Human apolipoprotein B cDNA clone isolation and demonstration that liver apolipoprotein B mRNA is 22 kilobases in length.

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 nicktranslation, and found to cross-hybridize strongly with the other four cDNA clones. The pB8 clone produces a fusion protein of -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 w22 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.Apolipoprotein B (apo B) is abundant in plasma, with a concentration of 0.7-1.0 mg/ml, and is found as a constituent of chylomicrons, very low density lipoproteins, and low density lipoproteins (LDL) (1). Most plasma apo B resides in LDL, a particle containing 75% lipid and 25% protein, almost all of which is apo B. LDL cholesterol levels are correlated with coronary disease susceptibility and recently the same association has been found for apo B levels (2). Human apo B is a glycoprotein that can be separated into two forms on NaDodSO4/PAGE, designated B-100 and B-48 (3). B-100 is made in liver, is necessary for the synthesis and secretion of hepatic-derived, triglyceride-rich lipoproteins, and appears to have a molecular mass of =550 kDa (3, 4). B-48 is synthesized in the small intestine, plays an important role in synthesis and secretion of intestinal-derived, triglyceriderich lipoproteins, and appears to have a molecular mass of 'z275 kDa (3, 4).The current report is concerned with the isolation of cDNA clones for human apo B. Recently, cDNA clones have been isolated for the other human apolipoproteins (5-11). In most cases, the method used relied on previously reported amino acid sequence data obtained by protein chemistry techniques. Sequences of amino acids specified by relatively unambiguous codons were identified and the corresponding mixture of oligonucleotides was synthesized. These were used as probes to screen liver cDNA libraries and appropriate clones were selected. Unfortunately, after delipidation of LDL, apo B is quite insoluble (12) and has resisted characterization by standard protein chemistry techniques. As a result, very little apo B primary amino acid sequence is known (13) and a different approach had to be taken for cloning apo B cDNA. Therefore, in the current study, apo B cDNA clones were isolated from an ...

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HMGIC, the gene for an architectural transcription factor, is amplified and rearranged in a subset of human sarcomas

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Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas

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