= Functional plastid-to-nucleus gene transfer experiment
<Experimental Reconstruction of Functional Gene Transfer from the Tobacco Plastid Genome to the Nucleus by Sandra Stegemann and Ralph Bock> (2006, Results, pp. 2870–2874) separated relocation of <DNA> from activation of a relocated <gene>. Engineered tobacco <chloroplasts> carried an nptII <gene> with nuclear expression signals beside an aadA <gene> with plastid expression signals. Selection on kanamycin recovered plants with the segment in the nuclear <genome>. Crosses then replaced the engineered <chloroplasts> with ordinary <chloroplasts>: the nuclear segment remained, but the plants were initially sensitive to spectinomycin and streptomycin. The transferred aadA <gene> was not yet functional in its new setting.
Leaf pieces were cultured on regeneration medium containing both drugs. Resistant regenerated plants supplied the functional readout. Using both drugs reduced selection of spontaneous plastid <mutations> conferring resistance mainly to one drug. Reciprocal crosses and selfing distinguished maternal plastid inheritance from nuclear <Mendelian segregation>; eight of sixteen initial candidates had the latter pattern. <Experimental Reconstruction of Functional Gene Transfer from the Tobacco Plastid Genome to the Nucleus by Sandra Stegemann and Ralph Bock>[Figure 1, Tables 1–2 and Results, pp. 2870–2871: selection, eliminated candidates and inheritance controls]{ref}
The molecular readout came from <RNA> hybridization and <DNA sequencing>. A <Northern blot> detected aadA <messenger RNA> in activated lines, with different transcript sizes. Thermal asymmetric interlaced <polymerase chain reaction> recovered upstream <DNA>; products present in activated lines but absent in original-line and ordinary-plant controls were sequenced. The mapped rearrangements connected aadA to the upstream nuclear <gene promoter>. Thus resistance, inheritance, <RNA> detection and changed <nucleotide sequences> supported activation of the transferred nuclear <gene>, rather than resistance alone. <Experimental Reconstruction of Functional Gene Transfer from the Tobacco Plastid Genome to the Nucleus by Sandra Stegemann and Ralph Bock>[Figures 3–4, Results, pp. 2872–2874, and “PCR and DNA sequencing”/“TAIL-PCR”, pp. 2876–2877]{ref}
\Image[/endosymbiosis-evidence-gene-transfer.png]
{title=Separate evidence for ancestry, <DNA> transfer and functional activation}
{description=Original evidence map and simplified experimental logic. Boxes summarize questions, readouts and controls; no measured <phylogenetic tree>, <nucleotide sequence>, deletion map or quantitative response is copied. <Organelle protein import> remains a separate question beyond this tobacco experiment.}
This engineered test demonstrates one route to functional nuclear <gene expression> after <plastid> <DNA> transfer. It does not establish a natural transfer rate, replay ancient <endosymbiosis> or demonstrate that the resulting <protein> is imported into a <chloroplast>. The authors explicitly identify acquisition of targeting information as an additional step not reproduced by this experiment. <Experimental Reconstruction of Functional Gene Transfer from the Tobacco Plastid Genome to the Nucleus by Sandra Stegemann and Ralph Bock>[Discussion, p. 2875: antibiotic selection and missing transit-peptide acquisition]{ref}
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