Gene. 42 (1986) 69-77

Elsevier

69

GENE 1546

Cloning and expression of the lepidopteran toxin produced by ~aciZ~~s t~uringie~sis var. t~uri~gie~- sis in Escherichia coli

(Recombinant DNA; plasmid size determination; pACYC; restriction analysis; colony hybridization, bio-

assay)

Alik Honigman ‘*, Galit Nedjar-Pazerini”, Aminadav Yawetz b, Uri Oron ‘, Silvia Schuster b, Meir Broza b and

Baruch Sneh b

(Received June 17th, 1985)

(Revision received September lXth, 1985)

(Accepted November 28th, 198.5)

SUMMARY

The BaciZlus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to

larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis

zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli,

we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated

pTN4. Following partial digestion of the B. th~r~ngiensjs plasmid pTN4 and cloning into the E. co/i pACYC 184

plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids

pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BumHI DNA fragments of B. thuringiensis var.

thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain bet-finer 17 15 has been shown

to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences

have been found between pTNG33 and the DNA of B. thu~ngie~is var. e~tomocidMs strain 24, which

exhibited insecticidal activity against S. littoralis similar to that of strain 3A.

* To whom correspondence and reprint requests should be

addressed.

.Abbreviations: Ap, ampicillin; bp, base pair(s); Cm, chloram-

phenicol; EtdBr, ethidium bromide; kb, 1000 bp; Lt,,, time

required for 501; mortality; PA, polyacrylamide; R, resistant; ‘,

sensitive; SDS, sodium dodecyl sulfate; SPYG medium, see

MATERIALS AND METHODS, section a; Tc, tetracycline;

TE buffer, see MATERIALS AND METHODS, section b; TEA

buffer, see MATERIALS AND METHODS, section c; UV,

ultraviolet

INTRODUCTION

The Gram-positive bacterium B. thuringiensis pro-

duces a proteinaceous parasporal crystal. The crys-

tal of most of the serotypes of this species are toxic

to lepidopteran larvae (Burgerjon and Martouret,

1971; Ignoffo et al., 1977; Izhar et al., 1979; Sneh

et al., 1981), while serotype 14 is toxic to dipteran

larvae (Goldberg and Margalit, 1976). Several

strains of B. thuringiensis exhibit effective insecticidal

70

activity against the larvae of Spodo~tera littoralis,

Boisd (Egyptian cotton leafworm, Sneh et al., 1981).

B. thuringiensis var. thuringiensis strain 3A is effective

against this insect and a variety of other important

pests such as H. zeae, H. virescens and Boarmia

selenuria (Cohen et al., 1983).

The repeating subunit which composes the crystal

is thought to be a major component of the spore coat

(Lecadet et al., 1972; Aronson and Pandey, 1978).

The isolation of sporulation-deficient mutants which

fail to produce spores and crystals (Spo _ Cry _,

Klier and Lecadet, 1976) suggests that crystal pro-

duction might be linked to sporulation.

Strains of B. thuringiensis harbor a large array of

plasmids. The different strains vary in number and

size of plasmids (Debabov et al., 1977; Gonzalez

and Carlton, 1980). Several reports in the last few

years indicate that the gene(s) coding for the toxin is

located on one or more plasmids (Stahly et al., 1978;

Schnepf and Whiteley, 1981; Gonzalez et al., 198 1)

and a possible duplicate gene on the bacterial chro-

mosome {Held et al., 1982; Gonzalez et al., 1982;

Klier et al., 1982). The size of the plasmid involved

in the production of the crystal protein appears to

vary among the different species according to various

reports (Gonzalez et al., 1981; Held et al., 1982;

Gonzalez et al., 1982). The present work describes

cloning of the nucleotide sequence coding for a toxic

polypeptide from B. thuri~lgie~sis strain 3A. This

gene is located on a 17-kb plasmid. This study

demonstrates that the gene(s) coding for the toxin

proteins of B. th~rjizgiensis var. thuringiensis strain

beriiner 17 15 and B. thuringiens~s var. thuringiensis

strain 3A share common antigens and that in both

strains the gene is located on similar size plasmids.

