Nicotiana chloroplast genome 7.Expression in E.col...
Summary.RuBPCase.the enzyme responsible for carboxylation and oxidation of RuBP in a wide variety of photosynthetic organisms，is the major protein found in the chloroplast.Here we present the first evidence for direct expression in E.coli and B.subtilis of tobacco and Chlamydomonas ct-DNA sequences coding for the LS of RuBPCase as demonstrated by a simple in situ immunoassay.
Key words：Nicotiana—Chlamydomonas—Chloroplast DNA—RuBPCase—E.coli—B.subtilis
Ribulose-1，5-bisphosphate carboxylase（RuBPCase）（Ec 4.1.1.39），the enzyme responsible for both CO2 fixation and evolution in a wide variety of photosynthetic organisms，is the major protein found in the chloroplast（Kung 1977）.In higher plants it occurs as an oligomer of eight large subunits（LS）（MW 53，000）and eight small subunits（SS）（MW 12，000-15，000）（Jensen and Bahr 1977）.The LS，containing the catalytic sites，are coded by the chloroplast genome（Chan and Wildman 1972）and are synthesized in the chloroplast（Gray and Kekwick 1973）.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（Chua and Schmidt 1978；Highfield and Schmidt 1978）.This enzyme provides an attractive model for studying nucleus-chloroplast cooperation and gene regulation in plants.Genes for the LS of RuBPCase from maize（Coen et al.1977），spinach（Bottomley and Whitfeld 1978；Erion et al.1981）and C.reinhardii（MaInoë et al.1979）have been expressed in vitro in coupled transcription-translation systems，and maize and wheat LS genes have been expressed in vivo in E.coli（Gatenby et al.1981）.Here we present the first evidence for direct expression in E.coli and B.subtilis of chloroplast DNA（ct-DNA）sequences from tobacco and Chlamydomonas coding for the LS of RuBPCase using a simple in situ immunoassay.
Present address*：Department of Cellular and Developmental Biology，Harvard University.16 Divinity Avenue，Cambridge.MA 02138.USA
Y.S.Zhu*，P.S.Lovett，D.M.Williams and S.D.Kung，本文发表于1984年“Theor Appl Genet.67：333-336.”
University of Maryland，Baltimore County，Department of Biological Science.5401 Wilkens Avenue，Catonsville，MD 21228，USA
Received May 20.1983；Accepted July 28，1983
Communicated by D.von Wettstein
A clone library of Nicotiana otophora ct-DNA has been constructed and mapped with respect to Bam HI and Smal sites（Zhu et al.1982）.Unique restriction fragments generated by cleavage of the ct-DNA with Sma I.SalI.Hind Ⅲ or Eco RI contain the intact LS gene suitable for expression studies，whereas Bam HI and PstI cut within the gene.This was determined by using the spinach LS gene as a probe previously inserted into plasmid pBR322（pJEA4）by Erion et al.（1981）.Figure 1A shows the identification of the tobacco LS gene by the hybridization of 32 P-labeled pJEA4 to N.otophora ct-DNA digested with Sma I.SalI，PstI，HindⅢ，EcoRI and BamHI.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 in E.coli using pBR325 and pBR322 as the respective vectors（see Figure 1 B）.Cloned fragments Sal 6（in plasmid PRCZ2）and Hind 2（in plasmid PRCZ1）hybridized with32P-labeled spinach LS gene probe，confirming that both clones contain the LS gene，although these fragments are considerably larger than the LS gene itself（1.3-1.6 kb）.(https://www.daowen.com)
The LS gene from Chlamydomonas reinhardii was used for cloning in Bacillus.This LS gene was previously cloned in plasmid pBR322（pLM401）by L.Metz（personal communication，see Figure 2 A）.Plasmid PRCZ3（see Figure 2 B）was constructed by substituting the 0.7 kb EcoRI-HindⅢ region of the B.subtilis expression plasmid pPL608（Williams et al.1981）with the EcoRI-HindⅢ fragment（1.15 kb and 0.75 kb）spanning the anterior region of the Chlamydomonas LS gene.This substitution is clearly illustrated with the dotted lines in Figure 2 between A and B.Two contiguous Hind Ⅲ fragments（0.75 kb and 1.25 kb）of pLM401（see Figure 2 A），which contain the major posterior region of the LS gene，were inserted into the HindⅢ site of plasmid pPL608，generating pRCZ4.This insertion is also marked with the dotted lines in Figure 2 between A and C.Both recombinant plasmids were transformed into B.subtilis strain IS53 with selection for the neomycin-resistance trait specified by pPL608.
