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

Abstract

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 ...

Mechanism of nuclear DNA replication in radicles of germinating cotton

“…With prolonged labeling in eukaryotic cells, radioactivity also appears in larger size molecules of discrete size, indicating that replication occurs discontinuously (10)(11)(12)15) through defined intermediates. These data suggest that replication in eukaryotes is initiated through multiple replication sites (9,16,17).In a previous publication, we demonstrated that replicating nuclear DNA in germinating cotton is associated with the nuclear membrane (18). In this communication we provide evidence that the nuclear-membrane-associated DNA replicates through short single-strand "Okazaki-type" (19) intermediates of discrete size.…”

“…In a previous publication, we demonstrated that replicating nuclear DNA in germinating cotton is associated with the nuclear membrane (18). In this communication we provide evidence that the nuclear-membrane-associated DNA replicates through short single-strand "Okazaki-type" (19) intermediates of discrete size.…”

“…RESULTS AND DISCUSSION In previous communications, we established that during germination of Pima cotton at 340, at approximately 36 hr, the incorporation of radioactive precursors into nuclear DNA reaches a maximum value (20,21 replication of nuclear DNA which appeared to be associated with the nuclear membrane (18). To determine the mechanism of DNA replication, nuclear DNA was isolated at 36 hr of germination and analyzed with biochemical and electron microscopic technique.…”

“…Nuclear DNA from germinating radicles was isolated for hydroxyapatite and sucrose gradient analysis as previously described (18 Martinson and Wagenaar (24). After equilibration, the single-and double-stranded DNAs were eluted from the column with (K)PO4 buffers at 600 as described by Miyazawa and Thomas (25).…”

Pressuromodulation at the cell membrane as the basis for small molecule hormone and peptide regulation of cellular and nuclear function

“…As a result of remaining at the cell membrane-to-aqueous interface, the steroids exert their molecular effects at the alpha helix-based CM receptors, whereby, the endogenous corticosteroids primarily exert their effects at the juxtaposed classical GR (97 kDa) and MR (110 kDa) (MR-classical GR complex) [ 9 ], by binding to the Mineralocorticoid Receptor (MR) portion of the classical glucocorticoid cum mineralocorticoid receptor complex for which portion they have greater binding affinities [ 9 , 10 ], while the endogenous sex steroids bind to the Estrogen Receptor (ER) (65 kDa), progestorone receptor (PR) and androgen receptor (AR), in order of greatest to least potency steroid receptor respectively (MR-classical GR > ER > PR > AR) [ 11 , 12 ]. Moreover, the molecular arrangement of hydrophilic groups attached directly to the immediate steroid backbone is insufficient to induce endocytosis of the respective hormone-receptor complexes, therefore, both classes of steroids are CM receptor pressuromodulators for the specific cell types expressing their receptors vis a vis ligand-bound receptor pressuromodulation of the specific receptor’s microtubular network (MR-classical GR, ER, PR or AR)-linked to the receptor’s nuclear chromatin DNA (MR-classical GR, ER, PR or AR) at the level of the nuclear membrane (NM) [ 13 ]: The intracellular microtubular network is immobile [ 14 ], as opposed to the intracellular F-Actin network, which mobilizes [ 14 ], in response to CM receptor-mediated pressuromodulation.…”