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Gene
1998 Mar 27;2101:127-34. doi: 10.1016/s0378-1119(98)00052-3.
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XL43 and XL75: two novel RING finger-containing genes expressed during oogenesis and embryogenesis in Xenopus laevis.
Perrin K
,
Lacroix JC
.
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This article reports on the isolation of two new Xenopus genes that encode two putative zinc finger proteins. The XL43 and XL75 proteins belong to the RBCC family, and also contains the rfp-like domain. XL43 and XL75 RNAs are found in the ovary, and myc-tagged proteins are detected in mRNA injected oocytes. Whole-mount in situ hybridization of embryos revealed that these two clones are expressed exclusively in neurectodermic and mesodermic tissues. The data suggest that XL43 and XL75 genes could play an important role in the frog's early development, perhaps as a transcription factor. This RBCC family, a subclass of the RING finger family, comprises proteins with known cellular transformation properties. All members possess, besides one RING finger motif, one or two B-boxes, each having a pair of zinc fingers, and a coiled coil domain (Borden, K.L. B. et al., 1996. Proc. Natl. Acad. Sci. USA 93, 1601-1606). Among this group, some members possess, besides the RING-B box-coiled coil (RBCC) motifs, a long C-terminal domain referred to as the rfp-like domain. Although its effective role has not been elucidated yet, this last domain could play an important role by binding a ligand (Bellini, M. et al., 1993. EMBO J. 12, 107-114).
Fig. 1. Nucleotide and deduced amino acid sequences of XL43 (Fig. 2) and XL75 (Fig. 3) determined from pBluescript cDNA clones. Five regions
are shown, from the N-terminal to C-terminal end: the zinc finger motifs RING, B1 and B2 are underlined and the conserved cysteine and histidine
residues possibly involved in metal binding formation are circled. The coiled coil motif is boxed, whereas the rfp-like domain is double underlined.
In the XL43 clone, the polyadenylation sequence is boxed. The XL43 and XL75 sequences are available under GenBank accession no. U63817
(XL43) and U63818 (XL75).
Fig. 2. Northern blot analysis of XL43 and XL75 mRNAs. Ovarian
total RNA (10 mg) of either Xenopus or Pleurodeles was separated on
formaldehyde–agarose gel, transferred to a 0.2 mm nitrocellulose mem-
brane, and hybridized with cDNAs transcribed in vitro from clone
XL43 and XL75. Bands at #2.5 kb (XL75) and #2.1 kb (XL43) are
seen in RNA from Xenopus but not Pleurodeles. A positive control
was conducted in the same conditions with a PwA33 probe, which
hybridizes as expected with a #2.7 kb band in RNA from Pleurodeles but not Xenopus (Bellini et al., 1993).
Fig. 3. Diagrammatic representation of the conserved zinc fingers, coiled coil domain and the rfp-like domain in the RBCC-containing family.
PwA33 (Bellini et al., 1993), Xnf7 (Reddy et al., 1991), SSA/Ro (Chan et al., 1991), Rfp (Takahashi and Cooper, 1987), AFP (Chu et al., 1995),
Staf50 (Tissot and Mechti, 1995) and RFB30 (Henry et al., 1997) possess a unique B box. Rpt-1 (Patarca et al., 1988) presents a frameshift
mutation. In T18/TIF1 (Miki et al., 1991), PML/Myl (de The´ et al., 1991) and h.Efp (Inoue et al., 1993) there are two B box-like zinc finger
domains, B1 and B2. Based on statistical analysis (Reddy et al., 1992), B2 is shown aligned with the Xnf7 and PwA33 B box.
Fig. 4. Capped in vitro transcripts from linearized 6-myc-XL43 or
6-myc-XL75 clones were injected into Xenopus or Pleurodeles oocytes
A positive control was realized by injecting 6-myc-kinase transcripts.
GV and cytoplasmic proteins were extracted 48 h later, electrophoresed
a 8% polyacrylamide gel and transferred to a PVDF filter. Myctagged
products were detected with mAb 9E10 by chemoluminescence.
A unique band appears in both GV and cytoplasm lanes for myc-
XL43 with an unexpected Mr of 32 000. Myc-XL75 protein is detected
in the cytoplasmic lane with a Mr of 69 000.
Fig. 5. Localization of XL43 and XL75 mRNA in early Xenopus embryos by whole-mount in situ hybridization. (A) Antisense riboprobes. 1:
Lateral view of a yolk plug stage embryo (St. 11.5) hybridized with XL75 probe. The dorsal ectodermic region is essentially labelled and the yolk
plug is totally negative. 2: Dorsal view of a neurula (St. 18) labelled with XL75 probe. XL75 mRNA is localized in the neurectoderm and neural
plate. 3,4: Lateral view of tailbud stages (St. 32–33) hybridized with XL43 probe (3) and XL75 (4) probe. In each case, mRNA is present essentially
in mesodermic tissues, in the head, somites and notochord. (B) Sense probes. Lateral view of a yolk plug stage embryo (St. 11.5) hybridized with
XL75 probe (1), a neurula (St. 19) labelled with XL43 probe (2), and a tailbud stage (St. 33) hybridized with XL75 probe (3). Only background
staining is observed.
xelaev18032656m (___) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anteriorleft, dorsal up.
trim7 (tripartite motif containing 7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anteriorleft, dorsal up.
