Note: Descriptions are shown in the official language in which they were submitted.
13213G4
HOECHST AKTIENGESELLSCHAFT HOE 87/F 333 J Dr. KL/mu
:
Specification
:i
Phosphinothricin-res;stance yene acti~e ~n plant~r ar4d ~ts ~se
5 C~Adian Pat~nt Applic~tion S~rl~l No. 545,037 propo~ pbo~-
phinothricin (PTC)-resistance gene which can be obtained
from the total DNA of ~
DS~ 40736 tgeneral collection) or DSM 4112 tdeposition `~.
under the Budapest Treaty), which has been selected for
phosphinothricyl-alanyl-alan;n~ (PTT)-resistance, by cut-
tin~ with ~amH$, cloning of a fragment 4.0 kb in size, and
selection ~or PTT-resistance, as ~ell as the use of this
~ene for the production of PTC-resistant pLants, as PTT-
resistance marker in bacteria and PTC-resistance ~arker
in plant cells. ~he aamHI fragment ~hich is 4 kb in size
and on ~hich the resistance g~ne is located is defined in
; detail b~ a restrictlon map (Figur~ 1 of Canadian P~tent Appliration
5eri~1 No. 545,037).
ZO
The position of ths coding region has been located ~ore accurately by
cloning part-regions of this 4 kb fragment. It emerged from this that the
resistance gene is located on the 1.6 kb SstII-SstI fragment (positions
0.55 ~o 2.15 in Fig. 1 of Canadian Patent Application Serial No. 545,037).
Digestion with BglII results in a fragment 0.8 kb in size ~hich confers
PTT-resistance after incorporation ~n a plasmid and transformation of S.
lividans. This resistance depends on the N-acetylation of PTC. Hence the
resistance gene codes for ~in acetyltr~nsferase.
The DNA s~qu~nc~ of th~ abovem~ntion~d 0.8 kb fr&g~nt i~ reproducad i~
C~n~d~ Patent ApplicAtion Seri~l No. 545,037.
I~ is possible to deter~ine fro~ the sequence the s~art
codon and the open reading frame of the ~ene sequence.
The ~ast nucleotide ii5 part of ~he stop codon ~GA~
Genes from Streptomycetes have a very high praportion of ~ :
G ~ C, the adenine SA3 ~ thymine (T) : guanine ~6) ~ ~
-`` 1321364
- 2 -
cytosine ~C) ratio being about 30 : 70. The proportion
of GC in plant genes is far lo~er, being about 50~. For
this reason, in a fureher development of the inventive
idea, the DNA sequence of the resistance gene has been
S optimized~ by de novo synthesis, to a codon usage favor-
able for plant RNA poly~eras~ II.
The lnv~lltloll r~l~t~ to ~ mod~f ~ tlon ~f the r~al~t~nce g~ wh~ch iLB
1~ prQpos~d in C~n~diAn P~tant Appl~c~tion Serial ~o. 545,037,
namely an adaptat;on eo th~ codon usage in p~ants. The
corresponding amino acid sequence is depicted in the annex.
~urther embodiments of the invention are defined in the
patent c~aims or are expLained hereinafter.
As is kno~n, the genetics code is degenerate, i.~. on~y
2 amino acids are coded for by a single tripletO where-
as the remaining 18 genetically codable amino acids are
assigned to 2 to 6 triplets. Thus, theoretically, a
~O ~;de;variety of:codon~-combinations-can be chosen for the
synthesis of the gen~. Since the said rel~tive propor-
~ion of the indiv;dua~ nucleotides in the total DNA se-
quence exerts an influence, it was used as one of the
criteria on ~hich the sequence optimi2ation ~as based.
The following ~odifi~ations ~ere ~ade tc the sequenced
gene: `
1~ The Streptomycetes gene stare codon GTG (posit;on
258-260 in ~he sequence in ~he additionaL applica-
tion) ~as replaced by the start codon ATG ~h;ch is
used by plant RNA poly~erase II.
