Tlsty T D, Adams P
Lineberger Cancer Research Center, University of North Carolina, Chapel Hill 27599-7295.
Exp Cell Res. 1990 May;188(1):164-8. doi: 10.1016/0014-4827(90)90293-j.
The purpose of this study is to determine the kinetics of the replication of intrachromosomal versus extrachromosomal amplified dihydrofolate reductase (DHFR) genes. Previous studies reported that the DHFR gene, when carried intrachromosomally on a homogeneously staining region, replicates (as a unit) within the first 2 h of the S phase of the cell cycle. We wished to determine if the extrachromosomal location of the amplified genes carried on double minute chromosomes effects the timing of their replication. Equilibrium cesium chloride ultracentrifugation was used to separate newly replicated (BUdR-labeled) DNA from bulk DNA in a synchronized cell population. Hybridization with the cDNA for the DHFR gene allowed us to determine the period of time within the cell cycle in which the DHFR DNA sequences were replicated. We found that, in contrast to intrachromosomal dihydrofolate reductase genes that uniformly replicate as a unit at the beginning of the S phase of the cell cycle, dihydrofolate reductase genes carried on double minute chromosomes (DMs) replicate throughout the S phase of the cell cycle. These results suggest that control of replication of extrachromosomal DNA sequences may differ from intrachromosomal sequences.
本研究的目的是确定染色体内与染色体外扩增的二氢叶酸还原酶（DHFR）基因的复制动力学。先前的研究报道，当DHFR基因位于染色体上的均匀染色区域时，它会在细胞周期S期的前2小时内（作为一个单元）进行复制。我们希望确定携带在双微小染色体上的扩增基因的染色体外定位是否会影响其复制时间。利用平衡氯化铯超速离心法，从同步化细胞群体中的大量DNA中分离出新复制的（溴脱氧尿苷标记的）DNA。与DHFR基因的cDNA杂交，使我们能够确定细胞周期中DHFR DNA序列进行复制的时间段。我们发现，与在细胞周期S期开始时作为一个单元均匀复制的染色体内二氢叶酸还原酶基因不同，携带在双微小染色体（DMs）上的二氢叶酸还原酶基因在细胞周期的整个S期都进行复制。这些结果表明，染色体外DNA序列复制的控制可能与染色体内序列不同。
