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J. Biol. Chem., Vol. 275, Issue 47, 36934-36941, November 24, 2000
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From the
Received for publication, April 24, 2000, and in revised form, August 24, 2000
We identified a novel gene of Drosophila
melanogaster, Male-specific IDGF (MSI), encoding a
transmembrane signaling molecule with exclusive expression in the
testis. This molecule (MSI) contains a single transmembrane domain and
has 35% amino acid identity with insect-derived growth factor (IDGF),
a soluble growth factor for embryonic cells of the flesh fly,
Sarcophaga peregrina. When MSI was exogenously expressed in
Schneiders's line 2 cells, it was shown to be localized on the cell
surface and exhibits growth factor activity, suggesting that MSI is a
membrane-bound extracellular signaling molecule. Gene expression
studies revealed that MSI mRNA was restricted to mature primary
spermatocytes, whereas MSI was detected in the cells at the later
developmental stages. Analysis using four meiotic arrest mutants,
aly, can, mia, and sa
suggested that MSI is involved in spermiogenesis, the final
differentiation step of spermatogenesis. These results suggest that
MSI is an extracellular signaling molecule participating in
spermatogenesis and is a new member of the IDGF family.
Spermatogenesis is a complex multistep differentiation program
leading to the development of highly specialized haploid male gametes
from primordial diploid cells. This differentiation program starts with
the formation of spermatogonia from germ line stem cells followed by
mitotic divisions to produce primary spermatocytes. Primary
spermatocytes then undergo meiotic divisions to give rise to haploid
spermatid cells followed by the final differentiation step,
spermiogenesis, to produce motile spermatozoa.
In Drosophila, the process of spermatogenesis has been
extensively studied at the cellular level (1-3), and genetic analysis has revealed several loci that are required for the spermatogenic processes (3-5). Furthermore, recent molecular biological studies have
identified several genes that function in spermatogenesis, including
Insect-derived growth factor
(IDGF)1 is the first soluble
invertebrate growth factor to be identified, being purified from the
conditioned medium of an embryonic cell line of the flesh fly,
Sarcophaga peregrina (13). This factor had no significant sequence similarity with any other growth factors so far characterized, but did show homology with atrial gland granule-specific antigen (AGSA)
of Aplysia californica (about 25% sequence identity), a secretory protein with unknown function (14). Here, we describe the
identification of a novel Drosophila gene,
Male-specific-IDGF (MSI), which is expressed
exclusively in testis and encodes a protein termed MSI, which is
assigned as a novel member of the IDGF family. Data suggest that MSI is
an extracellular signaling molecule required for spermatogenesis.
cDNA Cloning of MSI--
PCR was done using Drosophila
melanogaster genomic DNA as a template. DNA primers were designed
from amino acid sequences of Sarcophaga IDGF that showed
relatively strong homology with Aplysia AGSA. The nucleotide
sequences of the primers for the initial PCR were 5'-TA(T/C) ATI AGI
AGI ATG CC(T/C/A/G) AA(A/G) GG-3' and 5'-CCI GCI AC(A/G) TCI
GG(A/G) TA(T/C) TT-3', which correspond to the IDGF amino acid
sequences of 127YISSMPKG134 and
338KYPDFVAG345, respectively. The primers
for the nested PCR were 5'-ATI CGI CTI ACI TA(T/C) (C/A)G(T/C/ A/G)
GA(T/C) AA-3' and 5'-ATI AG(T/C) TTI GAI CC(A/G/T) AT (A/G) AA(A/G)
TC-3', which correspond to 151IRLTYRDN158 and
306DFIGSKLI313, respectively. The 620-bp DNA
fragment of the nested PCR product was amplified. The sequence analysis
showed that it encoded a peptide with 162 amino acid residues split by
a 132-bp intron. Because this peptide showed significant sequence
homology with part of IDGF, this 620-bp DNA fragment was used as a
probe for subsequent cDNA cloning. The probe was labeled with
[ Transfection of the Plasmid to Schneider's Line 2 Cells--
Schneider's line 2 cells (17) were cultured in
Schneider's insect medium (Sigma) containing 10% (v/v)
heat-inactivated fetal bovine serum (FBS), streptomycin (0.5 mg/ml),
and penicillin G (120 units/ml) at 27 °C.
