Method for the detection and quantitative determination of nitrosomonas strains in wastewaters or soils

For the detection and quantitative determination of nitrosomonas strains in wastewaters and soils, a gene probe is used which, by virtue of its complementary sequences, only hybridizes with parts of the genome of nitrosomonas strains from wastewater or soil samples and does not produce a positive hybridization signal with parts of the genome of other bacteria and the hybridized nucleic acid is quantitatively determined by means of a known label of the gene probe and thus provides a direct measure of the content of nitrosomonas strains in the wastewater or soil sample.

BACKGROUND OF THE INVENTION 
On account of its toxicity to fish, but above all on account of its 
contribution to the eutrophication of waters, ammonium is a substance 
which should be completely removed from wastewaters. Whereas there are 
numerous industrial processes for the removal of high concentrations of 
ammonium, the removal of ammonium in ppm concentrations can only be 
economically achieved by biological processes. This is done by two 
specialized groups of bacteria which derive their energy for the cell 
metabolism from the oxidation of ammonia to nitrite (ammonia oxidizers) 
and further to nitrate (nitrite oxidizers). The ammonia oxidizers use 
CO.sub.2 as the sole carbon source while nitrite oxidizers use additional 
carbon sources. The nitrate formed can be reduced to nitrogen by a number 
of heterotrophic bacteria at low oxygen concentrations and can therefore 
be completely removed (see for example (1)). 
However, disadvantages attending the microbial elimination of nitrogen lie 
in the low growth rates of the nitrificants. CO.sub.2 is the sole carbon 
source so that cell growth is minimal. In addition, many organic 
compounds, such as isothiocyanates, amines, phenols and 
nitrogen-containing heterocycles inhibit the growth of ammonia-oxidizing 
bacteria. As a result, the biological elimination of nitrogen in 
industrial effluent treatment plants is seriously reduced. 
Since nitrificants cannot be rapidly and quantitatively determined by 
microbiological culture methods, there have hitherto been no possibilities 
for recognizing changes in the quantity of nitrificants in wastewater 
populations and for achieving an optimal treatment capacity by 
corresponding control measures. 
SUMMARY OF THE INVENTION 
According to the invention, it has been possible to solve this problem 
through the construction of gene probes for these ammonia-oxidizing 
bacteria. Using these gene probes, nitrificants in complex biomasses, such 
as are present in wastewaters and soils, can be quantitatively determined 
from the concentration of nucleic acid. In the gene probe test, the gene 
probe and complementary target DNA from nitrificants are specifically 
duplexed (hybridized). The gene probe is produced either synthetically (up 
to 100 oligonucleotides) or biologically on the basis of the sequence 
described in the following and is provided with a label (radioactivity, 
dye, enzyme). Its addition to a sample material containing nitrificants, 
for example nucleic acid lysates from aeration sludge or soils, results in 
hybridization by the complementary sequences of gene probe and nitrificant 
DNA. This hybridization reaction can be carried out in solution or with 
nucleic acids fixed to carriers (nitrocellulose, nylon membranes, beads). 
The hybridized nucleic acid is quantitatively evaluated through the 
labeling of the gene probe and thus provides a direct measure of the 
nitrificant content of the biomass of the wastewater or soil. 
The present invention also extends to gene probes isolated by the same 
method and to modified variants of the gene probes described in the 
invention which have the features and properties essential for carrying 
out the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The main applications for the new gene probes are in the testing and 
monitoring of nitrificant concentrations in the biological elimination of 
nitrogen in waste water treatment plants. 
The detection limit for the detection of nitrificants with the new gene 
probes is in the range of 10.sup.5 to 10.sup.6 bacteria. 
The detection sensitivity can be distinctly improved by amplification 
methods, such as for example the PCR method (polymerase chain reaction). A 
detection limit of 10 to 100 bacteria can be achieved by amplification on 
the basis of the gene probe sequences described in the invention. 
The invention is illustrated by the following Examples. 
