From the Department of Biochemistry and Molecular Biology, the University of Georgia, Athens, Georgia 30602-7229
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ABSTRACT |
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Casein kinase II (CKII) of Saccharomyces
cerevisiae contains two distinct catalytic subunits, and
', that are encoded by the CKA1 and -2 genes, respectively. We have constructed conditional alleles of the
CKA1 gene. In contrast to cka1
cka2ts strains, which exhibit a defect in both
G1 and G2/M cell cycle progression,
cka1ts cka2 strains continue to
divide for three cell cycles after a shift to restrictive temperature
and then arrest as a mixture of budded and unbudded cells with a
spherical morphology. Arrested cells exhibit continued growth, a
nonpolarized actin cytoskeleton, delocalized chitin deposition, and a
significant fraction of multinucleate cell bodies, confirming the
presence of a cell polarity defect in cka1ts
strains. The presence of budded as well as unbudded cells in the
arrested population suggests that CKII is required for maintenance rather than establishment of cell polarity, although a role in both
processes is also possible. The terminal phenotype of
cka1ts strains bears a strong resemblance to
that of orb5 strains of Schizosaccharomyces
pombe, which carry a temperature-sensitive CKII catalytic subunit
mutation, but the underlying mechanism appears to be different in the
two cases. These results establish a requirement for CKII in cell
polarity in S. cerevisiae and provide the first evidence
for functional specialization of CKA1 and
-2.
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INTRODUCTION |
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Casein kinase II (CKII)1
is an essential, highly conserved, acidic-directed protein kinase that
is ubiquitous in eukaryotic organisms (1-4). The enzyme is composed of
catalytic () and regulatory (
) subunits that associate to form an
2
2 holoenzyme. Distinct isoforms of one
or both subunits occur in various species, and the three subunits of
the mammalian enzyme (
,
', and
) associate to form distinct
2
2,
'2
2,
and
'
2 tetramers in vivo (5). Whether
these distinct forms have unique functions is unknown. In
vitro, CKII activity is inhibited by polyanions and activated by
polycations, but the mechanism of regulation of the enzyme in
vivo remains to be defined. CKII phosphorylates a broad spectrum
of nuclear and cytoplasmic substrates (6) and has been implicated in
the regulation of fundamental cellular processes, including
transcription, translation, morphogenesis, and cell cycle regulation.
Consistent with a role in the latter process, misregulated expression
of CKII activity in lymphocytes of transgenic mice results in the
stochastic production of lymphoma and, when combined with
c-myc, leukemia (7).
Molecular/genetic techniques have been used to dissect the
physiological role of CKII and its subunits in both budding and fission
yeast. Purified CKII of Saccharomyces cerevisiae is composed of four subunits, ,
',
, and
', which are encoded by the
CKA1, CKA2, CKB1, and CKB2
genes, respectively (8). The enzyme is exclusively tetrameric, but
which of the nine possible isoforms occur has not been definitively
established. Disruption of either catalytic subunit gene yields no
obvious phenotype on normal media, but disruption of both is lethal (9,
10). Cells depleted of CKII activity arrest as a mixed population of
budded and unbudded cells, with a significant proportion of the budded
fraction displaying an elongated morphology (10). The continued growth
of the arrested cells suggests a cell cycle defect, and analysis of
temperature-sensitive alleles of the cka2 gene confirms that
CKII function is required for cell cycle progression at two points in
the cell cycle, one in G1 and one in G2/M (11).
Depletion of CKII activity also results in flocculation, a
lectin-mediated, Ca2+-dependent cell-cell
aggregation. The significance of this is unknown, but mutations in the
global transcriptional repressor, Ssn6/Tup1, display a similar
phenotype (12, 13). Rather surprisingly, disruption of CKB1
and/or CKB2 results in no overt phenotype on normal media;
however, cells lacking either gene display specific sensitivity to
Na+ and Li+ (14, 15).
