Assignment of FGF12, the human FGF homologous factor 1 gene, to chromosome 3q29-->3qter by fluorescence in situ hybridization.
Explore the source record for details and available documents.
Blotting, Southern↗
Biomedical subjects
I M Chiu
Publications and source records attributed to I M Chiu.
Amphibian FGF-1 is structurally and functionally similar to but antigenically distinguishable from its mammalian counterpart.
Recent studies have shown that fibroblast growth factors (FGF) play an important role in the diverse cellular mechanisms involved with vertebrate development. One system which has received a great deal of attention is the developing limb in part because of the extensive epithelial-mesenchymal interactions that take place during this process. Because it closely parallels the developmental process of the limb and is a model for wound repair, the phenomenon of amphibian limb regeneration has been used to investigate the role of FGF in these processes. We have recently reported on the cloning and functional characterization of an FGF receptor (FGFR) isolated from amphibian regenerative tissue. In this report, we describe the isolation and characterization of an FGF-1 molecule from the newt, Notophthalmus viridescens. Amino acid sequence comparisons indicate that the newt FGF-1 exhibits between 79 to 83% identity with FGF-1 from mammalian and avian species. The full length cDNA of the newt FGF-1 was cloned into a prokaryotic expression vector and purified from E. coli. Although the newt FGF-1 shares a high degree of primary amino acid sequence similarity with other FGF-1 molecules, the recombinant protein was not detected in a Western blot analysis using a polyclonal antibody directed against mammalian FGF-1. Despite the antigenic divergence, the newt FGF-1 was capable of binding to NIH/3T3 and Chinese hamster ovary cells overexpressing mammalian and amphibian FGFRs with dissociation constants comparable to those reported for mammalian FGF-1. Newt FGF-1 could also be cross-linked to receptors on the surface of NIH/3T3 cells. In addition, it elicits a mitogenic response in NIH/3T3 cells indistinguishable from human recombinant FGF-1.
3T3 Cells↗
A 720-kb yeast artificial chromosome contig spanning human chromosome 5q31.3-32.
Explore the source record for details and available documents.
Chromosome Mapping↗
Differential regulation of human fibroblast growth factor 1 transcripts provides a distinct mechanism of cell-specific growth factor expression.
Four variants of fibroblast growth factor 1 (FGF-1) mRNA, FGF-1.A, -1.B, -1.C, and -1.D, originate from four discrete promoters of the gene. Each promoter is coupled with its 5'-untranslated exon. These four promoters are separated by as much as 70 kbp in the FGF-1 locus. The present study indicates that expression of these transcripts in different cell types is regulated by distinct mechanisms. FGF-1.C mRNA requires de novo protein synthesis and de novo transcription for expression and processing, and this mRNA increases acutely in response to TGF-beta and serum stimulation. The serum-induced FGF-1.C correlates with a marked increase in protein level. In addition, FGF-1.C mRNA also increases significantly (more than 100-fold) in response to phorbol 12-myristate 13-acetate. FGF-1.D mRNA is uniquely superinduced by serum in the presence of cycloheximide and displays delayed early kinetics, suggesting that this mRNA does not require de novo protein synthesis for expression. In sharp contrast to the FGF-1.C and -1.D mRNAs, FGF-1.B mRNA levels do not increase in response to serum or phorbol 12-myristate 13-acetate and are in fact slightly down-regulated. Furthermore, FGF-1.B mRNA is stable and appears to have a long half-life (> 12 h). Thus, the unique cell-specific regulation of these FGF-1 transcripts and subsequent protein synthesis indicate that each transcript may have a distinct role in development, normal cellular processes, and, upon aberrant regulation, disease. In support of these conclusions, multiple FGF-1 transcripts in normal, fetal, and diseased tissues, containing mixed cell types, were detected. Our results suggest that FGF-1 transcripts FGF-1.C and -1.D arising from promoters 1C and 1D are specific and are potential markers for proliferation of certain cells, whereas transcripts FGF-1.A and -1.B arising from promoters 1A and 1B are specific for maintenance and survival of cells, particularly cardiac and neuronal cells. Together, these data provide evidence for a biological function for multiple promoter usage of a single gene. The discrete mechanisms for expression of the FGF-1 gene further underscore the biological significance of this growth factor.
Cell Line↗
Characterization of the 1B promoter of fibroblast growth factor 1 and its expression in the adult and developing mouse brain.
