ELECTRON-MICROSCOPIC VISUALIZATION OF DELETION MUTATIONS
ELECTRON-MICROSCOPIC VISUALIZATION OF DELETION MUTATIONS
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DOI：
10.1073/pnas.60.1.243
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发表时间：
1968-01-01
影响因子：
11.1
通讯作者：
DAVIDSON, N
中科院分区：
文献类型：
--
作者：
DAVIS, RW;DAVIDSON, N
X噬菌体的缺失突变体是由Kellenberger、Zichichi和Weigle发现的。这些缺失可以通过基因重组实验绘制出来。目前尚不清楚这样获得的重组图是否给出了缺失的真实物理位置。本文描述了一种用电子显微镜绘制缺失突变的方法，从而获得缺失的物理位置。基本原理如下。来自野生型病毒和缺失突变体的DNA分子的混合物受到链解离，然后再退火条件。所得到的制剂含有各种类型的一些双链再生分子和一些异双链分子。在后者中，一条链是野生型DNA，另一条链是缺失突变型DNA。因此，在缺失发生的地方，每个异双工应该在野生型DNA链中包含一个单链环。DNA分子的双链区域的轮廓长度可以用电子显微照片相当精确地测量出来。在制备电子显微镜网格的条件下，单链环被折叠成类似于MAlacHattie、Ritchie、Thomas和Richardson在T2噬菌体DNA中观察到的“灌木”。2这种灌木的轮廓长度最多只能粗略估计，但灌木相对于双链区域的位置可以精确测量。材料和方法。- CI点、突变体XC26和缺失突变体xb~ b5C1、Xb5c2 （Kellenberger, Zichichi, and Weigle’）和Xb221c26 （HuskeV3）由Dr. RobertHuskey获得。噬菌体通过标准方法成体纯化，包括密度梯度离心在CsCl中条带。最后，噬菌体溶液在0.01—1 M\gSO4, 0.01条件下进行透析。3I tris缓冲液（pH 7）。典型的异双相制备方法如下：Tengl为0.1。11乙二胺四乙酸酯（EDTA）（pH 8.2）， 15,；L (5)在一个6 × 50 mm的小试管中，按顺序加入46.6 M1的H20、5.1 IAI的XC26噬菌体溶液（A260= 10.0）、3.4 4ul的Xb5c2溶液（A260= 14.2）和10ul的1n NaOH。将混合物（pH 13）在室温下孵育10分钟，然后在冰浴中冷却。碱性溶液导致噬菌体裂解和DNA解离成单链。2 31 NaH2PO4（终体积100，/l）加入10,1，使溶液中和。通过将溶液加热到70℃30秒，使DNA部分还原，然后快速冷却（这些条件被选择为超过50%的还原，最小的单链断裂和最小的高阶聚集）。除了加入噬菌体悬浮液的体积不同，使两种噬菌体类型的数量相同，总噬菌体A260= 1.0（约10”1）。在对照实验中，只有一种噬菌体DNA存在。用碱性蛋白膜技术制备电镜栅格，并用铀酰盐染色。7将含有0.5 4g/ml DNA和0.1 mg/ml细胞色素c的溶液，在0.5 I1乙酸铵，0.001 41 EDTA （pH 7.9）中，涂抹在0.15 31乙酸铵（pH 6.5）上。细胞色素c- dna混合膜是在用3% w/v制备的膜覆盖（小于2天）的网格上拾取的
Deletion mutants of X coliphage were discovered by Kellenberger, Zichichi, and Weigle.'The deletions can be mapped by genetic recombination experiments. It is Iiot known if the recombination maps thus obtained give the true physical position of the deletions. The present paper describes a method for mapping deletion mutations with the electron microscope, thus obtaining the physical posi-tion of the deletion. The basic principle is as follows. A mixture of DNA mole-cules from wild-type virus and from the deletion mutant is subjected to strand dissociation followed by reannealing conditions. The resulting preparation con-tains some double-stranded renatured molecules of each type and some hetero-duplexes. In the latter, one strand is wild-type DNA and the other is deletion mutant-type DNA. Each heteroduplex should thus contain a single-stranded loop in the wild-type DNA strand at the point where the deletion occurs. The contour lengths of double-stranded regions of a DNA molecule can be rather accurately measured in electron micrographs. Under the conditionls used to pre-pare the electron microscope grids, the single-stranded loops are collapsed into" bushes" similarto those observed in T2 phage DNA by MAlacHattie, Ritchie, Thomas, and Richardson. 2 The contour length of such a bush can only be roughly estimated at best, but the bush position with respect to the double-stranded regions can be accurately measured. Materials and Mlethods.-The CI point, mutant XC26 and the deletion mutants xb~ b5C1, Xb5c2 (Kellenberger, Zichichi, and Weigle'), and Xb221c26 (HuskeV3) were obtained from Dr. RobertHuskey. Phage were grownand purified by standard methods, including banding in CsCl by density gradient centrifugation. 4 The phage solution was finally dialyzed against 0.01--1 M\gSO4, 0.01. 3I tris buffer (pH 7). A typical heteroduplex preparation was made as follows. Tengl of 0.1. 11 ethylene-diaminetetraacetate (EDTA)(pH 8.2), 15,. l of 5. f NaCl, 46.6, M1 of H20, 5.1 IAI of XC26 phage solution (A260= 10.0), 3.4 4ul of Xb5c2 solution (A260= 14.2), and 10, ul of 1 N NaOH were added, in the order given, to a small test tube (6 X 50 mm). The mixture (pH 13) was incubated at room temperaturefor 10 min and then chilled in an ice bath. The alkaline solution causes lysis of the phage and dissociation of the DNA into single strands. The solution was neutralized by addition of 10, 1 of 2 31 NaH2PO4 (final volume, 100,/l). The DNA was partially renatured byheating the solution to 70'C for 30 sec and then quickly cooled (these conditions were selected to give over 50% renaturation, minimal single-strand scissions, and minimal higher-order aggregation'). All other preparations were done similarly except that the volume of phage suspension added was varied to give equal numbers of the two phage types with a combined A260= 1.0 (ca. 10" 1 total phage). In control experiments only one typeof phage DNA was present. Grids for electron microscopy were prepared by the basic protein film technique6 and stained with uranyl salts. 7 A solution containing 0.5, 4g/ml of DNA and 0.1 mg/ml of cytochrome c, in 0.5 I1 ammonium acetate, 0.001 41 EDTA (pH 7.9), was spread onto 0.15 31 ammonium acetate (pH 6.5). The cytochrome c-DNA-mixed film was picked up on grids freshly covered (less than 2-days old) with films prepared from 3% w/v