From the Department of Chemistry, Graduate School of
Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, the
§ Department of Molecular and Cellular Biology, Institute
for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, and
¶ Core Research for Evolutional Science and Technology (CREST),
Japan Science and Technology Corporation (JST), Japan
Received for publication, January 17, 2001, and in revised form, January 31, 2001
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ABSTRACT |
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The endoplasmic reticulum (ER) has a mechanism to
block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent
degradation in the cytosol when they cannot achieve correct folding
and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for
the degradation of misfolded glycoproteins, the precise mechanism for
this recognition remains obscure. Here we report a new
The endoplasmic reticulum
(ER)1 is the site of entry
for proteins destined for the secretory pathway. The ER provides an
optimized environment for correct maturation, including correct
folding, oligomerization, N- and O-linked
glycosylation, and disulfide bond formation of proteins imported into
the ER (1, 2). Several components, including molecular chaperones and
folding enzymes that mediate these processes, have been identified in the ER. Shortage or defects of these components as well as mutations in
secretory proteins and environmental stress tend to result in failure
and therefore misfolding of secretory proteins. The misfolded proteins
are retained in the ER and are subjected to retrial for correct
maturation with the aid of molecular chaperones and folding enzymes.
However if this process is not successful, prolonged retention of the
misfolded proteins in the ER eventually leads to their degradation (3,
4). The ER-associated degradation (ERAD) requires the
retrotranslocation of misfolded proteins through the Sec61 channel from
the ER lumen to the cytosol and subsequent degradation by the 26 S
proteasome located in the cytosol (5-9).
For ERAD of aberrant proteins, the question of how proteins that are
misfolded and to be degraded are identified and targeted for the
retrotranslocation system has not been resolved. In yeast Saccharomyces cerevisiae, slow removal of In the present study, we identified a new Plasmids, Strains, and Culturing Conditions--
Standard
recombinant techniques were employed using an Escherichia
coli strain TG1 (supE hsd ERAD Assays--
Metabolic labeling of yeast cells with
Tran35S-label (ICN) and preparation of cell extracts were
performed as described previously (19). Immunoprecipitation was
performed as described by Nishikawa et al. (20). Yeast
microsome fractions were prepared from wild-type cells and
Fluorescence Microscopy--
Immunofluorescent staining of yeast
cells was performed as described previously (20) with minor
modifications. Incubation with the primary and the secondary antibodies
was performed as follows: 1) 1:250 dilution of the rabbit anti-BiP
polyclonal antibodies (21) and 1:250 dilution of the 16B12 mouse
monoclonal antibody and 2) 1:100 dilution of the
rhodamine-conjugated goat anti-rabbit IgG antibody, 1:100 dilution of
the fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody.
Mnl1p Is an ER Protein--
MNL1 (YHR204W) on yeast
chromosome VIII encodes an
To assess the functions of Mnl1p, we first analyzed its subcellular
location by indirect immunofluorescent microscopy. Mnl1p was tagged at
the C terminus with the HA epitope for recognition by the monoclonal
antibody 16B12. Cells that express the HA-tagged version of Mnl1p from
a multicopy plasmid were fixed, permeabilized, and subjected to
staining with the anti-HA antibody. Staining with the anti-HA antibody
showed perinuclear staining with tubular extensions in the cytoplasm
(Fig. 1B, panel b). This staining is typical for
yeast ER proteins, and nearly identical staining was observed with the
anti-BiP antibodies (Fig. 1B, panel a). Cells
that contain a multicopy plasmid without expression of the HA-tagged
Mnl1p did not show such staining (data not shown). These results
indicate that Mnl1p resides exclusively in the ER.