2. ~ithin the gene, ~he Strepto~ycetes gene codons ~ere
changed in such a ~ay that they resu~ted in codons
suitable in p~ant ~enes ~6/G ratio~.
3. The TGA stop codon ~as placed at the end of the se-
. quence to termin~te the translation processO
The beginning and end o~ the gene sequence ~ere
provid~d ~ith protruding ends of restriction sites
in order to be ~ble to amplify the gene and li~ate
æ
.. . -. . ........... .............. .. ,.,,.. .. ,
- .... . ~ . .. , . . ; - ..
~32~
. .
3 --
~ it between plant regulation sequences. ~
5. Palindromic sequences were reduced to a minimum. ~:
Description of the Drawin~
The DNA sequence I according to the invention twith the
S corresponding amino acid sequence) i~ depicted in Figure
1 . '
Three internal un;que cleavage sites for the res~riction
enzymes XbaI ~position 152), BamHI (312~ and XmaI (436)
make possible ehe subcloning o~ part-sequences ~hich can
be incorporated in ~ell-inves~igated cloning vectors such
as, for example, pUC18 or pUC19. In addition, a number
of other unique recognition sequences for restr;ction
en~ymes ~er~ incorporated within the gene~ and these, on
the one hand, provide access to part-sequences o~ acetyl-
transferase and, on the other hand, allo~ mod;f;cat;ons
to b~ ~ade:
Restriction enzyme Sut after nucleotide ~o.
BspMII 11
SacII
EcoRV 74
HpaI S0
AatII 99
BstXI 139
ApaI 232
Sc~I 272
AvrII 30~
AflII 336
StuI 385
~ssHII 449
FokI 4~7
BglI 536
BgLIl 550
The construction of p~rt-sequences by chemical synthesis
and enzy~atic li~a~ion reactions is carrieid out in a
~anner kno~n per se ~EP-A Q,133,282, 0,136,472, 0,1559590,
: . , -, ::
132~ 3~4
- 4
0~161~SD4, 0,163,249, 0,171,024, 0,173,149 or 0,177,827
Deta;ls, such as restriction analyses, ligation of DNA
fragments and transformation of plasmids in E. coli, are
described a~ length in the textbook of Maniatis (Mole-
cular Cloning, Maniatis et al., Cold Spring Harbor,
1982)~
The gene sequence which has been cloned in this way is
then in~roduced ;nto plan~s, under the control of plant
regulation signals, and its expression is induced.
EP-A 0~122,791 revie~s known methodsO In this way are
obtained PTC-res;stan~ plant cells (i.e~ a selection
feature for transformed cells is available), plants or
parts of plants and seeds.
Some embodiments of the invention are explained in detail
in the examples ~hich follo~. Unless other~ise indica-
ted, percentage data therein relate to weight.
`- - 20 Examples -- - -
The follo~ing media were used:
a) for bacteria:
25YT medium: O.5X yeast extract, 0.8% Bacto tryp-
tone, 0.5% NaCl
LP medium: 0.5X yeast extractr 1% Bacto tryptone,
1X NaCl
as solid mediu~: addition of 1.5X agar to each
b) for plants:
M+S medium: see Mur~shige and Skoog, Physiologica
Plantarum 15 (19~2) 473
2MS medium: M~S medium containing 2% sucrose
MSC10 medium: M~S medium containing 2% sucrose~ 500 mg/l
cefotaxime, 0.1 mg/l naphthylacetic
acid (NAA), 1 mg/l benzylaminopurine
(~AP), 100 mg/l kanamyc;n
MSC15 medium: M+S medium containing 2% sucrose,
,. : . :, . ,
~ .
~3213~
-- 5
500 mg/l cefotaxime~ 100 mg/l kanamycin.
