Isolation of Metaphase Chromosomes from HeLa Cells

Overview

Journal J Cell Biol

Specialty Cell Biology

Date 1966 Oct 1

PMID 5339564

Citations 11

Authors

J A Huberman

G Attardi

Affiliations

Soon will be listed here.

Abstract

The authors have developed a method for large-scale isolation of metaphase chromosomes from HeLa cells. The distinguishing feature of this method is the use of a pH sufficiently low (about 3) to stabilize the chromosomes against mechanical damage. Many milligrams of fairly pure, morphologically intact chromosomes can be isolated in 8 hr or less of total working time. The isolated chromosomes contain about 2.0 mg of acid-soluble protein, 2.7 mg of acid-insoluble protein and 0.66 mg of RNA for each milligram of DNA. The RNA bound to the isolated chromosomes consists mainly of ribosomal RNA, but there is also a significant amount of 45S RNA.

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References

Lin H, Chargaff E . METAPHASE CHROMOSOMES AS A SOURCE OF DNA. Biochim Biophys Acta. 1964; 91:691-4. DOI: 10.1016/0926-6550(64)90032-5. View

Boss J . Mitosis in cultures of newt tissues. IV. The cell surface in late anaphase and the movements of ribonucleoprotein. Exp Cell Res. 1955; 8(1):181-7. DOI: 10.1016/0014-4827(55)90055-0. View

Scherrer K, LATHAM H, Darnell J . Demonstration of an unstable RNA and of a precursor to ribosomal RNA in HeLa cells. Proc Natl Acad Sci U S A. 1963; 49:240-8. PMC: 299789. DOI: 10.1073/pnas.49.2.240. View

Burton K . A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956; 62(2):315-23. PMC: 1215910. DOI: 10.1042/bj0620315. View

Dupraw E . Evidence for a 'folded-fibre' organization in human chromosomes. Nature. 1966; 209(5023):577-81. DOI: 10.1038/209577a0. View

STEELE W, Okamura N, Busch H . EFFECTS OF THIOACETAMIDE ON THE COMPOSITION AND BIOSYNTHESIS OF NUCLEOLAR AND NUCLEAR RIBONUCLEIC ACID IN RAT LIVER. J Biol Chem. 1965; 240:1742-9. View

Bendich A, Borenfreund E, Hutchison D . STUDIES ON THE ISOLATION OF METAPHASE CHROMOSOMES. J Cell Biol. 1963; 19:59-69. PMC: 2106868. DOI: 10.1083/jcb.19.1.59. View

KAUFMANN B, McDonald M, Gay H . Enzymatic degradation of ribonucleoproteins of chromosomes, nucleoli and cytoplasm. Nature. 1948; 162(4125):814. DOI: 10.1038/162814a0. View

Dupraw E . THE ORGANIZATION OF NUCLEI AND CHROMOSOMES IN HONEYBEE EMBRYONIC CELLS. Proc Natl Acad Sci U S A. 1965; 53(1):161-8. PMC: 219449. DOI: 10.1073/pnas.53.1.161. View

10.

Penman S . RNA metabolism in the HeLa cell nucleus. J Mol Biol. 1966; 17(1):117-30. DOI: 10.1016/s0022-2836(66)80098-0. View

11.

FRENSTER J, Allfrey V, Mirsky A . REPRESSED AND ACTIVE CHROMATIN ISOLATED FROM INTERPHASE LYMPHOCYTES. Proc Natl Acad Sci U S A. 1963; 50:1026-32. PMC: 221266. DOI: 10.1073/pnas.50.6.1026. View

12.

KIRBY K . Some new solvent systems for the paper chromatography of nucleic acid degradation products. Biochim Biophys Acta. 1955; 18(4):575-6. DOI: 10.1016/0006-3002(55)90157-8. View

13.

Georgiev G, Samarina O, Lerman M, Smirnov M, SEVERTZOV A . BIOSYNTHESIS OF MESSENGER AND RIBOSOMAL RIBONUCLEIC ACIDS IN THE NUCLEOLOCHROMOSOMAL APPARATUS OF ANIMAL CELLS. Nature. 1963; 200:1291-4. DOI: 10.1038/2001291a0. View

14.

Cantor K, Hearst J . Isolation and partial characterization of metaphase chromosomes of a mouse ascites tumor. Proc Natl Acad Sci U S A. 1966; 55(3):642-9. PMC: 224200. DOI: 10.1073/pnas.55.3.642. View

15.

Love R . Distribution of ribonucleic acid in tumor cells during mitosis. Nature. 1957; 180(4598):1338-9. DOI: 10.1038/1801338a0. View

16.

LOWRY O, ROSEBROUGH N, FARR A, RANDALL R . Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265-75. View

17.

SIBATANI A, de Kloet S, Allfrey V, Mirsky A . Isolation of a nuclear RNA fraction resembling DNA in its base composition. Proc Natl Acad Sci U S A. 1962; 48:471-7. PMC: 220802. DOI: 10.1073/pnas.48.3.471. View

18.

LEVINTOW L, Darnell Jr J . A simplified procedure for purification of large amounts of poliovirus: characterization and amino acid analysis of Type 1 poliovirus. J Biol Chem. 1960; 235:70-3. View

19.

Von Bertalanffy L, Masin M, Masin F . A new and rapid method for diagnosis of vaginal and cervical cancer by fluorescence microscopy. Cancer. 1958; 11(5):873-87. DOI: 10.1002/1097-0142(195809/10)11:5<873::aid-cncr2820110503>3.0.co;2-b. View

20.

Brawerman G, Gold L, EISENSTADT J . A RIBONUCLEIC ACID FRACTION FROM RAT LIVER WITH TEMPLATE ACTIVITY. Proc Natl Acad Sci U S A. 1963; 50:630-8. PMC: 221238. DOI: 10.1073/pnas.50.4.630. View