Genetically engineered isolation of nitrosomonas gene probes 
The genomic nucleic acid from the strain Nitrosomonas europaea ATCC 19718 
was isolated by a preparative lysozyme/SDS nucleic acid isolation method. 
To construct gene probes, the genomic DNA was cleaved with the restriction 
enzyme BamHI into 1-15 kb large DNA fragments, linked to BamHI-linearized 
pBR322 plasmid vector by genetic engineering and transformed into 
competent E. coli cells AG1. 
Ampicillin-resistant clones were isolated the next day and recombinant 
clones containing DNA of nitrosomonas were identified through the 
sensitivity to tetracycline of bacteria transformed with the plasmid 
vector. The cloning experiments were carried out by the genetic method 
described by Maniatis (2). 
The plasmid DNA was isolated from the recombinant clones by an analytical 
nucleic acid isolation method and the size of the nitrosomonas DNA 
incorporated was determined by gel-electrophoretic separation. The exact 
size of clones containing incorporated nitrosomonas DNA in the 1-15 kb 
range was determined by cleavage of the plasmids with the restriction 
enzyme BamHI and by gel-electrophoretic separation of the linearized 
plasmids. Clones with the exact insert sizes of nitrosomonas DNA are 
listed in Table 1. 
By reversed-phase hybridization, it was possible to determine which of the 
isolated nitrosomonas gene probes had the broadest detection spectrum for 
nitrosomonas. To this end, the individual gene probes were subjected to 
gel-electrophoretic separation in agarose gel and the gene probe DNA was 
subsequently transferred from the gel to a nitrocellulose filter and 
fixed. The genomic nitrosomonas DNA was suitably labeled (P.sup.32 -ATP, 
biotin, digoxigenin, enzyme) and used as a gene probe in Southern blotting 
with the gene probes fixed to a nitrocellulose filter. 
A 6 kb gene probe SPN 323.13 was identified and produced a very strong 
hybridization signal in the Southern blotting hybridization test. This 
gene probe was analyzed for its nitrosomonas specificity by a dot blotting 
hybridization test. The experiments showed that the gene probe SPN 323.13 
is specific for nitrosomonas and has the broadest detection spectrum for 
nitrosomonas of all the gene probes tested. This gene probe was used for 
the further development of shorter gene probes and chemically synthesized 
oligonucleotide gene probes. 
Genetically engineered development of nitrosomonas gene probes 
Other shortened gene probes were constructed on the basis of the 6 kb gene 
probe SPN 323.13 (FIG. 1). 
The gene probe was first molecular-biologically characterized by cleavage 
with various restriction enzymes. The linear restriction map of the gene 
probe is shown in both orientations in FIG. 2. 
The 1.4 kb ClaI fragment, the 1.7 kb ClaI-HindIII fragment and the 2.5 kb 
SalI-BamHI fragment were subcloned into the vectors pBR 322 and pSK 
Bluescript (Stratagene). 
Recombinant clones containing the individual gene fragments were isolated 
and molecular-biologically characterized. After cleavage with the 
corresponding restriction enzymes and gel-electrophoretic separation, the 
individual gene fragments were isolated by electroelution. These gene 
probes were then labeled with the usual labeling substances (P.sup.32 ATP, 
biotin, digoxigenin, enzymes) and their specificity was determined in the 
gene probe test. 
By virtue of its high specificity for nitrosomonas strains and its 
nevertheless very broad detection spectrum for nitrosomonas, the 1.7 kb 
gene probe SPN 366.1 was identified as particularly suitable for the 
detection of nitrosomonas. 
For further optimization and synthesis of oligonucleotide gene probes, this 
gene probe was first molecular-biologically characterized by cleavage with 
restriction enzymes. The linear restriction map of the b 1.7 kb gene probe 
SPN 366.1 is shown in FIG. 3. 
To shorten the 1.7 kb gene probe SPN 366.1, the 0.2 kb, 0.3 kb, 0.5 kb and 
0.7 kb gene fragments formed by cleavage with the restriction enzymes 
HindIII, AccI, PstI and ClaI and ClaI were subcloned into the vector pSK 
Bluescript (Stratagene). 
