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The uneven distribution of maternal mRNAs in unfertilized eggs and the unequal inheritance of these molecules by dividing blastomeres may be one mechanism for determining cell fate during embryogenesis. Complementary DNA (cDNA) clones corresponding to maternal mRNAs localized to specific regions of the Xenopus laevis egg have been previously identified and cloned [Rebagliati et al., Cell 42(1985) 769-777]. The maternal mRNA, An1, was originally identified as being localized to the animal hemisphere of X. laevis eggs and early embryos. We describe here the two proteins encoded by two An1 mRNA isoforms which we designate An1a and An1b. These mRNAs are both approximately 3.0 kb long and are concentrated in the animal hemisphere of unfertilized eggs. The predicted amino acid (aa) sequences encoded by An1a and An1b correspond to 76.9 and 78.6 kDa, respectively, and are 88% identical. Both proteins contain a single N-terminal ubiquitin (Ub)-like domain (50% identical to X. laevis Ub) and a putative Zn(2+)-binding region near the C terminus. Unlike Ub polyproteins and most Ub fusion proteins, the N-terminal Ub-like domain found in the An1 proteins does not undergo proteolytic processing. In contrast to earlier studies showing that the An1 mRNA represents a strictly maternal transcript, we report that both related An1 transcripts are found in later embryonic stages and in all adult tissues tested.
Fig. I. Restriction maps of Anfa and Anlb cDNA clones and alignment
of the two deduced proteins. (A) Restriction maps of Ada and An/b
are shown. The nearly full-length Ada and Anlb cDNAs are approximately
2.9 kb and 2.8 kb and encode proteins predicted to be 76.9 kDa
and 78.5 kDa, respectively. (B) An alignment of the two proteins
encoded by the Ada and Anfb mRNAs according to the algorithm of
Needleman and Wunsch (1970) is shown. Solid vertical lines indicate
exact aa matches; single and double dots indicate conservative replacements.
The shaded box (aa 28-103) shows the region of Ub homology
found in both proteins. The open box (aa 625-693 in An la; aa 633-701
in Anl b) indicates homology to the PvPR3 protein from Phaseolus
vulgaris which includes the putative Zn 2â-finger-metal-binding domain
common to both proteins. Underlined aa are possible PEST sequences
identified using the PEST-FIND program (Rogers et al., 1986). Ada
and Anlb have GenBank accession Nos. LO8474 and L08475. Methods:
cDNA clones were isolated from a phage hgt IO oocyte cDNA library
by screening with the original 650-bp An1 isolate (Rebagliati et al.,
1985). Additional clones were obtained from the same oocyte library by PCR (Friedman et al., 1990) and RT-coupled PCR amplification of
oocyte RNA (Kawasaki, 1990). DNA sequences were obtained for both
strands by the dideoxynucleotide chain-termination procedure (Sanger
et al., 1977) using [a-âS]dATP and the Sequenaseâ Version 2.0 kit
(Tabor and Richardson, 1987). All nt and aa sequences were analyzed
using the sequence analysis software package of the Genetics Computer
Group, Madison, WI (Devereux et al., 1984).
Fig. 2. Comparison of Anla (a) and Anlb (b) Ubil sequences to
Xenopus Ub (u) and Anla and Anlb C-terminal sequences to the
C-terminal sequences of PvPR3 (p) from Phaseolus vulgaris. (A) The aa
sequences of Anla and An1 b (aa 28-104) compared to the 76 aa of
Xenopus Ub. Solid vertical lines indicate exact aa matches; single and
double dots indicate conservative replacements according to the
method of Needleman and Wunsch (1970). Both Anl isoforms have
about 50% identity to Xenopus Ub. Considering conservative substitutions,
Anla is 65% similar, and Anlb is 67% similar. (B) The aa
sequences of Anla (aa 625-693) and Anlb (633-701) compared to the
PvPR3 protein from Phaseolus vulgaris (aa 70-137). Both An I proteins
have about 50% identity to this novel protein. This C-terminal region
in all three proteins encodes a possible metal-binding domain.
Fig. 4. The Ubil domains of An la and An I b do not undergo proteolytic
processing in rabbit reticulocyte lysates. Separation of in vitro rabbitreticulocyte-
synthesized proteins by SDS-PAGE is shown in panel A.
Translation of sequences encoding the full-length Anla protein are in
lane I. A mutant form of the Anla protein that lacks the N-terminal
Ubil domain is in lane 2; this translation product serves as a size standard
for the remaining C-terminal polypeptide of a proteolytically processed
protein. Panel B (lane I) shows the separation of translation
products from a mRNA that contains two contiguous Ub-encoding
sequences (Agell et al., 1988) on a 0.1 M phosphate:6 M urea/O.l%
SDS/l 5% polyacrylamide gel system (Ley, 1984). The results indicate
that the diubiquitin polypeptide was proteolytically processed to form
a Ub monomer (about 8.5 kDa). No unprocessed Ub dimers. expected
to be about I7 kDa, were detected. Lanes 2 and 3 demonstrate that
small molecular weight polypeptides from the translation of pSP64TAnla
(lane 2) and pSP64T-AnlaAUbil (lane 3) were not detected. The
asterisk at about I4 kDa indicates globin protein. Methods: The An/
sequences translated in vitro were cloned into the translation vector
pSP64T (Krieg and Melton, 1984). The plasmids pSP64T-Anla and
pSP64T-AnlaAUbil were used in a Promega (Madison, WI) TNTTM
SP6 transcription-translation reticulocyte The insert
in pSP64T-AnlaAUbil the first nt of An/a coding
and therefore not encode N-terminal Ubil
This insert pSP64T was by PCR of the
codingsequencesusingtheSprimer(S-CCCCAG ATCTACCATGG
CAATTAATACACGCAGAGTGCCG)andthe3âprimer(Sâ-CCCAG
AGATCTAGATTTTTGGAAGTTTG).TheSâoligoprimerencodesa
Proââ4+Ala replacement and also encodes a consensus eukaryotic
translation initiation site just prior to this new Alaâ@â(Kozak, 1984a.b).
Both of these primers contain an engineered BgllI site which facilitated
cloning of the PCR-generated fragments into an unique Bg/ll site in
pSP64T. Approximately I pg of each circular plasmid was added
directly to the reticulocyte lysates (25 pl reaction volume) containing
45 pCi of [âSJmethionine with a specific activity of II20 Ciimmol
(present in Tran3âS-label), After incubation for 2 h at 30 C, the translation
products were separated by 0.1% SDS- 10% PAGE (Laemmli.
1970) or by a 0.1 M phosphate/6 M urea/O.l% SDS/15% polyacrylamide
gel system (Ley, 1984). The gel was fixed in 50% methanol/IO%
acetic acid for 30 min. soaked in distilled water for 30 min, Fluoro-
HanceTM for 30 min. dried, and exposed to Kodak XAR-5 film at
- 70â c.
