r

Gene, 3 (1978) 177--189 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

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HYBRID PLASMIDS CONTAINING THE a r a B A D GENES OF Escbericbia coli B/r

(Cloning in pBR317; ColE1; genetic complementation and recombination; BamI, E c o R I , PstI restriction endonucleases)

DONALD A. KAPLAN, LAWRENCE GREENFIELD, THOMAS BOONE and GARY WILCOX

Department o f Bacteriology and Molecular Biology Institute, University of California, Los Angeles, CA 90024 (U.S.A.) (Received November 22nd, 1977) (Revision received February 6th, 1978) (Accepted February 9th, 1978)

SUMMARY

The DNA fragments generated by restriction endonuclease B a m I which contain the araCBAD genes from E.coli B/r have been cloned. The DNA fragments containing ara genes were identified by a comparison of the BarnI fragments of ),h80dara phages containing different ara deletion mutations. The ara genes were cloned into the plasmid pBR31 ~, a derivative of ColE1. The cloned DNA fragments, were analyzed by digestion with pairs of restriction endonuc leases to determine the molecular weight of the chimeras and to identify the cloned ara DNA fragments. The cloned ara fragments were also identified by genetic complementation and recombination tests.

INTRODUCTION

Regulation of the L-arabinose operon, ara3AD, by the product of the araC gene in E. coil has been extensively studied (Englesberg and Wilcox, 1974). The recent development of recombinant DNA techniques greatly extends the kinds of experiments which can be designed to study gene regulation (Sinshe!mer, 1977). In the past few years a number of reports have appeared describing DNA cloning using a variety of cloning vectors. One of the most useful vectors described, a derivative of ColEI, is pBR317 (Boyer et al., 1977; see Fig. 4). It contains genes which confer on the host resistance to ampicillin ~ d tetracycline, There is a single B a m l restriction endonuclease Site in a gene conferring tetracycline resistance. Thus, if foreign DNA is inserted in the B a m I site, the resulting clone will be resistant to ampicillin and sensitive to tetracycline. We have used pBR317 as a vehicle

177

178

for cloning BamI fragments of a ),h80dara phage. Cloned DNA containing ara genes was identified by comparison with the BamI restriction map of ~h80dara DNA and by genetic complementation and recombination experiments. MATERIALS AND M ~ O D S

Bacterial and phage strains Escheriehia coil RE1 (F- leu pro thi lac Y strr hsdR hsdM), RE1 carrying the plasmid pBE317 and EY13 gal end (RI) were obtained from Herb Boyer (University of California, San Francisco). An F* derivative of RE1 was obtained from Dan Ray (University of Califomm, Los Angeles). Providencia stuardii and Bacillus anyloliquefacienz were obtained from J. Abelson (University of California, San Diego). RR1 am-strains were made by P1 transduction by selection for/eu ÷ as described by Englesberg et al., 1965). The ~h8Odara transducing phages, ~,h80dara ÷, ~,h80dam~ 719, ~h80dara~, 766, ?,h80dara~ 718 and ?.h80dara~ 735 have been previously described (Wilcox et al., 1974). DNA was prepared from these phages by the methods described in Wilcox et al. (1971), except that the final dialysis was against 0.01 M Tris--acetate, pH 8.0. Plasmids The plasmid pBR317 was prepared by the method of Clewell and Helinski (1969) after chloramphenicol-induced amplification (Clewell, 1972). Genetic complementation and recombination tests The RE1 ara-strains were transformed with plasmid DNA and Apr clones were selected as described below. A culture of a purified Ap r clone was grown overnight in TYE ampicillin media, serially diluted and 0.1 ml o£ each dilution plated on a minimal supplemented L-arabinose ampicillin (20 ~g/nfl) agar plate and a TYE ampici!!in plate. If complementation occurred, then the number of Ara÷ colonies should equal the number of Ap r colonies in the overnight culture. Recombination had occurred if the ratio of Axa*/ Aprcolonies was around 10-s and significantly above the reversion frequency of the non-transformed parent strain. The araC766 mutation is a deletion and araB24 and D139 are point mutations (Englesberg and Wilcox, 1974). Isolation of restriction enzymes The restriction enzymes PstI (Smith et al., 1976), BamI (Wilson and Young, 1975) and EcoRI (Green et al., 1974) were all isolated in the following manner. Three liters of cells were grown in TYE (per liter; 15 g tryptone, 10 g yeast extract, and 5 g NaCI) at 37°C to late log phase, harvested (yield = 15 g) and resuspended in 30 ml of Buffer A (0.1 M KPO4, pH 7.4, 0.01 M 2-mercaptoethanol, 10 -4 M EDTA). The cells were disrupted by passage through a French press at 10000 psi and the cell debris removed by centrifugation at 10 000 rev./min for 15 rain in a Sorvall SS34 rotor. The

