Late replication of the DNA associated with the nuclear membrane

Kay, Robert R.; Haines, Michael E.; Johnston, Irving R.

doi:10.1016/0014-5793(71)80358-7

Cited by 44 publications

(22 citation statements)

References 18 publications

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“…In contrast, most (6,7,8) [but not all (9)] biochemical studies show that newly replicated DNA is preferentially associated with the nuclear envelope. Newly replicated DNA is often (6,(10)(11)(12), but not always (13,14), localized in the interphase after phenol or chloroform-isoamyl alcohol extraction.…”

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confidence: 98%

Sites of Replication of Chromosomal DNA in a Eukaryotic Cell

Fakan

Turner

Pagano

et al. 1972

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

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In mouse cells (line P815), newly synthesized DNA labeled for [20][21][22][23][24][25][26][27][28][29][30] sec during exponential growth is found by electron microscope autoradiography at sites throughout the cell nucleus. These sites are relatively more concentrated in the peripheral region of the nucleus (averaged over a random population of S-phase cells), probably reflecting a higher local concentration of DNA in this region. Newly synthesized DNA is not preferentially associated with purified nuclear envelopes, but is found in a fraction of the chromosomal deoxynucleoprotein whose buoyant density in CsCl after formaldehyde treatment is about 1% lower than that of the deoxynucleoprotein peak.Kinetics experiments suggest that this material is a precursor of mature deoxynucleoprotein; it may represent regions of deoxynucleoprotein containing replicating DNA and the additional proteins involved in DNA replication. Other complexes of newly replicated DNA that are found in the interphase after phenol extraction of nuclei are formed during the extraction procedure, probably due to the partially single-stranded nature of replicating DNA, and do not appear to exist in vivo.The many studies on the location of replication sites of chromosomal DNA in the eukaryotic nucleus have led to divergent conclusions. Electron microscope autoradiography (1-3) shows that replication sites of chromosomal DNA are distributed throughout eukaryotic nuclei, and do not appear to be associated with any specific morphological structure. The peripheral replication sites in cells released from synchronizing inhibitors (4) may not represent normal S-phase initiation (5).In contrast, most (6,7,8) [but not all (9)] biochemical studies show that newly replicated DNA is preferentially associated with the nuclear envelope. Newly replicated DNA is often (6, 10-12), but not always (13,14), localized in the interphase after phenol or chloroform-isoamyl alcohol extraction. It sometimes associates preferentially with crystals of detergent in sucrose gradients (7,30). The interpretation of these observations has been influenced by models of membrane-associated replication of bacterial (15) and phage (16) DNA; the phage DNA-membrane association may, however, be related to transcription rather than replication (17).We present here studies designed to resolve these discrepancies and to identify by two independent methods, electron microscope autoradiography and cell fractionation, the sites of chromosomal DNA synthesis in exponentially growing mouse cells. Eukaryotic chromosomal DNA replicates at 0.5 (18) to 1-2 (19) Mum/min; within the nucleus, therefore, a labeled precursor newly incorporated into DNA could be displaced as much as 1 Mm away from its site of incorporation after 1 min. We have used labeling periods of 20-30 sec, which are about the lower practical limit for present techniques of electron microscope autoradiography; labeled molecules incorporated at the beginning of this period are unlikely to be displaced more than 0.5 ,m from the...

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confidence: 98%

Sites of Replication of Chromosomal DNA in a Eukaryotic Cell

Fakan

Turner

Pagano

et al. 1972

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

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“…In most preparative methods, the nuclear membrane fragments are then separated from non membraneous nuclear components by making use of the clearly lower buoyant density of membrane lipoproteins by sedimentation or flotation in concentration gradients of sucrose (continuous or discontinuous), sorbitol, or CsCI (Franke, 1966(Franke, , b, 1967aKashnig and Ka1!per, 1969;Zbarsky et al, 1969;Franke et al, 1970;Kay et al, 1971Kay et al, , 1972Zentgraf et al, 1971;Agutter, 1972;Fakan et al, 1972;Matsuura and Ueda, 1972;Monneron et al, 1972;Moore and Wilson, 1972;Price et al, 1972). Methods for preparing nuclear membrane fractions have been reported for various plant tissues such as onion root tip or leaves (Franke, 1966b), for the macronuclei of the ciliate, Tetrahymena pyriformis (Franke, 1967 a, b;Eckert, 1972), for mouse, rat, rabbit, and pig liver (Franke, 1967a, b;Zbarsky et al, 1967Zbarsky et al, , 1969Bornens, 1968;Kashnig and Kasper, 1969;Berezney et al, 1970Franke et al, 1970;Kartenbeck et al, 1971Kartenbeck et al, , 1973Agutter, 1972;Kay et al, 1972;Monneron et al, 1972;Price et al, 1972), for avian erythrocytes (Harris and Brown, 1971;Zentgraf et al, 1971) for rat and calf thymus (Matsuura and U eda, 1972;J arasch et al, 1973), for rat prostate gland (Moore and Wilson, 1972), and for a series of tumor cells (Zbarsky et al, 1967Comes and Franke, 1970;…”

Section: J\:\mentioning

confidence: 99%

“…though at proportions varying according to the specific isolation method Berezney et al, 1970Franke et al, 1970;Kay et al, 1971Kay et al, , 1972Zentgraf et al, 1971;Fakan et al, 1972;Monneron et al, 1972;Franke et al, 1973b;see, however, Kashnig and Kasper, 1969). Since Kubinski et al (1972) have demonstrated that isolated ER and nuclear membranes are capable of in vitro binding of DNA and component deoxyribonucleotides, one must recognize as potential artifacts the random associations of nuclear DNA with the isolated membranes during the course of preparation.…”