The 0.2 kb, 0.3 kb, 0.5 kb and 0.7 kb gene probes were isolated from the 
corresponding constructs by electroelution after cleavage with restriction 
enzymes and gel-electrophoretic separation in agarose gel. The gene probes 
were labeled (P.sup.32 ATP, biotin, digoxigenin-UTP, enzyme) by the random 
prime labeling method and subsequently tested for their specificity and 
sensitivity in the gene probe test. It was found that the gene probes SPN 
391.7 (0.2 kb), SPN 397.1 (0.3 kb), SPN 391.3 (0.5 kb) and SPN 391.16 (0.7 
kb) did not show any significant differences in regard to specificity and 
sensitivity of nitrosomonas detection and were all equally suitable for 
use as gene probes. The gene probe tests with the gene probes SPN 391.7, 
397.1, 391.3 and 391.16 are shown by comparison with the 1.7 kb gene probe 
SPN 366.1 in Table 2. 
Sequencing of the gene probe SPN 366.1 
The 1.7 kb gene probe was sequenced on the basis of the gene probe SPN 
366.1 (1.7 kb) and the 0.2 kb, 0.3 kb, 0.5 kb and 0.7 kb gene probes. 
Sequencing was carried out by the Sanger dideoxy chain termination method 
(b 5). The nucleotide sequence of the 1.7 kb gene probe SPN 366.1 is shown 
in the attached sequence listing. 
Chemical synthesis of oligonucleotide gene probes 
Oligonucleotide gene probes were chemically synthesized on the basis of the 
existing sequence of the 1.7 kb gene probe SPN 366.1 by the amidite method 
of S. L. Beaucage and M. H. Caruthers (6) . 
By gene probe tests with various oligonucleotides from the region of the 
1.7 kb gene sequence of the gene probe 366.1, it was found that 15 mer-100 
mer oligonucleotide gene probes from any regions of the 1.7 kb gene probe 
can be used for the gene probe test. 
The following oligonucleotide gene probes proved to be particularly 
suitable in the gene probe test: bases 1-53, bases 1315-1365, and bases 
1610-1663 of SEQ ID No. 1. 
Carrying out the gene probe test 
To determine the specificity and sensitivity of the gene probes, a gene 
probe test was carried out with digoxigenin DUTP labeled gene probes using 
the Boehringer/Mannheim digoxigenin test kit. 
Labeling the nitrosomonas gene probes 
The gene probes were labeled with digoxigenin-dUTP by the random prime 
method of Feinberg and Vogelstein (3). Before the random prime labeling, 
the gene probes were cut out from the corresponding recombinant plasmids 
with restriction enzymes. The linearized gene probes were separated from 
the linearized plasmid vector by gel electrophoresis in 0.8% agarose gel. 
The gene probe DNA was cut out from the agarose gel and the gene probe was 
isolated from the agarose block by electroelution. The gene probe DNA was 
then further purified by extraction with phenol and precipitation with 
ethanol. Before labeling with digoxigenin-dUTP, the gene probe DNA was 
denatured by heating for 10 mins. in a water bath to 100.degree. C. and 
rapid cooling on ice/NaCl. For labeling, 1 .mu.g denatured gene probe DNA, 
2 .mu.l hexanucleotide mixture and 2 .mu.l dNTP labeling mixture were 
combined, made up to 19 .mu.l with sterile twice-distilled water and 1 
.mu.Klenow enzyme was added. Labeling with digoxigenin-dUTP was carried 
out for 60 mins. at 37.degree. C. The reaction was then stopped by 
addition of 2 .mu.l EDTA solution 0.2 mol/l pH 8 and the labeled DNA was 
precipitated with 2.5 .mu.l LiCl 4 mol/l and 75 .mu.l precooled ethanol 
(-20.degree. C.). After 30 mins. at -70.degree. C., the DNA precipitate 
was centrifuged off at 12,000 g and washed with cold ethanol, 70%, dried 
in vacuo and dissolved in 50 .mu.l tris-HCl 10 mmol/l, EDTA 1 mmol/l pH 8. 
