Human apolipoprotein B cDNA clone isolation and demonstration that liver apolipoprotein B mRNA is 22 kilobases in length.
- 1 October 1985
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 82 (20), 6825-6829
- https://doi.org/10.1073/pnas.82.20.6825
Abstract
An expression library made in plasmids pUC8 and pUC9 with mRNA derived from the human hepatoma cell line HepG2 was screened with a rabbit antiserum to human low density lipoprotein (LDL). Approximately 12,000 clones were screened and five positives were identified. The cDNA inserts were all 1500-1600 base pairs in length. The insert from one clone, pB8, was isolated, labeled by nick-translation, and found to cross-hybridize strongly with the other four cDNA clones. The pB8 clone produces a fusion protein of .apprxeq. 37.5 kDa that reacts in electrophoretic transfer blot analysis with rabbit anti-human LDL. This reactivity can be abolished by pretreatment of the antiserum with purified human LDL, p = 1.025-1.050 g/ml. A pB8-derived probe was used to demonstrate that apolipoprotein B (apo B) mRNA is present in HepG2 cells and liver extracts but not in HeLa cells or extracts from small intestine, heart, aorta, spleen, brain, skeletal muscle, lung, kidney, or ovary. RNA transfer blot analysis revealed that HepG2 cell apo B mRNA was .apprxeq. 22 kilobases in length. These cDNA clones should allow the isolation of the apo B gene and ultimately the elucidation of the primary structure of this protein.This publication has 30 references indexed in Scilit:
- The determination of molecular weight of proteins by gel permeation chromatography in organic solvents.Journal of Biological Chemistry, 1984
- Apolipoprotein B variant derived from rat intestine.Proceedings of the National Academy of Sciences, 1980
- Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons.Proceedings of the National Academy of Sciences, 1980
- Isolation and partial characterization of a polypeptide belonging to apolipoprotein B from low-density lipoproteins of human plasmaBiochemistry, 1980
- Association of coronary atherosclerosis with hyperapobetalipoproteinemia [increased protein but normal cholesterol levels in human plasma low density (beta) lipoproteins].Proceedings of the National Academy of Sciences, 1980
- Isolation of biologically active ribonucleic acid from sources enriched in ribonucleaseBiochemistry, 1979
- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, 1979
- Studies of the cyanogen bromide fragments of the apoprotein of human serum low density lipoproteinsBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1978
- The cyanogen bromide peptides of the apoprotein of low density lipoprotein (apoB): Its molecular weight from a chemical viewBiochemical and Biophysical Research Communications, 1978
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970