High molecular mass forms of basic fibroblast growth factor are initiated by alternative CUG codons.

Abstract

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.

Angiogenesis induced in muscle by a recombinant adenovirus expressing functional isoforms of basic fibroblast growth factor

“…The sizes of the specific bands are in general agreement with the molecular weights described by others. 18,19 In our experiments, we were unable to detect the 23K species. Either the CTG initiating codon is rarely chosen by the translation machinery, or the gel does not allow a thorough resolution, making the 22.5K and 23K appear as a doublet.…”

“…The position of each band is in agreement with the molecular weights described by others. 18,19 Similar quantities of protein were found in the bands corresponding to the 18 kDa isoform in lane 6 and to the 16 kDa bFGF standard protein (20 ng). ELISA quantification of bFGF found in extracts from Ad-RSVbFGF-infected 293 or C2 cells (15 and 7 ng/10 6 cells/24 h, respectively) confirmed the results obtained in Western blots.…”

“…18 In addition to the ATG-initiated coding region of the gene, this sequence includes in its 5Ј end a 71 nucleotide long region containing several CTGs normally used as alternative initiation codons, resulting in the production of larger isoforms containing nuclear localization sequences. The 3Ј untranslated region contains only the first polyadenylation signal of the bFGF gene with a relatively small portion (984 nucleotides) of the very long 3Ј sequence (4.8 kb) existing in the cDNA and which is probably involved in regulation of mRNA stability.…”

“…The 3Ј untranslated region contains only the first polyadenylation signal of the bFGF gene with a relatively small portion (984 nucleotides) of the very long 3Ј sequence (4.8 kb) existing in the cDNA and which is probably involved in regulation of mRNA stability. 18 The human bFGF cDNA sequence was positioned under the control of the RSV promoter. The replication-deficient recombinant Ad-RSVbFGF was obtained by in vivo homologous recombination in 293 cells between the recombinant plasmid and viral DNA.…”

“…16 The E1-defective virus Ad-RSVbFGF, expressing the bFGF cDNA under the control of the promoter of the Rous sarcoma virus (RSV) was constructed as follows. The plasmid pAd-RSVbFGF was obtained after subcloning of a XhoI/EcoRV fragment (1.62 kb) from the pSCT40 expression vector containing the sequence from nucleotide 295 to 1919 of the human bFGF cDNA isolated by Prats et al, 18 into the plasmid pAd-LTRIX cleaved by SalI and EcoRV. 39 The recombinant virus Ad-RSVbFGF was obtained by homologous recombination, plaque-purified twice and amplified about four to six times to prepare larger stocks as described.…”