Process for the isolation and characterization of a gene enzyme system for inactivation of the herbicide phenmedipham and transfer of the gene into plants to produce herbicide-tolerant plants

A process for the isolation and characterization of a gene enzyme system for the inactivation of the herbicide phenmedipham, wherein the enzyme is a carbamate hydrolase of Arthrobacter oxidans, which is responsible for the cleavage of the carbamate bond between the benzene rings of phenmedipham. This process includes the isolation of the carbamate hydrolase, the identification of the amino acid sequence of two BrCN cleavage peptides of the carbamate hydrolase, the synthesis of oligonucleotides for specific determination of the carbamate hydrolase sequence by hybridization and identification of the coding region, cloning and specifying the nucleotide sequence of the carbamate hydrolase gene from Arthrobacter oxidans.

FIELD OF THE INVENTION 
The present invention relates to a process for the isolation and 
characterization of a gene enzyme system for the inactivation of the 
herbicide phenmedipham and transfer of the gene into plants to produce 
herbicide-tolerant plants. The enzyme is a carbamate hydrolase of 
Arthrobacter oxidans, which is responsible for the cleavage of the 
carbamate bond between the benzene rings of phenmedipham which is the 
common name for the herbicide methyl 
3-m-tolylcarbamoyloxyphenyl-carbamate. 
BACKGROUND OF THE INVENTION 
In practice it is often necessary to use several herbicides or herbicide 
mixtures to combat various weeds. These problems can be avoided by a 
biotechnical change to the plant in which resistance to a non-selective 
herbicide is introduced. 
The production of herbicide-tolerant plants is now coming more to the 
foreground in the plant protection area. 
In order to produce herbicide-tolerant plants, it is necessary first to 
have a process for the isolation and subsequent purification of an enzyme 
which is able to inactivate a herbicide, e.g. by metabolism, and then to 
have a process for characterizing the gene enzyme system containing the 
DNA sequence that codes for the active enzyme. After these steps the DNA 
sequence of the gene can be transferred into plants. 
Such a process for the isolation and subsequent characterization of a gene 
enzyme system which can inactivate phenmedipham and the transfer of this 
gene enzyme system into plants was not previously known. 
SUMMARY OF THE INVENTION 
It has now been found that a carbamate hydrolase can be isolated from some 
microorganisms, such as Arthrobacter oxidans, which is responsible for the 
hydrolysis of the carbamate bond between the two benzene rings of 
phenmedipham. The hydrolysis of this bond leads to herbicidally inactive 
compounds such as methyl 3-hydroxyphenylcarbamate and meta-toluidine, 
according to the following reaction: 
##STR1## 
DETAILED DESCRIPTION OF THE INVENTION 
For isolation and subsequent purification of a gene enzyme system which can 
hydrolyse phenmedipham according to the above described reaction, 
microorganisms of Arthrobacter oxidans are cultivated in a nutrient 
medium. The carbamate hydrolase responsible for the cleavage of 
phenmedipham is isolated by ultrasound cell destruction, centrifugation 
and purification by anion exchange chromatography, gradient elution, 
ammonium sulphate precipitation and FPLC separation until electrophoretic 
homogeneity is achieved. From the purified carbamate hydrolase, two 
peptides are isolated after BrCN cleavage whose sequence can be estimated 
by Edman degradation. According to the sequence information of these 
peptides, oligonucleotides can be synthesized which can be used as 
hybridization probes for the detection of the carbamate hydrolase gene. 
In all of the mentioned isolates of the soil bacteria Arthrobacter oxidans, 
plasmids could be detected after lysis of the cells and extraction of the 
nucleic acids. 
For the species Arthrobacter oxidans P52, it can be shown that the 
carbamate hydrolase is coded by one plasmid. 
With the loss of the plasmid pHP52 the properties of this species to be 
able to hydrolytically cleave phenmedipham is lost. Carbamate hydrolysis 
activity cannot be biochemically established in the plasmid-free 
derivative of the species P52. 
The plasmid pHP52 can be preparatively isolated and mapped with restriction 
endonucleases. The electrophoretically separated restriction fragments can 
be transferred onto membrane filters and hybridized with the 
oligonucleotides. From the data from various blot-hybridizations, it 
appears that the carbamate hydrolase gene is localized on a PstI 
restriction fragment with a size of 3.3 kb. 
This fragment can be preparatively isolated from the plasmid pHP52 and 
inserted in the PstI position of the vector pUC19C (Yanish-Perron, C., 
Vieira, J. & Messing (1985) Gene 33, 103 ff). After transformation of E. 
coli DH5.alpha. with the ligation preparation, two types of recombinant E. 
coli clones are obtained (pp52Pst and pp52Pst inv.) which contain the 
carbamate hydrolase gene in different orientations to the Lac-promoter of 
the vector (see FIG. 5). 
The carbamate hydrolase can be functionally expressed in the presence of 
the inducer isopropyl-.beta.-D-thiogalactopyranoside from cultures of the 
clone of the type E. coli DH5.alpha. (pp52Pst). In protein extracts of 
clones of the type E. coli DH5.alpha. (pp52 inv.) which contain the 
carbamate hydrolase gene in inverse orientation to the Lac-promoter, no 
expression of the carbamate hydrolase gene can be detected. 
The nucleotide sequence of the carbamate hydrolase gene can be determined 
according to the method of Sanger et al. (Sanger, F., Niclen, S. & 
Coulson, A. (1977), Proc. Natl. Acad. Sci, USA 74, 5463-5468). 
15 sub-clones arising from the cloning of 3.3 kb long PstI restriction 
fragments can be constructed in the single strain DNA bacteriophages M13 
mp 18 and mp 19 (Messing, J. (1983) Methods in Enzymol, 101, 20-78). In 
FIG. 6 an extract restriction map of the coded area is represented from 
which the sequencing strategy can be seen. 
The established nucleotide sequence (Seq ID No. 7) with the protein 
sequence thus derived is illustrated in FIG. 7 (Seq ID No. 6). The amino 
acid sequences of both established BrCN-splitting peptides (see example 4) 
can be identified in the same reading frame as the DNA level. This reading 
frame ends with a TGA translation stop codon (see FIG. 7 (Seq ID No. 6), 
nucleotide positions 1789-1791) and begins very probably with a GTG-start 
codon (FIG. 7 (Seq ID No. 6)--nucleotide positions 310-312). Altogether a 
reading frame of 1479 base pairs results. Upstream of the putative 
GTG-start codon, a region with significant homology to the consensus 
sequence for E. coli ribosome binding sites ("Shine-Dalgarno Box") can be 
established (see FIG. 7 (Seq ID No. 6)), nucleotide positions 298-302). 
After the nucleotide sequence of the carbamate hydrolase gene has been 
determined, the construction of plasmids for the expression of carbamate 
hydrolase in plants can be carried out (see Example 9). For this the 
chimeric carbamate hydrolase gene on plasmids can be transferred from E. 
coli to Agrobacterium tumefaciens and from Agrobacterium tumefaciens to 
the target plants (see Example 10). The operation of the carbamate 
hydrolase gene in transformed plants can be shown by spraying transformed 
and untransformed plants with phenmedipham (see Examples 11 and 12). 
On the Aug. 25, 1987 the following micro-organisms were deposited at the 
German Collection of Microorganisms (DSM) in Gottingen, Germany. 