The present study elucidates the molecular structure of a murine fibroblast growth factor 1 (FGF-1) promoter and describes its distribution in the adult and developing mouse brain. A cDNA clone coding for FGF-1 was isolated from a mouse brain cDNA library. Nucleotide sequence analysis revealed that the clone contained, in addition to the protein coding region, an untranslated exon (FGF-1B) 34 base pairs upstream of the translation start codon ATG. The mouse cDNA clone corresponded to the sole FGF-1 transcript in the brain. An RNase protection assay was used to map the transcription start site of the 1B promoter. The sequences upstream from the major transcription initiation site lacked consensus TATA or CAAT boxes. In situ hybridization with cRNA probes specific for the 1B transcript showed the message to be restricted largely to sensory and motor nuclei in the brainstem, and to the ventral spinal cord and cerebellum. Although occasional brainstem nuclei were labeled at low levels by embryonic day 18, the majority of nuclei became detectable autoradiographically during postnatal weeks 1 and 2, and adult levels of grain density were reached during the 3rd and 4th postnatal weeks. FGF-1B mRNA was expressed in phylogenetically older brain regions, which are involved primarily in processing information from exteroceptive sensory mechanoreceptors and in motor control. The relatively late developmental expression suggests a role for FGF-1 in neuronal maturation, rather than in neurogenesis.
Aging↗
Cloning and characterization of a novel upstream untranslated exon of the mouse Fgf-1 gene.
Fibroblast growth factor 1 (FGF-1 or aFGF), is the prototype member of the heparin-binding growth factors which are capable of angiogenesis in vivo. FGF-1 has been implicated in atherosclerosis, cancer, wound repair and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the etiopathogenesis of inflammation and cancer, we have undertaken steps to isolate and characterize the mouse Fgf-1 gene. Southern blotting and sequence analysis displayed considerable conservation within the coding and upstream untranslated regions of Fgf-1 in human, mouse, hamster, rat and bovine. By using primers derived from the 5'-untranslated exon of a rat prostate-specific Fgf-1 cDNA, a 220-bp product was amplified from mouse genomic DNA via PCR. Sequence analysis of this amplicon showed that there was 80% similarity with the corresponding region of the rat FGF-cDNA sequence. Primers designed from this amplicon and the Fgf-1 coding region were used to isolate multiple overlapping genomic clones spanning the entire mouse Fgf-1 gene. Sequencing analysis of the genomic sequence upstream from this novel 5'-untranslated exon did not reveal typical TATA, CCAAT sequences. It appears that the occurrence of multiple untranslated exons for FGF-1 is a highly conserved theme for this gene across species.
Animals↗
Cloning and characterization of the mouse Fgf-1 gene.
Fibroblast growth factor 1 (FGF-1 or aFGF), is a mitogen for a variety of mesoderm- and neuroectoderm-derived cells, as well as an angiogenic factor in vivo. It has been implicated in angiogenic diseases including atherosclerosis, cancer and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the pathobiology of inflammation, we have isolated and characterized the mouse Fgf-1 gene. Southern blot analysis of mouse genomic DNA using the mouse Fgf-1 cDNA as a probe revealed that mouse FGF-1 is encoded by a single copy gene. Comparison of the available mouse Fgf-1 cDNA sequence with newly obtained genomic sequence allowed us to establish the exon/intron boundaries. The mouse Fgf-1 coding region is comprised of three protein coding exons, which we determined to be separated by an 11.4-kb and a 4.9-kb intron. The elucidation of the mouse Fgf-1 coding region revealed great similarity between the mouse and human Fgf-1 gene structure.
Amino Acid Sequence↗
The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells.
Androgen-induced growth factor (AIGF or FGF-8) was originally isolated from the conditioned medium of an androgen-dependent Shionogi carcinoma, SC-3, cell line. It shares structural similarity with other members of the FGF family. The temporal and spatial expression patterns of the FGF-8 gene suggest its involvement in gastrulation, regionalization of the brain, and organogenesis of the limb and face as an embryonic epithelial factor. In the adult, expression of FGF-8 is restricted to gonads including testes and ovaries. Since FGF-8 is identified as a corroborating gene in MMTV-induced mammary tumors in Wnt-1 transgenic mice and because FGF-8 manifested its autocrine mitogenic activity in SC-3 cells, it is possible that aberrant expression of FGF-8 may be present in human cancers which are hormone dependent. However, very little is known about human FGF-8. To determine whether FGF-8 plays a role in human breast cancer, we have isolated the full-length cDNA from SK-BR-3 breast cancer cells. We have also isolated the corresponding genomic DNA in a P1 cloning vector. The FGF-8 gene has been mapped to chromosome 1Oq24 using both somatic cell hybrid genetic analysis and fluorescence in situ hybridization. Finally, we show that FGF-8 gene expression in a human breast cancer cell line, MDA-MB-231, is inducible by androgen. The findings presented here will facilitate our understanding of the molecular mechanism underlying hormone-responsive breast and prostate cancers.
