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DEVELOPMENTAL DYNAMICS 211:123–130 (1998)
Characterization of Cellular Nucleic Acid Binding Protein
From Xenopus laevis: Expression in All Three Germ Layers
During Early Development
IRWIN L. FLINK,1* IRA BLITZ,2 AND EUGENE MORKIN1,3,4
of Medicine, University of Arizona Health Sciences Center, University Heart Center, Tucson, Arizona
2Department of Developmental and Cell Biology, and the Developmental Biology Center, University of California,
Irvine, California
3Department of Physiology, University of Arizona Health Sciences Center, Tucson, Arizona
4Department of Pharmacology, University of Arizona Health Sciences Center,
Tucson, Arizona
1Department
ABSTRACT
The Xenopus CNBP homologue
(XCNBP) has been cloned from stage 14 neurula.
XCNBP encodes a 18.4-kDa protein containing
seven highly conserved zinc finger (Zn-finger)
repeats (CX2CX4HX4CX2), with sequence similarity to human, mouse, rat, and yeast CNBP. A
unique feature of XCNBP is that it contains a 10
amino acid (aa) deletion in the linker region
between Zn-fingers 1 and 2, immediately downstream from an alternatively spliced exon of human CNBP isoforms. A similar deletion is found
in mouse and yeast CNBP proteins. The deleted
region lacks potential PEST and casein kinase II
phosphorylation sites. Because CNBP proteins
from a variety of species contain deletions in a
similar region, these results suggest that the
pattern of alternative processing of CNBP isoforms is highly conserved among metazoa and
unicellular eukaryotes. XCNBP RNA is initially
maternally derived and is widely expressed
throughout early development at the gastrula,
neurula, and tailbud stages. At the early gastrula
stage, XCNBP is expressed in ectodermal, endodermal, and mesodermal germ layers. Previous
data have demonstrated the presence of CNBP in
the cytoplasm and nucleus. The interactions of
CNBP with single-stranded DNA and RNA suggest that CNBP may serve dual functions in
transcriptional and translational regulation in a
wide variety of tissues during development. Dev.
Dyn. 1998;211:123–130. r 1998 Wiley-Liss, Inc.
Key words: alternative processing; in situ hybridization; single stranded DNA; transcriptional and translational regulation; zinc finger
INTRODUCTION
The physiological and biochemical properties of cellular nucleic acid binding protein (CNBP) are largely
unknown. Initially, CNBP was postulated to function as
a negative transcriptional factor through its strong
interaction with a sterol regulatory element (SRE)
r 1998 WILEY-LISS, INC.
found within the promoter region of genes involved in
the coordinate control of cholesterol metabolism (Rajavashisth et al., 1989). Although addition of sterols has
been associated with altered levels of CNBP mRNA in
cell culture (Rajavashisth et al., 1989; Torres et al.,
1994), recent evidence suggests that CNBP is not
involved in the maintenance of cholesterol levels. Specifically, in cotransfection studies, CNBP expression
does not repress hydroxymethylglutaryl coenzyme A
(HMG CoA) reductase, the rate-limiting enzyme in
cholesterol biosynthesis. Changes in dietary cholesterol
do not alter CNBP mRNA expression (Warden et al.,
1994). Additionally, a basic-helix-loop-helix-leucine zipper transcriptional factor, SREBP-1, that interacts
specifically with the SRE found within the low-density
lipoprotein (LDL) has been cloned (Briggs et al., 1993;
Yokoyama et al., 1993). Overexpression of SREBP-1
was found to activate transcription of reporter genes
containing SRE, suggesting that CNBP may not interact with this domain in vivo.
