Browsing by Author "Brockly, F."
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- Molecular cloning, sequencing, and expression in Escherichia coli of human preprourokinase cDNAPublication . Jacobs, P.; Cravador, A.; Loriau, R.; Brockly, F.; Colau, B.; Chuchana, P.; Van Elsen, A.; Herzog, A.; Bollen, A.A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.
- Porcine D-amino acid oxidase: determination of the mRNA nucleotide sequence by the characterization of genomic and cDNA clonesPublication . Jacobs, P.; Brockly, F.; Massaer, M.; Loriau, R.; Guillaume, J. P.; Ciccarelli, E.; Heinderyckx, M.; Cravador, A.; Biemans, R.; Van Elsen, A.; Herzog, A.; Bollen, A.Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for D-amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824-8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3'-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1-151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal and significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.