Adenocarcinoma↗
A recombinant PCR approach requiring only three non-chimeric primers to generate a minigene of interest.
The recombinant PCR allows construction of chimeric molecules. Here we describe this approach utilizing non-chimeric primers. Unlike previous recombinant PCR methods, this approach eliminates the need of multiple sets of primers and multiple rounds of PCR making it an economical and expeditious alternative. We have used this approach to generate an FGF-1 minigene.
Base Sequence↗
Conservation of ligand specificity between the mammalian and amphibian fibroblast growth factor receptors.
We have previously cloned and sequenced a newt keratinocyte growth factor receptor (KGFR) cDNA which exhibited a unique spatial and temporal expression pattern in the regenerating newt limb. In this report, we further characterize the biochemical and functional properties of this newt KGFR. A stable Chinese hamster ovary transfectant overexpressing the newt KGFR was capable of binding both 125I-fibroblast growth factor-1 (FGF-1) and 125I-FGF-7 but not 125I-FGF-2, indistinguishable from the human KGFR. Scatchard analysis and cross-linking studies further support the conclusion that FGF-1 and FGF-7 are the ligands for the newt KGFR. In addition to their ability to bind to FGFs, both the human and the newt KGFR are also capable of repressing differentiation in mouse MM14 myoblasts. MM14 cells express FGFR1 and are repressed from differentiation by FGF-1, FGF-2, and FGF-4 but not FGF-7. Co-transfection of MM14 cells with either a human or newt KGFR expression construct conferred a response to FGF-7 as determined by a human alpha-cardiac actin/luciferase reporter construct. The response to FGF-7 was similar to the endogenous FGF response as FGF-7 prevented MM14 myoblasts from undergoing terminal differentiation. Thus, both the human and the newt KGFRs transduce signals similar to those transduced via the endogenous mouse FGFR1. Together these data indicate that this newly isolated newt KGFR is a functional receptor as it binds two FGF family members with high affinity and mediates signaling in skeletal muscle myoblasts. Because the binding pattern of the newt KGFR is similar to the pattern observed for its mammalian counterpart, it emphasizes the strict conservation that this ligand/receptor system has undergone through evolution.
3T3 Cells↗
Different fibroblast growth factor 1 (FGF-1) transcripts in neural tissues, glioblastomas and kidney carcinoma cell lines.
We have previously reported the tissue specific distribution of four different FGF-1 transcripts containing alternative 5' untranslated exons spliced to the first protein coding exon. The predominant transcript in brain is FGF-1.B and in kidney FGF-1.A. Others have shown, by in situ hybridization and immunohistochemical analysis, that expression of FGF-1 in the brain is exclusively in neural cells but not in glial cells. Here we have examined the distribution of FGF-1.B and FGF-1.A transcripts in glioblastoma and retinal tissues and in kidney carcinoma cell lines. Our results show that FGF-1.B is the predominant transcript in neural derived tissues including both the diabetic retina and normal retina tissues. Surprisingly, FGF-1.B transcript is highly expressed in glioblastoma tissues. In contrast, a normal brain glial cell line, CHII, expresses very low levels of FGF-1 mRNA. These results strongly implicate the role of FGF-1 in the etiology of glioblastoma. We also examined several kidney carcinoma derived cell lines for the expression of FGF-1 mRNA. Most of these kidney cell lines do not express any FGF-1 transcripts. An interpretation by deduction is that kidney adenocarcinomas are derived from cortex but medulla has been reported as the site of FGF-1 synthesis. Of the kidney derived cell lines which are positive for FGF-1 message, only one expressed FGF-1.A transcript. The data may suggest that the establishment of kidney cell lines results in a switch of promoter usage from the 1.A seen in kidney tissue. Similarly, culturing of glioma cell lines may result in a switch from FGF-1.B seen in glioma tissues to FGF-1.D seen in most glioma cell lines. Continued studies of the FGF-1 transcripts, their functional promoters and their tissues distribution will provide insight into the potential role of FGF-1 in cell growth, tissue differentiation and malignant transformation.
