Dani G M, Zakian V A
Proc Natl Acad Sci U S A. 1983 Jun;80(11):3406-10. doi: 10.1073/pnas.80.11.3406.
Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells. However, these plasmids are unstable, being rapidly lost from cells growing without selection. Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome. Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids. Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth. Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis. Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid. Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids. However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids. This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability. These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis.
含有自主复制序列（ARSs）的环状重组DNA质粒在转化的酵母细胞中以染色体外形式维持。然而，这些质粒不稳定，在无选择条件下生长的细胞中会迅速丢失。尽管酵母着丝粒DNA（CEN）的存在可提高此类质粒的稳定性，但与真正的酵母染色体相比，即使是CEN质粒也会以较高速率丢失。天然酵母染色体是线性分子；因此，我们探究了线性化是否能提高重组DNA质粒的稳定性。通过使用纤毛原生动物嗜热四膜虫的染色体外核糖体DNA（rDNA）末端作为“端粒”，在体外构建了有和没有酵母CEN的线性质粒。这些线性质粒能高效转化酵母，并在有丝分裂生长过程中作为线性染色体外分子维持。此外，含有CEN的线性质粒也能通过减数分裂传递：这些质粒在第一次减数分裂时主要以2+:2-的比例分离，表明嗜热四膜虫rDNA末端在酵母减数分裂过程中可提供端粒功能。不含CEN的线性质粒在有丝分裂中的稳定性与可比的环状质粒相当。因此，嗜热四膜虫rDNA末端对ARS质粒的有丝分裂稳定性没有明显的正向或负向影响。然而，含有CEN的线性质粒在有丝分裂细胞中的稳定性比环状CEN质粒低三到四倍。这种稳定性的降低并非由于着丝粒本身的功能变化；相反，CEN质粒线性化对其有丝分裂稳定性有直接的不利影响。这些结果可能反映了着丝粒和端粒位置存在空间限制，在有丝分裂过程中必须满足这些限制才能实现染色体的稳定分离。