Figure 1A Restriction patterns（left）and hybridization to 32 P-labelled spinach LS gene（right）of N.otophora ct-DNA.10 μg of purified ct-DNA（Rhodes and Kung 1981；Kolodner and Tewari 1975）were digested with SmaI，SalI，PstI HindⅢ.EcoRI and BamHI and subjected to electrophoresis in a 1%agarose gel（channels A→F，respectively）.DNA fragments in the gel were transferred by blotting to nitrocellulose filter paper（Smith and Summers 1980）.The plasmid pJEA4 DNA was prepared according to Lovett and Keggins（1979），and labelled with［α-32P］dATP by the nicktranslation reaction（Maniatis et al.1975）.Hybridization and autoradiography were made according to Southern（1975）.On the right（a-f）with size marker is an autoradiograph of the gel shown on the left，after hybridization whth the spinach LS gene.B Cloning of Hind 2 and Sal 6 restriction fragments of N.otophora ct-DNA in E.coli and hybridization to 32P-labelled spinach LS gene.10 μg of N.otophora ct-DNA was digested with Hind Ⅲ or SalI，and electrophoresed through 0.8%low-gelling-temperature agarose according to Herrmann et al.（1980）.The purified ct-DNA fragment Hind 2 was ligated to Hind Ⅲ-digested pBR322，and fragment Sal 6 was ligated to SalI-digested pBR325 according to Bolivar and Backman（1979）.The hybrid plasmids were transformed into CSR603 or HB101 cells as described by Dagart and Ehrlich（1979）.AmprTcs recombinants were selected and the ct-DNA inserts were analysed.Channels 1，2，3，and 4 contain HindⅢ digests of 1 λDNA；2 pBR322；3 and 4 PRCZl（Hind 2 fragment in pBR322 cloned in HB101 and CSR603 respectively）.Channels 5，6，and 7 contain SalI digests of 5 and 6，PRCZ2（Sal 6 fragment in pBR325 cloned in HB101 and CSR603，respectively）；7 pBR325.Channels 8，9，10 and 11 are autoradiograms demonstrating the hybridization of 32 P-labelled spinach LS gene to gel channels 3，4，5 and 6
Figure 2A-C Cloning of the C.reinhardii LS gene in B.subtilis.A C.reinhardii ct-DNA fragment（3.2 kb）was inserted into EcoRI and BamHI sites of pBR322 and cloned in HB101 as recombinant plasmid pLM401 by L.Metz.It contains the LS gene as marked by the arrow indicating the direction of transcription according to Metz's data.The central region of this gene is located in a 0.75 kb HindⅢ fragment.The 5'and 3'ends are located within the 1.15 kb EcoRIHind Ⅲ fragment and 1.25 kb Hind Ⅲ fragment respectively.B Construction of PRCZ3：10 μg of pPL608 DNA was digested with Hind Ⅲ for 2 h at 37℃，then partially digested with EcoRI for 5 min at room temperature.A plasmid fragment containing the 4.3 kb EcoRI-Hind Ⅲ region plus the 0.25 kb EcoRI promoter fragment as marked by the split bar，was separated and isolated by low-gelling-temperature agarose gel electrophoresis.The anterior portion of the Chlamydomonas LS gene（1.15 kb and 0.75 kb）in Figure 2 A was obtained by partial Hind Ⅲ digestion of the EcoRI linear form of PLM401.The deleted form of pPL608 and anterior portion of the Chlamydomonas LS gene were ligated and introduced into B.subtilis IS53 as described by Lovett and Keggins（1979）.C Construction of PRCZ4：Plasmids PLM401 and pPL608 were digested with Hind Ⅲ and ligated，and transformed into B.subtilis IS53.Neon，Cmsrecombinants were selected and the inserts were analysed.A clone PRCZ4 containing 0.75 kb and 1.25 kb Hind Ⅲfragments was obtained
For detection of the LS gene product a simple in situ immunoassay was employed（Anderson et al.1980）.The clones CSR603（PRCZ1）and CSR603（PRCZ2）containing 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 cells in the plates were lysed with chloroform vapor and lysozyme.A channel was made in the center of the plate into which was added antiserum against the LS of RuBPCase.Incubation was continued for another day at room temperature.A sharp immunoprecipitation line was formed between CSR603（PRCZ1）and the central channel demonstrating that this clone has produced the LS polypeptide（see Figure 3 A）.B.subtilis cells carrying PRCZ3 and PRCZ4 and control cells containing the vector pPL608 were similarly cultured on N-Z bottom agarose plate，lysed with chloroform vapor and lysozyme，and overlayered with molten agarose mixed with the antiserum against LS.Incubation continued for one week at 4℃.Colonies harboring both recombinant plasmids showed an immunoprecipitation ring.whereas the control cells exhibited no immunoreaction（data not shown）.This result was confirmed by immunoprecipitation of extracts of these clones with antiserum against LS in an Ouchterlony double diffusion test（Figure 3B）.Figure 3 B shows expression in B.subtilis of anterior portion of the LS gene from Chlamydomonas and in E.coli of the LS genes from spinach，tobacco，and Chlamydomonas.As it is expected，the smaller incomplete polypeptide made by the anterior portion of the LS gene from Chlamydomonas（well 6）migrated faster as compared to the complete LS polypeptide made by the entire LS genes from spinach，tobacco，and Chlamydomonas（wells 1-5）in E.coli.The sizes of the complete LS of tobacco and spinach are similar（52-53，00 daltons，Erion et al.1981）.Based on the intensity of immunoprecipitation line of a standard，the amount of LS produced in each cell was approximated to be 0.1 pg.
