Evolution of glutamine synthetase in vertebrates: multiple glutamine synthetase genes expressed in rainbow trout (Oncorhynchus mykiss)
1 Department of Zoology, University of Guelph, Guelph, Ontario, N1G 2W1,
Canada
2 Biochemistry and Microbiology, University of Victoria, PO Box 3055,
Victoria, BC V8W 3P6, Canada
* Present address: Biology Program, College of Science and Management,
University of Northern British Columbia, Prince George, BC, Canada V2N
4Z9
Author for correspondence (e-mail:
patwrigh{at}uoguelph.ca)
Accepted 10 January 2003
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Summary |
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Key words: salmonid, L-glutamate:ammonia ligase, tetraploidization, zebrafish, Takifugu, brain, intestine, rainbow trout, Oncorhynchus mykiss, ammonia, nitrogen, mRNA
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Introduction |
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The functional enzyme is composed of eight identical subunits, with some
microheterogeneity between the subunits, possibly due to post-translational
modifications (Smirnov et al.,
2000). Mammals appear to possess only a single GSase gene,
although pseudogenes for GSase have been noted
(Kou and Darnell, 1989
;
Wang et al., 1996
).
Elasmobranch fishes and birds also display a single GSase gene, albeit
producing different transcripts for mitochondrial and cytosolic isozymes
(Laud and Campbell, 1994
;
Pu and Young, 1989
;
Campbell and Smith, 1992
).
In fish, GSase is a multifunctional enzyme, just as the product glutamine
has many different metabolic roles. GSase is critical in the detoxification
process of the highly mobile and toxic ammonia (for reviews, see
Korsgaard et al., 1995;
Ip et al., 2001
). Neural
tissues are particularly sensitive to ammonia and, not surprisingly, GSase
activity is typically high in the brain
(Webb and Brown, 1976
;
Peng et al., 1998
;
Wang and Walsh, 2000
),
although liver can also be an important site of ammonia detoxification
(Jow et al., 1999
;
Iwata et al., 2000
).
The enzyme is also key to the `fish type' ornithine urea cycle, with
glutamine as the N-donor substrate for the initial step catalysed by carbamoyl
phosphate synthetase III (CPSase III) (for a review, see
Anderson, 2001). Consequently,
GSase is usually colocalised with CPSase III in the mitochondria of fish that
have a functional urea cycle (Casey and
Anderson, 1982
), but in some cases GSase is present in both
cytosol and mitochondria (Anderson and
Walsh, 1995
; Felskie et al.,
1998
). The regulation of urea synthesis, at least in the
facultatively ureogenic marine toadfish Opsanus beta, is upstream of
the urea cycle, and present attention is focused on GSase. Stimulation of
ureagenesis in O. beta by confinement or crowding is accompanied by a
multifold induction of hepatic (cytosolic) GSase activity
(Walsh et al., 1994
;
Julsrud et al., 1998
), mRNA
levels and protein concentration (Kong et
al., 2000
).
Fish GSase sequences have been reported in the marine toadfish O.
beta (Walsh et al.,
1999), the sleeper Bostrichthys sinensis
(Anderson et al., 2002
), and
the spiny dogfish shark Squalus acanthias
(Laud and Campbell, 1994
).
Additional sequence information on GSase genes from several fish species have
been recorded in GenBank. A follow up study in O. beta has revealed a
second gene, expressed primarily in the gills, which shares relatively low
nucleotide and amino acid sequence similarity (approx. 73%) with the original
toadfish GSase cDNA from liver (Walsh et
al., 2003
). GSase genes have not been isolated in any salmonid
species. The common ancestor of all salmonids is believed to have undergone a
tetraploidization event (duplication of the diploid set of chromosomes)
between 25100 mya (Allendorf and
Thorgaard, 1984
). In salmonids, therefore, one can expect two
genes (i.e. up to four alleles in a single fish at two different chromosome
locations or loci) instead of the single gene (i.e. up to two alleles at one
locus) found in diploid fish. Studies of GSase gene expression in salmonid
species may be complicated if the two genes are differentially expressed.
