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J Biol Chem, Vol. 274, Issue 26, 18567-18573, June 25, 1999
From the Division of Biological Sciences, Graduate School of
Science, Hokkaido University, Sapporo 060-0810, Japan
We determined the complete nucleotide sequences
of the The nitric oxide
(NO)1/cGMP signaling pathway
plays a critical role in various physiological phenomena (1, 2). The
second messenger, cGMP, is synthesized by guanylyl cyclase (GC), which is present in two forms, a membrane form and a soluble form (3). The
soluble form of GC is a heme-containing heterodimer composed of In vasodilation, soluble GC activated by NO derived from the
endothelium induces relaxation of vascular smooth muscle through cGMP-dependent protein kinase I (1, 16). As a neuronal
messenger, NO affects synaptic plasticity via generation of cGMP in the
hippocampus and olfactory bulb in mammals (17-19). Soluble GC
localized in the inner segments of photoreceptor cells is activated by
NO (20) and modulates synapses between cone and horizontal cells (21). The NO/cGMP signaling pathway is also expected to participate in
synaptogenesis (22) and synaptic suppression in a neuromuscular junction (23). Despite the rapid accumulation of information concerning
the functional importance of the NO/cGMP signaling pathway, there have
been only a few reports related to the regulation of the soluble GC
gene expression. It has been demonstrated that cAMP causes decreases in
the level of mRNA for The Animals and Embryos--
Mature adults of the orange-red variety
of medaka O. latipes were purchased from a dealer and kept
in indoor tanks as described previously (29). Naturally spawned and
fertilized eggs were collected and cultured in distilled water
containing 0.00006% methylene blue at 27 °C. The developmental
stage is expressed in days, and the day of fertilization is referred to
as Day 0. Hatching usually occurred at Day 10.
Isolation of Genomic Clones for OlGCS- 3'-Rapid Amplification of cDNA Ends (3'-RACE)--
Total RNA
was extracted from Day 9 embryos according to the acid guanidinium
thiocyanate-phenol-chloroform extraction method (31). The first strand
synthesis and first PCR were performed using the 3'-Full RACE Core Set
(Takara Shuzo Co., Ltd.). The specific primers for
OlGCS- Primer Extension Analysis--
Total RNA was extracted from the
adult medaka fish brain as described above. Poly(A)+ RNA
was isolated using Oligotex-dT30 Super (Roche) according to the
manufacturer's protocol. The oligonucleotides used for the primer
extension experiments were as follows: PE-L1 for
OlGCS- Southern Blot Hybridization--
Genomic DNA was isolated from
the adult medaka brain as described (28). The genomic DNA (10 µg) was
digested overnight with BamHI, EcoRV, and
HindIII (Takara Shuzo Co., Ltd.) and then separated on a
0.7% agarose gel. The DNA was transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech) using 0.4 M NaOH as
the transferring solution and hybridized with a 32P-labeled
cDNA probe amplified by PCR (nucleotide positions 1290 to 1518 for
OlGCS- Plasmid Construction--
To construct the fusion genes for the
promoter analysis, the 5'-upstream regions of
OlGCS-
The PCR product containing the 5'-upstream regions of
OlGCS- Microinjection of Fusion Gene Constructs and Detection of
GFP--
To prevent hardening of the chorion after fertilization,
medaka fish fertilized eggs were incubated in 20 mM
CAPS-NaOH (pH 10.5) containing 2 mM glutathione (Wako Pure
Chemicals Co., Ltd.) at 4 °C for several hours (32). Micropipettes
were made by the horizontal puller (PN-3, Narishige) from a siliconized
and sterilized 1 × 90-mm fiber-filled glass capillary tube (GD-1,
Narishige). The DNA solution (10 ng/µl) was injected into the
cytoplasm of both blastomeres of a medaka fish 2-cell stage embryo
using a micropipette. The injected embryo was cultured as described
above. GFP fluorescence was detected under a fluorescence microscope (IX70, Olympus).
