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

Abstract

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...

DNA changes involving repeated sequences in senescing soybean (Glycine max) cotyledon nuclei

“…If this differential loss is confined to repetitive sequences only, as noted for differential replication of repeated sequences in isolated soybean Physiol, Plant. 64, 1985 nuclei (Caboche and Lark 1981), is not known. The loss of DNA sequences, such as ribosomal and coding unique DNA, requires further investigation.…”

Isolation and characterization of a gene encoding meso-diaminopimelate dehydrogenase fromGlycine max

“…The cell culture was grown exponentially in MS medium as previously described (10). Protoplasts were prepared by a modification of the method of Caboche and Lark (7). Aliquots of exponentially growing cultures were allowed to settle for 15 30 minutes.…”

Michel Caboche, an outstanding plant molecular and cell biologist

“…He studied the growth in culture at low density of Nicotiana plumbaginifolia protoplasts, a condition for the selection of mutants in vitro, and showed that this growth was only possible if the concentration of auxin, a plant hormone necessary for the divisions, was limited [2]. In 1979-1980 he did a postdoctoral fellowship in the laboratory of Karl Gordon Lark, at the University of Utah, where he studied DNA replication in cultured soybean cells [3]. Back at the Laboratoire de Biologie Cellulaire in Versailles, he developed with Alain Deshayes, Pierre Rouzé and Luis Herrera-Estrella the di-rect transfer of genes by fusion of liposomes containing a plasmid DNA with tobacco protoplasts [4,5].…”

“…Il étudie la croissance de protoplastes de Nicotiana plumbaginifolia en culture à faible densité, condition pour la sélection de mutants in vitro et montre que cette croissance n'est possible que si l'on limite la concentration en auxine, hormone végétale nécessaire aux divisions [2]. Il réalise en 1979-1980 un stage postdoctoral dans le laboratoire de Karl Gordon Lark, à l'université d'Utah, où il étudie la réplication de l'ADN dans les cellules de soja en culture [3]. De retour au Laboratoire de Biologie Cellulaire de Versailles, il développe avec Alain Deshayes, Pierre Rouzé et Luis Herrera-Estrella le transfert direct de gènes par fusion de liposomes contenant l'ADN d'un plasmide avec des protoplastes de tabac [4,5].…”