Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family.

We isolated the cDNA encoding a novel member of the fibroblast growth factor (FGF) family from rat embryos by homology-based polymerase chain reaction. The FGF-related cDNA encodes a protein of 215 amino acids (∼24 kDa), which has a conserved ∼120-amino acid core with ∼30-60% amino acid sequence identity with the FGF family. This protein with a hydrophobic amino terminus appears to be a secreted protein. The cDNA was translated in a coupled in vitro transcription-translation system. The molecular mass of the translation product was observed to be ∼26 kDa. The expression of the FGF-related mRNA in the rat embryo and adult tissues was determined by Northern analysis and in situ hybridization. The mRNA was expressed in several discrete regions of the embryo. In adult tissues, the mRNA was preferentially expressed in the lung. The expression profile of the FGF-related mRNA was different from those of other FGF family mRNAs. As this protein is the 10th documented protein related to FGFs, we tentatively term this protein FGF-10.

We isolated the cDNA encoding a novel member of the fibroblast growth factor (FGF) family from rat embryos by homology-based polymerase chain reaction. The FGF-related cDNA encodes a protein of 215 amino acids (ϳ24 kDa), which has a conserved ϳ120-amino acid core with ϳ30 -60% amino acid sequence identity with the FGF family. This protein with a hydrophobic amino terminus appears to be a secreted protein. The cDNA was translated in a coupled in vitro transcription-translation system. The molecular mass of the translation product was observed to be ϳ26 kDa. The expression of the FGF-related mRNA in the rat embryo and adult tissues was determined by Northern analysis and in situ hybridization. The mRNA was expressed in several discrete regions of the embryo. In adult tissues, the mRNA was preferentially expressed in the lung. The expression profile of the FGF-related mRNA was different from those of other FGF family mRNAs. As this protein is the 10th documented protein related to FGFs, we tentatively term this protein FGF-10.
aFGF and bFGF are polypeptides with multiple biological activities in vivo and in vitro, including roles in angiogenesis, mitogenesis, cellular differentiation, and repair of tissue injury (1,2). Although the physiological significance of most FGFs remains to be elucidated, FGFs appear to play important roles as peptide growth factors in both developing and adult tissues. Although members of the FGF family are of different sizes, they have a conserved ϳ120-amino acid residue core with ϳ30 -70% amino acid sequence identity (1)(2)(3)(4)(5)(6)(7)(8)(9). To isolate the cDNA encoding a novel FGF, we examined rat embryo cDNA amplified by the polymerase chain reaction with primers specific for conserved amino acid sequences within the FGF family. Recently, we isolated the cDNA encoding a novel member of the FGF family from rat embryos by homology-based polymerase chain reaction. The nucleotide sequence of the cDNA revealed the complete amino acid sequence of a novel member of the FGF family. Here, we report the structure of this novel FGF and the expression of the mRNA encoding the FGF.

EXPERIMENTAL PROCEDURES
Preparation of RNA from Rat Embryos and Adult Rat Tissues-RNA was prepared from Wistar rat embryos (embryonic day 14, E14) and adult rat tissues by the acid guanidinium thiocyanate/phenol/chloroform extraction method (10). Poly(A) ϩ RNA was prepared using oligo(dT)-cellulose (Collaborative Research Inc., type 2).
Isolation and Analysis of Rat FGF Family cDNAs-For cDNA synthesis, rat embryo poly(A) ϩ RNA (5 g) was incubated for 60 min at 37°C in a reaction mixture (20 l) containing 300 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), 15 units of human placenta RNase inhibitor (Wako Pure Chemicals, Japan), and 0.5 g of random hexadeoxynucleotide primer. To amplify the FGF family cDNAs, the polymerase chain reaction (PCR) was performed for 30 cycles in a reaction mixture (25 l) containing an aliquot of the above cDNA solution, 0.05 unit/l Taq DNA polymerase (Wako Pure Chemicals, Japan), and 5 pmol/l each of the sense and antisense degenerate primers representing all possible codons corresponding to the consensus amino acid sequences of mouse FGF-3 and FGF-7, YL-AMNK and YNTYAS, respectively (11,12). The reaction product was fractionated by electrophoresis on an 8% polyacrylamide gel. The amplified DNA of the expected size (ϳ110 base pairs) was eluted from the gel by electrophoresis and was cloned into the pGEM-T DNA vector (Promega Co.). The nucleotide sequence of the cloned DNA was determined with a DNA sequencer model 373A (Applied Biosystems, Inc.). To determine the entire coding region of the cDNA, the cDNA was analyzed by the rapid amplification of cDNA ends method (13).
cDNA-directed Protein Synthesis-The poly(A) region (58 base pairs) of the pSP64 poly(A) vector DNA (Promega) was added at the 3Ј end of the FGF-related cDNA containing the entire coding region (645 base pairs) and 3Ј-noncoding region (49 base pairs). The cDNA containing the poly(A) region was inserted at the site downstream of the 5Ј leader sequence of pSPUTK vector DNA (Stratagene). cDNA-directed protein synthesis was performed in a coupled in vitro transcription-translation system (TNT SP6 Coupled Reticulocyte Lysate System, Promega) using the pSPUTK containing the cDNA as a template and [ 35 S]L-methionine (37 TBq/mmol) (ICN Biomedicals) in the presence or absence of canine pancreatic microsomal membranes (Promega). The translation products were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide (15%) gel electrophoresis, transferred onto a nitrocellulose membrane, and then detected with a radioimaging analyzer (BAS 2000, Fuji Photo Film Co., Japan).
Northern Analysis-Aliquots of poly(A) ϩ RNA (each 12 g) from rat embryos and adult rat tissues were resolved on a denaturing agarose gel (1%) containing formaldehyde and transferred to a nitrocellulose * This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D79215.

