Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
Bac~ the Inv ntion
For some time it has been known that DNA
(deoxyribonucleic acid) molecules contain the instruckions
for the assembly and organization of living systems. The
nucleotide bases along the DNA molecule chain, arranged in
specific sequences sometimes referred to as "genetic
codes", specify the structure of the thousands of proteins
that make up cells. The information in the DNA molecules
must first be transcribed into the complementary sequence
of messenger ribonucleic acid ImRNA). The messenger RNA
directs the assembly of amino acids into the specific
linear sequence characteristic of a particular protein
through a process called translation.
Another kind o~ RNA, called tRNA, carries or transfers
individual amino acids from the ~ree state inside the cell
to the mRNA in the sequence coded in the mRNA.
The -translation process begins when a particular tRNA
attaches itself to a particular amino acid in a reaction
catalyzed by an enzyme called arninoacyl~tRN~ synthetase.
~ach type oE synthetase is specific for one of the 20
dif~erent amino acids found in proteins. For example,
leucyl-tRNA synthetase selectively binds itselE to both the
amino acid leucine and to the tRNA for leucine. Similarly,
the other tRNA molecules are attached to their
corresponding amino acids by their corresponding
synthetase.
~Z~
Each tRNA mo]ecule included three special nucleotide
bases, called the anti-codon, which interacts with three
complementary codon bases in the messenger RNA. Thus, the
tRN~ molecule weakly attaches itselE at the corresponding
location along the mRNA chain. The next codon site along
the mRNA chain accepts the corresponding tRNA anti-codon.
The amino acid on the first tRNA is cleaved from its tRNA
and attaches to the free end of the amino acid on the
second tRNA. Sequentially, additional tRNA : amino acid
molecules ~arrying their amino acids attach to subsequenk
mRN~ sites, with the growing chain, cleaving and
reattaching to each next tRNA amino acid, each such shift
adding one amino acid to the chain.
Eventually, a specific polypeptide c,hain corresponding
to the coding in the original ~NA is formed. The above
discussion is limited to a discussion of the growth of the
chain by translaton, since the method o~ thi3 invention is
involved in only that portion oE proteln production. Other
ste~ps and mechanisms, such as those for staxting chain
growth, are not relevant to the method of this invention.
The process Aescribed above can be adap-ted to the
manufacture of a number of different beneficial
polynucleotides, such as those described in my earlier
U. S. Patents ~,358,586 and 4,401,759. Analogs of
medically or commercially important enzymes, hormones, or
proteins, more powerful than the corresponding natural ones
may be produced in large amounts by cloning and applying my
methods listed below khey may be commercially or medically
important proteins, enzymes, peptlde hormones,
antibiotics, peptides, peptides modified after cloning
helpful in fixation of nitrogen, fermentation adjuncts in
9~
speciEic feedstocks, or the like.
Peptide analogs built to order may be obtained by
application of my methods listed in this application
(examples of endorphin analogs made in cloning process are
listed below~, regaxdless of the sequence o~ DNA cloned,
and regardless of whether the DN~ sequence was synthesized
e.g. to produce insulin A & B chain, or somatostatin; or
the DNA gene was captured by deoxynucleotide as in my
endorphin, or any other state of the art method of arriving
at DNA that is la-ter cloned. Also, the structure o~ the
original DNA can be modified, or selected anti-codons may
be spliced into an mRNA chain to replace or add to the
natural anti-codon sequence. The resulting protein will
then be modified when the tRNA complementary to the new
anti-codon inserts its amino acid at that point into the
protein chain being produced.
These prior known methods for producing selected
proteins, while use~ul, have several disadvantageq. Solid
state t~chniques are slow and expensive. Cloning, while
2~ eEfective with "natural" proteins such as endorphin,
interferon and insulin, has not in the past been capable of
producing clones of analog proteins.
OEten, it is desirable to produce analogs of
beneficial proteins, such as endorphin, with improved
properties by replacing certain selected amino acids in the
protein chain. For example, analogs of endorphin have been
produced by the solid state method that have varlous
combinations of certain desirable properties. The solid
state method, unEortunakely/ is a very 510w process oE
assembliny the protein one amino acid at a time. However,
the analogs of endorphin so produced have been found to
produce grea~er relief from painr longer-lasting pain
relief, be more lipophilic or lipophobic in cértain
portions of the molecule and be more resistant to enzymatic
degradation (as is the case with D-ala-2-endorphin) when
compared with natural endorphin. Also, analogs may be
beneficial when patients become refractory to the effects
of ~he natural endorphin.