CNBP has also been shown to interact with a repressor domain in the human cardiac b-myosin heavy chain
(b-MHC) gene (Flink and Morkin, 1995a). The human
CNBP gene encodes two alternatively processed isoforms, a and b (Flink and Morkin, 1995a). CNBPa may
function as a negative transcriptional regulator by
binding to the promoters of target genes containing
guanine-rich sequences related to the SRE that are
Abbreviations used: aa, amino acid; bp, base pair(s); β-MHC,
β-myosin heavy chain; Byr3, yeast CNBP protein; CNBP, cellular
nucleic acid binding protein; CNBPa and CNBPβ, human cellular
nucleic acid binding protein isoforms; XCNBP, Xenopus cellular nucleic
acid binding protein; HMG CoA, hydroxymethylglutaryl coenzyme A;
nt, nucleotide(s); LDL, low-density lipoprotein; PCR, polymerase
chain reaction; rp, ribosomal protein(s); RT, reverse transcriptase;
SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; SRE, sterol
regulatory element; UTR, untranslated region; XPO, Xenopus posterior gene; Zn-finger, zinc finger.
Grant sponsor: Arizona Disease Control Research Commission;
Grant number: 9411; Grant sponsor: National Institutes of Health;
Grant number: PO1 HL20984.
*Correspondence to: Irwin L. Flink, University of Arizona Health
Sciences Center, University Heart Center, 1501 N. Campbell Avenue,
Tucson, AZ 85724. E-mail: iflink@u.arizona.edu
Received 15 May 1997; Accepted 24 October 1997
124
FLINK ET AL.
found within the human β-MHC promoter (Rajavashisth et al., 1989; Flink and Morkin, 1995a). CNBPa was
shown to downregulate β-MHC gene expression in
cotransfection studies using cultured heart cells. By
contrast, CNBPb appears to have a null function,
because this isoform is incapable of regulating b-MHC
transcription. The fact that CNBP preferentially binds
single-stranded DNA (Rajavashisth et al., 1989; Flink
and Morkin, 1995a; Michelotti et al., 1995) and has
strong homology with RNA binding retroviral nucleocapsid proteins (Rajavashisth et al., 1989) suggests that
CNBP may also interact with RNA. A recent study
(Pellizzoni et al., 1997) has demonstrated that CNBP
participates in a protein complex that interacts with
the untranslated region (UTR) of ribosomal protein
mRNAs. Although localization of CNBP to the cytoplasm (Warden et al., 1994) is consistent with these
data, the purification of CNBP from nuclear extracts
(Michelotti et al., 1995) and its possible role in gene
repression also suggest that CNBP may have dual roles
in translational and transcriptional control.
The human CNBP gene extends about 6.9 kbp (Flink
and Morkin, 1995b) and is located on the long arm of
chromosome 3 (Lusis et al., 1990). Southern blot analysis demonstrates that the human CNBP gene is a single
copy consisting of five exons, four of which contain
coding information for two alternatively spliced products, CNBPa and β. The β isoform arises from the
differential utilization of an internal 5’ donor site
located within exon 2. In the mouse, two genetic loci
have been identified on chromosomes 5 and 6 (homologous to human chromosome 3) (Warden et al., 1994)
that encode multiple cDNAs homologous to human
CNBPa and β (Flink and Morkin, 1995a).
CNBP is expressed in a wide variety of tissues,
including heart, liver, kidney, lung, and brain, with the
highest levels being found in testes and adrenal glands
(Rajavashisth et al., 1989). Approximately equal
amounts of a and b isoforms are present in heart and
liver (Flink and Morkin, 1995a). Although the role of
CNBP in mammals remains unclear, studies in yeast
have shown that Byr3, a protein highly homologous to
CNBP, can suppress sporulation defects in cells that
lack ras1 (Xu et al., 1992). Additionally, a gene found in
Xenopus, called posterior, contains a zinc finger (Znfinger) homologous to CNBP. The protein product of
posterior is expressed during early development starting after the midblastula transition (Sato and Sargent,
1991).
To date, no information is available concerning the
expression pattern of CNBP during development. In
this report, the Xenopus homologue of CNBP (XCNBP)
has been cloned, and its homology among various
species and its developmental expression pattern are
described. XCNBP is highly homologous to the b isoform of CNBP found in humans, suggesting that similar patterns of alternative processing have been conserved within these two species. Northern blot and in
situ hybridization studies show that CNBP is detected
in the unfertilized egg, where it is maternal in origin,
and is expressed in cells derived from early ectoderm,
endoderm, and mesoderm during development. CNBP
continues to be expressed during late embryogenesis in
a wide variety of cell types.