A plasmid that drives the expression of MSI was constructed by
amplifying the DNA fragment of MSI cDNA (nucleotides 1 to 1686 in
Fig. 1A) by PCR and ligating the PCR product downstream of the Drosophila actin 5C promoter. The transfection of
the plasmid to Schneider's line 2 cells was performed as described by
Hisahara et al. (18). Briefly, cells were seeded at a
density of about 3.5 × 105 cells/well in a 6-well
plate and cultured overnight. The medium was then changed to serum-free
medium (Life Technologies, Inc.). The constructed plasmid DNA (1 µg) and 4 µl of Cellfectin reagent (Life Technologies, Inc.) were
mixed. The mixture was added to each well, and incubation was continued
for 4 h. The medium was then changed to serum-containing
Schneider's insect medium and cultured for a further 2 days. The
expression of MSI was examined by immunodetection. About one-tenth of
the cells was transfected reproducibly under these conditions.
Preparation of Antibody against MSI--
A 50-µg aliquot of
the synthetic peptide CVDEEFYNLWRNYHSQP was conjugated to keyhole
limpet hemocyanin and injected into an albino rabbit with complete
Freund's adjuvant. Three booster injections were given before
harvesting the antiserum. In this peptide, V to P corresponded to
residues 119 (Val) to 144 (Pro) of MSI. The N-terminal Cys was added to
couple this peptide to Sepharose in preparation for purification of the
antibody by affinity column chromatography.
Cell Fractionation and Immunoblotting--
Fractionation of the
transfected Schneider's line 2 cells was performed as follows: cells
were homogenized in 10 volumes of phosphate-buffered saline (PBS)
containing 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml
leupeptin, and 100 ng/ml pepstatin. The homogenate was centrifuged at
1000 × g for 10 min, and the supernatant was then
subjected to ultracentrifugation at 100,000 × g for 30 min. The resulting supernatant and precipitate were used as the
cytosolic fraction and membrane fraction, respectively.
Immunoblotting was performed essentially as described by Burnette (19).
Briefly, proteins separated by SDS-polyacrylamide gel electrophoresis
were transferred onto a polyvinylidene difluoride membrane filter,
which was then treated with 0.5 µg/ml anti-MSI antibody solution
followed by peroxidase-linked anti-rabbit donkey IgG diluted 1:5000.
The filter was then treated with ECL Western blotting detection
reagents (Amersham Pharmacia Biotech) and exposed to x-ray film.
Immunofluorescence Staining--
Immunofluorescence staining of
the transfected Schneider's line 2 cells under non-fixative conditions
was carried out as described by Hori et al. (20). Briefly,
the cells were suspended in 10 ng/ml anti-MSI antibody solution and
kept on ice for 1 h. They were then washed three times with PBS
containing 10% (v/v) FBS, and suspended in FITC-labeled secondary
antibody solution for 40 min. The cells were then washed, transferred
to 12-well multitest slides, and examined with a fluorescence
microscope. Immunofluorescence staining under fixative
conditions was performed as described by Hisahara et al.
(18). Briefly, the transfected cells on coverslips were fixed in 4%
(v/v) paraformaldehyde in PBS for 10 min and blocked in 1% (w/v) skim
milk, 0.1% (v/v) Triton X-100 in PBS for 10 min. Then the cells were
successively treated with 10 ng/ml anti-MSI antibody and FITC-labeled
secondary antibody (Dako Corp.). Then they were washed three times with
PBS, mounted in 50% (v/v) glycerol containing 2.5% (v/v)
1,4-diazabicyclo[2.2.2]octane, and their fluorescence was examined
under a fluorescence microscope.
For whole mount immunostaining of testes, testes from adult flies were
fixed with 4% (v/v) formaldehyde, 1% (v/v) Nonidet P-40, 0.1% (v/v)
Triton X-100 in PBS for 1 h, then followed with the protocol as
described above. We used yw strain in immunofluorescence study because of its low level of natural fluorescence.