______________________________________ 
Arthrobacter oxidans 
P 16/4/B (DSM 4038) 
Arthrobacter oxidans 
P 67 (DSM 4039) 
Arthrobacter oxidans 
P 75 (DSM 4040) 
Arthrobacter oxidans 
P 11/1/-b (DSM 4041) 
Arthrobacter oxidans 
P 15/4/A (DSM 4045) 
Arthrobacter oxidans 
P 21/2 (DSM 4046) 
Arthrobacter oxidans 
P 52, containing 
(DSM 4044). 
the plasmid pHP52 
Abbreviations 
DEAE Diethylaminoethyl 
FPLC Fast protein/peptide/polynucleotide 
liquid Chromatography 
SDS Sodium lauryl sulphate 
DTT Dithiothreitol 
1 .times. SSC 0.15 M NaCl 0.015 M trisodium citrate 
pH 7.0 
1 .times. Denhardt 
0.02% (w/v) Bovine serum albumin 
(Sigma, Fraction V) 
0.02% (w/v) Ficoll 400 
0.02% polyvinylpyrrolidone 
MCS Multiple cloning site 
Abbreviation for restriction endonucleases 
Bm = BamHI, Bs = BstEII, CL = ClaI, EV = EcoRV, 
HII = HindII, Kp = KpnI, Nc = NcoI, Nd = NdeI, Nh = NheI, 
Ps = PstI, PvI = PvuI, PvII = PvuII, Sc = SacI, 
Sp = SphI, St = StuI, Xb = XbaI. 
______________________________________

EXAMPLE 1 
Isolation of microorganisms that possess the ability to inactivate the 
herbicide phenmedipham. 
To identify microorganisms which possessed the ability to inactivate the 
herbicide phenmedipham by metabolism, various microorganisms were 
screened. As source for the microorganisms, soil samples from various 
locations (field test sites which had been treated several times with 
phenmedipham), and also from settling sediment, were used. Selection 
criteria for the identification of microorganisms which can carry out a 
carbamate cleavage, were as follows. 
a) Growth in a nutrient medium with phenmedipham as a single carbon or 
nitrogen source. 
b) Breaking down of phenmedipham to highly water soluble compounds 
according to the following reaction. 
##STR2## 
From the large number of microorganisms obtained from the soil samples, 
which were capable of cleavage of phenmedipham, seven representatives were 
chosen which clearly showed a breakdown. These soil bacteria which are all 
representatives of the Arthrobacter species and within this species, the 
sub-species of oxidans, were cultivated in culture broths containing a 
synthetic medium (M9-medium) having the following composition: 
1.0 g/l NH.sub.4 Cl 
0.25 g/l MgSO.sub.4 .multidot.7H.sub.2 O 
3.0 g/l KH.sub.2 PO.sub.4 
7.0 g/l Na.sub.2 HPO.sub.4 .multidot.2H.sub.2 O 
2.0 g/l Glucose 
and 0.5 g/l NACl. 
The M9 medium, in addition, contained 1 mg/l thiamine (vitamin Bi) as well 
as trace elements which were added in the form of a stock solution (1 ml/l 
M9-medium). The trace element stock solution contained: 
0.5 Boric acid 
0.04 g/l CuSO.sub.4 .multidot.5H.sub.2 O 
0.2 g/l FeCl.sub.3 .multidot.6H.sub.2 O 
0.4 g/l MnSO.sub.4 .multidot.7H.sub.2 O 
0.4 g/l ZnCl.sub.2 
0.2 g/l (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 .multidot.4H.sub.2 O 
For shaking cultures in liquid mediums, the synthetic medium was 
supplemented by 0.1% casamino acids (Difco.RTM.). 
The soil bacteria were incubated in this M9 medium at 28.degree. C. with 
good aeration until the end of the logarithmic growth phase. For enzyme 
purification, a total of 10 litres of medium were inoculated with a 
stationary pre-culture (1:100). 
By HPLC analysis of the culture broth it was shown that in the cultures of 
Arthrobacter oxidans, the desired cleavage of phenmedipham to the 
herbicidally inactive products was achieved. 
EXAMPLE 2 
Isolation and purification of the carbamate hydrolase from Arthrobacter 
oxidans. 
The isolation and subsequent purification of the carbamate hydrolase to 
electrophoretic homogeneity was carried out over a six stage purification 
process. From 6 litres of an end logarithmic culture of Arthrobacter 
oxidans (pHP52) (DSM No. 4044), 0.5-1 mg carbamate hydrolase was 
reproducibly isolated. For isolation of the carbamate hydrolase, the cells 
were harvested by centrifugation (7000.times.g) and resuspended in about 
40 ml of decomposition buffer (10 mM sodium phosphate pH 6.8/1 mM DTT). 
The cell suspension was disrupted by ultrasound and homogenized at the 
same time. The homogenate was then centrifuged for 45 minutes at 
40000.times.g, at 4.degree. C. The sediment was removed and the 
supernatant, equilibrated with 100 mM Tris-HCl pH 7.2/100 mM NaCl/1 mM 
DTT, was applied to a DEAE Sephacel column (column diameter 2.6 mm, height 
of the gel bed 20.5 cm, column volume about 100 ml). Before application to 
the column, the cell extract was diluted at a ratio of about 1:10 with 
starting buffer (100 mM Tris/100 mM NaCl/1 mM DTT). The column was then 
washed with starting buffer in order to remove the unbound material. The 
carbamate hydrolase was then eluted with a linear gradient 100 mM 
NaCl--500 mM NaCl (5.times.column volume). The enzymatically active 
fractions were pooled and treated with dry ammonium sulphate 
(NH.sub.4).sub.2 SO.sub.4 to an end concentration of 33% of the saturated 
solution. The resulting protein precipitate was sedimented by 
centrifugation (20000.times.g/30 mins) and discarded. The supernatant was 
treated with solid ammonium sulphate to an end concentration of 60% of the 
saturated solution and stirred for about 12 hours at 0.degree. C. The 
sedimented protein was collected by centrifugation (20000.times.g/30 mins) 
and dissolved in about 1 ml starting buffer, treated with 10% (w/v) 
saccharose and loaded to a Sephacryl S-300 column. The gel filtration was 
carried out at a flow rate of 2.5 cm/h (elution buffer--: start buffer). 
The column had a diameter of 2.6 cm, a height of 95 cm and a volume of 475 
ml. The enzymatically active fraction was then worked up on an FPLC column 
(mono Q HR 5/5: anion exchange). Gradient elution 100 mM NaCl--: 300 mM 
NaCl; flow rate: 0.5 ml/mm; application volume 2 ml). The unbound protein 
was separated by isocratic elution with 19 ml starting buffer. (Gradient 
elution 100 mM NaCl--: 300 mM NaCl in 20 ml with 100 mM Tris/HCl pH 7.2/1 
mM DTT). 
The enzymatically active fractions were concentrated by ultra-filtration 
after electrophoretic analysis of the purity (SDS-polyacrylamide-gel 
electrophoresis by the method of Lammli), using an Amicon.RTM., Centrikon 
10 concentrator, and put on a FPLC gel-filtration column (Superose 6-HR 
10/30, Pharmacia) (flow rate 0.2 ml/min: application volume 100 ul: 
eluent: 100 mM Tris/HCl pH 7.2/10 mM NaCl). 