1. Chemical synthesis of a single stranded oligonucleo-
t;de
The synthesis of fragment II, one of the four part-
fragments I - IVD started from the terminal oLigonuc-
leotide IIc (nucleotide No~ 219 to 31Z ;n the coding
strand of DNA sequence I). For the solid-phase syn-
thes;s, the nucleoside at the 3' end, that is to say
guanosine (nucleotide No. 312) ;n ~he present case,
is covalently bonded via ~he 3'-hydroxyl group to a
support. The support material ;s CPG (controlled
pore glass) funct;onalized w;th long-chain amino
alkyl raclicals. Otherw;se, the syn~hesis follows
the known (from the Said EP_A~) methOdS.
The plan of synthesis is ind;cated in DNA sequ~nce
2a ~.I.-~annex~7-!*wh;ch-lothe.r~i.se-^~ror~re-spor~ds-:to.:DNA se-
quence I.
2~ Enzymatic linkage of the single-stranded oligonucleo-
tides to give gene ~ragment II
For the phosphorylat;on of the oligonucleotides at
the 5' end, 1 nmol of each Qf oligonucleotide~ IIb
and IIc was treated with S nmol of adenos;ne tr;phos-
phate and 4 un;ts of T4 polynucl~otide k;nase ;n
20 ~l of 50 mM tris-HCl buffer ~pH 7.6), 10 mM mag-
nesium chloride and 1Q mM dithiothreitol (DTT) at
37G for 30 m;nutes~ The enzyme ;s inactivated by
heating at 95C for 5 minutes. Oligonucleotides
IIa and IId, which form the "protrud;ng" sequence ;n
DNA fragment II, are not phosphorylated~ Th;s pre-
vents the formation, dur;ng the subsequent l;ga~;on,
of larger subfragments than correspond to DNA frag-
ment II.
.. . .
132~36~
-- 6 --
O~igonucleotides II (a-d) are ligated to give sub-
fragment II as follows: 1 nmol of each of oligonuc-
leotides IIa and IId and the 5'-phosphates of IIb
and IIc are together dijsolved in 45 ~l of buffer
containing 50 mM tr;s-HCl tpH 7.6), 20 mM magnesium
chloride, 25 mM potassium chloride and 10 mM DTT.
For the annealing of the oligonucleotides correspond-
ing to DNA fragm~nt II, the solution of the oligo-
nucle3tides is heated at 95C for 2 minutes and
then slowly ~ooled (2-3 hours) to 20C. Then, for
the enzymaeic Linkage, 2 ~l o~ 0O1 M DTT, 8 ~l of
2.5 ~M adenosine triphosphate (pH 7) and 5 ~l o~ T4
DNA ligase (2000 un;ts~ arY added, and the mixture
is incubated at 22C for 1b hsurs.
The gene fragment II is puri~ied by geL el~ctropho-
resis on a 10X Polyacrylamide gel ~ithout addition
of urea, Z0 x 40 cm, 1 ~m thick), ~he marker sub-
stance used b~;ng ~X 174 DNA (from ~RL) cut ~ith
- ?3 ~ -Hinfl,:or p~R3~2 cut:~ with -HaelII.
6ene fragm~nts I, III and IV are prep~red analogous-
ly, although the "protruding" sequences are, before
th~ annealin~, converted into the 53-phosphates be-
cause no l;gat;on step is necessary.
3. Preparat;on of hybrid plas~ids containin~ gene frag-
m~nts I, II, III and IV.
a~ Incorporation of gene frag~nt I in pUC18
Th~ commercially available Plasmid pUC1~ is opened
in a known ~anner using the restriceion endo nucLea-
ses SalI and XbaI in accordance ~ith the manufact-
ure~s' instructions. The digest;on mixtur~ is frac-
t;onated by el~ctrophoresis ;n a kno~n manner on a
1% agaras~ gelO and the fragmen~s are visuali~ed
by staining ~ith ethidium bro~ide~ The plasmid
band (about 2.6 kb) is then cut out of She agarose
~32~36~ :
-- 7
gel and removed from the agarose by electro-
elut;on.