The objective of this study was to isolate and characterize the GSase
gene(s) in the rainbow trout Oncorhynchus mykiss. Specifically, we
analysed what effects the tetraploidization event in the evolution of salmonid
fishes may have exerted on GSase genes and expression. Also, we wondered
whether the negligible mitochondrial GSase activity in the trout
(Korte et al., 1997) is in any
way reflected in the gene structure of trout GSase.
Surprisingly, we obtained multiple, and not the expected two, GSase gene sequences. A phylogeny of bony fish genes was reconstructed, based on a nucleotide alignment of the coding regions. To study allelic relationships, we compared non-coding sequences, while the total number of GSase genes in trout was estimated through Southern blot analysis. Finally, we showed differential expression of multiple GSase genes in tissues of adult rainbow trout.
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Materials and methods |
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Analysis of expressed sequence tag and pufferfish genomic
databases
The GenBank expressed sequence tag (EST)
(www.ncbi.nlm.nih.gov)
and the DOE Joint Genome Institute Fugu Genome
(www.jgi.doe.gov/fugu)
databases were searched for sequences with similarity to the deduced amino
acid sequence of an identified trout GSase gene (Onmy-GS01, accession
no. AF390021) through tBlastn searches. All EST sequences were downloaded and
analysed as described below to establish sets of contiguous sequence
information.
Sequence analysis
DNA sequence was edited and contiguous sequences were assembled using the
Sequencher program (GeneCodes Corp., Ann Arbor, MI, USA) (contiguous sequences
or `contigs' are overlapping segments of DNA). Alignments were constructed
using the program MacVector 7.0 (Oxford Molecular Group). Gene phylogenies
were estimated from a DNA alignment of the coding sequence using the
maximum-likelihood method of the computer programs Paup 4.0b8a (Sinauer
Associates Inc., Sunderland, MA, USA). 200 bootstrap trees were constructed to
estimate confidence in the branch topology. Intron 4, 3'-UTR and
pairwise dot-plot analyses were conducted on the computer program BioEdit
(Hall, 2001).
Southern blot
10 µg of total genomic DNA was digested to completion with either the
AluI or HinfI restriction enzymes. The resulting fragments
were size separated on a 1% agarose gel and blotted onto Hybond+ membrane
using a Pharmacia vacuum blotter (Amersham-Pharmacia, Baie d'Urfé, PQ,
Canada). Two GSase probes were produced by amplification from cloned fragments
of the Onmy-GS01 (CA0509) and Onmy-GS02 (CA0508) genes with
the primers GLUL-Onmy*e4f1/GLUL-Onmy*3UR1 and GLUL*e6f2/GLUL-Onmy*3UR3,
respectively. These products were isolated from the agarose gel as above and
labelled with 32P-dCTP (Amersham-Pharmacia) using a random priming
kit (Invitrogen/Gibco-BRL). Southern blots were prehybridised and hybridised
separately overnight at 62°C in 40 ml of Westneat solution (7% SDS, 1 mmol
l1 EDTA, 1% BSA, 0.25 mol l1
Na2HPO4). Membranes were washed twice for 10 min at room
temperature and once at 62°C with a 2x SSC, 0.1% SDS solution,
followed by a final wash at 62°C for 1020 min with a 0.5x
SSC, 0.1% SDS solution. Hybridisation signals were detected on a Molecular
Dynamics Phosphor-Imager (Amersham-Pharmacia).
Northern analysis
To study the distribution of glutamine synthetase mRNA in trout tissue,
total RNA was isolated from O. mykiss kidney, heart, liver, gill,
intestine, white muscle, red muscle and brain by the guanidine isothyocyanate
method using Trizol (Invitrogen/Gibco-BRL). Total RNA (10 µg/lane) was
separated in a 1.5% agarose gel in the presence of 1 mol l1
formaldehyde, and transferred to nylon membranes (Hybond-N, Amersham). The
membranes were initially probed using 32P-labeled random-primed cDNA probes
for two glutamine synthetase mRNAs: Onmy-GS01 (198 bp) and
Onmy-GS04 (180 bp), and hybridized at 65°C. Final
washing was in 40 mmol l1 Na2HPO4, pH
7.2, 1% SDS, 1 mmol l1 EDTA at 65°C. The membranes were
then stripped using 0.1x Denhardt's solution, 5 mmol
l1 Tris-HCl, pH 8.0, and 2 mmol l1 EDTA at
65°C for 1 h and reprobed with the other two glutamine synthetase mRNAs:
Onmy-GS02 (208 bp) and Onmy-GS03 (162 bp). The membrane was
once again stripped, and finally probed with a trout ß-actin gene
(283bp).