179

resulting supernatant was made 1 M in NaC1 and applied directly, 20 ml at a time, to an agarose A.5M column (2.5 X 50 cm) equilibrated in Buffer A containing 1 M NaCI. The column was monitored by A260 and A280 measurements and protein-containing fractions were pooled and dialyzed against Buffer B (10 mM KPO4, pH 7.4, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and 10% glycerol). The dialyzate was applied to a phosphocellulose column (2.5 × 10 cm) which was equilibrated with Buffer B, washed with 100 ml of Buffer B and eluted with a 500 ml linear 0 to 1 M NaCI gradient in Buffer B. Every other fraction was assayed as described by Green et al. (1974) by adding 5 gl of the fraction to be tested to 15 gl of Restriction Buffer (100 mM Tris--HC1, pH 7.5, 50 mM NaCI, 5 mM MgCI2 ) containing 0.5 gg of ~h8Odara DNA. Samples were incubated at 37°C for 15 rain and the reaction stopped by the addition of 5 gl of a solution containing 30% glycerol, 5% SDS, and 0.025% bromphenol blue. Samples were then applied to a 1.2% agarose slab gel in Tris--borate buffer composed of 5.5 g boric acid, 10.8 g Tris, 0.93 g EDTA per liter. Gels were run at 17 5 V for 90 rain, then stained for 5 rain in a solution of ethidium bromide (0.5 gg/ml) and visualized with ultraviolet light (300 nm). Active fractions were pooled and dialyzed against Buffer C (25 mM KPO4, pH 7.4, 1 mM EDTA, 7 mM 2-mercaptoethanol, and 1 mM NaN3 ). The dialyzate was applied to a hydroxyapatite column (2.5 × 10 cm) equilibrated in Buffer C and washed with 100 ml of Buffer C. Enzyme was eluted with a 200 ml gradient from 25 mM KPO4 to 500 mM KPO4 in Buffer B. Fractions were assayed as above and fractions containing significant activity were pooled and dialyzed against Storage Buffer (10 rnM Tris-HCI, pH 7.5, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and 50% glycerol) for 16 h. Enzymes were stored at-20°C.

Cloning: restriction, ligation, transformation pBR317 plasmid DNA (12.5 gg) and )~h8Odara DNA (33.5 gg) were mixed in a total volume of 100/~1 and restricted with 100 units of BamI for 7 h at 37 ° C. A 5 gl sample of the restricted DNA analyzed by electrophoresis on a 1.2% agarose gel, indicated complete digestion of the plasmid and phage DNA. The remaining 95 gl of DNA was heated to 65°C for 10 rain and an equal volume of ligase buffer (120 mM Tris--HCl, pH 7.5, 10 mM dithiothreitol, and 0.8 mM adenosine triphosphate) was then added along with 0.1 units of T4 DNA ligase (New England Biolabs). This mixture was kept at 14 ° C for 18 h and then used for transformation. Transformation was carried out as previously described (Kaplan et al., 1977a).