Hybridization with nitrosomonas gene probes 
For the hybridization experiments, the nitrosomonas-DNA-containing nucleic 
acid extracts from the biomass of wastewaters or soils were first 
denatured into the DNA single strands by heating for 10 minutes to 
100.degree. C. and rapid cooling on ice/NaCl. Nitrocellulose membranes 
were pretreated by swelling in water and 20.times.SSC (NaCl 3 mol/l, Na 
citrate 0.3 mol/l pH=7) and dried. Nylon membranes were used without any 
pretreatment. The denatured nucleic acid extracts were applied to the 
nitrocellulose or nylon membranes using a Schleicher & Schell Minifold II 
filtration unit and then fixed by baking in vacuo for 1 h at 80.degree. C. 
or by UV crosslinking for 5 mins. using a UV transilluminator (nylon 
membrane). 
The nylon/nitrocellulose filters were sealed in a plastic bag containing 20 
ml hybridizing solution (5.times.SSC; blocking reagent 0.5%; N-lauroyl 
sarcosine, Na salt 0.1%; SDS 0.02%) and prehybridized for 1 hour at 
68.degree. C. The prehybridizing solution was then replaced by 2.5 ml 
hybridizing solution (same composition) containing freshly denatured gene 
probe DNA (100 ng). The hybridization batch was incubated for 2 hours at 
68.degree. C. The filters were then washed for 2.times.5 mins. at room 
temperature with 50 ml 2.times.SSC; SDS 0.1% and then again for 2.times.15 
mins. at 68.degree. C. with 0.1.times.SSC 0.1% SDS. The filters were 
directly used for the detection of the hybridized DNA or were stored in 
air-dried form for subsequent detection. 
Detection of the hybridized nitrosomonas DNA 
An immunological detection reaction was carried out for quantitative 
detection of the hybridized nitrosomonas DNA. An antibody conjugate with 
coupled alk. phosphatase was used which binds to the hybridized 
digoxigenin-labeled DNA. The color reaction was started at an alkaline pH 
by addition of the colorless 5-bromo-4-chloro-3-indolyl phosphate and 
nitroblue tetrazolium. The blue precipitate formed was quantitatively 
evaluated after 2-12 hours with a Shimadzu CS430 densitometer. The 
following buffers were used for the detection reaction 
Buffer 1: Tris/HCl 100 mmol/l; NaCl 150 mmol/l pH 7.5 
Buffer 2: 0.5% solution of blocking reagent in buffer 1 
Buffer 3: Tris/HCl 100 mmol/l; NaCl 100 mmol/l, MgCl.sub.2 50 mmol/l pH 
9.5 
Buffer 4: Tris/HCl 10 mmol/l, EDTA 1 mmol/l pH 8 Dye solution (freshly 
prepared) 45 .mu.l NBT and 35 .mu.l X-phosphate were added to 10 ml buffer 
3. 
The nitrocellulose/nylon filters were washed for 1 minute in buffer 1, 
incubated for 30 minutes with 100 ml buffer 2 and rewashed with buffer 1. 
The antibody conjugate was diluted in a ratio of 1:5,000 in buffer 1 and 
the filters were incubated for 30 minutes with approx. 20 ml of the dilute 
antibody conjugate solution. Unbound antibody conjugate was removed by 
2.times.15 mins. washing with 100 ml buffer 1 and the filters were 
subsequently equilibrated for 2 mins. with 20 ml buffer 3. The filters 
were then incubated with 20 ml dye solution in darkness in a sealed 
plastic bag. The color intensity of the individual slot blots was 
determined by densitometry by comparison with a co-applied nitrosomonas 
DNA standard. 