Deletion of MNL1 Causes a Defect in ERAD of CPY*--
The trimming
of N-linked oligosaccharides is critical for
proteasome-dependent ERAD of misfolded glycoproteins. For
example in yeast, removal of
CPY is known to receive distinct posttranslational modifications in
different cellular compartments on its transport pathway to the vacuole
(25, 26). On translocation across the ER membrane, a prepro form of CPY
(prepro-CPY) receives proteolytic cleavage of the signal sequence and
addition of four N-linked oligosaccharide chains to generate
a 67-kDa ER form, p1CPY. In the Golgi complex, p1CPY is converted to a
69-kDa form, p2CPY, by addition of mannose to the N-linked
oligosaccharide chains (26). After reaching the vacuole, p2CPY becomes
a 61-kDa mature form, mCPY (26, 27). When wild-type cells or
We also noticed that p1CPY* in
A possible explanation for the glycosyl modifications of p1CPY* in
Mnl1p Is Not Involved in ERAD of
In parallel with these in vitro degradation assays, we
followed the degradation of p In the present study, we have characterized the functions of
Mnl1p, an The fate of CPY* was followed in detail when its degradation was
impaired by the deletion of the MNL1 gene. Whereas CPY* was partly retained as p1CPY*, the ER form to be degraded, a part of p1CPY*
molecules were converted to the early-Golgi form, p1'CPY*, containing
-mannosidase-like protein, Mnl1p
(mannosidase-like protein), in the
yeast ER. Mnl1p is unlikely to exhibit
1,2-mannosidase activity,
because it lacks cysteine residues that are essential for
1,2-mannosidase. However deletion of the MNL1 gene
causes retardation of the degradation of misfolded carboxypeptidase Y,
but not of the unglycosylated mutant form of the yeast
-mating
pheromone. Possible roles of Mnl1p in the degradation and in the
ER-retention of misfolded glycoproteins are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,2-mannose
from the middle branch of the protein-bound oligosaccharide, or
formation of Man8GlcNAc2, has been shown to be
critical for the degradation of misfolded carboxypeptidase Y (CPY), a
vacuolar glycoprotein (10). This is consistent with the reports that
inhibition of
-mannosidase trimming stabilizes specific misfolded
glycoproteins in the mammalian ER (11, 12). On the basis of these
observations, it has been proposed that the trimming of the
glycoprotein-bound oligosaccharide may well function as the biological
timer for the onset of the glycoprotein degradation that prevents
permanent residence of misfolded glycoproteins in the ER (2). This
naturally suggests the presence of a
Man8GlcNAc2-binding lectin involved in ERAD of
misfolded glycoproteins, which remains to be identified (10).
-mannosidase-like protein,
Mnl1p (mannosidase-like protein),
in the yeast ER. Although it shows some homology with Mns1p, yeast
1,2-mannosidase, Mnl1p is unlikely to exhibit the
1,2-mannosidase
activity, because it lacks cysteine residues that are essential
for the
1,2-mannosidase activity (13). However deletion of the
MNL1 gene resulted in retardation of the degradation of
misfolded CPY, but not of the unglycosylated mutant form of the
yeast
-mating pheromone (
Gp
F). Possible roles of Mnl1p in ERAD
and the ER retention of misfolded glycoproteins will be discussed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 thi
(lac-proAB) F' [tra
36 proAB+ lacIq
lacZ
M15]). Yeast strains used in this study were SEY6210
(MAT
ura3 leu2 trp1 his3 lys2 suc2) (14) and
KYSC1 (MAT
prc1-1 ura3 leu2 trp1 his3 lys2
suc2).2 Yeast strain
BJ3505 (MATa pep4::HIS3 prb1 lys2
trp1 ura3 gal2 can1) was used for preparation of the cytosol (9).
Cells were grown in YPD medium containing 1% yeast extract, 2%
polypeptone, and 2% glucose. A sulfate-free synthetic minimal medium
(15) was used for metabolic labeling of yeast cells. The gene for the C-terminally HA (influenza hemagglutinin) epitope-tagged version of
Mnl1p was cloned by PCR from the yeast genomic DNA. The amplified DNA
was subcloned into a yeast multicopy plasmid pYO326 (16) to generate
pKNM1, which expresses the Mnl1p-HA fusion protein. A null allele of
MNL1 was constructed by the PCR-based gene disruption using
the HIS3 gene of Candida glabrata (a gift from
Dr. Satoshi Harashima) as described previously (17, 18). The
mnl1 strains derived from SEY6210 and KYSC1 were named
SNY1079 and SNY1080, respectively.
mnl1 cells (9) and the yeast cytosol S100
fraction from yeast BJ3505 cells. 35S-Labeled mutant
prepro-
factor lacking the three consensus glycosylation sites
(
Gpp
F) (9) was synthesized in vitro using a yeast
cell-free translation. The in vitro ERAD assay was performed
as described previously (9).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase-like protein, which is 796 amino acids long, with a calculated molecular weight of 92,206. The
predicted amino acid sequence of Mnl1p shows 25% identity with Mns1p,
yeast
1,2-mannosidase that converts glycosyl residues from
Man9GlcNAc2 to
Man8GlcNAc2 in the ER (Fig.
