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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
Borrelia burgdorferi Polynucleotides and Sequences
Field of the Invention
The present invention relates to the field of molecular biology. In
particular, it relates to,
among other things, nucleotide sequences of Borrelia burgdorferi, contigs,
ORFs, fragments,
probes, primers and related polynucleotides thereof, peptides and polypeptides
encoded by the
sequences, and uses of the polynucleotides and sequences thereof, such as in
fermentation,
polypeptidc production, assays and pharmaceutical development, among others.
Statement as to RibJhts to Inventions Made Under
Federally-Sponsored Research arid Development
Part of the work performed during development of this invention utilized U.S.
Government funds. The U.S. Government may have certain rights in the invention
- DE-FC02-
95ER61962; DE-FC02-95ER61963; and NAGW 2554.
Background of the Invention
Spirochetes are a family of motile, unicellular, spiral-shaped bacteria which
share a
number of structural characteristics. Three genera of the spirochetes are
pathogenic in humans:
(a) Treponerna, which includes the pathogens that cause syphilis (T.
pallidum), yaws (T.
per-tenue), and pima (T. carateum); (b) Borreliu, which includes the pathogens
that cause
epidemic and endemic relapsing fever and Lyme disease; and (c) Leptospira,
which includes a
wide variety of small spirochetes that cause mild to serious systemic human
illness (Koff, A. B.
and Rosen, T. J. Arn. Acud. Dermutol. 29:519-535 ( 1993)).
Lyme borreliosis, more commonly known as Lyme disease, is presently the most
common human disease in the United States transmitted by an arthropod vector.
Centers for
Disease Control, Morbid. Mortal. Weekly Rep. 44:590-591 ( 1995). Further,
infection of house-
hold pets, such as dogs, is a considerable problem. The causative agent of
this affliction is the
spirochete Borrelia burgdorferi, which is generally transmitted to mammalian
hosts by feeding
ticks. Barbour, A. and Fish, D. Science 260:1610-1616 ( 1993). Once the
bacteria pass through
the skin they disseminate and produce a variety of clinical manifestations.
Diagnosis of this
disease is often made serologically by the identification of antiborrelial
antibodies. Hilton, E. et
al., J. Clin. Microbiol. 35:774-776 ( 1997).
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
While initial symptoms often include a rash at the infection point, Lyme
disease is a
multisystemic disorder that may include arthritic, carditic, and neurological
manifestations.
While antibiotics are currently used to treat active cases of Lyme disease, B.
bur-gdorferi appears
to be able to persist even after prolonged antibiotic treatment. Further, B.
burgdorfer-i can persist
for years in a mammalian host even in the presence of an active immune
response. Straubinger,
R. et al., J. Clin. Microbiol. 35:111-116 ( 1997); Steere, A., N. Engl. J.
Med. 321:586-596
( 1989).
Animal models have proven useful for studying the progression of Lyme disease,
methods for preventing this disease, and immunological responses to antigenic
challenges with
B. burgdorferi proteins. Garcia-Monoco, J. et al., J. Infect. Dis. 175:1243-
1245 (1997). Using
a canine model, Starubinger, R. et al., Infect. Immun. 65:1273-1285 (1977),
demonstrated that
B. burgdorferi migrates into joints and induces up-regulation of interleukin-8
in synovial
membranes. Similarly, B. burgdorferi induction of interleukin-8 production has
been
demonstrated in cultured human endothelial cells. Burns, M. et al., Infect.
Immun. 65:1217-
1222 ( 1997).
Antigenic heterogeneity has been postulated as a mechanism used by B.
burgdorferi for
evasion of host immune responses. Schwan, T. et al., Can. J. Microbiol. 37:450-
454 (i991).
In support of this mechanism, antigenic variation has been described with
other pathogenic
bacteria. Hagbloom, P. et al., Nature 315:156-158 (1985). Further, cassette
type genetic
recombination of genes encoding B. burgdorferi surface proteins has been shown
to decrease the
antigenicity of these organisms to antibodies generated against strains which
have not undone the
same recombination. Zhang, J. et al., Cell 89:275-285 ( 1997).
A number of different types of Lyme disease vaccines have been tested and
shown to
induce immunological responses. Whole-cell B. burgdorferi vaccines have been
shown to
induce both immunological responses and protective immunity in several animal
models.
Reviewed in Wormser, G., Clin. Infect. Dis. 21:1267-1274 ( 1995). For example,
dogs
inoculated with a chemically inactivated whole-cell vaccine primarily develop
antibodies to outer
surface membrane proteins of the administered organism. Further, passive
immunity has been
also demonstrated in animals using B. burgdorferi specific antisera.
Similarly, passive immunity
is conferred human by the administration of sera obtained from Lyme disease
patients.
While whole-cell Lyme disease vaccines confer protective immunity in animal
models,
use of such vaccines presents the risk that responsive antibodies will be
generated which cross
react with human antigens. Reviewed in Wormser, G., supra. This problem is at
least partly the
result of the production of B. burgdorferi specific antibodies which cross-
react with hepatocytes
and both muscle and nerve cells. B. bc~rgdor'eri heat shock proteins and the
41-kd flagellin
subunit are believed to contain the antigens against which these cross-
reactive antibodies are
generated.
It is clear that the etiology of diseases mediated or exacerbated by B.
burgdorferi genes,
and that characterizing the genes and their patterns of expression would add
dramatically to our
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
understanding of the organism and its host interactions. Knowledge of B.
burgdorferi genes
and genomic organization would dramatically improve understanding of disease
etiology and lead
to improved and new ways of preventing, ameliorating, arresting and reversing
diseases.
Moreover, characterized genes and genomic fragments of B. burgdorferi would
provide reagents
for, among other things, detecting, characterizing and controlling B.
burgdnrferi infections.
There is a need therefore to characterize the genome of B. burgdorferi and for
polynucleotides
and sequences of this organism.
SUMMARY OF THE INVENTION
The present invention is based on the sequencing of fragments of the Bnrrelia
burgdorferi
genome. The primary nucleotide sequences which were generated are provided in
SEQ 1D
NOS:1-1SS.
The present invention provides the complete nucleotide sequence of the
Borrelia
burgdorferi chromosome and 1 S4 contigs representing the majority of the
sequence of the B.
1 S burgdorferi extrachromosomal elements, all of which are listed in tables
below and set out in the
Sequence Listing submitted herewith, and representative fragments thereof, in
a form which can
be readily used, analyzed, and interpreted by a skilled artisan. In one
embodiment, the present
invention is provided as contiguous strings of primary sequence information
corresponding to the
nucleotide sequences depicted in SEQ ID NOS: 1-1SS.
The present invention further provides nucleotide sequences which are at least
9S%,
96%, 97%, 98%, and 99%, identical to the nucleotide sequences of SEQ ID NOS:I-
1SS, ORF
IDs and corresponding ORFs.
The nucleotide sequences of SEQ ID NOS:1-1SS, ORF ID or ORF within, a
representative fragment thereof, or a nucleotide sequence which is at least
9S% identical to said
2S nucleotide sequence may be provided in a variety of mediums to facilitate
its use. In one
application of this embodiment, the sequences of the present invention are
recorded on computer
readable media. Such media includes, but is not limited to: magnetic storage
media, such as
floppy discs, hard disc storage medium, and magnetic tape; optical storage
media such as CD-
ROM; electrical storage media such as RAM and ROM; and hybrids of these
categories such as
magnetic/optical storage media.
The present invention further provides systems, particularly computer-based
systems
which contain the sequence information herein described stored in a data
storage means. Such
systems are designed to identify commercially important fragments of the
Borrelia burgdorfera
genome.
3S Another embodiment of the present invention is directed to fragments of the
Borrelia
burgdorferi genome having particular structural or functional attributes. Such
fragments of the
Borrelia burgdnrferi genome of the present invention include, but are not
limited to, fragments
which encode peptides, hereinafter referred to as open reading frames or ORFs,
fragments which
modulate the expression of an operably linked ORF, hereinafter referred to as
expression
CA 02304925 1999-12-17
WO 98/58943 PCT/LJS98/12764
4
modulating fragments or EMFs, and fragments which can be used to diagnose the
presence of
Borrelia burgdorferi in a sample, hereinafter referred to as diagnostic
fragments or DFs.
Each of the ORF IDs and ORFs in fragments of the Borrelia burgdnrferi genome
disclosed in Tables I-6, and the EMFs found 5' prime of the initiation codon,
can be used in
numerous ways as polynucleotide reagents. For instance, the sequences can be
used as
diagnostic probes or amplification primers for detecting or determining the
presence of a specific
microbe in a sample, to selectively control gene expression in a host and in
the production of
polypeptides, such as polypeptides encoded by ORFs of the present invention,
particular those
polypeptides that have a pharmacological activity.
The present invention further includes recombinant constructs comprising one
or more
fragments of the Borrelia burgdor~eri genome of the present invention. The
recombinant
constructs of the present invention comprise vectors, such as a plasmid or
viral vector, into
which a fragment of the Borrelia burgdorferi has been inserted.
The present invention further provides host cells containing any of the
isolated fragments
of the Borrelia burgdorferi genome of the present invention. The host cells
can be a higher
eukaryotic host cell, such as a mammalian cell, a lower eukaryotic cell, such
as a yeast cell, or a
procaryotic cell such as a bacterial cell.
The present invention is further directed to isolated polypeptides and
proteins encoded by
ORFs of the present invention. A variety of methods, well known to those of
skill in the art,
routinely may be utilized to obtain any of the polypeptides and proteins of
the present invention.
For instance, polypeptides and proteins of the present invention having
relatively short, simple
amino acid sequences readily can be synthesized using commercially available
automated peptide
synthesizers. Polypeptides and proteins of the present invention also may be
purified from
bacterial cells which naturally produce the protein. Yet another alternative
is to purify
polypeptide and proteins of the present invention from cells which have been
altered to express
them.
The invention further provides methods of obtaining homologs of the fragments
of the
Borrelia burgdorferi genome of the present invention and homologs of the
proteins encoded by
the ORFs of the present invention. Specifically, by using the nucleotide and
amino acid
sequences disclosed herein as a probe or as primers, and techniques such as
PCR cloning and
colony/plaque hybridization, one skilled in the art can obtain homologs.
The invention further provides antibodies which selectively bind polypeptides
and
proteins of the present invention. Such antibodies include both monoclonal and
polyclonal
antibodies.
The invention further provides hybridomas which produce the above-described
antibodies. A hybridoma is an immortalized cell line which is capable of
secreting a specific
monoclonal antibody.
The present invention further provides methods of identifying test samples
derived from
cells which express one of the ORFs of the present invention, or a homolog
thereof. Such
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
methods comprise incubating a test sample with one or more of the antibodies
of the present
invention, or one or more of the DFs of the present invention, under
conditions which allow a
skilled artisan to determine if the sample contains the ORF or product
produced therefrom.
In another embodiment of the present invention, kits are provided which
contain the
necessary reagents to carry out the above-described assays.
Specifically, the invention provides a compartmentalized kit to receive, in
close
confinement, one or more containers which comprises: (a) a first container
comprising one of the
antibodies, or one of the DFs of the present invention; and (b) one or more
other containers
comprising one or more of the following: wash reagents, reagents capable of
detecting presence
of bound antibodies or hybridized DFs.
Using the isolated proteins of the present invention, the present invention
further provides
methods of obtaining and identifying agents capable of binding to a
polypeptide or protein
encoded by one of the ORFs of the present invention. Specifically, such agents
include, as
further described below, antibodies, peptides, carbohydrates, pharmaceutical
agents and the like.
Such methods comprise steps of: (a)contacting an agent with an isolated
protein encoded by one
of the ORFs of the present invention; and (b)determining whether the agent
binds to said protein.
The present genomic sequences of Bnrrelia bur~dor~eri will be of great value
to all
laboratories working with this organism and for a variety of commercial
purposes. Many
fragments of the Borrelia burgdnrferi genome will be immediately identified by
similarity
searches against GenBank or protein databases and will be of immediate value
to Borrelia
burgdorferi researchers and for immediate commercial value for the production
of proteins or to
control gene expression.
The methodology and technology for elucidating extensive genomic sequences of
bacterial and other genomes has and will greatly enhance the ability to
analyze and understand
chromosomal organization. In particular, sequenced contigs and genomes will
provide the
models for developing tools for the analysis of chromosome structure and
function, including the
ability to identify genes within large segments of genomic DNA, the structure,
position, and
spacing of regulatory elements, the identification of genes with potential
industrial applications,
and the ability to do comparative genomic and molecular phylogeny.
DESCRIPTION OF THE FIGURES
FIGURE 1 is a block diagram of a computer system ( 102) that can be used to
implement computer-based systems of present invention.
FIGURE 2 is a schematic diagram depicting the data flow and computer programs
used
to collect, assemble, edit and annotate the contigs of the Borrelia
burgdorferi genome of the
present invention. Both Macintosh and Unix platforms are used to handle the AB
373 and 377
sequence data files, largely as described in Kerlavage et ul., Proceedira~s of
the Twenty-Sixth
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
Annual Hawaii International Conference on System Sciences, 585, IEEE Computer
Society
Press, Washington D.C. ( 1993). Factura (AB) is a Macintosh program designed
for automatic
vector sequence removal and end-trimming of sequence files. The program Loadis
runs on a
Macintosh platform and parses the feature data extracted from the sequence
files by Factura to the
Unix based Borrelia burgdorferi relational database. Assembly of contigs (and
whole genome
sequences) is accomplished by retrieving a specific set of sequence files and
their associated
features using Extrseq, a Unix utility for retrieving sequences from an SQL
database. The
resulting sequence file is processed to trim portions of the sequences with a
high rate ambiguous
nucleotides. The sequence files were assembled using TIGR Assembler, an
assembly engine
designed at The Institute for Genomic Research {TIGR ) for rapid and accurate
assembly of
thousands of sequence fragments. The collection of contigs generated by the
assembly step is
loaded into the database with the lassie program. Identification of open
reading frames (ORFs) is
accomplished by processing contigs with zorf. The ORFs are searched against B.
burgdorferi
sequences from GenBank and against all protein sequences using the BLASTN and
BLASTP
programs, described in Altschul et al., J. Mol. Biol. 215: 403-410 { 1990).
Results of the ORF
determination and similarity searching steps were loaded into the database. As
described below,
some results of the determination and the searches are set out in Tables 1-6.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention is based on the sequencing of fragments of the Borrelia
burgdorferi
genome and analysis of the sequences. The primary nucleotide sequences
generated by
sequencing the fragments are provided in SEQ ID NOS: 1-155. (As used herein,
the "primary
sequence" refers to the nucleotide sequence represented by the IUPAC
nomenclature system.)
SEQ ID NOS:1-155
In addition, the present invention provides the nucleotide sequences of SEQ ID
NOS: 1-
155, or representative fragments thereof, in a form which can be readily used,
analyzed, and
interpreted by a skilled artisan.
As used herein, a "representative fragment of the nucleotide sequence depicted
in SEQ ID
NOS:1-155" refers to any portion of the SEQ ID NOS: 1-155 which is not
presently
represented within a publicly available database. Preferred representative
fragments of the
present invention are Borrelia burgdor feri open reading frames ( ORFs )
represented by ORF
IDs, expression modulating fragments (EMFs) and diagnostic fragments
(DFs)which can be used
to diagnose the presence of Borrelia hurgdorferi in sample. A non-limiting
identification of
preferred representative portions are provided in Tables 1-6 as ORF IDs. As
discussed in detail
below, the information provided in SEQ ID NOS:1-155 and in Tables 1-6 together
with routine
cloning, synthesis, sequencing and assay methods will enable those skilled in
the art to clone and
sequence all "representative fragments" of interest, including ORFs encoding a
large variety of
Borrelia bur~dorferi proteins.
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7
The present invention is further directed to nucleic acid molecules encoding
portions or
fragments of the nucleotide sequences described herein. Fragments include
portions of the
nucleotide sequences of Table I-b (ORF IDs) and SEQ ID NOS:I-155, at least 10
contiguous
nucleotides in length selected from any two integers, one of which
representing a 5' nucleotide
position and a second of which representing a 3' nucleotide position, where
the first nucleotide
for each nucleotide sequence in SEQ ID NOS:1-155 is position 1 (therefore, the
sequence
postions for each ORF ID is determined by the numbering of the SEQ ID
comprising the ORF
ID). That is, every combination of a 5' and 3' nucleotide position that a
fragment at least 10
contiguous nucleotides in length could occupy is included in the invention. At
least means a
fragment may be 10 contiguous nucleotide bases in length or any integer
between 10 and the
length of an entire nucleotide sequence of SEQ ID NOS:I-155 minus 1.
Therefore, included in
the invention are contiguous fragments specified by any 5' and 3' nucleotide
base positions of a
nucleotide sequences of SEQ ID NOS:I-155 wherein the contiguous fragment is
any integer
between 10 and the length of an entire nucleotide sequence minus 1.
Further, the invention includes polynucleotides comprising fragments specified
by size,
in nucleotides, rather than by nucleotide positions. The invention includes
any fragment size, in
contiguous nucleotides, selected from integers between 10 and the length of an
entire ORF ID or
SEQ ID NO:, minus 1. Preferred sizes of contiguous nucleotide fragments
include 20
nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides. Other preferred
sizes of contiguous
nucleotide fragments, which may be useful as diagnostic probes and primers,
include fragments
50-300 nucleotides in length which include, as discussed above, fragment sizes
representing each
integer between 50-300. Larger fragments are also useful according to the
present invention
corresponding to most, if not all, of the nucleotide sequences shown in Tables
1-6 (ORF IDs)
and SEQ ID NOS:1-155. The preferred sizes are, of course, meant to exemplify
not limit the
present invention as all size fragments, representing any integer between 10
and the length of an
entire nucleotide sequence minus 1, of each ORF ID and SEQ ID NO:, are
included in the
invention.
The present invention also provides for the exclusion of any fragment,
specified by 5'
and 3' base positions or by size in nucleotide bases as described above for
any ORF ID or SEQ
ID NOS:I-155. Any number of fragments of nucleotide sequences in ORF IDs or
SEQ ID
NOS:1-155, specified by 5' and 3' base positions or by size in nucleotides, as
described above,
may be excluded from the present invention.
While the presently disclosed sequences of SEQ ID NOS: 1-155 are highly
accurate,
sequencing techniques are not perfect and, in relatively rare instances,
further investigation of a
fragment or sequence of the invention may reveal a nucleotide sequence error
present in a
nucleotide sequence disclosed in SEQ ID NOS: I-155. However, once the present
invention is
made available (i.e., once the information in SEQ ID NOS: I-155 and Tables 1-6
has been made
availablej, resolving a rare sequencing error in SEQ ID NOS: 1-155 will be
well within the skill
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
of the art. The present disclosure makes available sufficient sequence
information to allow any of
the described contigs or portions thereof to be obtained readily by
straightforward application of
routine techniques. Further sequencing of such polynucleotide may proceed in
like manner using
manual and automated sequencing methods which are employed ubiquitous in the
art. Nucleotide
sequence editing software is publicly available. For example, Applied
Biosystem's (AB)
AutoAssembler can be used as an aid during visual inspection of nucleotide
sequences. By
employing such routine techniques potential errors readily may be identified
and the correct
sequence then may be ascertained by targeting further sequencing effort, also
of a routine nature,
to the region containing the potential error.
Even if all of the very rare sequencing errors in SEQ ID NOS: 1-155 were
corrected, the
resulting nucleotide sequences would still be at least 95% identical, nearly
all would be at least
99% identical, and the great majority would be at least 99.9% identical to the
nucleotide
sequences of SEQ ID NOS: I-155.
