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Cloning and expression of cDNA encoding Xenopus laevis bone morphogenetic protein-1 during early embryonic development.
Maéno M
,
Xue Y
,
Wood TI
,
Ong RC
,
Kung HF
.
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The Xenopus laevis DNA fragment encoding a protein homologous with human bone morphogenetic protein-1 (BMP-1) was amplified by polymerase chain reaction (PCR) from cDNA derived from stage 26 (st.26) embryonic RNA. Subsequently this fragment was used as a probe to isolate cDNA clones by screening of a X. laevis st.24 embryonic cDNA library. Two partial clones (22 and 63) were obtained and the missing 5'-end of the clone 22 was extended by the anchored PCR technique. The nucleotide sequence of the resulting clone (22AN) contained an open reading frame coding for a protein with 707 deduced amino acids. Three sizes of mRNA (2.9, 5.2 and 6.6 kb) were detected in blastula (st.9) and early gastrula (st.10) embryos, and in hatched tadpole (st.40), but little or no expression was observed in morula (st.7) and late gastrula (st.12) embryos, suggesting a physiological role(s) of X.laevis BMP-1 in normal embryonic development.
Fig. 1. Restriction maps of clones 22,63, and 22AN. Open box in 22AN
represents the predicted translated region. E, EcoRI; X, XhoI; S, SmaI;
Sa, Sucl; K, KpnI; H, HincII. The putative domain structure including
the metallo-protease domain (M), the EGF-like domain (E), and three
repeats of the complement lr/ls-like domains (BllB3) is shown below.
The triangles below mark the potential glycosylation sites. The aa numbers
are indicated above. cDNA was synthesized from 5 ug of total
RNA from St.26 embryos (Nieuwkoop and Faber, 1967) using the
Superscript Preamplification Kit (BRL). The reaction mixture (20 ~1)
consisted of 50 ng random hexamers/20 mM TrisHCl (pH 8.4)/50 mM
KCl/2.5 mM MgClJO.1 mg BSA per ml/10 mM DTT/O.S mM dNTP/
200 units Superscript reverse transcriptase. The PCR was then performed
in a 50 ~1 reaction mixture containing 10 mM TrisHCl
(pH 8.3)/50 mM KCl/1.5 mM MgCl,/O.OOl% gelatin/O.2 mM
dNTP/lOO mM primers (5’-AATTGGATCCATCTCCATYGGMAAGAAYTGYGAMAAGTT,
P2 in Fig. 2; and 5’-AATTAAGCTTAGAATGTGTTCCGMGCRTARTGCATRATG,
P4 in Fig. 2; where Y = C
or T, M = A or C; and R = A or G), 10 ul of the cDNA reaction mixture
described above, and 2.5 units of Tuq polymerase. The amplified product
was separated on an agarose gel, and the resulting ethidium bromide-
stained band was excised from the gel. Approximately l/l00 of
the DNA sample was again amplified with the same set of primers. The
PCR product was cut with Hind111 and BarnHI and cloned in Bluescript
(Stratagene, La Jolla, CA, USA).
Fig. 2. The nt and deduced aa sequences of clone 22AN (see Fig. 1). Potential glycosylation sites are indicated by triangles. The primer regions used
for PCR amplification are underlined (Pl-P4). Consensus sequences of the metallo-protease and Asp/Asn hydroxylation sites are indicated by
diamonds and circles, respectively. Conserved Cys residues found in human complement Clr and Cls, human BMP-1, and Drosophila tolloid are
boxed. The possible polyadenylation signals are shown with double-underlined boxes. The GenBank accession number for the sequence reported in
this article is L12249.
Fig, 3. Northern blot analysis of Xenopus mRNA encoding BMP-1 (A)
and EF1a (B) in Xenopus embryos. The embryos were staged according
to Nieuwkoop and Faber (1967). Total RNA from the different embryonic
stages were extracted by the guanidine thioc~nate-phenolchloroform
method (Chomczynski and Sacchi, 1987) using a RNA isolation
kit (Stratagene). Poly(A)+RNA was separated on an oligo(dT)
column, denatured with 6 M glyoxal at 50°C for 1 h. electrophoresed
in a 1.2% agarose gel, and transferred to a nylon membrane (Ausubel
et al.. 1987). Hybridization was performed in Hybrisol I (Oncor) at
42 ‘C with 2.2-kb EcoRI fragment of clone 22 labeled with [r-“*P]dtTP
by nick translation, and the filters were washed at 60 C with
0.1 x SSC/O.l% SDS, and exposed to Kodak XAR-5 film at -70 C.
Sizes (kb) of marker RNAs and of XBMP-1 mRNAs are shown at the
left and right margins, respectively. The same filters were rehybridized
with the Sstl-PstI fragment (378 bp) of EFlcl (Krieg et al., 1989). SSC
is 0.15 M NaCl/O.OIS M Na,citrate, pH 7.6.