RESULTS AND DISCUSSION
Molecular Cloning of a Xenopus Homologue
of Mammalian CNBP
A Xenopus lgt11 embryo (stage 14 [neurula]) cDNA
library was screened under moderately stringent conditions with a CNBPa probe (Rajavashisth et al., 1989)
encoding Zn-finger domains 3 through 7. Seven positive
plaques were identified, and the clone containing the
longest insert was analyzed. The nt sequence demonstrates strong homology to human CNBPa (65.9%) and
CNBPβ (66.4%). The deduced amino acid (aa) sequence
encodes a protein containing 168 aa (Mr 18.4 kD). The
ATG translation initiation triplet and TAA termination
codon are located at nt positions 150-152 and 654-656,
respectively. A polyadenylation signal is found 792 nt
downstream from the translation termination codon at
nt position 1446. A unique feature of XCNBP mRNA is
that it contains a long poly-T stretch in the 5’-UTR
beginning at nt position 53, which is not present within
either of the human CNBP isoforms. Translation of the
antisense strand of XCNBP encodes a putative protein
of 144 residues (Mr 15.1 kD). A computer search of the
Swiss-Prot data base did not reveal any homology of
this sequence to known proteins.
A greater degree of homology between XCNBP and
the human CNBP isoforms is observed at the aa level
than in their nt sequences. Thus, the deduced aa
sequence of XCNBP is 94.1- and 92.9% identical with
CNBPa and CNBPβ, respectively. Compared with
CNBPa and β, XCNBP contains a Thr to Ser substitution within the first Zn-finger (aa 12, Fig. 1) and two
additional substitutions within each of the linker regions between Zn-finger domains 3 and 4 (aa 82-83) and
4 and 5 (aa 107-108). Additionally, XCNBP has a Gly
insertion (aa 30); Gly to Met (aa 29) and Gly to Ser (aa
32) substitutions in the linker region between Znfingers 1 and 2; and a deletion of a Pro residue
corresponding to aa position 51 of CNBPa. A deletion of
10 aa occurs within XCNBP, immediately downstream
from a region that is removed by alternative processing
of human CNBPβ and the corresponding mouse and
yeast CNBP mRNAs. The seven aa deletion in CNBPβ
may be responsible for its null function in regulation of
b-MHC gene expression.
Figure 1 shows aa sequence alignments of XCNBP,
human CNBP a and β, a mouse isoform (Warden et al.,
1994), and yeast Byr3 (Xu et al., 1992). These comparisons suggest that Xenopus, mouse, and yeast cDNAs
correspond more closely to CNBPβ, because each of
these proteins contain a deletion within the first linker
CELLULAR NUCLEIC ACID BINDING PROTEIN
125
Fig. 1. Pairwise aa sequence alignments of XCNBP, human CNBPa
(Rajavashisth et al., 1989) and β (Flink and Morkin, 1995a), MCNBP15
(mouse CNBP, Warden et al., 1995), and Byr3 (Xu et al., 1992) (aa are
denoted in single-letter code). Positions of sequence identity are indicated
by a vertical line. Gaps introduced to generate this alignment are
represented by dashes. XCNBP, MCNBP15, and Byr3 contain a deletion
in the linker region between Zn-finger domains 1 and 2 in a similar position
as the deletion present within CNBPβ (deletion corresponds to aa
positions 36-42 of CNBPa). Other isoforms of mouse are similar to human
CNBPa and β (data not shown, see Warden et al., 1995). Rat CNBP
(Yasuda et al., 1995) is identical to human CNBPa (data not shown).