As discussed elsewhere herein, polynucleotides of the present invention
readily may be
obtained by routine application of well known and standard procedures for
cloning and
sequencing DNA. Detailed methods for obtaining libraries and for sequencing
are provided
below, for instance. A wide variety of Borrelia bur~dorferi strains that can
be used to prepare B.
burgdorferi genomic DNA for cloning and for obtaining polynucleotides of the
present invention
are available to the public from recognized depository institutions, such as
the American Type
Culture Collection ( ATCC ). While the present invention is enabled by the
sequences and other
information herein disclosed, the B. burgdor~eri strain that provided the DNA
of the present
Sequence Listing, has been deposited with the ATCC, 10801 University Blvd.
Mantissas, VA
20110-2209, as Deposit No. 202012, on 8 August 1997. The ATCC Deposit is
provided merely
as a convenience to those of skill in the art. Reference to the deposit is not
a waiver of any rights
of the inventors or their assignees in the present subject matter.
The nucleotide sequences of the genomes from different strains of Borrelia
burydorferi
differ somewhat. However, the nucleotide sequences of the genomes of all
Borrelia burgdor~feri
strains will be at least 95% identical, in corresponding part, to the
nucleotide sequences provided
in SEQ ID NOS: 1-155 and the ORF IDs within. Nearly all will be at least 99%
identical and the
great majority will be 99.9% identical.
The present application is further directed to nucleic acid molecules at least
90%, 95%,
9b°~o, 97%, 98% or 99% identical to a nucleic acid sequence shown in
SEQ ID NOS: 1-155 and
the ORF IDs within. The above nucleic acid sequences are included irrespective
of whether they
encode a polypeptide having B. burgdorferi activity. This is because even
where a particular
nucleic acid molecule does not encode a polypeptide having B. burgdorferi
activity, one of skill
in the art would still know how to use the nucleic acid molecule, for
instance, as a hybridization
probe. Uses of the nucleic acid molecules of the present invention that do not
encode a
polypeptide having B. burgdorferi activity include, inter alia, isolating a B.
burgdorferi gene or
allelic variants thereof from a DNA library, and detecting B. burgdorferi mRNA
expression from
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
biological or environmental samples, suspected of containing B. burgdorferi by
Northern Blot,
PCR, or similar analysis.
Preferred, are nucleic acid molecules having sequences at least 90%, 95%, 96%,
97%,
98% or 99% identical to the nucleic acid sequence shown in SEQ ID NOS: 1-155,
the ORF IDs,
and the ORF within each ORF ID, which do, in fact, encode a polypeptide having
B. burgdorferi
protein activity. By "a polypeptide having B. burgdorferi activity" is
intended polypeptides
exhibiting activity similar, but not necessarily identical, to an activity of
the B. bur-~dorferi
protein of the invention, as measured in a particular biological assay
suitable for measuring
activity of the specified protein.
Due to the degeneracy of the genetic code, one of ordinary skill in the art
will immediately
recognize that a large number of the nucleic acid molecules having a sequence
at least 90%, 95%,
96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in SEQ ID
NOS: 1-I55,
the ORF IDs, and the ORF within each ORF ID, will encode a polypeptide having
B. burgdor~eri
protein activity. In fact, since degenerate variants of these nucleotide
sequences all encode the
same polypeptide, this will be clear to the skilled artisan even without
performing the above
described comparison assay. It will be further recognized in the art that, for
such nucleic acid
molecules that are not degenerate variants, a reasonable number will also
encode a polypeptide
having B. burgdorferi protein activity. This is because the skilled artisan is
fully aware of amino
acid substitutions that are either less likely or not likely to significantly
effect protein function
(e.g., replacing one aliphatic amino acid with a second aliphatic amino acid),
as further described
below.
The biological activity or function of the polypeptides of the present
invention are
expected to be similar or identical to polypeptides from other bacteria that
share a high degree of
structural identity/similarity. Tables 1, 2, 4, and 5 lists accession numbers
and descriptions for
the closest matching sequences of polypeptides available through Genbank. It
is therefore
expected that the biological activity or function of the polypeptides of the
present invention will
be similar or identical to those polypeptides from other bacterial genuses,
species, or strains listed
in Tables I, 2, 4, and 5.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical"
to a reference nucleotide sequence of the present invention, it is intended
that the nucleotide
sequence of the polynucleotide is identical to the reference sequence except
that the
polynucleotide sequence may include up to five point mutations per each 100
nucleotides of the
reference nucleotide sequence encoding the B. burgdorferi polypeptide. In
other words, to
obtain a polynucleotide having a nucleotide sequence at least 95% identical to
a reference
nucleotide sequence, up to 5% of the nucleotides in the reference sequence may
be deleted,
inserted, or substituted with another nucleotide. The query sequence may be an
entire sequence
shown in SEQ ID NOS: 1-155, an ORF ID, or the ORF within each ORF ID, or any
fragment
specified as described herein.
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
As a practical matter, whether any particular nucleic acid molecule or
polypeptide is at
least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the
presence
invention can be determined conventionally using known computer programs. A
preferred
method for determining the best overall match between a query sequence (a
sequence of the
5 present invention) and a subject sequence, also referred to as a global
sequence alignment, can be
determined using the FASTDB computer program based on the algorithm of Brutlag
et al. See
Brutlag et al. ( 1990) Comp. App. Biosci. 6:237-245. In a sequence alignment
the query and
subject sequences are both DNA sequences. An RNA sequence can be compared by
first
converting U's to T's. The result of said global sequence alignment is in
percent identity.
10 Preferred parameters used in a FASTDB alignment of DNA sequences to
calculate percent
identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining
Penalty=30,
Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size
Penalty=0.05,
Window Size=500 or the lenght of the subject nucleotide sequence, whichever is
shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3'
deletions,
not because of internal deletions, a manual correction must be made to the
results. This is
because the FASTDB program does not account for 5' and 3' truncations of the
subject sequence
when calculating percent identity. For subject sequences truncated at the 5'
or 3' ends, relative to
the query sequence, the percent identity is corrected by calculating the
number of bases of the
query sequence that are 5' and 3' of the subject sequence, which are not
matchedlaligned, as a
percent of the total bases of the query sequence. Whether a nucleotide is
matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then subtracted
from the percent identity, calculated by the above FASTDB program using the
specified
parameters, to arrive at a final percent identity score. This corrected score
is what is used for the
purposes of the present invention. Only nucleotides outside the 5' and 3'
nucleotides of the
subject sequence, as displayed by the FASTDB alignment, which are not
matchedlaligned with
the query sequence, are calculated for the purposes of manually adjusting the
percent identity
score.
For example, a 90 nucleotide subject sequence is aligned to a 100 nucleotide
query sequence to
determine percent identity. The deletions occur at the 5' end of the subject
sequence and
therefore, the FASTDB alignment does not show a matched/alignment of the first
10 nucleotides
at 5' end. The 10 unpaired nucleotides represent 10% of the sequence (number
of nucleotides at
the 5' and 3' ends not matched/total number of nucleotides in the query
sequence) so 10% is
subtracted from the percent identity score calculated by the FASTDB program.
If the remaining
90 nucleotides were perfectly matched the final percent identity would be 90%.
In another
example, a 90 nucleotide subject sequence is compared with a 100 nucleotide
query sequence.
This time the deletions are internal deletions so that there are no
nucleotides on the 5' or 3' of the
subject sequence which are not matched/aligned with the query. In this case
the percent identity
calculated by FASTDB is not manually corrected. Once again, only nucleotides
5' and 3' of the
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subject sequence which are not matched/aligned with the query sequence are
manually corrected
for. No other manual corrections are to made for the purposes of the present
invention.
COMPUTER RELATED EMBODIMENTS
The nucleotide sequences provided in SEQ ID NOS: 1-155, including ORF IDs and
corresponding ORFs, a representative fragment thereof, or a nucleotide
sequence at least 95%,
preferably at least 96%, 97%, 98% or 99%, and most preferably at least 99.9%
identical to said
nucleotide sequences may be "provided" in a variety of mediums to facilitate
use thereof. As
used herein, provided refers to a manufacture, other than an isolated nucleic
acid molecule,
which contains a nucleotide sequence of the present invention, a
representative fragment thereof,
or a nucleotide sequence at least 95°l0, preferably at least 99% and
most preferably at least 99.9°~0
identical to a polynucleotide of the present invention. Such a manufacture
provides a large
portion of the Borrelia bur~,~dorferi genome and parts thereof (e.g., a
Borrelia burgdorferi open
reading frame (ORF)) in a form which allows a skilled artisan to examine the
manufacture using
l5 means not directly applicable to examining the Borrelia burgdorferi genome
or a subset thereof as
it exists in nature or in purified form.
In one application of this embodiment, a nucleotide sequence of the present
invention can
be recorded on computer readable media. As used herein, "computer readable
media" refers to
any medium which can be read and accessed directly by a computer. Such media
include, but are
not limited to: magnetic storage media, such as floppy discs, hard disc
storage medium, and
magnetic tape; optical storage media such as CD- ROM; electrical storage media
such as RAM
and ROM; and hybrids of these categories, such as magnetic/optical storage
media. A skilled
artisan can readily appreciate how any of the presently known computer
readable mediums can be
used to create a manufacture comprising computer readable medium having
recorded thereon a
nucleotide sequence of the present invention. Likewise, it will be clear to
those of skill how
additional computer readable media that may be developed also can be used to
create analogous
manufactures having recorded thereon a nucleotide sequence of the present
invention.
As used herein, "recorded" refers to a process for storing information on
computer
readable medium. A skilled artisan can readily adopt any of the presently know
methods for
recording information on computer readable medium to generate manufactures
comprising the
nucleotide sequence information of the present invention.
A variety of data storage structures are available to a skilled artisan for
creating a
computer readable medium having recorded thereon a nucleotide sequence of the
present
invention. The choice of the data storage structure will generally be based on
the means chosen
to access the stored information. In addition, a variety of data processor
programs and formats
can be used to store the nucleotide sequence information of the present
invention on computer
readable medium. The sequence information can be represented in a word
processing text file,
formatted in commercially- available software such as WordPerfect and
Microsoft Word, or
represented in the form of an ASCII mile, stored in a database application,
such as DB2, Sybase,
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12
Oracle, or the like. A skilled artisan can readily adapt any number of data-
processor structuring
formats (e.g., text file or database) in order to obtain computer readable
medium having recorded
thereon the nucleotide sequence information of the present invention.
Computer software is publicly available which allows a skilled artisan to
access sequence
information provided in a computer readable medium. Thus, by providing in
computer readable
form the nucleotide sequences of the present invention (e.g. SEQ ID NOS: 1-
155), a
representative fragment thereof, or a nucleotide sequence at least 95%,
preferably at least 96%,
97%, 98%, 99% and most preferably at least 99.9% identical to a sequence of
the present
invention (e.g. SEQ ID NOS: 1-155) enables the skilled artisan routinely to
access the provided
sequence information for a wide variety of purposes.
The examples which follow demonstrate how software which implements the BLAST
(Altschul et al., J. Mol. Binl. 215:403-410 ( 1990)) and BLAZE (Brutlag et
al., Comp. Chern.
7 7:203-207 ( 1993)) search algorithms on a Sybase system was used to identify
open reading
frames (ORFs) within the Borrelia burgdorferi genome which contain homology to
ORFs or
IS proteins from both Borrelia burgdorferi and from other organisms. Among the
ORFs discussed
herein are protein encoding fragments of the Burrelia burgdorferi genome
useful in producing
commercially important proteins, such as enzymes used in fermentation
reactions and in the
production of commercially useful metabolites.
The present invention further provides systems, particularly computer-based
systems,
which contain the sequence information described herein. Such systems are
designed to identify,
among other things, commercially important fragments of the Borrelia
burgdorferi genome.
As used herein, "a computer-based system" refers to the hardware means,
software
means, and data storage means used to analyze the nucleotide sequence
information of the present
invention. The minimum hardware means of the computer-based systems of the
present
invention comprises a central processing unit (CPU), input means, output
means, and data
storage means. A skilled artisan can readily appreciate that any one of the
currently available
computer-based system are suitable for use in the present invention.
As stated above, the computer-based systems of the present invention comprise
a data
storage means having stored therein a nucleotide sequence of the present
invention and the
necessary hardware means and software means for supporting and implementing a
search means.
As used herein, "data storage means" refers to memory which can store
nucleotide
sequence information of the present invention, or a memory access means which
can access
manufactures having recorded thereon the nucleotide sequence information of
the present
invention.
As used herein, "search means" refers to one or more programs which are
implemented
on the computer-based system to compare a target sequence or target structural
motif with the
sequence information stored within the data storage means. Search means are
used to identify
fragments or regions of the present genomic sequences which match a particular
target sequence
or target motif. A variety of known algorithms are disclosed publicly and a
variety of
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13
commercially available software for conducting search means are and can be
used in the
computer-based systems of the present invention. Examples of such software
includes, but is
not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA). A skilled
artisan can
readily recognize that any one of the available algorithms or implementing
software packages for
conducting homology searches can be adapted for use in the present computer-
based systems.
As used herein, a "target sequence" can be any DNA or amino acid sequence of
six or
more nucleotides or two or more amino acids. A skilled artisan can readily
recognize that the
longer a target sequence is, the less likely a target sequence will be present
as a random
occurrence in the database. The most preferred sequence length of a target
sequence is from
about 10 to 100 amino acids or from about 30 to 300 nucleotide residues.
However, it is well
recognized that searches for commercially important fragments, such as
sequence fragments
involved in gene expression and protein processing, may be of shorter length.
As used herein, "a target structural motif," or "target motif," refers to any
rationally
selected sequence or combination of sequences in which the sequence(sj are
chosen based on a
three-dimensional configuration which is formed upon the folding of the target
motif. There are a
variety of target motifs known in the art. Protein target motifs include, but
are not limited to,
cnzymic active sites and signal sequences. Nucleic acid target motifs include,
but are not limited
to, promoter sequences, hairpin structures and inducible expression elements
(protein binding
sequences).
A variety of structural formats for the input and output means can be used to
input and
output the information in the computer-based systems of the present invention.
A preferred
format for an output means ranks fragments of the Borrelia burgdorferi genomic
sequences
possessing varying degrees of homology to the target sequence or target motif.
Such
presentation provides a skilled artisan with a ranking of sequences which
contain various
amounts of the target sequence or target motif and identifies the degree of
homology contained in
the identified fragment.
A variety of comparing means can be used to compare a target sequence or
target motif
with the data storage means to identify sequence fragments of the Borrelia
burgdorferi genome.
In the present examples, implementing software which implement the BLAST and
BLAZE
algorithms, described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990),
is used to identify
open reading frames within the Borrelia burgdorferi genome. A skilled artisan
can readily
recognize that any one of the publicly available homology search programs can
be used as the
search means for the computer-based systems of the present invention. Of
course, suitable
proprietary systems that may be known to those of skill also may be employed
in this regard.
Figure I provides a block diagram of a computer system illustrative of
embodiments of
this aspect of present invention. The computer system 102 includes a processor
106 connected to
a bus 104. Also connected to the bus 104 are a main memory 108 (preferably
implemented as
random access memory, RAM) and a variety of secondary storage devices 110,
such as a hard
drive 112 and a removable medium storage device 114. The removable medium
storage device
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74
1 14 may represent, for example, a floppy disk drive, a CD-ROM drive, a
magnetic tape drive,
etc. A removable storage medium 116 (such as a floppy disk, a compact disk, a
magnetic tape,
etc. ) containing control logic and/or data recorded therein may be inserted
into the removable
medium storage device 114. The computer system 102 includes appropriate
software for reading
the control logic and/or the data from the removable medium storage device
114, once it is
inserted into the removable medium storage device 114.
A nucleotide sequence of the present invention may be stored in a well known
manner in
the main memory 108, any of the secondary storage devices 110, and/or a
removable storage
medium 116. During execution, software for accessing and processing the
genomic sequence
(such as search tools, comparing tools, etc.) reside in main memory 108, in
accordance with the
requirements and operating parameters of the operating system, the hardware
system and the
software program or programs.
BIOCHEMICAL EMBODIMENTS
Other embodiments of the present invention are directed to isolated fragments
of the
Borrelia burgdorferi genome. The fragments of the Borrelia burgdosferi genome
of the present
invention include, but are not limited to fragments which encode peptides,
hereinafter open
reading frames (ORFs), fragments which modulate the expression of an operably
linked ORF,
hereinafter expression modulating fragments (EMFs) and fragments which can be
used to
diagnose the presence of Borrelia burgdorferi in a sample, hereinafter
diagnostic fragments
(DFs).
As used herein, an "isolated nucleic acid molecule" or an "isolated fragment
of the
Borrelia burgdorferi genome" refers to a nucleic acid molecule possessing a
specific nucleotide
sequence which has been subjected to purification means to reduce, from the
composition, the
number of compounds which are normally associated with the composition.
Particularly, the
term refers to the nucleic acid molecules having the sequences set out in SEQ
ID NOS: 1-155, to
representative fragments thereof as described above including ORF IDs and
ORFs, to
polynucleotides at least 95%, preferably at least 96%, 97%, 98%, or 99% and
especially
preferably at least 99.9% identical in sequence thereto, also as set out
above.
A variety of purification means can be used to generate the isolated fragments
of the
present invention. These include, but are not limited to methods which
separate constituents of a
solution based on charge, solubility, or size.
In one embodiment, Borrelia hurgdorferi DNA can be enzymatically sheared to
produce
fragments of 15-20 kb in length. These fragments can then be used to generate
a Borrelia
bur~dorferi library by inserting them into lambda clones as described in the
Examples below.
Primers flanking, for example, an ORF, such as those enumerated in Tables 1-6
can then be
generated using nucleotide sequence information provided in SEQ ID NOS: 1-155.
Well known
and routine techniques of PCR cloning then can be used to isolate the ORF from
the lambda DNA
library or Borreliu burydorferi genomic DNA. Thus, given the availability of
SEQ ID NOS:1-
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155, the information in Tables 1-6, and the information that may be obtained
readily by analysis
of the sequences of SEQ ID NOS:1-155 using methods set out above, those of
skill will be
enabled by the present disclosure to isolate any ORF-containing or other
nucleic acid fragment of
the present invention.
5 The isolated nucleic acid molecules of the present invention include, but
are not limited to
single stranded and double stranded DNA, and single stranded RNA. For purposes
of
numbering and reference to polynucleotide and polypeptide sequences the entire
sequence of each
sequence of SEQ ID NOS:I-155 is included with the first nucleotide being
position 1.
Therefore, for reference purposes the numbering used in the present invention
is that provided in
10 the sequence listing for SEQ ID NOS:1-155.
As used herein, an open reading frame (ORF), means a series of nucleotide
triplets
coding for amino acid residues without any termination codons and is a
sequence translatable into
protein. Further, unless specified, the term "ORF" for each ORF ID is defined
by the termination
codon at the 3' end and the 5' most methionine codon, at the 5' end, in frame
with said 3'
15 termination codon. Unless specified, the term "ORF" also refers to a
particular polypeptide
sequence defined by the ORF polynucleotide sequence, wherein the N-terminus is
defined by the
5' most methionine codon in frame with the termination codon at the 3' end of
the ORF ID and
the C-terminus is defined by the last codon before the said 3' termination
codon. As used herein,
an ORF ID represents a sequence without any internal termination codons
flanked by termination
codons.
