From the University of Nice, INSERM U470, 06108 Nice, France
Received for publication, July 24, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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We identified a new member of the phosducin-like
(PhLP) protein family that is predominantly, if not exclusively,
expressed in male and female germ cells. In situ analysis
on testis sections and analysis of purified spermatogenic cell
fractions evidenced a stage-specific expression with high levels of RNA
and protein in pachytene spermatocytes and round spermatids. Three
mRNA species were detected, which correspond to different
polyadenylation sites and vary in abundance during germ cell
maturation. Only low levels of RNA were detected in whole ovary
extracts, but expression of the protein became detectable within hours
after hormonal induction of superovulation. The gene
(Mgcphlp) is located on mouse chromosome 5 in the immediate
vicinity of the Clock locus. The predicted amino acid
sequence shows extensive similarities not only with the known mammalian
PhLP proteins but also with the yeast phosducin-like protein Plp2,
required for the production and growth of haploid cells. Expression of
the murine protein was found to complement the defect of a yeast
plp2 Phosducin, a protein highly expressed in the retina and pineal
gland, has been considered as playing a role in retinal
phototransduction by interacting with the The mammalian phosducin and phosducin-like proteins are expressed
ubiquitously (5). Whether tissue-specific homologues exist remains an
open question. In the course of screening a mouse testis cDNA
library, we have identified a novel germ cell-specific phosducin-like
protein, designated MgcPhLP (for "mouse germ cell-specific phosducin-like protein"), which exhibits significant similarities to
both the mouse phosducin and phosducin-like proteins. Expression is
strictly restricted to the male and female germ cells in a regulated
manner depending on the stage of germ cell maturation. The murine gene
complemented the defect of a yeast plp2 Mice--
Mice used in all experiments were C57BL/6 × DBA/2 F1. Eggs were collected from either naturally ovulated females or
after hormonal induction of superovulation according to standard
procedures (6).
Cell Cultures and Preparation of Germ Cells--
Cultures of the
15P-1-established Sertoli cell line and primary Sertoli cell cultures,
preparation of total germ cells, and fractionation by elutriation
centrifugation were performed as described previously (7).
RNA Analysis--
Total RNAs were extracted and analyzed by
Northern blot hybridization as described previously (7). Dot blot
hybridization was performed on Mouse RNA MasterblotTM (Clontech).
DNA Sequencing--
Purified PCR products were first subcloned
in pGEM-T Easy vector System I (Promega, catalog no. A1360). The
purified plasmids were then sequenced with a DNA sequencing system kit
(Big Dye Terminator, PerkinElmer Life Sciences, catalog no. 4303149)
according to manufacturer's instructions. Sequences were analyzed with
the ABI PRISM 310 genetic analyzer (PerkinElmer Life Sciences).
Reverse Transcription and PCR Amplification--
Quantitation of
RNA amounts in cell extracts was done by comparison with
Gapdh reverse transcripts amplified as an internal standard
in the same reaction mixture. Total RNA (1 µg) prepared using the
total RNA isolation kit (Roche Molecular Biochemicals) was
reverse-transcribed using Moloney murine leukemia virus (MuLV) reverse transcriptase according to the supplier's instructions. PCR
amplification was performed using Taq DNA polymerase (Roche Molecular Biochemicals). The following pairs of oligonucleotide primers
were used (RACE and polymerization conditions according to the
supplier's instructions; Roche Molecular Biochemicals). 5' end
analysis: RT, Rev0, 5'-gccagtgtcatcttctcatatgg-3'; first round, Rev-1,
5'-ggcctcctgctgtaagcg-3' plus AAP from kit; and second round of PCR,
Rev-2, 5'-gcgcaacaccatctcttc-3' plus AUAP from kit. 3' end analysis: RT
with oligo(dT), first round, E11, 5'-gttggagcgatacagagtg-3' plus
AAP from kit; and second round of PCR, E12, 5'-cattgcagatatgatggtg-3'
plus AUAP from kit. The 3'-RACE products were then isolated after
hybridization with oligonucleotide primer F01,
5'-gtgacactgaggctaagtag-3'. To routinely follow Mgcphlp expression in testis and ovary, two pairs of oligonucleotide primers (common to the three isoforms) were used, namely Phos-1
(5'-ctggaaatcagtatgtgaatg-3') and Phos-2 (5'-ctgtatcgctccaacttctga-3'),
and Mgc271 (5'-cggttacaggaatggaaagc-3') and Mgc483
(5'-atgcagctattcacgatggc-3'). In yeast DNA, detection of the
PLP2 wild type allele was performed using primers Plp2-s (5'-gataaagacttgtcggatttgg-3') and Plp2-r
(5'-gtaatgcaattttctctcttccc-3'); and detection of the
URA3::plp2,plp2 In Situ Hybridization--
In situ hybridization was
performed as described previously (8) using single-stranded
digoxigenin-labeled RNA probes corresponding to sense or antisense
Mgcphlp messengers.
