Characterization of a cDNA clone coding for the beta chain of bovine fibrinogen.

Abstract

Recombinant plasmids containing bovine cDNA have been screened with a radiolabeled cDNA enriched for bovine fibrinogen. A number of plasmids containing cDNAs for fibrinogen were identified by this assay. One 66,124, 52,306, and 46,474, respectively (4). During the coagulation process, thrombin removes fibrinopeptides A and B from the amino termini of the a and 13 chains, respectively. This leads to the formation of fibrin monomers which are subsequently polymerized and crosslinked to form a tough, insoluble, fibrin clot (5,6).The amino acid sequences of the a, 13, and y chains of human fibrinogen have been determined (7-11). A high degree of homology among the three chains suggests that they may have been derived from a common ancestral gene (11). A distended polydomainal structure linked by interdomainal supercoiled ahelices has been proposed (12-15).Fibrinogen is synthesized in hepatic parenchymal cells (16) where the a, 13, and 'y chains are synthesized from individual mRNAs (17). Little is known, however, about the subsequent processing, assembly, and secretion of this protein into the plasma. The biosynthesis of fibrinogen is stimulated during inflammation, injury, or bacterial infection, and this can lead to a substantial increase in its plasma level (18). The mechanism for this increase has not been established. A number of questions regarding the structure, function, and biosynthesis of fibrinogen can be examined with cDNA probes to specific mRNAs. Accordingly, we have developed methods for the isolation and characterization of recombinant plasmids containing cDNA coding for the three chains of this protein.In the present report, we describe the properties of a plasmid containing a cDNA coding for the ,B chain. MATERIALS AND METHODSEnzymes. The Klenow fragment of Escherichia coli polymerase I and T4 polynucleotide kinase were purchased from P-L Biochemicals. All restriction endonucleases were purchased from Bethesda Research Laboratories (Rockville, MD), except Sau 3A which was purchased from New England BioLabs. Bovine fibrinogen was purchased from Calbiochem.Construction of Recombinant Plasmids. Reverse transcription of enriched bovine liver mRNA, synthesis of doublestranded cDNA, homopolymeric G/C tailing, construction of recombinant plasmids by using pBR322 as vector, and cloning into E. coli K-12, strain RRI, have been reported (19). The cloning was performed under P3-EK1 conditions in compliance with the revised (1978) guidelines for recombinant DNA research from the National Institutes of Health.Screening of Recombinant Plasmids. Bovine liver polysomes were prepared as described (17,20). mRNA for fibrinogen was enriched by immunoadsorption with Staphylococcus aureus cells and affinity-purified antibodies to bovine fibrinogen (21). Radiolabeled cDNA was prepared from the enriched mRNA and was used as a probe in a solution hybridization screening procedure as described (19).Restriction Endonuclease Mapping. The locations of restriction enzyme sites on the inserted DNA were determined by ...

Characterization of adsorbed protein films by time of flight secondary ion mass spectrometry

“…It is interesting to view the correlation between the loadings and the scores in view of the primary structure of these four proteins. The amino acid composition of each of the proteins used in this study was collected from various sources18–23 and is given in Table III. For a protein that has a significantly higher concentration of a particular amino acid, the peaks from that amino acid will be located in the loadings plot as the protein is in the scores plot.…”

The Utility of DNA Sequences of an Intron from the β-Fibrinogen Gene in Phylogenetic Analysis of Woodpeckers (Aves: Picidae)

“…To design primers, we compared the published exon sequences for chicken (Weissbach et al, 1991), human (Chung et al, 1983), rat (Eastman and Gilula, 1989), bovine (Chung et al, 1981), and lamprey (Bohonus et al, 1986) b-fibrinogens. Following alignment, highly conserved regions were found in the coding regions flanking five of the seven introns found in human b-fibrinogen and potential primers were designed for each using the primer design program Oligo 4.0.…”

MultiCoil: A program for predicting two‐and three‐stranded coiled coils

“…The construction of the twostranded coiled-coil database is discussed in Berger et al The three-stranded coiled-coil database was constructed from 42 sequences. The proteins in the three-stranded database have been characterized in the following references: laminins (families A, Bl, and B2 chains) (Pikkarainen et al, 1987(Pikkarainen et al, , 1988Sasaki et al, , 1988Monte11 & Goodman, 1988Hunter et al, 1989Hunter et al, , 1992Engel, 1992;Kallunki et al, 1992;Nomizu et al, 1992;Beck et al, 1993;Porter et al, 1993;Gerecke et al, 1994;Vuolteenaho et al, 1994;Wewer et al, 1994), fibrinogens (families a, j?, and y chains) (Chung et al, 1981(Chung et al, , 1983a(Chung et al, , 1983bCrabtree & Kant, 1982;Rixon et al, 1983;Crabtree et al, 1985;Strong et al, 1985;Bohonus et al, 1986;Koyama et al, 1987;Wang et al, 1989;Pastori et al, 1990;Weissbach & Grieninger, 1990;Pan & Doolittle, 1992), K. lactis heat shock factor (Peteranderl & Nelson, 1992), S. cerevisiae heat shock transcription factor (Sorger & Nelson, 1989), influenza virus hemagglutinin (Bullough et al, 1994), Moloney murine leukemia virus ( Fass et al, 1996), fibritin encoded by Wac gene for bacteriophage T4 and K3 (Efimov et al, 1994), gp17 leg protein in bacteriophages T7 and T3, and macrophage scavenger receptor protein (Conway & Parry, 1991).…”