MATERIALS AND METHODS

(a) Bacterial strains, plasmids and media

E. c&K-12(recA supE44m’r- (DR-100; Laban

and Cohen, 198 1) was used as the recipient for trans-

formation with the various plasmids. The B. thu-

ringiensis var. thuringiensis strain 3A was the same as

used in a previous study {Cohen et al., 1983). B. thu-

ringiensis strain berliner 17 15 was kindly provided by

Dr. R. Kalfon. For cloning experiments in E. coli,

plasmid pACYC184 (Chang and Cohen, 1978) was

used. B. thuringiensis var. entomocidus strain 24 was

isolated and described by Sneh et al. (1981).

Bacterial cultures were grown in L broth (1”;

Bacto tryptone, 0.5% yeast extract, l”/, NaCl) or on

LB agar plates (1.5 “/, agar). Cells transformed by

ApR, TcR, or CmR plasmids were seiected on

LB agar plates supplemented with either 100 @g/ml,

or 20 pg/ml or 10 pg/ml of the corresponding an-

tibiotics. For plasmid isolation, the bacteria were

grown in SPYG medium (Spizizen medium supple-

mented with 0.1 y0 yeast extract and 0.19/, glucose;

Spizizen, 1958).

(b) Plasmid isoiation from B. ?~~~~~~je~s~s

The isolation of plasmids from B. thuringiensis was

carried out by a slightly modified, rapid alkaline lysis

method (Birnboim and Doly, 1979). Overnight bac-

terial cultures were diluted to A 650 0.2 in one liter of

SPYG medium. The cells were grown, vigorously

shaken, and harvested at A,,,, 1.2-1.5. The sedi-

mented cells were resuspended in 20 ml of 257”

sucrose and 100 mg of lysozyme. After 30 min at

37”C, 40 ml of 1 ?/a SDS and 0.2 N NaOH were

added and the preparation was kept on ice for 5 min.

Then 30 ml of 5 M K. acetate pH 4.8 were added

and the mixture was kept on ice for 30 min. The

mixture was then centrifuged at 11000 x g for

20 min; the supematant was passed through cheese-

cloth and then mixed with 0.8 vol. of isopropanol

and stored overnight at -20°C. The mixture was

centrifuged at 11000 x gfor 1 h and the pellet resus-

pended in 15 ml of TE buffer (10 mM Tris, 1 mM

EDTA, pH 8). Solid NH,. acetate to a final concen-

tration of 2.5 M was added, mixed well and then the

mixture was placed on ice for 20 min. The pre-

paration was centrifuged at 12000 x g for 20 min,

and the supernatant was ethanol-precipitated. The

plasmid preparation was banded in a CsCl-EtdBr

gradient.

The CsCl-purified B. thuringiensis plasmid mixture

was cleaved with SalI, for better separation between

the various plasmids, and subjected to 0.5% low-

gelling-temperature agarose (Sigma agarose ty-

pe VII) electrophoresis. Individual DNA bands

were purilied as described by Weislander (1979); the

extraction of the DNA from the agarose was carried

out in presence of 0.5 mM NaCl.

71

(c) Agarose gel electrophoresis

Unless otherwise stated, gel electrophoresis was

performed in agarose type II (Sigma, St. Louis, MO,

USA) at concentrations of OS-0.7% (depending on

the sizes of the molecules to be resolved) using a

horizontal gel apparatus. Electrophoresis of the

DNA was carried out at loo-120 V for 4-5 h in

TEA buffer (30 mM Tris, 20 mM Na. acetate, and

2 mM EDTA adjusted to pH 7.8 with acetic acid).

(d) Hybridization procedures

The colony hybridization method described by

Grunstein and Hogness (1975) was used for selection

of clones. The transfer of DNA fragments to cellu-

lose nitrate sheets was described by Southern (1975).

The DNA probes were labeled with 32P according to

Maniatis et al. (1975). Hybridization was carried out

as previously described (Honing, 198 1).

(e) Western blot

Protein extracts were subjected to SDS-7.5-15:/i

PA gels. Following electrophoresis the proteins were

blotted to nitrocellulose filters and reacted with anti-

bodies made against purified toxin crystals of B. thu-

rirtgiensis strain 3A and iodinated protein A follow-

ing the procedure described by Towbin et al., 1979.

Protein size markers (BRL) were used to determine

the size of proteins.