Plasmid isolation Purified AprTc s clones were grown to an As40 of 1.5 in TYE ampicillin (20 #g/ml ~mpicillin) media. Chloramphenicol was then added to a final concentration of 250/~g/ml and the cells were incubated an additional 12 h at

180

37°C to allow for plasmid amplification as described by Clewell (1972). A cleared lysate was prepared as described by Guerry et al. (1973). The cleared lysate was extracted with phenol, ethanol-precipitated and resuspended in 0.2 ml of 10 mM Tris--HCl, pH 7.5. Analysis o f DNA with restriction endonucleases Plasmid DNA (5--10/~g) in a final volume of 100/~1 was incubated at 37°C for 6--16 h. For restriction of DNA with EcoRI, PstI, or SalI the reaction mixture contained 100 mM Tris--HCl, pH 7.5, 50 mM NaCI and 5 mM MgCI~ ; for restriction of DNA with BamI the buffer contained 6 mM Tris-HCI, pH 7.4, 6 mM MgCI2,6 mM 2-mercaptoethanol, and 50 mM NaCI. The reactions were stopped by the addition of 30/A of a glycerol
RESULTS Physical map o f the araCBAD region o f kh8Odara phage The structure of the kh80dara phage is shown in Fig. 1. The order of the araCBAD genes with respect to phage genes was originally determined by Gottesman and Beckwith (1969). Since the initial work with the ara transducing phage, a number of ara deletion mutations have been recombined into the phage (Wilcox et al., 1974). We have confirmed the structure of the phage and determined the length of the ara deletion mutations by electron microscopy of heteroduplexes. The results, summarized in Fig. 1, are consistent with what is known from both genetic and biochemical studies on the ara system (Englesberg and Wilcox, 1974). Thus, deletions 719 and 766 which removearaC are closer to the end of the phage than deletions 718 and 735 which remove araOIBA and araBA, respectively. We estimate that

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araA 718 removes 1800 base pairs, araA 719 removes 1000 base pairs, araA735 removes 1900 base pairs and araA 766 removes 1000 base pairs. The results are summarized in Fig. 1. ~766,~

7t8

17t91

:

•

,iCl,i

~.~.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'H.'H.

t I

0

i i

. i,

735

, J

B I A IDI

t .,I

I.

2

,

I

t I,,

4

I

I

6

i

•

8

I

I

I0

t I

I

t2

Fig. 1. Physical map of the left end of ~,h80dara ÷ bacteriophage. The arrows indicate BamI restriction sites. Each division on the scale represents one kilobase. The numbers represent deletions and the bars represent the extent of the deletions. C,B,A, and D represent the genes involved in the utilization of L-arabinose. The double horizontal line represents DNA. r ) / l l l i H , , ~80 DNA;I" I , bacterial DNA.

B a m I restriction endonuclease sites on the kh8Odara phages Analysis of the fragments obtained by digestion of ~,h80dara phages with BamI is shown in Fig. 2. The ~h8Odara + phage, lane b, yields nine restriction fragments when digested to completion with B a m I (the smallest fragment is not visible in the photograph). The molecular weights of the bands are: 1=7.8.106 , 2=5.5.106 , 3=4.9-106 , 4=2.8.10 ~ , 5=1.9.106 , 6=1.8.106 , 7=1.7 • 106 , 8=1.6"106 and 9=0.2.106. The total molecular weight of the fragments, 28.5-106 is in reasonable agreement with the molecular weight of 30-106 for ~,h80dara ÷ DNA calculated from contour length measurements in the electron microscope. Some of the restriction fragments can be assigned to specific regions of the araCBAD genes by comparison of the restriction fragments generated by digestion of ~,h80dara phages containing deletion mutations. Lanes c and d in Fig. 2 are digestions of ~,h80dara~ 766 and kh80dara~ 719. It can be seen that band 4 is absent in both of these phage DNAs and that a new band appears corresponding to a molecular weight of 2.1-106 . This new band is 0.7.106 daltons smaller than band 4 which corresponds to the amount of DNA removed in deletions 766 and 719 (see Fig. 1). Examination of the pattern from ~h8OdaraA 735, lane f, shows that only band 6 is missing. It cannot be seen in Fig. 2 but on a 1.2% agarose gel a new band corresponding to a molecular weight of 0.5.106 d~ltons appears. This is 1.3.106 daltons smaller than band 6, the size of deletion 735. Thus, there are B a m I sites on either side of araA "719, araA 766 and araA 735. By comparison of the physical map (Fig. 1) with the size of the fragments, there should be a BamI site to the left of ara~ 766, a site between araA 719 and ara~ 735, and a site to the right of araz~ 735. Consistent with the above assignment of B a m I sites is the restriction pattern of araA 718. Band 6 and band 4 are absent and a new band of 3.2.106 daltons is present. Thus, araA 718 has removed the BamI site between ara~