The active protein fractions which result from this step are 
electrophoretically homogenous. 
The isolated enzyme is active in buffered solutions (i.e. buffers 
conventionally used in biochemical systems, such as phosphate buffers, 
Tris buffers etc; pH 6.8). Co-factors or metal ions are not necessary for 
the reaction. A sensitivity against SH reagents is also not seen. The 
optimum pH of the enzyme is 6.8. 
The molecular weight of the carbamate hydrolase is in the range of 50-60, 
preferably 53-57 kd, both under denaturing/dissociating conditions (SDS 
gel electro-phoresis) as well as under native conditions (gel filtration). 
From this it follows that the carbamate hydrolase is a monomeric protein. 
The isoelectric point of the carbamate hydrolase is at pI=6.2. 
EXAMPLE 3 
Process for detecting the carbamate hydrolase. 
For a quick and sure determination of enzyme activity during the 
purification of the crude protein extracts, an in vitro enzyme test was 
developed. The test is based on the ability of the enzyme to change the 
highly water insoluble phenmedipham into a soluble hydrolysis product. For 
this, solid phenmedipham was suspended in water and micronised by 
ultrasound. This micro-suspension was then poured, with stirring at 
50.degree. C., into an agarose solution and this mixture put into a petri 
dish before it solidified, where it formed into a turbid gel matrix. The 
enzyme solution was then put into wells which had been punched in the 
solid matrix. After incubation of the test plates for 2-4 hours at 
30.degree. C., the enzyme activity ws demonstrated by observing clear 
zones in the matrix which had been made opaque by the phenmedipham. 
EXAMPLE 4 
Identification of the amino acid sequence of two BrCN cleaving peptides and 
synthesis of oligonucleotides for specific evidence of the carbamate 
hydrolase gene by hybridization. 
Resulting from the purified carbamate hydrolase, two peptides were isolated 
after BrCN cleavage, whose partial sequence was established by Edman 
degradation. 
BrCN Peptide I (Seq ID No. 1): 
H.sub.2 N-Ser-Asp-Glu-Phe-Ala-Asn-Leu-Asp- 
Arg-Trp-Thr-Gly-Lys-Pro-Phe-Val-Asp(Val)- 
Gly(His)-Leu-Asp-Glu-Val-Ala-Val-COOH 
BrCN Peptide II (Seq ID No. 2): 
N.sub.2 H-Glu-His-Thr-Lys-Phe(Val)-Asn(Gly)-Glu-Arg(Cys)- 
Pro-Leu-Ala-Phe-Tyr-Pro-Val-Phe-Asn-Glu-COOH 
According to the amino acid sequence information of these peptides, 
oligonucleotides were synthesized which could be used as hybridization 
probes for the detection of the carbamate hydrolase gene: 
Oligonucleotide I (Seq ID No. 3) (17 mer "mixed probe") contains as the 
single strand DNA fragment, the sequence information of the BrCN peptide I 
amino acid position 10-15 (complementary strand). 
.vertline.A .vertline.C .vertline.A .vertline.A .vertline. 
5'-AA .vertline.GGG .vertline.TTT .vertline.GCC .vertline.GGT .vertline.CC 
A-3' 
.vertline.C .vertline. .vertline.C .vertline.C .vertline. 
.vertline.T .vertline. .vertline.T .vertline.T .vertline. 
Phe.vertline.Pro .vertline.Lys .vertline.Gly .vertline.Thr .vertline.Tr 
Oligonucleotide II (Seq ID No. 4) (42 mer) contains as a single strand DNA 
fragment, the sequence information of the BrCN peptide I amino acid 
position 8-21 (complementary strand). The codon selection was carried out 
under the assumption of a guanine(G) and cytosine(C) rich DNA sequence 
(this takes into consideration guanine(G) and cytosine(C) nucleotides 
before adenine(A) and thiamine(T) nucleotides on the third position of the 
triplets). 
5'-CTG .vertline. GTC .vertline. CAG .vertline. GCC .vertline. GTC 
.vertline. CAC .vertline. GAA .vertline. 
Gln .vertline. Asp .vertline. Leu .vertline. Gly .vertline. Asp 
.vertline. Val .vertline. Phe .vertline. 
CGG .vertline. CTT .vertline. GCC .vertline. GGT .vertline. CCA 
.vertline. GCG .vertline. 
Pro .vertline. Lys .vertline. Gly .vertline. Thr .vertline. Trp 
.vertline. Arg .vertline. 
3' GTC .vertline. 
Asp .vertline. 
Oligonucleotide III (Seq ID No. 5) contains as the single strand DNA 
fragment sequence, information of the BrCN peptide II (complementary 
strand). 
5'- .vertline.TTC .vertline.GTT .vertline.GAA .vertline.GAC .vertline.CGG 
.vertline.GTA .vertline.GAA .vertline.CGC .vertline. 
.vertline.Glu .vertline.Asn .vertline.Phe .vertline.Val .vertline.Pro 
.vertline.Tyr .vertline.Phe .vertline.Ala .vertline. 
By using these oligonucleotides it was possible to localize the carbamate 
hydrolase gene within the plasmid pHP52 by hybridization. For this, the 
plasmid DNA was cleaved with restriction endonucleases and the resulting 
fragments were separated by agarose gel electrophoresis and then 
transferred according to the method of E.M. Southern (J. Mol. Biol. 98, 
503-17 (1975)) in single strand form on membrane filters (Gene Screen 
Plus.TM. hybridizing membrane, Du Pont de Nemours/NEN Research Products). 
The oligonucleotides were end marked by use of T4-polynucleotide kinase 
(Boehringer Mannheim) and .lambda.-.sup.32 P!-adenosine-5'-triphosphate 
(&gt;5000 Ci/mmol, Du Pont de Nemours/NEN Research Products) using the method 
of R. B. Wallace and C. G. Miyada, Methods in Enzymology 152, 432-442 
(1987) and treated without further purification for the hybridization. 
The hybridization was carried out using standard processes (P. J. Mason & 
J. G. Williams in "Nucleic Acid Hybridization" p. 113-160 (1985) B. D. 
Hames & S. J. Higgins Hrsg. IRL Press Oxford, Washington, D.C.). Under the 
conditions 6.times.SSC, 10.times.Denhardt, 0.5% w/v SDS and 100 u/ml t RNA 
(Beckerhefe, Boehringer Mannheim), as well as 10 ng/ml marked 
oligonucleotides I/II/III at 41.degree. C. (=6 hours), a specific 
hybridization can be achieved. The detection of the hybrids was carried 
out by autoradiography (T Maniatis, E F Fritsch & J Sambrook, "Molecular 
Cloning", Cold Spring Harbor Laboratory (1982)). 
EXAMPLE 5 
Isolation and characterization of the plasmid pHP52 from Arthrobacter 
oxidans P52. 
For isolation of plasmid pH52 from Arthrobacter, the alkali extraction 
method of Birnboim and Doly (Birnboim H. C. & Doly J. (1979) Nucl. Acid 
Res., 7, 1513-1523) was used, with a modification by Brandsch and Decker 
(Brandsch, R. & Decker, K. (1984) Arch. Microbiol. 138, 15-17). For 
plasmid preparation, the bacteria were cultivated in 6 litres of LB-medium 
comprising: 
______________________________________ 
Bacto-trypton (Difco .RTM.) 
