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In his account of the 1973 work, Stanley Norman Cohen describes purified R6-5 plasmid DNA cut with EcoRI, reassociated and joined by DNA ligase. The mixture entered calcium chloride-treated Escherichia coli; plates containing antibiotics selected biological colonies showing antibiotic resistance. The recovered pSC102 carried three R6-5 fragments and kanamycin antibiotic resistance. The team then joined material from pSC102 to the carrier pSC101 providing tetracycline antibiotic resistance, recovering pSC105 with both forms of antibiotic resistance.^[ref]

Stained agarose gel electrophoresis, gradient centrifugation and electron microscope comparisons characterized recovered plasmids. Parental and mixture lanes in Figure 3 supplied fragment comparisons; pSC101 alone gave one EcoRI fragment, while pSC105 combined its carrier fragment with the resistance-bearing fragment. This supported joined, propagating genetic material beyond selection alone. The comparisons did not determine every nucleotide sequence or establish a universal construction efficiency.^[ref]

The interesting inference combines two kinds of evidence: a recoverable phenotype identifies descendants worth examining, and the extracted DNA tests a structural explanation. The fragment comparisons are molecular scientific controls; they answer a different objection from a control for background growth. The hypothetical diagrams here illustrate that logic without reproducing the historical fragment lengths or biological colony counts.

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. ^[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. ^[ref]

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. ^[ref]