Alternative Splicing↗
Functional characterization of the brain-specific FGF-1 promoter, FGF-1.B.
Expression of alternatively spliced human FGF-1 (or aFGF) transcripts is regulated in a tissue-specific manner via multiple promoters. To identify the cis-regulatory elements in the brain-specific FGF-1.B promoter, we constructed a series of promoter deletions fused to the luciferase reporter gene and transfected into an FGF-1.B positive glioblastoma cell line, U1240MG, and a 1.B negative cell line, U1242MG. Results of transient transfections indicate three elements that are involved in the positive regulation of FGF-1.B expression. The core promoter is located in a 40-base pair region (between -92 and -49), and two regulatory regions (RR-1 and RR-2) are located within the 540-base pair region 5' to the major transcription start site (defined as +1). Electrophoretic mobility shift assays and footprinting analysis have identified sequence-specific binding sites in RR-1 and RR-2. Mutants of RR-2 abolished binding to nuclear proteins and showed diminished luciferase reporter activity. The effects seen are specific for the U1240MG cell line, supporting a role for RR-2 in the tissue-specific regulation of FGF-1.B. Southwestern analysis using an oligonucleotide probe derived from RR-2 (nucleotides -489 to -467) further identified a 37-kDa protein that is present in nuclear extracts from U1240MG and brain but not from U1242MG.
Base Sequence↗
Human fibroblast growth factor 1 gene expression in vascular smooth muscle cells is modulated via an alternate promoter in response to serum and phorbol ester.
We have previously isolated the human FGF-1 gene in order to elucidate the molecular basis of its gene expression. The gene spans over 100 kbp and encodes multiple transcripts expressed in a tissue- and cell-specific manner. Two variants of FGF-1 mRNA (designated FGF-1.A and 1.B), which differ in their 5' untranslated region, were identified in our laboratory. Recently, two novel variants of FGF-1 mRNA (designated FGF-1.C and 1.D) have been isolated. In this study we used RNase protection assays to demonstrate expression of FGF-1.D mRNA in human fibroblasts and vascular smooth muscle cells and to show that promoter 1D has multiple transcription start sites. A single-strand nuclease-sensitive region has also been identified in the promoter 1D region that may have implications in chromatin conformation and transcriptional regulation of this promoter. Using Northern blot hybridization analyses, a previous study demonstrated a significant increase of FGF-1 mRNA levels in cultured saphenous vein smooth muscle cells in response to serum and phorbol ester. Here we confirm these results by RNase protection analysis and show that FGF-1.C mRNA is significantly increased in response to these stimuli. RNase protection assays indicate that promoter 1C has one major start site. The phorbol ester effect suggests that a protein kinase C-dependent signalling pathway may be involved in this phenomenon. Our results point to a dual promoter usage of the FGF-1 gene in vascular smooth muscle cells. Thus, normal growing cells primarily utilize promoter 1D. In contrast, quiescent cells, when exposed to serum or phorbol ester, utilize a different FGF-1 promoter, namely promoter 1C. Overall, these phenomena suggest mechanisms for increased production of FGF-1 that may play a role in inflammatory settings, wound healing, tissue repair, and neovascularization events and processes via autocrine and paracrine mechanisms. Our findings suggest that different FGF-1 promoters may respond to different physiological conditions and stimuli, in reference to the cell type or tissue milieu, resulting in ultimate production of the FGF-1 protein.
Animals↗
Re-programming of expression of the KGFR and bek variants of fibroblast growth factor receptor 2 during limb regeneration in newts (Notophthalmus viridescens).
