Gene, 4 (1978) 25--36 25 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
AMPLIFICATION OF THE RESPIRATORY NADH DEHYDROGENASE OF £scherichia coli BY GENE CLONING (Restriction endonucleases; EcoRI; HindIII; BamI: NADH-ubiquinone oxidoreductase; amplifiable plasmids; membrane proteins) I'G. YOUNG, A. JAWOROWSKI and M.I. POULIS Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra, A.C. T. 2601 (Australia) (Received April 10th, 1978) (Accepted May 22nd, 1978)
SUMMARY
A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Esvherichiacolifrom unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI an(;~HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F wasused to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane, Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane t o b e i n c r e ~ d t o 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior t o their purification. INTRODUCTION
The ability to isolate and amplify specific genes by cloning onto amplifiable plasmids has many useful applications, The hybrid plasmids generated can serve asc0nvenient sources of large amounts of specific DNA for use in DNA sequencing and studies on the r e l a t i o n of gene expression. In addition, beAbbreviations: Apr, ampicillin resistance; Tcr, tetracycline resistance.
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cause of their high copy number they can also be u~;ed to amplify particular proteins in vivo. In the case of bacteria such as E. coli where a wide variety of mutants are available to enable direct selection of hybrid plasmids by complementation, gene cloning is a potentially valuable first step in the isolation of any protein or enzyme. Hybrid plasmids derived from ColE1 are maintained at 10--20 copies per chromosome and strains carrying hybrid plasmids may overproduce enzymes by up to ].5-fold (Hershfield et al., ].974; Vapnek eta]., 1976; Wickner et al., ].976; Raetz et al., ].977; Steffen and Scldeif, ].977). Most studies to date have been with soluble enz3mnes, where the level of overproduction is probably largely determined by the regulation of the gene concerned. In the case of membrane proteins additional constraints may apply. For some membrane proteins the number of available sites for insertion may be limited or they may need to be assembled with the other proteins of a complex before insertion into the membrane. Cloning is most conveniently carried out if the particular genes of interest have already been enriched, for exmaple by the preparation of a specialized transducing phage. At the present time since many genes are not yet available in an enriched form, methods which allow cloning of genes from complex sources of DNA such as bacterial chromosomes are particularly useful (Clarke and Carbon, 1976; Borck et al., 1976; Collins et al., ].976; Kozlow et al., 1977). In the present work a relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase* from total E. coil DNA. Cells carrying the hybrid plasmid were shown to produce 8--].0 fold elevated levels of the enzyme in their cytoplasmic membrane. The enzyme levels were further increased to 5 0 - 6 0 times normal by increasing the plasmid copy number with chloramphenicol. MATERIALS AND METHODS
Media, chemicals, enzymes The mineral salts medium used and the concentration of supplements have been de~Lcribed previously (Stroobant et al, 1972). The complete medium was brain-heart infusion (Oxoid). The chemicals and enzymes used were obtained from the following sources: ethidium bromide, agarose, lysozyme, Sigma Chemical Co. (St. Louis, MO, USA); CsCI, BDH Chemicals Ltd. (Poole, England); EcoRI, BamI, HindIII restriction endonucleases, Miles Research Products (Indiana, USA); and T4 DNA ligase, New England Biolabs (Beverly,
*We are using the term resp~atory NADH dehydrogenase to refer to the enzyme which chain c a t a l yof~~~ - coU_Wehaemeasured ' u n s f e r ° v f reducingequiv'~lents t h e e n z m efr°m i n telNADHt° ,f il ubiquin°ne in the respiratory ,i i. Yi r m o "~ NADH,ubiquinone 0xidoreauctas~ activity, This enzyme is absent from ndh mutants (Young and ~ l a c e , 1976) which are deficient in NADH oxidase and is distinct from the NADH-ferricyanide oxidoreductase described by Dancey et al. (197 6).
27 MA, USA). Ubiquinone-1 was the generous gift of Hoffmann LaRoche Co. (Basle, Switzerland). The NADH dehydrogenase used as a marker for SDS gel electrophoresis was a highly purified preparation prepared as described elsewhere (Jaworowski and Young, manuscript in preparation).
Bacterial strains and plasmids Strains canting the plasmids pSF2124 (So et al., 1975) and pGM706 (Hamer and Thomas, 1976) were kindly supplied by J. Langridge. Strain C600rk-mk ÷ was kindly supplied by D.W. Ribbons. Strain AN595 (thi his ilv trp rpsL)(Young and Wallace, 1976) was used as the source of E. coil DNA. The other other strains used were derived from IY12 (thi his ilv trp rpsL ndh) or its isogenic ndh* transductant IY13 (thi his i~v trp rpsL). Strain IY34 (derived from IY13) carries pSF2124 and IY35 (derived from IY12) carries the hybrid plasmid pIY1 which was constructed using EcoRI and possesses a 1.6 Mdal DNA fragment. Isolation of DNA Chromosomal DNA from E. coli was prepared as described by Marmur (1961). For the isolation of plasmid DNA 30 ml cultures were growr~ in glucose--mineral salts medium containing 0.5% casamino acids and the plasmid DNA amplified using chloramphenicol (Clewell, 1972). The cells were harvested and a cleared lysate prepared according to the method of Clewell and Helinski (1969) except that 0.3% w/v Triton X-100 was used instead of brij and deoxycholate. The DNA was purified using a CsCl-ethidium bromide density gradient (Clewell, 1972), the ethidium bromide removed by isopropanol extraction and the DNA dialyzed overnight against buffer (2 mM Tris HCI, 2 mM EDTA, 1 mM NaCI, pH 8.0). Restriction and ligation The following restriction buffers were used: EcoRI, 100 mM Tris-HCI, 50 mM NaCI, 10 mM MgCI2, pH 7.5; BamI, 6 mM Tris. HCI, 5 mM NaC1, 6 mM MgCI2, 0.1 mg/ml gelatin, pH 7.4; HindIII, 7 mM Tris-HCI, 60 mM NaC1, 7 mM MgCI2, pH 7.4. The ligase buffer used was 5 mM Tris-HCI, 5 mM MgCI2, 10 mM DTT, 50/~g/ml gelatin, 66/~M ATP, pH 8.0. Preparation of hybrid plasmid pools The chromosomal and plasmid DNAs were cleaved at 37 ° C with either EcoRI, SamI or HindIII and the restriction enzymes inactivated by heating for 10 rain at 65 ° C. pSF2124 was digested with EcoRI and BamI and pGM706 with HindIII. The progress of the endonuclease digestions were followed using agarose gel electrophoresis and the incubations were continued until digestion appeared complete. Digested chromosomal DNA (9 ~g) from AN595 (thi his ilv trp rpsL) was ligated for 15 h at 11 ° C with digested plasmid DNA (3/~g) using T4 ligase in a total volume of 175/d. The ligated DNA (10 ~g) was transformed into approx. 2-101° cells of an F ÷ derivative of c e 0 0 r k - m k ÷ using the
28
method of Lederberg and Cohen (1974), The transformec~ cells were diluted 1/10 in complete medium and grown overnight at 37 ° C. ?~ds culture was then diluted 1 / 5 i n fresh medium containing 25,/zg/ml ampicillin and the culture incubated a further 1 5 h a t 37 ° C to select for Ap r. The cells were concentrated and stored at 4° C for short periods, prior to use. For long-term storage, the cells were lyophilized.