Isolation and Characterization of Medaka Fish
OlGCS-
In a previous study, the nucleotide sequence of a part of the
3'-noncoding region of the cDNA for
OlGCS- Analysis of the 5'-Upstream Regions of OlGCS-
Comparison of the nucleotide sequence of the 5'-upstream region of
OlGCS- Genomic Southern Hybridization--
In a previous study we
performed a Southern hybridization analysis using medaka fish genomic
DNA to examine whether OlGCS- Promoter Analysis of OlGCS-
Roles of the 5'-upstream region of OlGCS-
Although ABBG-1 and ABBG-2 contain the BFP gene
driven by the 5'-upstream region of OlGCS- In this study we demonstrated that the 5'-upstream region of
OlGCS- Sequence analysis revealed that a number of E-boxes, which are known to
play a critical role in nerve and muscle differentiation (34), are
present in the 5'-upstream region of each gene. This is consistent with
our detection of GFP fluorescence in the brain of Day 4 embryos
injected with the 5'-upstream region of the
OlGCS- It has previously been demonstrated that the mRNA level of each
subunit gene is decreased by cAMP (24, 25). In this regard, it should
be noted that a cAMP-response element is present in the 5'-upstream
region of OlGCS- The nucleotide sequences of the 5'-upstream region of
OlGCS- Adenylyl cyclase, which synthesizes another second messenger, cAMP,
also has two catalytic domains punctuated by a membrane-spanning domain
(41). The primary structure of each catalytic domain of adenylyl
cyclase is conserved in those of the soluble and membrane forms of GCs
(5). It has been demonstrated that adenylyl cyclase as well as soluble
GC require the two catalytic domains for cyclase activity (41) and that
changes in a couple of the amino acids in the catalytic domain of
adenylyl cyclase cause a functional change in adenylyl cyclase from
cAMP production to cGMP production (42). This suggests that soluble GC
is evolutionarily related to adenylyl cyclase, although their forms are
quite different. In this study, we determined the complete structure of
OlGCS- The soluble form of GC is present as a heterodimer, and the
coexpression of both subunits is required for generating enzyme activity (13, 14). It has been reported that two functionally related
genes such as collagen IV We are grateful to the staff members of the
Research Center for Molecular Genetics and the Center for Experimental
Plants and Animals, Hokkaido University for facilitating the use of the equipment for radiolabeling probes and culturing medaka fishes.
*
This work was supported in part by Grants-in-Aid for
Scientific Research 08454267 and 10102001 from the Ministry of
Education, Science, Sports, and Culture of Japan and Grant 11039 from
the Akiyama Foundation.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) AB022280 (OlGCS-
The abbreviations used are:
NO, nitric oxide;
GC, guanylyl cyclase;
OlGCS, Oryzias latipes
soluble GC;
PCR, polymerase chain reaction;
RACE, rapid amplification
of cDNA ends;
GFP, green fluorescent protein;
bp, base pair(s);
CAPS, 3-(cyclohexylamino)propanesulfonic acid;
BFP, blue fluorescent
protein.
Tandem Organization of Medaka Fish Soluble Guanylyl Cyclase
1 and 
1 Subunit Genes
IMPLICATIONS FOR COORDINATED TRANSCRIPTION OF TWO SUBUNIT
GENES*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subunit gene
(OlGCS-1) and the 
1 subunit
gene (OlGCS-1) of medaka fish soluble
guanylyl cyclase. In the genome, OlGCS-1 and
OlGCS-1 are organized in tandem. The two
genes are only 986 base pairs apart and span approximately 34 kilobase
pairs in the order of OlGCS-1 and
OlGCS-1. The nucleotide sequence of a large
part of the 5'-upstream region of OlGCS-1 is
complimentarily conserved in that of
OlGCS-1. To analyze the promoter activity of
each gene, a fusion gene construct in which the 5'-upstream region was
fused with the green fluorescent protein gene was injected into medaka
fish 2-cell embryos. When the fusion gene containing the
OlGCS-1 upstream region was injected, green
fluorescent protein fluorescence was detected in the embryonic
brain. The 5'-upstream region of OlGCS-1
alone was insufficient for the reporter gene expression in the embryos.
