A physical map of Nicotiana tabacum plastid DNA including the location of structural genes for ribosomal RNAs and the large subunit of ribulose bisphosphate carboxylase/oxygenase

Abstract

1) Tobacco plastids contain a homogeneous population of double-stranded circular DNA molecules, 101 Megadalton (160 kbp) in circumference. In neutral CsCl equilibrium gradients, this DNA displays a density of 1.697 g · cm(-3) which is equivalent to an average base composition of 37.7 mole-% G+C. 2) A restriction endonuclease fragment map of the tobacco plastid chromosome is presented for the enzymes Bgl I, Sal I, Xho I and Pvu II which together dissect the DNA molecule into about 60 fragments. The map was derived by sequential digestion employing the previously described Seaplaque technique. The tobacco plastid chromosome has an anatomy similar to that of many other higher plants; the circular DNA is segmentally organized into two unique sequence segments of approximately 24 and 95 kbp separated on each side by a large inverted duplication of at least 20.4 kbp. 3) Saturation and blot hybridization showed that the genes for the 16S and 23S pt-rRNAs are duplicated. Each copy of the inverted repeat contains one set of rRNA genes; about 26 kbp (short distance) separate the sets from each other. 4) Cloned fragments of spinach ptDNA nick translated to high specific activity in vitro were used to probe the location of the large subunit gene of ribulose bisphosphate carboxylase/oxygenase on tobacco ptDNA. A 3.5 kbp-long fragment of the large single-copy region of the tobacco chromosome is complementary to structural sequences of the spinach gene. 5) Mapping and hybridization data suggest that the tobacco and spinach ptDNAs share striking similarities in anatomy and sequence.