Plant Physiol. (1996) 1 1 2: 99-1 04
Salt-Sensitive Mutants of Chlamydomonas reinhardfii
lsolated after lnsertional Tagging’
Rafael Prieto, José M. Pardo, Xiaomu Niu, Ray A. Bressan, and Paul M. Hasegawa*
Center for Plant Environmental Stress Physiology, 1 165 Horticulture Building, Purdue University,
West Lafayette, Indiana 47907-1 165
~
~
~
adaptation: to isolate genes that are differentially upregulated by NaCl or osmotic stress or to characterize the
expression and function of genes that, based on physiological or biochemical rationale, are likely to be involved in
NaCl tolerance (e.g. ATPases). However, the majority of
genes that have been identified encode proteins that are
essential for housekeeping functions, are apparently required to a greater extent during stress adaptation, or are
induced as part of the general stress response and have
little orno likelihood of being involved in NaCl adaptation
(Serrano and Gaxiola, 1994; Bohnert et al., 1995). We believe, as others have suggested (Haro et al., 1993; Serrano
and Gaxiola, 1994), that based on results from previous
efforts alternative strategies for identifying molecular determinants of plant adaptation to NaCl should be pursued
if one is to realize the goal of obtaining crops with sustained harvest indices in saline environments.
One alternative strategy is to identify NaC1-tolerance
determinants by selecting stress-sensitive mutants and isolating the wild-type allele of the mutant gene. This approach allows direct identification of the genes that alter
NaCl stress responses, since it does not screen for genes
solely based on expression that is induced as a result of
stress imposition. The unicellular alga Chlamydomonas reinkardtii is a logical organism on which to implement this
research approach. ChZamydomonas is a plant model system
that is particularly amenable for research on cellular processes with the genetic advantages of efficient identification of recessive mutants, intricate genetic dissection of
mutants, map-based cloning, etc. (Ferris and Goodenough,
1994). Furthermore, efficient transformation of CkZamydomonas has facilitated the use of insertional tagging methods
to generate mutants, to isolate functional genes, and, subsequently, to complement mutant phenotypes (Schnell and
Lefebvre, 1993; Tam and Lefebvre, 1993).
In this research, we report the isolation of the NaClsensitive Chlamydomonas mutants by insertional mutagenesis with a Nitl-containing plasmid. A11 mutants
are defective in a single recessive gene (or closely linked
genes). Genetic analyses indicated that four of the mutants are defective in independent loci, three resulting in
Na+ (and Li+) sensitivity ( s o d l , sod2, and sod3) and one
resulting in Li+ (but not Na+) sensitivity ( l i t l ) . The s o d l ,
sod3, and litl mutations are linked to a functional copy of
N i t l . Growth analyses on media supplemented with different osmotica indicated that Sod2 and Sod3 are involved in Na+ and K+ homeostasis; S o d l functions in
~
We describe the isolation of salt-sensitive Chlamydomonas reinhardtii mutants by insertional mutagenesis using the nitrate reductase ( N i t l )gene. The plasmid pMN24, containing Nitl, was used for
transformation of 305CW15 (nitl cw75 mt+), and transformants
were selected for complementation of the nit- phenotype. From
6875 nit+ colonies, four transformants (S4, S18, S46, and S66) were
isolated that exhibited both Na+ and li+ sensitivity (sod-), and
another transformant (S33) was selected that exhibited sensitivity to
Li+ but not Na+ (lit-) based on relative growth comparisons with
the wild-type strain. S33, S46, and S66 were no more growth
inhibited by sorbitol than was 305CW15. In comparison, S4 and S18
exhibited substantial growth inhibition in medium supplemented
with sorbitol. Cenetic analyses indicated that the salt-sensitive mutants were each defective in a single recessive gene. The mutant
genes in S4 (sodl), S33 (litr), and S66 (sod3) are linked to a
functional copy of Nitl and are presumably tagged with a pMN24
insertion.