1 ~g of plasmid, opened with XbaI and SalI, is
then ligated with 10 ng of DNA fragment I at 16C
overnight.
b) Incorporation of gene fragment II in pUC18.
In analogy to 3), pUC18 is cut open ~ith XbaI and
eamHI and ligated ~ith gene fragment II which
has previously heen ph~sphorylated at the protru-
ding ends as described in Exa-ple 2.
c~ Incorporation of gene fragmen~ III in pU~18
In analogy to a)~ pUC18 is cut open ~;th BamHI
and XmaIII and ligated with gene fragment III.
20~ d3 :In~orpora~--ion~o~;~gen~:~ragment.-IV--in pUC18
In analogy to a), pUC18 is cut ~ith YmaIII and
SalI and ligated wi~h ~ene fragmer,t IV.
Construction of the co~plete gene and cloning in a
pUC plasmid
a) Transformation and amplification of gene fragments
I - IY
The hybrid plasmids obtained ;n this way are
transformed into E. coli. For this purpose~ the
. ~
strain E. coli K 12 is made competent by treat-
-
ment with a 70 mM calciu~ chLoride soLu~ion, and
the suspension of the hybrid pLasmid in 10 mM
tr;s-HCl buffer (pH 7.5), which is 70 mM in cal-
cium chioride, is added~ The transformed strains
are selected as is customary, utiLizing the anti-
biot;c resis~ances or sensitivities conf~rred by
~2~6~
-- 8 ~
the plasmid, and the hybrid vectors are amplified.
After the cells have been killed, the hybrid plas-
mids are ;solated and cut open ~ith the restric-
t;on enzymes or;ginally used, and gene fragments
I, II, III and IV are isolated by gel electropho-
resis.
b) Linkage of gene fragments I, II, III and IV to
give the total gene
Subfragments I and II obtained by amplification
are linked as follows. 100 ng of each of the
isolated fragments I and II are dissolved togeth-
er in 10 ~l of buffer containing 50 mM tris-HCl
(pH 7.6), 20 mM magnesium chloride and 10 mM DTT,
and this solution is heated at 57C for 5 minutes.
After the solution has cooled to room temperature,
1 ~l of 10 mM adenosine triphosphate (pH 7) and
1 ~l of T4 ligase (400 units) are added~ and the
20 ~ mixt~re is incuba~-ed ~t-room~tempe-rature for 16
hours. After subsequent cutting with the restric-
~ion enzy~es SalI and BamHI, the desired 312 bp
fragment (nucleotides 1-312, SalI-~amHI) is puri-
fied by gel electrophoresis on an 8% polyacryl-
~5 amide gel, the warker substance used being ~X 174
RF DNA tfro~ 9RL~ cut with the restriction enzyme
H3eIII .
Gene frag~ents III and IV are linked together in
the same way, there being obtained after purifi-
cation a 246 bp fragment (nucleotides 313-558~
Ba~HI~SalI~ The marker used for the gel electro-
phoresis is pBR322 cut with the restriction en~
zyme MspI.
To contruct the total gene (DNA sequence I), 15 ng
of the 312 bp fragment and 12 ng of the 246 bp
fragment are ligated, as described above, with
1 ~9 of the commercially available plasmid pUC18
~3~3~
_ 9 _
which has previously been cut open with the rest-
riction enzyme SalI and enzymatically dephosphory-
lated at the ends. After transformation and ampli-
fication (as described in Example 4a), the correct
clone having the 558 bp fragment corresponding to
DNA sequence I is identified by SalI digestion~
5. Transformation of the hybrid plasmids
Cumpetent E. coli cells are transformed with 0u1 to
~ .