Protein D represents a newly identified isoform of XCNBP (Pellizzoni et
al., 1997). Protein D results from cleavage at a PEST site located
immediately preceding the NH2-terminal leucine residue. PEST sequences correspond to aa 42-55 of CNBPa. The remaining aa sequence
of protein D is identical to XCNBP, excluding the NH2-terminal leucine and
proline residues. Note that gaps in each protein are located only in linker
regions positioned between Zn-finger domains. The nt sequence was
deposited in the GenBank database under accession no. U20977.
domain. Additionally, aa sequence alignments show
that the aa residues comprising the seven Zn-finger
domains of CNBP are highly conserved in Xenopus,
human, mouse, rat, and yeast. Each of the finger
domains of CNBP are similar to the single Zn-finger
found within Xenopus posterior (Xpo) (Sato and Sar-
126
FLINK ET AL.
Fig. 2. Alignment of the Zn-finger domains of XCNBP, human CNBPa,
CNBPβ, Byr3, and Xpo. Each protein contains seven highly conserved
Zn-finger repeats (CX2CX4HX4C) except Xpo, which contains only a
single Zn-finger. Each protein also contains sequence similarity to the
finger domains of the family of retroviral nucleocapsid nucleic acid binding
proteins. The consensus aa sequence for the seven Zn-finger domains of
XCNBP, CNBPa, CNBPβ, MCNBP15 (mouse CNBP), and Byr3 is shown.
Mouse CNBP contains the consensus Zn-finger repeat found within
human CNBP. C-C-H-C aa, which are in tetrahedral coordination with
Zn21, are in bold, and identical residues are underlined. Shown at the
bottom is the consensus Zn-finger aa sequence motif of the proteins
described above.
gent, 1991) (Fig. 2). The consensus sequence of CNBP
Zn-finger domains is illustrated in Figure 2.
Temporal and Spatial Expression of CNBP
mRNA During Xenopus Early Embryogenesis
The onset and temporal expression pattern of CNBP
mRNA was determined during early Xenopus embryogenesis by Northern blot hybridization to staged total
embryonic RNAs using random-primed, full-length
XCNBP cDNA as probe (Fig. 3). A major band of ,1.5
kbp was obtained that corresponds to the size of the
XCNBP cDNA and human CNBP found in heart (Flink
and Morkin, 1995a) and liver (Rajavashisth et al.,
1989). A second prominent transcript ,3.5 kbp and a
minor 4.5 kbp band also were observed that appeared to
follow the expression pattern of the major 1.5 kbp
transcript, suggesting that they may represent additional isoforms of CNBP or partially processed message. The 1.5 kbp mRNA for XCNBP is first detected in
the unfertilized egg, demonstrating that it is maternally deposited during oogenesis. At the four-cell stage,
XCNBP expression is maintained at about the same
level. After this period, XCNBP mRNA appears to
diminish at the 64-cell to the midblastula stage. At
early gastrulation through late neurula, XCNBP mRNA
increases greater than the level found in the unfertilized egg and the four-cell stage until a steady-state is
reached, after which expression slightly diminishes at
the midtailbud stage and then becomes increasingly
greater at the late tailbud and tadpole stages. The
protooncogene, c-Src, was used as an internal control to
ensure equal RNA loading. Levels of c -Src do not
change during development (Collett and Steele, 1992).
To determine the spatial distribution of XCNBP
transcripts in early Xenopus embryos, whole mount in
Fig. 3. Northern blot hybridization to staged embryos. The 1.5 kbp
transcript corresponds to the size of human CNBP. The larger 3.5 kbp
mRNA may represent unprocessed message or another isoform of
XCNBP, because it follows the same pattern of expression as the 1.5 kbp
transcript. XCNBP appears to be maternally deposited at the unfertilized
egg stage. Transcripts are also observed from the four-cell through the
midtailbud stages. Transcription may be increased during the late tailbud
and tadpole stages.
