Molecular cloning of the gene sequences of a major apoprotein in avian very low density lipoproteins.
Molecular cloning of the gene sequences of a major apoprotein in avian very low density lipoproteins.
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禽类极低密度脂蛋白中主要脱辅基蛋白基因序列的分子克隆。
DOI：
10.1021/bi00565a027
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Means,AR
中科院分区：
文献类型：
--
作者：
Chan,L;Dugaiczyk,A;Means,AR
Lawrence Chan,* Achilles Dugaiczyk, and Anthony R. Means摘要：ApoVLDL-II是禽极低密度脂蛋白（VLDL）的主要载脂蛋白。雌性激素治疗可显著刺激其在公鸡肝脏中的合成[Chan， L., Jackson， R. L " O 'Malley， B. W " & Means， A. R.(1976) J. Clin。[j].中国投资管理，2016,33(2):1 - 7。我们通过以下程序从雌激素处理的公鸡肝脏中部分纯化了apoVLDL-II mrna：总核酸提取物，寡聚(dT)-纤维素层析，Sepharose 4B层析，重复寡聚(dT)-纤维素层析和蔗糖梯度超离心。将部分纯度（25-30%）的mRNA合成双链互补DNA (ds cDNA)，并插入质粒pBR322的Pstl位点。嵌合质粒在大肠埃希氏杆菌RRI菌株中转化扩增，通过直接集落转移和部分纯[32P]-apoVLDL-II cDNA探针原位杂交筛选克隆。对阳性克隆的dna进行分离，并采用杂交捕获无细胞翻译技术进行进一步研究。其中一个克隆pVLIO抑制apoVLDL-II mRNA的翻译，进一步通过DNA部分测序进行了表征。发现它含有编码载脂蛋白ldl - ii的62-75个氨基酸的核苷酸。将缺口翻译的[32P] pVLIO Hhal/Hphl片段与apoVLDL-II mRNA杂交导致J？0fi/2的5.6 X 10“3与>杂交完成90%。当使用类似的镍翻译探针时，在单次注射（2mg）己烯雌酚（DES）之前和之后12小时，在公鸡肝脏RNA中定量apoVLDL-II mRNA序列。激素处理导致在DES后12小时内apoVLDL-II特异性序列浓度增加12000倍。相比之下，这些序列在从这些动物的胸肌分离的RNA样品中未被检测到（高达3 × 103）。雌激素处理的公鸡已被用作研究类固醇激素作用机制以及脂蛋白合成分子方面的模型系统。在这种动物中，雌激素显著刺激脂蛋白合成，主要涉及极低密度脂蛋白（VLDL） 1 （Hillyard等，1956；Luskey等，1974；Chan等，1976,1977）。禽VLDL主要有两种载脂蛋白，占VLDL蛋白的90%以上。它们被命名为apoVLDL-I和apoVLDL-II。血浆中这两种蛋白的水平都明显受到雌激素的刺激。ApoVLDL-II已纯化至均匀性，并测定了其一级序列（Chan et al., 1976a, b; Jacksonet al., 1977）。已分离出apoVLDL-II mRNA，并对其翻译产物进行了表征。后者被发现比血浆蛋白大23个氨基酸，在其氨基端含有一个高度疏水的信号序列（Chan et al., 1978, 1980）。禽类肝脏mRNA的体外翻译实验证明，雌激素治疗可诱导apoVLDL-II mRNA的快速积累（Chan等人，1976a, 1978, 1979, 1980）。
Lawrence Chan,* Achilles Dugaiczyk, and Anthony R. Means abstract: ApoVLDL-II is a major apoprotein in avian very low density lipoproteins (VLDL). Its synthesis in the cockerel liver is markedly stimulated byestrogen treatment [Chan, L., Jackson, R. L „O’Malley, B. W „& Means, A. R.(1976) J. Clin. Invest. 58, 368-379]. We have partially purified apoVLDL-II mRNAfrom the liver of estrogen-treated cockerels by the following procedures: total nucleic acid ex-tract, oligo (dT)-cellulose chromatography, Sepharose 4B chromatography, repeat oligo (dT)-cellulose chromatography, and sucrose gradient ultracentrifugation. A double-stranded complementary DNA (ds cDNA) was synthesized from the partially (25-30%) pure mRNA and inserted into the Pstl site of the plasmid pBR322. Amplification of the chimeric plasmids was accomplished by transformation in Escherichiacoli RRI strain, and clones were screened by direct colony transfer and in situ hybridization by using the partially pure [32P]-apoVLDL-II cDNA probe. The DNAs of positive clones were isolated and further studied by the hybrid-arrested cell-free translation technique. One clone, pVLIO, which inhibited the translation of apoVLDL-II mRNA, was further characterized by DNA partial sequencing. It was found to contain nu-cleotides which code for amino acids 62-75 of apoVLDL-II. Hybridization of a nick-translated [32P] pVLIO Hhal/Hphl fragment to the apoVLDL-II mRNA resulted in a J? 0fi/2 of 5.6 X 10 “3 with> 90% completion of hybridization. When a similar nick-translated probe was used, apoVLDL-II mRNA sequences were quantified in cockerel liver RNA before and 12 h after a single injection (2 mg) of diethylstilbestrol (DES). Hormone treatment resulted in a 12000-fold increase in the concentration of apoVLDL-II specific sequences within 12 h after DES. In contrast, such sequences were not detected (up to a Rot of 3 X 103) in RNA samples isolated from thebreast muscles of these animals. e estrogen-treated cockerel has been used as a model system for investigations into the mechanisms of steroid hormone action as well as the molecular aspects of lipoprotein synthesis. In this animal, estrogen administration markedly stimulates lipoprotein synthesis, involving mainly very low density lipoproteins (VLDL) 1 (Hillyard et al., 1956; Luskey et al., 1974; Chan et al., 1976, 1977). There are two major apoproteins in avian VLDL which account for over 90% of the proteins in VLDL. They have been designated apoVLDL-I and apoVLDL-II. The plasma levels of both proteins are markedly stimulated byestrogen. ApoVLDL-II has been purified to homogeneity, and its primary sequence has been determined (Chan et al., 1976a, b; Jacksonet al., 1977). The mRNA for apoVLDL-II has been isolated and its translation product characterized. The latter was found to be larger than the plasma protein by 23 amino acids, containing a highly hydrophobic signalsequence at its amino terminus (Chan et al., 1978, 1980). Estrogen treatment was shown to induce a rapid accumulation of apoVLDL-II mRNA, as demonstrated by in vitro translation assays of avian hepatic mRNA (Chan et al., 1976a, 1978, 1979, 1980).