Tables 1-6 list ORF IDs in the Borrelia burgdorferi genomic contigs of the
present
invention that were identified as putative coding regions by the GeneMark
software using
organism-specific second-order Markov probability transition matrices. It will
be appreciated that
other criteria can be used, in accordance with well known analytical methods,
such as those
discussed herein, to generate more inclusive, more restrictive, or more
selective lists.
The B. burgdorferi genome consists of one large linear chromosome containing
approximately two thirds of its genetic material and multiple extrachromosomal
elements
(approximately 15) containing the remaining one third of its genetic material.
SEQ ID NO:1
(Contig ID 1 ) is the complete sequence of the large linear B. burgdorferi
chromosome. SEQ ID
NOS:?-155 (Contig ID 2-155 respectively) are fragments (contigs) of the
extrachromosomal
elements. Tables 1-3 below relate only to SEQ ID NO:1. Tables 4-6 relate to
the
extrachromosomal elements (SEQ ID NOS:2-155).
Table 1 sets out ORF IDs in the Borrelia burgdorferi chromosome of the present
invention that cover a continuous region of at least 50 bases are 95°~0
or more identical (by
BLAST analysis using default parameters) to a nucleotide sequence available
through GenBank
in July, 1997.
Table 2 sets out ORF IDs in the Borrelia burgdorferi chromosome of the present
invention that are not in Table 1 and match, with a BLASTP probability score
of 0.01 or less, a
polypeptide sequence available through GenBank in July, 1997.
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16
Table 3 sets out ORF IDs in the Borrelia burgdorferi chromosome of the present
invention that do not match significantly, by BLASTP analysis, a polypeptide
sequence available
through GenBank in July, 1997.
Table 4 sets out ORF IDs in the Borrelia burgdor feri extrachromosomal element
contigs
of the present invention that over a continuous region of at least 50 bases
are 95% or more
identical (by BLAST analysis) to a nucleotide sequence available through
GenBank in July,
1997.
Table 5 sets out ORF IDs in the Borrelia bur~dor~'eri extrachromosomal element
contigs
of the present invention that are not in Table 1 and match, with a BLASTP
probability score of
0.01 or less, a polypeptide sequence available through GenBank in July, 1997.
Table 6 sets out ORF IDs in the Borrelia burgdoyeri extrachromosomal element
contigs
of the present invention that do not match significantly, by BLASTP analysis,
a polypeptide
sequence available through GenBank in July, 1997.
In each table, the first and second columns identify the ORF ID by,
respectively, contig
number and ORF ID number within the contig; the third column indicates the
first nucleotide of
the ORF ID, counting from the 5' end of the contig strand; and the fourth
column indicates the
last nucleotide of the ORF ID, counting from the 5' end of the contig strand.
In Tables 1, 2, 4 and 5, column five, lists the Reference for the closest
matching
sequence available through GenBank. These reference numbers are the database
accession
numbers commonly used by those of skill in the art, who will be familiar with
their
denominators. Descriptions of the nomenclature are available from the National
Center for
Biotechnology Information. Column seven provides the BLAST identity score from
the
comparison of the ORF ID and the homologous gene; and column nine indicates
the length in
nucleotides of the highest scoring segment pair identified by the BLAST
identity analysis.
The concepts of percent identity and percent similarity of two polypeptide
sequences is
well understood in the art. For example, two polypeptides 10 amino acids in
length which differ
at three amino acid positions (e.g., at positions i, 3 and 5) are said to have
a percent identity of
70%. However, the same two polypeptides would be deemed to have a percent
similarity of
80% if, for example at position 5, the amino acids moieties, although not
identical, were
"similar" (i.e., possessed similar biochemical characteristics). As is known
in the art,
substitution of one amino acid for a "similar" amino acid is a conservative
substitution.
Generally, proteins are highly tolerant of conservative substitutions. Many
programs for analysis
of nucleotide or amino acid sequence similarity, such as fasta and BLAST
specifically list percent
identity of a matching region as an output parameter. Thus, for instance,
Tables l, 2, 4 and 5
herein enumerate the percent identity and similarity of the highest scoring
segment pair in each
ORF and its listed relative. Further details concerning the algorithms and
criteria used for
homology searches are provided below and are described in the pertinent
literature highlighted by
the citations provided below.
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17
It will be appreciated that other criteria can be used to generate more
inclusive and more
exclusive listings of the types set out in the tables. As those of skill will
appreciate, narrow and
broad searches both are useful. Thus, a skilled artisan can readily identify
ORFs in contigs of the
Borrelia burgdorferi genome other than those listed in Tables 1-6, such as
ORFs which are
overlapping or encoded by the opposite strand of an identified ORF in addition
to those
ascertainable using the computer-based systems of the present invention.
As used herein, an "expression modulating fragment," EMF, means a series of
nucleotide
molecules which modulates the expression of an operably linked ORF or EMF.
As used herein, a sequence is said to "modulate the expression of an operably
linked
sequence" when the expression of the sequence is altered by the presence of
the EMF. EMFs
include, but are not limited to, promoters, and promoter modulating sequences
(inducible
elements). One class of EMFs are fragments which induce the expression or an
operably linked
ORF in response to a specific regulatory factor or physiological event.
EMF sequences can be identified within the contigs of the Borrelia burydorferi
genome
by their proximity to the ORFs provided in Tables 1-6. An intergenic segment,
or a fragment of
the intergenic segment, from about 10 to 200 nucleotides in length, taken from
any one of the
ORFs of Tables 1-6 will modulate the expression of an operably linked ORF in a
fashion similar
to that found with the naturally linked ORF sequence. As used herein, an
"intergenic segment"
refers to fragments of the Borrelia burydorferi genome which are between two
ORF(s) herein
described. EMFs also can be identified using known EMFs as a target sequence
or target motif
in the computer-based systems of the present invention. Further, the two
methods can be
combined and used together.
The presence and activity of an EMF can be confirmed using an EMF trap vector.
An
EMF trap vector contains a cloning site linked to a marker sequence. A marker
sequence encodes
an identifiable phenotype, such as antibiotic resistance or a complementing
nutrition auxotrophic
factor, which can be identified or assayed when the EMF trap vector is placed
within an
appropriate host under appropriate conditions. As described above, a EMF will
modulate the
expression of an operably linked marker sequence. A more detailed discussion
of various marker
sequences is provided below. A sequence which is suspected as being an EMF is
cloned in all
three reading frames in one or more restriction sites upstream from the marker
sequence in the
EMF trap vector. The vector is then transformed into an appropriate host using
known
procedures and the phenotype of the transformed host in examined under
appropriate conditions.
As described above, an EMF will modulate the expression of an operably linked
marker
sequence.
As used herein, a "diagnostic fragment," DF, means a series of nucleotide
molecules
which selectively hybridize to Bnrrelia bur~dorJeri sequences. DFs can be
readily identified by
identifying unique sequences within contigs of the Borrelia burgdorferi
genome, such as by
using well-known computer analysis software, and by generating and testing
probes or
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18
amplification primers consisting of the DF sequence in an appropriate
diagnostic format which
determines amplification or hybridization selectivity.
The sequences falling within the scope of the present invention are not
limited to the
specific sequences herein described, but also include allelic and species
variations thereof. Allelic
and species variations can be routinely determined by comparing the sequences
provided in SEQ
ID NOS:1-155, ORF IDs and ORFs within, a representative fragment thereof, or a
nucleotide
sequence at least 99% and preferably 99.9% identical to SEQ ID NOS: 1-155, ORF
IDs and
ORFs within, with a sequence from another isolate of the same species.
Furthermore, to
accommodate codon variability, the invention includes nucleic acid molecules
coding for the same
amino acid sequences as do the specific ORFs disclosed herein. In other words,
in the coding
region of an ORF, substitution of one codon for another which encodes the same
amino acid is
expressly contemplated.
Any specific sequence disclosed herein can be readily screened for errors by
resequencing
a particular fragment, such as an ORF, in both directions (i.e., sequence both
strands).
Alternatively, error screening can be performed by sequencing corresponding
polynucleotides of
Borrelia burgdorferi origin isolated by using part or all of the fragments in
question as a probe or
pnmer.
Each of the ORF IDs and ORFs of the Borrelia burgdorferi genome disclosed in
Tables 1-
6, and the EMFs found 5' to the ORF IDs, can be used as polynucleotide
reagents in numerous
ways. For example, the sequences can be used as diagnostic probes or
diagnostic amplification
primers to detect the presence of a specific microbe in a sample, particularly
Borrelia burgdorferi.
Especially preferred in this regard are ORF IDs and ORFs such as those of
Tables 3 and 6, which
do not match previously characterized sequences from other organisms and thus
are most likely
to be highly selective for Borrelia hurgdorferi. Also particularly preferred
are ORF IDs and
ORFs that can be used to distinguish between strains of Borrelia burgdorferi,
particularly those
that distinguish medically important strain, such as drug-resistant strains.
In addition, the fragments of the present invention, as broadly described, can
be used to
control gene expression through triple helix formation or antisense DNA or
RNA, both of which
methods are based on the binding of a polynucleotide sequence to DNA or RNA.
Triple helix
formation optimally results in a shut-off of RNA transcription from DNA, while
antisense RNA
hybridization blocks translation of an mRNA molecule into polypeptide.
Information from the
sequences of the present invention can be used to design antisense and triple
helix-forming
oligonucleotides. Polynucleotides suitable for use in these methods are
usually 20 to 40 bases in
length and are designed to be complementary to a region of the gene involved
in transcription, for
triple-helix formation, or to the mRNA itself, for antisense inhibition. Both
techniques have been
demonstrated to be effective in model systems, and the requisite techniques
are well known and
involve routine procedures. Triple helix techniques are discussed in, for
example, Lee et al.,
Nucl. Acids Res. 6:3073 ( 1979); Cooney et cil., Science 241:456 ( 1988); and
Dervan et al.,
Science 251:1360 ( 1991 j. Antisense techniques in general are discussed in,
for instance, Okano,
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19
J. Neurochem. 56:560 ( 1991 ) and OlibJodeoxynucleotides ns Antisense
Inhibitors of Gene
Expression, CRC Press, Boca Raton, FL (1988)).
The present invention further provides recombinant constructs comprising one
or more
fragments of the Borreliu burgdorferi genomic fragments and contigs of the
present invention.
Certain preferred recombinant constructs of the present invention comprise a
vector, such as a
plasmid or viral vector, into which a fragment of the Borreliu burgdorferi
genome has been
inserted, in a forward or reverse orientation. In the case of a vector
comprising one of the ORF
IDs or ORFs of the present invention, the vector may further comprise
regulatory sequences,
including for example, a promoter, operably linked to the ORF ID or ORF. For
vectors
comprising the EMFs of the present invention, the vector may further comprise
a marker
sequence or heterologous ORF ID or ORF operably linked to the EMF.
Large numbers of suitable vectors and promoters are known to those of skill in
the art and
are commercially available for generating the recombinant constructs of the
present invention.
The following vectors are provided by way of example. Useful bacterial vectors
include
phagescript, PsiX 174, pBluescript SK, pBS KS, pNHBa, pNH 16a, pNH 18a, pNH46a
(available from Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRITS
(available from
Pharmacia); pQE vectors (available from Promega). Useful eukaryotic vectors
include pWLneo,
pSV2cat, pOG44, pXTI, pSG (available from Stratagene) pSVK3, pBPV, pMSG, pSVL
(available from Pharmacia).
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol
transferase) vectors or other vectors with selectable markers. Two appropriate
vectors are
pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ,
T3, T7, gpt,
lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV
thymidine
kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-
I. Selection of
2_5 the appropriate vector and promoter is well within the level of ordinary
skill in the art.
The present invention further provides host cells containing any one of the
isolated
fragments of the Borreliu burgdorferi genomic fragments and contigs of the
present invention,
wherein the fragment has been introduced into the host cell using known
methods. The host cell
can be a higher eukaryotic host cell, such as a mammalian cell, a lower
eukaryotic host cell, such
as a yeast cell, or a procaryotic cell, such as a bacterial cell.
A polynucleotide of the present invention, such as a recombinant construct
comprising an
ORF of the present invention, may be introduced into the host by a variety of
well established
techniques that arc standard in the art, such as calcium phosphate
transfection, DEAE, dextran
mediated transfection and electroporation, which are described in, for
instance, Davis, L. et ul.,
BASIC METHODS IN MOLECULAR BIOLOGY ( 1986).
A host cell containing one of the fragments of the Borreliu burKdorferi
genomic fragments
and contigs of the present invention, can be used in conventional manners to
produce the gene
product encoded by the isolated fragment (in the case of an ORF) or can be
used to produce a
heterologous protein under the control of the EMF.
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The present invention further provides isolated polypeptides encoded by the
nucleic acid
fragments of the present invention or by degenerate variants of the nucleic
acid fragments of the
present invention. By "degenerate variant" is intended nucleotide fragments
which differ from a
nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide
sequence but, due to
5 the degeneracy of the Genetic Code, encode an identical polypeptide
sequence.
Preferred nucleic acid fragments of the present invention are the ORF IDs
depicted in
Tables 2, 3, 5 and 6, and ORFs witin, which encode proteins.
A variety of methodologies known in the art can be utilized to obtain any one
of the
isolated polypeptides or proteins of the present invention. At the simplest
level, the amino acid
10 sequence can be synthesized using commercially available peptide
synthesizers. This is
particularly useful in producing small peptides and fragments of larger
polypeptides. Such short
fragments as may be obtained most readily by synthesis are useful, for
example, in generating
antibodies against the native polypeptide, as discussed further below.
In an alternative method, the polypeptide or protein is purified from
bacterial cells which
15 naturally produce the polypeptide or protein. One skilled in the art can
readily employ well-
known methods for isolating polypeptides and proteins to isolate and purify
polypeptides or
proteins of the present invention produced naturally by a bacterial strain, or
by other methods.
Methods for isolation and purification that can be employed in this regard
include, but are not
limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-
exchange
20 chromatography, and immuno-affinity chromatography.
The polypeptides and proteins of the present invention also can be purified
from cells
which have been altered to express the desired polypeptide or protein. As used
herein, a cell is
said to be altered to express a desired polypeptide or protein when the cell,
through genetic
manipulation, is made to produce a polypeptide or protein which it normally
does not produce or
which the cell normally produces at a lower level. Those skilled in the art
can readily adapt
procedures for introducing and expressing either recombinant or synthetic
sequences into
eukaryotic or prokaryotic cells in order to generate a cell which produces one
of the polypeptides
or proteins of the present invention.
The polypeptides of the present invention are preferably provided in an
isolated form, and
preferably are substantially purified. A recombinantly produced version of the
B. burgdorferi
polypeptide can be substantially purified by the one-step method described by
Smith et al. ( 1988)
Gene 67:31-40. Polypeptides of the invention also can be purified from natural
or recombinant
sources using antibodies directed against the polypcptides of the invention in
methods which are
well known in the art of protein purification.
The invention further provides for isolated B. burgdorferi polypeptides
comprising an
amino acid sequence selected from the group including: (a) the amino acid
sequence of a full-
length B. burgdorferi polypcptidc having the complete amino acid sequence from
the first
methionine codon to the termination codon of each sequence listed in SEQ ID
NOS:I-155,
wherein said termination codon is at the end of each SEQ ID NO: and said first
methioninc is the
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21
first methionine in frame with said termination codon; and (b) the amino acid
sequence of a full-
length B. burgdorferi polypcptide having the complete amino acid sequence in
(a) excepting the
N-terminal methionine.
The polypeptides of the present invention also include polypeptides having an
amino acid
sequence at least 80% identical, more preferably at least 90% identical, and
still more preferably
95%, 96%, 97%, 98°l0 or 99% identical to those described in (a) and (b)
above.
The present invention is further directed to polynucleotides encoding portions
or
fragments of the amino acid sequences described herein as well as to portions
or fragments of the
isolated amino acid sequences described herein. Fragments include portions of
the amino acid
sequences described herein at least 5 contiguous amino acid in length and
selected from any two
integers, one of which representing an N-terminal position and another
representing a C-terminal
position. The initiation codon of the ORFs of the present invention is
position 1. The initiation
codon (positon 1 ) for purposes of the present invention is the first
methionine codon of each
ORF ID which is in frame with the termination codon at the end of each said
sequence. Every
combination of a N-terminal and C-terminal position that a fragment at least 5
contiguous amino
acid residues in length could occupy, on any given ORF is included in the
invention, i.e., from
initiation codon up to the termination codon. "At least" means a fragment may
be 5 contiguous
amino acid residues in length or any integer between 5 and the number of
residues in an ORF,
minus l . Therefore, included in the invention are contiguous fragments
specified by any N-
terminal and C-terminal positions of amino acid sequence set forth in SEQ ID
NOS:1-155 or
Tables 1-6 wherein the contiguous fragment is any integer between 5 and the
number of residues
in an ORF minus 1.
Further, the invention includes polypeptides comprising fragments specified by
size, in
amino acid residues, rather than by N-terminal and C-terminal positions. The
invention includes
any fragment size, in contiguous amino acid residues, selected from integers
between 5 and the
number of residues in an ORF, minus I . Preferred sizes of contiguous
polypeptide fragments
include about 5 amino acid residues, about 10 amino acid residues, about 20
amino acid residues,
about 30 amino acid residues, about 40 amino acid residues, about 50 amino
acid residues, about
100 amino acid residues, about 200 amino acid residues, about 300 amino acid
residues, and
about 400 amino acid residues. The preferred sizes are, of course, meant to
exemplify, not limit,
the present invention as all size fragments representing any integer between 5
and the number of
residues in a full length sequence minus 1 are included in the invention. The
present invention
also provides for the exclusion of any fragments specified by N-terminal and C-
terminal
positions or by size in amino acid residues as described above. Any number of
fragments
specified by N-terminal and C-terminal positions or by size in amino acid
residues as described
above may be excluded.
The above fragments need not be active since they would be useful, for
example, in
immunoassays, in epitope mapping, epitope tagging, to generate antibodies to a
particular portion
of the protein, as vaccines, and as molecular weight markers.
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22
Further polypeptides of the present invention include polypeptides which have
at least
90% similarity, more preferably at least 95% similarity, and still more
preferably at least 96%,
97%, 98010 or 99% similarity to those described above.
A further embodiment of the invention relates to a polypeptide which comprises
the amino
acid sequence of a B. burgdorferi polypeptide having an amino acid sequence
which contains at
least one conservative amino acid substitution, but not more than 50
conservative amino acid
substitutions, not more than 40 conservative amino acid substitutions, not
more than 30
conservative amino acid substitutions, and not more than 20 conservative amino
acid
substitutions. Also provided are polypeptides which comprise the amino acid
sequence of a 13.
burbl~lorferi polypeptide, having at least one, but not more than 10, 9, 8, 7,
6, 5, 4, 3, 2 or 1
conservative amino acid substitutions.
