Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80%o of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 x 106 to 140 x 106 daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 x 106 daltons and another of 7 x 106 daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 x 106 and 7 x 106 daltons.Recently, it was discovered that pea roots have double-stranded extrachromosomal DNA (ex-DNA), with a modal size of 25 x 106 daltons, that is present in cells of the meristematic tip and remains associated with them as they differentiate from the G2 phase and occupy the elongation zone (22). Electron microscopic studies (D. Krimer and J. Van't Hof, submitted for publication) further showed that ex-DNA is of two forms: a double-stranded replicative form (RF), heterogeneous in length with a modal size of 10 to 15 ,um, and a single-stranded form (SF), with an average size of 3.8 ,um, that is replicated on the RF molecules by strand displacement. These observations raised several questions about the processes responsible for the appearance of RF DNA and about the time of replication of SF DNA relative to when RF DNA became extrachromosomal. Since ex-DNA has the same buoyant density as nuclear DNA, it was suspected that it was once chromosomal and was made extrachromosomal sometime during the process of DNA maturation. In the present experiments we addressed these questions. We examined the replication and maturation of chromosomal DNA in cells known to be responsible for the ex-DNA, determined when the molecules of 25 x 106 daltons become extrachromosomal, and measured the replication of nascent chains on the ex-DNA. These measurements, however, required further details about the cells from which the ex-DNA was obt...

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.

No abstract