While such analogs have been produced by solid sta-te
methods, those methods have a number of problems.
Solid state methods of "stitching" individual amino
acids together in the proper order.are severely limited by
the many steps and extended time involved in linking one
amino acid to the next to form the growing peptide chain
and the low yield at each step. Also, isolating the
desired peptide free from the many impurities i8 difEicult.
Summ~ Invention
The above-noted problems, and others~ are overcome by
a method which basically comprises the steps of selecting
an mRNA molecule capable of producing an unmodified protein
which has characteristics which are to be improved,
selecting an amino acid substitution to be made in the
protein chain ordinarily produced by said mRNA through
transla-tion, determining the codon along the mRNA chain
which ordinarily provides tlle normal or first amino acid to
be replaced by a second amino acid, modifying the -tRNA
having the anti~codon which would ordinarily complement the
~L2~
codon at that site to ordinarily deliver the firc~t amino
acid thereto in a way which will cause it to instead
.
deliver the second amino acid thereto and finally
conducting translation of said mRNA in the presence of the
modified tRNA and the appropriate tRNA for the other
required amino acids, whereby an analog of the protein
ordinarily produced by said mRNA results. The modified
protein will have the second amino acid in place of the
first amino acid.
The required modification of the tRNA can be
accomplished in several ways. Either a difEerent amino
acid may be substituted for a natural amino acid in a given
protein (eOg., alanine-2 for glycine-2 in endorphin) or an
amino acid analog may be substituted for the natural amino
acid in the protein (e.g., p-chlorophenylalanine for
phenylalanine in position 4 of endorphin).
One method of accomplishing the above-described
modification of the selected tRNA is misacylation. Ilere,
an x tRNA(y) is formed where ~Ix~ and llyll are amino acids so
that llxl1 is inserted into khe protein where the mRNA codon
calls Eor "y". Misacylation is facilitated by
dimethylsulfoxide/ cacodylate and methanol, as detailed
below.
Another method for modifying the selected tRNA is
changing the anti-codon of the selected tRNA to the anti-
codon of the tRNA carrying another amino acid by chemical
means such as the use of bisulfite, as detailed below.
Also, one may synthesize tRNA(y) with the anti-codon for
the tRNA corresponding to amino acid "x", so that the tRN~
will deliver amino acid "y" in place of "x" as the protein
is grown through translation.
Where the second amino acid is an analog oE the Elrst,
and not totally difEerent, the tRNA which ordinarily
carries the ~irst amino acid can be modified by "forcing"
it to accept and carry the second. Often, this can be
accomplished by merely mixing the analog (e.g., p-
chlorophenylalanine) into -the translation mixture in place
of the ordinarily called-for natural amino acid le.g.,
phenylalanine). In other cases, it is desirable to load
the analog into the corresponding tRNA prior to adding it
10 to the translation solution.
Also, an x-tRNA(x) can be modified to y-tRNA (x) by
chemical or enzymatic means such as the conversion of cys-
tRNA(cys) to ala-tRNA(cys) by reduction of cystein in the
presence of Raney nickel.
One may cleave tRNA into 2 approximately equal halves
neither half containing anti-codon; and add one of the 57-
60 other anti-codons to one of the tRNA halves. The
original half plus the other with its altered anti-codon
may now be allowed to snap together.
Incuba-tion of t-RNA CC-OH (lacking terminal A group)
with preaminoacylated PIP2 di(adenosine 5prime) diphosphate
may be accomplished in the presence of purified RNA ligase
transferred an aminoacyladenylate to the 3' terminus of the
shortened t-RNA in yields 33-79% also t-RNA CC-OH (2.5 to 4
A260 units) may be incuba-ted with 14 A256 units of
aminoacylated diadenosine diphosphate in presence of 10.5-
18 units of RNA ligase. After 4-8 hours of incubation, the
desired aminoacyl tRNA may be separated from unreacted
starting material by chromatography on BD cellulose.