Purification of the MgcPhLP Protein--
Mgcphlp
cDNA was inserted into the pGEX-5X vector (Roche Molecular
Biochemicals) by taking advantage of a BamHI site in the amino-terminal region (amino acid 5), generating a GST
(glutathione S-transferase) fusion gene in which expression
was induced in Escherichia coli cultures during overnight
growth at 25 °C in the presence of 0.1 mM
isopropyl-1-thio- Immunological Techniques--
GST-MgcPhLP fusion protein,
prepared bound to glutathione-Sepharose beads as described above, was
eluted by phosphate-buffered saline containing 20 mM
reduced glutathione and used to raise antiserum in rabbits. The
resulting antiserum recognized both GST and MgcPhLP in Western blot
analysis (data not shown). Specific antibodies, prepared by depleting
the rabbit serum with Sepharose beads carrying the GST protein, were
used as primary antibodies in immunofluorescence and Western
immunotransfer analyses.
Western transfers were obtained from mouse organs minced and boiled in
Laemmli's buffer by a standard procedure (9); and mouse oocytes were
collected and treated in the same way. Secondary antibody was a mouse
monoclonal anti-rabbit IgG Fc fragment antibody coupled to horseradish
peroxidase (Sigma). Staining was performed by the ECL technique
(Amersham Pharmacia Biotech).
Immunoprecipitation of 14-3-3 and associated proteins was performed
using 14-3-3 Yeast Strain, Media, and Plasmid--
The mutant S. cerevisiae strain
(plp2::URA3,plp2 A New Phosducin-like mRNA--
During the course of an
unrelated expression-based screen of a mouse testis library, we
identified a cDNA uniquely expressed in germ cells. Fig.
1 shows 818 base pairs of cDNA
sequence with an open reading frame of 240 amino acids, encoding a
putative protein of 27.824 kDa, designated MgcPhLP, which exhibits
30-36% amino acid identity to the mouse and bovine phosducins and to the rat phosducin-like protein in a 143-amino acid core region (Fig.
2). Important amino acid residues
conserved between the phosducin and phosducin-like proteins of various
species (human (11), rat (12), mouse (13)) are also conserved in
MgcPhLP protein. As documented in more detail under
"Evolutionary Conservation," significant similarities were also
detected with two phosducin-related proteins of the yeast S. cerevisiae.
A computer search indicated that in the mouse genome, the
Mgcphlp gene is located within a 204-kb segment of
chromosome 5 (GenBankTM accession number AF146793),
which also includes the Clock locus and has been designated
Pdcl2 (GenBankTM accession number AAD30564). The
existence of two potential proteins, named protein B and PDCL2
(corresponding, respectively, to amino acids 3-204 and 3-195 of
MgcPhLP) had been predicted from the genomic DNA sequence. The complete
nucleotide sequence derived from the initial cDNA clone and the 5'-
and 3'-RACE products now allows us to determine the correct position of
the initiating methionine and thus the complete amino acid sequence.
An analysis of mouse EST libraries showing identities with
Mgcphlp sequences (Unigene cluster Mm.143764 plus additional
clones) strongly suggested that the gene was at least preferentially
expressed in the testis. All identities were found in testis-derived
library sources and none among clones derived from other tissues, with the best match found in a purified spermatocyte library
(BG100925).