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Results |
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Analysis of sequences databases
To compare a wider range of GSase cDNAs among fish species, we searched the
DOE Joint Genome Institute Fugu Genome and GenBank EST databases for
entries with significant similarity to the Onmy-GS01 amino acid
sequence. Two putative genes were found in the Fugu Genomic databank, JGI
Genscan Gene Model nos. 15768 and 18692. The predicted amino acid sequences
are shown in Fig. 2. Of these
two genes, JGI15768, lacks exon 2 and possesses its first splice site 3 codons
downstream from the exon 2/3 border, predicted by comparison to the other fugu
GSase-like genes (JGI-18692) and other vertebrate GSase genomic organisations
(van de Zande et al., 1990;
Pu and Young, 1989
;
Kou and Darnell, 1989
).
103 eligible zebrafish (Danio rerio, Dare) entries were identified during a tBlastn search using the NCBI web interface (February, 2002). Two different contiguous sequences, supported by multiple entries (Fig. 2), were compiled from these EST entries. For both sequences, Dare-GS01, the consensus sequence of 22 entries, and Dare-GS02, the consensus sequence of 73 entries, a complete CDS region was determined. These sequences were reported in the Third Party Annotation GenBank database, accession numbers BK000047 and BK000048, respectively. The quality of eight remaining sequences was deemed too low to be used in the third party consensus of the zebrafish genes. No evidence for additional GSase genes was noted in these entries.
A similar analysis was conducted on the portion of the EST database (March, 2001) containing entries for the tetraploid amphibian Xenopus laevis (Xela). The tBlastn search produced 107 EST entries with significant similarity to the Onmy-GS01 peptide sequence. 44 of these entries matched the previously reported Xenopus GSase gene (accession no. d50062). Evidence for at least two additional sequences was found (Fig. 2). A second full-length CDS sequence (Xela-GS03, accession no. BK000049) was identified, based on the consensus of 44 EST entries. A partial sequence based on two entries, Xela-GS02 (accession no. BK000050), was also noted. The relationship of Xela-GS02 among the vertebrate GSase sequences based on this partial CDS (<1/2 of CDS) is not well supported in phylogenetic analyses (not shown) and further sequence information will be required prior to phylogenetic inference.
Sequence comparison
In the four fish cDNAs for which 5' UTR information was determined or
deduced, Omny-GS01, Onmy-GS02, Dare-GS01 and Dare-GS02, a
common initiation codon (Met) is present (position 1,
Fig. 2). Upstream of this
position no alternative Met codon is encountered prior to the first in-frame
stop codon.
Using the Salmonella typhimurium GSase X-ray crystallography
structure (Gill and Eisenberg,
2001) and a pairwise alignment with vertebrate GSases (not shown),
we can tentatively determine the homologous active site residues in trout
GSase. 15 of the 16 residues identified in Salmonella are identified,
bolded and marked in Fig. 2,
and show complete conservation within the bony fishes. Between
Salmonella and trout, only two of 15 residues are substituted
(positions 194 and 246), one of these maintaining the physicochemical nature
of the residue (246: fish, N; Salmonella, H). An examination of
vertebrate sequences at the active site positions
(Fig. 2) indicates a high
degree of conservation, with sequence variation found at only three
positions.
Phylogenetic analysis
A maximum likelihood phenogram, based on the alignment of CDS nucleotides
(positions 1371 in Fig.
2), was constructed (Fig.
3). This phylogeny shows strong bootstrap support (>70%) for
four evolutionary branches or clades, one containing all mammalian and avian
genes, a second containing all teleostean genes, a third containing
Xenopus and a single fugu sequence, JGI-15768, and a fourth
containing the shark sequences. The relationship among these four clades is
not as well supported (62%). Among the bony fish genes, the present phylogeny
suggests two rainbow trout lineages, an Onmy-GS01/03 and an
Onmy-GS02/04 clade. These clades are highly divergent and do not form
a monophyletic clade (i.e. share a common ancestor prior to sharing an
ancestor with genes from an other species), suggesting that most of the
variation arose prior to speciation.