Transfer of hybrid plasmids The celts containing the hybrid plasmids and the recipient IY12 (F-, ndh rpsL) were grown to early logarithmic phase at 37 ° C in complete medium. Equa ~,volumes of the two cultures were mixed and the mating allowed to proceed for I h at 37 ° C with slow shaking to allow transfer of the hybrid plasmids by F-mobilization. The cells were then washed once and selection made for complementation of ndh (i.e. growth on mannitol (Young and Wallace, 1976)) and resistance t o ampicillin (25 #g/ml) andstreptomycin (100
Containment levels The work described was carried out under conditions corresponding to P1 containment levels of the N.I.H. guidelines (1976). ,
Eleetrophorasis of DNA
~
Electrophoresis was carried out on 3 mm thick 0.8% agarose gels containi~g 0. 5 ~g/ml ethidium bromide using a horizontal slab gel apparatus. The buffer used was 40 mM Tris- HCI, 5 mM sodium acetate, I mM EDTA: pH adjusted to 7.8 with glacial acetic acid (Sharp et al., 1973); gels were run at room temperature at either 150 mA constant current for about 3 h or 15 mA overnight. DNA samples were diluted with an equal volume of dye mixture (14.5% sucrose, 0.045% bromophenol blue, 50 mM EDTA, pH 7.8) prior to loading. Gels were examined under UV light using a ~ s i l l u m i n a t o r (Ultraviolet Products, San Gabriel, Calif.) and photographed using a Polaroid type 55 P/N film through a red filter.
Measurement of culture turbidity Culture turbidities were measured using a Klett--Summerson colorimeter fitted with a blue filter and are expressed i~,Klett units.
Amplification of the NADH dehydrogenase Strain IY35 which possesses the hybrid plasmid pIY1 carrying ndh was used for these experiments. Amplification was achieved by using two different treatments designed to increase the plasmid copy numberand thenallowing the treated cells to grow briefly soas t o aliow expression o f the ndh genes on the plasmids. For amplification by amino acid starvation, 10 litre cultures of IY35 were grown in supplemented mineral salts medium containing limiting isoleucine
29
and valine (both 0.1 raM). This concentration of isoleucine/valine limits growth at a Klett of 100 (mid log phase) and the cessation of growth is sharply defined. The cultures were incubated under conditions of amino acid starvation for 1, 2 and 4 h, respectively, at 37°C with aeration and stirring. After the required time cell growth was restarted by the addition of 0.3 mM isoleucine and valine. Cells were allowed to grow for 1 generation and then harvested. For chloramphenicol amplification, 10 litre cultures were grown in sdpplemented mannitol-mineral salts medium containing 0.1% casamino sacids at 37 ° C. Chloram~henico! w,~ . . . . .~. .d. ~c _~.,.~' coi~eent~ion of 50/~g/ml when the Klett had reached 100. After 4 to 9 h incubation with aeration and stirring the cells were harvested and washed with a small volume of sterile minimal medium, then used to reinoculate a further 10 litres of growth medium. Cells were grown at 37 ° C with aeration and stirring and harvested after one generation.
Preparation of membranes and enzyme assays The preparation of membranes and the determination of oxidase ~'ates have been described previously (Wallace and Young, 1977) except that 0.8 mM NADH and 20 mM D-lactate were used for oxidase measurements. NADH dehydrogenase activity of membrane preparations was estimated at 30 ° C by measuring the ubiquinone dependent oxidation of NADH at 340 nnfi in ~ 1 ~ ! reaction mixture containing 20 mM N-Tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid, 120/~M NADH, 50/JM ubiquinone-1, 3 mM KCN and 40 ~M FAD.
8DS-polyacry lamide gel elec~ophoresis SDS-polyacrylamide gel e]ectrophoresis was performed on vertical slab gels (10 × 14 cm) of 10% polyacrylamide with a stacking gel of 4.5% polyacrylamide using an apparatus similar to that described by Studier (1973). The discontinuous SDS buffer system of Laemmli (1970) was used. Gels were run at a constant current of 30 mA for 3--4 h at room temperature. Membranes and purified enzyme samples were heated at 100 ° C for 3 min in SDS sample buffer (Laemmli, 1970). Gels were fixed at 65 ° C for 20 rain in 10% trichloracetic acid and stained with Coomassie Brilliant Blue R250 (0.1% w/v in destainer) for 20 min at 65 ° C then destained at room temperature using a mixture of ethanol/glacial acetic/water (25:8:67 v/v/v). RESULTS
Mutant strains of K coli have been described which are affected in the NADH dehydrogenase complex of the respiratory chain (Young and Wallace, 1976). These mutants (designated ndh) appear to be affected in the structural gene or genes coding for the NADH dehydrogenase. We wished to clone the ndh genes both to provide a highly enriched source of the ndh genes for further studies in vitro and to amplify the enzyme levels so as to allow convenient purification.
30
In preliminary experiments it was found that ndh strains gave much lower frequencies of transformation with ColE1 plasmids, than did standard strains such as C600rk-mk*. In order to avoid having to transfer ndh into C600 to try and improve the frequency of transformation, it was decided to use Fmobilization for the transfer of hybrid plasmids to ndh recipients. The smaller cloning vectors such as pMB9, pBR313 and pBR322 are not mobilized at high frequency by F (Bolivar et al., 1977; I.G. Young, unpublished data) but experiments showed that pSF2124 (ColE1-Apr) and pGM706 (ColE1-AprTcr) were both efficiently mobilized by F. After I h matings approx. 5% and 16% of recipients had received the plasmids pGM706 and pSF2124 respectively. pSF2124 has single restriction sites for EcoRI and BamI and pGM706 for HindIH and SaII. Thus several different restriction enzymes could be used for cloning to try and overcome the problem that the gene to be cloned may carry sites of restriction for particular enzymes. Both pSF2124 and pGM706 carry Apr which allows convenient selection of cells carrying the plasmids. Apr is not inactivated by insertion of DNA fragments at the various restriction sites. Cloning of the ndh gene Whole chromosomal DNA from E. coli was digested separately with the restriction endonucleases EcoRI, BamI and HindIII to produce three pools of DNA fragments with complementary single stranded ends. pSF2124 DNA was also digested with EcoRI and separately with BamI, while pGM706 DNA was cleaved with HindIII. The corresponding chromosomal and plasmid DNA digests were ligated with T4 ligase, the ligated DNA transformed into an F ÷ derivative of C600 rk-mk ÷ and selection made for Ap r in liquid medium. Hybrid plasmids complementing ndh were selected from the pools by Fmediated transfer to an ndh recipient selecting for both Ap r and complementation of ndh. The hybrid plasmids pIYl(derived from pSF2124) and pIY2 (derived from pGM706) were isolated from the EcoRI and HindIII hybrid plasmid pools respectively. The plasmid DNA was amplified using chloramphenicol: purified on CsCl-ethidium bromide density gradients and used to transform ndh mutants so as to verify complementation of ndh by the hybrid plasmids. The hybrid plasmid DNAs were also examined on agarose gels before and after cleavage with the appropriate restriction endonucleases (Fig.l). The two hybrid plasmids both showed a faster migrating band corresponding to covalently closed plasmid DNA and a slower migrating open circular DNA band. After digestion with EcoRI and HindIII respectively, pIY1 and pIY2 each gave two bands, the higher molecular weight band in each case being the parental plasmid (linear form) and the lower molecular weight band corresponding to the cloned DNA. By comparison with molecular weight standards (), DNA digested with EcoRI or HindIII) the cloned fragments in pIY1 and pIY2 were estimated to be 1.6 and 4.6 Mdal respectively.