Assay for the Growth Factor Activity--
Large scale
preparation of transfected Schneider's line 2 cells for the
measurement of the growth factor activity of MSI was achieved by using
a 75-cm2 tissue culture flask. Assay of the growth
factor activity was carried out as described before (13). The
transfected Schneider's line 2 cells were suspended in
serum-containing Schneider's insect medium at a density of
107 cells/ml, and 100 µl of the cell suspension was
poured into each well of a 96-well microtiter plate. The cells were
incubated for 1 day at 27 °C. Then, 10 µl of
[methyl-3H]thymidine (3.7 MBq/ml, Amersham
Pharmacia Biotech, 925 GBq/mmol) was added and incubation was continued
for 1 more day. The medium was discarded, and the cells were washed
thoroughly and solubilized with 150 µl of 0.5 N NaOH
solution, and 150 µl of ice-cold 60% (v/v) trichloroacetic acid was
added to the cell lysate. The acid-insoluble radioactive material was
trapped on a glass fiber filter (Whatman, GF/C), and the radioactivity
was measured (21).
Results were expressed as the mean ± S.E. The probability of
statistical differences between experimental groups was determined by
Student's t test, as indicated.
Northern Analysis and RT-PCR--
Total RNA was extracted from
D. melanogaster at various developmental stages by the
guanidine-thiocyanate method (22). RNA (20 µg) was separated on a
1.2% (w/v) agarose gel containing formaldehyde, blotted onto
GeneScreen Plus (PerkinElmer Life Sciences) in 10 × SSPE (1 × SSPE: 180 mM NaCl, 10 mM
NaH2PO4, 1 mM EDTA, pH 7.4). Hybridization was performed in 50% (v/v) formamide, 5 × SSPE, 1× Denhardt's solution (0.02% (w/v) each of Ficoll-400, bovine serum
albumin, and polyvinylpyrrolidone), and 0.2 mg/ml single-stranded salmon sperm DNA solution for 16 h at 42 °C. The radiolabeled probe was the same as for the cDNA cloning (620-bp DNA). The filter was subsequently hybridized with an 18 S rRNA probe to estimate the
amount of RNA loaded.
RT-PCR was carried out using an RT-PCR kit (Stratagene). The
oligonucleotide primers used to detect MSI mRNA were 5'-GTT AAC TTG
GAA CAG GAC TTC GAG-3' and 5'-TTC GAT ACT GAA ACT AAG GCC TCC GCC-3'.
As an internal control, the ribosomal protein 49 (rp49) mRNA (23)
was detected using oligonucleotide primers (5'-GAT CGA TAT GCT AAG CTG
TCG CAC-3' and 5'-CTC CTT GCT TCT TGG AGG AGA CGC-3'). The resulting
PCR products were electrophoresed in 2% (w/v) agarose gels, blotted
onto GeneScreen Plus (PerkinElmer Life Sciences), and detected by
hybridization with the radiolabeled oligonucleotide probes. These
probes were 5'-AGT CCT CAG CGG GCG GGA TGT GAG G-3' for MSI
and 5'-CAC AAA TGG CGC AAG GGT A-3' for rp49, respectively.
Other Methods--
Whole mount in situ hybridization
to squashed testes was performed essentially as described by Endo
et al. (11). Chromosomal location of the MSI was
determined according to the Drosophila laboratory manual
(24).
cDNA Cloning of Drosophila IDGF--
IDGF is a growth factor
of Sarcophaga embryonic cells. To gain more insight into the
biological role of IDGF, we intended to isolate cDNA of
Drosophila IDGF. For this, we first amplified a possible
genomic fragment of the Drosophila IDGF gene using degenerate primers designed after the amino acid sequences conserved between IDGF and AGSA. Then, we screened the cDNA library of adult Drosophila using this DNA fragment as a probe and finally
isolated one cDNA clone. The nucleotide and deduced amino acid
sequences of this cDNA clone are shown in Fig.
1A.
This clone contained one open reading frame of 561 amino acid residues.
Comparison of the deduced amino acid sequence of this protein with
those in the data bases revealed that this protein has significant
homology only with IDGF and AGSA, with overall amino acid identity
between IDGF and this protein being 35%. We termed this protein
Male-specific IDGF (MSI) for the reason presented below.