To determine the number of plasmids in B. thu-

ringiemis strain 3A (to be referred to as 3A) we

employed two methods: (a) restriction enzyme ana-

lysis and (b) UV irradiation of plasmids in the

presence of EtdBr (Gonzalez and Carlton, 1980).

The analysis of the restriction enzyme cleavage pro-

duct presented in Fig. 1 is based mainly on cleavage

of purified DNA bands from low-gelling-tempera-

ture agarose following agarose gel electrophoresis.

The analysis ofthe results suggests that B. thuringien-

sis strain 3A carries at least three different plasmids.

1 2 3 4 5878

RESULTS AND DISCUSSION

(a) Analysis of the plasmid content of B. ~~u~~~g~e~-

sis strain 3A

B. ~~~~~~g~e~s~~ strains differ from each other in

their plasmid array. The number of plasmids and

their sizes vary from one strain to another. Agarose

gel electrophoresis of the plasmid mixture shows a

complex band pattern. This pattern results, in part,

from the fact that each plasmid can exist in three

conformations as well as in multimers. Moreover,

the various plasmids in each strain differ from each

other also in their copy number.

Fig. 1. Analysis of the plasmids of B. r~~r~ngje~~~ strain 3A. The

uncleaved total plasmid mixture was analysed by I Y. agarose gel

electrophoresis (supercoiled in lane 1). Following cleavage of the

3A plasmids with restriction enzyme Sal1 (lane 2) or EroRI

(lane 4), pTNl (band A), produces bands E and F, respectively;

bands G and I-1 are the products of double digestion with

Sull -c EcoRI (lane 3). pTN2 (band B) is linearized following

restriction with Sal1 (lane 2) or with EcoRI (lane 4) (bands D

and I). Double digestion with Sal1 + EcoRI gives rise to band J

(lane 3). pTN3 appears in lane 1 in its supercoiled and circular

relaxed form (bands C). This plasmid is linearized following

digestion with SalI, band L, and is cleaved more than twice with

EcuRI, generating a 16kb DNA fragment, band K (we can not

account for the missing 1 kb). hollowing d~~uble-digestion with

Ec<jRi + Salt, the cleavage products of pTN3 migrate together

with the cleavage products of pTN2 (band J). pTNGI2,

pTNG22. and pACYCl84 cleaved with EcoRI were applied to

slots 5,6 and 7, respectively. EDNA cleaved with Hind111 served

as size markers (lane 8).

72

The cleavage of the plasmid mixture with either

SnfI or EcoRI reveals that the smallest plasmid,

pTN1 (Fig. 1, lane 1, band A of supercoiled DNA),

generates in each case a linear molecule of about 5 kb

(lane 2, band E and lane 4, band F). Double di-

gestion of 3A with EcoRI + Sal1 leads to the

appearance of two DNA fragments derived from

pTN1 (bands G and H1 lane 3).

The DNA band B (lane 1) consists of a piasmid of

about 7.5 kb, designated pTN2. The plasmid pTN2

is cleaved by Sal1 and EcoRI (bands D and 1, re-

spectively). These two sites must be very close to

each other since double digestion with EcoRI + SafI gives rise to a DNA band with a size similar to the

products of each enzyme separately (band J, lane 3).

A

The plasmid of about 17 kb, designated pTN3

(band C in lane 1) is cleaved once with Sal1 to give

rise to band L (lane 2) and at least twice with EcctRI

to generate a DNA band of about 16 kb (band K,

lane 4) and probably other DNA bands which we

could not resolve on this gel. The Sal1 restriction

enzyme cleaves within the 16-kb EcoRI fragment at

about 8 kb from two EcoRI sites to give rise to two

DNA bands of about 8 kb each which migrate

together with the linear pTN2 molecules (band J,

lane 3). The two faint bands under and above

band C in lane 1 may represent various forms of the

plasmids described above, since we cannot assign

any DNA bands following rest~ction enzyme cleav-

age to these bands. The identification of plasmid

B 12345 6

c

a

Fig. 2. Hybridization ofpTNG.13 to DNA ofvarious R. ~hwi~gierrsis strains. Purified plasmids from strains 3A, 24 and herliner I71 5 wcrc

subjected to O.S”, agarose electrophoresis (panel A: lanes I. 3 and 5, respectively). The BumHI cleavage products of these plasmids

are presented in panel A, (lanes 2, 4 and 6, respectively). The DNA bands were transferred to nitrocellulose filters (Southern, 1975)

and hybridized to [“P]pTNG33. Following autoradiography (panel B) it could be seen that pTNG33 hybridized to the pTN3 supcr-

coiled (a), open circle (b) and the linear molecules of 14 kb (c). The DNA of pTNG33 hybridized to the same size DNA molecule of

strains 3A (lanes I and 2) and her&w 171.5 (lanes 5 and 6) but did not hybridize to strain 24 plasmids (lanes 3 and 4). I DNA cleaved

with Hind111 served as size markers (not shown).

