| Popis: |
Fibrinogen is coded by three separate genes located in a 50kb region of chromosome 4 and organized in a α - β - γ orientation with an inversion of the gene 3- A human genomic library was constructed using the EMBL4 phage and screened with cDNA probes coding for human fibrinogen Aα, Bβ and γ chains. Clones, covering the fibrinogen locus,were identified, and their organization was analyzed by means of hybridization and restriction mapping. Among these clones one recombinant phage containing the β gene and large 5’ and 3’ -flanking sequences was isolated.To identify the regulatory sequences Dpstream from the human β gene, a 1.5 kb fragment of the immediate 5’-flanking region was sequenced. The SI mapping experiments revealed three transcription initiation sites. PotentialTATA and CAAT sequences were identified upstream the initiation start points at the positions -21 and -58 from the first initiation start point.Comparison of this sequence with that previously reported for the same region upstream from the human γ gene revealed no significant homology which suggests that the potential promoting sequences of these genes are different. In contrast, comparison of the 5’flanking regions of human and rat β genes showed more than 80% homology for 142 bp upstream from the gene. This highly conserved region is a potential candidate for a regulatory sequence of the human β gene.To verify this activity, a β fibrinogen minigene was constructed by deletion of the internal part of the normal gene and including 3.4kb of the 5’flanking region and 1.4kb of the 3’flanking region. The minigene was transfected into HepG2, a human hepatoma cell line, to show whether the 5’flanking region of the human fibrinogen gene contains DNA sequences sufficient for efficient transcription in HepG2. Constructions of several parts of the sequenced 5’flanking region of the human β gene with the gene of the chloramphenical acetyl transferase have been also transfected in the HepG2 cells to determine the specificity of the gene expression and to localize the sequences controlling the transcription of the gene. |