Environmental stresses are among the most limiting factors to plant productivity; salinity is one of the most detrimental (Boyer, 1982).The deleterious consequences of salt
in plant cells result from hyperosmotic shock and ionic
disequilibria that are caused by high concentrations of ions
in the externa1 solution (Niu et al., 1995). When salinity
results from excess NaC1, the homeostasis of not only Na+
and C1- but also K+ and Ca2+ is disturbed. Plant survival
and growth are very dependent on adaptations that expeditiously reestablish ionic homeostasis, thereby minimizing the duration of cellular exposure to the disequilibria of
ions. Among the adaptations that presumably affect ion
homeostasis are transport processes that control ion fluxes
across the plasma membrane or facilitate vacuolar ion compartmentation and regulatory molecules in NaC1-signaling
cascades that modulate these processes (Binzel et al., 1988;
Niu et al., 1995). Consequently, we assume that the genes
that are essential for the establishment and maintenance of
intracellular ion homeostasis are among the genes that will
increase the NaCl tolerance of plants.
To date, two general approaches have been used most
extensively to identify the molecular determinants of NaCl
This research was supported by a Ministerio de Educacion y
Ciencia de Espaiía fellowship to R.P. This is journal article no.
15021 of the Purdue University Agricultura1 Experiment Station.
* Corresponding author; e-mail hasegawaQsage.cc.purdue.edu;
fax 1-317-494-0391.
99
Prieto
etal.
Plant Physiol. Vol. 112, 1996
RESULTS
is
or
on
and
not
was
2).The
in
of
the
or
I
in
to
to
of
by
ion
astheCl~or
the
dinot
butno
or
inthe
at
not
KNO,
NH4C1
and
The
asthe
10mM
and
asthe
and0.5gof
Chlamydomonas cells were transformed with pMN24 using the procedure described by Kindle (1990). 305CW15
cells (5 X 107) were vortexed in the presence of 1 /xg of
pMN24 DNA, PEG-8000, siliconized glass
beads (1 mm in diameter) for 30 s. Cells were spread onto
solid medium containing NO3~ sole nitrogen source
nit"1" colonies were isolated. nit"1" transformants
were screened for their capacity to grow on solid medium
containing NH4C1 nitrogen source supplemented with either 150 mM NaCl or 30 HIM LiCl.
5%
and
to
or30mM
and
1).The
hadn
an
theionwas
ofthe
orn
and
in
Transformation of Chlamydomonas and Isolation of Nitl
Insertional Mutants
to
and
a
for
The
ina
305as
PAR
at
of
the
not
is
was
ous
knl
DNA
and
as
of
Chlamydomonas reinhardtii strains 6145c (mt~), 21gr (mt+),
and 305CW15 (nitl cw!5 mt+) were kindly supplied by Dr.
Emilio Fernandez (Department of Biochemistry and Molecular Biology, University Cordoba, Spain) have been
characterized previously (Sosa et al., 1978; Fernandez and
Matagne, 1984, 1986; Harris, 1989). The nitl mutation in the
nitrate reductase structural gene prevents 305CW15 from
using NO3~ nitrogen source, cw!5 confers cellwall-less phenotype that facilitates uptake transformation. Strain 305 (nitl mt~) was isolated from nit~
segregants of the cross 305CW15 X 6145c (mt~), and 305d
(nitl mt~) isolated from strain spontanekanamycin-resistant mutant that defective single
dominant nuclear gene (data shown). pMN24 plasmid, which contains Chlamydomonas nitrate reductase
gene (Nitl) (Fernandez et al., 1989), was provided by Dr.
Emilio Fernandez (University Cordoba). Cells were
grown 26°C under saturating either liquid
agar-solidified minimal medium containing 10 mM NH4C1
or 4 mM KNO3 as the nitrogen source (Harris, 1989). Cell
growth was estimated in liquid medium from optical density measurements (Harris, 1989) or by determination of
chlorophyll content (Arnon, 1949; Prieto Fernandez,
1993).
150mM
asthe
and
and
Chlamydomonas Strains, Culture Media, Growth
Conditions Plasmids
After the transformation of 305CW15 with pMN24, 6875
nit"1" colonies were isolated. These transformants were
screened their capacity grow solid medium containing NH4+ nitrogen source supplemented
with either NaCl LiCl. These concentrations of NaCl and LiCl were not lethal to 305CW15 cells but
were growth inhibiting. Four isolates (S4, S18, S46, and S66)
exhibited Na + - Li+-sensitive phenotype (sod*)
another (S33) Li + - Na+-sensitive (lit")
phenotype (Fig. other nit"1" transformants
exhibit any greater growth reduction in medium supplemented with NaCl LiCl than parental strain.