Hydropathy analysis revealed that MSI contains a single hydrophobic
region with 17 amino acid residues, which could potentially form a
helical transmembrane domain (Fig. 1B). No other structural motifs were identified in the predicted amino acid sequence of MSI. As
shown in Fig. 1C, MSI shows homology with IDGF and AGSA in
its long C-terminal region following the predicted transmembrane domain, suggesting that the long C-terminal region is an extracellular domain and short N-terminal region is an intracellular domain. As IDGF
and AGSA are thought to be humoral factors, the transmembrane structure
of MSI is unique. Possibly, MSI is a membrane-bound extracellular
signaling molecule and a novel member of the IDGF family rather than an
IDGF homologue of Drosophila.
Expression of MSI in Schneider's Line 2 Cells--
Because MSI
was suggested to be a transmembrane protein, we examined its cellular
localization by transiently expressing it in Schneider's line 2 cells,
which have no endogenous expression of MSI. For this, MSI cDNA was
expressed in these cells under the control of the Drosophila
actin 5C promoter, and the cells were subjected to immunodetection
using a specific antibody raised against the peptide derived from MSI.
Results from the immunoblotting experiment are shown in Fig.
2A. MSI was recovered
exclusively in the membrane fraction, when the cells were fractionated
into the membrane and cytosolic fractions, while co-expressed
Growth Factor Activity of MSI--
We next examined whether MSI
exhibits growth factor activity, like IDGF. For this,
[methyl-3H]thymidine incorporation of
MSI-transfected Schneider's line 2 cells was compared with
that of the control cells. As shown in Fig.
3, thymidine incorporation of
MSI-transfected cells was significantly higher than that of
the mock transfected cells at a cell density of 107
cells/ml (p < 0.01), where the cells were confluent.
Almost the same results were obtained with medium containing 5% and
12% FBS when cell density was 107 cells/ml. In contrast,
no appreciable enhancement of cell growth was detected at lower cell
densities. Possibly, contact of membrane-bound MSI to adjacent
cells may not be sufficient under these conditions. In addition, no
growth factor activity was detected in the conditioned medium of the
transfected cells (data not shown). These results suggest that MSI in
fact exhibits growth factor activity through cell-to-cell interaction.
The enhancement rate of 1.5- to 2-fold may be reasonable, considering
the fact that the ratio of MSI-expressing cells is about 10% and that
MSI is a transmembrane protein.
MSI Is a Testis-specific Protein--
To determine when and where
MSI functions, we performed gene expression analyses. Northern blot
analysis throughout the life cycle of Drosophila revealed
that the MSI mRNA is expressed in pupae and adult males, in
association with the development of adult organs (Fig.
4). The signal detected in pupae is
likely to be from males, because its expression in the adult is
restricted to the male. These results suggest that the tissue
synthesizing MSI is testis. Results of RT-PCR to test whether
expression is testis-specific or not are shown in Fig.
5. MSI mRNA was detected in the
testes of adult males but not in the carcass after removal of the
testes, indicating that MSI is expressed exclusively in testis.
To investigate the cells expressing this molecule, in situ
hybridization with testis squashes was performed. Fig.
6 shows that the mature primary
spermatocytes are the cells that express MSI mRNA (Fig.
6B, open arrowheads). However, the signal was
absent in the apical tip of the testis, where the stem cells and
spermatogonial cells are localized. Nor was the signal detected in the
developing primary spermatocytes that are localized between
spermatogonial cells and primary spermatocytes (Fig. 6B,
solid arrowheads). No such signals were detected with a
sense probe (Fig. 6C).
Next, the distribution of MSI was examined by immunofluorescence
staining. Testes were successively treated with antibody and
FITC-conjugated second IgG. The specificity of the antibody was
confirmed by immunoblotting, where it gave a single band with testis
homogenate (Fig. 7A). In
contrast to in situ hybridization result, fluorescence was
detected in the regions where spermatocytes at much later developmental
stages, including spermiogenesis, are present (Fig. 7B).