1.

24 3

5. .6 . 7 8

(3) Fig. 2. Electrophoresis of Barn1 restricted hh8Odara DNA in 1% agarose M. Phage DNA samples (5 pg) were restricted with 2 units of Barn1 in a total volume of 50 ~1 for 18 h at 37” C. (a) and (g) 0.5 pg of EcoRI restricted nonglucosylated T4 DNA; (b) 0.5 pg of Ah80dara+ DNA; (c) 0.5 pg of hh80daraA 766 DNA; (d) 0.5 pg of hh80daraA 719 DNA; (e j 0.5 c(g of hh80dara8 718 DNA; (f) 0.5 pg of hh80daraA 735 DNA. Fig. 3. Electrophoresia of Barn1 restricted pBR317-hh80dara chimeras in 1% agarose gel. Each cleared lysate was phenol extracted, ethanol precipitated and resuspended in Barn1 restriction buffer. 15 ~1 of DNA was incubated with LOunits of BamI for 18 h at 3 ?” C prior to electrophoresis in a 1.0% agarose gel. Lanes b-f and h-l, clones l-10, respectively; lanes, a, g and m, 0.2 pg of hhSOdara+DNA restricted with BamI.

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719 and araA 735 fusing bands 4 and 6 together to create a new band. The molecular weight of this new band corresponds to the sum of the molecular weights of bands 4 and 6 minus the molecular weight of the region corresponding to araA 718. Properties o f the kh80dara-pBR317 chimeras Restriction, ligation and transformation were carried out as described in MATERIALS AND METHODS. Plasmids from AprTcs clones were initially characterized by electrophoresis on agarose gels. Cleared lysates were prepared as described in MATERIALS AND METHODS and gel electrophoresis carried out in 0.6% agarose. The AprTcs clones contained plasmids which migrated more slowly than pBR317 suggesting that different BamI fragments had been cloned. The results are summarized in Table I, column 1. The cleared lysates were phenol-extracted, ethanol-precipitated, treated with BamI, and subjected to electrophoresis in 0.6% agarose gels. The results are shown in Fig. 3. It can be seen that BamI fragments are generated which correspond in size to the B a m I fragments of the kh8Odara phage. The ~h8Odara ÷ DNA digested with BamI (lanes a, g, and m) contains faint bands which are not seen in Fig. 2, lane b because larger quantities of kh8Odara ÷ DNA were run on the gel in Fig. 3. The faint bands are DNA fragments from },h80 helper phage which is present to some extent in all preparations of kh8Odara ÷ DNA. It can be seen in Fig. 2 that helper phage DNA fragments have been cloned and are present in the chimeras represented in lanes b, f, and k. The pBR317-kh80dara chimeras could have the general structures shown in Fig. 4. In Model 1, single or multiple copies of the same or different fragments are arranged in tandem on the same plasmid. In Model 2, there are only single or multiple copies of a given fragment on a plasmid and a single cell has been co-transformed by two different chimeras. This is the least likely model but it has been observed previously (Kaplan et al., 1977a). The EcoRI, PstI and SalI restriction sites on pBR317 make it possible to distinguish between the two models and determine the molecular weight of the chimeras by restricting simultaneously with either Bali and E c o R I , or SalI and PstI. The use of two sets of enzymes not only provides independent confirmation of molecular weights but is useful for mapping when the inserts themselves contain Bali, PstI of EcoRI sites. The double digestions are shown in Figs. 5a and 5b and the results are summarized in Table I. The ability to distinguish between the two models in Fig. 4 depends on (1) the molecular weight of the fragment(s) cloned relative to the molecular weights of the EcoRI-SalI fragments or the PstI-SalI fragments and (2) the accuracy with which we can estimate molecular weight from gel electrophoresis. If the molecular weight estimates are accurate to + 10% then all of the chimeras analyzed are of the type depicted in Model 1 in Fig. 4.

