Replication of DNA by Nuclei Isolated from Soybean Suspension Cultures

Román, Ruth; Caboche, Michel; Lark, Karl G.

doi:10.1104/pp.66.4.726

Cited by 14 publications

(12 citation statements)

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“…The in vitro replication of DNA by isolated soybean nuclei has been described (8). The system required the four deoxynucleotide triphosphates, ATP, and Mg2+, and it appeared to synthesize high molecular weight DNA which could be joined in vitro and which manifested the characteristics of replicative, as opposed to repair, synthesis.…”

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“…[32P]DNA synthesized in vitro, although about half of the DNA synthesized in vitro occurs as unjoined nascent Okazaki pieces about 500 nucleotides long (8). ] Whereas 55% of the in vivo labeled DNA remained denatured (not bound to hydroxylapatite) at Cot < 100, less than 20% of the in vitro 32P-labeled DNA remained.…”

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“…These procedures have been described in detail (8). Briefly, protoplasts were isolated under low osmotic stress (0.2 M KCl) from exponentially growing cells.…”

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“…Nuclei were then lysed in 50 mM Tris'HCl, pH 7.8/100 mM EDTA/150 mM NaCl/5 mg of NaDodSO4 per ml (TNES buffer) and incubated for 30 min at 65°C in the presence of proteinase K (200 ,ug/ml). DNA was then purified by isopycnic centrifugation in a CsCl gradient as described (8). DNA-containing fractions were dialyzed overnight against 50 liter and time in sec) were calculated for each sample after correction to standard salt conditions (0.12 M phosphate buffer).…”

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“…Standard incubations of nuclei for DNA synthesis were done at 30'C for 15 min. Autoradiography of nuclei was performed as described (8).…”

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Preferential replication of repeated DNA sequences in nuclei isolated from soybean cells grown in suspension culture.

Caboche¹,

Lark²

1981

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

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Repeated sequences comprise a large fraction of the DNA of higher eukaryotes (1). Recently it has become possible to amplify specific sequences in cultured mamalian cells by subjecting them to continuously increasing selection pressure. In these systems it has been found that enzyme level reflects the frequency of the corresponding structural nucleotide sequence (2, 3). Genomic DNA from plants also contains a large proportion of repetitious DNA (4, 5). Recently, we have found that the proportion of classes of repeated nucleotide sequence containing ribosomal genes can be altered during selective growth of soybean cells in tissue culture (6). This alteration may be similar to the selected changes reported in mammalian cell culture. Such results suggest that higher organisms might adapt to selective pressures by altering the frequency of specific genes and thereby the level of proteins within the cell.Eukaryotic DNA is replicated as groups of tandemly arranged replication units; within a group, some units may be replicated earlier than others (7). If incomplete replication structures (bubbles) are essential intermediates in gene amplification, the time of replication of specific sequences relative to their neighbors may influence the probability of altering their copy number.The in vitro replication of DNA by isolated soybean nuclei has been described (8). The system required the four deoxynucleotide triphosphates, ATP, and Mg2+, and it appeared to synthesize high molecular weight DNA which could be joined in vitro and which manifested the characteristics of replicative, as opposed to repair, synthesis. We now report that a large part of this replication involves specific repeated nucleotide sequences and that the replication in vitro may be altered by pretreatment with hormones in vivo. was obtained from P-L Biochemicals. Benzyladenine and kinetin were purchased from Sigma and 6-(y, y-dimethylallylamino)purine (21P) was from GIBCO. MATERIALS AND METHODS Materials. [methyl-3H]ThymidineCell Cultures and Media. Suspension cultures of the soybean cell line SB-1 were grown in B5 medium (9) containing 2,4-dichlorophenoxyacetic acid (1 mg/liter) and 0.2% casein hydrolysate as described (10).Isolation of Nuclei and Assay Conditions for DNA Biosynthesis. These procedures have been described in detail (8). Briefly, protoplasts were isolated under low osmotic stress (0.2 M KCl) from exponentially growing cells. These were then lysed by treatment with the nonionic detergent Triton X-100 and nuclei were collected by sedimentation. Isolation of nuclei and[32P]TTP incorporation were at pH 8.0 to prevent the action of a potent endonuclease, specific for single-stranded DNA and active below pH 7.5. Standard incubations of nuclei for DNA synthesis were done at 30'C for 15 min. Autoradiography of nuclei was performed as described (8).Preparation of Cytoplasmic Extracts. Cytoplasmic extracts from soybean cells were supernatant fractions obtained after sedimentation of nuclei from lysed protoplast preparations. These were clarified b...

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confidence: 99%

“…These procedures have been described in detail (8). Briefly, protoplasts were isolated under low osmotic stress (0.2 M KCl) from exponentially growing cells.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…Standard incubations of nuclei for DNA synthesis were done at 30'C for 15 min. Autoradiography of nuclei was performed as described (8).…”

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confidence: 99%

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