30 Additionally, Vse of aminoacyl tRNA containincJ non-cognate
amino acid or analog may be used to insert a non-coynate
amino acid or analog into a peptide chain, in place of the
normal amino-acid.
Detailed Description of the Invention
Details of the invention, and of certain preferxed
embodiments thereof, will be further understood upon
reference to the following description of the method of
this invention. All parts and percentages are by weight
unless otherwise indicated.
In preparing analogs oE useful proteins which have
improved properties, the first step is selec-ting analogs
for pharmaceutical testing which are likely to be
beneficial. Typical systems for investigating the likely
pharmaceutical characteristics of a yet to be built protein
have been described by P~ Gund et. al., Science (1980) 208~
1425 and others. Typical of these is the Crystnet System
where typical functions are network access to a protein
data base, large molecular display in three dimensions and
drug-enzyme doclcing studies. Similarly the MMS-X Sys-tem
provides conformational analysis, receptor mapping and
protein crystal fitting.
Using these known characteristics oE proteins and
amino acids, one or more substitutions which appear likely
to produce improved properties are selected. Typical
selections might be certain analogs of "natural" endorphin
and enkephalin. The following analogs appear to be
favorable: 3,4Cl2 phenylalanine 4; D-alanine 2 met 5
enkephalin; D-alanine 2, 3CF3 4CL phenylalanine 4; met 5
enkephalin and their corresponding endorphins~
SufEicient of the selected enkephalin and endorphin
analogs are then synthesized by solid state techniyues to
test their analgesic properties by any conventional test,
such as the rat tail flick test, comparing the analog with
"natural" endorphin and enkephalin whether administered
intravenously, inter-muscularly or by injection into the
spinal column. The most promising analog candidates are
then produced on a macro scale by the amino acid insertion
and cloning methods of this invention, using any suitable
combination of my methods as set forth above.
One method for inserting ~-amino acids into proteins
is as follows. I have found that D-alanin 2 met 5
enkephalin is a rnuch more effective analgesic and has a
greater resistance to enzymatic degradation when compared
to "natural" met-enkephalin.
Typical conditions for forming D-alanine tRNA (ala)
are as follows: A reaction mixture (0.4 ml) is prepared
containing about 100 mm 2-mercaptoethamol, about 100
micrograms/ml BSA, about 0.25 mM D-alanine, 2 to 15 A260
units E. coli tRNA and an excess (about 4 units) E. coli
tRNA synthetase. ~eaction is allowed to proceed for about
10 minutes. The D-alanine tRNA (ala) is isola-ted by acid
precipitation and filtration in the manner described by
Calendar and Berg, Biochemistry, 5, 1690 (1966).
The D-alanine is incorporated into the protein in a
translation mixture containing the D-alanine tRNA (ala)
made above. Cloning then is carried out by any of -the
techniques clescribed in my above referenced ~-~h~ U.S.
Patents~p~te~ , with D-ala tRN~(ala) added, and as
sole source of alanine in the cloning mixture.
In another amino acid substl-tution technique, tRNA may
be misacylated by non-cognate amino acids in the range of
about 1 to 0.1% under the conditions present in in vivo
translation. Yields may be increased up to about 70-80%
loading by non cognate amino acids by control of in vitro
conditions which include the use of solvents such as
dimethylsulfoxide, methanol, cacodylate ion, pH ratio of
synthetase to tRNA and temperature. Generallyl a p~l o~
about 8~25 to 8.75 is optimum for misacylation. Optimum
10 results are also obtained with temperatures in the 30-37
degree C. range. There is almost a linear increase in
misacylation as dimethylsulfoxide or ethanol is increased
to about 20~, while over 20% misacylation decreases.
The length of the protein chain is controlled by the
insertion of a stop signal. The mRNA's that contain codons
UGA, UAA, and UAG will translate all codons lnto
corresponding amino acids stitched into the protein chain
up to but not including the codon for termination; no
further ~mino acid~ will be added thereafter. Eor
~() insertion o stop codons UGA, UAA, and UAG, the
corresponding deoxynucleotides must be inserted at the
appropriate places into the DNA to be cloned. For example,
if met enkephalin is to be produced in a clone terminating
in methionine 5, the following deoxynucleotides are
typical of those which may be used:
d-TCA CAT GAA CCC CCC GTA
d-TTA CAT GAA CCC CCC GTA
d-CTA CAT GAA CCC CCC GTA
If beta-endorphin is to be produced Erom a clone the
termination signals need to be inserted termina-ting
endorphin at gln 31. Deoxynucleotide probes which
~2~
accomplish tha-t purpose have the general Eormula:
d-TCA xTG zCC xTT xTT yTG
d-TTA xTG zCC xTT xTT yTG
d-CTA xTG zCC xTT xTT yTG
wherein "x" is C or T, "y" G or A and "z" is A, G, C or T.