Specificity of the gene probes 
The specificity of the gene probes was analyzed by the described gene probe 
test. The nucleic acid was extracted from characterized gram-negative and 
gram-positive bacteria, including bacteria which degrade aromatic halogen 
compounds, aromatic nitro compounds, aromatic amino compounds, alkyl 
sulfonic acids and aryl sulfonic acids, and various nitrosomonas isolates 
and a dot blot hybridization test was carried out with the described gene 
probes to determine which bacterial lysates produced a positive 
hybridization signal (Table 2). 
Through the experiments, it was found that the developed nitrosomonas gene 
probes hybridized specifically with all nitrosomonas lysates and did not 
produce any hybridization signals with other bacterial lysates. 
Use of nitrosomonas gene probes for detecting and quantifying nitrosomonas 
in waters/wastewaters 
For detecting and quantifying nitrosomonas strains in waters/wastewaters, 
the total nucleic acid was first isolated from the centrifuged 
water/wastewater samples. 150 .mu.l 10.5M EDTA and 150 .mu.l twice-dist. 
H.sub.2 O and 3 .mu.l SDS, 20%, were added to 50 mg moist biomass which 
was then incubated in a water bath for 60 seconds at 100.degree. C. and, 
immediately afterwards, was placed in an ice/salt bath for 1 minute. 600 
.mu.l Tris-saturated phenol was then added for the first extraction with 
phenol, followed after mixing by centrifugation for 5 minutes at 5,000 G. 
The extraction with phenol was repeated with the upper aqueous DNA phase. 
Small amounts of phenol were removed by subsequent extraction with ether. 
The ether phase was removed, the DNA was precipitated with isopropanol and 
was then centrifuged off at 5,000 G in a tabletop centrifuge. The DNA 
pellet was washed with 70% ethanol. The DNA pellet was then taken up in 
220 .mu.l TE buffer and, as described with reference to the gene probe 
test procedure, was fixed to nitrocellulose or nylon membranes and then 
hybridized with the described gene probes. 
For quantifying, nitrosomonas DNA standard was applied in concentrations of 
250 ng to 3.5 ng corresponding to cell numbers of 2.5.times.10.sup.6 to 
3.5.times.10.sup.4 nitrosomonas cells. The positive hybridization reaction 
was evaluated on the basis of the color intensity of the 
5-bromo-4-chloro-3-indolyl nitroblue tetrazolium complex in a Shimadzu 
CS930 densitometer. The concentration of the nitrosomonas-specific DNA 
respectively the nitrosomonas cell titer in the sample material was 
determined by comparison with the slot blots of the nitrosomonas DNA 
standard. The detection limit of the described detection method was 
10.sup.5 -10.sup.6 nitrosomonas bacteria. 
Use of nitrosomonas gene probes for detecting and quantifying nitrosomonas 
strains in soils 
For detecting and quantifying nitrosomonas bacteria in soils, the nucleic 
acid of bacteria present in the soil was isolated by the method of Torsvik 
and Marmur (4). 
100 ml TE buffer (Tris/HCl 10 mmol/l, EDTA 1 mmol/l pH 8) were added to 10 
g soil, the sample was thoroughly mixed and the soil was subsequently 
separated from the bacterial extract by filtration. The bacteria were 
separated from the filtrate by centrifugation at 5,000 g. The bacterial 
fraction was washed once with 100 ml 0.1M Na.sub.4 P.sub.2 O.sub.7 (pH 7) 
and once with 100 ml 0.15M NaCl, 10 mM EDTA (Saline EDTA) and, after 
centrifugation at 5,000 g, was resuspended in 25 ml Saline EDTA. By 
addition of 1 mg/ml lysozyme and subsequent incubation for 30 minutes at 
37.degree. C., the bacteria were lysed with sodium dodecyl sulfate (SDS) 
in a final concentration of 1%. In order to remove most of the humic 
substances still present in the soil, further purification can be achieved 
by ion exchange chromatography and hydroxylapatite chromatography or 
pronase, RNase treatment and extraction with phenol. 