RESULTS AND DISCUSSION
Isolation of FGF-related cDNA from Rat Embryos--Members of the FGF family have conserved a ϳ120-amino acid residue core with ϳ30 -70% amino acid sequence identity. The amino acid sequences corresponding to amino acids 96 -101 (YLAMNK) and 126 -131 (YNTYAS) of FGF-3 are identical with those of the corresponding regions of FGF-7 (11,12). Thus, we designed degenerate oligonucleotide primers representing all possible codons corresponding to the consensus sequences to isolate the cDNA for a novel member of the FGF family by the polymerase chain reaction (PCR).
FGFs are abundantly present in embryos (5-9). Therefore, cDNA synthesized from rat embryo (E14) poly(A) ϩ RNA was amplified by PCR using these degenerate oligonucleotide primers. DNA of the expected size (ϳ110 base pairs), which was the major amplified product (data not shown), was cloned. Forty clones were isolated, and their nucleotide sequences were determined. Thirty-six clones had sequences homologous to those of the FGF family. Among the FGF-related cDNA clones, 11 had sequences highly homologous to those of the mouse FGF-7 or FGF-3 cDNA (11,12), indicating that they were rat homologues of these genes. Another 25 clones had an identical sequence that was similar to but distinct from those of the nine known members of the FGF family, suggesting that these clones encode a novel member of the FGF family. To determine the entire coding region of the novel FGF-related cDNA, the cDNA clone covering the entire coding region was isolated by the rapid amplification of cDNA ends method (13).
Structure of the FGF-related Protein-The nucleotide sequence of the coding region of the novel FGF-related cDNA is shown in Fig. 1. The translation initiation site was assigned to the translation initiation codon, ATG, at nucleotides 1-3, because the translation termination codon, TAA, was found at a site (nucleotides Ϫ108 to Ϫ106) upstream from the initiation codon in the same reading frame (data not shown). The translation termination site was assigned to the termination codon, TAG (nucleotides 646 -648). Thus, the cDNA was found to contain a coding region of 645 nucleotides.
The nucleotide sequence of the coding region allowed elucidation of the complete amino acid sequence (215 amino acids) of a protein with a conserved ϳ120-amino acid core (amino acids 82-173 and 184 -212) with ϳ30 -60% amino acid sequence identity with the FGF family (1-9) (Fig. 1). As this protein is the 10th documented protein related to FGFs, we propose the tentative designation FGF-10. The highest sequence identity (ϳ60%) was observed with FGF-3 and FGF-7 (Fig. 2). FGF-1, FGF-2, and FGF-9 have no typical signal sequence in their amino termini (1,2,4), and so the pathway of release and sequestration of these FGFs is not clear. In contrast, other FGFs, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, and FGF-8, have typical signal sequences and are secreted proteins (3,(5)(6)(7)(8)(9). FGF-10 also has a hydrophobic amino terminus (ϳ40 amino acids), which may serve as a signal sequence, and appears to be a secreted protein (Fig. 1). The signal sequence cleavage site was predicted to be a site between amino acid positions 36 (C) and 37 (Q) by the method of von Heijne (14) (Fig. 1). Two cysteine residues are well conserved in the FGF family, and these amino acids correspond to residues 91 and 157 in the FGF-10 sequence. Although a cysteine residue was found at position 157, a serine instead of a cysteine residue was found at position 91. The same substitution was also observed in the FGF-8 sequence (3). The most characteristic feature in the FGF-10 sequence was a serine-rich domain (amino acid positions 51-69) in the amino-terminal region. A similar domain was also found in the amino-terminal region of the FGF-5 sequence (7), although the significance of the domain remains to be elucidated.
FGF-10 cDNA-directed Protein Synthesis-FGF-10 cDNA was translated in a coupled in vitro transcription-translation system. The translation products were resolved by SDS-polyacrylamide gel electrophoresis. A major translation product of ϳ26 kDa was detected (Fig. 3). We assumed that the translation product was FGF-10, as the molecular mass of FGF-10 was calculated to be ϳ24 kDa by the above assignment. However, the translation efficiency of the cDNA was greatly reduced in the presence of canine pancreatic microsomal membranes. Then, we could not detected any translation products processed by the membranes (data not shown).