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confidence: 99%

“…(b) Mass isolations of nuclear membrane material usually start from a purified fraction of isolated nuclei. The nuclei are then fragmented and/ or extracted with combinations of diverse treatments: (i) rapid chromatin swelling in hypotonic solutions (Franke 1966a(Franke , b, 1967aZbarsky et al, 1967Zbarsky et al, , 1969Franke and Kartenbeck, 1969;Price et al, 1972; reviewed by Zbarsky, 1972a, b); ( ii) vigorous homogenization, shearing and sonication (Franke, 1966a(Franke, , b, 1967aBornens, 1968;Kashnig and Kasper, 1969;Zbarsky et al, 1969;Comes and Franke, 1970;Harris and Agutter, 1970;Harris and Brown, 1971;Zentgraf et al, 1971;Agutter, 1972;Fakan et al, 1972;Moore and Wilson, 1972;J arasch et al, 1973); (iii) limited digestion of the chromatin with deoxyribonuclease Kay et al, 1971Kay et al, , 1972Zentgraf et al, 1971;Matsuura and Ueda, 1972); (iv) destabilization with chelating agents such as citrate (Bornens, 1968;Kashnig and Kasper, 1969) or in high salt concentrations Franke et al, 1970;ZentFig. 2 Special nucleocytoplasmic separation zone of the primary (giant) nucleus in the rhizoid of the green alga, Acetabularia mediterranea.…”

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Structures and Functions of the Nuclear Envelope

Franke¹,

Scheer²

1974

The Cell Nucleus

153

150

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“…Friedman and Mueller (8) suggested that replicating DNA in mammalian cells is attached to some nuclear site; this idea was supported by workers, using electron microscopic autoradiography, who concluded that DNA replication in mammalian cells is at least initiated at the nuclear envelope (9-11). Use of the same technique, however, has also shown that DNA replication is not initiated at the nuclear membrane in Chinese hamster cells (12, 13) or mouse cells (14) and that any synthesis which appears to be membrane-bound occurs late in the S period and is believed to represent heterochromatin replication (12,15 (24) while chromatin attachment to the nuclear membrane in Triticum may occur only in specialized cells (25). We describe experiments using biochemical and electron microscopic techniques which indicate that the nuclear DNA and newly synthesized DNA in cotton (Gossypium barbadense) radicle tips is associated with nuclear membranes during germination.…”

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Chromatin and DNA synthesis associated with nuclear membrane in germinating cotton.

Clay¹,

Katterman²,

Bartels³

1975

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

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The synthesis of nuclear DNA and possible attachment sites of chromatin in the cells of cotton (Gossypium barbadense) radicles during germination was investigated. Biochemical analysis of nuclear membrane fragments or Sarkosyl-magnesium-membrane complexes indicates that the DNA, including newly replicated DNA, is attached to the nuclear membranes during periods of active synthesis. Electron micrographs of nuclear membrane fragments indicate a physical association between chromatin fibers and the memnbranes. The attachment site appears to be proteinaceous, since the chromatin is released by protein degradative enzymes as evidenced by biochemical techniques and electron microscopic observations. Short-term labeling results in incorporation into a membrane-associated product indistinguishable from the bulk of nuclear DNA. DNA polymerase activity is also associated with nuclear membrane preparations in which [3H]thymidine triphosphate is incorporated into an acid-insoluble, DNase-sensitive product.In recent years, considerable research has been conducted concerning the involvement of membranes in the replication of DNA. Initial investigations on bacterial cells have strongly indicated that the point of attachment of DNA during replication is permanently bound to the cellular membranes (1-4). The concept of DNA synthesis associated with membranes was further enhanced by the discovery of membranebound DNA polymerase activity (5, 6). It is, therefore, generally accepted that the constituents necessary for DNA.synthesis in prokaryotes appear to be associated with cell membranes during DNA replication (7).The proposal that DNA replication in eukaryotes involves an attachment of chromosomes to the nuclear membranes has not, however, gained universal acceptance. Friedman and Mueller (8) suggested that replicating DNA in mammalian cells is attached to some nuclear site; this idea was supported by workers, using electron microscopic autoradiography, who concluded that DNA replication in mammalian cells is at least initiated at the nuclear envelope (9-11). Use of the same technique, however, has also shown that DNA replication is not initiated at the nuclear membrane in Chinese hamster cells (12, 13) or mouse cells (14) and that any synthesis which appears to be membrane-bound occurs late in the S period and is believed to represent heterochromatin replication (12,15 (24) while chromatin attachment to the nuclear membrane in Triticum may occur only in specialized cells (25). We describe experiments using biochemical and electron microscopic techniques which indicate that the nuclear DNA and newly synthesized DNA in cotton (Gossypium barbadense) radicle tips is associated with nuclear membranes during germination. MATERIALS AND METHODSSeeds of Pima cotton, Gossypium barbadense L., cultivar Pima S-4, were germinated and surface sterilized as described (26). After 36 hr of germination when DNA synthesis is maximum (26), 40 seedlings were each incubated for 90 min with 250 AiCi of [3H]thymidine (13 Ci/mmol).Isolation of ...

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Late replication of the DNA associated with the nuclear membrane

Cited by 44 publications

References 18 publications

Sites of Replication of Chromosomal DNA in a Eukaryotic Cell

Sites of Replication of Chromosomal DNA in a Eukaryotic Cell

Structures and Functions of the Nuclear Envelope

Chromatin and DNA synthesis associated with nuclear membrane in germinating cotton.

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