184

A.

E~ Eco RI

Eco RI

..

8OmZ~¢/

°e,,~ C

Eco RI

E¢o RI

Fig. 4. Possible structures of the pBR317-~,hS0dara ÷ chimeras. (A) Restriction endonuclease cleavage sites on pBR317 (Boyer ~t al., 1977) (B) A cell contains only one kind of chimera which contains single (n or m = 1) or multiple (n or m greater than 1) copies of Xn and Ym. (C) A cell contains more than one kind of chimera.

Genetic tests T h e s e chimeras which contain all or p a r t o f the aragBAD genes should be able to c o m p l e m e n t or r e c o m b i n e w i t h specific ara m u t a t i o n s . The experim e n t s summarized in Table II d e m o n s t r a t e the following: (1) p K G 1 0 3 contains a functional araC gene since 100% o f the clones which contain t h e plasmid are A m ÷ in strains containing a deletion of araC, (2) p K G l l 0 conrains t h a t part o f araD which is altered in aruD139. T h e r e is a 50--200-fold increase in Ara ÷ colonies over b a c k g r o u n d indicating r e c o m b i n a t i o n has occurred, (3) p K G 1 6 0 contains arab because r e c o m b i n a t i o n is observed w i t h araB24. DISCUSSION The restriction endonuclease BamI cleaves in at least t w o sites within t h e araCBAD region in E. coli B/r. One o f these is located near t h e border bet w e e n t h e araA and araD genes and t h e o t h e r is located b e t w e e n the arab

185

TABLE I SUMMARY O F PHYSICAL AND GENETIC P R O P E R T I E S O F THE C H I M E R A S Molecular weight of chimera Clone number

Plasmid designation

BamI a

1 2 3 4 5 6 7 8 9 10 11

pKG101 pKG103 pKG106 pKG110 pKG113 pKG114 pKG115 pKG125 pKG127 pKG128 pKG160

6.1 8.2 9.9 7,0 7.1 6.1 9.8 9.9 7.3 -7.0 f

ara genes e

Sal~ EcoRI

SalI c PstI

CCL d

6.1 8.2 9.6 6.9 7.1 6.0 9.9 ~ 9.6 6.9 11.9 --

5.9 8.2 9.5 7.1 6.1 5.9 9.8 9.5 6.5 12.7 --

6.1 8.1 9.5 7.0 7.1 6.2 10.0 9.8 7.3 13.8 7.0

-araC -araD ------araBA

a,b,CDete~mined from data in Figs. 3, 5a, 5b, respectively for clones 1--10. d Determined f r o m electrophoresis o f a crude cleared lysate (CCL) in a 0.6% agarose gel. eDetermined f r o m data in Table II. fDetermined f r o m electrophoresis in a 1% agarose gel o f p K G 1 6 0 DNA restricted with BamI. TABLE II TEST F O R G E N E T I C COMPLEMENTATION OR RECOMBINATION Chromosomal genotype

araC766 araB24 araD139

Plasmid pKG103

pKG110

pKG160

pBR317

1(C) 0 --

--4.10 -s (R)