Expression of Mgcphlp in the Testis--
Northern blot analysis on
a limited series of tissues detected expression only in the testis
(Fig. 3A). A larger screen
performed by dot blot analysis on a membrane spotted with poly(A)+ RNA
from 22 mouse organs (Fig. 3B) again showed expression only
in the testis. Mgcphlp RNA was not detected in any of the
somatic organs tested. The same conclusion was reached using the most
sensitive RT-PCR assay, which could not detect Mgcphlp RNA
sequences in two somatic tissues (Fig. 3C). The RNA was
present in purified germ cells (see below and Fig. 5) but not in
either Sertoli cells, since primary cultures were negative (Fig.
3D), or in cells of the Sertoli line 15P-1 (not shown).
Although expression of the gene cannot be excluded either in yet
another tissue, or in a minor fraction of cells, possibly under
specific physiological conditions, it would appear to occur mostly in
testicular germ cells. In fact, as shown in a subsequent section,
Mgcphlp is also expressed in female germ cells during a
limited period of meiotic maturation.
In whole testis extracts, three mRNA species were detected.
Analysis of cDNA ends by 5'- and 3'-RACE revealed that these RNAs share the same 5' extremity but differ by their 3' ends. Three major
polyadenylation sites were identified, corresponding to three canonical
polyadenylation signals in the genomic sequence (Fig.
4). As shown in Fig. 3D,
distribution of the three isoforms significantly differed between the
pachytene spermatocyte, the round spermatid and the elongated spermatid
fractions prepared by elutriation centrifugation. During post-natal
development, RNA was first detected at 20 days post-partum, and
the maximum level of expression was reached at day 30. The midsize
mRNA was the only one identified at day 20, whereas the other two
isoforms appeared only at the later time points.
Stage-specific expression in the testis was confirmed by in
situ hybridization. The probe consisted of the entire coding
sequence of Mgcphlp and thus detected all three mRNA
isoforms. As shown in Fig. 5, expression
was detected in pachytene to round spermatids at all spermatogenesis
stages.
Expression of MgcPhLP was examined at the protein level by using a
rabbit antiserum raised against a GST fusion protein (see "Experimental Procedures"). Immunofluorescence staining of purified fractions of male germ cells was clearly positive from the meiotic to
late haploid stages of spermatogenesis, and the protein was also
present in the mature spermatozoa of epididymal sperm. It was not
detectable by Western blotting at 10 days post-partum but was
clearly present in the testis at day 18 (Fig. 5, B-F).
Expression in Ovary--
Despite the facts that no EST sequences
corresponding to Mgcphlp were present in ovarian libraries
from various species and that RNA was not detected by dot blot
hybridization (Fig. 3B), low levels of expression were
evidenced by RT-PCR analysis in total extracts from adult ovary (Fig.
6A). Western blot analysis (Fig. 6B) failed to detect expression of the protein in
extracts from either ovary or unfertilized eggs. It was, however,
detected in fertilized eggs (Fig. 6C). Taking into account
the smaller proportion of germ cells in the female gonad, these results
did not exclude the possibility of stage-specific expression during meiotic maturation. This was shown to be the case by Western blot analysis following injection of human chorionic gonadotropin as part of
a superovulation regime (6) (Fig. 6D). The protein was
detected as early as 3 h after hormone injection, a time
corresponding to the nuclear breakdown step of preovulatory
meiotic maturation.
Association with 14-3-3 Protein(s)--
The 14-3-3 family includes
a series of closely related proteins that bind phosphorylated
components of signal transduction pathways and modulate their
interactions (reviewed in Ref. 14). Several of them, prominently the
14-3-3 Evolutionary Conservation--
The Mgcphlp coding
sequence appears to have been relatively well conserved throughout
evolution. Coding regions of the murine gene are 87-92% identical at
the nucleotide level to human EST sequences contained in Unigene
cluster Hs.223712, derived exclusively from germ cells or testes. The
cluster sequences are located in human chromosome 4q11 region, again in
close proximity to the human homologue of the mouse Clock
gene. In the S. cerevisiae genomic sequence, two genes,
PLP1 and PLP2, encode proteins with a clear
similarity to the mammalian phosducins and phosducin-like sequences
(4). Alignments of phosducin-like sequences (Fig. 2) show that the
murine MgcPhLP protein is more closely related to Plp2. One may note
that, in the amino-terminal part of the sequences, an 11-amino acid
region implicated in G Complementation of a Yeast plp2 Mutant by the Murine Mgcphlp
Gene--
A yeast strain bearing mutations in the TRP1 and
URA3 nutritional markers and in which one PLP2
allele has been replaced by URA3
(plp2::URA3,plp2 A Meiotic Function Conserved from Yeast to Mammals?--
It was
initially reported (4) that haploid derivatives could not be grown from
the mixture of meiotic products generated by the diploid heterozygous
genotype (plp2::URA3,plp2 Phosducins are regulators of G protein activity in the retina, and
the phosducin-like proteins are considered to be potential ubiquitous
regulators of G As is the case for the other phosducin-related proteins, the function
of the protein at the molecular level remains largely to be
established. A possibly significant feature in this respect is its
association with at least one of the proteins of the 14-3-3 family.
