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. 1970 Oct 1;47(1):132–139. doi: 10.1083/jcb.47.1.132

SEPARATION OF NUCLEI REPRESENTING DIFFERENT PHASES OF THE GROWTH CYCLE FROM UNSYNCHRONIZED MAMMALIAN CELL CULTURES

O Wesley McBride 1, Elbert A Peterson 1
PMCID: PMC2108399  PMID: 5513550

Abstract

Nuclei have been isolated from unsynchronized cultures of Chinese hamster fibroblasts after varying intervals of growth following the incorporation of thymidine -3H for 20 min. These nuclei were fractionated by unit gravity sedimentation in a stabilizing density gradient of sucrose, and fractions were analyzed for the concentration of nuclei, DNA, and radioactivity. A more rapidly sedimenting population of nuclei in the G2 phase of the cell cycle was separated from a group of nuclei in the G1 phase, and nuclei in progressive stages of DNA synthesis (S phase) were distributed between these two regions. The fractionation of intact cells by sedimentation according to their position in the cell cycle was found to be less satisfactory than the corresponding separation of nuclei. This probably results from the continuous accumulation of mass within individual cells throughout the entire cell cycle, whereas most of the mass of a nucleus is replicated during a relatively narrow interval of the total cell cycle.

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Selected References

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  1. Albrecht C. F. Fractionation of rat liver nuclei by sucrose gradient centrifugation. Exp Cell Res. 1968 Feb;49(2):373–378. doi: 10.1016/0014-4827(68)90187-0. [DOI] [PubMed] [Google Scholar]
  2. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. ELKIND M. M., SUTTON H. Radiation response of mammalian cells grown in culture. 1. Repair of X-ray damage in surviving Chinese hamster cells. Radiat Res. 1960 Oct;13:556–593. [PubMed] [Google Scholar]
  5. Enger M. D., Tobey R. A. RNA synthesis in Chinese hamster cells. II. Increase in rate of RNA synthesis during G1. J Cell Biol. 1969 Jul;42(1):308–315. doi: 10.1083/jcb.42.1.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. FALZONE J. A., BARROWS C. H., YIENGST M. J. Fractionation of rat liver nuclei into diploid and tetraploid DNA classes by continuous density gradient sedimentation. Exp Cell Res. 1962 Mar;26:552–561. doi: 10.1016/0014-4827(62)90160-x. [DOI] [PubMed] [Google Scholar]
  7. Gallwitz D., Mueller G. C. Histone synthesis in vitro by cytoplasmic microsomes from HeLa cells. Science. 1969 Mar 21;163(3873):1351–1353. doi: 10.1126/science.163.3873.1351. [DOI] [PubMed] [Google Scholar]
  8. HAM R. G. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp Cell Res. 1963 Feb;29:515–526. doi: 10.1016/s0014-4827(63)80014-2. [DOI] [PubMed] [Google Scholar]
  9. KILLANDER D., ZETTERBERG A. QUANTITATIVE CYTOCHEMICAL STUDIES ON INTERPHASE GROWTH. I. DETERMINATION OF DNA, RNA AND MASS CONTENT OF AGE DETERMINED MOUSE FIBROBLASTS IN VITRO AND OF INTERCELLULAR VARIATION IN GENERATION TIME. Exp Cell Res. 1965 May;38:272–284. doi: 10.1016/0014-4827(65)90403-9. [DOI] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Littlefield J. W., Jacobs P. S. The relation between DNA and protein synthesis in mouse fibroblasts. Biochim Biophys Acta. 1965 Dec 9;108(4):652–658. doi: 10.1016/0005-2787(65)90061-4. [DOI] [PubMed] [Google Scholar]
  12. Mendelsohn J., Moore D. E., Salzman N. P. Separation of isolated Chinese hamster metaphase chromosomes into three size-groups. J Mol Biol. 1968 Feb 28;32(1):101–112. doi: 10.1016/0022-2836(68)90148-4. [DOI] [PubMed] [Google Scholar]
  13. Miller R. G., Phillips R. A. Separation of cells by velocity sedimentation. J Cell Physiol. 1969 Jun;73(3):191–201. doi: 10.1002/jcp.1040730305. [DOI] [PubMed] [Google Scholar]
  14. PATTERSON M. S., GREENE R. C. MEASUREMENT OF LOW ENERGY BETA-EMITTERS IN AQUEOUS SOLUTION BY LIQUID SCINTILLATION COUNTING OF EMULSIONS. Anal Chem. 1965 Jun;37:854–857. doi: 10.1021/ac60226a017. [DOI] [PubMed] [Google Scholar]
  15. PUCK T. T., CIECIURA S. J., FISHER H. W. Clonal growth in vitro of human cells with fibroblastic morphology; comparison of growth and genetic characteristics of single epithelioid and fibroblast-like cells from a variety of human organs. J Exp Med. 1957 Jul 1;106(1):145–158. doi: 10.1084/jem.106.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. PUCK T. T., SANDERS P., PETERSEN D. LIFE CYCLE ANALYSIS OF MAMMALIAN CELLS. II. CELLS FROM THE CHINESE HAMSTER OVARY GROWN IN SUSPENSION CULTURE. Biophys J. 1964 Nov;4:441–450. doi: 10.1016/s0006-3495(64)86794-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Penman S. RNA metabolism in the HeLa cell nucleus. J Mol Biol. 1966 May;17(1):117–130. doi: 10.1016/s0022-2836(66)80098-0. [DOI] [PubMed] [Google Scholar]
  18. Peterson E. A., Evans W. H. Separation of bone marrow cells by sedimentation at unit gravity. Nature. 1967 May 20;214(5090):824–825. doi: 10.1038/214824a0. [DOI] [PubMed] [Google Scholar]
  19. Robbins E., Borun T. W. The cytoplasmic synthesis of histones in hela cells and its temporal relationship to DNA replication. Proc Natl Acad Sci U S A. 1967 Feb;57(2):409–416. doi: 10.1073/pnas.57.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Scharff M. D., Robbins E. Synthesis of ribosomal RNA in synchronized HeLa cells. Nature. 1965 Oct 30;208(5009):464–466. doi: 10.1038/208464a0. [DOI] [PubMed] [Google Scholar]
  21. Spalding J., Kajiwara K., Mueller G. C. The metabolism of basic proteins in HeLa cell nuclei. Proc Natl Acad Sci U S A. 1966 Nov;56(5):1535–1542. doi: 10.1073/pnas.56.5.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. TERASIMA T., TOLMACH L. J. Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells. Exp Cell Res. 1963 Apr;30:344–362. doi: 10.1016/0014-4827(63)90306-9. [DOI] [PubMed] [Google Scholar]

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