Membrane associated DNA (“M‐band”) in growing and confluent 3T3 cells

Freienstein, C.M.; Freitag, H. Eckehard; Süß, R.

doi:10.1016/0014-5793(73)80644-1

Cited by 8 publications

(3 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We also obtained the latter result with L cells and WI38. This is in agreement with the work of others using mammalian cells (Freienstein et al, 1973; Hanaoka and Yamada, 1971). It has been shown that shearing procedures, such as vortexing, reduce the amount of DNA in the sarkosyl M-band preparations (Yamada and Hanaoka, 1973;Hildebrand and Tobéy, 1973).…”

Section: Resultssupporting

confidence: 93%

“…troversial. Some biochemical and cell-fractionation studies have suggested the existence of a DNA-nuclear membrane complex (Freienstein et al, 1973;Barrieux et al, 1973; Yantada and Hanaoka, 1973;Hildebrand and Tobey, 1973;Infante et al, 1973; Cabradilla and Toliver, 1975) and that replicating cells contain a greater amount of membrane-bound DNA than quiescent cells (Hildebrand and Tobey, 1973). However, other studies suggest that such DNA-nuclear membrane complexes may be either the results of artifacts during preparation (Fakan et al, 1972;Huberman et al, 1973), true complexes but not the site of DNA replication in the nuclei (Comings and Okada, 1973;Franke et al, 1973), or only partly concerned with such replication (O'Brien et al., 1973).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro

Infante¹,

Firshein²,

Hobart³

et al. 1976

Biochemistry

View full text Add to dashboard Cite

A DNA-nuclear membrane complex has been isolated by two different methods from the nuclei of cultured mouse fibroblast (3T3) cells. One method, utilizing the detergent sarkosyl (sodium lauroyl sarkosinate), yields a DNA-nuclear membrane complex (the M band), which contains virtually all of the DNA in the nuclei. However, treatment of the M band by sonication, vortexing, or freeze-thaw reduces the amount of DNA in the complex by approximately 50-80%, depending upon the phase of the cell cycle from which the complex was extracted. The remaining DNA is tightly bound to the nuclear membrane and resists further shearing procedures. Over 90% of the choline-labeled phospholipid present in nuclei is also found in these sheared M bands. The percentage of DNA associated with the nuclear membrane varies during the cell cycle and correlates well with the onset, continuation, and cessation of DNA synthesis. Thus, although DNA-membrane complexes can be detected throughout the cell cycle, the percentage of DNA bound to membrane increases during late G1 and S and decreases during G2. In addition, there are distinct qualitative differences in the type of DNA present in the membrane fraction, with a more highly d(A-T) rich DNA being present in confluent (G0) cells than in cells during the S phase. This d(A-T) rich DNA may be related to the mouse satellite DNA identified by others. The M band can be separated into two DNA-nuclear membrane subfractions by centrifugation through a continuous sucrose gradient. The relative proportions of these two subfractions depend upon the percentage of sarkosyl present in the M band prior to centrifugation, with complete removal of sarkosyl resulting in a very large increase in the sedimentation velocity of the complex and in the formation of only one fraction. Evidence that this is a complex of DNA with membrane is given by the finding that DNA is dissociated from the complex with Pronase, deoxycholate, or high levels of sarkosyl. Removal of virtually all of the DNA with DNase from this rapidly sedimenting complex does not dissociate any of the phospholipid which still sediments rapidly as a single band. A second method, which yields a DNA-membrane fraction from nuclei, utilizes sedimentation of lysed nuclei to equilibrium in CsCl density gradients. This low-density CsCl fraction contains only 10-15% of the total DNA, but contains most of the nascent DNA, which may be chased into a membrane-free fraction. The DNA-membrane fraction from CsCl gradients possesses properties in common with the M-band fraction and can be converted into an M band. DNA membrane complexes from sucrose gradients, as well as the crude M-band preparation and a non-membrane-associated DNA fraction from nuclei can synthesize DNA in vitro without the addition of an external DNA template or DNA polymerase. In contrast to the activity in the non-membrane-associated DNA fraction, the membrane-associated polymerase activity is strongly stimulated by adenosine triphosphate and is unaffected by ethidium bromide...

show abstract

Section: Resultssupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro

Infante¹,

Firshein²,

Hobart³

et al. 1976

Biochemistry

View full text Add to dashboard Cite

show abstract

“…(Comings & Kakefuda 1968;H anaoka & Y am ada 1971;Mizuno, Stoops & Peiffer 1971;Yoshida, M odak & Yagi 1971;Hatfield 1972;O 'Brien, Sanyal & Stanton 1972;Infante et al 1973). These data are very con troversial (Williams & Ockey 1970;Kay, Haines & Johnston 1971;Deumling & Franke 1972;Fakan, Turner, Pagano & Hancock 1972;Freienstein, Freitag & Suss 1973). We have tested rat liver nuclei from partially hepatectomized rats in in vitro systems allowing the incorporation…”

Section: Enzymes Associated With Nuclear Membranesmentioning

confidence: 98%

One-step isolation and characterization of nuclear membranes

Monneron

1974

Phil. Trans. R. Soc. Lond. B

View full text Add to dashboard Cite

Purified fractions of nuclei from rat liver and isolated calf thymocytes are treated with 0.5 M MgCl 2 or with an alkaline buffer, pH 8.5. Such procedures dissociate nuclear membranes from the chromatin. The membranes are simply floated in a gradient. The recovery in the membrane band obtained by the Mg 2+ technique is 55 to 60 %. Freeze-etching studies of the membrane pellets provide a good way to check their purity, pores being very conspicuous. A 5'-nucleotidase and an ATPase are detected on the nuclear membranes of thymocytes, as well as a still undescribed 3'-nucleotidase. However, no marker enzymes for the nuclear membrane have been found. The topology of sugar residues on the nuclear membranes is studied by means of lectins. Concanavalin A binds in large amounts to nuclear envelopes, both on the inner and outer leaflets, but not to pores.

show abstract

DNA synthesis associated with a DNA-nuclear-membrane complex from rat liver

Sinha

Mizuno

1977

Cell Tissue Res.

View full text Add to dashboard Cite

Ultrastructural analysis of M-band from nuclei of rat liver showed small amounts of chromatin, fragments of inner nuclear membrane, some amorphous nuclear material, and nucleopores. The outer nuclear membrane with its associated ribosomes was removed by Sarkosyl during the preparation of M-band sample. Morphological features of nucleopores and the inner nuclear membrane were confirmed by freez-fracture technique. The gross chemical composition of the M-band was similar to that of nuclear-membrane fractions prepared by other techniques. The M-band contained the greatest proportion of newly-labeled DNA and also supported DNA synthesis in vitro. Electron-microscopic autoradiography of the M-band showed localization of silver grains of thymidine-3H presumably over newly synthesized DNA. The DNA synthesis could not be attributed to spurious attachment of DNA polymerase to M-band during its isolation. It was partially removed from the M-band by treatment with 0.5 M KC1, phospholipase A or C; and completely, by the action of pancreatic DNase. DNA synthesis was greater in M-band fractions isolated from nuclei of 24-hour regenerating liver.

show abstract

Membrane associated DNA (“M‐band”) in growing and confluent 3T3 cells

Cited by 8 publications

References 7 publications

A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro

A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro

One-step isolation and characterization of nuclear membranes

DNA synthesis associated with a DNA-nuclear-membrane complex from rat liver

Contact Info

Product

Resources

About