When the OlGCS-1 upstream region was located
upstream of the OlGCS-1-green fluorescence
protein fusion gene, the reporter gene was expressed in the brain and
trunk region of the embryos. These results suggest that the
5'-upstream region of OlGCS-1 can
affect the expression of OlGCS-1. It is
therefore possible that the expression of
OlGCS-1 and OlGCS-1 is coordinated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits (4) and is activated by NO and CO (1). Both subunits
possess a catalytic domain in the C-terminal position; the primary
structure of which is conserved in the membrane form of GC and adenylyl
cyclase (5). However, it is known that each subunit of soluble GC has
no catalytic activity by itself (3). To date, two isoforms for each
subunit of soluble GC have been reported in mammals (6).
GCS-1 and GCS-1
cDNA have been characterized in bovines, rats, and humans (5,
7-10), and GCS-2 and
GCS-2 have been isolated by homology
screening from a human fetal brain cDNA library and a rat kidney
cDNA library, respectively (11, 12). Coexpression of the
1 subunit with the 1 subunit in COS cells
generates an active heterodimer (5, 13, 14), whereas coexpression of
the 2 subunit with the 1 subunit
generates a less active enzyme (11). Recently, it has been demonstrated that the 2 subunit inhibits activation of the
1/1 heterodimer by NO (15).
1 subunits of soluble GC in
the rat fetal lung fibroblast (24) and for both subunits of soluble GC
in the rat aortic smooth muscle cell (25).
1 and 1 subunit genes of soluble GC
are colocalized in human and rat chromosomes (26, 27). This and the
fact that the coexpression of both subunits is essential for enzyme
activity imply that the expression of both genes is coordinated.
However, there has been no report on the genomic structure and
transcriptional regulation of soluble GC. Recently, we have isolated
cDNA clones encoding the 1 subunit
(OlGCS-1) and 1 subunit
(OlGCS-1) of soluble GC of the medaka fish,
Oryzias latipes (28). As a first step in investigating
whether the expression of both genes is coordinated, we determined the
genomic structure of OlGCS-1 and OlGCS-1 and report here that
OlGCS-1 and OlGCS-1
are organized in tandem in the medaka fish genome. We analyzed promoter
function of these genes by introducing promoter-green fluorescent
protein (GFP) fusion constructs into embryos. The results suggest that the 5'-upstream region of OlGCS-1 controls
expression of both OlGCS-1 and
OlGCS-1 genes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
OlGCS-1--
A genomic library of medaka fish (white
strain) constructed in the Lambda Fix II vector was purchased from
Stratagene and used for the isolation of genomic clones for
OlGCS-1 and
OlGCS-1. The cDNA fragments (nucleotide
positions 1290 to 2346 for OlGCS-1 cDNA
and nucleotide positions 241 to 1229 and 1833 to 2620 for OlGCS-1 cDNA) amplified by polymerase
chain reaction (PCR) were labeled with digoxigenin-dUTP using the
digoxigenin-High Prime (Roche Molecular Biochemicals) and used for
screening as probes. Finally, four positive clones were obtained from
approximately 1.35 × 106 recombinant phages. Phage
DNA was purified by using the QIAGEN Lambda kit (QIAGEN), and the
insert DNA was subcloned into pBluescript II KS(-) (Stratagene). The
sequence of the insert DNA was determined by the dideoxynucleotide
chain termination procedure (30) with an Applied Biosystems 373A or
PRISM 377 DNA sequencer and analyzed on DNASIS software (Hitachi
Software Engineering Co.).
1 used for amplification were as
follows: LF-1 for first PCR, 5'-GTGCAACTACTTGTATGTTTC-3' (identical to nucleotides 2267-2287); LF-2 for second PCR,
5'-TTATTGATGTCTGACAGCCTA-3' (identical to nucleotides 2304-2324); and
LF-4 for second PCR, 5'-GTGTGGGTTGTGGATAAAACT-3' (identical to
nucleotides 2522-2542). Second PCR was performed using a 1/50 volume
of the first PCR products as a template. The second PCR products were
subcloned into pBluescript II KS(-) (Stratagene), and the sequence of
the insert DNA was determined as described above.