Because the large number of gene products needed for
postmeiotic stages are thought to be supplied in the mature
primary spermatocytes by premeiotic transcription (2, 3, 25, 26),
MSI is likely to be a protein that functions in meiosis or
spermiogenesis.
Expression of MSI in Meiotic Arrest Mutants--
To examine the
relation between MSI and other genes, we investigated the
expression level of MSI mRNA in four meiotic arrest mutants,
aly6, can3,
mia1, and sa1. Lin
et al. (27) and White-Cooper et al. (28) showed
that the transcription of the genes involved in spermiogenesis, such as
fzo (12) and don juan (24) are greatly reduced in
all these four mutants, whereas that of the genes required for meiosis, such as twine (7-9) and boule (10) are not
affected in the aly6 mutant. As shown in Fig.
8, no appreciable transcript of
MSI was detected in any of the four mutants, indicating that
the expression of MSI is under the control of each of these
four genes. However, the gene expression in twine mutant was
not reduced (data not shown). These results suggest that MSI
is needed for spermiogenesis rather than for meiosis.
We mapped the locus of MSI at 75A on the left arm of
chromosome 3 by in situ hybridization to a polytene (data
not shown), where few deficiency strains or mutants that could evaluate
the function of MSI have been reported so far.
We identified a new member of the IDGF family from
Drosophila. Although MSI is a membrane-bound protein, it is
structurally related to secretory IDGF and AGSA, suggesting that these
three proteins belong to a common structural family. Of these, IDGF was
shown to be a growth factor of Sarcophaga embryonic cells. Possibly, IDGF interacts with a specific receptor on the surface of
these cells and stimulates their growth. By contrast, MSI was suggested
to be a transmembrane protein consisting of a 47-residue intracellular
domain and a long C-terminal extracellular domain with a 17-residue
transmembrane domain. By analogy with IDGF, MSI is likely to be a
membrane-bound signaling molecule and possibly interacts with other
cells with its extracellular domain. In fact, MSI expressed in
Schneider's line 2 cells was shown to localize on the surface and
exhibits growth factor activity when cells are kept in high density.
These results suggest that Schneider's line 2 cells contain
receptor(s) for MSI.
Gene expression studies revealed that MSI is a testis-specific protein,
and its mRNA was detected exclusively in mature primary spermatocytes ready to enter into meiosis. Because most transcription in spermatocytes is shut off upon entry into meiotic division in
spermatogenesis of Drosophila, it is known that mRNAs
for a large number of genes required for postmeiotic spermatid
differentiation are synthesized by premeiotic transcription and
deposited in mature primary spermatocytes (2, 3, 25, 26). In accordance with these previous reports, MSI was shown to be expressed in the
spermatocytes at the later developmental stages, suggesting that MSI
functions in postmeiotic differentiation. Although MSI exhibits growth
factor activity to embryonic cells such as Schneider's line 2 cells,
it might convey a signal in the testis, because the cells at
those developmental stages are differentiating rather than proliferating.
The evidence that MSI is not expressed in meiotic arrest
mutants so far tested suggests that it is likely to be a spermiogenic gene, according to the proposal of White-Cooper et al. (28). This is consistent with the expression pattern of MSI. The
facts that MSI is a testis-specific protein and its transcription is under the control of meiotic arrest genes suggest that MSI plays a role
in spermiogenesis.
In spermatogenesis of Drosophila, developing germ cells are
surrounded by cyst cells, and differentiation of the germ cells progresses in this delimited space (30, 3). Although the cyst cells are
required for the differentiation of the germ cells, the germ cells are
also known to regulate the fate of the cyst cells (31). This close
association of the germ cells and cyst cells suggests the presence of
signaling processes between these two cells. On the other hand,
signaling processes could also be present between two germ cells.
However, no molecules have been identified that mediate cell-to-cell
interaction in spermatogenesis. Judging from its IDGF-like structure
and in vitro activity, MSI could be a candidate for such a
molecule on the surface of postmeiotic spermatids, which transmits a
specific signal to adjacent cells to bring them into spermiogenesis.
