220
N. Satoh et al.
of random oligomers. They found a 20-bp palindrome as a possible consensus sequence that binds to the Brachyury DNA-binding domain. However, this proposed
sequence has not been reported yet from in vivo studies. Interestingly, the promoter
of As-T contained the palindrome between -170 and -ISO from the transcription
start site (Fig. 4). When this consenslls sequence was deleted from As-T/lacZ, the
lacZ expression completely diminished, suggesting that this consensus sequence is
essential for As-T expression. These results suggest that the T -binding sequence of
As-T is important for enhancement of the gene expression or autogenetic activation
of As-T itsel f.
Therefore, the results of the promoter elements of As-T described above was
quite in contrast to that of Ci-Bra. Therefore, we are examining the reporter gene
expression when As-T/lacZ is injected into Ciona eggs or when Ci-Bra/lacZ is
injected into Halocynthia eggs.
5.2 Downstream Control of the Ascidian Brachyury Gene
As described in section 4, Ci-Bra ectopic expression caused an ectopic differentiation of notochord cells in the endoderm and endodermal strand precursor cells.
Northern blot analysis revealed that at least a few hundred times Ci-Bra mRNAs
were present in Ci-tkh-controlled Ci-Bra overexpressing embryos. Takahashi et al.
(unpublished) were able to make a cDNA library of mRNA'l of Ci-Bra overexpressing
embryos subtracted with mRNAs of normal embryos. The subtractive library contained 923 cDNA clones. Sequencing about SOO bp of both S' and 3' ends of all of
these clones, revealed that S99 of 923 clones were independent. Then we examined
whether the corresponding genes were actually up-regulated by Ci-Bra
overexpression. Each of the S99 clones was dot-hybridized with filters loaded with
the same amount of total RNAs of control and Ci-Bra overexpressing embryos. As
a result, SOl of the S99 eDNA clones represented genes that were up-regulated by
Ci-Bra overexpression.
Finally, Hotta et al. (unpublished) examined the spatial expression of all of the
SOl clon,es by whole-mount in situ hybridization. As a result, transcripts of SO of
the SOl clones were specific and/or predominant to the notochord cells. Sequence
analysis of S' and 3' regions suggested that polypeptides encoded by the notochordrelated genes include aryl hydrocarbon receptor nuclear translocator, reticulocalbin
precursor, ras GTPase-activating-like protein, GTP-binding protein, protein tyrosine
phosphatase, myosin heavy chain (non-muscle type), extensin precursor, ezrin/
moesin/radixin, tensin, P-selectin precursor, agrin/HSPG, collagen al (XI) chain,
sulfate transporter, ATP-citrate (pro-S-)-lyase, sulfate adenylate transferase, Nacetyllactosamine synthase, prothrombinase precursor, calcium/calmodulin-dependent protein kinase type I, 3-hydroxyacyl-coa dehydrogenase type II, f3-transduction repeat-containing protein, coatomer a subunit, SCOI protein precursor, cytochrome P4S0 IIHI (PBlS). These genes may be associated with the construction of
asci dian notochord.
N. Satoh et al.
of random oligomers. They found a 20-bp palindrome as a possible consensus sequence that binds to the Brachyury DNA-binding domain. However, this proposed
sequence has not been reported yet from in vivo studies. Interestingly, the promoter
of As-T contained the palindrome between -170 and -ISO from the transcription
start site (Fig. 4). When this consenslls sequence was deleted from As-T/lacZ, the
lacZ expression completely diminished, suggesting that this consensus sequence is
essential for As-T expression. These results suggest that the T -binding sequence of
As-T is important for enhancement of the gene expression or autogenetic activation
of As-T itsel f.
Therefore, the results of the promoter elements of As-T described above was
quite in contrast to that of Ci-Bra. Therefore, we are examining the reporter gene
expression when As-T/lacZ is injected into Ciona eggs or when Ci-Bra/lacZ is
injected into Halocynthia eggs.
5.2 Downstream Control of the Ascidian Brachyury Gene
As described in section 4, Ci-Bra ectopic expression caused an ectopic differentiation of notochord cells in the endoderm and endodermal strand precursor cells.
Northern blot analysis revealed that at least a few hundred times Ci-Bra mRNAs
were present in Ci-tkh-controlled Ci-Bra overexpressing embryos. Takahashi et al.
(unpublished) were able to make a cDNA library of mRNA'l of Ci-Bra overexpressing
embryos subtracted with mRNAs of normal embryos. The subtractive library contained 923 cDNA clones. Sequencing about SOO bp of both S' and 3' ends of all of
these clones, revealed that S99 of 923 clones were independent. Then we examined
whether the corresponding genes were actually up-regulated by Ci-Bra
overexpression. Each of the S99 clones was dot-hybridized with filters loaded with
the same amount of total RNAs of control and Ci-Bra overexpressing embryos. As
a result, SOl of the S99 eDNA clones represented genes that were up-regulated by
Ci-Bra overexpression.
Finally, Hotta et al. (unpublished) examined the spatial expression of all of the
SOl clon,es by whole-mount in situ hybridization. As a result, transcripts of SO of
the SOl clones were specific and/or predominant to the notochord cells. Sequence
analysis of S' and 3' regions suggested that polypeptides encoded by the notochordrelated genes include aryl hydrocarbon receptor nuclear translocator, reticulocalbin
precursor, ras GTPase-activating-like protein, GTP-binding protein, protein tyrosine
phosphatase, myosin heavy chain (non-muscle type), extensin precursor, ezrin/
moesin/radixin, tensin, P-selectin precursor, agrin/HSPG, collagen al (XI) chain,
sulfate transporter, ATP-citrate (pro-S-)-lyase, sulfate adenylate transferase, Nacetyllactosamine synthase, prothrombinase precursor, calcium/calmodulin-dependent protein kinase type I, 3-hydroxyacyl-coa dehydrogenase type II, f3-transduction repeat-containing protein, coatomer a subunit, SCOI protein precursor, cytochrome P4S0 IIHI (PBlS). These genes may be associated with the construction of
asci dian notochord.