Binding may be mediated by the RSSVP motif (amino acids 119-123, Fig.
1), which resembles the sites of interaction identified in other
14-3-3-binding proteins (14). 14-3-3 binds phosphorylated serine
residues in a number of proteins active in signal transduction. In
retinal photoreceptors, 14-3-3 is considered as regulating the
binding of phosducin to G Two phosducin-related genes were recently described in yeast,
PLP1 and PLP2 (4). MgcPhLP displays a
greater amino acid similarity with PLP2 than with
PLP1. The inability of a plp2 Mgcphlp is included in the 204-kb DNA fragment of the
Clock locus (GenBankTM accession number
AF146795). Taking into account that phosducin, which is expressed
abundantly in the retina, has been considered to be involved in signal
phototransduction cascades, one might speculate that Mgcphlp
expression could be part of the same type of signaling cascade
initiated by dark/light stimuli. In the mouse, the ovulation cycle is
known to be dependent on light periodicity, and circadian periods have
been reported for the ovarian melatonin and rhythm of cAMP
accumulation. Increase in Mgcphlp expression within hours
after induction of superovulation by human chorionic gonadotropin
injection clearly points to hormonal regulation. It is clear, however,
that beyond such speculations, the function of the mouse protein in
germ cell differentiation will require the use of site-directed and/or
temporally controlled mutagenesis technologies.
mutant. We propose that MgcPhLP/Plp2 proteins exert
a function in germ cell maturation that is conserved from yeast to mammals.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits of G proteins
and thereby modulating their signaling functions (1). A partially
similar, widely expressed phosducin-like protein
(PhLP)1 has been identified,
which also inhibits G
function (2, 3). Two related genes
identified in the yeast Saccharomyces cerevisiae were
designated PLP1 and PLP2 (4). The Plp1 protein was shown to bind efficiently the G
subunits. Binding of Plp2 was
also evidenced but with a lesser affinity. On the other hand, genetic
analysis evidenced the role of Plp2 in the generation of viable haploid cells.
mutant, suggesting an evolutionarily conserved function in meiotic
and/or post-meiotic cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutated
allele was performed using Plp2-s and Ura3-r
(5'-tcatctcttccacccatgtc-3').
-D-galactopyranoside. Bacteria
from 500 ml of culture were washed and extracted in 50 ml of 100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 2 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and 50 µg/ml of both leupeptin and
aprotinin, by sonication (six times for 10 s) at 4 °C.
Extract was clarified by high-speed centrifugation (30 min,
30,000 rpm, 4 °C in a Beckman Ti-50 rotor), and submitted
batchwise to adsorption on glutathione-Sepharose beads (0.50 ml packed
volume, prewashed with extraction buffer) with gentle agitation for
2 h at 4 °C. Beads were washed five times at 4 °C with 1 ml
of 20 mM Hepes, pH 7.5, 1.0 M NaCl, 1 mM EDTA, 2 mM dithiothreitol, 0.1% Nonidet P-40 and four times with proteolysis buffer (20 mM Hepes,
pH 7.5, 100 mM NaCl, 2 mM CaCl2, 2 mM dithiothreitol, 10% glycerol, and 20 µg/ml bovine
serum albumin). Biotinylated factor Xa (Roche Molecular Biochemicals)
was added (10 µg in 1 ml final volume), and reaction was allowed to
proceed for 2 h at 4 °C under gentle agitation.