1, 5'-AAGACAGATGCGCTCGAG-3'
(complementary to nucleotides 97-114) and PE-S3 for
OlGCS-1, 5'-ATGCTGAGATTGTCGGTGTT-3'
(complementary to nucleotides 2-21). The oligonucleotides were
hybridized with 2 µg of the brain poly(A)+ RNA and
extended by 200U SUPERSCRIPT II Reverse Transcriptase (Life
Technologies, Inc.) in 45 mM Tris-HCl (pH 8.3) containing 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTP, 1.85 MBq of
[-32P]dCTP at 42 °C for 1 h. The sequence
reaction was carried out by using Sequenase version 2.0 for the labeled
dCTP kit (USB) and [-32P]dCTP. Primer-extended
products were treated with RNaseA and then separated on a 7 M urea, 6% polyacrylamide gel with sequence reaction
products. The radioactive signals were analyzed using a FUJIX
Bio-Imaging Analyzer BAS2000 (Fuji Photo Film).
1 cDNA) as described previously
(28).
1 (nucleotide positions 
3913 to +45)
and OlGCS-1 (nucleotide positions 1014 to
+175) were amplified by PCR. The primer sets used for amplification
were as follows: OlGCS-1,
5'-CTCAAGCTTCGAGCAAGGGTTTATGCAGAG-3' and
5'-CGGGATCCCGTCAAATGATCAGCAAGAAGC-3'; OlGCS-1, 5'-CGCGGGCCCGAGTGCAATAAATGCTGTCAT-3'
and 5'-CGGGATCCCGACAGATGCTGAGATTGTCG-3'. The PCR products were doubly
digested with HindIII and BamHI for OlGCS-1, and ApaI and
BamHI for OlGCS-1 and were
subcloned into the corresponding sites of the pEGFP-1 Promoter Reporter
vector (CLONTECH), and the resultant fusion gene
constructs were named AG-1 and BG-1, respectively.
1 was subcloned into the pEBFP vector
(CLONTECH), and the resultant fusion gene construct
was named AB-1. A DNA fragment containing the 5'-upstream
regions of OlGCS-1 and the blue fluorescent
protein (BFP)-coding region was excised from AB-1 by
digesting with HindIII and StuI and cloned into
the corresponding site of BG-1. The resultant fusion gene
construct was named ABBG-1. To construct a fusion gene that
lacked a part of the 5'-upstream region of
OlGCS-1 containing the putative TATA box, PCR
was performed with oligonucleotide primers 5'-CCGACAATCTCAGCATCTGT-3'
(identical to nucleotides +156 to +175 of
OlGCS-1) and 5'-GTTCACAGACCACAGTCGAT-3'
(complimentary to nucleotides 213 to 232 of
OlGCS-1). Both ends of the PCR products were
blunted by T4 polymerase and then self-ligated. The resultant fusion
gene construct was named ABBG-2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and OlGCS-1 Genes--
A medaka
fish genomic library was screened with cDNA fragments of
OlGCS-1 or OlGCS-1.
After repeated screening, four different clones (A2, A11, B5, B16) were
obtained. Restriction enzyme mapping of these clones demonstrated that
they overlap each other (Fig. 1). The
nucleotide sequence of the insert DNA of each clone was determined and
compared with that of the respective cDNA. As shown in Fig. 1 and
Table I,
OlGCS-1 consists of 9 exons and
OlGCS-1 consists of 13 exons. The GT-AG rule
was conserved for all splice sites except exon/intron 8 in
OlGCS-1 (Table I). In the medaka fish
genome, OlGCS-1 and
OlGCS-1 are 986 bp apart and organized in
tandem. The two genes span approximately 34 kilobase pairs in the order
of OlGCS-1 and
OlGCS-1.
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Fig. 1.
Genomic structure of
OlGCS- 1 and
OlGCS-1. The exons are indicated
by open boxes (noncoding regions) or solid boxes
(coding region). The exons of OlGCS-1 and
OlGCS-1 are indicated as a and
b, respectively, followed by a number. Four isolated clones
(A2, A11, B5, B16) and restriction enzyme sites are shown below the
scheme of the genomic structure. E, EcoRI;
H, HindIII; kbp, kilobase pairs.
