Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA.
Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA.
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克隆编码部分鸡前白蛋白原的双链 cDNA。
DOI：
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发表时间：
1978
影响因子：
4.8
通讯作者：
R. Deeley
中科院分区：
文献类型：
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作者：
J. Gordon;A. T. Burns;J. Christmann;R. Deeley
提出了一种克隆鸡原白蛋白部分编码双链cDNA分子的方案。该方法不需要纯mRNA或cDNA，具有广泛的适用性。在小麦无生殖细胞翻译系统中由总胍合成的蛋白经免疫沉淀鉴定为Mr = 72,000的鸡原白蛋白。盐酸提取，鸡肝RNA。在用poly(U)-Sephadex G-10色谱法去除大部分核糖体RNA后，通过低盐、等速蔗糖梯度离心使白蛋白mRNA富集约2倍，直到它占mRNA序列的30%左右。将该mRNA制备的双链cDNA通过G-C尾链技术插入质粒PBR322的Pst 1位点，利用重组DNA转化大肠杆菌X1776。利用高度富集白蛋白cDNA序列的cDNA探针进行集落杂交鉴定含有假定白蛋白DNA序列的转化子。在RNA过剩的条件下，将部分纯化的RNA制备物与其cDNA杂交，使R0t值使只有最丰富的cDNA序列杂交，从而分离探针。未杂交的、不丰富的序列被随后的S1核酸酶酶切破坏。通过DNA-mRNA杂交阻滞的无细胞翻译确定了与这一丰富类别cDNA杂交的克隆的身份。将含有白蛋白的镍翻译质粒DNA与甲基汞琼脂糖凝胶分离并转移到重氮酶氧甲基纸上的肝脏总聚(A)+ RNA杂交，确定禽白蛋白mRNA的分子量为850,000。这个分子量相当于大约2600个核苷酸，或者比编码白蛋白前体多肽所需的长度长600个核苷酸。
A scheme is presented for cloning a double-stranded cDNA molecule that codes for a portion of chicken preproalbumin. This method, which does not require pure mRNA or cDNA, has widespread applicability. Chicken preproalbumin was identified as a Mr = 72,000 polypeptide by immunoprecipitation of proteins synehesized in a wheat germ cell-free translation system from total, guanidine.HCl-extracted, rooster liver RNA. After removal of the bulk of the ribosomal RNA by poly(U)-Sephadex G-10 chromatography, albumin mRNA was enriched approximately 2-fold by centrifugation through low salt, isokinetic sucrose gradients, until it represented about 30% of the mRNA sequences present. Double-stranded cDNA prepared from this mRNA was then inserted into the Pst 1 site of the plasmid PBR322 by the "G-C tailing" technique and the recombinant DNA was used to transform Echerichia coli stran X1776. Transformants containing putative albumin DNA sequences were identified by colony hybridization with a cDNA probe that was highly enriched for albumin cDNA sequences. This probe was isolated by hybridizing the partially purified RNA preparation to its cDNA, under conditions of RNA excess, to a R0t value such that only the most abundant cDNA sequences had hybridized. Unhybridized, less abundant, sequences were destroyed by subsequent S1 nuclease digestion. The identity of clones that hybridized to this abundant class cDNA was established by DNA-mRNA hybrid-arrested cell-free translation. Hybridization of nick-translated, albumin-containing, plasmid DNA to total liver poly(A)+ RNA, that had been separated on methyl mercury agarose gels and transferred to diazobenzyloxymethyl paper, established that avian albumin mRNA has a molecular weight of 850,000. This molecular weight corresponds to approximately 2,600 nucleotides, or 600 nucleotides longer than the size required to code for the preproalbumin polypeptide.