Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus.
Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus.
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DOI：
10.1016/0042-6822(85)90012-1
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发表时间：
1985-10
期刊：
影响因子：
3.7
通讯作者：
G. Darai;H. Delius;Jill Clarke;Heiko Apfel;Paul Schnitzler;R. Flügel
中科院分区：
文献类型：
--
作者：
G. Darai;H. Delius;Jill Clarke;Heiko Apfel;Paul Schnitzler;R. Flügel
建立了鱼类淋巴囊肿病病毒（FLDV）基因组的一个完整基因文库。用EcoRI、BamHI、EcoRI/BamHI和EcoRI/HindIII酶切FLDV DNA，用T4 DNA连接酶将所得片段插入pACYC 184或pAT 153质粒载体的相应位点。由于FLDV DNA在CpG序列处高度甲基化（Daraiet等，1983; Wagneret等，1985），需要大肠杆菌GC-3菌株来扩增含有FLDV DNA片段的重组质粒。选择携带重组质粒的细菌菌落。通过用不同的限制性内切酶消化重组质粒DNA，并通过重组质粒DNA与病毒DNA的杂交筛选所有克隆的片段。该分析显示，代表100%病毒基因组的序列已被克隆。利用这些重组质粒，构建了BamHI、EcoRI、BestE Ⅱ和Pst Ⅰ限制性内切酶的基因组物理图谱。虽然FLDV基因组是线性的，但由于环状排列，限制性图谱是环状的。
A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved withEcoRI,BamHI,EcoRI/BamHI andEcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Daraiet at, 1983; Wagneret al., 1985), anEscherichia coliGC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed forBamHI,EcoRI,BestEII, andPstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.