Cloning and physical mapping of a gene fragment coding for a 64-kilodalton major late antigen of human cytomegalovirus.
Cloning and physical mapping of a gene fragment coding for a 64-kilodalton major late antigen of human cytomegalovirus.
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编码人类巨细胞病毒 64 千道尔顿主要晚期抗原的基因片段的克隆和物理定位。
DOI：
10.1073/pnas.81.15.4965
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发表时间：
1984
影响因子：
11.1
通讯作者：
Zaia,JA
中科院分区：
文献类型：
--
作者：
Pande,H;Baak,SW;Riggs,AD;Clark,BR;Shively,JE;Zaia,JA
我们已经分离出一个克隆，其中含有一个64千道尔顿的糖蛋白，是人巨细胞病毒（HCMV）的主要晚期抗原编码的基因片段。基于该糖蛋白（HCMVgp 64）的胰蛋白酶肽的氨基酸序列，合成了两组混合序列探针，一组由16个十七脱氧核糖核苷酸的混合物组成，另一组由32个二十脱氧核糖核苷酸的混合物组成。在质粒pBR 327中构建HCMV（Towne株）DNA的亚基因组文库，并用这些合成的寡脱氧核苷酸探针的32 P标记的等分试样筛选转化体。在15，000个克隆中有两个克隆产生强阳性信号。从阳性克隆中分离质粒DNA，并使用两种探针通过限制性酶切图谱和Southern印迹分析进行表征。质粒DNA含有2.3-内切酶插入片段，经Sau 3A酶切后产生800碱基对和1500碱基对的片段。只有800个碱基对的片段与混合探针杂交，DNA序列分析显示它含有与HCMVgp 64的胰蛋白酶肽的氨基酸序列相容的核苷酸序列。利用该32 P标记的800碱基对克隆DNA对HCMV DNA进行限制性作图研究，使我们能够将该基因片段定位在HCMV（Towne株）基因组长的独特区域中，约等于0.5-0.51图单位。
We have isolated a clone containing a gene fragment coding for a 64-kilodalton glycoprotein that is the major late antigen of human cytomegalovirus (HCMV). Based upon the amino acid sequence of a tryptic peptide of this glycoprotein (HCMVgp64), two sets of mixed-sequence probes, one consisting of a mixture of 16 heptadecadeoxyribonucleotides and the other a mixture of 32 icosadeoxyribonucleotides, were synthesized. A subgenomic library of HCMV (Towne strain) DNA was constructed in plasmid pBR327 and transformants were screened with 32P-labeled aliquots of these synthetic oligodeoxyribonucleotide probes. Two clones among 15,000 gave strong positive signals. Plasmid DNA was isolated from the positive clones and characterized by restriction mapping and Southern blot analysis using both probes. The plasmid DNA contained a 2.3-kilobase insert, which yielded an 800-base-pair and a 1500-base-pair fragment after Sau3A digestion. Only the 800-base-pair fragment hybridized to the mixed probes, and DNA sequence analysis revealed that it contains nucleotide sequences compatible with amino acid sequences of tryptic peptides of HCMVgp64. Restriction mapping studies of HCMV DNA using this 32P-labeled 800-base-pair cloned DNA have allowed us to locate this gene fragment in the long unique region of HCMV (Towne strain) genome at approximately equal to 0.5-0.51 map unit.