NICOTIANA TABACUM CHLOROPLAST DNA, STRUCTURE AND GENE CONTENT

Abstract

The physical structure of the chloroplast DNA of Nicotiana tabacum has been characterized. This chloroplast DNA like other chloroplast DNAs, can be isolated as covalently closed circular molecules on CsCl-ethidium bromide gradients. Electron microscopy was used to measure the contour lengths of nicked circular chloroplast DNA molecules. N. tabacum chloroplast DNA is 28.8 times the size of phi X174. This measurement agrees reasonably well with the 96 megadalton molecular weight obtained by restriction enzyme analysis. Digestion with Sal I restriction enzyme produces 10 fragments each of which are unique in molecular weight and range in size from 1.8 to 17.0 megadaltons. A map of these sites relative to the location of the 18 Sma I fragments has been constructed. A technique utilizing the separation of double digests in two dimensions is the primary source of mapping data. This technique has also assigned the location of most of the Xba I sites. Restriction mapping and hybridization experiments have revealed that not all of this 45 um circle is unique DNA. Approximately 15 megadaltons is present in two copies. These duplicated segments contain the genes for ribosomal RNAs and are arranged in an inverted orientation to each other. The mapping of 16S and 23S rRNA was accomplished by utilizing this restriction map and the Southern hybridization technique. The results described indicate that there may be a non-coding interruption (an intron) in the 23S gene. Further investigation is needed to confirm this conclusion. Hybridization of 125-iodine labeled 16S and 23S rRNA to various restriction enzyme digests has allowed mapping of all of the Kpn I, Xba I, Bam HI and Eco RI fragments which contain DNA sequences complimentary to these rRNAs. These data, combined with the Sal I, Sma I, Xba I restriction map, produces a total of 70 sites whose locations on chloroplast DNA have been determined. The coding capacity of the N. tabacum chloroplast DNA genome has been estimated with the utilization of a new technique for assaying gene numbers. This technique, called the ribosome binding method, utilizes the recently discovered phenomenon that single stranded DNA will substitute for messenger RNA in the in vitro formation of initiation complexes. Results of experiments in which the ribosome binding sites of denatured chloroplast DNA have been saturated indicate that the N. tabacum chloroplast DNA may contain the coding sequences for as many as 200 distinct polypeptides. Ribosome DNA complexes visualized by electron microscopy produce structures which contain an average of approximately 2 ribosomes per 10 megadaltons. In conclusion the experiments and characterizations described reveal that N. tabacum chloroplast DNA has many features which are common to several higher plant chloroplast DNAs.