By a poIypeptide having an amino acid sequence at least, for example, 95%
"identical" to
a query amino acid sequence of the present invention, it is intended that the
amino acid sequence
of the subject polypeptide is identical to the query sequence except that the
subject polypeptide
sequence may include up to five amino acid alterations per each 100 amino
acids of the query
amino acid sequence. In other words, to obtain a polypeptide having an anuno
acid sequence at
least 95% identical to a query amino acid sequence, up to 5% of the amino acid
residues in the
subject sequence may be inserted, deleted, (indels) or substituted with
another amino acid. These
alterations of the reference sequence may occur at the amino or carboxy
terminal positions of the
reference amino acid sequence or anywhere between those terminal positions,
interspersed either
individually among residues in the reference sequence or in one or more
contiguous groups
within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%,
95%, 96%,
97%, 98°l0 or 99% identical to the ORF amino acid sequences encoded by
the sequences of SEQ
ID NOS:I-155, as described hererin, can be determined conventionally using
known computer
programs. A preferred method for determining the best overall match between a
query sequence
(a sequence of the present invention) and a subject sequence, also referred to
as a global sequence
alignment, can be determined using the FASTDB computer program based on the
algorithm of
Brutlag et al., ( 1990) Comp. App. Biosci. 6:237-245. In a sequence alignment
the query and
subject sequences are both amino acid sequences. The result of said global
sequence alignment is
in percent identity. Preferred parameters used in a FASTDB amino acid
alignment are:
Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization
Group
Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size
Penalty=0.05, Window Size=500 or the length of the subject amino acid
sequence, whichever is
shorter.
If the subject sequence is shorter than the query sequence due to N- or C-
terminal
deletions, not because of internal deletions, the results, in percent
identity, must be manually
corrected. This is because the FASTDB program does not account for N- and C-
terminal
truncations of the subject sequence when calculating global percent identity.
For subject
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23
sequences truncated at the N- and C-termini, relative to the query sequence,
the percent identity is
corrected by calculating the number of residues of the query sequence that are
N- and C-terminal
of the subject sequence, which are not matched/aligned with a corresponding
subject residue, as a
percent of the total bases of the query sequence. Whether a residue is
matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then subtracted
from the percent identity, calculated by the above FASTDB program using the
specified
parameters, to arrive at a final percent identity score. This final percent
identity score is what is
used for the purposes of the present invention. Only residues to the N- and C-
termini of the
subject sequence, which are not matchcd/aligned with the query sequence, are
considered for the
purposes of manually adjusting the percent identity score. That is, only query
amino acid
residues outside the farthest N- and C-terminal residues of the subject
sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100
residue
query sequence to determine percent identity. The deletion occurs at the N-
terminus of the
subject sequence and therefore, the FASTDB alignment does not match/align with
the first 10
residues at the N-terminus. The 10 unpaired residues represent 10°l0 of
the sequence (number of
residues at the N- and C- termini not matched/total number of residues in the
query sequence) so
10~o is subtracted from the percent identity score calculated by the FASTDB
program. If the
remaining 90 residues were perfectly matched the final percent identity would
be 90%. In
another example, a 90 residue subject sequence is compared with a 100 residue
query sequence.
This time the deletions are internal so there are no residues at the N- or C-
termini of the subject
sequence which are not matched/aligned with the query. In this case the
percent identity
calculated by FASTDB is not manually corrected. Once again, only residue
positions outside the
N- and C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which
are not matched/aligned with the query sequence are manually corrected. No
other manual
corrections are to made for the purposes of the present invention.
The above polypeptide sequences are included irrespective of whether they have
their
normal biological activity. This is because even where a particular
polypeptide molecule does not
have biological activity, one of skill in the art would still know how to use
the polypeptide, for
instance, as a vaccine or to generate antibodies. Other uses of the
polypeptides of the present
invention that do not have B. burgdorferi activity include, inter alia, as
epitope tags, in epitope
mapping, and as molecular weight markers on SDS-PAGE gels or on molecular
sieve gel
filtration columns using methods known to those of skill in the art.
As described below, the polypeptides of the present invention can also be used
to raise polyclonal
and monoclonal antibodies, which are useful in assays for detecting B.
burgdnrferi protein
3.5 expression or as agonists and antagonists capable of enhancing or
inhibiting B. burgdorferi
protein function. Further, such polypeptides can be used in the yeast two-
hybrid system to
"capture" B. burgdorferi protein binding proteins which are also candidate
agonists and
antagonists according to the present invention. See, e.~., Fields et al. (
1989) Nature
340:245-246.
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24
Any host/vector system can be used to express one or more of the ORFs of the
present
invention. These include, but are not limited to, eukaryotic hosts such as
HeLa cells, CV-1 cell,
COS cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B.
subtili.s. The most
preferred cells are those which do not normally express the particular
polypeptide or protein or
which expresses the polypeptide or protein at low natural level.
"Recombinant," as used herein, means that a polypeptide or protein is derived
from
recombinant (e.g., microbial or mammalian) expression systems. "Microbial"
refers to
recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast)
expression
systems. As a product, "recombinant microbial"defines a polypeptide or protein
essentially free
of native endogenous substances and unaccompanied by associated native
glycosylation.
Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli,
will be free of
glycosylation modifications; polypeptides or proteins expressed in yeast will
have a glycosylation
pattern different from that expressed in mammalian cells.
"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.
Generally,
DNA segments encoding the polypeptides and proteins provided by this invention
are assembled
from fragments of the Borrelia burgdorferi genome and short oligonucleotide
linkers, or from a
series of oligonucleotides, to provide a synthetic gene which is capable of
being expressed in a
recombinant transcriptional unit comprising regulatory elements derived from a
microbial or viral
operon.
Recombinant expression vehicle or vector" refers to a plasmid or phage or
virus or
vector, for expressing a polypeptide from a DNA (RNA) sequence. The expression
vehicle can
comprise a transcriptional unit comprising an assembly of ( 1 j a genetic
regulatory elements
necessary for gene expression in the host, including elements required to
initiate and maintain
transcription at a level sufficient for suitable expression of the desired
polypeptide, including, for
example, promoters and, where necessary, an enhancer and a polyadenylation
signal; (2) a
structural or coding sequence which is transcribed into mRNA and translated
into protein, and (3)
appropriate signals to initiate translation at the beginning of the desired
coding region and
terminate translation at its end. Structural units intended for use in yeast
or eukaryotic expression
systems preferably include a leader sequence enabling extracellular secretion
of translated protein
by a host cell. Alternatively, where recombinant protein is expressed without
a leader or
transport sequence, it may include an N-terminal methionine residue. This
residue may or may
not be subsequently cleaved from the expressed recombinant protein to provide
a final product.
"Recombinant expression system" means host cells which have stably integrated
a
recombinant transcriptional unit into chromosomal DNA or carry the recombinant
transcriptional
unit extra chromosomally. The cells can be prokaryotic or eukaryotic.
Recombinant expression
systems as defined herein will express heterologous polypeptides or proteins
upon induction of
the regulatory elements linked to the DNA segment or synthetic gene to be
expressed.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other
cells under
the control of appropriate promoters. Cell-free translation systems can also
be employed to
CA 02304925 1999-12-17
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produce such proteins using RNAs derived from the DNA constructs of the
present invention.
Appropriate cloning and expression vectors for use with prokaryotic and
eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York ( 1989), the
disclosure of
5 which is hereby incorporated by reference in its entirety.
Generally, recombinant expression vectors will include origins of replication
and
selectable markers permitting transformation of the host cell, e.g., the
ampicillin resistance gene
of E. coli and S'. cerevisiae TRP1 gene, and a promoter derived from a highly
expressed gene to
direct transcription of a downstream structural sequence. Such promoters can
be derived from
10 operons encoding glycolytic enzymes such as 3- phosphoglycerate kinase
(PGK), alpha-factor,
acid phosphatase, or heat shock proteins, among others. The heterologous
structural sequence is
assembled in appropriate phase with translation initiation and termination
sequences, and
preferably, a leader sequence capable of directing secretion of translated
protein into the
periplasmic space or extracellular medium. Optionally, the heterologous
sequence can encode a
15 fusion protein including an N-terminal identification peptide imparting
desired characteristics,
e.g., stabilization or simplified purification of expressed recombinant
product.
Useful expression vectors for bacterial use are constructed by inserting a
structural DNA
sequence encoding a desired protein together with suitable translation
initiation and termination
signals in operable reading phase with a functional promoter. The vector will
comprise one or
20 more phenotypic selectable markers and an origin of replication to ensure
maintenance of the
vector and, when desirable, provide amplification within the host.
Suitable prokaryotic hosts for transformation include strains of E. coli, B.
subtilis,
Salmonella typhimurium and various species within the genera Pseudomonas and
Streptomyces.
Others may, also be employed as a matter of choice.
25 As a representative but non-limiting example, useful expression vectors for
bacterial use
can comprise a selectable marker and bacterial origin of replication derived
from commercially
available plasmids comprising genetic elements of the well known cloning
vector pBR322
{ATCC 37017). Such commercial vectors include, for example, pKK223-3
{available form
Pharrnacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (available from Promega
Biotec,
Madison, WI, USA). These pBR322 "backbone" sections are combined with an
appropriate
promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host
strain to an
appropriate cell density, the selected promoter, where it is inducible, is
derepressed or induced by
appropriate means {e.g., temperature shift or chemical induction) and cells
are cultured for an
additional period to provide for expression of the induced gene product.
Thereafter cells are
typically harvested, generally by centrifugation, disrupted to release
expressed protein, generally
by physical or chemical means, and the resulting crude extract is retained for
further purification.
Various mammalian cell culture systems can also be employed to express
recombinant
protein. Examples of mammalian expression systems include the COS-7 lines of
monkey kidney
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26
fibroblasts, described in Gluzman, Cell 23: 175 ( 1981 }, and other cell lines
capable of expressing
a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian expression vectors will comprise an origin of replication, a
suitable promoter
and enhancer, and also any necessary ribosome binding sites, polyadenylation
site, splice donor
_5 and acceptor sites, transcriptional termination sequences, and 5' flanking
nontranscribed
sequences. DNA sequences derived from the SV40 viral genome, for example, SV40
origin,
early promoter, enhancer, splice, and polyadenylation sites may be used to
provide the required
nontranscribed genetic elements.
Recombinant polypeptides and proteins produced in bacterial culture is usually
isolated by
initial extraction from cell pellets, followed by one or more salting-out,
aqueous ion exchange or
size exclusion chromatography steps. Microbial cells employed in expression of
proteins can be
disrupted by any convenient method, including freeze-thaw cycling, sonication,
mechanical
disruption, or use of cell lysing agents. Protein refolding steps can be used,
as necessary, in
completing configuration of the mature protein. Finally, high performance
liquid
chromatography (HPLC} can be employed for final purification steps.
The present invention further includes isolated polypeptides, proteins and
nucleic acid
molecules which are substantially equivalent to those herein described. As
used herein,
substantially equivalent can refer both to nucleic acid and amino acid
sequences, for example a
mutant sequence, that varies from a reference sequence by one or more
substitutions, deletions,
or additions, the net effect of which does not result in an adverse functional
dissimilarity between
reference and subject sequences. Particularly preferred in this regard are
conservative
substitutions, known to those of skill in the art. For purposes of the present
invention,
sequences having equivalent biological activity, and equivalent expression
characteristics are
considered substantially equivalent. For purposes of determining equivalence,
truncation of the
mature sequence (e.g., removal of leader sequence(s)) should be disregarded.
The invention further provides methods of obtaining homologs from other
strains of
Bnrrelia burgdorferi, of the fragments of the Borrelia burgdorferi genome of
the present
invention and homologs of the proteins encoded by the ORFs of the present
invention. As used
herein, a sequence or protein of Borrelia burgdorferi is defined as a homolog
of a fragment of the
Bnrrelia burgdor~'eri fragments or contigs or a protein encoded by one of the
ORFs of the present
invention, if it shares significant homology to one of the fragments of the
Borreliu burgdorferi
genome of the present invention or a protein encoded by one of the ORFs of the
present
invention. Specifically, by using the sequence disclosed herein as a probe or
as primers, and
techniques such as PCR cloning and colony/plaque hybridization, one skilled in
the art can obtain
homologs.
As used herein, two nucleic acid molecules or proteins are said to "share
significant
homology" if the two contain regions which possess greater than 85% sequence
(amino acid or
nucleic acid) homology. Preferred homologs in this regard are those with more
than 90%
homology. Especially preferred are those with 95°l0 or more homology.
Among especially
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27
preferred homologs those with 96, 97%, 98°l0, 99% or more homology arc
particularly
preferred. The most preferred homologs among these are those with 99.9%
homology or more.
It will be understood that, among measures of homology, identity is
particularly preferred in this
regard.
Region specific primers or probes derived from the nucleotide sequence
provided in SEQ
ID NOS: 1-155 or from a nucleotide sequence at least 95%, particularly at
least 96%, 97%, 98%
or 99%, especially at least 99.5% identical to a sequence of SEQ ID NOS: 1-155
can be used to
prime DNA synthesis and PCR amplification, as well as to identify colonies
containing cloned
DNA encoding a homolog. Methods suitable to this aspect of the present
invention are well
known and have been described in great detail in many publications such as,
for example, Innis
et al., PCR Protocols, Academic Press, San Diego, CA ( 1990)).
When using primers derived from SEQ ID NOS: 1-155 or from a nucleotide
sequence
having an aforementioned identity to a sequence of SEQ ID NOS:1-155, one
skilled in the art will
recognize that by employing high stringency conditions (e.g., annealing at 50-
60°C in 6X SSPC
and 50% formamide, and washing at 50- 65°C in O.SX SSPC) only sequences
which are greater
than 75% homologous to the primer will be amplified. By employing lower
stringency
conditions (e.g., hybridizing at 35-37°C in SX SSPC and 40-45%
formamide, and washing at
42°C in O.SX SSPC), sequences which are greater than 40-50% homologous
to the primer will
also be amplified.
When using DNA probes derived from SEQ ID NOS:1-155, or from a nucleotide
sequence having an aforementioned identity to a sequence of SEQ ID NOS: 1-155
, for
colony/plaque hybridization, one skilled in the art will recognize that by
employing high
stringency conditions (e.g., hybridizing at 50- 65°C in SX SSPC and 50%
formamide, and
washing at 50- 65°C in O.SX SSPC), sequences having regions which are
greater than 90%
homologous to the probe can be obtained, and that by employing lower
stringency conditions
(e.g., hybridizing at 35-37°C in SX SSPC and 40-45% formamide, and
washing at 42°C in O.SX
SSPC), sequences having regions which are greater than 35-45% homologous to
the probe will
be obtained.
Any organism can be used as the source for homologs of the present invention
so long as
the organism naturally expresses such a protein or contains genes encoding the
same. The most
preferred organism for isolating homologs are bacteria which are closely
related to Borrelia
burgdorferi.
ILLUSTRATIVE USES OF COMPOSITIONS OF THE INVENTION
Each ORF of the ORF IDs provided in Tables 1, 2, 4 and 5 is identified with a
function
by homology to a known gene or polypeptide. As a result, one skilled in the
art can use the
polypeptides of the present invention for commercial, therapeutic and
industrial purposes
consistent with the type of putative identification of the polypeptide. Such
identifications permit
one skilled in the art to use the Borrelia basrgdorferi ORFs in a manner
similar to the known type
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28
of sequences for which the identification is made; for example, to ferment a
particular sugar
source or to produce a particular metabolite. A variety of reviews
illustrative of this aspect of the
invention are available, including the following reviews on the industrial use
of enzymes, for
example, BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY HANDBOOK, 2nd
Ed., MacMillan Publications, Ltd. NY ( 1991 j and BIOCATALYSTS IN ORGANIC
SYNTHESES, Tramper et al., Eds., Elsevier Science Publishers, Amsterdam, The
Netherlands
( 1985). A variety of exemplary uses that illustrate this and similar aspects
of the present
invention are discussed below.
1. Biosynthetic Enzymes
Open reading frames encoding proteins involved in mediating the catalytic
reactions
involved in intermediary and macromolecular metabolism, the biosynthesis of
small molecules,
cellular processes and other functions includes enzymes involved in the
degradation of the
intermediary products of metabolism, enzymes involved in central intermediary
metabolism,
enzymes involved in respiration, both aerobic and anaerobic, enzymes involved
in fermentation,
enzymes involved in ATP proton motor force conversion, enzymes involved in
broad regulatory
function, enzymes involved in amino acid synthesis, enzymes involved in
nucleotide synthesis,
enzymes involved in cofactor and vitamin synthesis, can be used for industrial
biosynthesis.
The various metabolic pathways present in Borrelia bur~dorferi can be
identified based on
absolute nutritional requirements as well as by examining the various enzymes
identified in Table
1-6 and SEQ ID NOS:l-155.
Of particular interest are polypeptides involved in the degradation of
intermediary
metabolites as well as non-macromolecular metabolism. Such enzymes include
amylases,
glucose oxidases, and catalase.
Proteolytic enzymes are another class of commercially important enzymes.
Proteolytic
enzymes find use in a number of industrial processes including the processing
of flax and other
vegetable fibers, in the extraction, clarification and depectinization of
fruit juices, in the extraction
of vegetables' oil and in the maceration of fruits and vegetables to give
unicellular fruits. A
detailed review of the proteolytic enzymes used in the food industry is
provided in Rombouts et
al., Symbiosis 21:79 ( 1986j and Voragen et al. in Biocatalysts In
Agricultural Biotechnology,
Whitaker et al., Eds., American Chemical Society Symposium Series 389:93
(1989) .
The metabolism of sugars is an important aspect of the primary metabolism of
Borrelia
bur~dorferi. Enzymes involved in the degradation of sugars, such as,
particularly, glucose,
galactose, fructose and xylose, can be used in industrial fermentation. Some
of the important
sugar transforming enzymes, from a commercial viewpoint, include sugar
isomerases such as
glucose isomerase. Other metabolic enzymes have found commercial use such as
glucose
oxidases which produces ketogulonic acid (KGA). KGA is an intermediate in the
commercial
production of ascorbic acid using the Reichstein's procedure, as described in
Krueger et al.,
Biotechnology ~, Rhine et al., Eds., Verlag Press, Weinheim, Germany ( 1984).
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29
Glucose oxidise (GOD) is commercially available and has been used in purified
form as
well as in an immobilized form for the deoxygenation of beer. See, for
instance, Hartmeir et al.,
Biotechnology Letters T :21 ( 1979). The most important application of GOD is
the industrial
scale fermentation of gluconic acid. Market for gluconic acids which are used
in the detergent,
textile, leather, photographic, pharmaceutical, food, feed and concrete
industry, as described, for
example, in Bigelis et al., beginning on page 357 in GENE MANIPULATIONS AND
FUNGI;
Benett et al., Eds., Academic Press, New York ( 1985). In addition to
industrial applications,
GOD has found applications in medicine for quantitative determination of
glucose in body fluids
recently in biotechnology for analyzing syrups from starch and cellulose
hydrosylates. This
application is described in Owusu et al., BioclTem. et BioPhysica. Acta. 872:
83 ( 1986), for
instance.
The main sweetener used in the world today is sugar which comes from sugar
beets and
sugar cane. In the field of industrial enzymes, the glucose isomerase process
shows the largest
expansion in the market today. Initially, soluble enzymes were used and later
immobilized
I S enzymes were developed (Krueger et al., Biotechnology, The Textbook of
Industrial
Microbiology, Sinauer Associated Incorporated, Sunderland, Massachusetts (
1990)). Today, the
use of glucose- produced high fructose syrups is by far the largest industrial
business using
immobilized enzymes. A review of the industrial use of these enzymes is
provided by
Jorgensen, Starch 40:307 ( 1988).
Proteinases, such as alkaline serine proteinases, are used as detergent
additives and thus
represent one of the largest volumes of microbial enzymes used in the
industrial sector. Because
of their industrial importance, there is a large body of published and
unpublished information
regarding the use of these enzymes in industrial processes. (See Faultman et
al., Acid Proteases
Structure Function and Biology, Tang, J., ed., Plenum Press, New York ( 1977)
and Godfrey et
al., Industrial Enzymes, MacMillan Publishers, Surrey, UK ( 1983) and Hepner
et al., Report
Industrial Enzymes by 1990, Hel Hepner & Associates, London ( 1986)).