Strain 305CW15 was more sensitive to K"1" than to Na+
(whether included medium
the NO3~ salt) than two other wild-type strains (21gr or
6145c), particularly high concentrations (data
shown). Relative sensitivities of 305CW15 and the mutants
NaCl were determined analyses growth media
containing different concentrations NaCl (Fig.
growth of S4, S18, S46, and S66 was substantially inhibited
by NaCl, and, relative 305CW15, growth reduction
more pronounced at higher NaCl concentrations. Two
other nit+ isolates (e.g. SO and S2) that exhibited a slow
growth phenotype on minimal medium relative to the
parental strain were sensitive NaCl LiCl (data
shown). S33 was inhibited by NaCl to a comparable extent
as 305CW15.
Analyses data illustrated Table confirmed
sod" phenotype of S4, SI8, S46, and S66 when either Cl~ or
NO3~ was the anion. These mutants also exhibited some
for
MATERIALS AND METHODS
to
Isolation Insertional Mutants Sensitive NaCl LiCl
of
and
osmotic homeostasis, Litl involved more specifically in Li+ homeostasis.
not
100
and
Genetic Analyses Complementation Test
and
NH4C1 LiCl
+30mM
ofthe
NH 4 Cl+150mMNaCl
and
In
and
54,
150mM
mM
the
are
or
theopand
for
or
Figure 1. Nitl insertional mutants of C. reinhardtii are sensitive to
NaCl LiCl. Comparisons between nit" parental strain
305CW15 and strains obtained by insertional mutagenesis of Nitl
after screening NaCl LiCl sensitivity. each plate starting
at moving clockwise: 305CW15, S18, S33, S46,
S66. Illustrated are plates after 1 week of growth on medium supplemented with 4 mM NO3~ (A), supplemented with 10 mM NH4+
(B-D), supplemented with NaCl (C), supplemented with
30 LiCl (D).
and
or
150mM
to
was
20/ng
mM
on
Thesodanlit
and
Genetic analyses were performed by the random sporeplating method (Levine Ebersold, 1960; Fernandez
Matagne, 1984). phenotypes transformants were evaluated minimal medium containing
10 NH4C1 supplemented with either NaCl
30 mM LiCl, respectively. Complementation and segregation analyses were conducted on diploids isolated from
crosses between 305d (nit~ kn R ) and each of the mutants
(Ebersold, 1967; Fernandez and Matagne, 1984). Minimal
medium containing NO3~ as the nitrogen source and supplemented with mL"1 kanamycin used select
for diploid genotypes.
1 o1
Salt-Sensitive Mutants of Chlamydomonas reinhardtii
30
O
25
50
75
100
125
NaCl
(mM)
Chlamydomonas strains exhibit different
sensitivities to NaCI. Cells of 305CW15, sod- (S4, S18, S46, and
S66), or lit- (S33) strains in exponential growth were harvested and
transferred to media containing O, 25, 50, 75, 100, or 125 mM NaCI.
Figure 2. The sod- and lit-
Cell m a s throughout the growth cycle was monitored by AG6,,determinations (Harris, 1989). lllustrated are doubling times determined from a minimum of five absorbance measurements made
during the exponential growth phase. A, Absolute doubling times; B,
relative doubling time, where 1 .O is growth in medium without NaCI.
0,
305CW15; U,S4; O, S18; A, S33; A, S46; and O, S66.
sensitivity to K+, particularly when added as KNO,. S4 and
S18 were also sensitive to isosmotic concentrations of sorbitol. Consequently, it seems that S46 and S66 are ionsensitive mutants, whereas S4 and S18 are more sensitive to
osmotic stresses. The lit- mutant S33 was not sensitive to
other cations or sorbitol.
Genetic Analyses of sod and lit Genotypes
Genetic crosses between the mutants and strain 305d
(knl m t - ) were made to determine the number of genes
involved in the sod- or lit- phenotype and the linkage of
these with N i t l . No sod- or lit- segregants emerged from
the cross 305CW15 X 305d (Table 11) or sod- segregants
from S33 x 305d (data not shown); therefore, the sod- or
lit- phenotype was not due to differences in the genetic
background of the two parenta1 strains. Segregation ratios
of approximately 1:l for sod- or lit- for progeny of the
crosses between 305d and either S4, S18, S46, and S66
(Table 11) or 533 (Table 111), respectively, indicated that a
single locus or closely linked loci were involved. A mutation in one locus (or closely linked loci) is responsible for
both Na+ and Li+ sensitivities in S4, S18, S46, and S66,
since both phenotypes co-segregatedin the progeny resulting from the crosses of the mutants (data not shown). The
existence of a low frequency of nit+ sod+ (Table 11) or nit+
lit+ (Table 111) but not nit- sod- or nit- lit- recombinants
among the progeny from the crosses between 305d and S4,
566, or S33 indicated that there is more than one linked
functional Nitl gene in these mutants; however, at least one
of these co-segregates with the sod- or lit- phenotype. In
the crosses S18 X 305d and S46 X 305d a low but significant
number of both nit+ sod+ and nit- sod- recombinants
were recovered (Table 11). The lack of linkage to a functional copy of Nitl in S18 and S46 segregants is most
probably because Nitl either is no longer inserted in the
Sod gene or has been inactivated.