10 g/l 
Bacto-Yeast-Extract (Difco .RTM.) 
5 g/l 
NaCl 10 g/l 
______________________________________ 
to a cell density of OD.sub.550 =1.4 and harvested by centrifuging. 
The cells were resuspended in a total of 210 ml solution I (50 mM glucose; 
10 mM EDTA; 25 mM Tris/HCl, pH 8.0; 1 mg/ml lysosyme) and incubated for 1 
hour at room temperature. Lysis was carried out by addition of 360 ml 
solution II (0.2M NaOH; 1% SDS). After gentle but thorough mixing and 
subsequent incubation for 5 minutes at room temperature, followed by 
cooling on ice for 5 minutes, the mixture was neutralized by the addition 
of 180 ml solution III (2M Tris/HCl, pH 7.0/0.5M KCl. After incubation for 
1 hour on ice, the undissolved precipitate was separated by 
centrifugation. The plasmid DNA was precipitated from the clear 
supernatant by addition of 0.6 volumes isopropanol and, after an 
incubation of 15 minutes at room temperature, pelleted by centrifugation 
(15,000.times.g/30 minutes). The plasmid-containing precipitate was dried 
in vacuo and dissolved in 24 ml 10.times.TE buffer (100 mM Tris/HCl, pH 
8.0; 10 mM EDTA). This plasmid containing solution was then purified by 
isopycnic cesium chloride density gradient, centrifuging in the presence 
of Ethidium bromide (Maniatis T., Fritsch E. F. & Sambrook J. in 
"Molecular Cloning" (1982), Cold Spring Harbor N.Y.). 
Purified plasmid DNA was mapped by restriction analysis which cut the 
plasmid once or gave several fragments. These fragments were resolved by 
agarose gel electrophoresis (0.8% w/v). Molecular weight standards used in 
mapping plasmid DNA were Hind III or Hind III and EcoRI digested 
bacteriophage DNA. 
The restriction analysis data were consistent with a circular map of pHP52 
(FIG. 4). The size of the plasmid is the sum of individual restriction 
fragments. 
All the processes were carried out in this Example according to standard 
methods (cf. Maniatis T., Fritsch E. F. & Sambrook J. in "Molecular 
Cloning" Cold Spring Harbor, N.Y. (1982)). 
EXAMPLE 6 
Identification of the coding region of the carbamate hydrolase gene by 
oligonucleotide hybridization. 
By hybridization of the restriction fragments of the plasmid pHP52 
separated by gel electrophoresis and transferred on membrane filters with 
the 32p marked oligonucleotide described in Example 4, the position of the 
coding region of the carbamate hydrolase gene can be definitely correlated 
on the restriction map of the plasmid pHP52. In FIG. 5, the hybridizing 
area is enlarged. All three oligonucleotides hybridize with the central 
part of a PstI restriction fragment of size 3.3 kb. In FIG. 6, a detailed 
restriction map of the fragment is shown from which the exact positions of 
the hybridizing areas can be seen. 
EXAMPLE 7 
Cloning of the carbamate hydrolase gene in E. coli and demonstration of the 
genes' expression under Lac promoter control. 
For cloning the carbamate hydrolase gene in E. coli the vector pUC19 
(Yannish-Perron, C., Vieira, J., & Messing, J. (1985) Gene 33, 103ff) was 
used. The pUC19 DNA was linearised by cleavage with restriction nuclease 
PstI and treated with alkaline phosphatase. The DNA of the 3.3 kb long 
PstI restriction fragment of the plasmid pHP52 was isolated (after 
digesting the wild-type plasmid DNA with PstI) by preparative agarose gel 
electrophoresis. The linearised and dephosphorylated vector DNA and the 
3.3 kb long PstI fragment was then ligated with T4 DNA ligase. E. coil 
DH5.alpha. was transformed with the ligation mixture. 
Two types of clones were obtained which contained the fragment in different 
orientations to the transcription direction of the lac Z' gene of the 
vector pUC 19. These are the clones pp52 Pst and pp52 Pst inv. The 
restriction map of both clones is shown in FIG. 5. The clones of type E. 
coli pp52 Pst express carbamate hydrolase, after addition of the inductor 
isopropyl-.beta.-D-thiogalactopyranosid to the culture medium. Without 
inducer addition (repressed state of the Lac promoter) to logarithmic 
cultures of the clone pp52 Pst as well as by repressed and induced 
logarithmic cultures of the clone pp52 Pst inv in enzyme extracts, no 
enzyme activity was seen using the assays described in Example 3. 
This means that the carbamate hydrolase gene in clones of type pp52 Pst 
lies in the same transcription direction (5'-3' orientation) as the lac Z' 
gene of the vector. The Arthrobacter promoter is not or only slightly 
expressed in E. coli. 
EXAMPLE 8 
Nucleotide sequence of the carbamate hydrolase gene from the Arthrobacter 
oxidans (species P52) and the deduced protein sequence. 
The nucleotide sequence of the carbamate hydrolase gene was established by 
the method of Sanger (Sanger F., Nicklen S. & Coulson A. (1977) Proc. 
Natl. Acad. Sci. USA, 74, 5463-5468). 
For this, 15 sub-clones in the single stranded DNA bacteriophage M13 mpl18 
and M13 mpl19 from the pp52 PST DNA were constructed (Messing, J. (1983) 
Methods in Enzymol, 101, 20-78). After transfection of E. coli 
DH5.alpha.F', the sequence of the single stranded recombinant DNA was 
established. 
In FIG. 6, the sequencing strategy of the carbamate hydrolase gene is 
shown. Altogether the sequence of 1864 base pairs was established. 
In FIG. 7 (Seq ID No. 6), the established nucleotide sequence is shown with 
the deduced amino acid sequence of the carbamate hydrolase. The reading 
frame is clearly defined as described by the amino acid sequences of two 
BrCN cleavage peptides as described in Example 4. The reading frame 
finishes with a TGA stop codon (nucleotide position 1789-1791 in FIG. 7 
(Seq ID No. 6)). As a translation start codon, a GTG (nucleotide position 
310-312) is suitable. This gives the longest open reading frame of 1479 bp 
(=493 amino acids). All open reading frames which begin with the usual ATG 
start codons give no protein of suitable size (compared to the molecular 
weight determination of the protein). 
The hypothesis that translation starts from GTG (position 310-312) is 
further supported by the existence of a definite homologous region to the 
consensus sequence for ribosomal E. coli binding sites 7 bp upstream of 
the putative GTG start codon. 
All cloning steps were carried out by standard processes (cf Maniatis, T., 
Fritsch, E. F. & Sambrook, J. (1982) in "Molecular Cloning", Cold Spring 
Harbor, N.Y.). The sequencing reactions were carried out using 
Sequenase.RTM. DNA Sequencing Kits (United States Biochemical Corporation) 
according to information by the producer. The separation of the marked 
reaction products was carried in 6% w/v polyacrylamide/urea gel (Maxam, A. 
M. & Gilbert, W. (1980) Methods Enzymol. 65, 497-559). 
EXAMPLE 9 
Construction of plasmids for the expression of carbamate hydrolase in 
plants. 
a) Construction of intermediate vectors 
Plasmid DNA from pp52Pst (FIG. 8) is methylated using TaqI Methylase (M. 
TaqI) to make the single SaII restriction site inaccessible to HindII. 
