Localization of peptide growth factors in the nucleus.
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Animals↗
Biomedical subjects
H Prats
Publications and source records attributed to H Prats.
Potential oncogenic effects of basic fibroblast growth factor requires cooperation between CUG and AUG-initiated forms.
Normal adult bovine aortic endothelial cells were infected with various recombinant retroviruses expressing one, two, or three human basic fibroblast growth factor (bFGF) proteins normally synthesized by an alternative use of translation initiation codons. We show here that the constitutive expression of the AUG-initiated from (18 kDa) leads the transfected cells to form colonies in soft agar. The expression of the high molar weight (HMW) forms (22.5 and 21 kDa) initiated at one of the two CUG initiation codons allows cell immortalization, whereas the tumorigenic potential is reached when the three forms are constitutively expressed. Furthermore, we provide evidence that constitutive expression of (HMW) bFGF forms has a down-regulation effect on bFGF synthesis from the gene naturally active in parental endothelial cells.
Animals↗
Nuclear translocation of basic fibroblast growth factor.
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Amino Acid Sequence↗
Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor.
Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUG-initiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.
Amino Acid Sequence↗
Production and functional characterization of human recombinant FGF-6 protein.
The fibroblast growth factor (FGF) gene family to date comprises seven members and has been implicated in a wide range of physiological and biological processes, including angiogenesis, morphogenesis, and tumorigenesis. The FGFs are mitogens for a broad range of cells of various embryological origins and can act as differentiation factors. The FGFs can bind to tyrosine kinase and non-tyrosine kinase transmembrane receptors; the physiological basis for this is still unknown. In order to study more thoroughly the activities of FGF-6, we have constructed a bacterial expression vector by inserting FGF-6 complementary DNA sequences into the T7 RNA polymerase-based pET3a vector. The resulting construct is able to drive the expression of a high amount of FGF-6 protein in Escherichia coli, which can be solubilized and purified through heparin-Sepharose chromatography and high salt elution. The purified FGF-6 protein displays a strong mitogenic activity on BALB/c 3T3 cells and is able to morphologically transform these cells. By contrast, adult bovine aortic endothelial cells, which normally require the presence of FGF-2 for their growth, show only a limited mitogenic response that is highly dependent on heparin concentration.
3T3 Cells↗
The human basic fibroblast growth factor gene is located on the long arm of chromosome 4 at bands q26-q27.
Using in situ chromosomal hybridization, we have mapped the gene encoding the basic fibroblast growth factor to chromosome 4 at bands q26-q27. This location is on the same chromosome as that of a related gene, FGF.5, but on a distinct region.
Chromosome Banding↗
Protein kinase NII and the regulation of rDNA transcription in mammalian cells.
Transcription of ribosomal RNA genes is generally accepted to correlate with cell growth. Using primary cultures of adult bovine aortic endothelial (ABAE) cells, we have shown that transcription of rDNA in confluent cells falls to 5% of the transcription level in growing cells. Protein kinase NII appears to be a limiting factor to promote rDNA transcription in isolated nuclei of confluent cells. Protein kinase NII was detected by immunocytochemistry in the cytoplasm, nuclei and nucleoli of growing cells while it was no longer present in nucleoli of confluent cells. The kinase activity, in isolated nuclei, was estimated by endogenous phosphorylation of a specific substrate, nucleolin. A 10% residual activity was present in confluent cell nuclei compared to growing cell nuclei. Concomitantly, the transcription 'in vitro' of rDNA in the corresponding nuclei was also highly reduced (by 85%). Addition of exogenous protein kinase NII to confluent cell nuclei induced a strong increase in the phosphorylation of specific proteins including nucleolin. In parallel, the transcription of rDNA was increased by a factor of 5, to nearly the level observed in nuclei prepared from growing cells. These data suggest that, in confluent cells, factors necessary for rDNA transcription machinery are present but inactive in the nucleolus and that the phosphorylation of one or several of these factors (nucleolin, topoisomerase I,...) by protein kinase NII is a key event in the regulation of rDNA transcription.
Alkaline Phosphatase↗
Autocrine regulation of bovine retinal capillary endothelial cell (BREC) proliferation by BREC-derived basic fibroblast growth factor.
Fibroblast growth factors (FGFs) are mitogenic for bovine retinal capillary endothelial cells (BREC) seeded at a low density. Seeding BREC cells at a high density greatly reduces their requirement for basic FGF (bFGF) in order to proliferate actively. We show here that monolayers of BREC cells synthesize and release into the culture medium a growth factor, which on the basis of biological activity, heparin affinity, immuno-cross reactivity with anti-bFGF antibodies and mRNA analysis, has been identified as basic fibroblast growth factor. These data indicate that BREC cells are able to synthesize and release bFGF, which can act as a promoting-growth factor for these cells by a para- and/or autocrine mechanism. We suggest thus, that this para- and/or autocrine mechanism involving bFGF may play a key role in preretinal neovascularization, particularly in diabetic patients presenting a proliferative retinopathy.
Animals↗
High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons.
