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: firstname.lastname@example.org 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. REFERENCES Blitz IL, Cho KW-Y. Anterior neuroectoderm is progressively induced during gastrulation: The role of the Xenopus homeobox gene orthodenticle. Development 1995;121:993–1004. Briggs MR, Yokoyama C, Wang Z, Brown MS, Goldstein JL. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence. J. Biol. Chem. 1993;268:14490– 14496. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987;162:156–159. Church GM, Gilbert G. Genomic sequencing. Proc. Natl. Acad. Sci. USA 1984;81:1991–1995. Collett JW, Steele RE. Identification and developmental expression of Src1 mRNAs in Xenopus laevis. Dev. Biol. 1992;152:194–198. Flink IL, Morkin E. Alternatively processed isoforms of cellular nucleic acid binding protein interact with a suppressor of the human b-myosin heavy chain gene. J. Biol. Chem. 1995a;270:6959–6965. Flink IL, Morkin E. Organization of the gene encoding cellular nucleic acid binding protein. Gene 1995b;163:279–282. 130 FLINK ET AL. Frank D, Harland RM. Localized expression of a Xenopus POU gene depends on cell-autonomous transcriptional activation and inductiondependent inactivation. Development 1992;115:439–448. Gamer LW, Wright CV. Autonomous endodermal determination in Xenopus: regulation of expression of the pancreatic gene XlHbox 8. Dev. Biol. 1995;171:240–251. Harland RM. In situ hybridization: An improved whole-mount method for Xenopus embryos. Methods. Cell Biol. 1991;36:685–695. Keller R. Early embryonic development of Xenopus laevis. In: Kay BK, Peng BH, eds. Xenopus Laevis: Practical Uses in Cell and Molecular Biology. New York: Academic Press, 1991:61–113. Lusis AJ, Rajavashisth TB, Klisak I, Heinzmann C, Mohandas T, Sparkes RS. Mapping of the gene for CNBP, a finger protein, to human chromosome 3q13.3-q24. Genomics 1990;8:411–414. Michelotti EF, Tomonaga T, Krutzsch H, Levens D. Cellular nucleic acid binding protein regulates the CT element of the human c-myc protooncogene. J. Biol. Chem. 1995;270:9494–9499. Nieuwkoop PD, Faber J. Normal Table of Xenopus Laevis. Amsterdam: Garland Publishing, 1967. Pellizzoni L, Lotti F, Maras B, Amaldi P-P. Cellular nucleic acid binding protein binds to a conserved region of the 5’UTR of Xenopus laevis ribosomal protein mRNAs. J. Mol. Biol. 1997;267:264–275. Rajavashisth TB, Taylor A, Andalibi A, Svenson K, Lusis A. Identification of a zinc finger protein that binds to the sterol regulatory element. Science 1989;245:640–643. Rogers S, Wells R, Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis. Science 1986;234: 364–368. Sato SM, Sargent TD. Localized and inducible expression of Xenopusposterior (Xpo), a novel gene active in early frog embryos, encoding a protein with a ‘CCHC’ finger domain. Development 1991;112:747– 753. Tomonaga T, Levens D. Activating transcription from single stranded DNA. Proc. Natl. Acad. Sci. USA 1996;93:5830–5835. Torres SA, Johnson BH, Thompson EB. Oxysterol sensitive and resistant lymphoid cells: Correlation with regulation of cellular nucleic acid binding protein. J. Steroid Biochem. Mol. Biol. 1994;48: 307–315. Warden CH, Skaidrite KK, Purcell-Huynh D, Leete LM, Daluiski A, Diep A, Taylor BA, Lusis AJ. Mouse cellular nucleic acid-binding protein: A highly conserved family identified for genetic mapping and sequencing. Genomics 1994;24:14–19. Xu H-P, Rajavashisth T, Grewal N, Jung V, Riggs M, Rodgers L, Wigler M. A gene encoding a protein with seven finger domains acts on the sexual differentiation pathways of Schizosaccharomyces pombe. Mol. Biol. Cell 1992;3:721–734. Yasuda J, Mashiyama S, Makino R, Ohyama S, Sekiya T, Hayashi K. Cloning and characterization of rat cellular nucleic acid binding protein (CNBP) cDNA. DNA Res. 1995;2:45–49. Yokoyama C, Wang X, Briggs MR, Admon A, Wu J, Hua X, Goldstein JL, Brown MS. SREBP-1, a basic helix-loop-helix leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell 1993;75:187–197.