Then the methylated plasmid is cut with both restriction enzymes XbaI and 
HindII to release a 2.2 kb DNA fragment containing the open reading frame 
for carbamate hydrolase. This fragment is purified by preparative agarose 
gel electrophoresis. The vector plasmid pA5 (FIG. 8) is cut with XbaI and 
HindII and then ligated with the purified 2.2 kb DNA fragment, thus 
linking the 3'--end of the carbamate hydrolase coding region to the 
polyadenylation signal of the octopine synthetase (OCS) gene (Dhaese et 
al., EMBO J. 2:419, 1983). Competent cells of E. coli DH5alpha are 
transformed with the recombined DNA and clones are selected on LB-agar 
containing 100 .mu.g/ml carbenicillin. Clones containing the recombined 
plasmid pU014 (FIG. 8) are identified by restriction analysis of 
isolated plasmid DNA. 
Two oligonucleotides of the following sequence are synthesized by an 
automatic synthesizer: 
1) 5'-CTAGAGATCT CAACAATGGT TACCAGACCG ATCGCCCACA CCACCGCTGG G-3' (Seq ID 
No. 8) 
2) 5'-GTCCCCAGCG GTGGTGTGGG CGATCGGTCT GGTAACCATT GTTGAGATC-3' (Seq ID No. 
9) 
They represent complementary DNA strands which are able to reconstitute the 
N-terminal portion of the open reading frame of the carbamate hydrolase 
gene upstream (5') of the PpuMI restriction site. 
Both complementary oligonucleotides are mixed, phosphorylated by 
polynucleotide kinase and then annealed by shifting temperature from 
70.degree. C. to room temperature overnight. Plasmid DNA of pU014 is 
digested with XbaI and then ligated with the annealed oligonucleotide. 
This covalently links two oligonucleotides at their XbaI-compatible ends 
to both ends of the linearised plasmid. The linear ligation product is 
purified by preparative agarose gel electrophoresis and digested with 
PpuMI to remove the N-terminal part of the coding region. The linear DNA 
is then recircularized by ligase treatment. Competent cells of E. coli 
DH5alpha are transformed with the recombined DNA and clones are selected 
on LB-agar containing 50 .mu.g/ml carbenicillin. Clones containing the 
recombined plasmid pUP01015 (FIG. 9) are identified by restriction 
analysis of isolated plasmid DNA. The correct ligation of the 
oligonucleotide is verified by sequence analysis of purified pUP01015 DNA 
using the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. USA 
74:5463, 1977) modified for plasmid DNA as template (Chen and Seeburg, DNA 
4:165, 1985). 
b) Construction of plasmids for the cytoplasmatic expression of carbamate 
hydrolase. 
Plasmid DNA of pUP01015 is digested with both restriction enzymes XbaI and 
HindIII to create a DNA fragment that contains the whole recombinant 
coding region of the carbamate hydrolase together with the polyadenylation 
signal described above. The fragment is then ligated with the plant 
expression vector plasmid pA8 (A. v. Schaewen, Dissertation, FU Berlin, 
1989; FIG. 10) which was similarly treated with XbaI and HindIII. The 
ligation links the gene in correct orientation to the cauliflower mosaic 
virus (CaMV) 35S promoter (Paszkowski et al., EMBO J. 3:2717, 1984) 
contained in pA8. Competent cells of E. coli DH5alpha are transformed with 
the recombined DNA and clones are selected on LB-agar containing 25 
.mu.g/ml streptomycin. Clones containing the recombined plasmid pMCP01021 
(FIG. 10) are identified by restriction analysis of isolated plasmid DNA. 
Plasmid DNA of pMCP01021 is digested with both restriction enzymes EcoRI 
and HindIII to create a DNA fragment that contains the CaMV 35S promoter, 
the carbamate hydrolase coding region and the OCS polyadenylation signal. 
The fragment is then ligated with a EcoRI and HindIII cleaved vector 
plasmid pBIN19 that is part of the binary transformation system described 
by Bevan, Nucl. Acids Res. 12:8711, 1984. Competent cells of E. coli S17-1 
(Simon et al. Bio/Technology 1:784-791, 1983) are transformed with the 
recombined DNA and clones are selected on LB-agar containing 50 .mu.g/ml 
kanamycin. Clones containing the recombined plasmid pBCP01027 are 
identified by restriction analysis of isolated plasmid DNA. 
c) Construction of plasmids for the extracellular expression of carbamate 
hydrolase. 
Plasmid pA22 (FIG. 11) contains a synthetic intronless sequence that codes 
for the signal peptide of the proteinase inhibitor II(PI) from potato and 
can be attached to the N-terminus of other genes to direct the gene 
product into the endoplasmatic reticulum of the plant cell. To create an 
intermediate vector for targeting of carbamate hydrolase, the DNA fragment 
encoding the signal peptide is excised from plasmid pA22 by cleavage with 
restriction enzymes KpnI and XbaI and then ligated with a KpnI and XbaI 
cleaved plasmid pA8. Competent cells of E. coli DH5alpha are transformed 
with the recombined DNA and clones are selected on LB-agar containing 25 
.mu.g/ml streptomycin. Streptomycin resistant clones are then screened for 
the loss of plasmid pA22 on LB-agar containing 50 .mu.g/ml carbenicillin. 
Carbenicillin sensitive clones are analyzed by restriction analysis of 
isolated plasmid DNA and recombined plasmids containing the signal peptide 
encoding sequence are designated pmA 1017 (FIG. 11). 
Plasmid DNA of pUP01015 is digested with both restriction enzymes HindIII 
and BgIII to create a DNA fragment that contains the whole recombinant 
coding region of the carbamate hydrolase together with the polyadenylation 
signal described above. The fragment is then ligated with the plant 
expression vector plasmid pMA1017 which has been digested with BamHI and 
HindIII. The ligation links the gene in correct orientation to the 
cauliflower mosaic virus (CaMV) 35S promotor (Paszkowski et al., EMBO J. 
3:2717, 1984) and the proteinase inhibitor signal sequence described 
above. Competent cells of E. coli DH5alpha are transformed with the 
recombined DNA and clones are selected on LB-agar containing 25 .mu.g/ml 
streptomycin. Clones containing the recombined plasmid pMAP01022 (FIG. 12) 
are identified by restriction analysis of isolated plasmid DNA. 
Plasmid DNA of pMAP01022 is digested with both restriction enzymes EcoRI 
and HindIII to create a DNA fragment that contains the CaMV 35S promoter, 
the PI signal sequence linked in frame to the carbamate hydrolase coding 
region and the OCS polyadenylation signal. The fragment is then ligated 
with a EcoRI and HindIII cleaved vector plasmid pBIN19 (Bevan, Nucl. Acids 
Res. 12:8711, 1984). Competent cells of E. coli S17-1 (Simon et al. 
Bio/Technology 1:784-791, 1983) are transformed with the recombined DNA 
and clones are selected on LB-agar containing 50 .mu.g/ml kanamycin. 
Clones containing the recombined plasmid pBAP01027 are identified by 
restriction analysis of isolated plasmid DNA. 