Genetic crosses between the different mutant strains
were performed to determine whether the mutations are
allelic (Tables I1 and 111). Segregation ratios of approximately 1 sod+:3 sod- from the crosses involving S4, S46,
and S66 indicated that these strains are defective in three
unlinked loci, Sodl, Sod2, and Sod3, respectively (Table 11).
Similarly, the approximate 1 litf:3 lit- in crosses that included S33 means that this phenotype is mediated by a
single gene ( L i t l )unlinked to the Sod genes (Table 111). The
mutations causing the sod- phenotype in S4 and S18 are
allelic for Sodl, since no sod+ recombinants were recovered
from the cross (Table 11). Diploids carrying both wild-type
and mutant loci for sod or Zit (isolated from crosses with
305d) had the wild-type phenotype, indicating that a11
sod- and lit- phenotypes are recessive (data not shown).
DISCUSSION
In this report, we describe the isolation of Na+- and
Li+-sensitive Chlamydomonas mutants after insertional mutagenesis using the Nitl gene. Genetic analyses indicate
that the sod- phenotype of three mutants is due to unique
mutations in unlinked loci, Sodl, Sod2, and sod3, and the
lit- phenotype of another is due to a mutation in a separate
locus, Litl. The S o d l , Sod3, and Litl mutations are each
linked to a functional copy of the Nitl gene. This approach
of insertional mutagenesis has been used successfully to
isolate Chlamydomonas genes required for flagellar assembly and function (Tam and Lefebvre, 1993) and nitrate
assimilation (Schnell and Lefebvre, 1993). From the sequences flanking Nitl, probes can be synthesized and used
to isolate the wild-type allele. Since the mutations are
recessive, it is presumed that the wild-type gene will complement the mutant phenotype; i.e. the wild-type gene is a
functional determinant of NaCl tolerance. Sequence
matches of the gene product to homologs in other species
will provide insight about its function in NaCl adaptation.
Elucidation of the biochemical or physiological defect that
is the basis for NaCl sensitivity of a particular mutant may
further the identification of the process that is mediated by
the gene product. If we assume that insertion of exogenous
DNA into the 100-Mb Chlamydomonas genome occurs ran-
Prieto et al.
102
Plant Physiol. Vol. 11 2 , 1996
Table I. Growth characteristics of Chlamydomonas strains in response to osmotic and ionic stresses
Ten microliters of cell suspension (between 2 and 4 pg of chlorophyll) was inoculated onto solid medium. After 1 week, cells were harvested,
and the chlorophyll content of the cells was determined (Prieto and Fernandez, 1993). Chlorophyll content has been used as an indicator of
Chlamydomonas cell biomass. Wild type (305CW15) or mutant strains (S4, S18, S33, S46, or S66) were inoculated onto minimal medium with
4 mM KNO, or 1 O mM NH,CI supplemented with 100, 150, or 200 mM NaCI, KCI, NaNO,, or KNO,; 150, 225, or 300 mM sorbitol;or 1O, 20,
or 30 mM LiCI. The corresponding osmolalities of the solutes are listed in the first column. The data are expressed as chlorophyll content relative
to that of cells grown on medium with 10 mM NH,CI. The absolute chlorophyll values ( p g chlorophyll/mL) of cells grown on medium with NH,
as the nitrogen source were: 305CW15, 10.9; S4, 76.6; S18, 81.7; S33, 69.4; S46, 69.6; and S66, 95.