situ hybridization was performed using antisense and
sense probes corresponding to the same full-length
cDNA used in Northern analyses. Antisense XCNBP
probe, but not sense probe, detected RNA transcripts in
the early gastrula (stage 10.25-10.5) throughout the
animal pole (the prospective epidermis and neural
plate) and in the marginal zone that contains prospective mesoderm (Fig. 4A,B). XCNBP was not detected in
the vegetal pole, the prospective endoderm. Transcripts
localized to the yolk-rich vegetal hemisphere are typically more refractory to whole mount in situ hybridization (Frank and Harland, 1992; Blitz et al., unpublished observations). RT-PCR analyses of stage 10.25
embryos showed that XCNBP is present in the same
tissues as detected by whole mount in situ hybridization, but XCNBP was also detected in vegetal prospective endoderm (Fig. 5). Transverse sections of embryos
at stage 10.25-10.5 gastrulae confirm the consistent
staining pattern in ectoderm and mesoderm (data not
shown).
At the late neurula (stage 15; Fig. 4C,D) and tailbud
(stage 27; Fig. 4E,F) stages, XCNBP continues to be
uniformly expressed along the dorsal axis from anterior
to posterior. Additionally, the cement gland appears to
be unstained (Fig. 4E). Transverse sections of these
CELLULAR NUCLEIC ACID BINDING PROTEIN
Fig. 4. Temporal and spatial expression of CNBP mRNA during
Xenopus early embryogenesis. A-F: Whole mount in situ localization of
XCNBP. G,H: Transverse sections of C and E, respectively. A,B: Early
gastrula stage (10.25-10.5). These embryos are viewed from the animal
pole and side, respectively. Dorsal is to the right. The asterisk indicates
the blastocoele cavity, the arrowheads indicate the dorsal lip of the
blastopore, and the arrow indicates XCNBP expression in the animal cap
region (above the blastocoele cavity). XCNBP staining in the marginal
127
zone is shown by the bracket. In A and B, the embryo on the right is a
sense strand control. C,E: Late neurula (stage 15) and late tailbud (stage
27), respectively (antisense probe). D,F: Late neurula (stage 15) and late
tailbud (stage 27), respectively (sense probe). In C-F, dorsal is up and
anterior is to the right. Epi., epidermis; Endo., endoderm; E, eyes; FB,
forebrain; L.M., lateral mesoderm; MB, midbrain; N.T., neural tube; No.,
notochord; Som., somitic mesoderm.
128
FLINK ET AL.
Fig. 5. Localization of XCNBP to endodermal tissue by RT-PCR. A
(lateral cross-section) and B (vegetal pole) are schematic diagrams
depicting different dissected regions for preparation of RNA from stage
10.25 gastrulae. Dotted lines represent incisions used to remove embryo
fragments. Asterisks denote the dorsal lip. In A and B, dorsal is to the right
and ventral to the left. Numbers correspond to lanes of the RT-PCR gel
shown in C. Histone H4 (H4; loading control) shows relative ratio of RNA
in each PCR reaction. XCNBP appears to be expressed approximately
uniformly throughout stage 10.25. -RT; no reverse transcriptase (control),
shows that the PCR signal is due to amplification of RNA and not
contaminating genomic DNA; Embryo, RNA isolated from whole embryo;
AC, animal cap; DMZ, dorsal marginal zone; LMZ, lateral marginal zone;
VMZ, ventral marginal zone; VP, vegetal pole.
stages show that XCNBP is detected in a wide variety of
tissue types, including the neural tube, epidermis, and
somitic and lateral plate mesoderm (Fig. 4G,H). The
apparent reduction in expression of XCNBP RNA in the
notochord may be an artifact of reduced penetration of
the probe into deeper tissues under the proteinase K
conditions used (Fig. 4; see Experimental Procedures
for details). Because Northern analyses, under stringent hybridization conditions, demonstrated strong hybridization to a major band (similar in size to human
CNBP during early development through the late gastrula stage [Fig. 3]) and sense strand and other probes
(Xotx2; Blitz and Cho, 1995) used in side-by-side analyses demonstrated no detectable background staining
(results not shown), these data strongly suggest that
the staining pattern can be attributed to authentic
XCNBP transcripts.