"
a
31
b
c:
d
. L-..,,,~
I
I I
Fig.1. Electrophoresis of plasmid DNAs on 0.8% agarose gels. Samples: (a) pIY1; (b) pIY1 digested with EeoRI; (c) pIY2; (d) pIY2 digested with HindIII. About 1 ~g DNA was loaded except for sample (b) where 3 . g was loaded so that the 1.6 Mdal restriction fragment could be readily seen. The two bands present in (a) and (c) correspond to the covalently closed and open circular forms of the respective hybrid plasmid DNAs. The open circular bands are in each case closer to the origin.
Amplification or" the respiratory NADH dehydrogenase Since the EcoRI-derived plasmid pIY1 carried the smaller DNA fragment (1.6 Mdal) it was used for experiments on the amplification of enzyme levels. Cells containing the hybrid plasmid were found to possess 8--10 times the normal level of the NADH dehydrogenase (measured as NADI-I-ubiquinone oxidoreductase) in their membranes (Table I). Two different methods were used to attempt to further increase the levels of NADH dehydrogenase by increasing the plasmid copy number. It has been shown that when cellular protein synthesis is prevented by amino acid starvation or by treatment with chloramphenicol, chromosomal DNA synthesis ceases but ColE1 and its derivatives continue to replicate (Clewell, 1972).
32
TABLE I AMPLIFICATIONOF THE NADH DEHYDROGENASE •
Strain
Plasmid
Treatmenta
NADH dehy -b drogenmm
NADH o x i -b dase
IY12 IY13 IY34 IY35 rY35
--pSF2124 pIY1 pIY1
IY35
pIY1
---a m i n o acid starvation I h a m i n o acid starvation 2 h a m i n o a c i d Starvation 4 h chloramphenicol 4 h chloramphenicol 9 h
0.03 0.52 0.63 4.91 7.82 9.26 9.50 30.6 31.1
0.02 0.49 0.44 2.14 3.24 3.30 3.05 4.69 3.23
aUnless otherwise stated, cells were harvested in late log phase; procedures for amino acid starvation and treatment with chloramphenicol are described in methods. bActivities expressed as ~moles substrate oxidized/min/mg membrane protein. When cells carrying pIY1 were starved of amino acids for up to 4 h and then allowed to grow for I generation in complete medium the NADH dehydrogenase levels were increased to 1 5 - 1 8 times normal (Table I). A potentially more effective way of increasing plasmid copy number is to use chloramphenico~., but for the purpose of amplification of enzyme levels it was essential to establish conditions such that the inhibition of protein synthesis by chloramphenicol could be subsequently reversed. We wished to establish conditions whereby cells could be treated with chloramphenicol so as to increase plasmid copy number, then the chloramphenicol removed to allow the cells to grow again and express the ndh gene on the hybrid plasmid. It was found that the chloramphenicol concentration could be reduced to 50 #g/ml and still inhibit growth as effectively as higher concentrations. Cells were grown to mid log phase, incubated with chloramphenico: (50 #g/ml) for different lengths o f time then washed free of antibiotic and tested for their ability to grow normally in fresh medium. After treatment with chloramphenicol for 4 - 9 h the cells could grow again without excessive lag suggesting that protein synthesis was being effectively recovered in the majority of cells. Treatment with chloramphenicol for 4 - 9 h followed by growth for I generation in its absence resulted in the elevation of enzyme levels in the membrane to 5 0 - 6 0 fold normal (Table I). The amplification of the NADH dehydrogenase also led to an increase in the NADH oxidase levels (Table I) but appeared -to be quite specific in that the levels of succinate of D-lactate oxidase were unaffected(data not shown). Assay of the soluble fraction for NADH-ubiquinone oxidoreductase showed that no increase in activity had occurred (data n o t shown)suggesting that either all of the NADH dehydrogenase produced was present in the membrane fraction or any enzyme not incorporated into the membrane was inactive.
33
The respiratory NADH dehydrogenase has been solubilized from the membranes containing the highly elevated levels of the enzyme and purified to homogeneity (Jaworowski and Young, manuscript in preparation). Oil SDSpolyacrylamide gels it consists of a single polypeptide of molecular weight approx. 45 000. SDS-polyacrylamide gels of membranes from chloramphenicolamplified cells containing the hybrid plasmid pIY1 showed that the level of one polypeptide from the membrane fraction is dramatically increased (Fig.2). This polypeptide corresponds to the purified enzyme. -
k
e
?i~.... ~!i/i/~, ii~iii!/jii~i~if?if!~! • ~
' ~ i~ •
~, /
Fig. 2. SDS-polyacrylamide gels of membranes prepared from wild type and plasmid-conraining cells. Direction of running is from top to bottom. Samples: (a) and (e) purified NADH-dehydrogenase (see METHODS); (b) IY13 membranes; (c) IY34 membranes; (d) IY35 membranes after enzyme amplification using chloramphenicol. DISCUSSION
Sever~ laboratories have now reported the cloning of genes from total E. coil DNA. Clarke and Carbon (1976) established a colony bank of over 2000 clones carrying fragments of the E. coli genome on hybrid plasmids. They used sheared E. coU DNA and joined the fragments to ColE1 using the poly (dA.dT) "connector" method (Jackson et al., 1972; Lobban and Kaiser, 1973). Fmediated transfer of the hybrid plasmids from each clone was used to test for complementation of auxotrophic markers. Collins et al. (1976) and Koslov et
34
al. (1977) ligated together E c o R I digested plasmid and chromosomal DNAs and transformed the hybrid plasmids directly into point mutants. Borck et al. (1976) used restriction enzyme digests of total E. c o l i D N A to prepare specialized lambda transducmg phages in vitro, In t h e present work three different populations of hybrid plasmids were prepared by digesting plasmid and chromosomal DNA with EeoRI, BamI or H/ndHI, ligating the fragments and transforming into an F ÷ derivative of strain C600. The cloning vectors used carry drug resistance markers for ease of selection and are mobilized by F. F-mediated transfer to point mutants was used to select specific hybrid plasmids from the pools. This also allows examination of hybrid plasmids for the presence of other genes not selected for in the initial screening and overcomes the poor transformability of some strains. Selection of hybrid plasmids by complementation generally requires that the isolated fragment carries the particular gene intact toge~.,her with its promotor unless a plasmid promotor c ~ e used. The use of three different endonucleases gives a greater chance of obtaining a ~ n e n t active in complementation. Apart from the ndh gene, we have used the hybrid plasmid pools to clone several other genes concerned with membrane functions and the approach would seem to be generally useful for cloning of genes from E . coli in cases where no enriched form of the gene to be cloned is available. After primary cloning by this method the fragments can be recloned into smaller vectors such as pBR322 (Bolivar et al. 1977). The respiratory NADH dehydrogenase of £. coli although a major dehydrogenase of the respiratory chain is normally present in quite small quantities in the membrane (Fig.2). The 8--10 fold increased levels of the enzyme in cells carrying the hybrid plasmid can be attributed to the increased number of copies of the ndh gene since the plasmid pSF2124 is generally present at 1 0 12 copies per chromosome (So et al., 1975). The technique of further increasing plasmid copy number by a brief treatment with chloramphenicol followed by recovery of protein synthesis gave a 50--60-fold increase in enzyme levels in the present case and would appear to be generally applicable to the amplification of other proteins. It is of particular interest that the £. coli cytoplasmic membrane can accommodate considerably elevated levels of the NADH dehydrogenase. It would appear from the high NADH oxidase levels that the enzyme may be correctly positioned within the membrane such that it can interact with the other components of the respiratory chain. Thus the selective enrichment of proteins by the techniques of gene cloning and amplification may prove to be of great value not only for the isolation and purification of proteins which are normally present in small amounts in the cell but also ~or probing membrane structure and function, ACKNOWLEDGEMENTS
We are grateful to D.W. Ribbons for many helpful discussions on gene cloning and to G. Mayo for excellent technical assistance.