1 ~9 of the hybrid plasmid containing DNA sequence
I~ and are plated out on amplicillin-containing agar
plates. It is then possible to identify clones
~hich contain the correc~ly integrated sequences in
the plasmid by rapid DNA analysis (Maniatis loc.
cit.).
6. Fusion of the synthetic gene to regulation s;gnals
~hich are recognized in plants~
The optim;zed resistance gene ~hich had been provi-
ded at the ends with SalI cleavage sites was ligated
in the ~alI cLe3vage site of the polyl;nker sequence
of the plasm;d pDH51 (Pietrzak et al., Nucle;c Acids
Res. 14 (198b) 5857). The promoter and terminator
of the 35S transcript ~rom cauliflower mosaic virus,
which are recognized by the plant transcription appa-
ra~us, are located on th;s plasmid. The ligat;on
of the resistance gene resulted in it bein~ inserted
do~nstrea~ of the promoter and upstream of the termin
ator of the 35S transcript~ The correct orientation
of the gene ~as sonfirmed by restriction analyses.
The promo~er of the ST-LS1 gene from s~lan~m t~bero
sum (Eckes et al., Mol. 6en. Genet. 205 (1986) 14)
was likewise ~sed for the expression of the optim;zed
acetyLtransferase gene in plants.
. . . . . ...:
: ~ , . : - ~ . -
-: :, . ~. . . ....
`- ~3213~4
- 10 -
7. Insertion of the resistance gene having the regula-
tion sequences into Agrobacterium tu~efaciens
a~ Cointegrate method
The entire transcription uni~ comprising promoter~
optimized resistance gene and ter~inator (ExampLe
6) ~as cut out with the restriction enzyme EcoRI
and ligated in the EcoRI cleavage site of the
intermediary E. coli vector pMPK110 (Peter Eckes,
___
Thesis, Univ. Cologne, 19850 pages 91 et seqO).
This intermediary vector was necessary for the
transfer of the resistance gene ~ith its regu-
lation sequences into the Ti pLasmid of
Agrobacterium tumefaciens. This so-called conju-
gation ~as carried out by the method described
by Van Haute et al. (EMB0 J. 2 (1983~ 411). This
entailed the gene with its regulation signals be-
ing integrated in the Ti pLas~id by homoLogous
. ~''ZO . ~ tre.c~b.in-ati~on,.vi-a~the~,seqllenc.es of the standard
vector p9R322 ~hich are present in the pMPK110
vector and in the Ti plasmid pGV3850kanR (Jones
et al., EMBQ J~ 4 (1985) 2411
:
For this purpose~ 50 ~l of fresh liquid cultures
of each of the E. coli strains DH1 (hos~ strain
of the pMPK110 derivative) and GJ23 (Van Haute et
al., Nucleic Acids Res. 14 (1986) 5857) were mix-
ed on a dry YT-agar plate and incubated at 37C
for one hour. The bacteria ~ere resuspended in
3 ~l of 10 mM MgS04 and pLated out on antibiotic-
agar plates tspectinomycin 50 ~g/ml: selection
for pMPK110; tetracycline 1D ~g/ml: seLection for
R64drd11; kanamycin 50 ~g/ml: selec~ion for
pGJ28). The bacteria growing on the selective
agar plates contained the three plasmids and were
grown for the conjugation ~ith Agrobacteriu~ tume-
faciens in YT liquid medium at 37C. The Agro
bacteria were cultiva~ed in L~ medium at 28C.
.. . . ~ . . ::
~ ` 13 2 3 6
50 ~l of each bacterium suspension were mixed on
a dry YT-agar plate and ;ncubated at 28C fsr 12
to 16 hours. The bacteria were resuspended in
3 ml o~ 10 mM MgS04 and plated out on antib;otic
plates (erythromycin 0.05 g/l, chloramph~nicol
0.025 g/l: selection for the Agrobacter;um strain;
streptomycin 0.03 g/l and spectinomycin 0.1 g/L:
selection for integration of pMPK110 in the Ti
plasmid). Only ~grobacteria in which the pMPK110
derivative has been integrated into the bacterial
Ti plasmid by homologous recombination are able
to grow on the~e selected plates.
