Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts.

Abstract

The uptake and expression by plastids isolated from dark-grown cucumber cotyledons (etioplasts) of two pUC derivatives, pCS75 and pUC9-CM, respectively carrying genes for the large small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans or chloramphenicol acetyltransferase, is reported. Untreated etioplasts take up only 3% as much DNA as that taken up by EDTA-washed etioplasts after 2 hr of incubation with nick-translated [32P]-pCS75. The presence or absence of light does not affect DNA uptake, binding, or breakdown by etioplasts. Calcium or magnesium ions inhibit DNA uptake by 86% but enhance binding (23-200%) and breakdown (163-235%) of donor DNA by EDTA-treated etioplasts. Uncouplers that abolish membrane potential (delta psi), transmembrane proton gradient (delta pH), or both do not affect DNA uptake, binding, or breakdown by etioplast. However, both DNA uptake and binding are severely inhibited by ATP. Presumably this results from the hydrolysis of ATP, because the poorly hydrolyzable analog adenyl-5'-yl imidodiphosphate does not inhibit the uptake or binding of DNA by etioplasts. beta-Lactamase specified by the ampicillin resistance gene of pCS75 can be detected only in EDTA-treated etioplasts that have been incubated with the plasmid pCS75. After the incubation of EDTA-treated etioplasts with pCS75, immunoprecipitation using antiserum to the small subunit of ribulose bisphosphate carboxylase/oxygenase from A. nidulans reveals the synthesis of small subunits; these are smaller by 2 kDa than the cucumber small subunit encoded by the nuclear genome. Treatment of etioplasts with 10 mM EDTA shows a 10-min duration to be optimal for the expression of chloramphenicol acetyltransferase encoded by pUC9-CM. A progressive increase in the expression of this enzyme is observed with an increase in the concentration of pUC9-CM in the DNA uptake medium. The plasmid-dependent incorporation of [35S]methionine by EDTA-treated organelles declines markedly during cotyledon greening in vivo.

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

“…Therefore, early investigations of chloroplast transformation in vascular plants focused on the development of chloroplast systems capable of efficient, prolonged protein synthesis and expression of foreign genes [9]. The development of the gene gun as a transformation device by John Sanford (reviewed in Ref.…”

“…Selectable markers and reporters aadA Aminoglycoside-3′-adenylyltransferase [14] nptII Neomycin phosphotransferase [52] codA Cytosine deaminase [53] BADH Betaine aldehyde dehydrogenase [4] uidA β-glucoronidase [12] cat Chloramphenicol acetyl transferase [9,11] gfp Green fluorescent protein [24,54] aadA:gfp Selectable or screenable fusion protein [47] Plant traits: herbicide resistance aroA Glyphosate resistance [2,19] bar Bialaphos resistance [18,20] Insect resistance…”

Containment of herbicide resistance through genetic engineering of the chloroplast genome

“…Early investigations on chloroplast transformation focused on the development of intact chloroplasts capable of efficient and prolonged transcription and translation 7-9 and expression of foreign genes in isolated chloroplasts 10 . These experiments were done under the premise that it was possible to introduce isolated intact chloroplasts into protoplasts and regenerate transgenic plants 11 .…”

“…Another important advantage of chloroplast transformation is the higher levels of foreign gene expression due to a very high copy number (5000-10,000) of chloroplast genomes in plant cells. Because the transcriptional and translational machinery of the chloroplast is prokaryotic in nature, herbicide-resistant genes of bacterial origin can be expressed at extraordinarily high levels in chloroplasts.Early investigations on chloroplast transformation focused on the development of intact chloroplasts capable of efficient and prolonged transcription and translation 7-9 and expression of foreign genes in isolated chloroplasts 10 . These experiments were done under the premise that it was possible to introduce isolated intact chloroplasts into protoplasts and regenerate transgenic plants 11 .…”

Chloroplast Vector Systems for Biotechnology Applications

“…GUS, chloramphenicol acetyl transferase, and GFP have been used as plastid reporters (Daniell and McFadden, 1987;Daniell et al, 1990;Ye et al, 1990;Khan and Maliga, 1999). The enzymatic activity of GUS can be visualized by histochemical staining Daniell et al, 1991), whereas GFP is a visual marker that allows direct imaging of the fluorescent gene product in living cells.…”