ABSTRACT
A sensitive immunoassay was used to identify recombinant DNA plasmids carrying cDNA fragments of bovine caseins in the cDNA library from rRNA of bovine mammary glands. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter paper. Antigens covalently bound to the CNBr-activated paper or bound to the nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. Four clones were positive among 5400 bacterial clones of the cDNA library--al, b2, b5, h7. Molecular weights of chimeric proteins pre-beta-lactamase:casein synthesized in Escherichia coli were determined by immunoblotting. Colony hybridization and DNA sequence analysis showed that clone b5 contained cDNA fragment of bovine kappa-caseins and clone h7 cDNA fragment of beta-casein. The last clone was designated pKcas beta-7.
Subject(s)
Caseins/genetics , Cloning, Molecular , DNA, Bacterial/genetics , DNA/genetics , Genes , Animals , Base Sequence , Caseins/immunology , Cattle , DNA/analysis , Escherichia coli/genetics , Female , Immunoassay , Nucleic Acid Hybridization , Protein Biosynthesis , RNA, Messenger/geneticsABSTRACT
The bradykinin (BK) gene chemically synthesized and cloned into pBR 322 was used for the study of tissue localization and quantitation of mRNA coding for the BK precursor, kininogen. Poly(A+)-mRNAs from bovine liver, spleen, kidney, mammary gland and pancreas were used for dot-hybridization to [32P]DNA of the BK gene or to the plasmid-containing BK gene. The experimental results demonstrate that [32P]DNA of BK is hybridized only to the liver poly(A+)-RNA, which proves the liver to be the main kininogen mRNA-producing tissue. In other tissues, the kininogen mRNA synthesis is either altogether absent or its level is two orders of magnitude less as compared to the liver. Several approaches for the quantitation of the kininogen mRNA were developed. The amount of this mRNA was shown to be about 0.6% of the total cellular poly(A+)-RNA. Poly(A+)-RNA which is bound to BK DNA-cellulose and is enriched with BK-coding sequences was used for the study of the hybridization kinetics and translation in a cell-free system from rabbit reticulocytes. The polypeptides synthesized contain BK as was shown by the use of a bio-test in rat uterus.
Subject(s)
Kininogens/biosynthesis , Liver/metabolism , RNA, Messenger/biosynthesis , Animals , Bradykinin/genetics , Bradykinin/pharmacology , Cattle , Cloning, Molecular , DNA/genetics , Female , Genes , In Vitro Techniques , Nucleic Acid Hybridization , Plasmids , Poly A/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , Rats , Tissue Distribution , Uterine Contraction/drug effectsABSTRACT
A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-beta-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine chi-casein, clone 3-H7 contained a cDNA fragment of beta-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of alpha s1-casein.
Subject(s)
Caseins/genetics , Animals , Base Sequence , Caseins/immunology , Cattle , Cloning, Molecular , DNA/genetics , DNA, Recombinant , Immunologic Techniques , Nucleic Acid Hybridization , Protein BiosynthesisABSTRACT
The possibility of RNA-synthesis by E. coli RNA-polymerase using denatured DNA-templates from mouse liver, immobilized on nitrocellulose filters was shown. The size of RNA molecules, synthesized on immobilized templates was estimated by electrophoresis in polyacrylamide gel. The length of the RNA molecules was found to be about 30 nucleotides. Data from alkaline hydrolisys and sedimentation in sucrose density gradient suggest that there is no connection between the DNA-primer and the RNA-product, therefore the DNA-primer is not necessary for the initiation of RNA-synthesis.
