Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Gene
1996 Oct 10;1751-2:167-72. doi: 10.1016/0378-1119(96)00143-6.
Show Gene links
Show Anatomy links
cDNA cloning and its pronephros-specific expression of the Wilms' tumor suppressor gene, WT1, from Xenopus laevis.
Semba K
,
Saito-Ueno R
,
Takayama G
,
Kondo M
.
???displayArticle.abstract???
The Wilms' tumor suppressor gene, WT1, plays a crucial role during early urogenital development in mammals. To elucidate the function of WT1 in other vertebrates, we isolated the Xenopus WT1 homolog (XeWT1) from a testis cDNA library. Comparison of the XeWT1 protein with other WT1 proteins revealed that the zinc finger domain of XeWT1 is identical to that of the human WT1 except for the first two amino acid residues. An alternative splice II site, located between the third and the fourth zinc finger is also conserved. In the transcriptional regulatory region, however, three domains, a glycine stretch, a proline stretch and one alternative splice site which are present in the mammalian WT1, are not conserved in amphibians. The XeWT1 gene is expressed in testis and kidney, and whole mount in situ hybridization revealed that the onset of the WT1 gene expression coincides with the early stages of pronephros development in Xenopus. These findings implicate the involvement of WT1 protein during urogenital development in amphibians as well as in mammals.
Fig. 1. Primary structure of Xenopus WT1. (A) Nucleotide and
predicted aa sequence of Xenopus WT1. Nucleotide numbers are
indicated on the sequence, and aa are numbered at the right. An
in-frame stop codon in the 5'-non-coding region is underlined.
Oligonucleotides NI: 5'-CGCGGATCCGTACGGTCGGCATC-
(AGCT)GA(AG)AC-3', N2: 5'-CGCGGATCCAGATATTTTAAGCT-
(AGCT)TC(AGTC)CA-Y, Ch 5'-CGCCTCGAGCTGATGCAT-
(AG)TT(AG)TG(AG)TG-3' and C2: 5'-CGCCTCGAGGGTCATGTT(
CT)CT(CT)TG(AG)TG-3' were used for RT-PCR amplification
of the Xenopus WT1 sequence. PCR reactions were performed
using cDNA prepared from 0.05pg of poly(A)+RNA with the
following conditions: one cycle of 95°C for 5 min, 45°C for 2 min,
72°C for 3 min and 95°C for 2 rain, 35 cycles of 45°C for 1 min, 72°C
for 1.5 rain and 95°C for 0.5 min, and one cycle of 45°C for 2 rain,
72°C for 10 min and 5°C for 5 min. One tenth of the PCR
reaction mixture was used for the second round of PCR under
the same conditions as described above. The oligonucleotide,
Y-CA (CY) AC (AGCT) GG (AGCT) GA (AG) AA (AG) CC (AGCT)-3'
was used as a probe for Southern blot hybridization. Hybridization
and washing conditions were as described (KOster et al., 1988). The
positive fragment was cloned and sequenced to confirm the presence
of a WTl-like sequence. Using this DNA as a probe, Xenopus WT1
cDNA No. 65 was isolated from X. laevis testis cDNA library
(constructed with TimeSaver cDNA synthesis kit, Pharmacia Biotech
and Lambda ZAP II/EcoRI/Gigapack Cloning Kits, Stratagene).
Sequencing of both strands of the cDNA clone was carried out using
a deletion kit for kilo-sequencing (Takara Biochemicals) and an
automated DNA sequencer (Applied Biosystems). Sequence was
analyzed by the Genetyx-Mac program (Software Development Co.,
Ltd.). The nt sequence reported in this paper has been submitted to
the DDBJ, EMBL and GenBank nt sequence databases with accession
No. D82051. (B) +KTS and -KTS variants. Comparison of Xenopus
WT1 cDNA No. 65 (-KTS form) and cDNA No. 37 (+ KTS form).
Asterisks indicate identical aa and dashes indicate absence of a residue
at each position.
Fig. 2. Amino acid alignment of vertebrate WT1 proteins. Predicted
aa sequences of human (Gessler et al., 1990), mouse (Buckler et al.,
1991), rat (Sharma et al., 1992), chick (Kent et al., 1995), alligator
(Kent et al, 1995) and zebrafish (Kent et al., 1995) WT1 are compared.
Identical aa are indicated by dots and absent residues by dashes. Aa
residues labeled with asterisks are described in the text.
Fig. 3. Expression of the Xenopus WT1 gene in adult frog tissues.
Poly(A) + RNA was isolated from organs of adult frogs using ISOGEN
(Nippongene) and Oligotex-dT30 (Takara Biomedicals). 3pg of
poly(A)+RNAs were electrophoresed in 1% agarose gel containing
2.2 M formaldehyde and then subjected to blot hybridization as
described. A 1.5-kb NotI fragment of clone No. 65 was labeled with
3/p using Megaprime DNA labeling systems (Amersham). Arrow
indicates WT1 transcript. Bars indicate mobility of molecular weight
markers, 9.49, 7.46, 4.40, 2.37, 1.35 kb (RNA Ladder, Gibco BRL).
The autoradiogram was exposed for 5 days.
Fig. 4. Spatial and temporal distribution of Xenopus WT1 transcript
during embryogenesis. (A) Results from a tailbudembryo, stages 30,
26, 25 and 23 (from top to bottom): pronephros anlage was stained
in purple. (B) Tailbud, stage 30: both sides of pronephros were stained.
(C) Sense probe did not detect any specific regions. WT1 sense and
antisense probes were generated by in vitro transcription in the
presence of rUTP-digoxigenin (Boehringer Mannheim) using T3 and
T7 RNA polymerases (Promega). The length of both probes was
1.5 kb. Hybridization was performed according to the previously
described method (Harland, 1991), with the following modifications.
Wild type embryos were bleached with 6% hydrogen peroxide after
rehydration and then hybridized at 60°C in a buffer containing 50%
formamide, 5 × SSC, 1 mg/ml torula RNA, 100 mg/ml heparin,
1 xDenhardt's, 0.1% Tween 20, 0.1% CHAPS and 10mM EDTA.
After hybridization, embryos were washed at 60°C with 0.2 × SSC, at
60°C with 0.2 × SSC, and then at RT with maleic acid buffer (100 mM
maleic acid, 150 mM NaC1, pH 7.5). Alkaline phosphate-coupled anti-
DIG antibody and BM Purple (Boehringer Mannheim) were used for
detection of the probes. Stained embryos were postfixed in MEMPFA
and cleared with benzyl benzoate/benzyl alcohol (2:1) and
photographed.
