B.Expression of ct-DNA in E.coli and B.subtilis
We are interested in the expression of developmentally and environmentally regulated chloroplast genes in Nicotiana.As a first step in approaching this goal，we took advantage of the recombinant DNA techniques which make possible the expression of eukaryotic genes in bacteria.Efficient expression in bacteria of a non-bacterial gene requires the assistance of a native bacterial promoter and ribosomal binding sites.Since chloroplasts have procaryotic features，ct-DNA is assumed to be easily expressed in bacteria.In order to study the transcription of ct-DNA in bacteria a maxicell（CSR603）technique was employed.All the plasmids from the gene library containing ct-DNA inserts were subcloned in E.coli CSR603.Irradiation with UV destroys chromosomal DNA，whereas plasmids can survive.Therefore，whenever 35S-methionine is applied to irradiated cells，the plasmid-encoded polypeptides can be detected.Though a few chloroplast genes（in Bam 7，12 and 16）were expressed in the maxicells（as demonstrated by synthesizing 43，29 and 13.5 kb polypeptides），90%of the ct-DNA genome was not transcribed and translated［15］.The failure of most chloroplast genes to be expressed may be due to misrecognition of promoters and ribosome binding sites，or expression that is not strong enough to be detected by this method.In addition，some genes may be split by BamHI.One example is the gene for the LS of RUBPCase which is cleaved by BamHi to Bam 20 and Bam 24.
We are especially interested in the RuBPCase as it is the major protein in chloroplasts and a key enzyme in photosynthesis.In Nicotiana，as in other higher plants，it occurs as an oligomer of eight large subunits（LS）（MW 55，000）and eight small subunits（SS）（MW 15，000）.The LS，containing the catalytic sites，are coded by the chloroplast genome and are synthesized in the chloroplast.The SS，whose function remains uncertain，are coded by the nuclear genome and synthesized in the cytoplasm as a precursor of higher molecular weight（MW 20，000）that is cleaved and transported into the chloroplast.RuBPCase was first crystallized from N.tabacum，providing an attractive model for studying nucleus-chloroplast cooperation and gene regulation in plants［23］.
Although BamHI，as well as pstl，cuts within the LS gene，the unique restriction fragments generated by cleavage of ct-DNA with Smal，SaIl，Hindlll or EcoRI contain intact LS genes necessary for expression studies，as demonstrated by hybrid zation using the spinach LS gene as a probe［24］.The fragments Sal 6（14 kb）and Hind 2（11 kb）containing the intact LS gene，were isolated from total N.otophora ct-DNA and cloned into E.coli using pBR325 and pBR322 as the respective vectors.Cloned fragments Sal 6（in plas mid PRC22）and Hind 2（in plasmid PRCZI）were shown by hybridization to contain the LS gene.(https://www.daowen.com)
For detection of the LS gene product a simple in situ immunoassay was employed.The clones CSR603（PRCZI）and CSR603（PRC22），contaning the N.otophora LS gene，were inoculated and incubated on a N-Z bottom agarose plate overnight at 37℃.CSR603 cells containing pBR322 and pBR325 were used as controls.After incubation，the colonies were lysed on the plate with chloroform vapor and lysozyme.A channal was made in the center of the plate into which was added antiserum against the LS of RuBPCase.After incubaton for 1 day a sharp immunoprecipitation line was formed between CSR603（PRCZI）and the central channel，demonstrating that this clone had produced the LS polypeptide.This result was confirmed by immunoprecipitation of extracts of these clones with antiserum against LS in an Ouchterlony double diffusion test.The LS gene from spinach，N.tabacum，and Chlamydomonas were also expressed in E.coli as shown by this method［25］.
We provided the first demonstration of expression of Chlamydomonas LS gene in B.subtilis.This LS gene was previously cloned in pBR322（pLM 401）by L.Mets（personal communication）.Either the anterior or posterior portions of the LS genes were cloned into B.subtilis using pPL608 as the vector.Both portions were expressed in B.subtilis as demonstrated by in situ immunoassay and Ouchterlony assay［21］.
