Materials and Methods

Plant and Bacterial Strains.N.otophora was grown in a greenhouse for 2—3 months.The leaves were harvested for preparation of ct-DNA.Escherichia coli strains HB101 and CSR603（recAl，uvrA6，phr-1）were used as the host in transformation experiments.Plasmid pBR322 was cloning vector.

Isolation of Chloroplast and Plasmid DNA.N.otophora ct-DNA was isolated as described previously（Rhodes and Kung，1981）.Plasmid DNA was isolated and purified by centrifugation in CsCl/ethidium bromide equilibrium gradients（Kolodner and Tewari，1975）.

Cloning of ct-DNA in E.coli.The“shotgun”approach was employed in this study.10 μg of N.otophora ct-DNA and 2 μg of pBR322 DNA were digested with BamHI，then ligated according to the procedure of Bolivar and Backman（1979）.The total ligation mixture（50 μl）was used to transform 0.1 ml of competent HB101 or CSR603 cells（Pagert and Ehrlich，1979）.The ampicillin resistant transformants were screened for tetracycline sensitivity.Tetracycline sensitive transformants were lysed and plasmid DNA was partially purified.After digestion with BamHI，each was analyzed for their ct-DNA inserts on 1%horizontal agarose slab gel using BamHI digests of N.otophora ct-DNA and pBR322 DNA as markers.

Cloning of Specific ct-DNA Fragments in E.coli.N.otophora ct-DNA was digested with BamHI and electrophoresed on 0.8%low-gelling-temperature-agarose according to the method of Herrman et al.（1980）.Specific fragments of interest were cut from the gel，melted at 65℃，and isolated from agarose by phenol extraction and ethanol precipitation.The purified ct-DNA fragment was ligated to plasmid pBR322 pretreated with BamHI，then used to transform 0.1 ml of competent HB101 cells.(https://www.daowen.com)

Isolation of Probes for r-RNA and RuBPCase LS Genes.EcoRI-generated fragment 4（E4），and 8（E8）and 29（E29）from N.tabacum ct-DNA were isolated from low-gellingtemperature-agarose gel.The fragments E8，E4 and E29 corresponding to 16S，23S and SS r-RNA genes respectively（Sugiura and Kusuda，1979；Fluhr and Edelman，1981）were used as probes for r-RNA genes.Spinach RuBPCase LS gene probe was isolated from the clone PJEA 4 digested with BamHI and Aval（Erion et al.，1981；Weissbach，personal communication）.Then，this 2.0 kb ct-DNA fragment released from chimeric plasmid pBR322 after digestion contains whole RuBPCase LS gene.

Nick Translation and Hybridization.ct-DNA fragments for probing r-RNA and RuBPCase LS genes were labelled with［α-32 P］dATP by the nick-translation reaction（Maniatis et al.，1975）and hybridized to BamHI or SmaI fragments of N.otophora ct-DNA blotted on nitrocellulose paper（Southern，1975）.The localization of genes for 16S，23S，and 5S r-RNA and RuBPCase LS on the N.otophora ct-DNA was determined by autoradiography.

In vivo Protein Synthesis.All the BamHI fragments in cloned HB101 were subcloned in maxicell CSR 603.These subclones were individually examined for their ability to direct the synthesis of new polypeptides in CSR 603.After irradiation with UV light（0.5 JM-2S-1）the cells were fed with 35S-methionine.The newly synthesized polypeptides were identified by SDS polyacrylamide gel followed by autoradiography（Sancar et al.，1979）.