Organellar DNA synthesis in permeabilized soybean cells

Abstract

Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L-α-lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase α inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

“…We have optimized conditions for permeabilization of tobacco cells with La-lysophosphatidylcholine. The permeabilized cells are capable of uptake and incorporation of deoxynucleoside triphosphates into DNA, as reported by Weissbach and coworkers (30). This may be an ideal system to study in vivo replication of foreign DNA because of the fact that [32P]TP gets incorporated only into organellar DNA (30 …”

Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max

“…The soybean suspension culture used as a source of chloroplast is a cell line capable of photoautotrophic growth that we have found to be rich in actively replicating chloroplasts [2]. The purification procedure employed has been demonstrated to provide a high yield of chloroplasts that are free of contaminating mitochondria and nuclei [2,11].…”

“…The purification procedure employed has been demonstrated to provide a high yield of chloroplasts that are free of contaminating mitochondria and nuclei [2,11]. Lysis and extraction of the chloroplasts by treatment with high-salt buffer results in an extract that contains DNA and RNA polymerase activity as well as an appreciable amount of endogenous nucleic acids.…”

“…We have been investigating organellar DNA replication in a photoautotrophic soybean cell line that is remarkable in that the amount of chloroplast DNA per cell is extraordinarily high [ 1,2]. We are interested in an in vitro replication system similar to the ones reported for pea, petunia, and Chlamydomonas but based on chloroplast extracts from soybean cells.…”