pTN3 and its restriction products were verified by

the hybridization experiment of pTNG33 (a sub-

clone of pTN3, see next section) to various restric-

tion enzyme digestion products of 3A (Figs. 2 and 3).

Analysis of agarose gel electrophoresis patterns of

UV-irradiated plasmids in the presence of EtdBr

(Gonzalez and Carlton, 1980) supports the analysis

of the restriction enzyme mapping (results not

shown).

Determination of plasmid content by the method

described by Eckhardt (1978) and modified by

Gonzales et al., (198 1) revealed five additional large

DNA bands (60 kb and larger). These DNA bands

were not seen in the CsCl-EtdBr-purified plasmid

preparation. It is not clear to us whether these DNA

bands represent multimers of the three plasmids

described above or large plasmids that were lost

during plasmid purification.

Strain 3A was compared with strain bet-liner 1715

and B. thuringiensis strain 24 for their plasmid

content (Fig. 2, lanes 1, 3 and 5, respectively; see

Klier et al., 1982 for plasmids of berliner 1715).

13

Strain berliner 1715 possesses two DNA bands in

addition to those present in the plasmid preparation

of strain 3A, while strain 24 shares less common

bands with strain 3A. The restriction pattern of the

plasmids of 3A and berliner 17 15, following digestion

with various restriction enzymes (HindIII, EcoRI,

Sal1 or BamHI) demonstrates that strains 3A and

berliner 1715 share many common-size DNA frag-

ments (Figs. 2 and 3).

(b) Cloning of B. thuringiensis plasmids into E. coli

The plasmid mixture of B. thuringiensis strain 3A

was digested with EcoRI and ligated to the E. coli

plasmid pACYCl84 cleaved with EcoRI. Following

transformation of E. coli DRlOO, transformants

were selected on LB agar plates containing

20 pgTc/ml. The transformants were tested for

growth on an LB agar plate containing 10 pg Cm/ml.

Among the transformants analysed, four clones

contained the complete pTN 1 plasmid inserted into

B 1 2345678

Fig. 3. Hybridization ofpTNG33 to various restriction cleavage products ofB. thuringiensis strains 3A and herliner 1715 DNAs. Purified

plasmids of strains 3A and herher 1715 were cleaved with EcoRI (lanes 1 and 2, respectively), Sal1 (lanes 3 and 4). HirrdlII (lanes 5

and 6) and with SsrI (lanes 7 and 8). The cleavage products were separated on a O.joO agarose gel (panel A). Following blot hybridization

to [“PIprobe pTNG33 and autoradiography (panel B). it could be seen that the probe hybridized to the same cleavage products of the

two plasmid preparations: a I&kb EcY>RI DNA, 17.kb StrlI, 3.5-kb ffi?fdIII, and 5.5-kb Ss11 fragments (indicated on the margins).

pACYCl84, in either of the two possible orientations

(one such hybrid plasrnid, pTNG12, cleaved with

EcoRI is presented in Fig. 1, lane 5), and two clones

carried pTN2 (one such plasmid, pTNG22 is pres-

ented in Fig. 1, lane 6). Using a similar approach, we

constructed hybrid plasmids composed of

pACYC184 and pTN1 cleaved with restriction

enzyme Sal1 and inserted into the Sal1 restriction

site of pACYC184.

To clone the large pTN3 plasmid, the SalI- linearized DNA molecule was purified following

low-gelling-temperature agarose gel electrophoresis.

The DNA was partially cleaved with Sau3A, and

ligated to pACYC184 at the BarnHI cleavage site.