Osmotic and lonic Stress
305CW15
4 mM KNO,
10 m M NH,CI
<5
1o0
NaCl (mosmol)
s4
185
278
3 70
68
43
34
29
1o (S)"
<5
185
278
370
50
15
6
45
14 (T)b
19
37
56
96
91
93
62
45
24
150
225
300
69
58
40
44
31 (SI
16
183
275
362
53
23
7
12
<5 6)
<5
181
264
344
38
6
<5
14
<5 ( S )
<5
KCI (mosmol)
LiCl (mosmol)
Sorbitol (mosmol)
S18
s33
S46
S66
106
1O 0
<5
6)
NaNO, (mosmol)
KNO, (mosmol)
a
(S), Sensitive.
(T), Tolerant.
(S/T), Intermediate.
domly, that there is one integration event per transformant,
and that the average size of a gene is 5 kb (Adam et al.,
1993), the mutation frequency observed in this study is
consistent with the existence of 10 to 12 loci in the Cklamydomonas genome that are involved in NaCl and osmotic
stress tolerance.
The data in Table I indicate that Nat and Lit tolerance is
mediated by Sodl through more general osmotic (rather
than ion-specific) homeostasis processes, since S4 (or S18)
is equally sensitive to isosmotic levels of sorbitol and NaC1.
However, S4 also has characteristics of an ion-sensitive
mutant, since levels of Li', which would not constitute a
substantial osmotic stress (less than 56 mosmol), are
growth inhibitory. Sodl may be involved in osmoregulatory processes required for osmotic adjustment, such as the
synthesis or the accumulation of glycerol in the cytosol
(León and Galván, 1995). Recently, it has been determined
that genes involved in glycerol biosynthesis and Na+ / Lit
efflux are activated by both ionic and nonionic osmotic
signals, implying that components of the regulatory pathways are shared (Hirayama et al., 1995; Márquez and Serrano, 1996). A mutation in a gene encoding one of these
molecules would result in the sodl phenotype of sensitivity
to both nonionic and ionic osmotic agents. It is also possi-
ble that Sodl has a role in volume regulation, since a defect
in this process would make a cell extremely sensitive to
osmotic perturbation.
L i t l , SodZ, and Sod3 are more likely involved in attenuating ion toxicity, but the lack of Naf sensitivity of S33
implies that the Litl product does not function in ion
transport. Since Nat and Li+ are probably transported by
common systems, a transport mutation should affect sensitivity to both ions. Presumably, S33 is lit- because Litl
encodes a Li+-sensitive component of metabolic machinery
that is necessary for NaCl adaptation or functions in intracellular Li+ detoxification. A possible scenario can be envisaged using as examples the rice (RHL) and Arabidopsis
thaliana ( S A L 1 ) homologs of yeast HALZ (Glaser et al., 1993;
Murguía et al., 1995; Peng and Verma, 1995; Quintero et al.,
1996). These genes encode products that increase the tolerante of yeast to Na+ and Li+. Apparently, the 3'(2'),5'bisphosphate nucleotidase activity of this enzyme functions in the control of sulfate activation, which is
apparently essential during NaCl stress adaptation (Murguia et al., 1995; Peng and Verma, 1995). Furthermore, the
enzyme has inositol polyphosphate 1-phosphatase activity,
implicating an involvement in the phosphoinositidesignaling pathway that could result in the activation of the
Salt-Sensitive Mutan ts of Chlamydomonas reinhardtii
103
Table II. Segregation data from genetic crosses o f sodium-sensitive strains o f Chlamydomonas
Phenotypes were scored in media with KNO, (nit) and 150 mM NaCl (sod). NH,CI was used as the nitrogen source in media containing NaCI.
lllustrated are the number of recombinants assayed and the segregation ratios. Strains S18d (mt-), S46d (mt-), and S66d (mt-) were isolated as
nit+ sod- segregants of the crosses between 305d (knl, mt-) and S18 (mt+), and S46 (mt+) and S66 (mt+), respectively.
Cenetic Cross
305CW15 X 305d
S4 X 305d
S18 X 305d
S46 X 305d
S66 X 305d
S4 X S46d
S4 X S18d
S4 X S66d
S66 X S46d
a
Segregation
Recombinant
nit+ sod-
nit-:nit+
sod+ :sod-
189:O
(1 :O)
124:134
(1 :1 .08)
67:80
(1 :1.19)
73:70
(1 :0.96)
9 6 9 08
(1 :1.13)
18:75
(1 :4.17)
13:60
(1 :4.62)
22:76
(1 :3.45)
27:80
(1 :2.96)
189:O
(1 :O)
136:122
(1 :0.90)
73:74
(1 :1.01)
78:65
(1 :0.83)
1O1 :103
(1 :1.02)
24:69
(1 :2.88)
0:73
122
(P)
65
( P)
64
(P)
103
( P)
66
(0:l)
(P)
26:72
(1 :2.77)
33:74
(1 :2.24)
O
nit- sod+
nit+ sod+
189
O
O
12
O
15
9
6
1
5
O
9
3
O
O
13
22
4
O
25
8
2
nit- sod-
(P)"
124
(P)
58
(P)
72
(P)
96
( P)
15
(P)
60
72
(P)
72
(P)
(P), Parental phenotype.
