Download transcription initiation sites and genomic Southern blot (Fig. 7

transcription initiation sites and genomic Southern blot (Fig. 7)
suggest that there were more than two IGF-1 genes (IGF-1a and
IGF-1b) found in zebrafish.
2. One form proIGF-1
Ea-2 mRNA is expressed
Compared with that of other teleosts, the sequence analysis
of zebrafish preproIGF-1 cDNAs shows homologous to the IGF-1 Ea-2
form. For further elucidation of zebrafish IGF-1 genomic structure
in E domain, we have cloned IGF-1 prohormone cDNAs in the liver
by RT-PCR. After RT-PCR, two expected bands with size of 366 bp
(IGF-1 Ea-1) and 402 bp (IGF-1 Ea-2) were detected in all organs
and hatching embryos after electrophoresis on 2.5% agarose gels
stained with ethidium bromide (EtBr). At other developmental
stages, the RT-PCR products of embryo were detected only one band
of 402 bp. For further confirming the accuracy of bands, the
transferred blots were hybridized with 5’ end
32
P-labeled zebrafish
IGF-1 Ea-2 cDNA probe. Although the EtBr stained gels showed two
52
bands, the Southern blot data revealed only one form (IGF-1 Ea-2)
expression in all organs and at all embryo developmental stages
besides at zygote and cleavage periods (Fig. 2 a, b). In the
meantime, these two bands were also eluted from liver after gel
separation and further cloned into PCR-Blunt (Invitrogen) and
eleven clones were sequenced. The sequencing data showed 402 bp
and 366 bp represent zebrafish IGF-1 Ea-2 cDNAs and non-specific
sequence insert, respectively. Conclusively, IGF-1 Ea-2 mRNA was
a predominant form in embryo developmental stages and bisexual of
zebrafish.
In addition, the zebrafish IGF-1 Ea-2 transcripts were
regulated by growth hormone, prolactin, insulin and fasting. After
RT-PCR, the amplified products were detected two expected bands
of 366 bp and 402 bp by electrophoresis on 2.5% agarose gels in
all zebrafish organs and embryos after hatching, corresponding to
IGF-1 Ea-1 and Ea-2, respectively. At other developmental stages,
the RT-PCR products of embryo were detected only one band of 402
53
bp. For further confirming the accuracy of bands, the Southern blot
was hybridized with
32
P-labeled zebrafish IGF-1 Ea-2 cDNA. Although
the gel revealed two bands, the Southern blot data revealed the
IGF-1 Ea-2 expression in all organs and embryo at developmental
stages, and not detected at zygote and cleavage periods (Fig. 2.
a, b). In the meantime, these two bands were eluted from gel and
further cloned and sequenced to verify. The sequencing data showed
402 bp and 366 bp products represent the zebrafish IGF-1 Ea-2 cDNA
and an PCR artifact insert, respectively. Furthermore, total RNA
extracted from zebrafish liver after hormone (oGH, oPRL or bIns)
or fasting treatment, the RT-PCR amplified products revealed two
bands (366 and 402 bp) by electrophoresis on 2.5% gel followed by
Southern blotting with 5' end
32
P-labeled zebrafish IGF-1 Ea-2 cDNA
(Fig. 9). These two bands were also further analyzed by cloning
and sequencing as same as above. The sequencing data showed 402
bp and 366 bp represent the zebrafish IGF-1 Ea-2 cDNA and an PCR
artifact insert, respectively. All each RT-PCR group includes the
54
direct amplification from RNAs to serve as negative controls.
Conclusively,
zebrafish IGF-1
Ea-2 mRNA
was
only one
form
expression even the hormone treatment or fasting condition.
3. Biological
activity
of Synthetic
zfEa-2
polypeptide
Owing to only one form expression Ea-2 mRNA (exon 3 - 4 5) in zebrafish, we proposed the zebrafish Ea-2 polypeptide of
proIGF-1 Ea-2 may have some biological functions in zebrafish.
First, we have tested the hypothesis that synthetic polypeptide
of E domain would exhibit [35S]sulfate uptake in cartilage matrix.
For evaluation the [35S]sulfate uptake method is work, we
pretest the tilapia cartilage sulfation. The best sulfate uptake
region is located near the gill filament site (Fig. 10). The
dose-response of zfEa-2 polypeptide, hIGF-1, hEc and bIns on the
sulfation of tilapia cartilage is shown in Fig. 11. The activity
of zfEa-2 was examined in the gill [35S]sulfate uptake of zebrafish
with a maximum effect of 3-fold over control in vitro, whereas the
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