Goto C, Hayakawa T, Maeda S
Department of Entomology, University of California, Davis 95616, USA.
Virus Genes. 1998;16(2):199-210. doi: 10.1023/a:1007972108026.
In order to characterize the genome organization of Xestia c-nigrum granulovirus (XcGV), mapping of putative XcGV genes was performed by construction of lambda and M13 phage libraries followed by Southern blot and nucleotide sequencing analyses. Mapping of the lambda (32 clones covering the entire XcGV genome) and M13 (133 clones made by random cloning) phage library clones was carried out by hybridization of the labeled lambda phage clone DNAs to 1) Southern blotted XcGV genomic DNA fragments cleaved with EcoRI, BamHI, or HindIII, and 2) dot blotted M13 clone DNAs. All 133 M13 clone DNAs were sequenced, and coding possibilities were investigated by computer-assisted homology search; in total, about 43 kb of the genome was sequenced. Amino acid sequence homology searches of 67 M13 clones suggested that these GV DNAs coded for previously characterized genes identified in nucleopolyhedroviruses (NPVs) and GVs. These 67 M13 clones were classified into 25 gene homolog groups (including 29 putative genes) based on their homologies to NPV and GV genes. The remaining M13 clones, except one that encoded a putative metalloproteinase, did not possess deduced amino acid sequences with significant homology to proteins in gene databases. Complete nucleotide sequences of the putative XcGV DNA polymerase and Ac144 homolog genes confirmed the reliability of our speculation of putative genes based on the M13 clones sequencing analysis. In a comparison of relative locations of putative XcGV genes with locations of their homologs in NPVs, most XcGV genes were mapped close to the corresponding locations in NPV genomes. These results suggested that XcGV, compared to NPVs, had relatively conserved gene arrangements, although about 22 kb of 43 kb of DNA sequenced randomly in the XcGV genome consisted of sequences/genes non-homologous to those of previously characterized NPVs.
为了表征八字地老虎颗粒体病毒（XcGV）的基因组结构，通过构建λ噬菌体和M13噬菌体文库，随后进行Southern印迹和核苷酸测序分析，对假定的XcGV基因进行了定位。通过将标记的λ噬菌体克隆DNA与以下物质杂交，对λ噬菌体文库（32个覆盖整个XcGV基因组的克隆）和M13噬菌体文库（133个通过随机克隆获得的克隆）进行定位：1）用EcoRI、BamHI或HindIII切割的Southern印迹XcGV基因组DNA片段，以及2）点印迹的M13克隆DNA。对所有133个M13克隆DNA进行了测序，并通过计算机辅助同源性搜索研究了编码可能性；总共对约43 kb的基因组进行了测序。对67个M13克隆的氨基酸序列同源性搜索表明，这些GV DNA编码了先前在核多角体病毒（NPV）和GV中鉴定的基因。根据它们与NPV和GV基因的同源性，将这67个M13克隆分为25个基因同源组（包括29个假定基因）。其余的M13克隆，除了一个编码假定金属蛋白酶的克隆外，其推导的氨基酸序列与基因数据库中的蛋白质没有显著同源性。假定的XcGV DNA聚合酶和Ac144同源基因的完整核苷酸序列证实了我们基于M13克隆测序分析对假定基因推测的可靠性。在比较假定的XcGV基因的相对位置与其在NPV中的同源基因的位置时，大多数XcGV基因被定位在NPV基因组中的相应位置附近。这些结果表明，与NPV相比，XcGV的基因排列相对保守，尽管在XcGV基因组中随机测序的43 kb DNA中约有22 kb由与先前表征的NPV的序列/基因非同源的序列组成。