Na+ efflux system (Quintero et al., 1996). RHL and SAL1
are relatively insensitive to Na+ but are very sensitive to Li+
(Quintero et al., 1996). Any mutation that would reduce the
production of this enzyme or increase its sensitivity to Li+
would result in the phenotype exhibited by S33.
S46 and S66 are sensitive to Na+ and Li+ but not to
sorbitol. Thus, Sod2 and Soa3 seem to be involved in the
maintenance of ion homeostasis (Niu et al., 1995). Since
vacuolar compartmentation is not likely to be a significant
process for Na+ homeostasis in Chlamydomonas (Guillard,
1960; Reynoso and de Gamboa, 1982; Malhotra and Glass,
1995b), Sod2 and Sod3 likely function in the regulation of
Na+ flux across the plasma membrane, i.e. transport processes that mediate K+ acquisition (and control Na+ influx)
or Na+ efflux (Haro et al., 1993; Nakamura et al., 1993;
Mendoza et al., 1994). From our data, we were unable to
establish whether S46 or S66 have reduced capacity for K+
acquisition, analogous to the NaC1-sensitive mutant of A.
thaliana that is defective in high-affinity K+ transport (Wu
et al., 1996). Both high- and low-affinity K+ transport systems have been identified in Cklamydomonas that mediate
acquisition of this cation (Malhotra and Glass, 1995a,
199510). However, the Chlamydomonas trkl mutant (Polley
and Doctor, 1985), which is unable to grow on media with
low K+ levels (100 p ~ )is, apparently defective in a K + / H +
symporter that is required to flux the cation from the
chloroplast to the cytosol (Malhotra and Glass, 199513).The
chloroplast represents a K+ sink in Chlamydomonas that
contributes substantially to the capacity for sustaining
growth at low externa1 K+ levels. Alternatively, Sod2 or
Soa3 could be a co-transporter or a Na+ pump that effluxes
the cation across the plasma membrane against a thermodynamic gradient (Niu et al., 1995). Perhaps the most interesting possibility is that Sod2 or Sod3 encodes a regula-
Table 111. Segregation data from the genetic crosses o f the lithium-sensitive strain o f Chlamydomonas
Phenotypes were scored in media with KNO, (nit) or NH,CI supplemented with 30 mM LiCl (lit). Strains S4d (mt-), S46d (mt-), and S66d (mt-)
were isolated as nit+ sod- segregants of the crosses between 305d (knl, mt-) and S4, S46, and S66, respectively. lllustrated are the number of
recombinants assayed and the segregation ratios.
Cenetic Cross
S33 X 305d
S33 X S4d
S33 X S46d
S33 X S66d
a
(P), Parental phenotype.
Segregation
Recombinant
nit-:nit+
lit+:lit-
nit+ lit-
nit- lit+
nit+ lit+
nit- lit-
81 :91
(1:1.12)
16:62
(1 :3.88)
2357
(1 :2.48)
24:72
(1 :3.0)
86:86
(1 :1)
21 :57
(1 :2.71)
19:61
(1 :3.21)
30:66
(1 :2.20)
86
(P)"
57
(P)
54
(P)
66
( P)
81
( P)
16
5
O
5
O
16
3
7
24
6
O
Prieto et al.
104
tory molecule that, like yeast calcineurin, is p a r t of a signal
cascade that regulates Na+ influx a n d efflux across t h e
plasma m e m b r a n e (Haro e t al., 1993; N a k a m u r a e t al., 1993;
Mendoza e t al., 1994).
ACKNOWLEDCMENTS
We would like to thank Drs. Satomi Takeda and Emilio Fernández and members of the Hasegawa and Bressan laboratories for
technical and intellectual input throughout the course of this
research.
Received February 29, 1996; accepted May 31, 1996.
Copyright Clearance Center: 0032-0889/96/ 112/0099/06.
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