1A). Mns1p is a type II ER
transmembrane glycoprotein with its N-terminal hydrophobic segment
functioning as a signal-anchor sequence. The question of whether the
segment of hydrophobic residues 1-21 (Fig. 1A) functions as
a signal-anchor sequence to make Mnl1p a type-II membrane protein
should await future studies. The amino acid residues involved in the
substrate binding, Ca2+ binding, and catalytic activity in
Mns1p (22) are conserved in Mnl1p except for the two cysteine
residues (Cys340 and Cys385 in Mns1p)
that form a disulfide bridge and are essential for the
-mannosidase
activity (13, 22). The lack of the essential cysteine residues
in Mnl1p suggests that Mnl1p is unlikely to have the
-mannosidase
activity. The C-terminal part of Mnl1p shows no homology with any
proteins deposited in the data base.
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Fig. 1.
Properties of Mnl1p. A,
predicted amino acid sequence of yeast Mnl1p aligned to the sequence of
a homologous yeast -mannosidase, Mns1p. Identical amino acids are
boxed. Hydrophobic segments are indicated with
horizontal bars and putative N-linked
glycosylation sites with dots. Two conserved cysteine
residues that are essential for the
-mannosidase activity are
indicated with asterisks. B, localization of
Mnl1p by immunofluorescence microscopy. Cells of SEY6210/pKNM1 were
grown in SCD medium (minimal medium containing 2% (w/v) glucose and
0.5% (w/v) casamino acids) lacking uracil at 30 °C and analyzed by
double label immunofluorescence microscopy using anti-BiP polyclonal
antibodies and the 16B12 monoclonal antibody. Panels a,
b, and c show the same field of the fluorescent
images stained with the anti-BiP antibodies, and the 16B12 antibody,
and 4',6-diamidino-2-phenylindole, respectively. Bar, 2 µm.
1,2-glucose by glucosidase I and
glucosidase II and that of
1,2-mannnose by ER-
1,2-mannosidase,
Mns1p, to yield Man8GlcNAc2 are essential for
the efficient degradation of CPY*, mutated and therefore misfolded
carboxypeptidase Y (10, 23). Although Mnl1p is not expected to have the
-mannosidase activity, its homology with Mns1p raises the
possibility that Mnl1p is involved in the recognition of specific
oligosaccharide structures and/or ERAD of misfolded glycoproteins. We
thus analyzed the effects of deletion of the MNL1 gene on
ERAD of a model misfolded protein, CPY*, in vivo. The
mnl1 null mutant strain did not show any
detectable growth phenotypes as reported previously (24).
mnl1 cells were metabolically labeled with
35S-containing amino acids for 5 min at 30 °C, the p1
form of labeled CPY* was recovered as pellets by immunoprecipitation
(Fig. 2A, upper panel, chase 0 min). When the fate of labeled p1CPY* in wild-type cells was followed,
the amount of p1CPY* decreased rapidly without changing its molecular
mass during the chase period. This indicates that misfolded p1CPY* was
prevented from its exit from the ER for the Golgi complex and was
degraded efficiently with a half-life of 18 min at 30 °C (Fig. 2,
A and B). In contrast, the p1 form of labeled
CPY* in
mnl1 cells was degraded 3-fold more
slowly than in wild-type cells (Fig. 2B). This suggests that Mnl1p is, whether directly or indirectly, involved in ERAD of CPY*.
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Fig. 2.
Deletion of MNL1 causes
retardation of the degradation of CPY*. A, wild-type
cells (KYSC1, wt) and mnl1 cells (SNY1080,
mnl1) were pulse-labeled with 35S-containing
amino acids for 5 min at 30 °C and chased for the indicated times.
Cell extracts were prepared from the labeled cells and subjected to
immunoprecipitation with the anti-CPY antibodies. The
immunoprecipitated proteins were incubated with (+) or without (
)
0.17 unit/ml endoglycosidase H (EndoH) at 37 °C for
22 h and analyzed by SDS-PAGE and radioimaging with a Storm 860 image analyzer (Molecular Dynamics). p1, p1CPY*;
p1', p1'CPY*;
Gp1, a deglycosylated
form of p1CPY* and p1'CPY*. B, quantification of the results
shown in A (
EndoH). The amount of CPY* at
0-min chase is set to 100%. Data represent the average of the results
of two independent assays.
mnl1 null
mutant cells was converted to a higher molecular weight form, which was
designated as p1'CPY* and was observed as a smear on the SDS-PAGE gel,
during the chase period (Fig. 2A). When p1'CPY* was treated
with endoglycosidase H, it was converted to a deglycosylated form with
the same electrophoretic mobility as that for p1CPY* (Fig.