Phenylmethylsulfonyl fluoride was then added (to a final concentration
of 1 mM), and, after the beads were spun down, the
supernatant was cleared of protease by incubation for 1 h at
4 °C with streptavidin beads (50 µl packed volume, Roche Molecular Biochemicals). The latter were removed by centrifugation, and the final
supernatant was dialyzed against phosphate-buffered saline (400 ml, two
changes, 4 h, 4 °C). The final preparation was analyzed by
SDS-PAGE (see Fig. 6) and stored frozen at
70 °C. In parallel,
control buffer was prepared in the same way using bacteria that
express GST.
(H-8):sc-1657 mouse monoclonal
antibody according to the supplier's instructions (Santa Cruz
Biotechnology Inc.). This antibody reacts with all of the known
proteins of the 14-3-3 family.
) used in this study
was obtained from Dr. H. G. Dohlman (Yale University School of
Medicine, New Haven, CT). For expression in yeast, the full-length
Mgcphlp cDNA was inserted in the expression vector pRS314 (10).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence of Mgcphlp
cDNA and open reading frame of the corresponding protein
sequence.
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Fig. 2.
Amino acid sequence alignments of phosducin
and phosducin-related proteins. A, alignments were
generated by standard algorithms, first by pairwise comparison and then
by grouping all alignments to optimize the conserved positions.
Black boxes indicate similarities between MgcPhLP
(NM023508), mouse phosducin (L08075), rat phosducin-like (NM022247),
yeast Plp1 (YDR183w), and Plp2 (YOR281c) proteins
(GenBankTM accession numbers are in parentheses).
Shown in gray are similarities with the Plp2 amino acid
sequence. B, identical residues (percent) among the
phosducin and phosducin-like proteins.
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Fig. 3.
Mgcphlp RNA in somatic and
germinal tissues. A, Northern blot analysis of total RNAs
prepared from brain (br), heart (he), liver
(li), intestine (in), lung (lu),
spleen (sp), and adult testis (t). B,
RNA dot blot analysis of poly(A)+ RNA from 22 different mouse tissues
and 7 different control RNAs and DNAs indicated in the diagram (Mouse
RNA MasterblotTM, Clontech).
Hybridization was performed with a complete Mgcphlp cDNA
probe according to the manufacturer's instructions. C,
total RNA (1 µg) from testis, liver, and brain was
reverse-transcribed using oligo(dT) primers and amplified by PCR with
primers Mgc271 and Mgc483, with controls performed with cloned complete
Mgcphlp cDNA (C) or without reverse
transcriptase (RT ); M, molecular weight
markers. D, testis RNA at days 10, 20, 25, and 30 and at 2 months (Ad) after birth (d.p.p., days
post-partum). Sc, freshly isolated Sertoli cells (primary
culture); eS, rS, and Pac, 80-90%
pure elutriated preparations of, respectively, elongated spermatids,
round spermatids, and pachytene spermatocytes. Hybridization was
performed in succession with a complete Mgcphlp cDNA
probe and with a cDNA probe for the ubiquitous ribosomal S26
protein.
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Fig. 4.
Genomic organization of the
Mgcphlp locus and alternative polyadenylation
signals. The six exons of the gene were located on the basis of
cDNA sequence. Analysis of the RNA 3' ends evidenced three major
sites of polyadenylation (underlined) corresponding to
putative polyadenylation signals on the genomic sequence
(boxed; 7-30 RACE clones analyzed for each site).
Nucleotide numbers correspond to the sequence of the
Clock locus (AF146795).
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Fig. 5.
Stage-specific expression of
Mgcphlp in male germ cells. A,
in situ hybridization on testis sections of an antisense
riboprobe of the complete cDNA. Digoxigenin staining is shown in
panels 2 and 4 and superposition with Hoechst
33258 nuclear stain in panels 1 and 3. In
panels 5 and 6, the same experiment was performed
with the sense probe. P, pachytene spermatocytes;
rS, round spermatids; eS, elongated spermatids.
B D, immunofluorescence and nuclear staining of elutriation
purified pachytene spermatocytes (B), round spermatids
(C), and elongated spermatids (D). E,
control with secondary antibody only. Scale bars: 50 µm in
A and E, 5 µm in B, C, and
D. F, protein analysis during prepubertal
development. Western blot analyses of total protein extract (40 µg of
protein) were performed on testis extracts from three mice at
day 10 of post-natal development (before entry into meiosis),
from two mice at day 18 (end of first meiotic division), and from one
mouse after puberty (day 30).
