Isolation and characterization of cDNA clones for human apolipoprotein A-I.

Breslow, J L; Ross, David A.; McPherson, Joseph; Williams, Huw D.; Kurnit, David M.; Nussbaum, A. L.; Karathanasis, Sotirios K.; Zannis, Vassilis I.

doi:10.1073/pnas.79.22.6861

Cited by 125 publications

(64 citation statements)

References 39 publications

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“…Northern and dot blot of total cellular RNA were performed as described (36). For RNA hybridization, a specific human apo A-I cDNA probe was used (37). A mouse apo A-I cDNA fragment was cloned from mouse liver mRNA by reverse transcription and PCRamplification (sense primer: GAC AGC GGC AGA GAC TAT GTG TCC CAG TTT GAA; antisense primer: TGG CTT TCT CGC CAA GTG TCT TCA GGT GG).…”

Section: Methodsmentioning

confidence: 99%

“…Transcription run-on assays were performed as described by Nevins (40), using 100 l of nuclei. Equivalent counts of nuclear RNA labeled with [ ␣ -32 P]UTP (3,000 Ci/mmol) were hybridized for 36 h at 65 Њ C to 5 g human (37) and mouse apo A-I cDNAs immobilized on Hybond-C Extra filters (Amersham Corp.). The empty vector plasmid was used as a negative control.…”

Section: Methodsmentioning

confidence: 99%

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Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice.

Berthou¹,

Duverger²,

Emmanuel³

et al. 1996

J. Clin. Invest.

239

158

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The regulation of liver apolipoprotein (apo) A-I gene expression by fibrates was studied in human apo A-I transgenic mice containing a human genomic DNA fragment driving apo A-I expression in liver. Treatment with fenofibrate (0.5% wt/wt) for 7 d increased plasma human apo A-I levels up to 750% and HDL-cholesterol levels up to 200% with a shift to larger particles. The increase in human apo A-I plasma levels was time and dose dependent and was already evident after 3 d at the highest dose (0.5% wt/wt) of fenofibrate. In contrast, plasma mouse apo A-I concentration was decreased after fenofibrate in nontransgenic mice. The increase in plasma human apo A-I levels after fenofibrate treatment was associated with a 97% increase in hepatic human apo A-I mRNA, whereas mouse apo A-I mRNA levels decreased to 51%. In nontransgenic mice, a similar down-regulation of hepatic apo A-I mRNA levels was observed. Nuclear run-on experiments demonstrated that the increase in human apo A-I and the decrease in mouse apo A-I gene expression after fenofibrate occurred at the transcriptional level. Since part of the effects of fibrates are mediated through the nuclear receptor PPAR (peroxisome proliferator-activated receptor), the expression of the acyl CoA oxidase (ACO) gene was measured as a control of PPAR activation. Both in transgenic and nontransgenic mice, fenofibrate induced ACO mRNA levels up to sixfold. When transgenic mice were treated with gemfibrozil (0.5% wt/wt) plasma human apo A-I and HDL-cholesterol levels increased 32 and 73%, respectively, above control levels. The weaker effect of this compound on human apo A-I and HDL-cholesterol levels correlated with a less pronounced impact on ACO mRNA levels (a threefold increase) suggesting that the level of induction of human apo A-I gene is related to the PPAR activating potency of the fibrate used. Treatment of human primary hepatocytes with fenofibric acid (500 M) provoked an 83 and 50% increase in apo A-I secretion and mRNA levels, respectively, supporting that a direct action of fibrates on liver human apo A-I production leads to the observed increase in plasma apo A-I and HDLcholesterol. ( J. Clin. Invest. 1996. 97:2408-2416.)

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice.

Berthou¹,

Duverger²,

Emmanuel³

et al. 1996

J. Clin. Invest.

239

158

View full text Add to dashboard Cite

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“…26 NotI digestion of pNTC3CMV␤ deleted the lacZ gene and allowed insertion of apoAI cDNA (806 bp) 27 or LCAT cDNA (1322 bp), 28 keeping the CMV immediate-early promotor SV40 splice donor/acceptor and SV40 polyA signal flanking the inserted sequences ( Figure 1). To coexpress apoAI and LCAT, an AAV-based bicistronic vector was produced by inserting intervening internal ribosome entry site (IRES) sequences derived from poliovirus ( Figure 1).…”

Section: Resultsmentioning

confidence: 99%

Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

et al. 1998

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Plasma apolipoprotein AI (apoAI) and lecithin-cholesterolThe secretion of apoAI or LCAT by transduced cultures acyltransferase (LCAT) play important roles in reverse was two-to five-fold higher using AAV-based plasmid veccholesterol transport, promoting the removal of excess tors than conventional plasmid vectors. Additionally, cells cholesterol from peripheral cells and reducing formation of transfected with a bicistronic AAV-based vector containing atherosclerotic lesions. Gene augmentation of either apoAI an internal ribosome entry site (IRES) efficiently expressed or LCAT, or both, are thus attractive targets for prevention both apoAI and LCAT simultaneously. Furthermore, AAVor treatment of atherosclerosis. With the eventual aim of based vector sequences were retained by host cells, safe and efficient gene delivery to skeletal muscle, our whereas those of conventional plasmid vectors were lost. chosen secretory platform for systemic delivery of antiThese studies indicate that ectopic overexpression of atherogenic proteins, we have constructed conventional apoAI and LCAT in muscle tissue using AAV-based and AAV-based plasmid vectors containing human apoAI plasmid vectors might provide a feasible anti-atherogenic or LCAT cDNAs; their efficacy was tested by lipoplex transstrategy in vivo. fection of mouse C2C12 muscle cells or human 293 cells.

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“…Recently, cDNA clones have been isolated for the other human apolipoproteins (5)(6)(7)(8)(9)(10)(11). In most cases, the method used relied on previously reported amino acid sequence data obtained by protein chemistry techniques.…”

mentioning

confidence: 99%

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

Huang¹,

Bock²,

Feinstein³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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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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Isolation and characterization of cDNA clones for human apolipoprotein A-I.

Cited by 125 publications

References 39 publications

Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice.

Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice.

Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

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

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