In vitro study of illegitimate recombination: involvement of DNA gyrase.
In vitro study of illegitimate recombination: involvement of DNA gyrase.
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非法重组的体外研究：DNA 旋转酶的参与。
DOI：
10.1101/sqb.1981.045.01.054
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
T. Matsumoto
中科院分区：
文献类型：
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作者：
H. Ikeda;K. Moriya;T. Matsumoto
材料与方法体外重组试验。将噬菌体X DNA的体外包装系统用于重组试验（小林和Ikeda 1977）。从诱导的溶原性大肠杆菌制备标准包装混合物。coli KI 2：594（XcI 857 Dam 15 Flam 96 BSam 7）和594（XcI 857 Eam 4Sam 7）。还从含有细菌重组突变和病毒插入、红色和缺失突变的各种组合的溶原菌制备包装混合物。将携带氨苄青霉素抗性（Apr）决定簇的质粒DNA（约1 μ g/测定）加入到包装混合物中，并在28 ℃下温和振荡孵育90分钟。除DNA外，孵育混合物（35#1）含有1.5 × 109个诱导细胞、50 mM Tris-HCl（pH 7.4）、7 mM MgCl 2、7 mM KCl、7 mM NaCl、0.15 mM EDTA、10%二甲亚砜、7 mM亚精胺和1 mM ATP。通过加入1 ml含有10 μ g/ml胰DNA酶和一滴CHCl 3的SMC缓冲液（小林和Ikeda 1977）终止反应。通过低速和高速离心纯化所得噬菌体悬浮液，并测定噬斑形成和Apr转导能力。通过感染E. coli Ymel，涂布于含20 μ g/ml氨苄青霉素的琼脂平板上，进行异源双链体分析。通过Davis等人的方法分析异源双链分子。1971年，作了一些修改。通过CsCl密度梯度离心纯化噬菌体颗粒。将噬菌体悬浮液的等分试样（5 × 109个颗粒）与10#1的0.2M EDTA混合，用水调节至90#1，然后与10#1的1 M NaOH混合用于变性。在室温下10分钟后，向混合物中加入100 μ l甲酰胺和10 μ l 2 M Tris-HCl（pH 6.7），并在室温下孵育90分钟。将3#1份混合物与15#1甲酰胺、2.5#1 1 M Tris-HCl（pH 8.0）、1#1 0.25M EDTA、1.5 μ l 1 mg/ml细胞色素c混合，并用水调节至25#1的体积。将混合物铺展在水的下相上。用Parlodion包被的铜网格拾取蛋白单层，用90%乙醇脱水，用5· 10-5 M乙酸双氧铀染色30秒，并用己烷脱色。用Pt-Pd线对网格进行旋转阴影
MATERIALS AND METHODSIn vitro recombination assay. The in vitro packaging system for phage X DNA was used for the recombination assay (Kobayashi and Ikeda 1977). A standard packaging mixture was prepared from induced lysogens of E. coli KI2: 594 (XcI857Dam15Flam96BSam7) and 594 (XcI857Eam4Sam7). Packaging mixtures were also prepared from lysogens containing various combinations of bacterial rec mutations and viral int, red, and deletion mutations. A plasmid DNA carrying the ampicillin resistance (Ap r) determinant (about 1# g/assay) was added to the packaging mixture and incubated for 90 minutes at 28~ with gentle shaking. In addition to the DNA, the incubation mixture (35# 1) contained 1.5 x 109 induced cells, 50 mM Tris-HC1 (pH 7.4), 7 mM MgC12, 7 mM KCI, 7 mM NaCI, 0.15 mM EDTA, 10% dimethyl sulfoxide, 7 mM spermidine, and 1 mM ATP. The reaction was stopped by adding 1 ml of SMC buffer (Kobayashi and Ikeda 1977) containing 10# g/ml of pancreatic DNase and one drop of CHCI 3. The resulting phage suspension was purified by low-speed and high-speed centrifugation and assayed for plaque formation and Apr transduction capability. Apr transduction was carried out by infection to E. coli Ymel and spreading to a~, agar plate containing 20# g/ml of ampicillin.Heteroduplex analysis. Heteroduplex molecules were analyzed by the method of Davis et al.(1971) with some modifications. Phage particles were purified by CsCI density gradient centrifugation. An aliquot of phage suspension (5• l0 9 particles) was mixed with 10# 1 of 0.2 M EDTA, adjusted to 90# 1 with water, and then mixed with 10# 1 of 1 M NaOH for denaturation. After 10 minutes at room temperature, 100/d of formamide and 10# 1 of 2 M Tris-HCl (pH 6.7) were added to the mixture and incubated for 90 minutes at room temperature. A 3# 1 portion of the mixture was mixed with 15# 1 of formamide, 2.5# 1 of 1 M Tris-HC1 (pH 8.0), 1# 1 of 0.25 M EDTA, 1.5/tl of 1 mg/ml of cytochrome c and adjusted to a volume of 25# 1 with water. The mixture was spread onto a hypophase of water. The protein monolayer was picked up with a Parlodion-coated copper grid, dehydrated with 90% ethanol, stained with 5• 10-5 M uranyl acetate for 30 seconds and destained with hexane. Grids were rotary-shadowed with a Pt-Pd wire