Bacterial clones were selected by colony hybridiza-

tion to [a-32P]CTP-labeled pTN3 plasmid and by

the CmRTcS phenotype. The clones that hybridized

to DNA fragments of pTN3 were further analyzed

by agarose gel electrophoresis. Out of 250 positive

colonies, 18 clones that carried a DNA insert larger

than 1 kb were further purified, and extracts were

prepared for toxicity bioassay.

(c) Toxin synthesis in the E. coli clones

Neither of the E. coli clones carrying hybrid plas-

mids derived from pTN 1 or pTN2 showed any toxi-

city toward the 2nd-instar larvae of S. littoralis. Three clones derived from pTN3, pTNG35,

pTNG33, and pTNG31 exhibited toxicity towards

the larvae. Two clones, pTNG33 and pTNG35, were

more active (Lt,, = 2 days) than the third clone,

pTNG31, Lso = 4 days; Fig. 4). Restriction enzyme

analysis of the three recombinant plasmids,

pTNG33, pTNG35, and pTNG31, revealed that

these plasmids carry inserts of about 1700,560O and

5700 bp, respectively. Comparison of the electro-

phoretic mobility of several restriction fragments

revealed that the three hybrid plasmids share some

similar size bands (not shown). Since pTNG33

carried the shortest DNA insert that expressed toxin

activity, further analysis was carried out with this

plasmid.

(d) Identification of plasmids and restriction DNA fragments bar~ring the toxin gene

The various strains of B. thuringiensis differ in their

insecticidal activity against various lepidopteran lar-

3A

Days after treatment

Fig. 4. Insecticidal activity of B. rhuringiemis and E. coli clones.

These killing curves represent one typical experiment of several

performed. Cultures of E. coli clones were grown overnight in

LB containing 5 peg Cmjmi. Cells were harvested by centrifu-

gation for 10 min at 100~0 rev./mm (Sorval SS34 rotor) and

washed twice with saline solution (0.859,, NaCl). Cells of 53. thu-

rin,@:imsis strain 3A were grown as described by Sneh et al. (198 1).

All cultures of the E. co/i clones (at the same absorbance) were

concentrated l(K)-fold to a volume of 10 ml and lysed by freezing

and thawing and sonication (total sonication time 4 mm with

pulses of 30 s). The sonic&es were then centrifuged 30 min at

12000 rev.jmin (Sorval 5534 rotor) and the pellets were

resuspended in 2 ml distilled water. For the bioassay (agar

method), 2nd~instar larvae (ten larvae per dish, four dishes per

treatment) were starved for 18 h and then placed on an agar

medium as previously described (Yawetz et al., 1983). Purrtied

crystals (open circles) at a concentration that kiils loo”,, of the

larvae in two days and cell extract of pTNG22 (filled-in squares)

which show no killing effect were used as controls. The

Lt,,, values were derived from these curves for comparison of the

toxic activity of the various extracts prepared from E. coli carry-

ing pTNG31 (open squares), pTNG33 (filled in triangles), and

pTNG35 (open triangles). Percent mortalities up to two days

(dotted lines) were extrapolated.

vae. The number and size of the plasmids vary from

one strain to the other. The toxin gene was found to

be located, in many cases, on a large plasmid of more

than 45 kb (Schneff and Whiteiey, 1981; Klier et al.,

1982; Held et al., 1982). In spite of the differences in

toxin specificity and the different locations of the

genes coding for the toxin in various species, it was

shown that many of the toxin genes cross-hybridize

(Schneff and Whiteley, 1981). These results indicate

that these genes may share common nucleotide

sequences.

TABLE I

Plasmids used in this study

Plasmids Size (kb) and Source or reference

other properties

pTN1 5 “-5.5 b B. rhuringiensis 3A

(our collection)

pTN2 7.4 “-8.0 b B. thuringiensis 3A

(our collection)

pTN3 17b B. thuringiensis 3A

(our collection)

pACYC184 4.0 a E. cofi (Chang and

Cohen, 1978)

pTNGl1, pTNG12

pTNG13, pTNG14

pTNG2 1, pTNG22

pTNG3 1

pTNG33

pTNG35

9.5 a, A c,d, TcR This work

9.5 a, B ‘.“, TcR This work

12”, TcR This work

9.7 ‘,j, CmR This work

5.7p.‘, CmR This work

9.6 h,‘, CmR This work

a The size (kb) was determined by agarose gel electrophoresis.

b The size (kb) was determined by electron micrographic

measurements.

