A new method for the rapid isolation of chromosomes, mitotic apparatus, or nuclei from mammalian fibroblasts at near neutral pH

Wray, Wayne; Stubblefield, Elton

doi:10.1016/0014-4827(70)90656-7

Cited by 220 publications

(51 citation statements)

References 16 publications

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“…The poles of marine egg mitotic apparatuses have been separated from the rest of the structure and have been shown to be capable of acting as sites for initiation of microtubule as- Chromosomes were prepared by a modification of the method of Wray and Stubblefield (18). Mitotic cells were sedimented at 1000 X g (2000 rpm) for 3 min, and the cell pellet was resuspended in 10 ml of chromosome isolation buffer (CIB) containing 1.0 M hexylene glycol (2-methyl-2,4-pentanediol), 0.5 mM CaCl2, and 0.1 mM Pipes [piperazine-N-N'-bis(2-ethanesulfonic acid)], pH 6.9.…”

mentioning

confidence: 99%

Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells.

Telzer¹,

Moses²,

Rosenbaum³

1975

Proc. Natl. Acad. Sci. U.S.A.

126

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The kinetochores of isolated HeLa cell chromosomes attached to an electron microscope specimen grid, fixed in formaldehyde, and stained with alcoholic phosphotungstic acid are visible as dark, preferentially stained structures distinct from the chromatin with which they are associated. When unfixed chromosomes are immobilized by attachment to grids and incubated with chick brain tubulin, microtubules are observed to assemble onto the kinetochores. This demonstrates the competence of kinetochores in isolated chromosomes to act in vitro as microtubule assembly sites and suggests that they also possess this capacity in vivo. In addition, the results provide a possible means for isolating and characterizing kinetochores. The kinetochore is the site of attachment for the chromosomal microtubules of the mitotic apparatus. While the kinetochore may not be recognizable as a distinct, morphologically identifiable structure in some organisms (1) or may exist as a diffuse region along the chromosome in others (2, 3), in mammalian cells the kinetochore is a localized, welldefined area from which microtubules arise. Considerable detail of the fine structure of the mammalian kinetochore is known from the study of thin sections in the electron microscope (4-6); characteristically, the organelle consists of a tripartite, disc-shaped structure localized at the primary constriction of the chromosome. However, it is only recently that techniques have been defined which permit electron microscopic visualization of the kinetochore in whole mount chromosomes (7). By application of these techniques to surface spreads of the meiotic chromosomes of the locust (7), hamster (8), and man (9) as well as spreads of mitotic chromosomes of HeLa cells (10) the kinetochore has been revealed to be a preferentially stained structure distinct from the chromatin with which it is associated.There are various sites for the attachment and presumptive assembly of microtubules in eukaryotic cells; among these are the basal bodies of cilia and flagella, centrioles of the mitotic apparatus, and the kinetochores of chromosomes.With the development of methods for the in vitro assembly of brain tubulin (11), it has become possible to study the assembly of microtubules onto these sites. Isolated flagellar axonemes of Chiamydonas (12,13) and sea urchin sperm (12) as well as isolated basal bodies of Chlamydomonas (14) will act as initiating sites for the in vitro assembly of tubulin.Moreover, it has been demonstrated that brain tubulin will reversibly assemble and disassemble onto the isolated mitotic apparatuses of marine eggs (15) and rat kangaroo cells (16). The poles of marine egg mitotic apparatuses have been separated from the rest of the structure and have been shown to be capable of acting as sites for initiation of microtubule assembly (microtubule organizing centers; 17). However, it has not been demonstrated whether the kinetochores of chromosomes are also capable of initiating microtubule assembly, and an answer to this question would have s...

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mentioning

confidence: 99%

Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells.

Telzer¹,

Moses²,

Rosenbaum³

1975

Proc. Natl. Acad. Sci. U.S.A.

126

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“…Previous studies (18,27,28,30) have shown the influence of pH on the stability as well as on the physical structure of chromosomes. We found that when the pH of the PAB buffer was between pH 7.0 and 8.0, tighter chromosome peaks were produced with the effect restricted to a reduction of the CVs in HO fluorescence.…”

Section: Discussionmentioning

confidence: 95%

“…Various chromosome isolation procedures (6,16,18,(22)(23)(24)(26)(27)(28)(29) have been developed and adapted for generation of flow karyotypes. The factors influencing the quality of chromosome preparations using chromosome stabilization based on magnesium ions have been investigated previously (18,22,23).…”