In contrast to S. cerevisiae, Schizosaccharomyces pombe appears to have a single gene encoding each subunit of CKII (16). Temperature-sensitive mutations of the cka1+ gene encoding the catalytic subunit have been isolated in a screen for morphological mutants (17). These mutations (orb5) are recessive lethal and confer a spherical morphology at the restrictive temperature. Although arrested cells display aberrant actin and tubulin cytoskeletons, analysis of double mutants between orb5 and various cell division cycle mutants indicates that CKII is required not for cell polarity per se but for the re-establishment of polarized growth following cytokinesis. No defects in cell cycle progression were defined for these mutants, and orb5 cells in fact undergo two to three cell divisions prior to arrest. In contrast to S. cerevisiae, null mutations of the ckb1+ gene encoding the regulatory subunit confer a slow growth phenotype, cell-cell aggregation, and cold sensitivity (16). Such mutants also display an abnormal rounded morphology, reminiscent of the orb5 phenotype.
The rather disparate results obtained in the two systems make it difficult to construct a unified model of the physiological role of CKII. In S. cerevisiae, CKII is implicated in cell-cell aggregation, ion homeostasis, cell cycle regulation, and perhaps morphogenesis. In S. pombe, CKII is implicated in cell-cell aggregation and polarized cell growth. Although cell-cell aggregation and morphological abnormalities are common to both systems, CKII appears to be dispensable for cell growth in S. cerevisiae and for cell cycle progression in S. pombe. Furthermore, the morphological abnormalities observed in the two systems are opposite in sign, since the elongated phenotype observed in S. cerevisiae is indicative of hyperpolarization (18), whereas the spherical morphology of orb5 alleles reflects a loss of polarity (or the ability to grow in a polarized fashion). Although it is formally possible that the orb5 alleles are gain-of-function mutations, the recessive nature of these alleles makes this unlikely (17).
In the course of analyzing conserved elements in the N-terminal region
of the CKII subunit, we have constructed two temperature-sensitive alleles of the S. cerevisiae CKA1 gene. We report here that
cka1ts mutants display an apolar phenotype, in
marked contrast to the cell cycle phenotype displayed by
cka2ts strains. Although this phenotype is
similar in several respects to that observed in S. pombe
orb5 mutants, we find that in S. cerevisiae CKII
function is required not for polarized growth but for cell polarity
itself. Our results also provide genetic evidence for functional
specialization of the two catalytic subunit isoforms in budding
yeast.
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EXPERIMENTAL PROCEDURES |
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Strains, Media, and Growth Conditions--
Principal S. cerevisiae strains and plasmids used in this study are listed in
Table I. For routine work, yeast strains were grown at 29 °C in YPD
medium (1% yeast extract, 2% peptone, 2% D-glucose) or
in synthetic complete medium (SC) lacking specific components as
necessary (19). Routine molecular cloning was carried out in
Escherichia coli DH5 (CLONTECH).
E. coli strains were propogated at 37 °C in Luria broth
containing 50 µg/ml ampicillin as appropriate (20).
Plasmid Construction-- Routine molecular biology procedures were according to Ausubel et al. (20). Synthesis of oligonucleotide primers and DNA sequencing were carried out on Applied Biosystems instruments at the University of Georgia Molecular Genetics Instrumentation Facility.
To construct the cka1-Strain Construction-- Strains were constructed according to standard molecular/genetic procedures (19). Strains YAR105, YAR106, YAR107, and YAR12 were constructed by lithium acetate-mediated transformation of strain RPG41-1a (Table I) with plasmids pAR1, pAR2, pAR3, and pAR4, respectively. Cells prototrophic for both leucine and uracil were selected on the appropriate medium and colony-purified. Strains YAR108, YAR109, and YAR13 were generated from YAR105, YAR106, and YAR12, respectively, by eviction of the resident URA3 plasmid derived from RPG41-1a. Briefly, cells were grown nonselectively in YPD for 3 days at 29 °C and then plated on SC plates containing 0.75 mg/ml 5-fluoroorotic acid (5-FOA), which selects against the presence of the URA3 gene (22). 5-FOA-resistant cells were colony-purified by restreaking on the same medium.