Besides the gene for resistance to the antibiotic
kanamycin, uhich is active in plants and was al-
ready present from the outset, the PTC-resistance
gene was no~ also located on the Ti plasmid
pGV3850kanR. 8efore these Agrobacterium clones
vere used for transformation, a Southern blot
exper~ment ~-as c~rried-out~to~checkYwhether the
desired integration had taken pLace~
~'
b~ Binary vector method
The binary vector system described by Koncz et
al. (Mol. Gen. Genet. Z04 t1986) 383) ~as used.
The vector pPCV701 described by Koncz et al.
tPNAS 84 t1987) 131) ~as modified in the follow-
ing way: the restriction enzy~es BamHI and HindIII
were us~d to delete from the vector a fragment
on which are located, inter aLia, the TR1 and
TR2 promoters. The resulting pla~mid ~as recir-
cularized. Into the EcoRI cleavage site present
on this vector was inserted a fragment from the
vector pDH51 which i about 800 base-pairs in
length and on which were located the promoter and
terminator of the 35S transcript from cauliflower
mosaic virus (Pietrzak et al., Nucleic Acids Res.
1~ (1986~ 5358). The resulting plasmid pPCV801
` 1321~
- 12 -
had a unique SalI cleavage site bet~een the 35S
promoter and terminator. The optimized PTC-
resistanc~ gene ~as inserted into this cleavage
site. Its expression ~as now under the control
of the 35S transcript regulation sequences. ~ ;
Thi~ plasmid tpPCV8~1Ac~ ~as transformed into the
E. coli strain SM10 tSimon et ~ io~Technology 1
t1983) 784). For the transfer of the plasmid
pPCV801Ac into ~ , 50 ~l of
both the SM10 culture and a CS8 Agrobact~rium cult-
ur~ tGV3101~ Van L~rebekP et al.~ Nature 252 (1974)
16~) were mixed ~ith the Ti plasmid pMP90RK ~Konc~ :
et al., Mol. Gen. Genet. 204 ~1986) 383)
as helper plasmid on a dry YT-
agar plate, and the mixtur~ ~as incuba~ed a~ 28C
for about 16 hoursO The bacteria ~ere then resus-
pended in 3 mL of 1 mM M9504 ~nd plated out on
antibiotic plates (rifampicin 0.1 ~ selection
for GV3101, kanamycîn 0.025 g~l: selection for
pMP90RK, c~rben-ic~t W n 0~1 g/l: selection for
pPCV801Ac)~ Only Agrobact~ria ~hich contained both
plas~ids SpMP90RK and pPCY801Ac) ~re abl2 to grow
: on th~se pL~tes~ Be~ore these Agrobacteria ~ere
used for th~ plant transformation, 50uth~rn blot- :
ting ~as carri~d out to check that the plasmid
pPCV801Ac is present in its correct for~ in the
Agrobacteria~
,
30 8. Transformation of Nlcotina tabacum by Agrobacterium
~!~ :
~ .
The o~timized resistance gene ~as transferred into
~oba co plants using the so-called leaf disk trans- -~
~o~ma~ign method~
The Agrobacteria were cultured in 30 ml of L~ medium
containing the appropriate antibiotics at 28C, ~hak-
ing continuously (about 5 days). The bacteria ~ere
th@n sQdimented by centrifugation at 700Q rpm in a
~3~13~
- 13 -
Christ centr;fuge for 10 minutes, and were washed
once with 20 ml of 10 mM MsS04. After a further
centrifugation, the bacteria were suspended ;n 20 ml
of 10 mM MgS04 and transferred into a Petri dish.