Another class of commercially usable proteins of the present invention are the
microbial
lipases, described by, for instance, Macrae et al., Pl2ilosophical
Transactions of tl2e Chiral
Society of London 310:227 ( 1985) and Poserke, Journal of the American Oil
Chemist Society
61: 1758 (1984). A major use of lipases is in the fat and oil industry for the
production of neutral
glycerides using lipase catalyzed inter-esterification of readily available
triglycerides. Application
of lipases include the use as a detergent additive to facilitate the removal
of fats from fabrics in the
course of the washing procedures.
The use of enzymes, and in particular microbial enzymes, as catalyst for key
steps in the
synthesis of complex organic molecules is gaining popularity at a great rate.
One area of great
interest is the preparation of chiral intermediates. Preparation of chiral
intermediates is of interest
to a wide range of synthetic chemists particularly those scientists involved
with the preparation of
new pharmaceuticals, agrochemicals, fragrances and flavors. (See Davies et
al., Recent
Advances in the Generation of Chiral Intermediates Using Enzymes, CRC Press,
Boca Raton,
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Florida ( 1990)). The following reactions catalyzed by enzymes are of interest
to organic
chemists: hydrolysis of carboxylic acid esters, phosphate esters, amides and
nitrites,
esterification reactions, trans-esterification reactions, synthesis of amides,
reduction of alkanones
and oxoalkanates, oxidation of alcohols to carbonyl compounds, oxidation of
sulfides to
5 sulfoxides, and carbon bond forming reactions such as the aldol reaction.
When considering the use of an enzyme encoded by one of the ORFs of the
present
invention for biotransformation and organic synthesis it is sometimes
necessary to consider the
respective advantages and disadvantages of using a microorganism as opposed to
an isolated
enzyme. Pros and cons of using a whole cell system on the one hand or an
isolated partially
10 purified enzyme on the other hand, has been described in detail by Bud et
al., Chemistry in
Britain (1987), p. 127.
Amino transferases, enzymes involved in the biosynthesis and metabolism of
amino
acids, arc useful in the catalytic production of amino acids. The advantages
of using microbial
based enzyme systems is that the amino transferase enzymes catalyze the stereo-
selective
15 synthesis of only L-amino acids and generally possess uniformly high
catalytic rates. A
description of the use of amino transferases for amino acid production is
provided by Roselle-
David, Methods of Enzymology 136:479 ( 1987).
Another category of useful proteins encoded by the ORFs of the present
invention include
enzymes involved in nucleic acid synthesis, repair, and recombination.
2. Generation of Antibodies
As described here, the proteins of the present invention, as well as homologs
thereof, can
be used in a variety of procedures and methods known in the art which are
currently applied to
other proteins. The proteins of the present invention can further be used to
generate an antibody
which selectively binds the protein.
B. burgdorferi protein-specific antibodies for use in the present invention
can be raised
against the intact B. burgdorferi protein or an antigenic polypeptide fragment
thereof, which may
be presented together with a carrier protein, such as an albumin, to an animal
system (such as
rabbit or mouse) or, if it is long enough (at least about 25 amino acids},
without a carrier.
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mob) is
meant to
include intact molecules, single chain whole antibodies, and antibody
fragments. Antibody
fragments of the present invention include Fab and F(ab')2 and other fragments
including single-
chain Fvs (scFv) and disulfide-linked Fvs (sdFv). Also included in the present
invention are
chimeric and humanized monoclonal antibodies and polyclonal antibodies
specific for the
polypeptides of the present invention. The antibodies of the present invention
may be prepared
by any of a variety of methods. For example, cells expressing a polypeptide of
the present
invention or an antigenic fragment thereof can be administered to an animal in
order to induce the
production of sera containing polyclonal antibodies. For example, a
preparation of B.
burgdorferi polypeptide or fragment thereof is prepared and purified to render
it substantially free
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31
of natural contaminants. Such a preparation is then introduced into an animal
in order to produce
polyclonal antisera of greater specific activity.
In a preferred method, the antibodies of the present invention are monoclonal
antibodies
or binding fragments thereof. Such monoclonal antibodies can be prepared using
hybridoma
technology. See, e.~~., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold
Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:
MONOCLONAL
ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981). Fab and
F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such
as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
Alternatively, B. bur~dorferi
polypeptide-binding fragments, chimeric, and humanized antibodies can be
produced through the
application of recombinant DNA technology or through synthetic chemistry using
methods
known in the art.
Alternatively, additional antibodies capable of binding to the polypeptide
antigen of the
present invention may be produced in a two-step procedure through the use of
anti-idiotypic
antibodies. Such a method makes use of the fact that antibodies are themselves
antigens, and
that, therefore, it is possible to obtain an antibody which binds to a second
antibody. In
accordance with this method, B. bur~dor~eri polypcptide-specific antibodies
are used to
immunize an animal, preferably a mouse. The splenocytes of such an animal are
then used to
produce hybridoma cells, and the hybridoma cells are screened to identify
clones which produce
an antibody whose ability to bind to the B. hurgdorferi polypeptide-specific
antibody can be
blocked by the B. burgdorferi polypeptide antigen. Such antibodies comprise
anti-idiotypic
antibodies to the B. burgdnrferi polypeptidc-specific antibody and can be used
to immunize an
animal to induce formation of further B. burgdor~feri polypeptide-specific
antibodies.
Antibodies and fragements thereof of the present invention may be described by
the
portion of a polypeptide of the present invention recognized or specifically
bound by the
antibody. Antibody binding fragements of a polypeptide of the present
invention may be
described or specified in the same manner as for polypeptide fragements
discussed above., i.e,
by N-terminal and C-terminal positions or by size in contiguous amino acid
residues. Any
number of antibody binding fragments, of a polypeptide of the present
invention, specified by N-
terminal and C-terminal positions or by size in amino acid residues, as
described above, may also
be excluded from the present invention. Therefore, the present invention
includes antibodies the
specifically bind a particuarlly discribed fragement of a polypeptide of the
present invention and
allows for the exclusion of the same.
Antibodies and fragements thereof of the present invention may also be
described or specified in
terms of their cross-reactivity. Antibodies and fragements that do not bind
polypeptides of any
other species of Borr-elia other than B. burgdorferi are included in the
present invention.
Likewise, antibodies and fragements that bind only species of Borrelia, i.e.
antibodies and
fragements that do not bind bacteria from any genus other than Bnrrelia, are
included in the
present invention.
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32
3. Epitope-Bearing Portions
In another aspect, the invention provides peptides and polypeptides comprising
epitope-bearing portions of the B. burgdorferi polypeptides of the present
invention. These
epitopes are immunogenic or antigenic epitopes of the polypeptides of the
present invention. An
"immunogenic epitope" is defined as a part of a protein that elicits an
antibody response when the
whole protein or polypeptide is the immunogen. These immunogenic epitopes are
believed to be
confined to a few loci on the molecule. On the other hand, a region of a
protein molecule to
which an antibody can bind is defined as an "antigenic determinant" or
"antigenic epitope." The
number of immunogenic epitopes of a protein generally is less than the number
of antigenic
epitopes. See, e.g., Geysen, et al. ( 1983) Proc. Natl. Acad. Sci. USA 81:3998-
4002. Amino
acid residues comprising anigenic epitopes may be determined by algorithms
such as the the
Jameson-Wolf analysis or similar algorithms or by i~2 vivo testing for an
antigenic response using
the methods described herein or those known in the art.
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e., that
contain a region of a protein molecule to which an antibody can bind), it is
well known in that art
that relatively short synthetic peptides that mimic part of a protein sequence
are routinely capable
of eliciting an antiserum that reacts with the partially mimicked protein.
See, e.g., Sutcliffe, et
al., ( 1983) Science 219:660-666. Peptides capable of eliciting protein-
reactive sera are
frequently represented in the primary sequence of a protein, can be
characterized by a set of
simple chemical rules, and are confined neither to immunodominant regions of
intact proteins
(i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides
that are extremely
hydrophobic and those of six or fewer residues generally are ineffective at
inducing antibodies
that bind to the mimicked protein; longer, peptides, especially those
containing proline residues,
usually are effective. See, Sutcliffe, et al., .supra, p. 661. For instance,
18 of 20 peptides
designed according to these guidelines, containing 8-39 residues covering
75°70 of the sequence
of the influenza virus hemagglutinin HA I polypeptide chain, induced
antibodies that reacted with
the HA 1 protein or intact virus; and 12112 peptides from the MuLV polymerase
and 18/ 18 from
the rabies glycoprotein induced antibodies that precipitated the respective
proteins.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore useful
to raise antibodies, including monoclonal antibodies, that bind specifically
to a polypeptide of the
invention. Thus, a high proportion of hybridomas obtained by fusion of spleen
cells from
donors immunized with an antigen epitope-bearing peptide generally secrete
antibody reactive
with the native protein. See Sutcliffe, et al., .supra, p. 663. The antibodies
raised by antigenic
epitope-bearing peptides or polypeptides are useful to detect the mimicked
protein, and antibodies
to different peptides may be used for tracking the fate of various regions of
a protein precursor
which undergoes post-translational processing. The peptides and anti-peptide
antibodies may be
used in a variety of qualitative or quantitative assays for the mimicked
protein, for instance in
competition assays since it has been shown that even short peptides (e.g.,
about 9 amino acids)
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33
can bind and displace the larger peptides in immunoprecipitation assays. See,
e.g., Wilson, et
al., ( 1984) Cell 37:767-778. The anti-peptide antibodies of the invention
also are useful for
purification of the mimicked protein, for instance, by adsorption
chromatography using methods
known in the art.
Antigenic epitope-bearing peptides and polypeptides of the invention designed
according
to the above guidelines preferably contain a sequence of at least seven, more
preferably at least
nine and most preferably between about 10 to about 50 amino acids (i.e. any
integer between 7
and 50j contained within the amino acid sequence of a polypeptide of the
invention. However,
peptides or polypeptides comprising a larger portion of an amino acid sequence
of a polypeptide
of the invention, containing about 50 to about 100 amino acids, or any length
up to and including
the entire amino acid sequence of a polypeptide of the invention, also are
considered
epitope-bearing peptides or polypeptides of the invention and also are useful
for inducing
antibodies that react with the mimicked protein. Preferably, the amino acid
sequence of the
epitope-bearing peptide is selected to provide substantial solubility in
aqueous solvents (i.e., the
sequence includes relatively hydrophilic residues and highly hydrophobic
sequences are
preferably avoided); and sequences containing proline residues are
particularly preferred.
The epitope-bearing peptides and polypeptides of the present invention may be
produced
by any conventional means for making peptides or polypeptides including
recombinant means
using nucleic acid molecules of the invention. For instance, an epitope-
bearing amino acid
sequence of the present invention may be fused to a larger polypeptide which
acts as a carrier
during recombinant production and purification, as well as during immunization
to produce
anti-peptide antibodies. Epitope-bearing peptides also may be synthesized
using known methods
of chemical synthesis. For instance, Houghton has described a simple method
for synthesis of
large numbers of peptides, such as 10-20 mg of 248 different 13 residue
peptides representing
single amino acid variants of a segment of the HAl polypeptidc which were
prepared and
characterized (by ELISA-type binding studies) in less than four weeks
(Houghton, R. A. Proc.
Natl. Acad. Sci. USA 82:5131-5135 ( 1985)). This "Simultaneous Multiple
Peptide Synthesis
(SMPS)" process is further described in U.S. Patent No. 4,631,21 I to Houghton
and coworkers
( 1986). In this procedure the individual resins for the solid-phase synthesis
of various peptides
are contained in separate solvent-permeable packets, enabling the optimal use
of the many
identical repetitive steps involved in solid-phase methods. A completely
manual procedure
allows 500-1000 or more syntheses to be conducted simultaneously (Houghton et
al. ( 1985)
Proc. Natl. Acad. Sci. 82:5131-5135 at 5134.
Epitope-bearing peptides and polypeptides of the invention are used to induce
antibodies
according to methods well known in the art. See, e.g., Sutcliffe, et al.,
supra;; Wilson, et al.,
.supra;; and Bittle, et al. ( 1985) J. Gen. Virol. 66:2347-2354. Generally,
animals may be
immunized with free peptide; however, anti-peptide antibody titer may be
boosted by coupling of
the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin
(KLH) or tetanus
toxoid. For instance, peptides containing cysteine may be coupled to carrier
using a linker such
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34
as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides
may be
coupled to carrier using a more general linking agent such as glutaraldehyde.
Animals such as
rabbits, rats and mice are immunized with either free or carrier-coupled
peptides, for instance, by
intraperitoneal and/or intradermal injection of emulsions containing about 100
pg peptide or
_5 carrier protein and Freund's adjuvant. Several booster injections may be
needed, for instance, at
intervals of about two weeks, to provide a useful titer of anti-peptide
antibody which can be
detected, for example, by ELISA assay using free peptide adsorbed to a solid
surface. The titer
of anti-peptide antibodies in serum from an immunized animal may be increased
by selection of
anti-peptide antibodies, for instance, by adsorption to the peptide on a solid
support and elution
of the selected antibodies according to methods well known in the art.
Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a
protein that
elicit an antibody response when the whole protein is the immunogen, are
identified according to
methods known in the art. For instance, Geysen, et al., supra, discloses a
procedure for rapid
concurrent synthesis on solid supports of hundreds of peptides of sufficient
purity to react in an
ELISA. Interaction of synthesized peptides with antibodies is then easily
detected without
removing them from the support. In this manner a peptide bearing an
immunogenic epitope of a
desired protein may be identified routinely by one of ordinary skill in the
art. For instance, the
immunologically important epitope in the coat protein of foot-and-mouth
disease virus was
located by Geysen et al. ,supra with a resolution of seven amino acids by
synthesis of an
overlapping set of all 208 possible hexapeptides covering the entire 213 amino
acid sequence of
the protein. Then, a complete replacement set of peptides in which all 20
amino acids were
substituted in turn at every position within the epitope were synthesized, and
the particular amino
acids conferring specificity for the reaction with antibody were determined.
Thus, peptide
analogs of the epitope-bearing peptides of the invention can be made routinely
by this method.
U.S. Patent No. 4,708,781 to Geysen ( 1987) further describes this method of
identifying a
peptide bearing an immunogenic epitope of a desired protein.
Further still, U.S. Patent No. 5,194,392, to Geysen ( 1990), describes a
general method
of detecting or determining the sequence of monomers (amino acids or other
compounds) which
is a topological equivalent of the epitope (i.e., a "mimotope") which is
complementary to a
particular paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Patent
No. 4,433,092, also to Geysen ( 1989), describes a method of detecting or
determining a
sequence of monomers which is a topographical equivalent of a ligand which is
complementary
to the ligand binding site of a particular receptor of interest. Sinvlarly,
U.S. Patent No.
5,480,971 to Houghten, R. A. et al. ( 1996) discloses linear C,-C~-alkyl
peralkylated
oligopeptides and sets and libraries of such peptides, as well as methods for
using such
oligopeptide sets and libraries for determining the sequence of a peralkylated
oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus, non-peptide
analogs of the
epitope-bearing peptides of the invention also can be made routinely by these
methods. The
entire disclosure of each document cited in this section on "Polypeptides and
Fragments" is
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hereby incorporated herein by reference.
As one of skill in the art will appreciate, the polypeptides of the present
invention and the
epitope-bearing fragments thereof described above can be combined with parts
of the constant
domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins
5 facilitate purification and show an increased half-life ifi vivo. This has
been shown, e.g., for
chimeric proteins consisting of the first two domains of the human CD4-
polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins.
(EPA 0,394,827; Traunecker et al. ( 1988) Nature 331:84-86. Fusion proteins
that have a
disulfide-linked dimeric structure due to the IgG part can also be more
efficient in binding and
10 neutralizing other molecules than a monomeric B. burgdorferi polypeptide or
fragment thereof
alone. See Fountoulakis et al. ( 1995) J. Biochem. 270:3958-3964. Nucleic
acids encoding the
above epitopes of B. burgdorferi polypeptides can also be recombined with a
gene of interest as
an epitope tag to aid in detection and purification of the expressed
polypeptide.
15 4. Diagnostic Assays and Kits
The present invention further relates to methods for assaying Borrelia
infection in an
animal by detecting the expression of genes encoding Borrelia polypeptides of
the present
invention. The methods comprise analyzing tissue or body fluid from the animal
for
Borrelia-specific antibodies, nucleic acids, or proteins. Analysis of nucleic
acid specific to
20 Borrelia is assayed by PCR or hybridization techniques using nucleic acid
sequences of the
present invention as either hybridization probes or primers. See, e.g.,
Sambrook et al.
Molecular cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press,
2nd ed., 1989,
page 54 reference); Eremeeva et al. ( 1994) J. Clin. Microbiol. 32:803-810
(describing
differentiation among spotted fever group Rickettsiae species by analysis of
restriction fragment
25 length polymorphism of PCR-amplified DNA) and Chen et al. 1994 J. Clin.
Microbiol. 32:589-
595 (detecting B burgdorferi nucleic acids via PCR).
Where diagnosis of a disease state related to infection with Borrelia has
already been
made, the present invention is useful for monitoring progression or regression
of the disease state
whereby patients exhibiting enhanced Borrelia gene expression will experience
a worse clinical
30 outcome relative to patients expressing these genes) at a lower level.
By "biological sample" is intended any biological sample obtained from an
animal, cell
line, tissue culture, or other source which contains Borrelia polypeptide,
mRNA, or DNA.
Biological samples include body fluids (such as saliva, blood, plasma, urine,
mucus, synovial
fluid, etc.) tissues (such as muscle, skin, and cartilage) and any other
biological source suspected
35 of containing Borrelia polypeptides or nucleic acids. Methods for obtaining
biological samples
such as tissue are well known in the art.
The present invention is useful for detecting diseases related to Borrelia
infections in
animals. Preferred animals include monkeys, apes, cats, dogs, birds, cows,
pigs, mice, horses,
rabbits and humans. Particularly preferred are humans.
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Total RNA can be isolated from a biological sample using any suitable
technique such as
the single-step guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski et
al. ( 1987) Anal. Biochem. 162:156-159. mRNA encoding Borrelia polypeptides
having
sufficient homology to the nucleic acid sequences identified in SEQ ID NOS:1-
155 to allow for
hybridization between complementary sequences are then assayed using any
appropriate method.
These include Northern blot analysis, S I nuclease mapping, the polymerise
chain reaction
(PCR), reverse transcription in combination with the polymerise chain reaction
(RT-PCR), and
reverse transcription in combination with the ligase chain reaction (RT-LCR).
Northern blot analysis can be performed as described in Harada et al. ( 1990)
Cell
63:303-312. Briefly, total RNA is prepared from a biological sample as
described above. For
the Northern blot, the RNA is denatured in an appropriate buffer (such as
glyoxal/dimethyl
sulfoxide/sodium phosphate buffer), subjected to agarose gel electrophoresis,
and transferred
onto a nitrocellulose filter. After the RNAs have been linked to the filter by
a UV linker, the filter
is prehybridized in a solution containing formamide, SSC, Denhardt's solution,
denatured
salmon sperm, SDS, and sodium phosphate buffer. A B. burgdorferi
polynucleotide sequence
shown in SEQ ID NOS:I-155 labeled according to any appropriate method (such as
the
~2P-multiprimcd DNA labeling system (Amersham}) is used as probe. After
hybridization
overnight, the filter is washed and exposed to x-ray film. DNA for use as
probe according to the
present invention is described in the sections above and will preferably at
least 15 nucleotides in
length.