Any suitable amino acid may be inserted into a protein
in place of another amino acid to modify the properties of
the protein. Typical amino acids which may be used include
the 20 basic. amino acids and p-aminophenylalanine, p-
10 fluoro/ m-fluoro, o-fluoro phenylalanine, ethionine,
norleucine, seleno-methionine, azatryptphane, 2-thienyl
alanine, azetidine 2 carboxylic acid 1, 2, 4
triazolealanine, beta hydroxy aspartic acid lanthionine,
citrulline, sarcosine, 3, 5 di-iodo tyrosine, and alpha
amino isobutyric acid.
Another method fo.r synthesizing analogs by use o~ the
techniques oE my invention will be described for several
se1ected enkephalins and endorphins which ~ have found to
have superior propertiesO These analogs are D-ala2-4-
20 chlorophenylalanine-4-met enkephalin; D-ala2-3CF3, 4Cl
phenylalnine-4 met enkephalin; D-ala2-3, 4(CF3)2
phenylalanine-~ met enkephalin; and D ala2-3CF3, 4-NO2
phenylalanine-4 met enkephalin and their corresponding beta
endorphins.
I'he synthesis of these analogs is based on cloned
material from beef endorphin gene. There are basically
four steps involved in obtaining each of-the above-listed
analogs. First, the codon gly 2 is changed to ala 2. Next
the peptide is terminated at the end of the fifth amino
acld (met enkephalin) or -thirty~first amino acid (for beta
endorphin)O Next d-ala 2 is inserted in place of gly 2.
.
The fixst two tasks will be perfomed by the use of one
probe to produce enkephalin and two probes to produce
endorphin ~one probe to substitute alanine for glycene 2
and the other probe to terminate the peptide chain). These
probes are used in the manner described in my two ~a~
U. SO PatentSa-pp-l~ns listed above. The probes for
enkephalin are TTA CAT yAA zCC zGC yTA; TCA CAT yAA zCC zGC
10 yTA; and CTA CAT yAA zCC zGC yTA with the endorphin probes
being any of these plus TTA xTG zCC xTT xTT yTG; TCA xTG
zCC xTT yTA; CTA xTG zCC xTT xTT yT&; wherein "x" is C or
T, "y" G or A, and "z" is A, G, C or T and the underlined
triplet is the "stop" signal, with (in the first set of
sequences) the underlined single nucleotide identifying the
substitution of alanine 2 for glycene 2. The third step is
accomplished by preparing V-ala tRNA (ala) as described
above. Similarly, the Eourth step is accomplished by
substitutincJ the desired phenylalanine analog into the phe
20 tRNA (phe) in the cloning mixture, omitting al:L
phenylalanine thererom.
The following examples provide a specific preferred
embodiment of the method of my invention within the contex-t
of the more general method described in detail ahove.
These examples detail a preferred method of analog
production, together with preferred tests to assure that
the selected analog is actually produced. All parts and
percentages are by weiqht unless otherwise indicatedO
Example I
-
Extraction of m-RNA (endorphin) is performed by the
following method. Beef pituitary is removed from fresh
beeE carcasses at the abattoir. The pituitary is finely
minced and is mixed with about 0.1 m. per g. of Dulbecco's
medium (available from Gibco, Catalog No. 320). The
mixture is pureed in a Waring blender. To the puree is
added about 0.8 m./g. Dulbecco's medium, about 0.1 mg/g
fetal calf serum; about 0.5~ proteinase K, about 1
microliter per gram tissue of a radioac-tive amino acid (as
a tracer) and about 0.5% collagen. The mixture is
incubated for about 30 minutes at about 37C. Then
sufficient cycloheximide is added to produce an about 1
microM concentration and the mixture is incubated for about
two minutes. Solid cell matter is filtered out using
several layers of cheesecloth. Cells from the Eiltrate
are pelleted by centriEuging for about ten minutes at about
1000 G. The cells are washed with a mixture o~ RSB (a
rinse buffer containing about 10 mM Tri.s (hydroxymethyl
amino methene sulfonic acid), pH 7.4, about 10 mM NaCl,
about 3 mM MgC12) and about 1 mM cycloheximide. The cells
are then resuspended in RSB, then are lysed with 1/10 vol.