2A). This means that p1'CPY* arose from glycosyl
modifications of p1CPY*. As a control, we confirmed that the deletion
of MNL1 does not affect the exit of p1CPY from the ER for
the Golgi complex and for the vacuole (not shown).
mnl1 cells is that a part of misfolded p1CPY*
escaped the ER and received addition of
1
6- and/or
1
3-linked mannose to core oligosaccharides in the Golgi complex
(25, 28). To test if p1'CPY* had been modified by the Golgi enzymes,
reactivity of p1'CPY* to the anti-
1
6 mannose and the
anti-
1
3 mannose antibodies was examined. Wild-type cells
expressing CPY or CPY* and
mnl1 cells
expressing CPY* were metabolically labeled for 5 min and chased, and
the CPY or CPY* species were immunoprecipitated with the anti-CPY
antibodies from the cell extracts. The immunoprecipitated proteins were
subjected to the second-round immunoprecipitation with the antibodies
against CPY,
1
6 mannose, or
1
3 mannose. As shown in Fig. 4,
p1'CPY* in
mnl1 cells was immunoprecipitated with the anti-
1
6 mannose antibodies but not with the
anti-
1
3 mannose antibodies (Fig. 3,
lanes 14 and 15), whereas p1CPY* was not
recognized by these antibodies (Fig. 3, lane 13). p1CPY* in wild-type cells, which was retained in the ER and therefore not glycosylated by the Golgi enzymes, was not precipitated with the anti-
1
6 mannose or anti-
1
3 mannose antibodies (Fig. 3,
lanes 9 and 10). As a control, it was confirmed
that mCPY in wild-type cells, which had received carbohydrate
modification in the Golgi complex, were precipitated with these
antibodies (Fig. 3, lanes 4 and 5). These results
indicate that p1'CPY* was modified by the early Golgi enzymes in
mnl1 cells and that it is the intermediate form of the mannose addition in the Golgi complex. In other words, deletion of the MNL1 gene allowed exit of a fraction of
p1CPY*, which is strictly retained in the ER in wild-type cells, from the ER for at least the early Golgi cisternae (29).
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Fig. 3.
CPY* becomes an early-Golgi glycosylated form
in mnl1 cells. Wild-type cells
(KYSC1, wild-type) expressing CPY (CPY/MNL1), wild-type
cells expressing CPY* (CPY*/MNL1), and
mnl1
cells (SNY1080,
mnl1) expressing CPY*
(CPY*/
mnl1) were pulse-labeled with
35S-containing amino acids for 5 min at 30 °C and chased
for the indicated times. Cell extracts were prepared from the labeled
cells and subjected to immunoprecipitation with the anti-CPY antibodies
(1st antibody). The immunocomplexes were solubilized, and
aliquots were subjected to the second round of immunoprecipitation with
the indicated antibodies (2nd antibody; the 1-6
and 1-3 antibodies are specific for the
1, 6-, and
1,3-mannose linkages). The amounts of cell extracts for lanes
2-5, 7-10, and 12-15 were twice as much
as those for lanes 1, 6, and 11,
respectively. The immunoprecipitated proteins were analyzed by SDS-PAGE
and radioimaging. p1, p1CPY; p1', p1'CPY*;
p2, p2CPY; m, mCPY.
Gp
F--
We next asked if
Mnl1p is involved in ERAD of misfolded but unglycosylated proteins. For
this purpose, we performed in vitro export/degradation
assays with an unglycosylated mutant form (
Gp
F) of the yeast
-mating pheromone, pro-
-factor (p
F), as a substrate (8, 9, 30,
31). A radiolabeled precursor form of
Gp
F (
Gpp
F) was
translocated into microsomes prepared from the
mnl1 strain or from the isogenic wild-type
strain. Upon signal sequence cleavage, the resulting product,
Gp
F, becomes an ERAD substrate when the washed vesicles are
incubated in the presence of the cytosol and ATP (9). When this assay
was performed using microsomes prepared from
mnl1 cells, degradation of
Gp
F was as
efficient as that with wild-type microsomes at 30 °C (Fig.
4A).
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Fig. 4.
Deletion of MNL1 does not
affect the degradation of
Gp
F either in
vitro or in vivo. A,
in vitro ERAD assays for radiolabeled
Gpp
F.