Expression of FGF-10 mRNA in Embryonic and Adult Rat
Tissues-To examine the expression of FGF-10 in the rat embryo (E14), its mRNA was identified in the embryo poly(A) ϩ RNA by Northern analysis using a 32 P-labeled FGF-10 cRNA probe. The integrity of RNA was confirmed by electrophoresis on a denaturing agarose gel containing formaldehyde (data not shown). The labeled probe mainly hybridized to an mRNA of ϳ4.5 kilobases which is large enough to encode FGF-10 (Fig. 4).
To examine the region-specific expression of FGF-10 mRNA in the embryo, sagittal sections of the embryo were analyzed by in situ hybridization with an antisense 35 S-labeled FGF-10 cRNA probe, followed by microautoradiography. With the Poly(A) ϩ RNA (12 g each) was electrophoresed on a denaturing agarose gel (1%) containing formaldehyde. RNA was transferred onto a nitrocellulose membrane, and hybridization was performed with a 32 P-labeled rat FGF-10 cRNA probe. The positions of 28 S and 18 S RNAs are indicated as 28S and 18S. Lanes Em, Br, He, Lu, Ki, and In indicate poly(A) ϩ RNA from the embryo (E14) and the adult, brain, heart, lung, kidney, and intestine, respectively. probe, discrete labeling was observed in several regions of the embryo, including the posterior pituitary, the first cervical vertebra, sacral and coccygeal segments of the spinal cord, the duodenum, and the lung (Fig. 5). However, only diffused labeling was observed with a sense 35 S-labeled FGF-10 cRNA probe as a control (data not shown).
To examine the expression of FGF-10 mRNA in the adult rat tissues, poly(A) ϩ RNA from tissues including the brain, heart, lung, liver, kidney, and small intestine was also examined by Northern analysis using the 32 P-labeled FGF-10 cRNA probe. The integrity of RNA was also confirmed by electrophoresis on a denaturing agarose gel containing formaldehyde as described (15). The labeled probe strongly hybridized to an mRNA of ϳ4.5 kb in the lung (Fig. 4). The size of the hybridizing mRNA in the lung was similar to that in the embryo. The probe also weakly hybridized to an mRNA of the same size in the heart but not in the brain, liver, kidney, or small intestine. We also examined the expression of FGF-10 in other tissues including the thymus, stomach, pancreas, spleen, testis, and muscle by PCR with specific primers for the mRNA. However, no FGF-10 mRNA was detected in these tissues (data not shown).
FGF-1 and FGF-2 are widely expressed in developing and adult tissues (1,2). In contrast, most other FGFs are predominantly expressed in the embryo and show restricted expression patterns in the adult tissues as described below (3)(4)(5)(6)(7)(8)(9). FGF-3 is expressed in the embryo and in the adult brain and testis. FGF-4 is expressed in the embryo but not in the adult tissues. FGF-5 is expressed in the embryo and in the adult brain. FGF-6 is expressed in the embryo and in the adult heart, testis and muscle. FGF-7 is expressed in the embryo and the adult kidney and gastrointestinal tract. FGF-8 is expressed in the embryo and in the adult testis and ovaries. FGF-9 is expressed in the embryo and in the adult kidney and brain. FGF-10 is predominantly expressed in the embryo and the adult lung. The expression profile of FGF-10 is quite different from those of other members of the FGF family. Although the physiological significance of FGF-10 remains to be elucidated, FGF-10 appears to be a novel FGF which presumably has a unique physiological role.