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Estimating the number of GSase genes
The amino acid sequence similarities of Onmy-GS01 to
Onmy-GS03 and Onmy-GS02 to Onmy-GS04 could
represent allelic diversity. Alternatively, their similarities may reflect a
relatively recent duplication event. To investigate these hypotheses, we
conducted an analysis of the variation in intron 4. Primer pairs
GLUL-Onmy*e4f1/e5r1 or GLUL-Onmy*e4f2/e5r2 were used to amplify the fourth
intron of either Onmy-GS01/GS03 or Onmy-GS02/GS04 cDNAs,
respectively. These primer pairs designed for parts of exons 4 and 5, flanking
intron 4, were used to identify four unique intron sequences from a single
fish. The intron sequences were 146, 256, 120 and 113 bp for
Onmy-GS0104, respectively. In all cases, the locations of the
intron splice boundaries were identical to the locations identified in the
rodent, chicken (van de Zande et al.,
1990; Pu and Young,
1989
; Kou and Darnell,
1989
) and fugu (JGI-18692) GSase genes. Reflecting the
maximum-likelihood phylogeny, a dot-plot analysis shows a slightly higher
level of similarity between the Onmy-GS01/GS03 cDNAs sequences and
also between the Onmy-GS02/GS04 sequences
(Fig. 4). As expected, this
similarity is most noticeable around the splice boundaries. For most of the
remaining intron sequence no significant similarity exists, arguing against an
allelic relationship between the sequence pairs. The analysis of the
3'-UTR sequences agrees with the intron analysis. No significant
similarity is found between the 3'-UTR sequences of the two clades (i.e.
Onmy-GS01 or GS03 compared with Omny-GS02 or
GS04). Separate sequence alignments could be made between the
Onmy-GS01 and GS03 or the Onmy-GS02 and
GS04 3'-UTRs (not shown). In both cases, a large number of
substitutions and insertions or deletions (indels) were present;
GS01/GS03: 13 indels (involving 52 bp), 86% similar (324/377 bp,
excluding indels); GS02/GS04: 14 indels (involving 62 bp), 84%
similar (316/376 bp). Consequently, we must conclude that the similarity
within these gene sequence pairs reflects a recent gene duplication event.
|
To estimate the number of GSase genes present in trout, a Southern blot was hybridised concurrently with two probes made up of the Onmy-GS01 and Onmy-GS02 genes, respectively. In a single individual, 57 bands are visible in HinfI and AluI digests, respectively (not shown). HinfI and AluI restriction sites are present within the four sequence regions complementary to the Onmy-GS01/02 probes. The observed number of fragments is consistent with the expected number of fragments that should be detectable (i.e. >500 bp) upon digestion of genomic DNA with Hinfi and AluI, assuming all four sequences are present and an average intron size of 200 bp. Exact fragment expectations cannot be made without complete intron sequence information.
Northern analysis
The expression of the four GSase mRNAs in trout was studied by northern
analysis. Within each tissue, the intensity of the GS signal was compared to
that of ß-actin. Onmy-GS02 was expressed at higher levels than
Onmy-GS01, GS03 and GS04 in most tissues
(Fig. 5). In the brain and
gill, however, Onmy-GS01 was expressed at the highest level relative
to the other three transcripts. When comparing between tissues, expression
relative to ß-actin is not appropriate because ß-actin levels vary
in different tissues (Foss et al.,
1998). The amount of total RNA loaded appeared very consistent
between lanes. For each individual transcript, the order of the highest to the
lowest level of mRNA was: Onmy-GS01: brain, intestine, gill, liver,
kidney/red muscle, white muscle, heart; Onmy-GS02: brain >
intestine > liver > red muscle > kidney > white muscle > gill
> heart; Onmy-GS03: brain > intestine > liver > red
muscle > kidney > white muscle > gill > heart; Onmy-GS04:
brain > intestine > gill > liver > kidney > red muscle >
white muscle > heart. Overall, the highest level of expression of the total
of the four transcripts was in the brain, followed by (in descending order)
intestine > liver > red muscle > gill/kidney > white muscle >
heart.