EXAMPLE 10 
Transformation of tobacco with chimeric carbamate hydrolase genes. 
a) Transfer of recombinant carbamate hydrolase from E. coli to A. 
tumefaciens 
Strains of E. coli S17-1 containing chimeric carbamate hydrolase genes on 
plasmids pBCP01027 or pBAP01028 are grown at 37.degree. C. in liquid LB 
medium containing 50 .mu.g/ml kanamycin. Agrobacteria tumefaciens LBA4404 
(Bevan, Nucl. Acids Res. 12:8711, 1984) is grown at 28.degree. C. in 
liquid YEB medium (Yeast extract 1 g/l, beef extract 5 g/l, peptone 5 g/l, 
sucrose 5 g/l, sucrose 5 g/l, MgSO.sub.4 0.5 g/l). 0.4 ml samples of E. 
coli culture are centrifuged and the bacterial pellets are resuspended in 
0.4 ml YEB. Bacterial suspensions of E. coli in YEB and samples of 
Agrobacterium tumafaciens culture in YEB are then mixed in a 1:1 ratio in 
relation to cell density. Samples of 50-100 .mu.l from the mixtures are 
spotted onto LB-agar and incubated at 28.degree. C. for 6-16 hours. 
Bacterial mating mixtures that have grown on the agar are suspended in 
liquid M9-salts (6 g/l Na.sub.2 HPO.sub.4, 3 g/l KH.sub.2 PO.sub.4, 0.5 
g/l NaCl, 1 g/l NH.sub.4 Cl, 2 mM MgSO.sub.4, 0.1 mM CaCl.sub.2, 1 mM 
thiamine, HCl) and then plated in several dilutions onto M9-agar 
containing 2 g/l sucrose and 50 .mu.g/ml kanamycin. Plates are incubated 
at 28.degree. C. for several days until bacterial colonies have grown. 
These colonies are further purified by subsequent cultivation on the same 
medium. That these clones of A. tumefaciens LBA4404 contain recombinant 
plasmids pBCP01027 and pBAP01028 respectively is verified by restriction 
analysis of isolated plasmid DNA. 
b) Transfer of recombinant carbamate hydrolase from A. tumefaciens to 
tobacco. 
For transformation of tobacco, A. tumefaciens strains harboring carbamate 
hydrolase plasmids are grown overnight at 28.degree. C. in liquid YEB 
medium containing 50 .mu.g/ml kanamycin. Cells are centrifuged at 5000 g 
for 15 minutes and resuspended in the same volume of YEB without 
antibiotic. Nicotiana tabacum Wisconsin W38 plantlets are grown under 
sterile conditions on solid MS medium containing 20 g/l sucrose. Leaves 
are cut from those plants, dissected into pieces of around 1 cm.sup.2 and 
rinsed with the bacterial suspension. Leaf disks are then placed onto 
solid MS medium containing 20 g/l sucrose. After 2 days of incubation at 
room temperature in the dark, leaf disks are transferred to solid MS 
medium containing 16 g/l glucose, 1 mg/l benzylaminopurine, 0.2 mg/l 
naphthylacetic acid, 500 mg/l claforan and 50 mg/l kanamycin. Incubation 
is continued at 25.degree. C. under a daily regime of 16 hours light 
(photosynthetically active radiation=67.mu.EM-.sup.2 s-.sup.1) and 8 hours 
dark. The medium is changed every week until shoots appear. These are cut 
from the callus and transferred to MS medium containing 20 g/l sucrose and 
250 mg/l claforan. Incubation is continued under the same conditions until 
roots of 1-2 cm in length have formed and plants are transferred to soil. 
Total RNA isolated from leaves is analyzed by northern blot hybridization 
using the 1.8 kb EcoRI-HindIII fragment of pUP01015 as a labeled probe. 
Transformed plants synthesize a transcript of around 1.8 kb in size that 
specifically hybridizes with the carbamate hydrolase coding sequence. 
EXAMPLE 11 
Detection of transformed plants which are resistant to the herbicidal 
activity of phenmedipham. 
Transgenic plants are transferred to soil and grown in a growth chamber at 
25.degree. C. with a day/night rhythm of 16 hours light and 8 hours dark. 
No difference in growth can be seen between transformed and untransformed 
tobacco. Plants that have a leaf length of around 10 cm are sprayed with 
the herbicide Betanal.RTM. (active ingredient: 157 g/l phenmedipham). 
Doses corresponding to field application rates of 1 kg/ha, 3 kg/ha and 10 
kg/ha are used to distinguish between resistant plants and untransformed 
wildtype plants: Whereas 1 kg/ha is completely lethal for wildtype plants, 
transgenic plants which express the carbamate hydrolase gene show 
resistance levels between 1 kg/ha and 10 kg/ha (FIG. 13 and 14). 
The same spraying experiment is done by using Betanal.RTM. AM (active 
ingredient: 157 g/l desmedipham) as the herbicidal agent. 
EXAMPLE 12 
Analysis of herbicide detoxification in plants sprayed with phenmedipham. 
Transgenic tobacco plants expressing carbamate hydrolase and untransformed 
control plants are grown as described in Example 11 and sprayed with the 
herbicide Betanal.RTM. corresponding to 1 kg/ha phenmedipham. Normalized 
variable fluorescence is measured on intact leaves of sprayed plants as is 
described by Voss, Weed Science 32:675-680 (1984). The equipment used is a 
Kompakt Fluorometer RKF 1000 (Ingenieurburo F.U.R. Dr. M. Voss, Berlin). 
Measurement values before spraying are taken as 100% relative variable 
fluorescence. Subsequent measurements are performed in a time course of 2, 
4, 8, 24 hours and then every day up to 4 days. Relative variable 
fluorescence values of transgenic tobacco plants expressing carbamate 
hydrolase stay constantly higher than 90%; in contrast values from 
untransformed tobacco fall below 10% within the first 8 hours after 
spraying (FIG. 15). 
