Planta. 1978 Jan;143(3):241-53. doi: 10.1007/BF00391994.

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures.

Planta

D E Cress, P J Jackson, A Kadouri, Y E Chu, K G Lark

PMID: 24408461 DOI: 10.1007/BF00391994

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.

References

1. Annu Rev Biochem. 1969;38:569-604 - PubMed
2. Exp Cell Res. 1968 Apr;50(1):151-8 - PubMed
3. J Exp Med. 1954 Feb;99(2):167-82 - PubMed
4. Annu Rev Genet. 1975;9:245-84 - PubMed
5. J Mol Biol. 1963 Nov;7:534-40 - PubMed
6. J Mol Biol. 1965 Feb;11:373-90 - PubMed
7. Annu Rev Biochem. 1975;44:45-78 - PubMed
8. Planta. 1976 Jan;132(3):259-68 - PubMed
9. J Mol Biol. 1971 Jun 28;58(3):873-5 - PubMed
10. J Mol Biol. 1962 Jun;4:430-43 - PubMed
11. Exp Cell Res. 1977 Mar 1;105(1):15-26 - PubMed
12. J Mol Biol. 1968 Feb 14;31(3):579-94 - PubMed
13. Biochim Biophys Acta. 1975 Apr 2;383(4):410-20 - PubMed
14. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1004-12 - PubMed
15. Biochim Biophys Acta. 1971 Feb 11;228(3):595-609 - PubMed
16. Chromosoma. 1977 Jul 18;62(4):291-300 - PubMed
17. Bacteriol Rev. 1972 Sep;36(3):361-405 - PubMed
18. J Mol Biol. 1966 Jan;15(1):372-3 - PubMed
19. Exp Cell Res. 1975 Jun;93(1):95-104 - PubMed
20. Cold Spring Harb Symp Quant Biol. 1974;38:205-23 - PubMed
21. J Mol Biol. 1964 Aug;9:288-307 - PubMed
22. J Mol Biol. 1975 Dec 5;99(2):317-37 - PubMed
23. J Mol Biol. 1968 Mar 14;32(2):327-41 - PubMed

Publication Types

• Journal Article