Exon-intron organization of the medaka fish soluble GC 1 and
1 subunit genes
1, which should contain a
polyadenylation signal sequence, has not been determined (28). To
determine the nucleotide sequence of the 3'-end of
OlGCS-1, 3'-RACE was performed using total
RNA from the Day 9 embryos. The 3'-RACE product with LF-2 primer
contained the nucleotide sequence corresponding to that of intron 8 in
OlGCS-1. The 3'-RACE with LF-4 primer, which
was designed to cross the insert site of the intron 8, produced a
344-bp cDNA fragment. Each 3'-RACE product contained a putative
polyadenylation signal sequence, AATAAA, 24-18 bp upstream of poly(A).
1 and
OlGCS-1--
To determine the transcription initiation
sites of OlGCS-1 and
OlGCS-1, primer extension experiments were
performed using medaka fish brain poly(A)+ RNA (2 µg).
Using a specific primer for OlGCS-1, one
major and two minor bands were detected. The nucleotide corresponding
to the major band, which is located most upstream of the three bands, was assigned to the transcription initiation site for
OlGCS-1 (Fig.
2). In the same way, the nucleotide
corresponding to one detected band was assigned to the transcription
initiation site for OlGCS-1. A putative TATA
box, TATAGAA, is present 30-25 bp upstream of the transcription
initiation site of OlGCS-1 (Fig. 3). There is no TATA box in the
corresponding region of OlGCS-1, although a
TATA box consensus sequence, TATAATA, is present at 141-136 bp
upstream of the transcription initiation site for
OlGCS-1 (Fig. 2). A number of E-boxes are
present in the 5'-upstream regions of each gene (Fig.
4). Other known cis-regulatory
elements, AP1, MEF-2, GATA, Sp1, CREB, and C/EBP binding sequences, are
found in the 5'-upstream region of OlGCS-1.
On the other hand, there is one GATA, AP1, and two C/EBP binding
sequences in the 5'-upstream region of
OlGCS-1.
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Fig. 2.
The nucleotide sequences of the 5'-upstream
region of OlGCS- 1. The first
nucleotide of exon 1 was determined by the primer extension method and
numbered as +1. An open box indicates the exon, and the
initiation and stop codons are shaded. A putative TATA box
is double-underlined, and the consensus sequences of known
cis-regulatory elements are underlined.
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Fig. 3.
The nucleotide sequences of the 5'-upstream
region of OlGCS- 1. The first
nucleotide of exon 1 was determined by the primer extension method and
numbered as +1. An open box indicates the exon, and the
initiation and stop codons are shaded. A putative TATA box
is double-underlined, and the consensus sequences of known
cis-regulatory elements are underlined.
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Fig. 4.
Schematic drawing of fusion gene
construction. Fusion genes containing the 5'-upstream regions of
OlGCS- 1 or OlGCS-1
with the pEGFP-1 vector were constructed and named AG-1 and
BG-1, respectively. A putative TATA box and known
cis-regulatory elements found in the 5'-upstream region of
the respective genes are indicated schematically in the open
box. The conserved sequence regions between the 5'-upstream
regions of OlGCS-1 and
OlGCS-1 are indicated by arrows in
the direction from 5' to 3'. ABBG-1 and ABBG-2
are BG-1 derivatives in which the
OlGCS-1 upstream region connected with the
BFP gene was inserted upstream of the
OlGCS-1/OlGCS-1
intervening sequence. ABBG-2 contains a 380-bp deletion
(226/+154 of OlGCS-1). The transcription
initiation site of each gene is numbered as +1. The nucleotide number
in the 5'-upstream regions of both genes is indicated every 1000 bp.
1 with that of the intervening region
between OlGCS-1 and
OlGCS-1 demonstrated that the nucleotide
sequences of about 330 bp (1061 to 733 for
OlGCS-1, 370 to 701 for
OlGCS-1) are complimentarily conserved (Fig.
5). The conserved sequences are divided
into six highly conserved regions (I, 1046 to 977 for
OlGCS-1; II, 971 to 953 for
OlGCS-1; III, 942 to 888 for
OlGCS-1; IV, 850 to 840 for
OlGCS-1; V, 824 to 791 for OlGCS-1; VI, 753 to 742 for
OlGCS-1). Some of these regions contain
several consensus sequences of cis-regulatory elements, such
as an E-box and C/EBP, which could be used for the transcription of
both OlGCS-1 and
OlGCS-1.
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Fig. 5.