35
REFERENCES
Bolivar, F., Rodriguez, R.L., Greene P.J., Betlach, M.C., Heyneker, H.L. and Boyer, H.W., Construction and characterization of new cloning vehicles. II. A multipurpose cloning system, Gene, 2 (1977) 95--113. Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S. and Murray, N.E., The construction in vitro of transducing derivatives of phage lambda, Mol. Gen. Genet., 146 (1976) 199-207. Clarke, L. and Carbon J., A colony bank containing synthetic ColE1 hybrid plasmids representative of the entire E. coli genome, Cell, 9 (1976) 91--99. Clewell, D.B., Nature of ColE1 plasmid replication in Escherichia cofi in the presence of chloramphenicol, J. Bacteriol., 110 (1972)667--676. Clewell, D.B. and Helinski D.R., Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form, Proc. Natl. Acad. Sci. USA, 62 (1969) 1159--1166. Collins, C.J., Jackson, D.A. and De Vries, F.A., Biochemical construction of specific chimeric plasmids from colE1 DNA and unfractionated Escherichia coli DNA, Proc. Natl. Acad. Sci. USA, 73 (1976) 3838--3842. Dancey, G.F., Levine, A.E. and Shapiro, B.M., The NADH dehydrogenase of the respiratory chain of Escherichia coli, 1. Properties of the membrane bound enzyme, its solubilization, and purification to near homogeneity, J. Biol. Chem., 251 (1976) 5911--5920. Hamer, D.H. and Thomas, C.A., Molecular cloning of DNA fragments produced by restriction endonucleases, SalI and BamI, Proc. Natl. Acad. Sci. USA 73 (1976) 1537--1541. Hershfield, V., Boyer, H.W., Yanofsky, C., Lovett, M.A. and Helinski, D.R., Plasmid ColE1 as a molecular vehicle for cloning and amplification of DNA, Proc. Natl. Acad. Sci. USA, 71 (1974)3455--3459. Jackson, D . ~ , Symons, R.H. and Berg, P., Biochemical method for inserting nvw genetic information into DNA of Simian virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli, Proc. Natl. Acad. Sci. USA, 69 (1972) 2904--2909. Kozlov, J.I., Kalinina N.A., Gening, L.V., Rebentish, B.A., Strongin, A.Y., Bogush, V.G. and Debabov, V.G., A suitable method for construction and cloning hybrid plasmids containing EcoRI fragments of E. coli genome, Mol. Gen. Genet., 150 (1977) 211--219. Laemmli., U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680---685. Lederberg, E.M., and Cohen, S.N., Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid, J. Bacteriol., 119 (1974) 1072- _074. Lobban, P.E. and Kaiser, A.D., Enzymatic end-to-end joining of DNA molecules, J. Mol. Biol., 78 (1973) 453--471. Marmur, J., A procedure for the isolation of deoxyribonucleic acid from micro-organisms, J. biol. Biol., 3 (1961) 208--218. Raetz, C.R.H., Larson, T.J. and Dowhan, W., Gene cloning in the isolation of enzymes of membrane lipid synthesis: phosphatidylserine synthase overproduction in Escherichia coU, Proc. Natl. Acad. Sci. USA, 74 (1977) 1412--1416. Sharp, P..~, Sugden, B. and Sambrook, J., Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis, Biochemistry, 12 (1973) 3055--3063. So, M., Gill, 1~ and Falkow, S., The generation of a ColE1-Apr cloning vehicle which allows detection of inserted DNA. Mol. Gen. Genet., 142 (1975) 239--249. Steffen, D. and Schleif, R., In vitro construction of plasmids which result in overproduction of the protein product of the araC gene of Escherichia coli, Mol. Gen. Genet., 157 (1977) 341--344.
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Stroobant, P., Young, LG., and Gibson, F., Mutants of Ewherichia ¢oli K12 blocked in the final reaction of ubiquinone biosynthesis: characterization and genetic analysis, J. BacterioL, 109 (1972) 134--139. Studier, F.W., Analysis of bacteriophage T7 early RNA's and proteins on slab gels, J. Mol. BioL, 79 (1973) 237--248. Vapnek, D., Alton, N.K., Bassett, C . L and Kushner, 8,R., Amplification in Escherieh~z coli of enzymes involved in genetic recombination: construction of hybrid colE1 piesmids carrying the structural gene for exonuclease I, Proc. Natl. Aead. 8ei. USA, 73 (1976) 3492--3496. Wallace, KJ. and Young, LG., Role of quinones in electron transport to oxygen and nitrate in Esther[chin col[. Studies with a ubiA- menA- double quinone mutant, Biochim. Biophys. Acta, 461 (1977) 84--100. Wickner, S.I~, Wickner, R.B. and Raetz, C.R.H., Overproduction of d ~ gene products by Escherichia coli strains carrying hybrid ColE1 plasmids~ ~iochen~ Biophys. Res. Commun., 70 (1976) 389--396. Young, LG. and Wallace, B.J., Mutatio;,~ affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli, Biochim. Biophys. Acta, 449 (1976) 376--385. Communicated by W. 8zybalski.
