Abstract
Recently, we have found that defibrination of rats with Malayan pit viper venom induces a 10-38-fold increase in the levels of translatable fibrinogen mRNA in the liver. We have used this response to obtain cDNA clones for the three polypeptide chains of rat fibrinogen. A large cDNA library was created in pBR322 from induced rat liver polyadenylated RNA by the poly(dG, dC)-tailing method. Part of this library was screened using colony hybridization with [32P]cDNA prepared from induced and noninduced rat liver RNA. Colonies consistently giving a more intense signal with the induced [32P]cDNA were considered possible fibrinogen recombinants and were used for hybrid selection and translation of mRNA. In this way, cDNA clones for each of the three fibrinogen mRNA's were identified. Analysis of polyadenylated RNA by Northern blotting indicates that the three chains are synthesized from mRNA's of 2300, 2050, and 1950 nucleotides for the alpha, beta, and gamma chains, respectively. The fact tha each of the three chains has a separate mRNA indicates that the highly coordinated regulation of the three messages for rat fibrinogen does not occur by translation of a common cytoplasmic RNA.
Highlights
Induced rat liver polyadenylated RNA by the poly(dG, dC)-tailing method
Mo.; guanidine HCI was from Bethesda Research Laboratories Inc., Bethesda, Md.; oligo(dT)-cellulose was from Collaborative Research, Waltham, Mass.; rabbit reticulocyte lysate was both prepared as consistently giving a more intense signal with the in- described [6]and obtained from New England Nuclear, BostonMass.; duced [32P]cDNAwere considered possible fibrinogen CM-cellulose was from Whatman, Kent,England; cyanogen bromiderecombinants and were used for hybrid selection and translationof mRNAI.n this way, cDNA clones for each of the three fibrinogen mRNA’s were identified
Petersburg, Fla.; E . coli DNA polymerase, calf liver tRNA, and terminadl eoxynucleotidyl transferase were from Boerhinger Mannheim, Indianapolis, Ind.; SI nuclease was from Miles Laboratories, Kankakee, Ill.; Bio-Gel A-150 was from Bio-Rad, Richmond, Calif.; [”2P]dNTP’sZ(specific activity, 390-410 Ci/mmol) were from three chains has a separate mRNA indicates that the Amersham, Arlington Heights,Ill.; methyl mercury was from Venturi highly coordinatedregulation of the three messages for Chemicals; nitrocellulose filter paper BA 85 was from Schleicher and rat fibrinogen does not occur by translation of a com- Schuell, Keene, N
Summary
Induced rat liver polyadenylated RNA by the poly(dG, dC)-tailing method. Part of this library was screened using colony hybridization with [32P]cDNAprepared from induced and noninducedrat liver RNA. Cross-reacting antibodies were 25 pg/ml, [32P]dTTPwas the labeled base, and polyadenylated RNA removed by passing each affinity-purified antiserum over the other was incubated with 2 m methyl mercury for 10 min at room two columns. This was repeated until no detectable cross-reaction temperature [15] before addition to the reaction. Synthesis of Double-stranded cDNA-Thirty p1 of polymerase cleaved with Pst 1.After phenol extraction and ethanolprecipitation, reaction mixture were added to theabove tube at0 "C This mixture the cleaved plasmid DNA was resuspended in 10 n m Tris, 1 mM consisted of 92 pl of buffer (100 mM 4-(2-hydroxyethyl)-l-piperazine- EDTA, pH 8.0, and denatured by heating to 100 "C for 10 min. Fragments larger than 400 nucleotides. cDNA molecules were bacteria were slowly thawed on ice, and hybrid DNA (20 ng) in 100 inserted into the Pst site of pBR322 usingpoly(dC) (cDNA)
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