Chromosomal integration of the cka1-Cell Biology Protocols-- To assay temperature-sensitive growth, cells were cultured overnight in YPD at 25-29 °C, diluted as necessary, and then spotted in triplicate (2000 cells/10-µl spot) on a series of YPD plates. Plates were incubated at the desired temperatures for 4 days.
For generation of growth curves, cells were grown overnight in YPD at 25-29 °C and then inoculated into fresh YPD at a starting concentration of 1 × 105 cells/ml. Cultures were grown at permissive temperature (either 25 or 29 °C) with vigorous shaking (450 rpm). For temperature shift studies, parallel cultures were shifted to restrictive temperature (either 37 or 38.5 °C) when the culture reached a density of 5 × 105 cells/ml. To determine cell number, aliquots were removed at the indicated times, fixed with 3.7% formaldehyde, and counted in a hemacytometer. Viable cells were determined by plating an appropriate dilution of an unfixed aliquot on YPD at 25 °C. Percent viability was calculated as 100 × (number of colonies)/(cell number). To study cell morphology and budding pattern, formaldehyde-fixed cells from the growth curves were examined by phase contrast and Nomarski optics. Nuclear morphology was visualized by staining with DAPI (4',6'-diamidino-2-phenylindole) as described by Hanna et al. (11), and chitin distribution was visualized by staining with 0.1 mg/ml calcofluor as described by Pringle (23). Photographs were taken in a Zeiss IM 35 epifluorescence microscope fitted with Nomarski optics. F-actin was visualized by staining with rhodamine-conjugated phalloidin as described (24). Stained cells were examined by epifluorescence microscopy using the DeltaVision System (Applied Precision, Inc.). Digitized images were processed on a Macintosh computer running Adobe Photoshop. ![]() |
RESULTS |
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The Terminal Phenotype of cka1ts Mutants Is Distinct
from That of cka2ts Mutants--
Our studies were
initiated in an effort to analyze the function of the N-terminal region
of the CKII catalytic subunit in vivo. This region is well
conserved among different species (Fig. 1), with the highest conservation
concentrated in two six-residue motifs (corresponding to the sequences
SEARVY and EYWDYE of Cka1). We generated two deletion mutants of the
S. cerevisiae CKA1 gene, one of which
(cka1-2) removed the first element, and the
other of which (cka1-
3) removed both, together
with the intervening sequence (Fig. 1). The ability of these mutants to
complement a cka1 cka2 null background was then tested by
transforming each construct into RPG41-1a (Table
I) and plating the resultant strains on
5-FOA to eliminate the original rescuing plasmid present in this
strain. As shown in Fig. 2, the smaller
deletion mutant (cka1-
2) supported viability
in this assay (strain YAR106), whereas the larger deletion
(cka1-
3) did not (strain YAR107). YAR106
generated viable colonies at a frequency comparable with that of
strains rescued by either wild-type CKA1 (strain YAR105) or
CKA2 (strains YDH31 and YDH6) but exhibited a small colony
size, indicative of a reduced growth rate. This small colony phenotype
was reproducible, and subsequent analysis of this strain (designated
YAR109 after eviction of the URA3 plasmid) revealed a
doubling time of 120 min in liquid YPD at 29 °C, compared with 90 min for the isogenic wild-type control (YAR108). YAR109 also exhibited
constitutive flocculation (data not shown). Both of these phenotypes
are characteristic of reduced CKII activity (10, 11, 14). This and the
recessive nature of the mutation (data not shown) suggest that
cka1-
2 is a hypomorphic loss-of-function
allele.
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cka1ts Mutants Exhibit a Defect in Cell Polarity-- The spherical morphology conferred by temperature-sensitive cka1 mutants suggested that these mutants have a cell polarity defect. To test this proposition, we analyzed the distribution of nuclear chromatin, cell wall chitin, and actin in cka1ts and cka2ts strains grown at both permissive and restrictive temperature. At permissive temperature both mutants exhibited an essentially wild-type pattern (see below and data not shown).