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Fig. 6.
Expression in ovary and female germ
cells. A, total RNA (1 µg) from testis and adult
ovary was reverse-transcribed and amplified by PCR with primers Phos-1
and Phos-2 with control ( RT) performed in the absence of
reverse transcriptase. B, Western blot analysis of protein
extracts from somatic and germinal tissues. Ov, ovary;
K, kidney; Sp, sperm; T, testis;
M, molecular size markers. C, analysis by
Western blotting and ECL detection of the protein in unfertilized
oocytes and in fertilized eggs performed on 25 cells/lane.
D, Western blot analysis of ovary extracts prepared at times
0, 1, 3, and 24 h after induction of superovulation by human
chorionic gonadotropin (hCG). Testis extract and the
purified GST-MgcPhLP protein are shown for comparison.
protein, are expressed in a stage-dependent
manner in the spermatogenic differentiation pathway (15). As shown in
Fig. 7, a complex of the MgcPhLP protein with 14-3-3 protein(s) was evidenced by immunoprecipitation of testicular protein extracts with polyvalent anti-14-3-3 antibodies followed by Western blot analysis of the precipitated complexes with
anti-MgcPhLP antiserum. Further experiments are in progress to identify
more precisely the protein(s) present in these complexes.
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Fig. 7.
14-3-3 binding to MgcPhLP.
Immunoprecipitates prepared using the monoclonal antibody
14-3-3 (H-8):sc-1657 reacting with all of the
14-3-3 family members were analyzed by Western blot analysis for the
presence of MgcPhLP as shown in Fig. 6. Lane 1, testis
extract; lane 2, liver extract; lane 3, molecular
size markers.
binding (16) is completely conserved
between the phosducin proteins and Phlp1 but present neither in Plp2
nor in MgcPhLP. That would distinguish two groups of phosducin-like
proteins: on one hand, the mammalian phosducins with the region that
interacts with G
proteins, and on the other, the MgcPhLP and PLP2
proteins, with a distinct pattern of conserved amino acids. Other
discrete patches of pairwise similarities could also be observed in the
central and carboxyl-terminal parts of the sequences.
) was established
previously (4). The deletion of PLP2 prevented the recovery
of mutated haploid clones upon induction of sporulation in diploid
heterozygotes. The mutated strain was transformed with an expression
vector for Mgcphlp and TRP1 (see "Experimental
Procedures"), and the resulting colonies were induced to sporulate.
Isolated spores were tested in duplicate in media selective for either
URA3 (plp2
) or TRP1
(Mgcphlp). Haploid colonies were then revealed by replica
plating with yeast strains of either the a or
mating types carrying a mutation in the nutritional HIS gene
and selection in histidine-free medium. Haploid derivatives identified
as carrying both the URA3 (plp2D) allele and
TRP1 (Mgcphlp) were grown. Analysis by PCR
amplification confirmed the expected absence of the wild type
PLP2 and the presence of the Mgcphlp allele (Fig.
8A). These complemented
haploid clones were successfully grown for successive generations. As
expected from the published data, transfer of the empty vector did not result in the production of viable haploid cells.
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Fig. 8.
Complementation of the S. cerevisiae plp2 mutation by the
murine Mgcphlp gene. A, genotypes of
two representative yeast clones (clones 1 and 3) derived from spores
generated by a plp2::URA3,plp2
yeast
clone after transfer of the Mgcphlp expression vector. These
clones were checked to be haploid on the basis of their ability to
conjugate with reference a and
yeast strains.
PCR analysis was performed with primers Plp2-s and Plp2-r, generating a
525-bp product diagnostic of the wild type allele (WT) and a
1.3-kbp fragment from the deleted gene with the URA3
insert. B, loss of the mouse gene upon long term growth of
the complemented haploid clones. PCR analysis was performed with
primers Mgc271 and Mgc483 (lanes 1, Mgcphlp),
Plp2-s and Plp2-r (lanes 2, PLP2), and Plp2-s and
Ura3-r (lanes 3, URA3, plp2
). Panel
A, wild type diploid; panel B, diploid
Mgcphlp, plp2::URA3,plp2
;
panel C, haploid clone 1 early after isolation (same as in
panel A); panel D, clone 1 after 30 generations
in culture.
