Friedmann A, Broit M, Becker Y
IARC Sci Publ (1971). 1978(24 Pt 1):137-48.
DNA isolated from highly purified virions of herpes simplex virus type-1 (HF strain) was denatured by centrifugation in alkaline sucrose gradients. DNA molecules corresponding to intact single-stranded virion DNA (50 x 10(6) daltons) were isolated and adjusted to neutral pH. The DNA was annealed under conditions permitting reassociation of intact single-stranded molecules and studied by electron microscopy. Three classes of DNA molecules showing double-stranded sequences were observed: (a) fully double-stranded DNA molecules the size of the intact HSV DNA genome, namely 52 micron; (b) DNA hybrids with a region of partial double-strandedness ranging from 5 to 12 micron, plus long single strands; and (c) DNA hybrids with a double-stranded region of 32--40 micron, plus short single strands. (These results suggest that the alkali-resistant single-stranded HSV DNA molecules are composed of several subclasses that permit annealing of either the total genome or the S or L components.) The 5 micron double-stranded region probably constitutes the S component of HSV DNA and the sequences longer than 5 micron and shorter than 12 micron represent annealing of the repeat sequences on either or both sides of the S component. The double-stranded sequences with a length of 32--40 micron may represent the L component. Treatment of the annealed, partially double-stranded hybrid DNA molecules with S1 endonuclease to remove the single-stranded termini and centrifugation in neutral sucrose gradients yielded two distinct peaks. Centrifugation of fractions from the two peaks in caesium chloride density gradients showed that the small DNA component (possibly the S and the repeat sequences) had a higher buoyant density and the longer (possibly the L) DNA component had a lower density than the HSV DNA marker. Annealing of alkali-resistant viral DNA strands therefore provides a means of isolating the L, S and repeat sequence regions of HSV DNA.
从单纯疱疹病毒1型（HF株）的高度纯化病毒粒子中分离出的DNA，通过在碱性蔗糖梯度中离心进行变性处理。分离出与完整单链病毒粒子DNA（50×10⁶道尔顿）相对应的DNA分子，并将其调至中性pH值。在允许完整单链分子重新结合的条件下使DNA退火，并通过电子显微镜进行研究。观察到三类显示双链序列的DNA分子：（a）大小与完整HSV DNA基因组相同的完全双链DNA分子，即52微米；（b）具有5至12微米部分双链区域以及长单链的DNA杂种；（c）具有32 - 40微米双链区域以及短单链的DNA杂种。（这些结果表明，耐碱单链HSV DNA分子由几个亚类组成，这些亚类允许整个基因组或S或L成分退火。）5微米的双链区域可能构成HSV DNA的S成分，长度大于5微米且小于12微米的序列代表S成分两侧重复序列的退火。长度为32 - 40微米的双链序列可能代表L成分。用S1核酸内切酶处理退火后的部分双链杂种DNA分子以去除单链末端，并在中性蔗糖梯度中离心，产生了两个不同的峰。在氯化铯密度梯度中对来自这两个峰的级分进行离心，结果表明，小的DNA成分（可能是S和重复序列）具有较高的浮力密度，而较长的（可能是L）DNA成分的密度低于HSV DNA标志物。因此，耐碱病毒DNA链的退火提供了一种分离HSV DNA的L、S和重复序列区域的方法。