AMPLIFICATION OF THE RESPIRATORY NADH DEHYDROGENASE OF £scherichia coli BY GENE CLONING (Restriction endonucleases; EcoRI; HindIII; BamI: NADH-ubiquinone oxidoreductase; amplifiable plasmids; membrane proteins) I'G. YOUNG, A. JAWOROWSKI and M.I. POULIS Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra, A.C. T. 2601 (Australia) (Received April 10th, 1978) (Accepted May 22nd, 1978)
SUMMARY
A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Esvherichiacolifrom unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI an(;~HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F wasused to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane, Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane t o b e i n c r e ~ d t o 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior t o their purification. INTRODUCTION
The ability to isolate and amplify specific genes by cloning onto amplifiable plasmids has many useful applications, The hybrid plasmids generated can serve asc0nvenient sources of large amounts of specific DNA for use in DNA sequencing and studies on the r e l a t i o n of gene expression. In addition, beAbbreviations: Apr, ampicillin resistance; Tcr, tetracycline resistance.
26
cause of their high copy number they can also be u~;ed to amplify particular proteins in vivo. In the case of bacteria such as E. coli where a wide variety of mutants are available to enable direct selection of hybrid plasmids by complementation, gene cloning is a potentially valuable first step in the isolation of any protein or enzyme. Hybrid plasmids derived from ColE1 are maintained at 10--20 copies per chromosome and strains carrying hybrid plasmids may overproduce enzymes by up to ].5-fold (Hershfield et al., ].974; Vapnek eta]., 1976; Wickner et al., ].976; Raetz et al., ].977; Steffen and Scldeif, ].977). Most studies to date have been with soluble enz3mnes, where the level of overproduction is probably largely determined by the regulation of the gene concerned. In the case of membrane proteins additional constraints may apply. For some membrane proteins the number of available sites for insertion may be limited or they may need to be assembled with the other proteins of a complex before insertion into the membrane. Cloning is most conveniently carried out if the particular genes of interest have already been enriched, for exmaple by the preparation of a specialized transducing phage. At the present time since many genes are not yet available in an enriched form, methods which allow cloning of genes from complex sources of DNA such as bacterial chromosomes are particularly useful (Clarke and Carbon, 1976; Borck et al., 1976; Collins et al., ].976; Kozlow et al., 1977). In the present work a relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase* from total E. coil DNA. Cells carrying the hybrid plasmid were shown to produce 8--].0 fold elevated levels of the enzyme in their cytoplasmic membrane. The enzyme levels were further increased to 5 0 - 6 0 times normal by increasing the plasmid copy number with chloramphenicol. MATERIALS AND METHODS
Media, chemicals, enzymes The mineral salts medium used and the concentration of supplements have been de~Lcribed previously (Stroobant et al, 1972). The complete medium was brain-heart infusion (Oxoid). The chemicals and enzymes used were obtained from the following sources: ethidium bromide, agarose, lysozyme, Sigma Chemical Co. (St. Louis, MO, USA); CsCI, BDH Chemicals Ltd. (Poole, England); EcoRI, BamI, HindIII restriction endonucleases, Miles Research Products (Indiana, USA); and T4 DNA ligase, New England Biolabs (Beverly,
*We are using the term resp~atory NADH dehydrogenase to refer to the enzyme which chain c a t a l yof~~~ - coU_Wehaemeasured ' u n s f e r ° v f reducingequiv'~lents t h e e n z m efr°m i n telNADHt° ,f il ubiquin°ne in the respiratory ,i i. Yi r m o "~ NADH,ubiquinone 0xidoreauctas~ activity, This enzyme is absent from ndh mutants (Young and ~ l a c e , 1976) which are deficient in NADH oxidase and is distinct from the NADH-ferricyanide oxidoreductase described by Dancey et al. (197 6).
27 MA, USA). Ubiquinone-1 was the generous gift of Hoffmann LaRoche Co. (Basle, Switzerland). The NADH dehydrogenase used as a marker for SDS gel electrophoresis was a highly purified preparation prepared as described elsewhere (Jaworowski and Young, manuscript in preparation).
Bacterial strains and plasmids Strains canting the plasmids pSF2124 (So et al., 1975) and pGM706 (Hamer and Thomas, 1976) were kindly supplied by J. Langridge. Strain C600rk-mk ÷ was kindly supplied by D.W. Ribbons. Strain AN595 (thi his ilv trp rpsL)(Young and Wallace, 1976) was used as the source of E. coil DNA. The other other strains used were derived from IY12 (thi his ilv trp rpsL ndh) or its isogenic ndh* transductant IY13 (thi his i~v trp rpsL). Strain IY34 (derived from IY13) carries pSF2124 and IY35 (derived from IY12) carries the hybrid plasmid pIY1 which was constructed using EcoRI and possesses a 1.6 Mdal DNA fragment. Isolation of DNA Chromosomal DNA from E. coli was prepared as described by Marmur (1961). For the isolation of plasmid DNA 30 ml cultures were growr~ in glucose--mineral salts medium containing 0.5% casamino acids and the plasmid DNA amplified using chloramphenicol (Clewell, 1972). The cells were harvested and a cleared lysate prepared according to the method of Clewell and Helinski (1969) except that 0.3% w/v Triton X-100 was used instead of brij and deoxycholate. The DNA was purified using a CsCl-ethidium bromide density gradient (Clewell, 1972), the ethidium bromide removed by isopropanol extraction and the DNA dialyzed overnight against buffer (2 mM Tris HCI, 2 mM EDTA, 1 mM NaCI, pH 8.0). Restriction and ligation The following restriction buffers were used: EcoRI, 100 mM Tris-HCI, 50 mM NaCI, 10 mM MgCI2, pH 7.5; BamI, 6 mM Tris. HCI, 5 mM NaC1, 6 mM MgCI2, 0.1 mg/ml gelatin, pH 7.4; HindIII, 7 mM Tris-HCI, 60 mM NaC1, 7 mM MgCI2, pH 7.4. The ligase buffer used was 5 mM Tris-HCI, 5 mM MgCI2, 10 mM DTT, 50/~g/ml gelatin, 66/~M ATP, pH 8.0. Preparation of hybrid plasmid pools The chromosomal and plasmid DNAs were cleaved at 37 ° C with either EcoRI, SamI or HindIII and the restriction enzymes inactivated by heating for 10 rain at 65 ° C. pSF2124 was digested with EcoRI and BamI and pGM706 with HindIII. The progress of the endonuclease digestions were followed using agarose gel electrophoresis and the incubations were continued until digestion appeared complete. Digested chromosomal DNA (9 ~g) from AN595 (thi his ilv trp rpsL) was ligated for 15 h at 11 ° C with digested plasmid DNA (3/~g) using T4 ligase in a total volume of 175/d. The ligated DNA (10 ~g) was transformed into approx. 2-101° cells of an F ÷ derivative of c e 0 0 r k - m k ÷ using the
28
method of Lederberg and Cohen (1974), The transformec~ cells were diluted 1/10 in complete medium and grown overnight at 37 ° C. ?~ds culture was then diluted 1 / 5 i n fresh medium containing 25,/zg/ml ampicillin and the culture incubated a further 1 5 h a t 37 ° C to select for Ap r. The cells were concentrated and stored at 4° C for short periods, prior to use. For long-term storage, the cells were lyophilized.