The evidence described here raises the possibility that MSI has a
function in spermiogenesis as an extracellular signaling molecule. To
clarify the biological role of MSI in spermatogenesis, a
loss-of-function mutation of the MSI gene would be
helpful, and such strains could be generated by the technique
of insertional mutagenesis with single P elements (32, 33). The
identification of the receptor for MSI, if any, would be also helpful
to understand the function of MSI. We found three genes related to
MSI in the data base of Drosophila genome. Those
genes are likely to be for the new members of IDGF family, but their
functions are unknown.
We gratefully thank M. T. Fuller for
providing the four meiotic arrest mutants, M. Miura for his gift of
Schneider's line 2 cells, and J. Inoue for his gift of the DNA
fragment of Drosophila actin 5C promoter. We are also
grateful to K. Endo, H. Kanuka, and Y. Yagi for their courteous advice
on the experimental procedures and to K. Nakane for her great help.
*
This work was supported by Core Research for Evolutional
Science and Technology of the Japan Science and Technology Corporation and a grant-in-aid for scientific research from the Ministry of Education, Science, Sports 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB025255.
¶
To whom correspondence should be addressed: Tel.:
81-48-467-9437; Fax: 81-48-462-4693; E-mail:
natori@postman.riken.go.jp.
Published, JBC Papers in Press, August 30, 2000, DOI 10.1074/jbc.M003455200
The abbreviations used are:
IDGF, insect-derived
growth factor;
AGSA, atrial gland granule-specific antigen;
RT-PCR, reverse transcription-polymerase chain reaction;
bp, base pair(s);
FBS, fetal bovine serum;
PBS, phosphate-buffered saline;
FITC, fluorescein
isothiocyanate;
rp49, ribosomal protein 49.
Male-specific IDGF, a Novel Gene Encoding a
Membrane-bound Extracellular Signaling Molecule Expressed Exclusively
in Testis of Drosophila melanogaster*
,§,§,, and Graduate School of Pharmaceutical Sciences,
University of Tokyo, 7-3-1 Bunkyo-ku, Tokyo 113-0033 and the
§ Natori Special Laboratory, The Institute of Physical and
Chemical Research, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-tubulin, twine,
boule, degenerative spermatocyte-1 (des-1), and fuzzy onions (fzo). The
gene product of 2-tubulin is one of
the major components of the germ cell-specific cytoskeleton and is
essential for normal germ cell development (6). Other genes,
twine, boule, and des-1, are known to
be required for the meiotic division. The twine gene encodes
a Drosophila homologue of cdc25, a cell-cycle
phosphatase, which is essential for the onset of the first meiotic
division (7-9). The boule gene encodes a predicted
RNA-binding protein showing strong homology with the product of the
human DAZ gene, a candidate for the Y-chromosome azoospermia factor (10), whereas the des-1 gene product is a novel membrane protein required for the initiation of meiosis (11). The
fzo gene encodes a protein with GTPase motifs and regulates
mitochondrial fusion at the early postmeiotic stage (12). In contrast
to the extensive studies of spermatogenesis at the gene level, the
upstream regulatory factors for these genes, such as extracellular
signaling molecules, have not been well characterized.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP using a random primer labeling kit (Takara,
Kyoto). One hybridization-positive clone was isolated from 1.5 × 105 clones of a Drosophila adult cDNA
library (Stratagene) by plaque hybridization, and its complete sequence
was determined. Data base searches were done using the BLAST program
(15). A hydrophobicity/hydrophilicity plot of MSI was generated using
the GENETYX program (Software Development, Tokyo) with the parameters
described by Kyte and Doolittle (16). The nucleotide sequence of MSI
cDNA has been deposited in the GenBankTM/EBI Data Bank with
accession number AB025255.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
MSI is a new member of IDGF family with a
single transmembrane region. A, nucleotide and deduced
amino acid sequences of the cDNA for MSI. The amino acid residues
are numbered starting from the first Met, and nucleotide numbers from
the first letter of the Met codon. The asterisk shows the
termination codon. The putative transmembrane region is
boxed, and the polyadenylation signal is underlined.
B, hydrophobicity plot (16) of MSI. The arrow indicates
a predicted transmembrane region. C, comparison of the
deduced amino acid sequence of MSI with other members of IDGF family,
Sarcophaga IDGF and Aplysia AGSA. Gaps
were inserted for optimal matching. The amino acid numbers from the
first Met residue are shown on the left of each line. Solid
boxes indicate exact matches; open boxes indicate
predicted transmembrane region for MSI or leader sequences for IDGF and
AGSA.