S I mapping can be performed as described in Fujita et al. (1987) Cell 49:357-
367. To
prepare probe DNA for use in S 1 mapping, the sense strand of an above-
described B.
burgdorferi DNA sequence of the present invention is used as a template to
synthesize labeled
antisense DNA. The antisense DNA can then be digested using an appropriate
restriction
endonuclease to generate further DNA probes of a desired length. Such
antisense probes are
useful for visualizing protected bands corresponding to the target mRNA (i.e.,
mRNA encoding
Borrelia polypeptides).
Levels of mRNA encoding Borrelia polypeptides are assayed, for e.g., using the
RT-PCR method described in Makino et al. ( 1990) Technique 2:295-301. By this
method, the
radioactivities of the "amplicons" in the polyacrylamide gel bands are
linearly related to the initial
concentration of the target mRNA. Briefly, this method involves adding total
RNA isolated from
a biological sample in a reaction mixture containing a RT primer and
appropriate buffer. After
incubating for primer annealing, the mixture can be supplemented with a RT
buffer, dNTPs,
DTT, RNase inhibitor and reverse transcriptase. After incubation to achieve
reverse transcription
of the RNA, the RT products are then subject to PCR using labeled primers.
Alternatively, rather
than labeling the primers, a labeled dNTP can be included in the PCR reaction
mixture. PCR
amplification can be performed in a DNA thermal cyclcr according to
conventional techniques.
After a suitable number of rounds to achieve amplification, the PCR reaction
mixture is
electrophoresed on a polyacrylamide gel. After drying the gel, the
radioactivity of the appropriate
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37
bands (corresponding to the mRNA encoding the Borrelia polypeptides of the
present invention)
are quantified using an imaging analyzer. RT and PCR reaction ingredients and
conditions,
reagent and gel concentrations, and labeling methods are well known in the
art. Variations on the
RT-PCR method will be apparent to the skilled artisan. Other PCR methods that
can detect the
nucleic acid of the present invention can be found in PCR PRIMER: A LABORATORY
MANUAL (C.W. Dieffenbach et al. eds., Cold Spring Harbor Lab Press, 1995j.
The polynucleotides of the present invention, including both DNA and RNA, may
be
used to detect polynucleotides of the present invention or Borrelia species
including B.
bur~dorferi using bio chip technology. The present invention includes both
high density chip
arrays (>1000 oligonucleotides per cm') and low density chip arrays (<1000
oligonucleotides per
cm'). Bio chips comprising arrays of polynucleotides of the present invention
may be used to
detect Borrelia species, including B. hurgdorferi, in biological and
environmental samples and to
diagnose an animal, including humans, with an B. burgdorferi or other Borrelia
infection. The
bio chips of the present invention may comprise polynucleotide sequences of
other pathogens
including bacteria, viral, parasitic, and fungal polynucleotide sequences, in
addition to the
polynucleotide sequences of the present invention, for use in rapid
diffenertial pathogenic
detection and diagnosis. The bio chips can also be used to monitor an B.
burgdnrferi or other
Borrelia infections and to monitor the genetic changes (deletions, insertions,
mismatches, etc.) in
response to drug therapy in the clinic and drug development in the laboratory.
The bio chip
technology comprising arrays of polynucleotides of the present invention may
also be used to
simultaneously monitor the expression of a multiplicity of genes, including
those of the present
invention. The polynucleotides used to comprise a selected array may be
specified in the same
manner as for the fragements, i.e, by their 5' and 3' positions or length in
contigious base pairs
and include from. Methods and particular uses of the polynucleotides of the
present invention to
detect Borrelia species, including B. burgdorferi, using bio chip technology
include those known
in the art and those of: U.S. Patent Nos. 5510270, 5545531, 5445934, 5677195,
5532128,
5556752, 5527681, 5451683, 5424186, 5607646, 5658732 and World Patent Nos.
WO/9710365, WO/9511995, WO/9743447, WO/9535505, each incorporated herein in
their
entireties.
Biosensors using the polynucleotides of the present invention may also be used
to detect,
diagnose, and monitor B. burgdorferi or other Borrelia species and infections
thereof.
Biosensors using the polynucleotides of the present invention may also be used
to detect
particular polynucleotides of the present invention. Biosensors using the
polynucleotides of the
present invention may also be used to monitor the genetic changes (deletions,
insertions,
mismatches, etc.) in response to drug therapy in the clinic and drug
development in the
laboratory. Methods and particular uses of the polynucleotides of the present
invention to detect
Borrelia species, including B. burydor/eri, using biosenors include those
known in the art and
those of: U.S. Patent Nos 5721102, 5658732, 5631170, and World Patent Nos.
W097/35011,
WO/9720203, each incorporated herein in their entireties.
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38
Thus, the present invention includes both bio chips and biosensors comprising
polynucleotides of the present invention and methods of their use.
Assaying Borrelia polypeptide levels in a biological sample can occur using
any
art-known method, such as antibody-based techniques. For example, Bnrrelia
polypeptide
expression in tissues can be studied with classical immunohistological
methods. In these, the
specific recognition is provided by the primary antibody (polyelonal or
monoclonal) but the
secondary detection system can utilize fluorescent, enzyme, or other
conjugated secondary
antibodies. As a result, an immunohistological staining of tissue section for
pathological
examination is obtained. Tissues can also be extracted, e.g., with urea and
neutral detergent, for
the liberation of Bnrrelia polypeptides for Western-blot or dotJslot assay.
See, e.y.. Jalkanen,
M. et al. ( 1985) J. Cell. Biol. 101:976-985; Jalkanen, M. et al. ( 1987) J.
Cell . Biol.
105:3087-3096. In this technique, which is based on the use of cationic solid
phases,
quantitation of a Borrelia polypeptide can be accomplished using an isolated
Borrelia polypeptide
as a standard. This technique can also be applied to body fluids.
Other antibody-based methods useful for detecting Borrelia polypeptide gene
expression
include immunoassays, such as the ELISA and the radioimmunoassay (RlA). For
example, a
Borrelia polypeptide-specific monoclonal antibodies can be used both as an
immunoabsorbent
and as an enzyme-labeled probe to detect and quantify a Borrelia polypeptide.
The amount of a
Borrelia polypeptide present in the sample can be calculated by reference to
the amount present in
a standard preparation using a linear regression computer algorithm. Such an
ELISA is described
in Iacobelli et al. ( 1988) Breast Cancer Research and Treatment 11:19-30. In
another ELISA
assay, two distinct specific monoclonal antibodies can be used to detect
Borrelia polypeptides in a
body fluid. In this assay, one of the antibodies is used as the
immunoabsorbent and the other as
the enzyme-labeled probe.
The above techniques may be conducted essentially as a "one-step" or "two-
step" assay.
The "one-step" assay involves contacting the Borrelia polypeptide with
immobilized antibody
and, without washing, contacting the mixture with the labeled antibody. The
"two-step" assay
involves washing before contacting the mixture with the labeled antibody.
Other conventional
methods may also be employed as suitable. It is usually desirable to
immobilize one component
of the assay system on a support, thereby allowing other components of the
system to be brought
into contact with the component and readily removed from the sample.
Variations of the above
and other immunological methods included in the present invention can also be
found in Harlow
et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press,
2nd ed. 1988).
Suitable enzyme labels include, for example, those from the oxidase group,
which
catalyze the production of hydrogen peroxide by reacting with substrate.
Glucose oxidase is
particularly preferred as it has good stability and its substrate (glucose) is
readily available.
Activity of an oxidase label may be assayed by measuring the concentration of
hydrogen peroxide
formed by the enzyme-labeled antibody/substrate reaction. Besides enzymes,
other suitable
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39
labels include radioisotopes, such as iodine (''SI, '''I), carbon ('aC),
sulphur (35S), tritium (~H),
indium ("'ln), and technetium (''9"'Tc), and fluorescent labels, such as
fluorescein and
rhodamine, and biotin.
Further suitable labels for the Borrelia polypeptide-specific antibodies of
the present
invention are provided below. Examples of suitable enzyme labels include
malate
dehydrogenase, Borrelia nuclease, delta-5-steroid isomerase, yeast-alcohol
dehydrogenase,
alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase,
peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonucleasc,
ureasc, catalase,
glucose-6-phosphate dehydrogenase, glucoamylasc, and acetylcholine esterase.
Examples of suitable radioisotopic labels include ~H, "'In, ''SI, '~'I, ~'P,
;SS, 'aC, 5'Cr,
S~To, S~Co, SyFe, 'SSe, 'S'Eu, ''°Y, 6'Cu, 2"Ci, 2"At, 2''Pb, '"Sc,
'°yPd, etc. "'In is a preferred
isotope where in vivn imaging is used since its avoids the problem of
dehalogenation of the''SI
or "'I-labeled monoclonal antibody by the liver. In addition, this
radionucleotide has a more
favorable gamma emission energy for imaging. See, e.g., Perkins et al. ( 1985)
Eur. J. Nucl.
Med. 10:296-301; Carasquillo et al. ( 1987) J. Nucl. Med. 28:281-287. For
example, "'In
coupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTA has
shown little uptake
in non-tumors tissues, particularly the liver, and therefore enhances
specificity of tumor
localization. See, Esteban et al. (1987) J. Nucl. Med. 28:861-870.
Examples of suitable non-radioactive isotopic labels include'S'Gd, 55Mn,
"'ZDy, SzTr,
and s~Fe.
Examples of suitable fluorescent labels include an'S~Eu label, a fluorescein
label, an
isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin
label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.
Examples of suitable toxin labels include, Pseudomouas toxin, diphtheria
toxin, ricin,
and cholera toxin.
Examples of chemiluminescent labels include a luminal label, an isoluminal
label, an
aromatic acridinium ester label, an imidazole label, an acridinium salt label,
an oxalate ester label,
a luciferin label, a luciferase label, and an aequorin label.
Examples of nuclear magnetic resonance contrasting agents include heavy metal
nuclei
such as Gd, Mn, and iron.
Typical techniques for binding the above-described labels to antibodies are
provided by
Kennedy et al. (1976) Clin. Chim. Acta 70:1-31, and Schurs et al. (1977) Clin.
Chim. Acta
81:1-40. Coupling techniques mentioned in the latter are the glutaraldehyde
method, the
periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-
succinimide ester
method, all of which methods are incorporated by reference herein.
In a related aspect, the invention includes a diagnostic kit for use in
screening serum
containing antibodies specific against B. hurgdorferi infection. Such a kit
may include an
isolated B. burgdorferi antigen comprising an epitope which is specifically
immunoreactive wish
at least one anti-B. bur~jdorferi antibody. Such a kit also includes means for
detecting the
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binding of said antibody to the antigen. 1n specific embodiments, the kit may
include a
recombinantly produced or chemically synthesized peptide or polypeptide
antigen. The peptide
or polypeptide antigen may be attached to a solid support.
In a more specific embodiment, the detecting means of the above-described kit
includes a
S solid support to which said peptide or polypeptide antigen is attached. Such
a kit may also
include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the
antibody to the B. burgdorferi antigen can be detected by binding of the
reporter labeled antibody
to the anti-B. burydorferi polypeptide antibody.
In a related aspect, the invention includes a method of detecting B.
burgdorferi infection
10 in a subject. This detection method includes reacting a body fluid,
preferably serum, from the
subject with an isolated B. burgdor~eri antigen, and examining the antigen for
the presence of
bound antibody. In a specific embodiment, the method includes a polypeptide
antigen attached to
a solid support, and serum is reacted with the support. Subsequently, the
support is reacted with
a reporter-labeled anti-human antibody. The support is then examined for the
presence of
15 reporter-labeled antibody.
The solid surface reagent employed in the above assays and kits is prepared by
known
techniques for attaching protein material to solid support material, such as
polymeric beads, dip
sticks, 96-well plates or filter material. These attachment methods generally
include non-specific
adsorption of the protein to the support or covalent attachment of the protein
, typically through a
20 free amine group, to a chemically reactive group on the solid support, such
as an activated
carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated
plates can be used in
conjunction with biotinylated antigen(s).
The polypeptides and antibodies of the present invention, including fragments
thereof,
may be used to detect Borrclia species including B. bur~dorferi using bio chip
and biosensor
?5 technology. Bio chip and biosensors of the present invention may comprise
the polypeptides of
the present invention to detect antibodies, which specifically recognize
Borrelia species, including
B. burgdorferi. Bio chip and biosensors of the present invention may also
comprise antibodies
which specifically recognize the polypeptides of the present invention to
detect Borrelia species,
including B. burgdorferi or specific polypeptides of the present invention.
Bio chips or
30 biosensors comprising polypeptides or antibodies of the present invention
may be used to detect
Borrelia species, including B. burgdorferi, in biological and environmental
samples and to
diagnose an animal, including humans, with an B. burgdorferi or other Borrelia
infection. Thus,
the present invention includes both bio chips and biosensors comprising
polypeptides or
antibodies of the present invention and methods of their use.
35 The bio chips of the present invention may further comprise polypeptide
sequences of
other pathogens including bacteria, viral, parasitic, and fungal polypeptide
sequences, in addition
to the polypeptide sequences of the present invention, for use in rapid
diffenertial pathogenic
detection and diagnosis. The bio chips of the present invention may further
comprise antibodies
or fragcments thereof specific for other pathogens including bacteria, viral,
parasitic, and fungal
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41
polypeptide sequences, in addition to the antibodies or fragements thereof of
the present
invention, for use in rapid diffenertial pathogenic detection and diagnosis.
The bio chips and
biosensors of the present invention may also be used to monitor an B.
burgdorferi or other
Borrelia infection and to monitor the genetic changes (amio acid deletions,
insertions,
substitutions, etc.) in response to drug therapy in the clinic and drug
development in the
laboratory. The bio chip and biosensors comprising polypeptides or antibodies
of the present
invention may also be used to simultaneously monitor the expression of a
multiplicity of
polypeptides, including those of the present invention. The polypeptides used
to comprise a bio
chip or biosensor of the present invention may be specified in the same manner
as for the
fragements, i.e, by their N-terminal and C-terminal positions or length in
contigious amino acid
residue. Methods and particular uses of the polypeptides and antibodies of the
present invention
to detect Borrelia species, including B. burgdorferi, or specific polypeptides
using bio chip and
biosensor technology include those known in the art, those of the U.S. Patent
Nos. and World
Patent Nos. listed above for bio chips and biosensors using polynucleotides of
the present
invention, and those of: U.S. Patent Nos. 5658732, 5135852, 5567301, 5677196,
5690894
and World Patent Nos. W09729366, W09612957, each incorporated herein in their
entireties.
5. Screening Assay for Binding Agents
Using the isolated proteins of the present invention, the present invention
further provides
methods of obtaining and identifying agents which bind to a protein encoded by
one of the ORFs
of the present invention or to one of the fragments and the Borrelia
burgdorferi fragment and
contigs herein described.
In general, such methods comprise steps of:
(a) contacting an agent with an isolated protein encoded by one of the ORFs of
the
present invention, or an isolated fragment of the Bnrrelia burgdorferi genome;
and
(b) determining whether the agent binds to said protein or said fragment.
The agents screened in the above assay can be, but are not limited to,
peptides,
carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents
can be selected
and screened at random or rationally selected or designed using protein
modeling techniques.
For random screening, agents such as peptides, carbohydrates, pharmaceutical
agents and
the like are selected at random and are assayed for their ability to bind to
the protein encoded by
the ORF of the present invention.
Alternatively, agents may be rationally selected or designed. As used herein,
an agent is
said to be "rationally selected or designed" when the agent is chosen based on
the configuration
of the particular protein. For example, one skilled in the art can readily
adapt currently available
procedures to generate peptides, pharmaceutical agents and the like capable of
binding to a
specific peptide sequence in order to generate rationally designed antipeptide
peptides, for
example see Hurby et ul., "Application of Synthetic Peptides: Antisense
Peptides," in Svjithetio
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42
Peptides, A USei''s Guide, W. H. Freeman, NY ( 1992), pp. 289-307, and
Kaspczak et al.,
Biochemistry 28: 9230-8 ( I989), or pharmaceutical agents, or the like.
In addition to the foregoing, one class of agents of the present invention, as
broadly
described, can be used to control gene expression through binding to one of
the ORFs or EMFs
of the present invention. As described above, such agents can be randomly
screened or rationally
designed/selected. Targeting the ORF or EMF allows a skilled artisan to design
sequence
specific or element specific agents, modulating the expression of either a
single ORF or multiple
ORFs which rely on the same EMF for expression control.
One class of DNA binding agents are agents which contain base residues which
hybridize
or form a triple helix by binding to DNA or RNA. Such agents can be based on
the classic
phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl
or polymeric
derivatives which have base attachment capacity.
Agents suitable for use in these methods usually contain 20 to 40 bases and
are designed
to be complementary to a region of the gene involved in transcription (triple
helix - see Lee et al.,
Nucl. Acids Res. 6:3073 ( 1979); Cooney et al., Science 241:456 ( 1988); and
Dervan et al.,
Science 251: 1360 ( 1991 )) or to the mRNA itself (antisense - Okano, J.
Neurochem. 56: 560
(1991); Oligodeoxynucleotides as Antisense l~zhihitors of Gene Expression, CRC
Press, Boca
Raton, FL ( 1988)). Triple helix- formation optimally results in a shut-off of
RNA transcription
from DNA, while antisense RNA hybridization blocks translation of an mRNA
molecule into
polypeptide. Both techniques have been demonstrated to be effective in model
systems.
Information contained in the sequences of the present invention can be used to
design antisense
and triple helix-forming oligonucleotides, and other DNA binding agents.
6. Pharmaceutical Compositions and Vaccines
The present invention further provides pharmaceutical agents which can be used
to
modulate the growth or pathogenicity of Borrelia burgdorferi, or another
related organism, in
vivo or in vitro. As used herein, a "pharmaceutical agent" is defined as a
composition of matter
which can be formulated using known techniques to provide a pharmaceutical
compositions. As
used herein, the "pharmaceutical agents of the present invention" refers the
pharmaceutical agents
which are derived from the proteins encoded by the ORFs of the present
invention or are agents
which are identified using the herein described assays.
As used herein, a pharmaceutical agent is said to "modulate the growth
pathogenicity of
Borrelia bLCrgdorferi or a related organism, in vivo or in vitro," when the
agent reduces the rate of
growth, rate of division, or viability of the organism in question. The
pharmaceutical agents of
the present invention can modulate the growth or pathogenicity of an organism
in many fashions,
although an understanding of the underlying mechanism of action is not needed
to practice the
use of the pharmaceutical agents of the present invention. Some agents will
modulate the growth
by binding to an important protein thus blocking the biological activity of
the protein, while other
agents may bind to a component of the outer surface of the organism blocking
attachment or
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43
rendering the organism more prone to act the bodies nature immune system.
Alternatively, the
agent may comprise a protein encoded by one of the ORFs of the present
invention and serve as a
vaccine. The development and use of a vaccine based on outer membrane
components are well
known in the art.
As used herein, a "related organism" is a broad term which refers to any
organism whose
growth can be modulated by one of the pharmaceutical agents of the present
invention. In
general, such an organism will contain a homolog of the protein which is the
target of the
pharmaceutical agent or the protein used as a vaccine. As such, related
organisms do not need to
be bacterial but may be fungal or viral pathogens.