DAPI staining of nuclear chromatin at restrictive temperature is illustrated in Fig. 5. Control strain YPH499 displayed a pattern of nuclear staining typical of dividing cells, with unbudded cells containing a single nucleus and budded cells containing either a single nucleus or a nucleus in both mother and daughter (panel A). In contrast, both cka1ts strains, YAR109.1 (panel B) and YAR13 (data not shown), arrested as a mixed population of budded and unbudded spherical cells, as noted above; and many of these cells contained more than one nucleus, most often two, in a single cell body (Table II). Multiple nuclei were invariably well separated and generally located at the extreme periphery of the cell. A similar multinuclear phenotype is characteristic of mutants defective in either the establishment or maintenance of cell polarity in this organism (27, 28). The cka2ts strain, YDH13 (panel C), arrested as a mixture of unbudded and large budded cells, and these exhibited the array of nuclear morphologies described previously for the cka2-8 allele (11). Despite the identical amino acid replacement in the cka1-13 and cka2-13 alleles, multinuclear cells were completely absent. Collectively, these results imply that the nuclear cycle is at least partially uncoupled from the budding cycle in cka1ts mutants and that this behavior is specific to cka1.
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DISCUSSION |
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We report the construction of the first conditional alleles of the S. cerevisiae CKA1 gene. Analysis of these alleles reveals a role for CKII in cell polarity, a function not previously associated with CKII in this organism. Our results provide an interesting counterpoint to the role of CKII in cell polarity in other systems, notably S. pombe, and provide the first indication of functional specialization of the CKA1 and CKA2 genes in S. cerevisiae.
Although we have not analyzed the expression of the mutant protein, the
inviability of cka1-3 strains suggests that
the N-terminal region of the CKII catalytic subunit is essential
in vivo. A similar N-terminal extension is present in
cAMP-dependent protein kinase, where it forms a long
-helix that associates with both the large and small lobes of the
catalytic subunit (25). Veron et al. (33) have proposed that
this N-terminal helix, as well as the corresponding region of CKII, is
crucial in stabilizing the hinge region between the two lobes. Our
results are consistent with such a crucial structural role. The
viability of cka1-
2 strains suggests that the
first conserved motif (SEARVY of Cka1) is not essential for the
function of the N-terminal arm. However, we note that the six amino
acids immediately upstream of this motif bear a fortuitous similarity
to this sequence so that the motif is only partially disrupted in the
cka1-
2 allele (see Fig. 1). Complete
disruption of the motif might reveal that it is in fact essential.
The morphology and budding growth habit of S. cerevisiae are mediated by dramatic rearrangements of the actin cytoskeleton and secretory apparatus (34-36). Bud emergence involves the assembly of a ring of cortical actin patches at the presumptive bud site followed by polarized secretion into the region defined by the ring. The actin cytoskeleton, specifically actin cables terminating at the actin patches, is believed to target secretory vesicles to the site of bud growth (37, 38). Both the cortical actin patches and secretion are directed to the apex of the bud early in the budding cycle but become randomly distributed later, the ultimate shape of the bud being determined by the balance between these apical and isotropic modes of growth. These cytoskeletal and secretory rearrangements are themselves dependent upon proper regulation of cell polarity. Two cell division cycle genes CDC42, which encodes a Rho-type GTPase, and CDC24, which encodes a guanine nucleotide exchange factor, are central to the establishment of cell polarity in S. cerevisiae (27, 29). Temperature-sensitive mutants of either gene arrest as spherical, unbudded cells having an isotropic distribution of cortical actin patches as well as cell wall chitin. Cell growth is not halted but occurs isotropically; and the nuclear cycle continues, resulting in a high proportion of cells with two or more nuclei. A similar apolar phenotype is elicited by mutations in several other genes, including the bud emergence genes BEM1 and BEM2 (39, 40).
