Romanov G A, Vaniushin B F
Mol Biol (Mosk). 1977 Jul-Aug;11(4):941-9.
A simple and sensitive method for determining the selective eucaryotic DNA synthesis (amplification, ordered replication) amounting up to 10(-6)% of genome has been proposed. The essence of the method is measuring of the combined renaturation of total and in vivo labelled newly synthesized DNA. By means of a special computer program a theoretical investigation of amplification (from 50 to 2-fold) was accomplished, based on the experimentally found renaturation curve of rat liver DNA. A new method of graphical analysis of experimental results is also proposed based on the changes in the specific activities against Cot values. Some advantages of the new graphical method, i. e. greater sensitivity and reliability, simplicity of calculation of the amplification level and of the proportion of radioactivity in the amplified sequences are shown. The major patterns of the changes in the specific activities in different examples of amplification were revealed. Evidence is presented that the amplification necessarily brings about essential changes in the specific activities of denatured and renatured DNA fraction at high Cot values. On the basis of the method developed, the mode of liver DNA synthesis in normal and hormone-induced rats was established. Both in the normal and hydrocortison-induced rat livers no selectivity of DNA synthesis was found in the sense that hormonal induction of RNA synthesis is not a consequence of additional synthesis of DNA templates.
已提出一种简单且灵敏的方法,用于测定占基因组达10⁻⁶%的真核生物DNA的选择性合成(扩增、有序复制)量。该方法的本质是测量总DNA与体内标记的新合成DNA的联合复性。借助一个特殊的计算机程序,基于实验发现的大鼠肝脏DNA复性曲线,完成了对扩增(从50倍到2倍)的理论研究。还基于比活性相对于Cot值的变化,提出了一种实验结果的图形分析新方法。展示了这种新图形方法的一些优点,即更高的灵敏度和可靠性、扩增水平计算的简便性以及扩增序列中放射性比例计算的简便性。揭示了不同扩增实例中比活性变化的主要模式。有证据表明,在高Cot值下,扩增必然会导致变性和复性DNA组分的比活性发生本质变化。基于所开发的方法,确定了正常大鼠和激素诱导大鼠肝脏中DNA的合成模式。在正常大鼠和氢化可的松诱导的大鼠肝脏中,均未发现DNA合成具有选择性,即RNA合成的激素诱导并非DNA模板额外合成的结果。