As described with reference to the gene probe test procedure, the DNA was 
fixed to nitrocellulose or nylon membranes and the hybridization reaction 
was carried out with the Boehringer/Mannheim digoxigenin test kit. The 
nitrosomonas-specific DNA concentration or cell titer was quantitatively 
evaluated from the color intensity of the 5-bromo-4-chloro-3-indolyl 
nitroblue tetrazolium complex of the slot blots. 
TABLE 1 
______________________________________ 
Molecular characterization of nitrosomonas clones 
Clone code Vector Insert [KB] 
Strain 
______________________________________ 
256/15 pBR 322 2.7 Nitrosomonas 
256/32 pBR 322 5.8 Nitrosomonas 
256/36 pBR 322 2.3 Nitrosomonas 
256/39 pBR 322 1.3 Nitrosomonas 
258/21 pBR 322 3.9 Nitrosomonas 
258/22 pBR 322 5.9 Nitrosomonas 
258/23 pBR 322 3.3 Nitrosomonas 
258/27 pBR 322 21.6 Nitrosomonas 
258/29 pBR 322 6.0 Nitrosomonas 
258/33 pBR 322 3.4 Nitrosomonas 
258/34 pBR 322 7.1 Nitrosomonas 
258/35 pBR 322 7.5 Nitrosomonas 
323/9A pSPT 19 6.0 Nitrosomonas 
323/13B pSPT 19 6.0 Nitrosomonas 
322/9A pSK 6.0 Nitrosomonas 
322/10B pSK 6.0 Nitrosomonas 
______________________________________ 
Nitrosomonas DNA clones as BamHI fragments in the E. coli vector pBR 322 
into E. coli 5K or AG1. The genomic DNA was isolated from the strain 
Nitrosomonas europaea 9718 and cloned. The 6 kb gene probe from 258/29 was 
recloned in both orientations into the vectors pSPT19 and pSK Bluescript. 
TABLE 2 
______________________________________ 
Specificity of the nitrosomonas gene probe 
Degrada- 
Hybridization with 
Strain tion of 0.2kb 0.3kb 
0.5kb 
0.7kb 
1.7kb 
______________________________________ 
Nm. europaea 
Ammonia + + + + ++ 
Nm. spec. 41-3/BE 
Ammonia ++ ++ ++ ++ +++ 
Nm. spec. LA33 
Ammonia ++ ++ ++ ++ +++ 
Nm. spec. 41-3/GB 
Ammonia + + + + ++ 
Nm. spec. 41-3/RW 
Ammonia + + + + ++ 
Nm. spec. A 83 
Ammonia + + + + ++ 
Nm. spec. A 13 
Ammonia + + + + ++ 
N1. multiformis 
Ammonia - - - - - 
Nb. agilis Nitrite - - - - - 
Th. pantotropha 
Sulfur - - - - - 
Tb. novellus 
Sulfur - - - - - 
Tb. perometab 
Sulfur - - - - - 
Tb. acidophilus 
Sulfur - - - - - 
Alc. faccalis - - - - - 
Str. facalis - - - - - 
Staph. capitis - - - - - 
Kl. planticola - - - - - 
Microb. lacticum - - - - - 
E. coli 5k - - - - - 
P. spec 61-Tol4 
Toluene - - - - - 
P. syringae 50-16 
Sulfonic - - - - - 
acid 
P. putida 82-1 
Nitro- - - - - - 
benz. 
Ps. putida NCIB 
Naphtha- - - - - - 
12042 lene 
Br. spec. 233 
Poly- - - - - - 
cycles 
Alcal. spec. 
Benzene - - - - - 
67-1.4R4 
Ps. spec. 67-D3/2 
Benzene - - - - - 
M. spec. 1.2/2 
Di- - - - - - 
chlorob. 