The pharmaceutical agents and compositions of the present invention may be
administered
in a convenient manner, such as by the oral, topical, intravenous,
intraperitoneal, intramuscular,
subcutaneous, intranasal or intradermal routes. The pharmaceutical
compositions are
administered in an amount which is effective for treating and/or prophylaxis
of the specific
indication. In general, they are administered in an amount of at least about 1
mg/kg body weight
I 5 and in most cases they will be administered in an amount not in excess of
about 1 g/kg body
weight per day. In most cases, the dosage is from about 0.1 mg/kg to about 10
glkg body
weight daily, taking into account the routes of administration, symptoms, etc.
The agents of the present invention can be used in native form or can be
modified to form
a chemical derivative. As used herein, a molecule is said to be a "chemical
derivative" of another
molecule when it contains additional chemical moieties not normally a part of
the molecule. Such
moieties may improve the molecule's solubility, absorption, biological half
life, etc. The
moieties may alternatively decrease the toxicity of the molecule, eliminate or
attenuate any
undesirable side effect of the molecule, etc. Moieties capable of mediating
such effects are
disclosed in, among other sources, REMINGTON'S PHARMACEUTICAL SCIENCES ( 1980)
cited elsewhere herein.
For example, such moieties may change an immunological character of the
functional
derivative, such as affinity for a given antibody. Such changes in
immunomodulation activity are
measured by the appropriate assay, such as a competitive type immunoassay.
Modifications of
such protein properties as redox or thermal stability, biological half-life,
hydrophobicity,
susceptibility to proteolytic degradation or the tendency to aggregate with
carriers or into
multimers also may be effected in this way and can be assayed by methods well
known to the
skilled artisan.
The therapeutic effects of the agents of the present invention may be obtained
by
providing the agent to a patient by any suitable means {e.g., inhalation,
intravenously,
intramuscularly, subcutaneously, enterally, or parenterally). It is preferred
to administer the
agent of the present invention so as to achieve an effective concentration
within the blood or
tissue in which the growth of the organism is to be controlled. To achieve an
effective blood
concentration, the preferred method is to administer the agent by injection.
The administration
may be by continuous infusion, or by single or multiple injections.
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44
In providing a patient with one of the agents of the present invention, the
dosage of the
administered agent will vary depending upon such factors as the patient's age,
weight, height,
sex, general medical condition, previous medical history, etc. In general, it
is desirable to
provide the recipient with a dosage of agent which is in the range of from
about 1 pg/kg to 10
mg/kg (body weight of patient), although a lower or higher dosage may be
administered. The
therapeutically effective dose can be lowered by using combinations of the
agents of the present
invention or another agent.
As used herein, two or more compounds or agents are said to be administered
"in
combination" with each other when either ( 1 ) the physiological effects of
each compound, or (2)
the serum concentrations of each compound can be measured at the same time.
The composition
of the present invention can be administered concurrently with, prior to, or
following the
administration of the other agent.
The agents of the present invention are intended to be provided to recipient
subjects in an
amount sufficient to decrease the rate of growth (as defined above) of the
target organism.
The administration of the agents) of the invention may be for either a
"prophylactic" or
"therapeutic" purpose. When provided prophylactically, the agents) are
provided in advance of
any symptoms indicative of the organisms growth. The prophylactic
administration of the
agents) serves to prevent, attenuate, or decrease the rate of onset of any
subsequent infection.
When provided therapeutically, the agents) are provided at (or shortly after)
the onset of an
indication of infection. The therapeutic administration of the compounds)
serves to attenuate the
pathological symptoms of the infection and to increase the rate of recovery.
The agents of the present invention are administered to a subject, such as a
mammal, or a
patient, in a pharmaceutically acceptable form and in a therapeutically
effective concentration. A
composition is said to be "pharmacologically acceptable" if its administration
can be tolerated by a
recipient patient. Such an agent is said to be administered in a
"therapeutically effective amount"
if the amount administered is physiologically significant. An agent is
physiologically significant
if its presence results in a detectable change in the physiology of a
recipient patient.
The agents of the present invention can be formulated according to known
methods to
prepare pharmaceutically useful compositions, whereby these materials, or
their functional
derivatives, are combined in a mixture with a pharmaceutically acceptable
carrier vehicle.
Suitable vehicles and their formulation, inclusive of other human proteins,
e.g., human serum
albumin, are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES,
16th Ed., Osol, A., Ed., Mack Publishing, Easton PA ( 1980). In order to form
a
pharmaceutically acceptable composition suitable for effective administration,
such compositions
will contain an effective amount of one or more of the agents of the present
invention, together
with a suitable amount of carrier vehicle.
Additional pharmaceutical methods may be employed to control the duration of
action.
Control release preparations may be achieved through the use of polymers to
complex or absorb
one or more of the agents of the present invention. The controlled delivery
may be effectuated by
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a variety of well known techniques, including formulation with macromolecules
such as, for
example, polyesters, polyamino acids, polyvinyl, pyrrolidone,
ethylenevinylacetate,
methylcellulosc, carboxymethylcellulose, or protamine, sulfate, adjusting the
concentration of the
macromolecules and the agent in the formulation, and by appropriate use of
methods of
5 incorporation, which can be manipulated to effectuate a desired time course
of release. Another
possible method to control the duration of action by controlled release
preparations is to
incorporate agents of the present invention into particles of a polymeric
material such as
polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene
vinylacetate copolymers.
Alternatively, instead of incorporating these agents into polymeric particles,
it is possible to
10 entrap these materials in microcapsules prepared, for example, by
coaccrvation techniques or by
interfacial polymerization with, for example, hydroxymethylcellulose or
gelatine-microcapsules
and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug
delivery systems,
for example, liposomes, albumin microspheres, microemulsions, nanoparticles,
and
nanocapsules or in macroemulsions. Such techniques are disclosed in
REMINGTON'S
1 _5 PHARMACEUTICAL SCIENCES ( 1980).
The invention further provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions of the
invention. Associated with such containers) can be a notice in the form
prescribed by a
governmental agency regulating the manufacture, use or sale of pharmaceuticals
or biological
20 products, which notice reflects approval by the agency of manufacture, use
or sale for human
administration.
In addition, the agents of the present invention may be employed in
conjunction with
other therapeutic compounds.
25 7. Shot-Gun Approach to Megabase DNA Sequencing
The present invention further demonstrates that a large sequence can be
sequenced using a
random shotgun approach. This procedure, described in detail in the examples
that follow, has
eliminated the up front cost of isolating and ordering overlapping or
contiguous subclones prior
to the start of the sequencing protocols.
30 Certain aspects of the present invention are described in greater detail in
the examples that
follow. The examples are provided by way of illustration. Other aspects and
embodiments of
the present invention are contemplated by the inventors, as will be clear to
those of skill in the art
from reading the present disclosure.
ILLUSTRATIVE EXAMPLES
LIBRARIES AND SEQUENCING
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46
1. Shotgun Sequencing Probability Analysis
The overall strategy for a shotgun approach to whole genome sequencing follows
from
the Lander and Waterman (Landerman and Waterman, Gennmics 2: 231 ( 1988))
application of the
equation for the Poisson distribution. According to this treatment, the
probability, P0, that any
given base in a sequence of size L, in nucleotides, is not sequenced after a
certain amount, n, in
nucleotides, of random sequence has been determined can be calculated by the
equation PO = e-
m, where m is L/n, the fold coverage. For instance, for a genome of 2.8 Mb,
m=1 when 2.8
Mb of sequence has been randomly generated ( 1X coverage). At that point, PO =
e-I = 0.37.
The probability that any given base has not been sequenced is the same as the
probability that any
region of the whole sequence L has not been determined and, therefore, is
equivalent to the
fraction of the whole sequence that has yet to be determined. Thus, at one-
fold coverage,
approximately 37% of a polynucleotide of size L, in nucleotides has not been
sequenced. When
14 Mb of sequence has been generated, coverage is SX for a 2.8 Mb and the
unsequenced
fraction drops to .0067 or 0.67/0. SX coverage of a 2.8 Mb sequence can be
attained by
sequencing approximately 17,000 random clones from both insert ends with an
average sequence
read length of 410 bp.
Similarly, the total gap length, G, is determined by the equation G = Le-m,
and the
average gap size, g, follows the equation, g = L/n. Thus, SX coverage leaves
about 240 gaps
averaging about 82 by in size in a sequence of a polynucleotide 2.8 Mb long.
The treatment above is essentially that of Lander and Waterman, Genomics 2:
231
( 1988).
2. Random Library Construction
In order to approximate the random model described above during actual
sequencing, a
nearly ideal library of cloned genomic fragments is required. The following
library construction
procedure was developed to achieve this end.
Borrelia burgdorferi DNA is prepared by phenol extraction. A mixture
containing 200 pg
DNA in 1.0 ml of 300 mM sodium acetate, 10 mM Tris-HCI, 1 mM Na-EDTA, 50010
glycerol is
processed through a nebulizer (IPI Medical Products) with a stream of nitrogen
adjusted to 35
Kpa for 2 minutes. The sonicated DNA is ethanol precipitated and redissolved
in 500 p.l TE
buffer.
To create blunt-ends, a 100 ~1 aliquot of the resuspended DNA is digested with
5 units of
BAL31 nuclease (New England BioLabs) for 10 min at 30°C in 200 ~1 BAL31
buffer. The
digested DNA is phenol-extracted, ethanol-precipitated, redissolved in 100 ~1
TE buffer, and
then size-fractionated by electrophoresis through a 1.0% low melting
temperature agarose gel.
The section containing DNA fragments 1.6-2.0 kb in size is excised from the
gel, and the LGT
agarose is melted and the resulting solution is extracted with phenol to
separate the agarose from
the DNA. DNA is ethanol precipitated and redissolved in 20 ~1 of TE buffer for
ligation to
vector.
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47
A two-step ligation procedure is used to produce a plasmid library with
97°lo inserts, of
which >99% were single inserts. The first ligation mixture (50 ul) contains 2
pg of DNA
fragments, 2 ~,g pUCI8 DNA (Pharmacia) cut with SmaI and dephosphorylated with
bacterial
alkaline phosphatase, and 10 units of T4 ligase (GIBCO/BRL) and is incubated
at 14°C for 4 hr.
The ligation mixture then is phenol extracted and ethanol precipitated, and
the precipitated DNA is
dissolved in 20 pl TE buffer and electrophoresed on a I.0% low melting agarose
gel. Discrete
bands in a ladder are visualized by ethidium bromide-staining and UV
illumination and identified
by size as insert (I), vector (v), v+I, v+2i, v+3i, etc. The portion of the
gel containing v+I DNA
is excised and the v+I DNA is recovered and rcsuspended into 20 p! TE. The v+I
DNA then is
blunt-ended by T4 polymerise treatment for 5 min. at 37°C in a reaction
mixture (50 ul)
containing the v+I linears, 500 pM each of the 4 dNTPs, and 9 units of T4
polymerise (New
England BioLabs), under recommended buffer conditions. After phenol extraction
and ethanol
precipitation the repaired v+I linears are dissolved in 20 ~tl TE. The final
ligation to produce
circles is carried out in a 50 ~l reaction containing 5 ~tl of v+I linears and
5 units of T4 ligase at
14°C overnight. After 10 min. at 70°C the following day, the
reaction mixture is stored at -20°C.
This two-stage procedure results in a molecularly random collection of single-
insert
plasmid recombinants with minimal contamination from double-insert chimeras
(<1 %) or free
vector (<3%).
Since deviation from randomness can arise from propagation the DNA in the
host, E. coli
host cells deficient in all recombination and restriction functions (A.
Greener, Strategies 3 (I ):5
( 1990)) are used to prevent rearrangements, deletions, and loss of clones by
restriction.
Furthermore, transformed cells are plated directly on antibiotic diffusion
plates to avoid the usual
broth recovery phase which allows multiplication and selection of the most
rapidly growing cells.
Plating is carried out as follows. A 100 pl aliquot of Epicurian Coli SURE II
Supercompetent Cells (Stratagene 200152) is thawed on ice and transferred to a
chilled Falcon
2059 tube on ice. A I .7 p,l aliquot of I .42 M beta-mercaptoethanol is added
to the aliquot of cells
to a final concentration of 25 mM. Cells are incubated on ice for 10 min. A 1
~1 aliquot of the
final ligation is added to the cells and incubated on ice for 30 min. The
cells are heat pulsed for
sec. at 42°C and placed back on ice for 2 min. The outgrowth period in
liquid culture is
30 eliminated from this protocol in order to minimize the preferential growth
of any given
transformed cell. Instead the transformation mixture is plated directly on a
nutrient rich SOB
plate containing a 5 ml bottom layer of SOB agar (5% SOB agar: 20 g tryptone,
5 g yeast extract,
0.5 g NaCI, 1.5% Difco Agar per liter of media). The 5 ml bottom layer is
supplemented with
0.4 ml of 50 mg/ml ampicillin per 100 ml SOB agar. The 15 ml top layer of SOB
agar is
supplemented with 1 ml X-Gal (2%), 1 ml MgCl2 (I M), and 1 ml MgS04/100 ml SOB
agar.
The 15 ml top layer is poured just prior to plating. Our titer is
approximately 100 colonies/10 ~.l
aliquot of transformation.
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48
All colonies are picked for template preparation regardless of size. Thus,
only clones lost
due to "poison" DNA or deleterious gene products are deleted from the library,
resulting in a
slight increase in gap number over that expected.
3. Random DNA Sequencing
High quality double stranded DNA plasmid templates are prepared using a
"boiling bead"
method developed in collaboration with Advanced Genetic Technology Corp.
(Gaithersburg,
MD) (Adams et al., Science 252:1651 ( 1991 ); Adams et al., Nature 355: 632 (
1992)). Plasmid
preparation is performed in a 96-well format for all stages of DNA preparation
from bacterial
growth through final DNA purification. Template concentration is determined
using Hoechst
Dye and a Millipore Cytofluor. DNA concentrations are not adjusted, but low-
yielding templates
are identified where possible and not sequenced.
Templates are also prepared from two Borrelia burgdorferi lambda genomic
libraries. An
amplified library is constructed in the vector Lambda GEM-12 (Promega) and an
unamplified
library is constructed in Lambda DASH II (Stratagene). In particular, for the
unamplified lambda
library, Bnrrelia burgdorferi DNA (> 100 kb) is partially digested in a
reaction mixture (200 ul)
containing 50 ~g DNA, 1X Sau3AI buffer, 20 units Sau3AI for 6 min. at
23°C. The digested
DNA was phenol-extracted and electrophoresed on a 0.5% low melting agarose gel
at 2V/em for
7 hours. Fragments from 15 to 25 kb are excised and recovered in a final
volume of 6 ul. One
pl of fragments is used with 1 pl of DASHII vector (Stratagene) in the
recommended ligation
reaction. One pl of the ligation mixture is used per packaging reaction
following the
recommended protocol with the Gigapack II XL Packaging Extract (Stratagene,
#227711).
Phage are plated directly without amplification from the packaging mixture
(after dilution with
500 pl of recommended SM buffer and chloroform treatment). Yield is about 2.5x
103 pfu/ul.
The amplified library is prepared essentially as above except the lambda GEM-
12 vector is used.
After packaging, about 3.5x 104 pfu are plated on the restrictive NM539 host.
The lysate is
harvested in 2 ml of SM buffer and stored frozen in 7% dimethylsulfoxide. The
phage titer is
approximately 1 x 109 pfu/ml.
Liquid lysates ( 100 ~l) are prepared from randomly selected plaques (from the
unamplified library) and template is prepared by long-range PCR using T7 and
T3 vector-specific
pnmers.
Sequencing reactions are carried out on plasmid and/or PCR templates using the
AB
Catalyst LabStation with Applied Biosystems PRISM Ready Reaction Dye Primer
Cycie
Sequencing Kits for the M 13 forward (M 13-21 ) and the NT 13 reverse (M 13RP
1 ) primers (Adams
et al., Nature 368:474 ( 1994) ). Dye terminator sequencing reactions are
carried out on the
lambda templates on a Perkin-Elmer 9600 Thermocycler using the Applied
Biosystems Ready
Reaction Dye Terminator Cycle Sequencing kits. T7 and SP6 primers are used to
sequence the
ends of the inserts from the Lambda GEM-12 library and T7 and T3 primers are
used to sequence
the ends of the inserts from the Lambda DASH II library. Sequencing reactions
are performed
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49
by eight individuals using an average of fourteen AB 373 DNA Sequencers per
day. All
sequencing reactions are analyzed using the Stretch modification of the AB
373, primarily using a
34 cm well-to-read distance. The overall sequencing success rate very
approximately is about
85% for M 13-21 and M 13RP 1 sequences and 65% for dye-terminator reactions.
The average
usable read length is 485 by for M 13-21 sequences, 445bp for M 13RP 1
sequences, and 375 hp
Cor dye-terminator reactions.
Richards et al., Chapter 28 in AUTOMATED DNA SEQUENCING AND ANALYSIS,
M. D. Adams, C. Fields, J. C. Venter, Eds., Academic Press, London, ( 1994)
described the
value of using sequence from both ends of sequencing templates to facilitate
ordering of contigs
in shotgun assembly projects of lambda and cosmid clones. We balance the
desirability of both-
end sequencing (including the reduced cost of lower total number of templates)
against shorter
read-lengths for sequencing reactions performed with the M13RP1 (reverse)
primer compared to
the M13-21 (forward) primer. Approximately one-half of the templates are
sequenced from both
ends. Random reverse sequencing reactions are done based on successful forward
sequencing
reactions. Some M 13RP 1 sequences are obtained in a semi-directed fashion: M
13-21: sequences
pointing outward at the ends of contigs are chosen for M13RP1 sequencing in an
effort to
specifically order contigs.
4. Protocol for Automated Cycle Sequencing
The sequencing is carried out using ABI Catalyst robots and AB 373 Automated
DNA
Sequencers. The Catalyst robot is a publicly available sophisticated pipetting
and temperature
control robot which has been developed specifically for DNA sequencing
reactions. The Catalyst
combines pre-aliquoted templates and reaction mixes consisting of deoxy- and
dideoxynucleotides, the thermostable Taq DNA polymerise, fluorescently-
labelled sequencing
primers, and reaction buffer. Reaction mixes and templates are combined in the
wells of an
aluminum 96-well thermocycling plate. Thirty consecutive cycles of linear
amplification (i.e..,
one primer synthesis) steps are performed including denaturation, annealing of
primer and
template, and extension; i.e., DNA synthesis. A heated lid with rubber gaskets
on the
thermocycling plate prevents evaporation without the need for an oil overlay.
Two sequencing protocols are used: one for dye-labelled primers and a second
for dye-
labelled dideoxy chain terminators. The shotgun sequencing involves use of
four dye-labelled
sequencing primers, one for each of the four terminator nucleotide. Each dye-
primer is labelled
with a different fluorescent dye, permitting the four individual reactions to
be combined into one
lane of the 373 DNA Sequencer for electrophoresis, detection, and base-
calling. ABI currently
supplies pre-mixed reaction mixes in bulk packages containing all the
necessary non-template
reagents for sequencing. Sequencing can be done with both plasmid and PCR-
generated
templates with both dye-primers and dye- terminators with approximately equal
fidelity, although
plasmid templates generally give longer usable sequences.
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Thirty-two reactions are loaded per AB373 Scquencer each day, for a total of
960
samples. Electrophoresis is run overnight following the manufacturer's
protocols, and the data is
collected for twelve hours. Following electrophoresis and fluorescence
detection, the ABI 373
performs automatic lane tracking and base-calling. The lane-tracking is
confirmed visually. Each
5 sequence electropherogram (or fluorescence lane trace) is inspected visually
and assessed for
quality. Trailing sequences of low quality are removed and the sequence itself
is loaded via
software to a Sybase database (archived daily to 8lnm tape). Leading vector
polylinker sequence
is removed automatically by a software program. Average edited lengths of
sequences from the
standard ABI 373 are around 400 by and depend mostly on the quality of the
template used for
10 the sequencing reaction. ABI 373 Sequencers converted to Stretch Liners
provide a longer
electrophoresis path prior to fluorescence detection and increase the average
number of usable
bases to 500-600 bp.
