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Two different precursors for secretory polypeptides from the stomach of Xenopus laevis have been characterized by cDNA cloning. Both mature polypeptides are potential candidates for gastrointestinal growth factors. One, xP1, is the X. laevis homologue of the pS2 gene product consisting only of a single P-domain, whereas the second, xP4, is a novel polypeptide formed by four P-domains arranged in tandem. Northern analysis detected both transcripts in the stomach but not in the skin or the brain. In situ hybridizations localized the expression of both precursors in surface mucous cells of the gastric mucosa. With an antibody generated against the deduced C-terminal end of xP4, the mature polypeptide was investigated by Western analysis revealing N-glycosylation of xP4.
FIG. 1. Nucleotide sequence and translation of the xPl transcript as deduced from eDNA clones
pXSP-5' -3.3 (positions 1-258) and pXSP-3' -4.1 (positions 183-483). 'fhe potential cleavage site for
signal peptidase is indicated by an arrow. 'fhe conserved tryptophan residue in the P-domain is encircled, and the
polyadenylation signal and a restriction site are underlined. Also marked are the positions of the synthetic
oligonucleotides.
FIG. 2. Nucleotide sequence and
t ranslation of the xP4 transcript as
deduced from eDNA clones pXSP-
5'-1.27 (positions 1- 730} and
pXSP-3'-2.1 (pos itions 304-854).
'fhe potential cleavage site for signal 1 peptidase is indicated by an arrow. 'fhe ,
conserved tryptophan residue in each p. j
domain is encircled, the polyadenylation .
signal, restriction sites, and the potential ~
N-glycosylation site are underlined. Also :
marked are the positions of the synthetic ;
oligonucleotides. 'fhe sequence selected '
for the synthetic peptide (XGP-1) is in · :
dicated by a dotted line.
FIG. 3. Northern analysis. Hybridization of 20 ;.tg of total RNA
from X. laeu/$ skin (lanes a and d), stomach (lanes b and e), and
brain (lanes c and f) with the radioactively labeled inserts of eDNA
clone pXSP-3'·4.1 (encoding xPL; lanes a-c) or pXSP-3'-2.5 (encoding
xP4, similar pXSP-3'-2.1; lanes d-f). As a size marker, a RNA
ladder was used (purchased from Bethesda Research Laboratories).
FIG. 4. In situ hybridizations to
sections through fundic regions of
X. laevis stomach. A, 8, C, hybridi7.a·
tion with ' H-labeled single-stranded
cRNAs representing xP1 (from eDNA
clone pXSP-3' ·4.1}; exposure time, 14
days. D, E, F, hybridization with 32PIabeled
single-stranded cRNAs repre·
senting xP4 (from eDNA clone pXSP·
5' -1.26, similar pXSP-5' -1.27); exposure
time, 5 days. Positive signals (dark/ieki
illumination) were obtained with the an·
tisense probes (A, D), but not with the
sense probes (C, F). 8, E, corresponding
phase contrast pictures to A and D. Scale
Ftc. 5. SDS-polyacrylamide gel e lectrophoresis (15%) and
s ubsequent Wester n a na lysis. Lanes a, c, d, and e, X . laeuis
stomach extract. Lane b, X. laeuis stomach extract after digestion
with glycopeptidase-F. In lanes a- d, staining with antiserum XGP-1
is shown either directly (lanes a and b) or after preadsorption with
peptide XGP·l (lane c) or peptide XGP-2 (lane d), whereas lane e
represents reaction with preimmune serum.
F1G. 6. Comparison of a ll P-domains
from X. laevis ide ntified so
far (xPl; xP4; FlM-A.l, Hoffmann,
1988; p75 k , Gmnchl et al., 1990)
and human p82 (Jakolew et al.,
1984). Invariant amino acid residues
are enclosed in boxes. 'fhe 6 characteristic
cysteine residues are numbered.
Bars mark deletions to maximize homo I·
ogy.