Section: Discussionmentioning

confidence: 99%

Factors affecting flow karyotype resolution

Carter

2006

Cytometry Pt A

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Background: One of the major factors which influences the chromosome purity achievable particularly during high speed sorting is the analytical resolution of individual chromosome peaks in the flow karyotype, as well as the amount of debris and fragmented chromosomes. We have investigated the factors involved in the preparation of chromosome suspensions that influence karyotype resolution. Methods: Chromosomes were isolated from various human and animal cell types using a series of polyamine buffer isolation protocols modified with respect to pH, salt concentration, and chromosome staining time. Each preparation was analyzed on a MoFlo sorter (DAKO) configured for high speed sorting and the resolution of the flow karyotypes compared. Results: High resolution flow cytometric data was obtained with chromosomes optimally isolated using hypotonic solution buffered at pH 8.0 and polyamine isolation buffer (with NaCl excluded) between pH 7.50 and 8.0.

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“…Nuclei were isolated by a modification of the method of Wray and Stubblefield (6). The isolation buffer contained 1 mM CaCl2, 0.5 mM piperazine-NN'-bis(2-ethanesulfonic acid) monosodium monohydrate "PIPES" (Calbiochem) at pH 6.7, and 0.5 M hexylene glycol.…”

Section: Methodsmentioning

confidence: 99%

Enhancement of DNA Synthesis in a Mammalian Cell-Free System by Trypsin Treatment

Brown¹,

Stubblefield²

1974

Proc. Natl. Acad. Sci. U.S.A.

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DNA synthesis in a broken cellular preparation of Chinese hamster cells was enhanced approximately 10-fold by a brief trypsin treatment. Alphachywotrypsin also enhanced the, synthesis, whereas Pronase did not. The trypsin appears to be acting on a compopent of the nucleus. Evidence suggests that the trypsin is not removing protein from the DNA, but may be activating the system some other way.Much remains to be learned about DNA replication, especially in eukaryotic systems. The presence of histones, other chromosomal proteins, and the nuclear envelope provide extra complications for such studies 'in eukaryotes. Using cell culture systems, Taylor has shown that the nascent DNA is replicated in 6S units and then joined by a ligase (1). The same mechanism exists in prokaryotes (2). Recently it has been shown that a short chain of RNA is associated with the nascent DNA, and it has been suggested that this RNA must be synthesized before the DNA can be replicated (3). Silverman and Mirsky (4) have shown that exogenous DNA polymerase and RNA polymerase both have much less access to the DNA in isolated nuclei or chromatin than in isolated DNA.Using isolated nuclei, chromatin, and DNA, we have studied DNA synthesis using the endogenous DNA polymerase. In the course of such experiments we found a striking effect of trypsin on the rate of DNA synthesis. Suspecting at first that the effect might be due to the removal of histones from the DNA, we were surprised to find that this was not the explanation. The key experiments will be reported in this paper; a preliminary abstract was published earlier (5). MATERIALS AND METHODSCulture&. Chinese hamster fibroblast cultures (Don and Don-C) were grown as monolayers in McCoy's 5a medium supplemented with 10% fetal-calf serum (v/v).I8olation of Nuclei and(Cytosol&. Nuclei were isolated by a modification of the method of Wray and Stubblefield (6). The isolation buffer contained 1 mM CaCl2, 0.5 mM piperazine-NN'-bis(2-ethanesulfonic acid) monosodium monohydrate "PIPES" (Calbiochem) at pH 6.7, and 0.5 M hexylene glycol. Cells were either removed by a brief trypsin treatment or else scraped off in isolation buffer with a rubber policeman. In either case, the cells were washed once in 4 ml of isolation buffer (5 X 106 cells per ml) at 40 and then pelleted by centrifugation at 800 X g for 5 min at 4°. The pellet was then resuspended in isolation buffer at approximately 2 X 107 cells per ml. The nuclei were isolated by rapid 2432 passage of the suspension through a 22-gauge needle with the bevel placed against the side of the centrifuge tube. This was repeated until clean nuclei were obtained, as determined by phase microscopy. The resulting preparation is called the cellular lysate. The nuclei can be removed from the suspension by centrifugation at 800 X g for 5 min at 4°. In some cases the supernatant from this was' then centrifuged again at 100,000 X g at 4°. The supernatant from the high-speed centrifugation is termed the cytoplasmic cyto8ol. The pellet of nuclei was resuspende...

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A new method for the rapid isolation of chromosomes, mitotic apparatus, or nuclei from mammalian fibroblasts at near neutral pH

Cited by 220 publications

References 16 publications

Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells.

Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells.

Factors affecting flow karyotype resolution

Enhancement of DNA Synthesis in a Mammalian Cell-Free System by Trypsin Treatment

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