Transfer of hybrid plasmids The celts containing the hybrid plasmids and the recipient IY12 (F-, ndh rpsL) were grown to early logarithmic phase at 37 ° C in complete medium. Equa ~,volumes of the two cultures were mixed and the mating allowed to proceed for I h at 37 ° C with slow shaking to allow transfer of the hybrid plasmids by F-mobilization. The cells were then washed once and selection made for complementation of ndh (i.e. growth on mannitol (Young and Wallace, 1976)) and resistance t o ampicillin (25 #g/ml) andstreptomycin (100
Containment levels The work described was carried out under conditions corresponding to P1 containment levels of the N.I.H. guidelines (1976). ,
Eleetrophorasis of DNA
~
Electrophoresis was carried out on 3 mm thick 0.8% agarose gels containi~g 0. 5 ~g/ml ethidium bromide using a horizontal slab gel apparatus. The buffer used was 40 mM Tris- HCI, 5 mM sodium acetate, I mM EDTA: pH adjusted to 7.8 with glacial acetic acid (Sharp et al., 1973); gels were run at room temperature at either 150 mA constant current for about 3 h or 15 mA overnight. DNA samples were diluted with an equal volume of dye mixture (14.5% sucrose, 0.045% bromophenol blue, 50 mM EDTA, pH 7.8) prior to loading. Gels were examined under UV light using a ~ s i l l u m i n a t o r (Ultraviolet Products, San Gabriel, Calif.) and photographed using a Polaroid type 55 P/N film through a red filter.
Measurement of culture turbidity Culture turbidities were measured using a Klett--Summerson colorimeter fitted with a blue filter and are expressed i~,Klett units.
Amplification of the NADH dehydrogenase Strain IY35 which possesses the hybrid plasmid pIY1 carrying ndh was used for these experiments. Amplification was achieved by using two different treatments designed to increase the plasmid copy numberand thenallowing the treated cells to grow briefly soas t o aliow expression o f the ndh genes on the plasmids. For amplification by amino acid starvation, 10 litre cultures of IY35 were grown in supplemented mineral salts medium containing limiting isoleucine
29
and valine (both 0.1 raM). This concentration of isoleucine/valine limits growth at a Klett of 100 (mid log phase) and the cessation of growth is sharply defined. The cultures were incubated under conditions of amino acid starvation for 1, 2 and 4 h, respectively, at 37°C with aeration and stirring. After the required time cell growth was restarted by the addition of 0.3 mM isoleucine and valine. Cells were allowed to grow for 1 generation and then harvested. For chloramphenicol amplification, 10 litre cultures were grown in sdpplemented mannitol-mineral salts medium containing 0.1% casamino sacids at 37 ° C. Chloram~henico! w,~ . . . . .~. .d. ~c _~.,.~' coi~eent~ion of 50/~g/ml when the Klett had reached 100. After 4 to 9 h incubation with aeration and stirring the cells were harvested and washed with a small volume of sterile minimal medium, then used to reinoculate a further 10 litres of growth medium. Cells were grown at 37 ° C with aeration and stirring and harvested after one generation.
Preparation of membranes and enzyme assays The preparation of membranes and the determination of oxidase ~'ates have been described previously (Wallace and Young, 1977) except that 0.8 mM NADH and 20 mM D-lactate were used for oxidase measurements. NADH dehydrogenase activity of membrane preparations was estimated at 30 ° C by measuring the ubiquinone dependent oxidation of NADH at 340 nnfi in ~ 1 ~ ! reaction mixture containing 20 mM N-Tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid, 120/~M NADH, 50/JM ubiquinone-1, 3 mM KCN and 40 ~M FAD.
8DS-polyacry lamide gel elec~ophoresis SDS-polyacrylamide gel e]ectrophoresis was performed on vertical slab gels (10 × 14 cm) of 10% polyacrylamide with a stacking gel of 4.5% polyacrylamide using an apparatus similar to that described by Studier (1973). The discontinuous SDS buffer system of Laemmli (1970) was used. Gels were run at a constant current of 30 mA for 3--4 h at room temperature. Membranes and purified enzyme samples were heated at 100 ° C for 3 min in SDS sample buffer (Laemmli, 1970). Gels were fixed at 65 ° C for 20 rain in 10% trichloracetic acid and stained with Coomassie Brilliant Blue R250 (0.1% w/v in destainer) for 20 min at 65 ° C then destained at room temperature using a mixture of ethanol/glacial acetic/water (25:8:67 v/v/v). RESULTS
Mutant strains of K coli have been described which are affected in the NADH dehydrogenase complex of the respiratory chain (Young and Wallace, 1976). These mutants (designated ndh) appear to be affected in the structural gene or genes coding for the NADH dehydrogenase. We wished to clone the ndh genes both to provide a highly enriched source of the ndh genes for further studies in vitro and to amplify the enzyme levels so as to allow convenient purification.
30
In preliminary experiments it was found that ndh strains gave much lower frequencies of transformation with ColE1 plasmids, than did standard strains such as C600rk-mk*. In order to avoid having to transfer ndh into C600 to try and improve the frequency of transformation, it was decided to use Fmobilization for the transfer of hybrid plasmids to ndh recipients. The smaller cloning vectors such as pMB9, pBR313 and pBR322 are not mobilized at high frequency by F (Bolivar et al., 1977; I.G. Young, unpublished data) but experiments showed that pSF2124 (ColE1-Apr) and pGM706 (ColE1-AprTcr) were both efficiently mobilized by F. After I h matings approx. 5% and 16% of recipients had received the plasmids pGM706 and pSF2124 respectively. pSF2124 has single restriction sites for EcoRI and BamI and pGM706 for HindIH and SaII. Thus several different restriction enzymes could be used for cloning to try and overcome the problem that the gene to be cloned may carry sites of restriction for particular enzymes. Both pSF2124 and pGM706 carry Apr which allows convenient selection of cells carrying the plasmids. Apr is not inactivated by insertion of DNA fragments at the various restriction sites. Cloning of the ndh gene Whole chromosomal DNA from E. coli was digested separately with the restriction endonucleases EcoRI, BamI and HindIII to produce three pools of DNA fragments with complementary single stranded ends. pSF2124 DNA was also digested with EcoRI and separately with BamI, while pGM706 DNA was cleaved with HindIII. The corresponding chromosomal and plasmid DNA digests were ligated with T4 ligase, the ligated DNA transformed into an F ÷ derivative of C600 rk-mk ÷ and selection made for Ap r in liquid medium. Hybrid plasmids complementing ndh were selected from the pools by Fmediated transfer to an ndh recipient selecting for both Ap r and complementation of ndh. The hybrid plasmids pIYl(derived from pSF2124) and pIY2 (derived from pGM706) were isolated from the EcoRI and HindIII hybrid plasmid pools respectively. The plasmid DNA was amplified using chloramphenicol: purified on CsCl-ethidium bromide density gradients and used to transform ndh mutants so as to verify complementation of ndh by the hybrid plasmids. The hybrid plasmid DNAs were also examined on agarose gels before and after cleavage with the appropriate restriction endonucleases (Fig.l). The two hybrid plasmids both showed a faster migrating band corresponding to covalently closed plasmid DNA and a slower migrating open circular DNA band. After digestion with EcoRI and HindIII respectively, pIY1 and pIY2 each gave two bands, the higher molecular weight band in each case being the parental plasmid (linear form) and the lower molecular weight band corresponding to the cloned DNA. By comparison with molecular weight standards (), DNA digested with EcoRI or HindIII) the cloned fragments in pIY1 and pIY2 were estimated to be 1.6 and 4.6 Mdal respectively.