). One
could not on this basis distinguish between a function of the gene
during meiosis (or at an early stage of spore formation) and a general requirement for cellular growth. In view of the results shown in Fig.
8B, we may now conclude that the gene is not required for
growth. Genotyping of haploid clones that had been maintained in
culture for 30-40 generations after their isolation from the complemented parent strain showed that the mouse gene was eventually lost. Growth properties of the clones in the absence of a functional PLP2 gene remained, however, unaffected. It is therefore
most likely that the protein, not necessary for growth, was required for the establishment of the haploid state, a conclusion that is
consistent with the expression of the mouse gene being restricted to
the meiotic and early post-meiotic stages.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
signaling. We describe in this report a novel
phosducin-like mRNA specific of the meiotic and post-meiotic germ
cells, which shows significant amino acid sequence similarities to the
phosducin and phosducin-like proteins of various species. Searching
existing EST clones from both mouse and human suggested germ cell
specificity, because related sequences were found in EST libraries from
testes but not from other tissues. In the mouse, expression of
Mgcphlp was at least predominantly observed in male and
female germ cells, in both sexes at the meiotic and post-meiotic stages. It must be taken into account, however, that a detailed in situ analysis could not be performed on every possible
tissue, and therefore we cannot exclude the possibility that the gene might be expressed in a minor cell population and/or only during a
limited physiological or developmental period (as is in fact the case
in the ovary). In the male gonad, three Mgcphlp RNA isoforms were identified at different stages of differentiation. These three
specific mRNAs correspond to different polyadenylation sites in the
locus. It is interesting to note the presence in the vicinity of the
polyadenylation sites in genomic DNA of AU-rich sequences similar to
the sequences described as cytoplasmic polyadenylation elements
mediating polyadenylation and translation of messages during the
oocyte release from the meiotic block at ovulation and prior to
the activation of zygotic genome at the two cell stage (17, 18). The
presence of stage-specific isoforms of the Mgcphlp message
may thus reflect a translational control during meiotic and
post-meiotic maturation. The conclusions of RNA analysis were confirmed
by direct determination of the protein by polyclonal antibodies
specific for the mouse protein. This was especially informative in the
ovary, in which expression is normally limited to the small number of
oocytes undergoing meiosis but could readily be evidenced after
hormonal stimulation leading to superovulation.
by sequestering the phosphorylated phosducin molecules and blocking their binding. 14-3-3 was also recently shown to interact in the brain with a phosducin-like protein
(19, 20). The specificity of 14-3-3 binding and its relationship with
the phosphorylation of serine residues in MgcPhLP are currently being studied.
mutant to
generate viable haploid products was successfully complemented by
transfer of the mouse gene. Regarding the function of the yeast gene,
published data (4) have left two possibilities open; the Plp2 protein
could either be necessary for growth in general or specifically
required for the generation of haploid products, either during or after
meiosis. The observation that haploid clones bearing the
plp2
mutation could not be grown from a sporulating culture is compatible with both interpretations, thus making it impossible to evaluate the phenotype of the diploid homozygous mutant.
Our data, in fact, rather favor the hypothesis of a meiotic function.
We observed, upon long term growth of several of the complemented
haploid strains, a loss of the murine gene that did not impair their
growth ability. A meiotic function of the yeast gene is consistent with
the restricted meiotic and post-meiotic expression of
Mgcphlp in the mouse.
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ACKNOWLEDGEMENTS |
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We thank Rob Arkowitz for help in the design of yeast experiments and Yan Fantei and Mireille Cutajar for skilled technical help.
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FOOTNOTES |
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* This work was supported by grants from the Association pour la Recherche sur le Cancer (France).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 Immunology and Oncology, Centro Nacional
de Biotecnologia, Campus Universidad Autónoma, 28049 Madrid, Spain.
§ To whom correspondence should be addressed: INSERM U470, Faculté des Sciences, 06108 Nice cedex 2, France. Tel.: 33-492-07-64-11; Fax: 33-492-07-64-02; E-mail: fcuzin@unice.fr.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M207434200
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ABBREVIATIONS |
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The abbreviations used are: PhLP, phosducin-like protein; GST, glutathione S-transferase; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; EST, expressed sequence tag.
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