Carter B J, Khoury G, Denhardt D T
J Virol. 1975 Sep;16(3):559-68. doi: 10.1128/JVI.16.3.559-568.1975.
Cleavage of adenovirus-associated virus type 2 (AAV2) DNA linear duplex monomers with the restriction endonuclease R-EcoRI yielded three fragments, A, B, and C, having approximate mol wt of 1.6 X 10(6), 1.1 X 10(6), and 1.3 X 10(5), respectively. Radioactive labeling the 5' termini of AAV DNA before cleavage with R-EcoRI showed that A and B were terminal fragments and C was internal. Separation of the complementary strands of fragments A and B showed that A contained the 5' terminus of the minus strand and the 3' terminus of the plus strand, and conversely for fragment B. The physical map of the AAV R-EcoRI fragments can thus be unambiguously determined and is drawn with B at the left-hand and A at the right-hand end. On this map, transcription of stable AAV mRNA from the minus strand proceeds from left to right, beginning in fragment B and terminating in fragment A. The asymmetry in distribution of thymidine between the AAV DNA plus and minus strands is preferentially located in fragment A, which represents the right-hand half of the duplex molecule. These experiments enable preparative separation of all four single-strand termini of AAV DNA and provide a basis for orientation of fragment maps derived by cleavage with other restriction enzymes.
用限制性内切酶R-EcoRI切割2型腺相关病毒（AAV2）DNA线性双链单体，产生了三个片段，A、B和C，其分子量分别约为1.6×10⁶、1.1×10⁶和1.3×10⁵。在用R-EcoRI切割之前对AAV DNA的5'末端进行放射性标记，结果表明A和B是末端片段，C是内部片段。对片段A和B的互补链进行分离，结果显示A包含负链的5'末端和正链的3'末端，而片段B则相反。因此，可以明确确定AAV R-EcoRI片段的物理图谱，并将B画在左端，A画在右端。在该图谱上，从负链转录稳定的AAV mRNA从左向右进行，起始于片段B，终止于片段A。AAV DNA正负链之间胸苷分布的不对称性优先位于片段A中，片段A代表双链分子的右半部分。这些实验能够对AAV DNA的所有四个单链末端进行制备性分离，并为通过用其他限制性酶切割得到的片段图谱的定向提供了基础。