A 6.75-kilobase human hepatoma-derived basic fibroblast growth factor (bFGF) cDNA was cloned and sequenced. An amino-terminal sequence generated from a purified hepatoma bFGF was found to correspond to the nucleotide sequence and to begin 8 amino acids upstream from the putative methionine start codon thought to initiate a 154-amino acid bFGF translation product. This sequence suggests that a form of bFGF of at least 163 amino acids exists. The hepatoma cDNA was transcribed in vitro into RNA; in vitro translation of this RNA generated three forms of bFGF with molecular masses of 18, 21, and 22.5 kDa. By use of in vitro mutagenesis, it was found that the 22.5-kDa bFGF and possibly the 21-kDa form were initiated with CUG start codons. The 18-kDa bFGF was initiated with an AUG codon. By transfecting into COS cells human hepatoma bFGF cDNA and a construct from which the AUG initiator was eliminated, it was found that the higher molecular mass forms of bFGF were as biologically active as the 18-kDa form.
Amino Acid Sequence↗
Basic fibroblast growth factor enters the nucleolus and stimulates the transcription of ribosomal genes in ABAE cells undergoing G0----G1 transition.
The cellular action of growth factors, among them basic fibroblast growth factor (bFGF), is mediated by their interaction with a cell surface receptor, but the mechanism of transfer of mitogenic (or other) signals to the nucleus has not been identified. In this work, we show that bFGF is translocated to and accumulated in the nucleolus. Furthermore, the nucleolar localization of bFGF is correlated with a stimulation of transcription of ribosomal genes during G0----G1 transition induced by bFGF alone in adult bovine aortic endothelial cells (ABAE cells). Stimulation of ribosomal gene transcription is preceded by a significant increase of the major nonhistone nucleolar protein, nucleolin. In vitro, the growth factor has a direct effect on the enhancement of RNA polymerase I activity in isolated nuclei from quiescent sparse (G0) ABAE cells. The direct action of bFGF on the level of ribosomal gene transcription could correspond to an additional growth-signaling pathway, mediated by this growth factor.
Animals↗
The hexB mismatch repair gene of Streptococcus pneumoniae: characterisation, cloning and identification of the product.
A second gene involved in mismatch repair in Streptococcus pneumoniae, the hexB gene, has been characterised. The gene was cloned into a multicopy plasmid vector. The cloned hexB gene is expressed as judged by its ability to complement a chromosomal hexB- allele. Its direction of transcription and its functional limits were defined. Comparison of the proteins encoded by recombinant plasmids and by restriction fragments allowed us to identify an Mr 83,000 protein as the probable product of the hexB gene. We offer evidence that this gene together with the hexA gene is essential for repair of transition as well as frameshift mismatches.
Alleles↗
A plasmid vector allowing positive selection of recombinant plasmids in Streptococcus pneumoniae.
A new plasmid, pSP2, was constructed as a cloning vector for use in Streptococcus pneumoniae. It allows direct selection of recombinant plasmids, even for DNA fragments not homologous to the S. pneumoniae chromosome, as based on the failure to maintain long inverted repeats (LIRs) hyphen-free in bacterial plasmids. Plasmid pSP2 contains a 1.4-kb BamHI fragment ("hyphen") flanked by 1.9-kb LIRs. The removal of the 1.4-kb BamHI fragment followed by ligation creates a plasmid containing a 1.9-kb insert-free LIR; plasmids with such non-hyphenated LIRs were not established when transferred into S. pneumoniae. Replacement of the original 1.4-kb insert by other restriction fragments restored plasmid viability. Investigation of plasmid transfer by transformation suggests that intrastrand synapsis between the LIRs could occur, thus facilitating plasmid establishment (a process we call self-facilitation). Such an intrastrand synapsis could also account for rare occurrences of insert-inversion noticed upon transfer as well as for the formation of palindrome-deleted derivatives at low frequency. Plasmid pSP2 carries two selectable genes, tet and ermC, and can be used for cloning of fragments produced by a variety of restriction enzymes (BamHI, Bg/II, Bc/I or Sau3A, and Sa/I or XhoI).
Chromosome Mapping↗
Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product.
The hexA mismatch repair gene of Streptococcus pneumoniae has been cloned into multicopy plasmid vectors. The cloned hexA gene is expressed as judged from its ability to complement various chromosomal hexA- alleles. Its direction of transcription was defined and the functional limits were localized by original methods relying on homology-dependent integration of nonautonomous chimeric plasmids carrying chromosomal inserts into the chromosome. Comparison of the proteins encoded by recombinant plasmids and by restriction fragments allowed us to identify an Mr 94 000 protein as the probable product of the hexA gene.
Bacterial Proteins↗
Identification of Streptococcus pneumoniae mismatch repair genes by an additive transformation approach.
During transformation of Streptococcus pneumoniae, mismatch repair occurs on donor-recipient heteroduplexes harboring some mismatched base pairs. A few mutants defective in mismatch repair have been isolated and termed hex-. However, neither the number of genes involved nor their products have yet been identified. In an attempt to characterize such genes we have used an additive transformation approach--that is the inactivation of genes by insertion of chimeric plasmids. Pneumococcal DNA fragments were joined in vitro to a plasmid derivative of pBR325 that carries an erythromycin resistance determinant and does not replicate autonomously in S. pneumoniae. Ery-r transformants obtained with such a ligation mixture arise via homology-dependent integration of the chimeric plasmids into the chromosome. Hex- mutants have been selected among the ery-r population. Comparison of these mutants by Southern blot hybridization with a vector probe reveals that at least two genes are involved in mismatch repair.
Cloning, Molecular↗