Nonidet P40 (a non-ionic detergent available from Sigma)
and are allowed to settle for about five minutes with
gentle agitation. The mixture is then centrifuged for
about five minutes at about 900 G. and the supernatant is
collected. The supernatant is treated witll an equal volume
oE 5~ Triton X-100 (an ionic detergent from Sigma) and
agitated for about five minutes. The pH is lowered to
about 5.3 ~ith 2m E~Cl. The mixture is then centrifuged Eor
about five minutes at about 8000 G. The pellets produced
are crude polysomes. The polysomes are resuspended in a
* Trade mark
minimum volume o a Borate BufEer (about 0.1 M borate, pH
8.2, and 1% BSA, bovine serum albumin). Next the m-RNA
(endorphin) present in the polysome suspension is isolated
by antibody purification. A small affinity column of
Sepharose 4B (an exclusion chromatography resin, Pharacia
17-0120-01) is washed with anti-ACTH (an antibody that
binds selectively to adrenocriticotrophic hormone) with
about 0.1 M Hac (acetic acid) at about 4C. The colurnn is
prepaxed with about 5 volumes Borate Buffer. The polysome
suspension sample is then added to the affinity column
which is allowed to equilibrate overnight. Unreacted mRNA
is then washed out with Borate Buffer. The column is then
washed with 1 volume saline Triton ~uffer (l~BSA, 0.1 M
borate (pH 8.2), 0.2~ Triton X-100 and 0.3 M NaCl), then
with 0.1 volume of a 10/1 dilution Borate Buffer, then with
1 volume 0.1 M HAc. This is the main elution; a check of
the radioactive count will show the relative amount o~
enriched mRNA (endorphin) present. The column is khen
washed with about 1 volume of 0.001 M HCl, and finally wi.th
ahout 1 volume of 0.12 M HCl.
Exam~le II
Glutamine t-RNA (qlu) is prepared and purified as
follows. One unit (50 micrograms) t-RNA (glu), about 5
microliters *glutamine (6.25 nM), one unit synthetase (an
enzyme which transfers amino acid to its corresponding -t-
RNA), about 100 micrograms BSA, about 2 mM ATP (adenosine
triphosphate), about 70 nM MgC12 and about 10 mM beta-
mercaptoethanol are mixed together, then the mixture is
maintained at about 35C. For about two hours. About 100
microliters o~ this crude glutamine t-RN~ (glu) mixture is
mixed with about 660 microliters TCA (tri-chloroacetic
acid), a Sephadex G-200 buffer to make 1 volun-le, 1% KAc
13
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~o~
tpotassium acetate), pH 4.6, and a small amount of the
marker, Blue Dextrose. The mixture is applied to a
Sephadex G-200 column (an exchange chromatographic resin,
Pharmacia 17-oo81-01). The glutamine t-RNA (glu) appears
as a discrete peak where maxima are obtained Eor
radioactivity due to glutamine and color ~or t-RNA, thus
confirming tha'c they are combined.
Example III
The following method translates m-RNA (endorphin) to
10 produce an endorphin analog in which phe 4~18 is replaced
by pNH2 phe 4,18. A translation mixture is prepared
consisting of: 35g. total polysomal RMA enriched in m-RNA
(endorphin), (obtained in Example I) 100 lambda
reticulocyte lysate mixture; 30 lambda lOX aminoacid
mixture minus gly, tyr, phe; PNh2 phe in 30 lambda 100 X 25
lambda 60 mM fructose diphosphate; 60 lambda placental
ribonuclease inhibitor (300 u.); 85 larnbda KAc ~2M); 150
lambda *tyr (lmCi/l ml.); and 100 lambda *gly (1 mCi/]
ml.). This mixture is mixed gently and allowed to incubate
20 at about 30C. for about 90 minutes. The reaction is
stopped by placing the reaction vessel on an ice bath.