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Discussion |
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The two lineages of trout GSase cDNAs, Onmy-GS01/03 and
Onmy-GS02/04, are strongly supported in the phylogenetic analysis.
The genetic distance estimates between the members of each pair (0.0948 and
0.0825 substitutions/site, respectively) are greater than those observed
between mus and rattus (0.0798)
(Fig. 3). The divergence of rat
and mouse lineages is thought to have occurred between 1035 mya (see
Li, 1997). GSase genes have
been shown to evolve in a clock-like manner over a wide range of evolutionary
times (Pesole et al., 1991
).
Assuming a molecular clock for bony fish GSase genes, the
Onmy-GS01/03 and Onmy-GS02/04 values are, therefore,
consistent with the duplication of these loci (from 2 to 4) during the
ancestral tetrapoidization event estimated to have occurred 25100 mya
(Allendorf and Thorgaard,
1984
).
The presence of two GSase genes in zebrafish and fugu and four in trout
suggests that multiple, independent gene duplication events of GSase have
occurred within bony fishes. Taylor et al.
(2001) examined 27 groups of
orthologous genes (i.e. homologous genes, derived from a common ancestral
gene, which are found in different species), each including two zebrafish
genes as well as genes from Xenopus, chicken, mouse and man. They
concluded that a single genome duplication event in the ancestor of teleostean
fishes was the most parsimonious explanation for the duplicated gene copies
found in zebrafish. Our data are not inconsistent with the results of Taylor
et al. (2001
), which did not
examine orthologous loci of teleost species beyond zebrafish, but they do
indicate that the ancestral genome duplication event may have been specific to
the lineage leading to zebrafish and not ancestral to all teleosts. The
presence of the trout GSase sequences in a sister clade to that of the two
zebrafish sequences suggests the occurrence of a second ancestral
lineage-specific gene duplication event. Within the lineage containing the
trout sequences, two GSase genes are reported for B. sinensis
(Bosi, Fig. 2)
(Anderson et al., 2002
). Due to
their sequence similarity these genes appear to be alleles of a single locus;
however, the possibility of additional GSase genes in this species was not
excluded (Anderson et al.,
2002
). In the gulf toadfish, a single gene was reported that may
be differentially spliced leading to cytosolic and mitochondrial isozymes
(Walsh et al., 1999
).
Subsequent studies, however, have revealed the presence of a second cytosolic
O. beta gene (Walsh et al.,
2003
), supporting our proposal of multiple GSase duplication
events in ray-finned fishes.
Evidence for two GSase loci is found in the draft sequence of the fugu
genome. One of these sequences, JGI-18692 is clearly orthologous to the trout
Onmy-GS02/04 lineage. We predict that this is a functional GSase gene
in fugu. In contrast, the second locus, JGI-15768, is divergent from all other
bony fish genes. Interestingly, this gene lacks the second exon, which
contains a number of conserved active site residues
(Fig. 2), and may be a
pseudogene. Alternatively, it may have evolved a new function. In either case,
its relationship to the divergent amphibian GSase genes
(Fig. 3), and to a second
gill-specific GSase gene reported in O. beta
(Walsh et al., 2003) is
intriguing. Although not detected in the present study, the presence of
additional trout GSase genes that belong to this divergent lineage remains to
be fully tested. At least three GSase cDNAs are also present in the tetraploid
African clawed frog, Xenopus laevis. The surprising, although weakly
supported, phylogenetic position of the Xenopus genes
(Fig. 3) suggests these genes
are not the true orthologues of the other tetrapod genes or alternatively,
they may have undergone substantial evolutionary change. The presence of bony
fish genes within this clade suggests that the genes of this lineage are not
the true orthologues of the other vertebrate genes. More work, however, in
both fugu and Xenopus, is needed to determine if (1) both putative
fugu GSase-like genes identified from genomic sequence actually code for an
expressed functional protein, (2) there are additional orthologous GSase-like
genes in amphibians and (3) how the complete sequence of the
Xela-GS02 gene fits into the present phylogeny.