Our analysis of cka1-2 and a second
cka1ts allele, cka1-13, indicates
that cka1ts mutants have a defect in cell
polarity. This is in marked contrast to the cell cycle arrest phenotype
previously defined for cka2ts mutants (11). The
fact that two molecularly distinct cka1 alleles yield the
one phenotype and five distinct cka2 alleles (one of which
is identical in form to one of the cka1 alleles) yield the other strongly implies that these phenotypes are specific to the CKA1 and -2 genes themselves. The terminal
phenotype of cka1ts strains closely resembles
that of cdc24 or -42 mutants. In both cases,
arrested cells display a spherical morphology, continued growth, a
nonpolarized actin cytoskeleton, delocalized chitin deposition, and a
significant fraction of multinucleate cell bodies. Nevertheless, there
exists one important distinction: cdc24 or 42 mutants arrest as unbudded cells, consistent with their role in the
establishment of cell polarity (27, 29, 34), whereas cka1ts strains arrest as a mixture of budded as
well as unbudded cells. At face value, the latter result implies that
CKII is required for the maintenance of cell polarity rather than for
its establishment, although a requirement for CKII in both processes
would also be consistent with the observations. Although it is formally
possible that CKII is required only for establishment of polarity and
that both of the alleles we have examined are leaky, this seems
unlikely given the distinct nature of the alleles, the absence of any
change in the proportion of unbudded cells as arrest occurs (data not shown), and the constancy of the terminal phenotype at different restrictive temperatures.
Several genes that appear to be involved in the maintenance of cell polarity in S. cerevisiae have been identified, including RHO3 and RHO4, which encode partially redundant Rho-type GTPases (41, 42), and BOI1 and BOI2, which encode proteins that interact physically and genetically with BEM1 and genetically with RHO3 (43, 44). Although the mechanism of action of Rho3,4 and Boi1,2 is unknown, the terminal phenotype of rho3ts rho4 and of boi1 boi2 strains (which are also temperature-sensitive) resembles that of cka1ts strains, including the loss of cell polarity in budded as well as unbudded cells. We have tested the ability of multicopy CDC24, CDC42, BEM1, BOI1, and BOI2 to suppress cka1ts as well as cka2ts alleles and have also constructed cka1 boi1, cka2 boi1, cka2 boi2, as well as cka1ts boi1 and cka2ts boi2 double mutants. No genetic interactions were observed in any of these tests. Additional studies will be required to determine whether CKII interacts with any of these functions.
Given the apparent functional redundancy of CKA1 and -2 (9, 10), the distinct arrest phenotypes displayed by cka1ts (this work) versus cka2ts mutants (11) are paradoxical. As one way to resolve this paradox, we propose that Cka1 and -2 are both able to phosphorylate all essential CKII substrates but that, because of differing kinetic constants or different localizations, they do so with different efficiencies. Strains lacking one or the other gene would thus harbor a different spectrum of hypophosphorylated substrates, with the result that different functions initially become limiting when the activity of the remaining subunit is removed. Because a cka1ts mutant harbors a null allele of cka2 and vice versa, to explain the observed phenotypes it is necessary to postulate that cka1 preferentially phosphorylates substrates required for cell cycle progression and cka2, substrates required for cell polarity, although this is undoubtedly an oversimplification. Cka1 and Cka2 in fact differ at two well conserved positions (Lys-76 and Lys-80 of Cka1) which have recently been implicated in recognition of the +3 position of the protein substrate (45). Regardless of the underlying mechanism, the distinct phenotypes of cka1ts and cka2ts mutants provide the first evidence for functional specialization of CKA1 and -2.
The hyperpolarization displayed by cka2ts strains relative to the apolar phenotype of cka1ts strains is also surprising. However, such ambidextrous behavior is not unprecedented, even for a single gene and indeed for a single allele. For example, incubation of cdc24-4 cells at semipermissive temperature (33 °C) results in the stochastic production of cells with an elongated bud (29). Interestingly, such buds stain uniformly for chitin, as do those produced by cka2ts strains. The explanation for these and similar situations may lie in the complexity of the interactions regulating cell polarity in S. cerevisiae. These interactions include a network of Rho-type GTPases (40) as well as protein-protein interactions mediated by pleckstrin homology and SH3 domains (43). It is possible that there is cross-talk among these functions in various mutant backgrounds. The involvement of CKII in the cell cycle may also be relevant given the intimate connections that exist between cell cycle progression and cell polarity in S. cerevisiae (18, 46).