0 0 4-10 -s (R) 1-10 -v 2-10 -7 8-10 -v

No plasmid 0 0 6-10 -7

Each strain was transformed with the indicated plasmid and tested for recombination or c o m p l e m e n t a t i o n as described in Materials and Methods. The data are reported as the ratio of the n u m b e r of Ara ÷ colonies/Ap r colonies. R denotes recombination; C denotes complementation.

and araC genes. The sites were assigned on the basis of a comparison of the restriction fragments obtained from ara deletion mutations, by a comparison with the physical map of the kh8Odara phage, and by genetic complementa-

tion and recombination tests. The assignment of a fragment to a gene by genetic criteria agreed in every case with the assignment made by physical criteria. A BamI restriction site has been localized in the araI region in E. coli K12 (Hirsch and Schleif, 1977). Assuming that the BamI site in E. coli B/r is also in aral (see below), then the observed recombination rather than

186

(5A) complementation between the cloned araBA fragment and araB24 is explained. The cloned fragment in pKG160 does n o t contain a functional p r o m o t e r and read-through from a plasmid p r o m o t e r does not occur. The derivative of ColE1, pBR317, was a particularly useful cloning vehicle for our experiments. The single B a m I restriction site in the Tc r gene allows detection of inserts by scoring for a Tc s phenotype. This made it possible to carry out the initial selection and scoring in the absence of L-arabinose and thus may prevent selection for or against particular combinations or orientations of ara genes. The chimeric plasmid DNAs were analyzed with pairs of restriction endonucleases. This avoided any ambiguity that could result, for example,

187

(5B) Fig. 5. Electrophoresis in 1% agarose gel of pBR317-hh80dara chimeras digested with two restriction endonucleases. (A)SalI--EcoRI restriction; Lane a, EcoRI restriction of non-glucosylated T4 DNA; Lanes c--g and i--m, SalI--EcoRI restriction of clones 1--10, respectively; lane o, SalI--EcoRI restriction of pBR317. (B) SalI--PstI restriction. Lanes a and o, Eco RI restriction of non-glucosylated T4 DNA; lanes b--h, SalI--PstI restriction of clones 1--10, respectively; lanes I and m SalI--PstI restriction of pBR317.

from cloning a small fragment in tandem and provided an independent analysis of the molecular weight of the DNA. The use of a second set of restriction endonucleases can resolve ambiguities which result from the case where the cloned fragments have restriction sites for the enzymes being used

188

for the analysis or when restriction fragments of similar molecular weight are generated. An example of the latter is the result obtained in Fig. 5a, lanes e and k. Two fragments are generated, each with a molecular weight of approx. 4.8-10 ~ daltons. Although this can be deduced f r o m the intensity of the bands, the digestions with other combinations of enzymes (see Fig. 5B, lanes d and i), allowed an unambiguous estimation of the molecular weight of these plasmids. Three of the plasmids described have B a m I fragments containing a m genes: pKG103 contains araC intact since it can c o m p l e m e n t an araC deletion; pKG110 contains most if n o t all of araD and p K G 1 6 0 contains the araBA fragment. The cloned DNA fragments are being used to sequence parts of araC or araBAD and in particular the controlling site regions between the araB and araC genes. The B a m I site is located 45 base pairs from the start site of the araBAD m R N A (L. Greenfield and G. Wilcox, unpublished dat~). The failure of this fragment to complement araB24 suggests t h a t this fragment lacks specific sequences required in vivo for initiation of araBAD mRNA. In addition the cloned fragments are very useful for DNAR N A hybridization studies, for example, plasmid p K G 1 0 3 will hybridize araC m R N A b u t n o t araBAD m R N A and the reverse is true with pKG160. ACKNOWLEDGEMENTS This research was supported by National Science F o u n d a t i o n grant PCM 76-80445. Lawrence Greenfield was supported by training grant CA-0905603 from the National Cancer Institute, Thomas Boone by the NRSA training program in Genetics and Regulatory Mechanisms, and Gary Wilcox by an American Cancer Society Faculty Research Award. These experiments were performed under NIH guidelines which call for EK1, P1.