’ The orientation of insertion relative to the promoter of the

CmR gene is arbitrarily designated as A and B.

’ 4 kb of pACYC and 5.5 kb of pTN1.

e 4 kb of pACYC and 8.0 kb of pTN2.

’ 4 kb of pACYC and 5.7 kb of Sau3A DNA fragment of pTN3.

8 pACYC plus 1.7 kb of Sau3A DNA fragment of pTN3.

’ pACYC and 5.6 kb of Sau3A fragment derived from pTN3.

I Hybrid plasmids coding for the B. thuringiensis endotoxin gene.

The three B. thuringiensis strains, berliner 1715,24, and 3A, are all toxic to larvae of S. littoralis, but differ

in their activity. Strain 24 is the most active against

these larvae, strain berliner 1715 the least. Strain 3A

is closer to strain 24 in its insecticidal activity (B.S.,

unpublished results). The number and size of the

plasmids of strain 3A, however, is more similar to

that of strain berliner 1715 (Fig. 2). Surprisingly,

plasmid pTNG33 did not show any hybridization to

DNA fragments of strain 24 plasmids but did hyb-

ridize with the same efficiency and to the same DNA

bands originating from strains 3A and berliner 1715 (Figs. 2 and 3). DNA of pTNG33 hybridized to the

pTN3 plasmid (Fig. 2, lane l), as well as the 14-kb

BamHI, 17-kb SalI, 16-kb EcoRI, 3.5-kb HindUI,

and approx. 6.5-kb SstI fragments (Figs. 2 and 3). It

is clear from these results that pTNG33 hybridizes

to plasmid and cleavage products of the same size of

both strains berliner 1715 and 3A. Hybridization to

15

the same size DNA fragments of berliner 1715 plas-

mid digest was observed by Klier et al. (1982) using

strain berliner 1715 4-kb DNA probe isolated from

a toxin-producing E. coli clone, pBT-15-88.

As mentioned above, the location of the endotoxin

gene in strain berliner 1715 was localized by Klier

et al. (1982) on very high M, plasmids (more than

45 kb). However, in strain 3A we located this gene

on a much smaller plasmid. We demonstrated that

the nucleotide sequence from strain 3A which cross-

hybridized with the various restriction fragments

from the coding region of the toxin gene of strain

berliner 1715 hybridized to a plasmid of about

17 kb in both strains. Possibly, the discrepancy

between our results and previous reports derived

from differences in plasmid isolation techniques, or

differences in growth conditions. For instance, the

AB C

Fig. 5. Immunoautoradiograph of blotted proteins of B. thu-

ringiensis strains 3A and berliner 1715 (lanes A and B, respec-

tively) reacted with antibodies made against purified toxin

crystals of strain 3A. The purified 3A toxin proteins were applied

in lane C. The numbers at the side ofthe picture indicate the M,s

(in kDa).

16

high M, plasmids may represent multimers of smaller

plasmids that have been generated by recombination

between identical plasmids, or homologous regions

shared by different plasmids.

In any event, our results suggest that the nucleo-

tide sequence of the 1.7-kb DNA fragment in

pTNG33 is closely related to those of the toxin genes

in strains berliner 1715 and kurstaki HD-1 (for

similarity with the kurstuki strain, see Klier et al.,

1982). But some significant differences must exist

between these sequences, since these strains differ in

their specific toxicity towards S. littoralis (Sneh

et al., 1981; B.S., unpublished results).

Western blot experiments using antibodies pre-

pared against the purified crystals of strain 3A indi-

cated that these antibodies cross-reacted with the 68

and approx. 120-kDa proteins made by both strains;

3A and berliner 17 15 (Fig. 5).

(e) Conclusions

The results of this study suggest that the toxin

genes of various strains of B. thuringiensis may share

common sequences and differ in their toxic activity

(strain 3A vs. berliner 17 15), or have similar toxic

activity but share very little, if at all, common se-

quences (3A vs. 24).

ACKNOWLEDGEMENTS

We would like to thank Dr. R. Kalfun for strains

of B. thuringiensis. The help of Darlene White in

bringing this manuscript to its final form is also

appreciated. This work was made possible partially

by a grant from “Biotechnology Application, Israel.”

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Communicated by R.E. Yasbin