______________________________________ 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1722 bp 
(B) TYPE: Nucleotide 
(C) STRANDEDNESS: Double 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Genomic DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TTAGAAGTAATGAGCCCATGGCTATTTGCCGCGCATAAACAT42 
MetSerProTrpLeuPheAlaAlaHisLysHis 
TTTATCCAGTTCAGCCAGGCTGATTTCAAACCAGGTAGG81 
PheIleGlnPheSer GlnAlaAspPheLysProGlyArg 
TCGGCCGTGATTGCATTGATCCGAGCGCTCGGTTACTTC120 
SerAlaValIleAlaLeuIleArgAlaLeuGlyTyrPhe 
CATTTTGCGCAGAGTTCATTCATTTCAATCAGCGTTAA T159 
HisPheAlaGlnSerSerPheIleSerIleSerValAsn 
TGCCGGTTAGCGCGAACGGCACCGTGACAGGCCATGGTG198 
CysArgLeuAlaArgThrAlaProGlnAlaMetVal 
GCCAGTAATTCATTA CDACGGGCGGCAAGCAGTTGAGCG237 
AlaSerAsnSerLeuXaaArgAlaAlaSerSerAla 
GGATCGCCATTCCTGATTTCATCCAGCAGAGCACGTACC276 
GlySerProPheLeuIleSerSerSerArgAlaA rgThr 
AGTTTCGCATCAGCATGCTGCAGTGTGGCGGGGACTGTG315 
SerPheAlaSerAlaCysCysSerValAlaGlyThrVal 
CGTATAACAAGTGTGGTAGCGGACAGCGTGCTCACTTCA354 
ArgIleThrSer ValValAlaAspSerValLeuThrSer 
AAACACAGTTGCTGCAAAAGCGCCTGATTTTCCTCCACT393 
LysHisSerCysCysLysSerAlaPheSerSerThr 
GTCGCGATGTCGAGGCTATCTGCGTGAAATGTAA CCGGT432 
ValAlaMetSerArgLeuSerAlaAsnValThrGly 
ATCAGCAATCGGTTGTGCGGATAATACTTGTTGATCCAG471 
IleSerAsnArgLeuCysGlyTyrLeuLeuIleGln 
TTGTGTCTTCAA CTGCTCGTAGACAATGCGTTCGTGCGC510 
LeuCysLeuGlnLeuLeuValAspAsnAlaPheValArg 
GGCGTGCATGTCTACAATCACCAATCCTTTTTGGTTTTG549 
GlyValHisValTyrAsnHisGlnSerPhe LeuValLeu 
CGCCAGGATATAGATGCCGCGAAGTGCCCCAACGCAAAG588 
ArgGlnAspIleAspAlaAlaLysCysProAsnAlaLys 
CCCAGCGGGGGCATAGCCGAATTTTCATCGCTTTCTCCT627 
ProSerGl yGlyIleAlaGluPheSerSerLeuSerPro 
TCCCCGGTTTGTCGAGGTTGATTTTGTATGGCAGTGGCG666 
SerProValCysArgGlyPheCysMetAlaValAla 
CCGGATTCTCCGCCGGATAGAACCTGATAA AAGTTAAAA705 
ProAspSerProProAspArgThrLysLeuLys 
GGGTGCGCCACCCTTTCTGATGACAGCCGTGCTTGCCTG744 
GlyCysAlaThrLeuSerAspAspSerArgAlaCysLeu 
GGGTAGTT CAACGTCCACAGCCGGTGTAAAACCGGTGCG783 
GlyPheAsnValHisSerArgCysLysThrGlyAla 
CGTTGGATCAACAGATGCATCCTGCGTACCTGGCCACAC822 
ArgTrpIleAsnArgCysIleLeuArg ThrTrpProHis 
GGCCCCAACAGGAGAGGAGGATGCTACAGCCGAGCGGGG861 
GlyProAsnArgArgGlyGlyCysTyrSerArgAlaGly 
TAGAGCCAGCGCCTTGTGACGCCGTGGTAAATAAATTGG900 
S erGlnArgLeuValThrProTrpIleAsnTrp 
TGGATGGCCCGCTTCGGCGAAAGCGACTACGTTTCGTCG939 