The same spraying experiment is done by using Betanal.RTM. AM (active 
ingredient: 157 g/l desmedipham) as the herbicidal agent. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 9 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 24 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Arthrobacter - # oxidans 
(B) STRAIN: P52 
#ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ 
- Ser Asp Glu Phe Ala Asn Leu Asp Arg Trp Th - #r Gly Lys Pro Phe Val 
# 15 
- Xaa Xaa Leu Asp Glu Val Ala Val 
20 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 18 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: internal 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Arthrobacter - # oxidans 
(B) STRAIN: P52 
#ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ 
- Glu His Thr Lys Xaa Xaa Glu Xaa Pro Leu Al - #a Phe Tyr Pro Val Phe 
# 15 
- Asn Glu 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: YES 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Arthrobacter - # oxidans 
(B) STRAIN: P52 
#ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ 
# 17 A 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 42 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: YES 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Arthrobacter - # oxidans 
(B) STRAIN: P52 
#ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ 
# 42 ACGA ACGGCTTGCC GGTCCAGCGG TC 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 24 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: YES 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Arthrobacter - # oxidans 
(B) STRAIN: P52 
#ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ 
# 24TAGA ACGC 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1864 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
(A) NAME/KEY: RBS 
(B) LOCATION: 298..302 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 310..1791 
#/codon= (seq: "gta", aa: Val)N: 
/product=- # "carbamate hydrolase" 
/transl.sub.-- - #except= (pos: 310 .. 312, aa: Met) 
#"terminator (1789-1791)" 
- (ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- - #peptide 
(B) LOCATION: 310..1788 
#ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ 
- TCCTTGCCAG TCACGGCACC CCAGCCAACC CGGAAGTGGC ACCTGCTCGG GC - #ACATCGGT 
60 
- GCGAACGCTT CGTCCTGATT CCGATGCCAA CTGCTTGACG GCCGTGACAC AT - #ATGTAGCA 
120 
- TAGTCGCCTA GCATGGACCC GCAGCACACC TGCTGTCGGC TCCCGCGCTA TC - #CCCGACCA 
180 
- GCGCCGGTCA CGGGTAGTCC TCGTGAGAGG CACCAGAACG ACAACGGCGC AC - #TGTCCCGC 
240 
- AACACGGCCG TATAACCCCA CCGGGGTCCG CGCCCGAGCT AGTTCTGGCT CA - #ACCATAAG 
300 
- GAGAACCTC GTG ATT ACC AGA CCG ATC GCC CAC ACC - # ACC GCT GGG GAC 
348 
#Ile Ala His Thr Thr Ala Gly Asp 
# 10 
- CTC GGC GGT TGC CTT GAA GAC GGC CTG TAC GT - #G TTC CGA GGA GTG CCG 
396 
Leu Gly Gly Cys Leu Glu Asp Gly Leu Tyr Va - #l Phe Arg Gly Val Pro 
# 25 
- TAC GCC GAG CCG CCG GTC GGC GAC CTG CGG TG - #G CGG GCG GCG CGC CCG 
444 
Tyr Ala Glu Pro Pro Val Gly Asp Leu Arg Tr - #p Arg Ala Ala Arg Pro 
# 45 
- CAC GCC GGC TGG ACC GGC GTC CGC GAC GCC TC - #C GCG TAT GGT CCC TCG 
492 
His Ala Gly Trp Thr Gly Val Arg Asp Ala Se - #r Ala Tyr Gly Pro Ser 
# 60 
- GCG CCG CAA CCC GTG GAG CCT GGC GGC TCG CC - #G ATC CTT GGG ACA CAC 
540 
Ala Pro Gln Pro Val Glu Pro Gly Gly Ser Pr - #o Ile Leu Gly Thr His 
# 75 
- GGC GAC CCT CCG TTT GAC GAG GAC TGC CTG AC - #T CTC AAT CTT TGG ACC 
588 
Gly Asp Pro Pro Phe Asp Glu Asp Cys Leu Th - #r Leu Asn Leu Trp Thr 
# 90 
- CCG AAC CTC GAC GGC GGT AGC CGG CCG GTC CT - #C GTC TGG ATC CAT GGT 
636 
Pro Asn Leu Asp Gly Gly Ser Arg Pro Val Le - #u Val Trp Ile His Gly 
# 105 
- GGG GGC CTA CTA ACC GGC TCG GGA AAT CTA CC - #T AAC TAC GCG ACC GAT 
684 
Gly Gly Leu Leu Thr Gly Ser Gly Asn Leu Pr - #o Asn Tyr Ala Thr Asp 
110 1 - #15 1 - #20 1 - 
#25 
- ACC TTC GCC CGC GAC GGC GAC TTG GTA GGT AT - #C TCA ATC AAT TAC CGG 
732 
Thr Phe Ala Arg Asp Gly Asp Leu Val Gly Il - #e Ser Ile Asn Tyr Arg 
# 140 
- CTC GGG CCT CTT GGA TTC CTC GCA GGA ATG GG - #C GAC GAG AAT GTC TGG 
780 
Leu Gly Pro Leu Gly Phe Leu Ala Gly Met Gl - #y Asp Glu Asn Val Trp 
# 155 
- CTC ACC GAT CAG GTA GAG GCA CTG CGC TGG AT - #T GCA GAT AAC GTT GCT 
828 
Leu Thr Asp Gln Val Glu Ala Leu Arg Trp Il - #e Ala Asp Asn Val Ala 
# 170 
- GCC TTC GGT GGA GAC CCG AAC CGG ATC ACT CT - #C GTC GGT CAA TCA GGC 
876 
Ala Phe Gly Gly Asp Pro Asn Arg Ile Thr Le - #u Val Gly Gln Ser Gly 
# 185 
- GGG GCA TAC TCG ATC GCA GCG CTC GCC CAA CA - #C CCG GTC GCC CGT CAG 
924 
Gly Ala Tyr Ser Ile Ala Ala Leu Ala Gln Hi - #s Pro Val Ala Arg Gln 
190 1 - #95 2 - #00 2 - 
#05 
- CTG TTC CAC CGC GCG ATC CTA CAA AGC CCA CC - #A TTC GGG ATG CAA CCC 
972 
Leu Phe His Arg Ala Ile Leu Gln Ser Pro Pr - #o Phe Gly Met Gln Pro 
# 220 
- CAT ACA GTT GAA GAA TCG ACG GCA AGG ACG AA - #G GCC CTG GCC CGG CAT 
1020 
His Thr Val Glu Glu Ser Thr Ala Arg Thr Ly - #s Ala Leu Ala Arg His 
# 235 
- CTC GGG CAC GAT GAC ATC GAG GCC CTG CGC CA - #T GAG CCG TGG GAG AGG 
1068 
Leu Gly His Asp Asp Ile Glu Ala Leu Arg Hi - #s Glu Pro Trp Glu Arg 
# 250 
- CTG ATT CAA GGC ACG ATA GGC GTC CTG ATG GA - #A CAC ACC AAA TTT GGC 
1116 
Leu Ile Gln Gly Thr Ile Gly Val Leu Met Gl - #u His Thr Lys Phe Gly 
# 265 
- GAA TGG CCC CTG GCA TTC TAT CCG GTG TTC GA - #T GAG GCA ACG ATA CCT 
1164 
Glu Trp Pro Leu Ala Phe Tyr Pro Val Phe As - #p Glu Ala Thr Ile Pro 
270 2 - #75 2 - #80 2 - 
#85 