"
a
31
b
c:
d
. L-..,,,~
I
I I
Fig.1. Electrophoresis of plasmid DNAs on 0.8% agarose gels. Samples: (a) pIY1; (b) pIY1 digested with EeoRI; (c) pIY2; (d) pIY2 digested with HindIII. About 1 ~g DNA was loaded except for sample (b) where 3 . g was loaded so that the 1.6 Mdal restriction fragment could be readily seen. The two bands present in (a) and (c) correspond to the covalently closed and open circular forms of the respective hybrid plasmid DNAs. The open circular bands are in each case closer to the origin.
Amplification or" the respiratory NADH dehydrogenase Since the EcoRI-derived plasmid pIY1 carried the smaller DNA fragment (1.6 Mdal) it was used for experiments on the amplification of enzyme levels. Cells containing the hybrid plasmid were found to possess 8--10 times the normal level of the NADH dehydrogenase (measured as NADI-I-ubiquinone oxidoreductase) in their membranes (Table I). Two different methods were used to attempt to further increase the levels of NADH dehydrogenase by increasing the plasmid copy number. It has been shown that when cellular protein synthesis is prevented by amino acid starvation or by treatment with chloramphenicol, chromosomal DNA synthesis ceases but ColE1 and its derivatives continue to replicate (Clewell, 1972).
32
TABLE I AMPLIFICATIONOF THE NADH DEHYDROGENASE •
Strain
Plasmid
Treatmenta
NADH dehy -b drogenmm
NADH o x i -b dase
IY12 IY13 IY34 IY35 rY35
--pSF2124 pIY1 pIY1
IY35
pIY1
---a m i n o acid starvation I h a m i n o acid starvation 2 h a m i n o a c i d Starvation 4 h chloramphenicol 4 h chloramphenicol 9 h
0.03 0.52 0.63 4.91 7.82 9.26 9.50 30.6 31.1
0.02 0.49 0.44 2.14 3.24 3.30 3.05 4.69 3.23
aUnless otherwise stated, cells were harvested in late log phase; procedures for amino acid starvation and treatment with chloramphenicol are described in methods. bActivities expressed as ~moles substrate oxidized/min/mg membrane protein. When cells carrying pIY1 were starved of amino acids for up to 4 h and then allowed to grow for I generation in complete medium the NADH dehydrogenase levels were increased to 1 5 - 1 8 times normal (Table I). A potentially more effective way of increasing plasmid copy number is to use chloramphenico~., but for the purpose of amplification of enzyme levels it was essential to establish conditions such that the inhibition of protein synthesis by chloramphenicol could be subsequently reversed. We wished to establish conditions whereby cells could be treated with chloramphenicol so as to increase plasmid copy number, then the chloramphenicol removed to allow the cells to grow again and express the ndh gene on the hybrid plasmid. It was found that the chloramphenicol concentration could be reduced to 50 #g/ml and still inhibit growth as effectively as higher concentrations. Cells were grown to mid log phase, incubated with chloramphenico: (50 #g/ml) for different lengths o f time then washed free of antibiotic and tested for their ability to grow normally in fresh medium. After treatment with chloramphenicol for 4 - 9 h the cells could grow again without excessive lag suggesting that protein synthesis was being effectively recovered in the majority of cells. Treatment with chloramphenicol for 4 - 9 h followed by growth for I generation in its absence resulted in the elevation of enzyme levels in the membrane to 5 0 - 6 0 fold normal (Table I). The amplification of the NADH dehydrogenase also led to an increase in the NADH oxidase levels (Table I) but appeared -to be quite specific in that the levels of succinate of D-lactate oxidase were unaffected(data not shown). Assay of the soluble fraction for NADH-ubiquinone oxidoreductase showed that no increase in activity had occurred (data n o t shown)suggesting that either all of the NADH dehydrogenase produced was present in the membrane fraction or any enzyme not incorporated into the membrane was inactive.
33
The respiratory NADH dehydrogenase has been solubilized from the membranes containing the highly elevated levels of the enzyme and purified to homogeneity (Jaworowski and Young, manuscript in preparation). Oil SDSpolyacrylamide gels it consists of a single polypeptide of molecular weight approx. 45 000. SDS-polyacrylamide gels of membranes from chloramphenicolamplified cells containing the hybrid plasmid pIY1 showed that the level of one polypeptide from the membrane fraction is dramatically increased (Fig.2). This polypeptide corresponds to the purified enzyme. -
k
e
?i~.... ~!i/i/~, ii~iii!/jii~i~if?if!~! • ~
' ~ i~ •
~, /
Fig. 2. SDS-polyacrylamide gels of membranes prepared from wild type and plasmid-conraining cells. Direction of running is from top to bottom. Samples: (a) and (e) purified NADH-dehydrogenase (see METHODS); (b) IY13 membranes; (c) IY34 membranes; (d) IY35 membranes after enzyme amplification using chloramphenicol. DISCUSSION
Sever~ laboratories have now reported the cloning of genes from total E. coil DNA. Clarke and Carbon (1976) established a colony bank of over 2000 clones carrying fragments of the E. coli genome on hybrid plasmids. They used sheared E. coU DNA and joined the fragments to ColE1 using the poly (dA.dT) "connector" method (Jackson et al., 1972; Lobban and Kaiser, 1973). Fmediated transfer of the hybrid plasmids from each clone was used to test for complementation of auxotrophic markers. Collins et al. (1976) and Koslov et
34
al. (1977) ligated together E c o R I digested plasmid and chromosomal DNAs and transformed the hybrid plasmids directly into point mutants. Borck et al. (1976) used restriction enzyme digests of total E. c o l i D N A to prepare specialized lambda transducmg phages in vitro, In t h e present work three different populations of hybrid plasmids were prepared by digesting plasmid and chromosomal DNA with EeoRI, BamI or H/ndHI, ligating the fragments and transforming into an F ÷ derivative of strain C600. The cloning vectors used carry drug resistance markers for ease of selection and are mobilized by F. F-mediated transfer to point mutants was used to select specific hybrid plasmids from the pools. This also allows examination of hybrid plasmids for the presence of other genes not selected for in the initial screening and overcomes the poor transformability of some strains. Selection of hybrid plasmids by complementation generally requires that the isolated fragment carries the particular gene intact toge~.,her with its promotor unless a plasmid promotor c ~ e used. The use of three different endonucleases gives a greater chance of obtaining a ~ n e n t active in complementation. Apart from the ndh gene, we have used the hybrid plasmid pools to clone several other genes concerned with membrane functions and the approach would seem to be generally useful for cloning of genes from E . coli in cases where no enriched form of the gene to be cloned is available. After primary cloning by this method the fragments can be recloned into smaller vectors such as pBR322 (Bolivar et al. 1977). The respiratory NADH dehydrogenase of £. coli although a major dehydrogenase of the respiratory chain is normally present in quite small quantities in the membrane (Fig.2). The 8--10 fold increased levels of the enzyme in cells carrying the hybrid plasmid can be attributed to the increased number of copies of the ndh gene since the plasmid pSF2124 is generally present at 1 0 12 copies per chromosome (So et al., 1975). The technique of further increasing plasmid copy number by a brief treatment with chloramphenicol followed by recovery of protein synthesis gave a 50--60-fold increase in enzyme levels in the present case and would appear to be generally applicable to the amplification of other proteins. It is of particular interest that the £. coli cytoplasmic membrane can accommodate considerably elevated levels of the NADH dehydrogenase. It would appear from the high NADH oxidase levels that the enzyme may be correctly positioned within the membrane such that it can interact with the other components of the respiratory chain. Thus the selective enrichment of proteins by the techniques of gene cloning and amplification may prove to be of great value not only for the isolation and purification of proteins which are normally present in small amounts in the cell but also ~or probing membrane structure and function, ACKNOWLEDGEMENTS
We are grateful to D.W. Ribbons for many helpful discussions on gene cloning and to G. Mayo for excellent technical assistance.