The conserved nucleotide sequences between
the 5'-upstream region of OlGCS- 1 and
OlGCS-1. The nucleotide sequence
of the 5'-upstream region of OlGCS-1 was
compared with that of OlGCS-1. Six highly
conserved regions are indicated with open boxes and numbered
by roman numerals. The consensus sequences of known
cis-regulatory elements are shaded.
1 and
OlGCS-1 exist as a single copy (28). However,
the results were not clear because the cDNA fragments used as
probes crossed over many introns. Therefore, in this study we performed
a genomic Southern analysis using a different probe containing a single exon (exon 5 of OlGCS-1). As shown in Fig.
6, only one major band was detected in
each of the three lanes. The size of the band in each lane is
consistent with that of the DNA fragments obtained from the digestion
of genomic clones by the respective restriction enzymes. These results
suggest that OlGCS-1 is a single copy
gene.
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Fig. 6.
Genomic Southern hybridization. Medaka
fish genomic DNA (10 µg) digested with BamHI,
EcoRV, and HindIII was hybridized with a
32P-labeled DNA fragment corresponding to a part of exon 5 of OlGCS- 1. The positions of size markers are
indicated. kbp, kilobase pairs
1 and
OlGCS-1--
To examine whether the 5'-upstream region
of OlGCS-1 and the intervening region between
OlGCS-1 and OlGCS-1
have promoter activity, a fusion gene containing the 5'-upstream region
of each gene and the GFP gene was constructed and named AG-1
or BG-1, respectively (Fig. 4). The fusion gene was injected
into the cytoplasm of both blastomeres of medaka fish 2-cell stage
embryos. At 7 days after fertilization, the number of live embryos and
embryos with GFP fluorescence was counted (Table
II). GFP fluorescence was detected in the
brain and somite of the Day 4 embryos injected with AG-1
(Fig. 7A). At 7 days after
fertilization more than half (57.1%) of the injected embryos were
alive, and GFP fluorescence in these embryos, which tended to increase
as development proceeds, was detected in 14.8% of the live embryos
(Fig. 7, E and F). In the embryos injected with
BG-1, 74.3% of the embryos were alive at 7 days after
fertilization. But no GFP fluorescence was detected in any injected
embryos at this stage (Fig. 7C), suggesting that the
intervening region between OlGCS-1 and
OlGCS-1 alone is not sufficient for
detectable promoter activity.
Detection of GFP fluorescence in medaka fish embryos injected with the
fusion gene constructs containing the 5'-upstream regions of the
1 and/or 1 subunit genes
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Fig. 7.
Promoter analysis and expression of GFP in
medaka fish embryos injected with fusion genes. The fusion gene
construct, AG-1 or BG-1, was injected into the
cytoplasm of both blastomeres of 2-cell stage embryos. Injected embryos
were incubated and objected under a microscopy every day as described
under "Experimental Procedures." GFP fluorescence was detected in
the brain (arrow) and somite (arrowhead) of the
embryos injected with AG-1 (A, B,
E, and F). A, the Day 4 embryo;
B, the Day 4 embryo under the light scope; E, the
Day 7 embryo; F, GFP fluorescence in somite of the Day 7 embryo. No GFP fluorescence in the embryos injected with
BG-1 (C) and the uninjected embryos
(D) was seen at Day 4. A scale bar denotes 200 µm.
1
and the intervening region between the two subunit genes in
OlGCS-1 expression were investigated by using
a GFP-fusion construct (ABBG-1) in which the
OlGCS-1 upstream region connected to a
BFP-coding sequence were inserted upstream of the intervening region of
BG-1 (Fig. 4). GFP fluorescence was detected in the brain
and trunk region of embryos injected with ABBG-1 (Table II).
On the other hand, the reporter gene expression was not observed when a
short upstream region (from 226 to +154) of
OlGCS-1 containing a putative TATA box and a
transcription initiation site was removed from ABBG-1 (Table
II, ABBG-2). These results suggest that the 5'-upstream region of OlGCS-1 can activate transcription
of OlGCS-1 in conjunction with the
intervening region between OlGCS-1 and
OlGCS-1.