- CGC CAT CCG ATT GAG TCC ATT ATC GAT TCC GA - #C ATC GAA ATC ATC ATC 
1212 
Arg His Pro Ile Glu Ser Ile Ile Asp Ser As - #p Ile Glu Ile Ile Ile 
# 300 
- GGC TGG ACA CGC GAC GAG GGC ACT TTT CCG TT - #T GCC TTC GAC CCT CAG 
1260 
Gly Trp Thr Arg Asp Glu Gly Thr Phe Pro Ph - #e Ala Phe Asp Pro Gln 
# 315 
- GTT TCA CAG GCG GAT CGC GAT CAG GTC GAG TC - #A TGG TTG CAG AAG CGT 
1308 
Val Ser Gln Ala Asp Arg Asp Gln Val Glu Se - #r Trp Leu Gln Lys Arg 
# 330 
- TTC GGA GAC CAC GCC GCC TCG GCC TAC GAG GC - #T CAC GCC GGC GAC GGA 
1356 
Phe Gly Asp His Ala Ala Ser Ala Tyr Glu Al - #a His Ala Gly Asp Gly 
# 345 
- ACC AGT CCT TGG ACC GTT ATC GCC AAC GTT GT - #G GGC GAC GAG CTC TTT 
1404 
Thr Ser Pro Trp Thr Val Ile Ala Asn Val Va - #l Gly Asp Glu Leu Phe 
350 3 - #55 3 - #60 3 - 
#65 
- CAC AGC GCT GGG TAC CGG GTC GCG GAC GAA CG - #G GCA ACG CGC AGA CCG 
1452 
His Ser Ala Gly Tyr Arg Val Ala Asp Glu Ar - #g Ala Thr Arg Arg Pro 
# 380 
- GTA CGG GCC TAT CAG TTC GAC GTA GTC TCG CC - #C TTG TCG GAC GGA GCC 
1500 
Val Arg Ala Tyr Gln Phe Asp Val Val Ser Pr - #o Leu Ser Asp Gly Ala 
# 395 
- CTC GGC GCG GTC CAC TGC ATC GAA ATG CCG TT - #C ACA TTT GCC AAT CTC 
1548 
Leu Gly Ala Val His Cys Ile Glu Met Pro Ph - #e Thr Phe Ala Asn Leu 
# 410 
- GAC CGT TGG ACG GGG AAG CCG TTC GTG GAC GG - #C CTG GAT CCA GAC GTG 
1596 
Asp Arg Trp Thr Gly Lys Pro Phe Val Asp Gl - #y Leu Asp Pro Asp Val 
# 425 
- GTG GCT CGG GTG ACC AAC GTG TTG CAT CAG GC - #C TGG ATC GCA TTC GTC 
1644 
Val Ala Arg Val Thr Asn Val Leu His Gln Al - #a Trp Ile Ala Phe Val 
430 4 - #35 4 - #40 4 - 
#45 
- CGA ACG GGA GAC CCC ACG CAC GAC CAG TTG CC - #G GTG TGG CCA ACG TTC 
1692 
Arg Thr Gly Asp Pro Thr His Asp Gln Leu Pr - #o Val Trp Pro Thr Phe 
# 460 
- CGA GCG GAC GAC CCA GCG GTG TTG GTC GTC GG - #C GAC GAG GGA GCA GAG 
1740 
Arg Ala Asp Asp Pro Ala Val Leu Val Val Gl - #y Asp Glu Gly Ala Glu 
# 475 
- GTG GCG CGG GAT CTA GCG CGC CCG GAC CAC GT - #C AGC GTT CGG ACC CTA 
1788 
Val Ala Arg Asp Leu Ala Arg Pro Asp His Va - #l Ser Val Arg Thr Leu 
# 490 
- TGA GGGTCGCGGG TCGCCGGGGT CTTGAGGCCG GAGGGCCTCG CGTATGCAG - #T 
1841 
* 
# 1864GCCA GTT 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 493 ami - #no acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:7: 
- Met Ile Thr Arg Pro Ile Ala His Thr Thr Al - #a Gly Asp Leu Gly Gly 
# 15 
- Cys Leu Glu Asp Gly Leu Tyr Val Phe Arg Gl - #y Val Pro Tyr Ala Glu 
# 30 
- Pro Pro Val Gly Asp Leu Arg Trp Arg Ala Al - #a Arg Pro His Ala Gly 
# 45 
- Trp Thr Gly Val Arg Asp Ala Ser Ala Tyr Gl - #y Pro Ser Ala Pro Gln 
# 60 
- Pro Val Glu Pro Gly Gly Ser Pro Ile Leu Gl - #y Thr His Gly Asp Pro 
# 80 
- Pro Phe Asp Glu Asp Cys Leu Thr Leu Asn Le - #u Trp Thr Pro Asn Leu 
# 95 
- Asp Gly Gly Ser Arg Pro Val Leu Val Trp Il - #e His Gly Gly Gly Leu 
# 110 
- Leu Thr Gly Ser Gly Asn Leu Pro Asn Tyr Al - #a Thr Asp Thr Phe Ala 
# 125 
- Arg Asp Gly Asp Leu Val Gly Ile Ser Ile As - #n Tyr Arg Leu Gly Pro 
# 140 
- Leu Gly Phe Leu Ala Gly Met Gly Asp Glu As - #n Val Trp Leu Thr Asp 
145 1 - #50 1 - #55 1 - 
#60 
- Gln Val Glu Ala Leu Arg Trp Ile Ala Asp As - #n Val Ala Ala Phe Gly 
# 175 
- Gly Asp Pro Asn Arg Ile Thr Leu Val Gly Gl - #n Ser Gly Gly Ala Tyr 
# 190 
- Ser Ile Ala Ala Leu Ala Gln His Pro Val Al - #a Arg Gln Leu Phe His 
# 205 
- Arg Ala Ile Leu Gln Ser Pro Pro Phe Gly Me - #t Gln Pro His Thr Val 
# 220 
- Glu Glu Ser Thr Ala Arg Thr Lys Ala Leu Al - #a Arg His Leu Gly His 
225 2 - #30 2 - #35 2 - 
#40 
- Asp Asp Ile Glu Ala Leu Arg His Glu Pro Tr - #p Glu Arg Leu Ile Gln 
# 255 
- Gly Thr Ile Gly Val Leu Met Glu His Thr Ly - #s Phe Gly Glu Trp Pro 
# 270 
- Leu Ala Phe Tyr Pro Val Phe Asp Glu Ala Th - #r Ile Pro Arg His Pro 
# 285 
- Ile Glu Ser Ile Ile Asp Ser Asp Ile Glu Il - #e Ile Ile Gly Trp Thr 
# 300 
- Arg Asp Glu Gly Thr Phe Pro Phe Ala Phe As - #p Pro Gln Val Ser Gln 
305 3 - #10 3 - #15 3 - 
#20 
- Ala Asp Arg Asp Gln Val Glu Ser Trp Leu Gl - #n Lys Arg Phe Gly Asp 
# 335 
- His Ala Ala Ser Ala Tyr Glu Ala His Ala Gl - #y Asp Gly Thr Ser Pro 
# 350 
- Trp Thr Val Ile Ala Asn Val Val Gly Asp Gl - #u Leu Phe His Ser Ala 
# 365 
- Gly Tyr Arg Val Ala Asp Glu Arg Ala Thr Ar - #g Arg Pro Val Arg Ala 
# 380 
- Tyr Gln Phe Asp Val Val Ser Pro Leu Ser As - #p Gly Ala Leu Gly Ala 
385 3 - #90 3 - #95 4 - 
#00 
- Val His Cys Ile Glu Met Pro Phe Thr Phe Al - #a Asn Leu Asp Arg Trp 
# 415 
- Thr Gly Lys Pro Phe Val Asp Gly Leu Asp Pr - #o Asp Val Val Ala Arg 
# 430 
- Val Thr Asn Val Leu His Gln Ala Trp Ile Al - #a Phe Val Arg Thr Gly 
# 445 
- Asp Pro Thr His Asp Gln Leu Pro Val Trp Pr - #o Thr Phe Arg Ala Asp 
# 460 
- Asp Pro Ala Val Leu Val Val Gly Asp Glu Gl - #y Ala Glu Val Ala Arg 
465 4 - #70 4 - #75 4 - 
#80 
- Asp Leu Ala Arg Pro Asp His Val Ser Val Ar - #g Thr Leu 
# 490 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 51 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (vi) ORIGINAL SOURCE: 
(A) ORGANISM: Arthrobacter - # oxidans 
(B) STRAIN: P52 
#ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ 
# 51CAATGGT TACCAGACCG ATCGCCCACA CCACCGCTGG G 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 49 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
#ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ 
# 49GTGGG CGATCGGTCT GGTAACCATT GTTGAGATC 
__________________________________________________________________________