Stenger D C, Revington G N, Stevenson M C, Bisaro D M
Biotechnology Center, Ohio State University, Columbus 43210.
Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8029-33. doi: 10.1073/pnas.88.18.8029.
Agrobacterium-mediated inoculation of Nicotiana benthamiana plants with Ti plasmids containing tandem genome repeats derived from different strains of the gemini-virus beet curly top virus (BCTV) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants. When two putative plus-strand origins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant genotype. The genotype was dependent upon the arrangement of repeated parental genomes in the inocula. Sequencing across the junction between parental BCTV strains in the recombinant progeny allowed mapping of the plus-strand origin of replication to a 20-base-pair sequence within the conserved hairpin found in all geminivirus genomes. In contrast, when inocula contained tandemly repeated BCTV genome sequences but only a single conserved hairpin, a number of different progeny genotypes were simultaneously replicated in infected plants, a result expected if unit-length viral genomes were generated by random intramolecular recombination events. These results and other considerations indicate that geminivirus DNA replication occurs by a rolling-circle mechanism.
用含有源自双生病毒甜菜曲顶病毒（BCTV）不同菌株的串联基因组重复序列的Ti质粒，通过农杆菌介导接种本氏烟草植株，在系统感染的植株中产生了单位长度的重组子代基因组。当用作接种物的构建体中存在两个假定的正链复制起点时，有利于一种复制逃逸机制，该机制导致产生单一主要基因型的子代基因组。该基因型取决于接种物中重复亲本基因组的排列方式。对重组子代中亲本BCTV菌株之间的连接处进行测序，使得能够将正链复制起点定位到在所有双生病毒基因组中发现的保守发夹结构内的一个20个碱基对的序列上。相比之下，当接种物包含串联重复的BCTV基因组序列但只有一个保守发夹时，在受感染植株中同时复制了许多不同的子代基因型，如果单位长度病毒基因组是由随机分子内重组事件产生的，那么这一结果是可以预期的。这些结果及其他考虑因素表明，双生病毒DNA复制是通过滚环机制进行的。