We have previously shown, by in situ hybridization, that fibroblast growth factor receptor 2 (FGFR2) is present in the basal layer of wound epithelium during limb regeneration in newts (Notophthalmus viridescens). In contrast, FGFR1 expression is observed throughout the blastema mesenchyme but is distinctly absent from the wound epithelium (Poulin et al. [1993] Development 119:353-361). Sequence analysis revealed that we have isolated both the KGFR and bek variants of FGFR2. These two variants differ only in the second half of the last of their three (or two) Ig-like domains. In this report, we show the expression patterns of FGFR2 variants during limb regeneration by in situ hybridization. During the pre-blastema stages of regeneration, FGFR2 expression was observed in the basal layer of the wound epithelium and in the cells of the periosteum. The wound epithelial hybridization was observed when the KGFR-specific probe was used while the bek-specific probe hybridized to mRNA in the cells of the periosteum. As regeneration progresses to the blastema stages, KGFR expression continued to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. The bek-specific hybridization pattern observed at this stage corresponds specifically to the mesenchymal hybridization. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. Interestingly, there appears to be a dorsoventral gradient of the expression of both KGFR and bek variants of FGFR2, which are opposite each other at the later stages of regeneration. Thus, re-programming of expression of the two FGFR2 variants is required during the initial wound closure of limb regeneration. Remarkably, the expression patterns of KGFR and bek mimic those observed in the mouse limb bud during early embryonic development (Orr-Urtreger et al. [1993] Dev. Biol. 18:475-486). Moreover, our results suggest that the two FGFR2 variants have distinct roles in limb regeneration. Further investigation regarding the potential sources of the FGF ligands will help establish the roles that FGFs and FGFRs play in limb regeneration.
Amino Acid Sequence↗
Nucleotide sequences of two newt (Notophthalmus viridescens) fibroblast growth factor receptor-2 variants.
The FGF receptor tyrosine kinase family consists of four members. We report the sequence of two newt (Notophthalmus viridescens) FGFR2 cDNAs which were isolated from a forelimb blastema cDNA library and represent the newt cognates of two different isoforms of FGFR2, one homologous to bek the other to the KGFR.
Amino Acid Sequence↗
Construction of a yeast artificial chromosome contig encompassing the human acidic fibroblast growth factor (FGF1) gene: toward the cloning of the ANLL/MDS tumor-suppressor gene.
The region surrounding the human acidic fibroblast growth factor (FGF1) locus on chromosome 5q31 is of particular interest since it represents a critical region consistently lost in acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS) patients who have a demonstrable deletion of the distal portion of the long arm of chromosome 5. It is proposed that an ANLL/MDS leukemia suppressor gene resides on 5q31. We have previously shown that the gene is most likely localized between FGF1 and PDGFRB/CSF1R loci. The region has also been linked to at least four other genetic diseases, Treacher Collins syndrome, diastrophic dysplasia, limb-girdle muscular dystrophy, and an autosomal dominant deafness, by linkage analysis. Here, we describe yeast artificial chromosomes (YAC) spanning 450 kb around the FGF1 gene. Six YAC clones were isolated from a human YAC library and their restriction enzyme maps were determined. The overlap of the clones with each other and with FGF1 cosmid and phage clones was characterized. Three of the YAC clones were found to contain the entire FGF1 gene, which spans more than 100 kb. Proximal and distal ends of several of these YAC clones were isolated for further overlap cloning. The proximal ends of both Y2 and Y4 were localized to previously isolated FGF1 DNA by sequence analysis. The distal ends of these two clones also hybridized to a human-hamster hybrid containing chromosome 5 as the only human genetic material. These results suggest that these YAC clones represent colinear DNA around the FGF1 locus.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Alternative splicing of fibroblast growth factor 1 (FGF-1) transcripts: a cellular dilemma in determining exon selection and exclusion.
During reverse transcription and polymerase chain reaction (RT-PCR) of kidney RNA using fibroblast growth factor 1 (FGF-1) gene-specific primers, we amplified five fragments with the sizes of 507, 410, 310, 285, and 216 bp. Cloning of the 507-, 310-, and 216-bp fragments revealed that the latter two share the same sequences to the 507-bp DNA but missing 197 and 291 bp, respectively. Characterization of the corresponding genomic DNA sequences showed that the three cDNA are alternative splicing products of the same gene. We further showed that these three transcripts are also present in brain in similar proportions albeit in a much lesser extent. Repeated attempts to clone the 410-bp fragment resulted in isolation of three additional cDNA clones; each has the inclusion of a distinct novel exon. Because each novel exon is delimited by the splicing donor and acceptor sequences, these cDNA clones are not likely to be RT-PCR artifacts. Yet their mRNA levels are extremely low, because we could not detect the corresponding PCR products on ethidium bromide-stained gels nor by Southern hybridization. These novel exons may be routinely used in different tissues. The nature of the 410- and 285-bp products was shown to be heteroduplexing among the 507-, 310-, and 216-bp PCR products. Identification of the three novel FGF-1 exons, which we designate exons -1B, -1E, and -1F, will facilitate the studies of the mechanisms of regulated alternative splicing.
Alternative Splicing↗
Single primer-mediated polymerase chain reaction.
Explore the source record for details and available documents.
Base Composition↗