In addition to its role as a transcriptional regulator,
there is strong evidence that CNBP is involved in
translational control of ribosomal protein (rp) mRNAs
(Pellizzoni et al., 1997). This class of mRNAs encode up
to 80 different rp in Xenopus. The synthesis of rp
mRNAs is coordinately regulated at the translational
level by interaction of a common pyrimidine tract cis
element with a phosphoprotein called La and an accessory protease-sensitive factor. The interaction of CNBP
with GA- and GC-rich regions downstream from the
pyrimidine tract also may involve the protease-sensitive factor, which may coordinately modulate the functions of CNBP and La. Translational control of ribosomal protein, and possibly other transcripts rich in GC
content, is consistent with the cytoplasmic localization
of CNBP demonstrated by Western blotting of subcellular fractions (Warden et al., 1994). The role for CNBP as
a transcriptional regulator remains unresolved.
Support for CNBP as a transcriptional regulator
stems from the fact that it has been purified from
nuclear extracts (Michelotti et al., 1995) and its ability
to interact with promoter regions of genes encoding the
LDL receptor (Rajavashish et al., 1989), human b-MHC
(Flink and Morkin, 1995a), and CT-rich regions of the
c-Myc protooncogene (Michelotti et al., 1995). CNBP
may function as a transcriptional factor by virtue of its
strong affinity for recognition sequences in singlestranded DNA, which may tend to open doublestranded DNA. The interaction of CNBP with purinerich single-stranded DNA sequences could also result
from the unfolding of double-stranded DNA by the
interaction of heterogeneous ribonucleoproteins and
other macromolecules with pyrimidine-rich sequences
(Tomonaga and Levens, 1996). This would make the
complimentary purine-rich sequence more accessible
for binding. In vivo footprint analysis with strandspecific probes supports this notion and partly explains
regulation of the c-Myc promoter by single-stranded
binding proteins (Michelotti et al., 1995). G-rich RNA
motifs also appear to be target binding sites for CNBP
(Yasuda et al., 1995; Pellizzoni et al., 1997).
The longer version of CNBP in mouse and human
(CNBPa) contains a PEST sequence that may be susceptible to proteolytic cleavage (Fig. 1). Cleavage at this
site has been shown to occur in XCNBP in vivo (Pellizzoni et al., 1997), resulting in a shortened protein
beginning with an NH2-terminal leu residue (designated protein D in Fig. 1). Proteins that contain one or
more PEST regions have been associated with relatively short half-lives (Rogers et al., 1986). The region
corresponding to the deletion in Xenopus also contains
a consensus phosphorylation site for casein kinase II
(SLPD). The role of proteolytic and phosphorylation
sites as well as the significance of CNBP alternatively
processed isoforms are still unclear. However, differences in the affinities of CNBP isoforms for singlestranded DNAs have been observed (Flink and Morkin,
1995a; Michelotti et al., 1995).
The studies described here demonstrating the presence of CNBP in the unfertilized oocyte and its broad
expression pattern during embryogenesis, taken to-
CELLULAR NUCLEIC ACID BINDING PROTEIN
gether with previous data showing the association of
CNBP with ribosomal proteins and G-rich recognition
sequences of certain promoters, are consistent with the
idea that CNBP may serve a fundamental cellular role
at both the transcriptional and translation levels. Understanding how structural modifications among CNBP
isoforms influence DNA binding and protein-protein
interactions (Pellizzoni et al., 1997) to control regulation of transcription and translation must await further
studies.