35S-Labeled
Gpp
F was translocated into microsomes
prepared from wild-type (SEY6210, wt) and
mnl1
(SNY1079,
mnl1) cells at 20 °C for 50 min. Microsomes
containing
Gp
F were collected by centrifugation, washed once with
buffer 88 (20 mM HEPES-KOH, pH 6.8, 250 mM
sorbitol, 150 mM KOAc, 5 mM
Mg(OAc)2) (9), and resuspended in buffer 88. The microsomes
were further incubated in buffer 88 containing an ATP-regenerating
system (1 mM ATP, 50 µM GDP-mannose, 40 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase)
and the yeast cytosol (5 mg of protein/ml) at 30 °C. At the
indicated time points, proteins were precipitated with trichloroacetic
acid and subjected to SDS-PAGE in the presence of 4 M urea.
The upper band is
Gpp
F, which is bound to the
cytosolic surface of the microsomes and escaped ERAD. B,
quantification of the results shown in A. The amount of
Gp
F at 0-min incubation was set to 100%. Data represent the
average of the results of two independent assays. C,
in vivo ERAD assays for radiolabeled p
F. Wild-type
(SEY6210, wt) cells and
mnl1 (SNY1079,
mnl1) cells were pulse-labeled with
35S-containing amino acids for 5 min at 30 °C in the
presence of 10 µg/ml of tunicamycin, chased for the indicated times,
and subjected to immunoprecipitation with the anti-
F antibodies.
D, quantification of the results shown in C. The
amount of
Gp
F at 0-min chase was set to 100%. Data represent the
average of the results of two independent assays.
F in vivo by pulse-chase
experiments. The previous study showed that tunicamycin, an inhibitor
of the N-linked glycosylation in the ER, causes formation of
the unglycosylated and therefore misfolded
F precursor
(32).2 The unglycosylated p
F is rapidly degraded in a
Sec61p-dependent manner, suggesting that it is a substrate
for ERAD in vivo ((32); Fig. 4C).
Wild-type cells and
mnl1 cells were metabolically labeled for 5 min in the presence of tunicamycin, and p
F was recovered as
pellets by immunoprecipitation by the anti-
-factor antibodies. As
shown in Fig. 4B, unglycosylated p
F was rapidly degraded
in
mnl1 cells with nearly the same kinetics as
that for wild-type cells. Taken together, these results of the in
vivo as well as the in vitro degradation assays
strongly suggest that Mnl1p is not required for ERAD of
unglycosylated proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase-like protein in the ER. Deletion of the MNL1 gene led to retardation of ERAD of CPY*, a misfolded
glycoprotein, but not of
Gp
F, a misfolded unglycosylated protein,
suggesting that Mnl1p plays important roles in ERAD of misfolded
proteins with N-linked oligosaccharides. Then what is the
role of Mnl1p in ERAD? The lack of two cysteine residues
strongly suggests that Mnl1p does not have the
-mannosidase
activity. Mnl1p may be involved in the recognition of oligosaccharide
structures, including Man8GlcNAc2, which is
essential for ERAD substrates, of misfolded proteins. Indeed, a
recently identified mammalian counterpart of Mnl1p does not have the
mannosidase activity and probably functions as a lectin that
specifically recognizes the oligosaccharide structure of
Man8GlcNAc2 (34).
1
6 mannose. This indicates that at least some p1CPY* was released
from the ER to the Golgi complex. However, since
1
3 mannose was
not attached to p1'CPY* even after the prolonged chase, CPY* most
likely undergoes recycling through the early-Golgi cisternae into the
ER. Deletion of DER1 encoding an ER protein involved in ERAD
also leads to escape of some CPY* from the ER for the early-Golgi
compartment (33). Therefore ER-resident proteins that are involved in
ERAD are also responsible for retaining their substrates in the ER
probably by their affinity for misfolded proteins.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tohru Yoshihisa in our
laboratory for discussions; Dr. Akihiko Nakano for the antisera against
pF,
1
3 mannose, and
1
6 mannose; Dr. Jeffrey L. Brodsky
for the plasmid for in vitro translation of
Gpp
F; and
Dr. Satoshi Harashima for the HIS3 gene of C. glabrata.
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FOOTNOTES |
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* This work was supported by grants-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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. Tel.: 81-52789-2490; Fax: 81-52789-2947; E-mail: endo@biochem.chem.nagoya-u.ac.jp.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.C100023200
2 S. Nishikawa, S. W. Fewell, Y. Kato, J. L. Brodsky, and T. Endo, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
ER, endoplasmic reticulum;
ERAD, endoplasmic reticulum-associated
degradation;
CPY, carboxypeptidase Y;
CPY*, a mutated version of
carboxypeptidase Y;
p F, pro-
factor;
pp
F, prepro-
factor;
Gp
F, an unglycosylated mutant form of prepro-
factor;
Gpp
F, an unglycosylated mutant form of prepro-
factor;
HA, hemagglutinin;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis.
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