The phylogenetic analysis of vertebrate GSase cDNAs reported here is
similar to previous phylogenies (Pesole et
al., 1991; Laud and Campbell,
1994
; Walsh et al.,
1999
). Consistent with the original proposal of Pesole et al.
(1991
), independent origins
can be postulated for each of the dogfish, chicken and Gulf toadfish
mitochondrial GSase cDNAs. There is strong support for the inclusion of the
toadfish Opbe-af118103 cDNA within a monophyletic bony fish clade
(Fig. 3). The convergent
evolution of mitochondrial targeted GSases in several animals (see above),
including toadfish, is further supported by (1) the apparent lack of
mitochondrial GSase activity in the closely related O. tau and
Porichthys notatus (Anderson and
Walsh, 1995
), (2) the lack of a mitochondrial transport leader
sequence in all other bony fish GSase cDNAs fully characterised, and (3) the
dissimilarity of the kinetic properties (i.e. phenotype) between the dogfish
and toadfish mitochondrial isozymes
(Walsh, 1996
). Concerning the
latter, one would predict that if both fish mitochondrial isozymes had evolved
from a common mitochondrial isozyme, then they would share a similar
phenotype.
The function of multiple GSase genes in some bony fishes is intriguing. The
high degree of conservation of GSase genes throughout eukaryote and prokaryote
evolution (Pesole et al.,
1991), and the lack of variation noted at the inferred glutamate
binding site residues, suggest that these genes are all functioning in the
expected manner. Levels of Onmy-GS0104 gene expression differ
among tissues and would presumably lead to functional octamers of differing
subunit make-up. [We have also measured the levels of Onmy-GS01 and
GS02 mRNAs in adult trout tissues using semi-quantitative RT-PCR and
the ribonuclease protection assay, and observed a very similar pattern of mRNA
expression (P. Essex-Fraser, N. Bernier, B. Murray and P. Wright, unpublished
data). Given this assumption, two hypotheses may explain the retention of
multiple GSase genes in bony fishes: (1) the presence of a mutant subunit in
the functional octameric protein may disrupt the enzymatic function of the
entire protein and be selected against, i.e. a dominant-negative mutation
(Gibson and Spring, 1998
) and
(2) the various duplicated genes may develop specialised regulatory
subfunctions, i.e. the duplicationdegeneration complementation model
(Force et al., 1999
). The
multifunctional role of GSase and the tissue-specific expression patterns
reported here favour the second hypothesis. In bony fishes the regulation of
the various isozymes, i.e. octameric proteins of differing subunit make-up,
may be coupled to different developmental stages. GSase activity is detected
early in embryonic rainbow trout (Steele
et al., 2001
) and the levels of activity are much higher in whole
larvae relative to adult liver tissue
(Wright et al., 1995
). Studies
are underway to determine the pattern of mRNA expression of the four genes
(Onmy-GS0104) during early stages of rainbow trout
development. The data presented here suggest that GSase genes are
differentially expressed in different tissues and therefore possibly involved
in different metabolic pathways. In most teleosts, GSase activities are
highest in the brain (Webb and Brown,
1976
; Chamberlin et al.,
1991
; Peng et al.,
1998
) and this is also true in rainbow trout
(Mommsen et al., 2003
),
followed, in descending order by kidney > liver > intestine > white
muscle in adults (Korte et al.,
1997
). While this descending order of GSase activities is not
entirely consistent with the relative levels of total mRNA expression of the
four trout GSase genes in the present study, it is unclear how the differing
subunit make-up of the octameric protein would affect the GSase activity. Of
interest, the seemingly contradictory results between enzyme assay and mRNA
expression studies of GSase in other species (e.g.
Kong et al., 2000
), especially
when the full complement of coding genes was not identified, may also be
explained by the presence of multiple genes expressed at different levels in
each tissue. This study clearly emphasizes the need to fully identify the
number of genes coding for the protein of interest prior to the interpretation
of data showing changes in the levels of mRNA expression, and suggests a
complex interaction of the gene products of duplicated loci in multimeric
proteins, such as GSase.
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Acknowledgments |
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References |
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