大鼠DNA中5-溴脱氧尿苷非随机掺入的酶法测定

Biochemistry. 1976 Jul 13;15(14):3097-105. doi: 10.1021/bi00659a025.

Secondary cultures of normal rat embryo cells were synchronized by a double thymidine block and pulsed with 10(-7) M 5-[3H]bromodeoxyuridine (BrdUrd) OR 10(-7) M[3H]thymidine during an entire S phase (7.5 h). To examine the pattern of [3H]thymidine, DNA was immediately extracted and purified at the completion of the S phase, CsCl density gradient centrifugation revealed that substitution for thymine by bromouracil was less than 7%. Single-strand specific nucleases obtained from Aspergillus oryzae and Neurospora crassa were allowed to react with native and partially depurinated (24-29%) [3H]BrdUrd-labeled rat DNA samples, and the products were assayed by hydroxylapatite column chromatography. Approximately 4-6% of the native, nondepurinated rat DNA was hydrolyzed by both nucleases. However, 24-28% of the partially depurinated, [3H] thymidine-labeled rat DNA was hydrolyzed by both enzymes as determined by loss of mass as well as radioactivity. Whereas comparable levels of depurinated, [3H]BrdUrd-labeled DNA were physically hydrolyzed by both nucleases, nearly 65% of the radioactivity was not recovered. Native, as well as depurinated, enzyme-treated DNA samples were sequentially and preparatively reassociated into highly repetitive, middle repetitive, and nonrepetitive nucleotide sequence components. The absolute and relative specific activities of each subfraction of native [3H]thymidine-labeled DNA were comparable. [3H]BrdUrd was differentially concentrated in the middle repetitive sequences as compared to other reiteration frequency types. When depurinated, nuclease-treated DNA samples were similarly fractionated, [3H]thymine moieties were uniformly distributed thoughout all sequences. However, a differential loss of [3H]BrdUrd moieties was detected predominantly from the middle repetitive nucleotide fraction. Melting profiles of the renatured DNA samples were characteristic of each respective DNA subfraction regardless of isotopic precursor. These results suggest that [3H]BrdUrd may be differentially incorporated into A + T rich clusters of rat DNA, especially in the moderately repeated chromosomal elements.

正常大鼠胚胎细胞的传代培养物通过双胸腺嘧啶核苷阻断进行同步化处理，并在整个S期（7.5小时）用10⁻⁷ M 5-[³H]溴脱氧尿苷（BrdUrd）或10⁻⁷ M [³H]胸腺嘧啶核苷进行脉冲标记。为了检测[³H]胸腺嘧啶核苷的掺入模式，在S期结束时立即提取并纯化DNA，氯化铯密度梯度离心显示，溴尿嘧啶取代胸腺嘧啶的比例小于7%。将从米曲霉和粗糙脉孢菌获得的单链特异性核酸酶与天然的和部分脱嘌呤（24 - 29%）的[³H]BrdUrd标记的大鼠DNA样品反应，产物通过羟基磷灰石柱色谱法进行分析。两种核酸酶均可水解约4 - 6%的天然、未脱嘌呤的大鼠DNA。然而，通过质量损失以及放射性测定，两种酶均可水解24 - 28%的部分脱嘌呤的、[³H]胸腺嘧啶核苷标记的大鼠DNA。尽管两种核酸酶对相当水平的脱嘌呤的、[³H]BrdUrd标记的DNA进行了物理水解，但近65%的放射性未被回收。将天然的以及脱嘌呤的、经酶处理的DNA样品依次进行制备性复性处理，使其重新形成高度重复、中度重复和非重复核苷酸序列组分。天然的[³H]胸腺嘧啶核苷标记的DNA各亚组分的绝对和相对比活性相当。与其他重复频率类型相比，[³H]BrdUrd在中度重复序列中差异富集。当对脱嘌呤的、经核酸酶处理的DNA样品进行类似的分级分离时，[³H]胸腺嘧啶部分均匀分布于所有序列中。然而，主要从中度重复核苷酸组分中检测到[³H]BrdUrd部分的差异损失。无论同位素前体如何，复性DNA样品的解链图谱均为各相应DNA亚组分所特有。这些结果表明，[³H]BrdUrd可能差异掺入大鼠DNA富含A + T的簇中，尤其是在中度重复的染色体元件中。