About ten microliter samples are placed on Ecteola paper
strips which are then washed with KAc and HAc. The
resulting product on the strips at this point is ACTH/Beta
liptropin.
Example IV
Free endorphin analogs are produced by cleaving
ACTH/Beta liptropin with clostripain according to the
following method. Clostripain cleaves preferentially only
30 at arginine sites. I have found that this applies to the
endorphin precur.sors described above. Therefore, lysine
1~
~ groups need not be protected by citraconic anhydride or the
like. A sample of the protein products produced in Example
III is heated on a steam bath for about five minutes.
About 6 sample volumes of a sample buffer consisting of
about 0.1 M NaPO4 (pH 7.8~ and about 0.005 M DTT
(dithioerythreitol), is mixed with the sample and about
1/10 sample vol. of clostripain (12.38 mg/ml) is added
thereto. The mixture is heated on a steam bath for about
five hours at about 30C. The remains are then solubilized
10 in a buffer mixture of about 0.15 M pyridine and pH is
adjusted to about 3.04 wi~h HAc. Cleavage products are
then fractionated on a Sephadex G-75 (an exchange
chromatographic resin, Phaxmacia 17-0051-01~ column as
previously described. A pronounced peak of radioactive
peptide is obtained corresponding to the molecular weight
of endorphin (M 3500).
Example V
That the structure obtained is that of the desired
analog is proved by the following method. ~he first 8
20 amino acids are removed in order by Edman Degradation.
Phenylisothiocyanate (PITC, the Edman reagent) reacts with
the polypeptide to yield a phenylthiohydontain (PTH-)
derivative of the N-terminal acid that can be identified
chromatographically. The resulting polypeptlde (minus the
N-terminal amino acid~ is treated again by the same
procedure to identify the next amino acid. Each arnino acid
thus removed is placed separately on thin layer
chromatographic material. Control chromatographs are run
wi~h known samples of the amino acids which should be
30 present if my product structure is correct. The positions
of the samples produced above result in chrornatographs
identical with the control samples, confirming that the
* Trade rnark
endorphin analog having pNH2 phe 4,18 in place of the phe
4,18 is produced.
_ .
Exam~e VI
The following method translates m RNA (endorphin) to
produce an analog in which glu 8 is replaced by gln 8. A
translation mixture is prepared consisting of about 35
micrograms of total polysomal RNA enriched in m-R~A
(endorphin) prepared as detailed in Example I; 100 lambda
reticulocyte lysate mixture; 30 lambda 10x amino acid
mixture minus gly, tyr and glu;a 10-fold molar excessover
average amino acid (100x) of gln t-RNA (glu) prepared a~ in
Example II, 25 lambda 60nM fructose diphosphate, 60 lambda
placental ribonuclease inhibiter (300 u); 85 lambda KAc
(2~); 150 lambda *tyr (lmCi/ml); 100 lambda *gly (lmCi/ml).
This mixture is mixed gently and allowed to incubate at
about 30C. for about 90 minutes. The reaction is stopped
by placing the reaction vessel on an ice bath. About 10
microliter samples are placed on ECTEOLA paper strips which
are then washed with KAc and HAc. The resulting product on
the strips at this point is ACTH/Beta lipotropin. The
ACTH/Beta is then cleaved by clostripain as described in
Example IV. A pronounced radioactive peptide peak is
obtained corresponding to the molecular weight of
endorphin. The structure obtained is confirmed by the
me-thod described in Example V to be the endorphin analog
having glu 8 in place of gln 8.
Example_VII
The steps of Examples I-V are further repeated to
produce other analogs of endorphin as follows:
16
~a) to provide pCl phe in place of phe,
~h) to provide thiolysine in place of lys;
~c) to provide PNO2 in place of phe ~; and
~d) to provide ala2 in place of gly 2.
In each case, the test of Example V shows that the
desired analog is produced.
While the above description of the method of my
invention and of several preferred embodiments thereof
described certain specific analogs, amino acid
substitutions, temperatures, etc., these may be varied,
where suitable with similar results.
Other variations, applications and ramifications of my
invention will become apparent to those skilled in the art
upon reading this disclosure. These are intended to come
within the scope of this invention as defined in the
appended claims.
17
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