Association of p34cdc2 with replicating DNA.
Association of p34cdc2 with replicating DNA.
复制标题
p34cdc2 与复制 DNA 的关联。
DOI：
10.1101/sqb.1991.056.01.039
复制
发表时间：
1991
期刊：
影响因子：
--
通讯作者：
J. Roberts
中科院分区：
文献类型：
--
作者：
R. Fotedar;J. Roberts
材料和方法SV 40微型染色体的分离。复制反应的条件和细胞提取物的制备如前所述进行（Stillman和Gluzman 1985），不同之处在于50/t1反应含有100 ng pSV ori DNA和100 txg来自Manca细胞的胞质S-100。如Stillman（1986）先前所述，在琼脂糖凝胶上分析反应产物。对于SV 40 DNA的生物素化，将dq-TP减少至10 μ M，并包括6 μ M生物素-11-dUTP（ENZO）。我们发现，当使用生物素-11-dUTP时，SV 40微型染色体与链霉亲和素-生物素-纤维素的结合比使用生物素-21-dUTP或生物素-7-dATP（均来自BRL）时更好。如前所述（Stillman 1986）制备用于将DNA组装成染色质的Manca细胞核提取物（见图2B）。复制反应首先在4~ 12.5 × 2.5（ID）cm Sepharose 6 B（Pharmacia）柱上分级。用含有40 mM HEPES-KOH（pH 7.5）、8 mM NaCl、40 mM NaCl、lmM Dq-T、lmM ATE、0.5 μ g/ml亮抑酶肽和1 μ g/ml抑肽酶（均来自Boehringer）的柱缓冲液平衡Sepharose 6 B。收集500 μ l的级分，并合并含有微小染色体的级分。使用前，用含有ImM EDTA的柱缓冲液平衡100 μ 1（床体积）的生物素-纤维素（Pierce），用150 μ g链霉亲和素（Boehringer）孵育15-30分钟，并用柱缓冲液洗涤。然后将合并的SV 40微型染色体级分在该链霉亲和素-生物素-纤维素柱上通过8-10次。用柱缓冲液洗涤柱后，将与链霉抗生物素蛋白-生物素-纤维素珠结合的DNA用于进一步测定。在复制条件下含有胞质S-100但不含SV 40 DNA的对照反应也通过类似的Sepharose 6 B柱。收集SV 40微型染色体正常结合的级分，合并，并通过如上所述的链霉亲和素-生物素-纤维素柱。这些生物素-纤维素珠被用作对照，以显示蛋白质与SV 40 DNA结合的特异性，
MATERIALS AND METHODSIsolation of SV40 minichromosomes. Conditions for replication reactions and preparation of cell extracts were carried out as described previously (Stillman and Gluzman 1985), with the exception that 50/tl reaction contained 100 ng of pSV ori DNA and 100 txg of cytosolic S-100 from Manca cells. Reaction products were analyzed on agarose gels as described previously by Stillman (1986). For biotinylation of SV40 DNA, dq-TP was reduced to 10/~ M, and 6/zM biotin-11-dUTP (ENZO) was included. We found that SV40 minichromosomes bound to streptavidin-biotin-cellulose better when biotin-11-dUTP was used than when biotin-21-dUTP or biotin-7-dATP (both from BRL) was used. Nuclear extract from Manca cells used for assembly of DNA into chromatin (see Fig. 2B) was prepared as described previously (Stillman 1986). Replication reactions were first fractionated at 4~ over 12.5 x 2.5 (ID) cm Sepharose 6B (Pharmacia) columns. Sepharose 6B was equilibrated with column buffer containing 40 mM HEPES-KOH (pH 7.5), 8 mMMgC1 z, 40 mM NaCI, 1 mM Dq-T, I mM ATE 0.5 p~ g/ml leupeptin, and 1/zg/ml aprotinin (both from Boehringer). Fractions of 500/zl were collected, and the minichromosome-containing fractions were pooled. Before use, 100/~ 1 (bed volume) of biotin-cellulose (Pierce) was equilibrated with column buffer containing 1 mM EDTA, incubated with 150/~ g streptavidin (Boehringer) for 15-30 minutes, and washed with column buffer. Pooled SV40 minichromosome fractions were then passed 8-10 times over this streptavidin-biotin-cellulose column. After washing the column with column buffer, the DNA bound to streptavidin-biotin-celtutose beads was used for further assays. Control reactions containing cytosolic S-100 under replication conditions but without SV40 DNA were also passed over similar Sepharose 6B columns. Fractions where SV40 minichromosomes normally elute were collected, pooled, and passed through streptavidin-biotin-cellulose columns as discussed above, These biotin-cellulose beads have been used as a control to show specificity of binding of proteins to SV40 DNA,