ABSTRACT
Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 µm. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4-6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4-6S) and large (20-30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm(3). Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 µm.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.
ABSTRACT
Suspension cultures of Glycine max (L.) Merr. were grown at 22 and 33°. The doubling times of dividing cells were 35 and 25 h, respectively. G2 was 6.2 and 6.7 h, and S was 13.8 and 6.5 h. G1 was calculated as 13 and 10 h, respectively. These values were determined by labeling cells with (3)H thymidine and measuring the appearance of radioactive mitotic figures. Treatment with 5-fluorodeoxyuridine (FudR) inhibited DNA synthesis and, as a result, cells accumulated in S. Such cells were viable and, upon removal of the FudR, proceeded synchronously into mitosis. Treatment with 5-bromodeoxyuridine, following FudR synchronization, sensitized the cells to white light. Thus cells capable of synthesizing DNA could be killed. 20-30% of the cells in suspension cultures growing at 22 or 33° were not able to synthesize DNA. Nevertheless, these non-dividing (Q) cells were able to synthesize RNA and protein at a reduced rate. The proteins synthesized appear to be a particular subset of the proteins made by normal cells. The results are analyzed in relation to the use of this suspension cell culture system for isolating conditional lethal mutants of plant cells.
ABSTRACT
Weakness of F(1) plants is frequently found in hybrids between strains of Oryza breviligulata (wild) and O. glaberrima (cultivated rice) endemic to West Africa. A set of two complementary dominant weakness genes, W(1) and W(2), was found to control the observed F(1) weakness. Many breviligulata strains had W(1), while most of the glaberrima and semi-wild strains had W(2) or were free of both. In the weak F(1) plants, tissue differentiation in adventitious roots seemed to be disturbed. Modifier genes affecting the expression of the weakness genes appear to be present also.
