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We have previously isolated a cDNA coding for a RalB protein from a Xenopus maternal library. By mutagenesis of RalB and microinjection of its mRNA in embryos we show that RalB is involved in Xenopus early development. We have used the yeast two-hybrid system to screen a Xenopus maternal cDNA library in order to isolate proteins interacting to RalB. We have identified a full-length cDNA encoding a protein homologous to mammalian smgGDS. The Xenopus gene shows 84.6% identity with the mammalian counterpart and contains one additional armadillo repeat. The XsmgGDS mRNA is expressed from oogenesis up to late embryogenesis at a higher level than that in adult tissues. Thus RalB is another protein that interacts with smgGDS which suggests that RalB may be activated by a Ras independent pathway.
FIG. 1. Expression of Xenopus RalB (XRalB) in yeast. Expression
was assayed by its binding to RLIP76 positive control. Proteins
Ral were expressed as fusion proteins to the LexA DNA binding
domain in the pLex vector containing or not a nuclear localization
sequence (NLS). The pGAD vector encoded the Gal4 activation domain
alone or fused to RLIP76 (the full-length protein of RLIP1).
Yeast strain L40 was transfected with the indicated plasmid combinations
for the two-hybrid test.
FIG. 2. Alignment of the first three armadillo repeats of Xenopus,
human and bovin smgGDS sequence. Beneath the repeats for
comparison is shown a consensus sequence (A) of arm repeats or
smgGDS and at the bottom (B) is an universal arm repeat consensus
defined by the armadillo repeat contained in five proteins (14). Numbers
correspond to the numbers of armadillo repeat. Dotted line
above the sequences indicates the position of the insert which is
absent in the mammalian homologs. The underlined letters correspond
to amino acid matches to the smgGDS consensus sequence or
the universal arm repeat.
FIG. 3. Northern blot analysis of Xenopus transcripts at different developmental stages and in various tissues from adult animals. The
positions of the transcripts hybridizing to the XsmgGDS (A) probe (2.9 Kb) and to the EF1-alpha (B) control probe (1,7Kb) are indicated. The
expression of the translational elongation factor EF1-alpha starts at the mid-blastula transition during development (24).
FIG. 4. Interaction of XsmgGDS and XRLIP76 with mutant proteins
RalA and RalB. Strain L40 transformed with a pLex construct
containing various Ral cDNAs or a negative control (Lamin) were
mated with strain AMR70 transformed with pGAD construct containing
one of the partners selected from our two-hybrid screen or a
positive control (RLIP76).