EXPERIMENTAL PROCEDURES
Cloning of XCNBP
A PCR fragment of 376 bp, containing Zn-finger
domains 3 through 7 of CNBPa, was used as a 32Plabeled probe to screen a lgt11 cDNA library prepared
from Xenopus (stage 14, gift from Dr. Igor Dawid). The
library was plated at a density of <20 3 105 plaques/14
cm plate and transferred to nitrocellulose filters. Hybridization was carried out in buffer containing 50% formamide, 53 Denhardts solution, 63 SSC, 100 mg/ml
sheared herring DNA, 0.05% SDS, and 1 3 106 cpm/ml
32P-labeled PCR probe at 45°C. The PCR product was
amplified from nt positions 298 to 673 using two 21-mer
primers and CNBPa as template. Seven positive plaques
strongly hybridized with the probe. The longest clone
was purified, and lgt11 DNA was prepared by largescale liquid lysis and equilibrium banding for nt sequence determination. Sequence analyses were carried
out on both strands using synthetic primers and the
Applied Biosystems (Foster City, CA) Model 373A DNA
sequencing system. Sequence alignments and homologies were obtained using the software package PC/
GENE (Intelligenetics, Mountain View, CA).
Northern Analysis of Staged Embryos
Total embryonic RNA was isolated from Xenopus eggs
and embryos (25-50/developmental stage) by the method
of Chomzcynski and Sacchi (1987). Embryos were staged
according to Nieuwkoop and Faber (1967) and Keller
(1991). RNA was precipitated with isopropanol, and the
pellet was resuspended and reprecipitated with 2.5 M
LiCl overnight at 4°C to remove any contaminating
genomic DNA. Twenty micrograms of total embryonic
RNA was fractionated by gel electrophoresis on formaldehyde agarose gels and transferred to nitrocellulose
membrane. Hybridization was carried out using random-primed, full-length XCNBP probe at 60°C in 0.5 M
sodium phosphate and 7% SDS (Church and Gilbert,
1984). Membranes were washed at 60°C in 0.2 3 SSC,
0.2% SDS and subjected to autoradiography. RNA
molecular weight markers (Gibco BRL, Gaithersburg,
MD) were used to determine RNA size. The protooncogene, C-Src, was used as an internal control for RNA
loading (bottom panel).
Whole Mount In Situ Hybridization
Whole mount in situ hybridization was performed
essentially as described by Harland (1991), except that
129
proteinase K treatment was for 5 min and BM-purple
(Boehringer-Mannheim, Indianapolis, IN) was used as
the chromogenic substrate for color development. Embryos were embedded in polyethylene wax and sectioned as previously described (Blitz and Cho, 1995).
Embryos shown in Figure 4 were photographed in
methanol, except in B where embryos were ‘‘rendered
optically clear’’ in benzyl benzoate:benzyl alcohol (2:1).
Sense and antisense probes were synthesized using
pBluescript KS1-XCNBP and T3 and T7 polymerase,
respectively.
Localization of XCNBP in Endodermal Tissue
by RT-PCR
Oligodeoxynucleotides used for RT-PCR amplification of XCNBP were as follows: sense, 5’-TGA CGT GTA
AAA GGA GGA AAG GG-3’; antisense, 5’-TCT GCC
AAA TTG CTG CTG CC-3’. The amplified product is 216
bp extending from nt position 453 to nt position 668.
Oligonucleotides used for RT-PCR of Xenopus histone
H4 were as previously described (Blitz and Cho (1995).
Pigmented Xenopus embryos were cultured in 0.13
Barth’s saline at room temperature until early gastrula
stage 10.25. Animal cap tissue was removed near the
blastocoele floor as shown in Figure 5A, and the embryos were turned vegetal pole up (Fig. 5B). Marginal
zone and vegetal pole tissue fragments were dissected
in 0.13 Barth’s saline by making incisions as shown in
Figure 5B. Total cellular RNA was immediately isolated
from tissue fragments (20 embryos), and RT-PCR was
carried out for 24 cycles as previously described (Blitz
and Cho, 1995).
ACKNOWLEDGMENTS
The authors thank Dr. Igor Dawid for providing the
Xenopus lgt11 expression library prepared from stage
14 neurula, Dr. Robert Steele for providing the c-Src
cDNA, and Dr. Ken Cho for generously providing
support during the course of this project. Thanks is also
given to Wanda Hauglum, Xiang Zhou, and Niranjan
Maitra for excellent technical assistance.
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