The ability of cka1ts strains to proceed through several cell divisions in the apparent absence of CKII activity has one important implication for the function of CKII during the cell cycle (11), namely that the crucial cell cycle targets of CKII are not dephosphorylated in an obligatory manner at each cell cycle. This implies that CKII is not part of the cell cycle oscillator per se. An important caveat of course is that CKII activity is rapidly and fully inactivated in cka1ts strains.
The phenotype of the cka1ts alleles described here shares many features in common with that of orb5 alleles of S. pombe (17). Both types of mutant undergo several cycles of cell division prior to arrest, arrest is accompanied by a loss of viability, and arrested cells are spherical and have a non-polarized actin cytoskeleton. The tubulin cytoskeleton of orb5 strains is disorganized as well. Coupled with the cell-cell aggregation common to CKII mutants in both organisms, these similarities suggest that the physiological role of CKII in S. cerevisiae and S. pombe may be better conserved than was previously apparent. However, the mechanism underlying the development of the apolar phenotype appears to be different in the the two cases. Analysis of double mutants in S. pombe indicates that orb5 strains remain polarized but become deficient in the re-establishment of polarized growth following cytokinesis. In this model, the spherical morphology of orb5 strains is generated by continued rounds of cell division in the absence of growth, until the cell is approximately spherical and too small to divide again. The observed cytoskeletal disorganization is either illusory, due to the difficulty of identifying a polar distribution in a small spherical cell, or secondary. However, this model cannot, even in principle, explain the spherical morphology of S. cerevisiae cka1ts mutants, first, because growth is a prerequisite for cell division (budding) and, second, because the absence of growth cannot convert an oval cell into a spherical one. As noted above, our results suggest that the primary defect in cka1ts strains is a loss of cell polarity and that the spherical morphology is generated by subsequent isotropic growth. We do not at present have an explanation for the apparent difference in mechanism between the two systems.
CKII is also implicated in cell polarity in mammalian cells. Treatment of cultured neuroblastoma cells with antisense oligonucleotides directed against the CKII catalytic subunit inhibits serum deprivation-induced neuritogenesis, and this inhibition is correlated with the dephosphorylation of the microtubule-associated protein MAP1B at sites that are phosphorylated in vitro by purified CKII (47). The potential involvement of the tubulin cytoskeleton may be relevant to the orb5 phenotype in S. pombe, since disruption of microtubules affects cell morphology in S. pombe, and the microtubule cytoskeleton is perturbed in orb5 strains (17). In contrast, it appears unlikely that such an explanation underlies the loss of cell polarity in cka1ts strains since polarized growth is predominantly if not exclusively a function of the actin cytoskeleton in budding yeast (37, 38). Thus, although an effect of CKII on polarity is observed in phylogenetically diverse systems, the underlying mechanism may not be identical in all cases.
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ACKNOWLEDGEMENTS |
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We thank Michael Bender for use of the fluorescence microscope; Kelley Dawe for access to the DeltaVision System; Paul Fawcett for assistance in processing image files; Jeff Cole for assistance with some of the strain constructions, and Sricharan Bandhakavi for helpful discussions. We thank Erfei Bi and John Pringle for multicopy plasmids carrying CDC24 and -42, and we also thank Alan Bender for boi1 and boi2 strains and multicopy plasmids carrying BEM1, BOI1, and BOI2.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM33237 and American Cancer Society Grant VM-19.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.
Present address: Dept. of Biology, Merrimack College, North
Andover, MA 01845.
§ To whom correspondence should be addressed. Tel.: 706-542-1769; Fax: 706-542-1738; E-mail: glover{at}bscr.uga.edu.
1 The abbreviations used are: CKII, casein kinase II; DAPI, 4',6'-diamidino-2-phenylindole; 5-FOA, 5-fluoroorotic acid.
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REFERENCES |
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