REFERENCES

Boyer, H., Betlach, M., Bolivar, F., Rodriguez, R., Heyneker, H . , Shine, J. and Goodman, H., The construction of molecular cloning vehicles, in R, Beers and E. Basset (Eds.) Recombinant Molecules: Impact on Science and Society, Raven Press, New York, 1977, pp. 9--20. 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. Clewell, D.B., Nature of ColE1 plasmid replication in Escherichia coli in the presence of chloramphenicol, J. Bacteriol,, 110 (1972)667--676. Davis, R.W., Simon, M. and Davidson, N., Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, in S,P. Colowick and N,O. Kapl~ (Eds,)Method s in Enzymology, Vol. 21, Academic Press, New York, 1971, pp. 413--428, Englesbergi E, andWilcox, G., Regulation: positive control, Annu. Rev. Genet,, 8 ( 1 9 7 4 ) 219--241. ....... : Englesberg, E,, Irr, J,, Power, J. and Lee. N.. Positive control of enzyme synthesis by gene C in the L-arabinose system, J, Bacteriol., 90 (1965) 946--957.

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Gottesman, S. and Beckwith, J.R., Directed transposition of the arabinose operon: a technique for the isolation of specialized transducing bacteriophages for any Escherichia coli gene, J. Mol. Biol., 44 (1969) 117--127. Green, P.J., Betlach, M.C., Goodman, H.M. and Boyer, H., The EcoRI restriction endonuclease, in R.B. Wickner (Ed.) Methods in Molecular Biology, Vol. 7 Marcel Dekker, New York, 1974, pp. 87--111. Guerry, P., LeBlanc, D.J. and Falkow, S., General method for the isolation of plasmid deoxyribonucleic acid. J. Bacteriol., 116 (1973) 1064--1066. Hirsch, J. and Schleif, R., The araC promoter: transcription, mapping and inttmction with the araBAD promoter, Cell 11 (1977) 545--550. Kaplan, D.A. and Nierlich, D.P., Cleavage of nonglucosylated bacteriophage T4 deoxyribonucleic acid by restriction endonuclease EcoRI, J. Biol. Chem., 250 (1975) 2395-2397. Kaplan, D.A. and Wilcox, G., Horizontal slab gel electrophoresis of DNA, in G. Wilcox, J. Abelson and C.F. Fox (Eds.) Molecular Approaches to Eucaryotic Genetic Systems, Academic Press, New York, 1977, pp. 103--110. Kaplan, D.A., Greenfield, L. and Wilcox G., Molecular cloning of ~h8Odara restriction fragments with non-complementary ends, in G. Wilcox, J. Abelson and C.F. Fox (Eds.) Molecular Approaches to Eucaryotic Genetic Systems, Academic Press, New York, 1977(a), pp. 85--102. Kaplan, D.A., Russo, R. and Wilcox, G., An improved horizontal slab gel electrophoresis apparatus for DNA separation, Anal. Biochem., 78 (1977b) 235--243. Sinsheimer, R.L., Recombinant DNA, Annu. Rev. Biochem. 46 (1977) 415--438. Smith, D.E., Blattner, F.R. and Davies, J., The isolation and partial characterization of a new restriction endonuclease from Providencia stuartii, Nucl. Acids Res., 3 (1976) 343--353. Wilcox, G., Singer, J. and Heffernan, L. Deoxybribonucleic-ribonucleic acid hybridization studies on the L-arabinose operon of Escherichia co~i B/r, J. Bacteriol., 108 (1971) 1--4. Wilcox, G., Clemetson, K.J., Cleary, P. and Englesberg, E., Interaction of the regulatory gene product with the operator site in the L-arabinose operon of Escherichia coli, J. Mol. Biol., 85 (1974) 589--602. Wilson, G.A. and Young, F.E., Isolation of a sequence specific endonuclease (Bam~) from Bacillus amyloliquefaciens, J. Mol. Biol., 97 (1975) 123--125. Communicated by J. Carbon.