tissue and per
mg of liver protein significantly increased in fasted fish, if one considers
enzyme activity per gram of fish, arginase activities are similar between
fasted and fed fish (6 weeks). This is simply because the fish fed for 6 weeks
had more than doubled their body mass, whereas the mass of the fasted fish did
not change considerably. From another perspective, one can say that the level
of arginase activity was maintained in fasted fish relative to fed fish,
despite a large difference in body mass. From either angle, the arginase
response is not typical of other liver metabolic enzymes after a fast.
Although hepatic glutamate dehydrogenase activity also increased
(Jürss et al., 1987
),
lipogenic, glycolytic and gluconeogenic enzyme activities did not change or
decreased (Lin et al., 1977
;
Moon and Johnston, 1980
;
Jürss et al., 1987
).
In conclusion, our findings provide evidence for two distinct arginase
genes in rainbow trout, Onmy-ARG01 and Onmy-ARG02, both with
mitochondrial targeting sequences. Further, Onmy-ARG01 and
Onmy-ARG02 share sequence similarities to Type I and Type II arginase
genes isolated from amphibians and mammals but differ in their pattern of
tissue expression. This is an important piece of information in the arginase
evolutionary puzzle. Although arginase activities were previously localized to
mitochondria of teleost livers (Mommsen
and Walsh, 1989; Korte et al.,
1997
; Felskie et al.,
1998
), prior to our study there was little sequence information
available in teleosts to determine the number and type of arginase genes
present. With this sequence and expression data in hand, it will now be
possible to separate the functional significance of two distinct arginase
genes in the ammoniotelic rainbow trout.
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Acknowledgments |
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Footnotes |
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