Figure 3A Detection of the N.otophora LS polypeptide synthesis in E.coli by in situ immunoprecipitation reaction.Clones containing different ct-DNA inserts were cultured on N-Z bottom agarose plate containing ampicillin（20 μg/ml）in a 50 mm petri dish.After lysis and washing，a channel was made in the center of the plate and 100 μ1 of an antiserum against LS was loaded.Overnight incubation at room temperature allowed development of a zone of immunoprecipitation（marked by arrow）around CSR603（PRCZ1）（a），whereas control cells（b）containing plasmid pBR322 exhibited no reaction.B Detection of the synthesis of LS polypeptide of N.otophora，N.tabacum，spinach and C.reinhardii in E.coli or B.subtilis by Ouchterlony double diffusion.Five ml of cultures were grown in L broth at 37℃for 4 h.Cell pellets were washed with TES buffer（Lovett and Keggins 1979）and lysed by boiling in 0.1 ml of 2%SDS for 2 min.After removal of free SDS by acetone treatment，twenty μl of lysates were added to the outer holes.Twenty μl of antiserum against LS was loaded into the central hole.After incubation for one day at room temperature，the immunoprecipitates were observed in all clones containing LS gene except in control cells.
1.PRCZ1：Hind2（N.otophora）/pBR322/CSR603
2.PRCZ2：Sal 6（N.otophora）/pBR325/CSR603
3.pLM401：Chlamydomonas LS gene/pBR322/HB101
4.pJEA4：Spinach LS gene/pBR322/HB101
5.pHPE4：Eco R4（N.tabacum BR325/HB101
6.PRCZ3：anterior Chlamyclomonas LS gene/pPL608/IS53
The results presented here demonstrate direct expression in E.coli and B.subtilis of the LS genes from tobacco and Chlamydomonas.Previous studies demonstrated that expression of the maize LS gene in E.coli was initiated within its own promoter（Gatenby 1981）.Our results also indicate that the expression in E.coli of tobacco LS gene was directed by its promoter，since the LS gene and its promoter are located in the middle of the Hind 2 fragment（11 kb）.Furthermore，this promoter is able to initiate the expression in E.coli of the galactokinase gene on the promoter-less plasmid pK01（C.M.Lin and X.F.Kong，unpublished data）.
This is the first demonstration of expression of a plant gene in Bacillus.Hind Ⅲfragments（0.75 and 1.25 kb）cloned in PRCZ4 represent a large posterior part of the LS gene of C.reinhardii presumably lacking its own promoter.Its expression in Bacillus.which is stimulated by chloramphenicol（0.1 μg/ml）（data not shown），is apparently initiated by a strong promoter located whthin the 0.25 kb EcoRI fragment in pPL608（Williams et al.1981）（Figure 2C）.The unique HindⅢ site in pPL608 is located within a structural gene specifying a chloramphenicol-inducible，chloramphenicol acetyltransferase（CAT）.Based on the data of Williams et al.（1981）it is expected that the LS polypeptide in PRCZ4 is synthesized as a hybrid with the amino terminal portion of chloramphenicol acetyltransferase.Transcription of the LS gene in the clone PRCZ3，which contains the anterior portion of the LS gene（Figure 2B）is either initiated within its own promoter or within the plasmid promoter.
Acknowledgements.The authors are grateful to Drs.L.Metz and H.Weissbach for the generous gifts of their clones pLM401 and pJEA4 respectively.This investigation was supported by NIH grant CM22746-01，U.S.Dept of Agriculture cooperative agreement 58-3204-0-157 from the tobacco laboratory and NSF grants PCM 78-05755 and PCM 82-02701.