TrpMetAlaArgPheGlyGluSerAspTyrValSerSer 
ATGTACGTTGACATCCACCTGTTCAGG ATCGATCGCCAG978 
MetTyrValAspIleHisLeuPheArgIleAspArgGln 
ATACAGCACGAAAGCGGCATGACGATCAAGGTGCAGCAC1017 
IleGlnHisGluSerGlyMetThrIleLysValGlnHis 
ATCA CGATAGCTTCGCGCAGGGCATGGGTAATCAGCTTG1056 
IleThrIleAlaSerArgArgAlaTrpValIleSerLeu 
TCGCGGATGAAGCGTCCGTTAACAAAAAAATACTGCATG1095 
SerArgMetLysArgProLeuTh rLysLysTyrCysMet 
TCGCGGGTGGCGCGTGAATACGCGGGCAATGCCAGCATC1134 
SerArgValAlaArgGluTyrAlaGlyAsnAlaSerIle 
CCCTGCAAACCGATGCCGGCGGATTGTTCGTCCATCCAG1173 
ProCysLysProMetProAlaAspCysSerSerIleGln 
GTAGCCGNTCCGGCAAATTCCTCGCCAAGTACGGCTCCG1212 
ValAlaXaaProAlaAsnSerSerProSerThrAlaPro 
ATACGCTCTGCAGCCTCTGCTGC CTGCCAGTGCTGCGCA1251 
IleArgSerAlaAlaSerAlaAlaCysGlnCysCysAla 
GGTTTCCATTGTGCCGCAGCGTAAAGGTAATATCAGCGT1290 
GlyPheHisCysAlaAlaAlaArgTyrGlnArg 
GGGAAAGTGCCATCCGCCGAAAAACTTCTTCGCAGTGGG1329 
GlyLysValProSerAlaGluLysLeuLeuArgSerGly 
CAAACTCTGTAGCTTCTGTTTTAAGAAATTTGCGGCGGG1368 
GlnThrLeuLeuLeuP heGluIleCysGlyGly 
CAGGCAGGTTGAAAAACAGATCCCGGACTTCAACCGTAG1407 
GlnAlaGlyLysThrAspProGlyLeuGlnPro 
TGCCCGCCATGTGGGATGAAGGCTCCGGCGACATTAACG1446 
CysProProCysGlyMetLysAlaProAlaThrLeuThr 
TGTCCCCTCACTGCGGATTTCCCAGGCATGTTTGCCAGC1485 
CysProLeuThrAlaAspPheProGlyMetPheAlaSer 
GGGTTGATGACTGATGAGCGAC AAATACGAAACTGACGC1524 
GlyLeuMetThrAspGluArgGlnIleArgAsnArg 
GATACTGGCCAGCCCTTCCCCCCGGAATCCCAGGCTGGT1563 
AspThrGlyGlnProPheProProGluSerGlnAlaGly 
GATGCTGTGCAAATCCTCCTGGCTGGCAATTTTGCTGGT1602 
AspAlaValGlnIleLeuLeuAlaGlyAsnPheAlaGly 
TGCGTGACGTGTAAGTGCAAGCGGCAGTTCTTCTGCGGG1641 
CysValThrCysLysCys LysArgGlnPhePheCysGly 
AATGCCGCTGCCGTTATCGGTCACACGGATCAGTTTCAA1680 
AsnAlaAlaAlaValIleGlyHisThrAspGlnPheGln 
TCCACCCTGTGCGATATTGACCGTAATCTCAGTCGCACC1 719 
SerThrLeuCysAspIleAspArgAsnLeuSerArgThr 
GGC1722 
Gly 
Literature 
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Physiol, 30, 125-177 (1989) 
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Maniatis, Cold Spring Harbor Laboratory Press (1989) 
3. "A Technique for Radiolabeling DNA Restriction Endonuclease Fragments to 
High Specific Activity" A. P. Feinberg and B. Vogelstein, Anal Biochem 
132, 6 (1983) 
4. "Isolation of Bacterial DNA from Soil," V. L. Torsvik, Soil Biol. 
Biochem. 12, 10-21 (1980) 
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