1,
BFP fluorescence was not observed in embryos injected with these
constructs (data not shown). This might be because of much weaker
fluorescence intensity of BFP than that of the enhanced variant of GFP
used in this study.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 is essential for the expression of
OlGCS-1. On the other hand, the intervening
sequence between OlGCS-1 and
OlGCS-1 seemed not to be sufficient for
OlGCS-1 expression. Considering the tandem
organization of OlGCS-1 and
OlGCS-1, both genes might be cotranscribed as
a single polycistronic mRNA as in the mouse and human upstream of
the GDF gene (UOG-1) and the growth/differentiation factor-1
gene (GDF-1) (33). Alternatively, other regions such as the
5'-upstream region and/or intron of OlGCS-1
would affect the transcription of OlGCS-1.
Our results support the latter possibility. First, GFP fluorescence was
observed in embryos injected with the ABBG-2 construct in
which the 5'-upstream region of OlGCS-1 was
located upstream of the
OlGCS-1/OlGCS-1
intervening sequence followed by the GFP gene. Second, there is a
transcription initiation site for OlGCS-1 and
a TATA box consensus sequence, TATAGAA, 30-25 bp upstream of the
transcription initiation site for OlGCS-1. The reporter gene expression was abolished when the TATA box and the
transcription initiation site were removed from ABBG-1.
Therefore, basal transcription factors probably act on the intervening
region between OlGCS-1 and
OlGCS-1, and an enhancer in the
OlGCS-1 upstream region can affect the
promoter activity. The above could make it possible to temporally and
spatially coordinate the transcription of
OlGCS-1 and OlGCS-1
during the embryogenesis of the medaka fish.
1-GFP construct. Scholz et
al. (35) have demonstrated that NO synthase and NO-sensitive
guanylyl cyclase are broadly distributed in the central nervous system
of lobsters at hatching. The participation of the NO/cGMP signaling
pathway in synaptogenesis has also been reported (22). Detection of GFP
fluorescence in the brain of Day 4 embryos suggests a relation between
soluble GC and neuronal development during the embryogenesis of the
medaka fish. The ratio of embryos with GFP fluorescence to live embryos
7 days after fertilization was relatively low (14.8%). Although a
higher concentration of DNA solution was able to increase the number of
embryos with GFP fluorescence, it tended to cause morphological
abnormalities and/or death (data not shown).
1. This element may
participate in the regulation of transcription with a cAMP-response
element modulator, which is known to inhibit transcription by binding to the cAMP-response element (36). There is one GATA and one MEF-2
binding sequence in the 5'-upstream region of
OlGCS-1, and it has been suggested that these
sequences may participate in the differentiation of vascular smooth
muscle cells (37-39). Considering that soluble GC induces the
relaxation of vascular smooth muscle in mammals (1), these elements may
regulate the expression of both genes in the medaka fish. In addition,
others including Sp1, C/EBP, and AP1 in the 5'-upstream region of
OlGCS-1 and OlGCS-1
may also be involved in the regulation of expression of both genes for
soluble GC subunits in the medaka fish (40).
1 are highly conserved in relation to
that of the intervening region between
OlGCS-1 and OlGCS-1
in six different regions. Some of these regions have
cis-regulatory elements in common with each other,
suggesting that the highly conserved regions also participate in
coordinated transcription.
1 and
OlGCS-1, demonstrating that the two genes are
tandemly organized like a single gene. A comparison of the genomic
structure between soluble GC and adenylyl cyclase may clarify the
evolutionary relationship between both enzymes, although the genomic
structure of the latter enzyme has not yet been reported.
1 and 2 chain genes are coordinately expressed by a bidirectional promoter (43). In this regard, our results
presented here suggest the possibility of temporally and spatially
coordinated transcription of both subunit genes for soluble GC during embryogenesis.
ACKNOWLEDGEMENTS
FOOTNOTES
1) and AB022281
(OlGCS-1).
To whom correspondence should be addressed. Tel.: 81-11-706-4908;
Fax: 81-11-706-4851; E-mail: norio-s{at}sci.hokudai.ac.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, C.,
Wang, G.-X.,
Korth, M.,
Aszódi, A.,
Andersson, K.-E.,
Krombach, F.,
Mayerhofer, A.,
Ruth, P.,
Fässler, R.,
and Hofmann, F.
(1998)
EMBO J.
17,
3045-3051[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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