Leaves of ~isconsin 38 tobacco plants growing on 2MS
medium in sterile culture were used for the leaf
disk infection All the sterile cul~ur~s were main-
tained at 25 to 27C in a 16 hours light/8 hours
dark rhythm under ~hite light.
Tobacco leaves were cut off, and the leaf surfaces
were lacerated ~ith sandpapern After the laceration~
the leaves were cut into smaller p;eces and d;pped
;n the bacterium culture. The leaf pieces were then
transferred to M+S medium and maintained under nor-
mal culture conditions for two days. After the 2-
day ;nfect;on with the bacteria, the leaf p;eces
~ere ~ashed ;n liqu;d M~S medium and transferred to
MSC1Q-agar plates. Transformed shoots ~ere selected
-~ 20~ o~the;b-as.is of~ he-resist3nce~*0 the antib;otic
kanamycin which had also been transferred. The
first shoots became visibl~ 3 to 6 weeks later. In-
div;dual shoots ~ere further cultivated on MSC15
medium in glass jars. In ehe ~eeks which followed,
some of the shoots wh;ch had been cut off developed
roots at the site of the cut.
It was aLso possible to select transformed plants
directly on PTC-containing plant media. The presence
and the expression of the PTC-resistance gene was
demonstrated by DNA analysis (Southern blotting) and
RNA anaiysis ~Northern blotting) of the transformed
plants.
9. Demonstration of the PTC-res;stance of the transfor-
med plants
To check the funct;oning of ~he resistance gene in
transformed plants, leaf fragments from ~ransformed
,; : : .: ~
_ 141_
and non-transfor~ed plants were transferred to M-~S
nutrient ~edia containing 1 x 10 4 M L-PTC. The
fragments from non-transformed plants died, while
the fragments from transformed plants were able to
S regenerate new shoots. Transformed shoots took root
and grew ~ithout difficuLty on M~S nutrient media
containing 1 x 10 3 M L-PTC~ Transformed plants
were, from sterile conditions, potted in soil and
sprayed with 2 kg/ha and S kg/ha PTC. ~hereas non-
transformed plants did not survive this herbicide
~reatment, transformed plants showed no da~age brought
about by the herbicide. The appearance and gro~th
behavior of the sprayed transformed plants was at
least as good as that of unsprayed control plants.
10. Acetyltransferase assay to demonstrate acetylation
of PTC in transgenic PTC-res;stant plants
About 100 mg of leaf tissue from transgenic PTC-
-'~ ^ ?Z~.~'-,~,.,,, ~resistant;~tobacco~.pl-ants.~ rom~.n-on-t.rans.formed
tobacco plants were homogenized in a buf~er composed
of: 5Q mM tris-HCl, pH 7.5; 2 mM EDTA; 0.1 ~/ml
leupeptin; 0.3 mg/ml bovine seru~ album;n; 0.3 ~g/ml
DTT; 0.15 mg/ml phenyL~ethyLsulfonyl fluoride ~PMSF).
After subsequent centrifugation, 2n ~l of the clear
supernatant ~ere incubated with 1 ~l of 10 mM radio-
labe~L~d DrL-PTC and 1 ~l of 1U0 ~M acetyl-CoA at
37C for 20 minutesu 25 ~l of 1?% trichloroacetic
acid w~re then added to ~he reaction mixture, fol-
lo~ed by centrifugation~ 7 ~l of the supernatant were
transferred to a thin-layer chromatography pla~e and
subjPcted to ascending development twice in a mixture
of pyrid;ne : n-butanol : acetic acid : ~ater (50 : 75
: 15 : 60 parts by volume). PTC and acetyl-PTC were
separated ~rom one another in this way~ and could be
detected by autoradio~raphy. Non-transformed plants
exh;bited no conversion of PTC into acetyl-PTC, where-
as transgenic resistant plants were capabLe of this.
..
., .: :
: :: : . ~: : ~ -: -
:
- ~ ~ - :- ,. .
.
2~36~
- 15 -
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