35
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Bolivar, F., Rodriguez, R.L., Greene P.J., Betlach, M.C., Heyneker, H.L. and Boyer, H.W., Construction and characterization of new cloning vehicles. II. A multipurpose cloning system, Gene, 2 (1977) 95--113. Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S. and Murray, N.E., The construction in vitro of transducing derivatives of phage lambda, Mol. Gen. Genet., 146 (1976) 199-207. Clarke, L. and Carbon J., A colony bank containing synthetic ColE1 hybrid plasmids representative of the entire E. coli genome, Cell, 9 (1976) 91--99. Clewell, D.B., Nature of ColE1 plasmid replication in Escherichia cofi in the presence of chloramphenicol, J. Bacteriol., 110 (1972)667--676. Clewell, D.B. and Helinski D.R., Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form, Proc. Natl. Acad. Sci. USA, 62 (1969) 1159--1166. Collins, C.J., Jackson, D.A. and De Vries, F.A., Biochemical construction of specific chimeric plasmids from colE1 DNA and unfractionated Escherichia coli DNA, Proc. Natl. Acad. Sci. USA, 73 (1976) 3838--3842. Dancey, G.F., Levine, A.E. and Shapiro, B.M., The NADH dehydrogenase of the respiratory chain of Escherichia coli, 1. Properties of the membrane bound enzyme, its solubilization, and purification to near homogeneity, J. Biol. Chem., 251 (1976) 5911--5920. Hamer, D.H. and Thomas, C.A., Molecular cloning of DNA fragments produced by restriction endonucleases, SalI and BamI, Proc. Natl. Acad. Sci. USA 73 (1976) 1537--1541. Hershfield, V., Boyer, H.W., Yanofsky, C., Lovett, M.A. and Helinski, D.R., Plasmid ColE1 as a molecular vehicle for cloning and amplification of DNA, Proc. Natl. Acad. Sci. USA, 71 (1974)3455--3459. Jackson, D . ~ , Symons, R.H. and Berg, P., Biochemical method for inserting nvw genetic information into DNA of Simian virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli, Proc. Natl. Acad. Sci. USA, 69 (1972) 2904--2909. Kozlov, J.I., Kalinina N.A., Gening, L.V., Rebentish, B.A., Strongin, A.Y., Bogush, V.G. and Debabov, V.G., A suitable method for construction and cloning hybrid plasmids containing EcoRI fragments of E. coli genome, Mol. Gen. Genet., 150 (1977) 211--219. Laemmli., U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680---685. Lederberg, E.M., and Cohen, S.N., Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid, J. Bacteriol., 119 (1974) 1072- _074. Lobban, P.E. and Kaiser, A.D., Enzymatic end-to-end joining of DNA molecules, J. Mol. Biol., 78 (1973) 453--471. Marmur, J., A procedure for the isolation of deoxyribonucleic acid from micro-organisms, J. biol. Biol., 3 (1961) 208--218. Raetz, C.R.H., Larson, T.J. and Dowhan, W., Gene cloning in the isolation of enzymes of membrane lipid synthesis: phosphatidylserine synthase overproduction in Escherichia coU, Proc. Natl. Acad. Sci. USA, 74 (1977) 1412--1416. Sharp, P..~, Sugden, B. and Sambrook, J., Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis, Biochemistry, 12 (1973) 3055--3063. So, M., Gill, 1~ and Falkow, S., The generation of a ColE1-Apr cloning vehicle which allows detection of inserted DNA. Mol. Gen. Genet., 142 (1975) 239--249. Steffen, D. and Schleif, R., In vitro construction of plasmids which result in overproduction of the protein product of the araC gene of Escherichia coli, Mol. Gen. Genet., 157 (1977) 341--344.
36
Stroobant, P., Young, LG., and Gibson, F., Mutants of Ewherichia ¢oli K12 blocked in the final reaction of ubiquinone biosynthesis: characterization and genetic analysis, J. BacterioL, 109 (1972) 134--139. Studier, F.W., Analysis of bacteriophage T7 early RNA's and proteins on slab gels, J. Mol. BioL, 79 (1973) 237--248. Vapnek, D., Alton, N.K., Bassett, C . L and Kushner, 8,R., Amplification in Escherieh~z coli of enzymes involved in genetic recombination: construction of hybrid colE1 piesmids carrying the structural gene for exonuclease I, Proc. Natl. Aead. 8ei. USA, 73 (1976) 3492--3496. Wallace, KJ. and Young, LG., Role of quinones in electron transport to oxygen and nitrate in Esther[chin col[. Studies with a ubiA- menA- double quinone mutant, Biochim. Biophys. Acta, 461 (1977) 84--100. Wickner, S.I~, Wickner, R.B. and Raetz, C.R.H., Overproduction of d ~ gene products by Escherichia coli strains carrying hybrid ColE1 plasmids~ ~iochen~ Biophys. Res. Commun., 70 (1976) 389--396. Young, LG. and Wallace, B.J., Mutatio;,~ affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli, Biochim. Biophys. Acta, 449 (1976) 376--385. Communicated by W. 8zybalski.