-galactosidase, which is a cytosolic protein, was recovered in the
cytosolic fraction. The culture medium did not contain an appreciable
amount of MSI, indicating that it is not a secretory protein (data not
shown). Furthermore, immunofluorescence staining of the transfected
cells under non-fixative conditions revealed that this protein is
localized on the cell surface (Fig. 2, B and C).
When fixed cells were stained with the same antibody, patches were
detected along the edges of the cells (Fig. 2, D and
E). These results support the conclusion that MSI is a
transmembrane protein with its C-terminal side forming an extracellular
domain, because the epitope recognized by the antibody used here is
located in this domain.
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Fig. 2.
MSI is a cell surface membrane protein.
A, immunoblotting of transfected Schneider's line 2 cells. After the transient expression of MSI cDNA under the control
of Drosophila actin 5C promoter in Schneider's line 2 cells, the cells were fractionated into cytosolic fraction and membrane
fraction and subjected to immunoblotting using specific antibody for
MSI. As a control, -galactosidase, which is a cytosolic protein, was
co-expressed. T, C, and M indicate
total cell lysate, cytosol fraction, and membrane fraction,
respectively. B, immunofluorescence staining of transfected
Schneider's line 2 cells under non-fixative conditions with antibody
for MSI. C, bright-field microscopic view of B. D, immunofluorescence staining under fixative conditions.
E, DAPI staining of D to reveal DNA.
Arrowheads indicate fluorescent cells.
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Fig. 3.
MSI exhibits growth factor activity for
Schneider's line 2 cells. The incorporation of
[methyl-3H]thymidine into DNA of
MSI-transfected cells (solid bars) was compared
with that of control vector-transfected cells (open bars).
The cells at the indicated densities were incubated with
[methyl-3H]thymidine in Schneider's insect
medium containing FBS at the indicated concentrations, and then
incorporated radioactivity was measured. Note that the effect of MSI
was observed at the cell density of 107 cells/ml, in which
the cells were confluent. Data represent means ± S.E. of four
determinations. **p < 0.01 (Student's t
test).
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Fig. 4.
Expression of MSI is
associated with the development of adult male flies. Northern
analysis was performed using total RNA from embryos; first instar
larvae (1), second instar larvae (2), third
instar larvae (3); early pupae (E), late pupae
(L); 2-day-old and 6-day-old male (m) and female
(f) adults. The filter was rehybridized with an 18 S rRNA
probe for RNA loading control.
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Fig. 5.
MSI is exclusively expressed in adult male
testes. RT-PCR was performed using total RNA extracted from whole
bodies (1), testes (3), and carcass after removal
of testes (2) of adult males. As an internal control, a
fragment of the ribosomal protein 49 gene (rp49) transcript
was amplified.
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Fig. 6.
MSI mRNA is expressed in mature primary
spermatocytes. Whole mount in situ hybridization
to adult testes was performed with antisense (A,
B) and sense (C) probes transcribed from MSI
cDNA. B, a higher magnification view of the apical
region of the testis shown in A. Developing and mature
primary spermatocytes are indicated by solid and open
arrowheads, respectively. Bars represent 100 µm.
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Fig. 7.
MSI is present in the spermatocytes at the
late developmental stages in the testis. A,
immunoblotting of testis homogenate with the antibody for MSI. The
antibody recognized a single band of 66 kDa, which is consistent with
the predicted molecular mass. B-D, immunofluorescence to
whole mount adult testes stained with the antibody for MSI
(B) or control normal antibody (C). D,
bright field of B. Arrows indicate the apical tip
of testis. Bars represent 100 µm.
View larger version (38K):
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Fig. 8.
MSI expression is absent in the meiotic
arrest mutants. Northern analysis was carried out with total RNA
extracted from adult males of the wild type (WT) and from
aly6, can3,
mia1, and sa1 mutants. The filter
was rehybridized with an 18 S rRNA probe for RNA loading control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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