INFORMATICS
15 1. Data Management
A number of information management systems for a large-scale sequencing lab
have been
developed. (For review see, for instance, Kerlavage et al., Proceedings of the
Twenty-Sixtlz
Annual Hawaii International Confererzce on System Sciences, IEEE Computer
Society Press,
Washington D. C., 585 ( 1993)) The system used to collect and assemble the
sequence data was
20 developed using the Sybase relational database management system and was
designed to
automate data flow wherever possible and to reduce user error. The database
stores and
correlates all information collected during the entire operation from template
preparation to final
analysis of the genome. Because the raw output of the ABI 373 Sequencers was
based on a
Macintosh platform and the data management system chosen was based on a Unix
platform, it
25 was necessary to design and implement a variety of multi- user, client-
server applications which
allow the raw data as well as analysis results to flow seamlessly into the
database with a
minimum of user effort.
2. Assembly
30 An assembly engine {TIGR Assembler) developed for the rapid and accurate
assembly of
thousands of sequence fragments was employed to generate contigs. The TIGR
assembler
simultaneously clusters and assembles fragments of the genome. In order to
obtain the speed
necessary to assemble more than 104 fragments, the algorithm builds a hash
table of 12 by
oligonucleotide subsequences to generate a list of potential sequence fragment
overlaps. The
35 number of potential overlaps for each fragment determines which fragments
are likely to fall into
repetitive elements. Beginning with a single seed sequence fragment, TIGR
Assembler extends
the current contig by attempting to add the best matching fragment based on
oligonucleotide
content. The contig and candidate fragment are aligned using a modified
version of the Smith-
Waterman algorithm which provides for optimal gapped alignments (Waterman, M.
S., Methods
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51
in Enzymnlogy 164:765 ( 1988)). The contig is extended by the fragment only if
strict criteria for
the quality of the match are met. The match criteria include the minimum
length of overlap, the
maximum length of an unmatched end, and the minimum percentage match. These
criteria are
automatically lowered by the algorithm in regions of minimal coverage and
raised in regions with
a possible repetitive element. The number of potential overlaps for each
fragment determines
which fragments are likely to fall into repetitive. elements. Fragments
representing the boundaries
of repetitive elements and potentially chimeric fragments are often rejected
based on partial
mismatches at the ends of alignments and excluded from the current contig.
TIGR Assembler is
designed to take advantage of clone size information coupled with sequencing
from both ends of
each template. It enforces the constraint that sequence fragments from two
ends of the same
template point toward one another in the contig and are located within a
certain range of base
pairs (definable for each clone based on the known clone size range for a
given library). The
process resulted in 155 contigs as represented by SEQ ID NOs: I-I55.
3. Identifying Genes
The predicted coding regions of the Borrelia burgdorferi genome were initially
defined
with the program GencMark, which finds ORFs using a probabilistic
classification technique.
The predicted coding region sequences were used in searches against a database
of all nucleotide
sequences from GenBank (July, 1997), using the BLASTN search method to
identify overlaps
of 50 or more nucleotides with at least a 95% identity (using default
parameters). Those ORFs
with nucleotide sequence matches are shown in Table 1. The ORFs without such
matches were
translated to protein sequences and compared to a non-redundant database of
known proteins
generated by combining the Swiss-prot, PIR and GenPept databases. ORFs that
matched a
database protein with BLASTP probability less than or equal to 0.01 are shown
in Table 2. The
table also lists assigned functions based on the closest match in the
databases. ORFs that did not
match protein or nucleotide sequences in the databases at these levels are
shown in Table 3.
ILLUSTRATIVE APPLICATIONS
1. Production of an Antibody to a Borrelia burgdorferi
Protein
Substantially pure protein or polypeptide is isolated from the transfected or
transformed
cells using any one of the methods known in the art. The protein can also be
produced in a
recombinant prokaryotic expression system, such as E. coli, or can be
chemically synthesized.
Concentration of protein in the final preparation is adjusted, for example, by
concentration on an
Amicon filter device, to the level of a few micrograms/ml. Monoclonal or
polyclonal antibody to
the protein can then be prepared as follows.
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52
2. Monoclonal Antibody Production by Hybridoma Fusion
Monoclonal antibody to epitopes of any of the peptides identified and isolated
as
described can be prepared from murine hybridomas according to the classical
method of Kohler,
G. and Milstein, C., Nature 256:495 ( 1975) or modifications of the methods
thereof. Briefly, a
mouse is repetitively inoculated with a few micrograms of the selected protein
over a period of a
few weeks. The mouse is then sacrificed, and the antibody producing cells of
the spleen
isolated. The spleen cells are fused by means of polyethylene glycol with
mouse myeloma cells,
and the excess unfused cells destroyed by growth of the system on selective
media comprising
aminopterin (HAT media). The successfully fused cells are diluted and aliquots
of the dilution
placed in wells of a microtiter plate where growth of the culture is
continued. Antibody-
producing clones are identified by detection of antibody in the supernatant
fluid of the wells by
immunoassay procedures, such as ELISA, as originally described by Engvall, E.,
Meth.
En;,ymol. 70:419 ( 1980), and modified methods thereof. Selected positive
clones can be
expanded and their monoclonal antibody product harvested for use. Detailed
procedures for
monoclonal antibody production are described in Davis, L. et al., Basic
Methods in Molecular
Biology, Elsevier, New York. Section 21-2 (1989).
3. Polyclonal Antibody Production by Immunization
Polyclonal antiserum containing antibodies to heterogenous epitopes of a
single protein
can be prepared by immunizing suitable animals with the expressed protein
described above,
which can be unmodified or modified to enhance immunogenicity. Effective
polyclonal antibody
production is affected by many factors related both to the antigen and the
host species. For
example, small molecules tend to be less immunogenic than others and may
require the use of
carriers and adjuvant. Also, host animals vary in response to site of
inoculations and dose, with
both inadequate or excessive doses of antigen resulting in low titer antisera.
Small doses (ng
level) of antigen administered at multiple intradermal sites appears to be
most reliable. An
effective immunization protocol for rabbits can be found in Vaitukaitis, J. et
al., J. Clin.
Endocrinol. Metab. 33:988-991 ( 1971 ).
Booster injections can be given at regular intervals, and antiserum harvested
when
antibody titer thereof, as determined semi-quantitatively, for example, by
double
immunodiffusion in agar against known concentrations of the antigen, begins to
fall. See, for
example, Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental
Immunology, Wier,
D., ed, Blackwell ( 1973). Plateau concentration of antibody is usually in the
range of 0. 1 to 0.
2 mg/ml of serum (about 12M). Affinity of the antisera for the antigen is
determined by
3S preparing competitive binding curves, as described, for example, by Fisher,
D., Chap. 42 in:
Manual of Clinical Immunology, second edition, Rose and Friedman, eds., Amer.
Soc. For
Microbiology, Washington, D. C. (1980)
Antibody preparations prepared according to either protocol are useful in
quantitative
immunoassays which determine concentrations of antigen-bearing substances in
biological
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53
samples; they are also used semi- quantitatively or qualitatively to identify
the presence of antigen
in a biological sample. In addition, antibodies are useful in various animal
models of
pneumococcal disease as a means of evaluating the protein used to make the
antibody as a
potential vaccine target or as a means of evaluating the antibody as a
potential immunotherapeutic
or immunoprophylactic reagent.
4. Preparation of PCR Primers and Amplification of DNA
Various fragments of the Borrelin hurgdorferi genome, such as those of Tables
1-6 and
SEQ ID NOS: 1-155 can be used, in accordance with the present invention, to
prepare PCR
primers for a variety of uses. The PCR primers are preferably at least 15
bases, and more
preferably at least 18 bases in length. When selecting a primer sequence, it
is preferred that the
primer pairs have approximately the same G/C ratio, so that melting
temperatures are
approximately the same. The PCR primers and amplified DNA of this Example find
use in the
Examples that follow.
5. Isolation of a Selected DNA Clone From B. burgdorferi
Three approaches arc used to isolate a B. burgdorferi clone comprising a
polynucleotide
of the present invention from any B. burgdorJeri genomic DNA library. The B.
burgdorferi
strain B31PU has been deposited as a convienent source for obtaining a B.
hurgdorferi strain
although a wide varity of strains B. burgdorferi strains can be used which are
known in the art.
B. bc~rgdorferi genomic DNA is prepared using the following method. A 20m1
overnight
bacterial culture grown in a rich medium (e.g., Trypticase Soy Broth, Brain
Heart Infusion broth
or Super broth), pelleted, fished two times with TES (30mM Tris-pH 8.0, 25mM
EDTA, 50mM
NaCI), and resuspended in 5ml high salt TES (2.5M NaCI). Lysostaphin is added
to final
concentration of approx 50ug/ml and the mixture is rotated slowly 1 hour at
37C to make
protoplast cells. The solution is then placed in incubator (or place in a
shaking water bath) and
warmed to 55C. Five hundred micro liter of 20% sarcosyl in TES (final
concentration 2%) is
then added to lyre the cells. Next, guanidine HCl is added to a final
concentration of 7M (3.698
in 5.5 ml). The mixture is swirled slowly at 55C for 60-90 min (solution
should clear). A CsCI
gradient is then set up in SW41 ultra clear tubes using 2.Om1 5.7M CsCI and
overlaying with
2.85M CsCI. The gradient is carefully overlayed with the DNA-containing GuHCI
solution.
The gradient is spun at 30,000 rpm, 20C for 24 hr and the lower DNA band is
collected. The
volume is increased to 5 ml with TE buffer. The DNA is then treated with
protease K (10 ug/ml)
overnight at 37 C, and precipitated with ethanol. The precipitated DNA is
resuspended in a
desired buffer.
In the first method, a plasmid is directly isolated by screening a plasmid B.
burgdorferi
genomic DNA library using a polynucleotide probe corresponding to a
polynucleotide of the
present invention. Particularly, a specific polynucleotide with 30-40
nucleotides is synthesized
using an Applied Biosystems DNA synthesizer according to the sequence
reported. The
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54
oligonucleotide is labeled, for instance, with ~ZP-y-ATP using T4
polynucleotide kinase and
purified according to routine methods. (See, e.g., Maniatis et al., Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY ( 1982).) The
library is
transformed into a suitable host, as indicated above (such as XL-1 Blue
(Stratagene)) using
techniques known to those of skill in the art. See, e.g., Sambrook et al.
MOLECULAR
CLONING: A LABORATORY MANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel
et al., CURRENT PROTOCALS IN MOLECULAR BIOLOGY (John Wiley and Sons, N.Y.
1989). The transformants are plated on I .5% agar plates (containing the
appropriate selection
agent, e.g., ampicillin) to a density of about 150 transformants (colonies)
per plate. These plates
are screened using Nylon membranes according to routine methods for bacterial
colony
screening. See, e.g., Sambrook et al. MOLECULAR CLONING: A LABORATORY
MANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel et al., CURRENT
PROTOCALS
IN MOLECULAR BIOLOGY (John Wiley and Sons, N.Y. 1989) or other techniques
known to
those of skill in the art.
Alternatively, two primers of I S-25 nucleotides derived from the 5' and 3'
ends of a
polynucleotide of SEQ ID NOS:1-I55 are synthesized and used to amplify the
desired DNA by
PCR using a B. burgdorferi genomic DNA prep as a template. PCR is carried out
under routine
conditions, for instance, in 25 ~1 of reaction mixture with 0.5 ug of the
above DNA template. A
convenient reaction mixture is 1.5-5 mM MgCl2, 0.01 % (w/v) gelatin, 20 ~M
each of dATP,
dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase.
Thirty five
cycles of PCR (denaturation at 94°C for 1 min; annealing at 55°C
for 1 min; elongation at 72°C
for 1 min) are performed with a Perkin-Elmer Cetus automated thernial cycler.
The amplified
product is analyzed by agarose gel electrophoresis and the DNA band with
expected molecular
weight is excised and purified. The PCR product is verified to be the selected
sequence by
subcloning and sequencing the DNA product.
Finally, overlapping oligos of the DNA sequences of SEQ ID NOS:I-155 can be
chemically synthesized and used to generate a nucleotide sequence of desired
length using PCR
methods known in the art.
6(a). Expression and Purification Borrelia polypeptides in E. coli
The bacterial expression vector pQE60 is used for bacterial expression of some
of the
polypeptide fragements of the present invention. (QIAGEN, Inc., 9259 Eton
Avenue,
Chatsworth, CA, 9131 1 ). pQE60 encodes ampicillin antibiotic resistance
("Ampr") and contains
a bacterial origin of replication ("ori"), an IPTG inducible promoter, a
ribosome binding site
("RBS"), six codons encoding histidine residues that allow affinity
purification using nickel-
nitrilo-tri-acetic acid ("Ni-NTA") affinity resin (QIAGEN, Inc., supra} and
suitable single
restriction enzyme cleavage sites. These elements are arranged such that an
inserted DNA
fragment encoding a polypeptide expresses that polypeptide with the six His
residues (i.e., a "6
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X His tag") covalently linked to the carboxyl terminus of that polypeptide.
The DNA sequence encoding the desired portion of a B. burgdorferi protein of
the
present invention is amplified from B. burgdorferi genomic DNA using PCR
oligonucleotide
primers which anneal to the 5' and 3' sequences coding for the portions of the
B. bcergdorferi
5 polynucleotide shown in SEQ ID NOS:1-155. Additional nucleotides containing
restriction sites
to facilitate cloning in the pQE60 vector are added to the 5' and 3'
sequences, respectively.
For cloning the mature protein, the 5' primer has a sequence containing an
appropriate
restriction site followed by nucleotides of the amino terminal coding sequence
of the desired B.
bur~dorferi polynucleotide sequence in SEQ ID NOS: l-I55. One of ordinary
skill in the art
10 would appreciate that the point in the protein coding sequence where the 5'
and 3' primers begin
may be varied to amplify a DNA segment encoding any desired portion of the
complete protein
shorter or longer than the mature form. The 3' primer has a sequence
containing an appropriate
restriction site followed by nucleotides complementary to the 3' end of the
polypeptide coding
sequence of SEQ ID NOS: I-155, excluding a stop codon, with the coding
sequence aligned with
15 the restriction site so as to maintain its reading frame with that of the
six His codons in the pQE60
vector.
The amplified B. burgdorferi DNA fragment and the vector pQE60 are digested
with
restriction enzymes which recognize the sites in the primers and the digested
DNAs are then
ligated together. The B. burgdorferi DNA is inserted into the restricted pQE60
vector in a manner
20 which places the B. burgdorferi protein coding region downstream from the
IPTG-inducible
promoter and in-frame with an initiating AUG and the six histidine codons.
The ligation mixture is transformed into competent E. coli cells using
standard procedures
such as those described by Sambrook et al., supra.. E. coli strain M 15/rep4,
containing multiple
copies of the plasmid pREP4, which expresses the lac repressor and confers
kanamycin
25 resistance ("Kanr"), is used in carrying out the illustrative example
described herein. This strain,
which is only one of many that are suitable for expressing a B. bur~dorferi
polypeptide, is
available commercially (QIAGEN, Inc., supra). Transformants are identified by
their ability to
grow on LB agar plates in the presence of ampicillin and kanamycin. Plasmid
DNA is isolated
from resistant colonies and the identity of the cloned DNA confirmed by
restriction analysis,
30 PCR and DNA sequencing.
Clones containing the desired constructs are grown overnight ("O/N") in liquid
culture in
LB media supplemented with both ampicillin ( 100 ~tg/ml) and kanamycin (25
~.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of approximately
1:25 to 1:250. The cells
are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-~3-D-
35 thiogalactopyranoside ("IPTG") is then added to a final concentration of 1
mM to induce
transcription from the lac repressor sensitive promoter, by inactivating the
lacI repressor. Cells
subsequently are incubated further for 3 to 4 hours. Cells then are harvested
by centrifugation.
The cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCI, pH
8. The cell
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56
debris is removed by centrifugation, and the supernatant containing the B.
burgdorferi
polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin column
(QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin
with high affinity
are purified in a simple one-step procedure (for details see: The
QIAexpressionist, 1995,
QIAGEN, Inc., supra). Briefly the supernatant is loaded onto the column in 6 M
guanidine-HCI,
pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8,
then washed
with 10 volumes of 6 M guanidine-HCl pH 6, and finally the B. burgdor~eri
polypeptide is
eluted with 6 M guanidine-HCI, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-
buffered saline
(PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI. Alternatively, the
protein could be
successfully refolded while immobilized on the Ni-NTA column. The recommended
conditions
are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCI,
20% glycerol, 20
mM Tris/HCI pH 7.4, containing protease inhibitors. The renaturation should be
performed over
a period of 1.5 hours or more. After renaturation the proteins can be eluted
by the addition of
250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS
or 50 mM
sodium acetate pH 6 buffer plus 200 mM NaCI. The purified protein is stored at
4° C or frozen at
-80° C.
The polypeptide of the present invention are also prepared using a non-
denaturing protein
purification method. For these polypeptides, the cell pellet from each liter
of culture is
resuspended in 25 mls of Lysis Buffer A at 4°C (Lysis Buffer A = 50 mM
Na-phosphate, 300
mM NaCI, 10 mM 2-mercaptoethanol, 10% Glycerol, pH 7.5 with 1 tablet of
Complete EDTA-
free protease inhibitor cocktail (Boehringer Mannheim #1873580) per 50 ml of
buffer).
Absorbance at 550 nm is approximately 10-20 O.D./ml. The suspension is then
put through
three freeze/thaw cycles from -70°C (using a ethanol-dry ice bath) up
to room temperature. The
cells are lysed via sonication in short 10 sec bursts over 3 minutes at
approximately 80W while
kept on ice. The sonicated sample is then centrifuged at 15,000 RPM for 30
minutes at 4°C. The
supernatant is passed through a column containing I.0 ml of CL-4B resin to pre-
clear the sample
of any proteins that may bind to agarose non-specifically, and the flow-
through fraction is
collected.
The pre-cleared flow-through is applied to a nickel-nitrilo-tri-acetic acid
("Ni-NTA")
affinity resin column (Quiagen, Inc., supra). Proteins with a 6 X His tag bind
to the Ni-NTA
resin with high affinity and can be purified in a simple one-step procedure.
Briefly, the
supernatant is loaded onto the column in Lysis Buffer A at 4°C, the
column is first washed with
10 volumes of Lysis Buffer A until the A280 of the eluate returns to the
baseline. Then, the
column is washed with 5 volumes of 40 mM Imidazole (92°lo Lysis Buffer
A / 8% Buffer B)
(Buffer B = 50 n>NI Na-Phosphate, 300 mM NaCI, 10% Glycerol, 10 mM 2-
mercaptoethanol,
500 mM Imidazole, pH of the final buffer should be 7.5). The protein is eluted
off of the column
with a series of increasing Imidazole solutions made by adjusting the ratios
of Lysis Buffer A to
Buffer B. Three different concentrations are used: 3 volumes of 75 mM
Imidazole, 3 volumes of
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
57
150 mM Imidazole, 5 volumes of 500 mM Imidazole. The fractions containing the
purified
protein are analyzed using 8 %, 10 % or 14% SDS-PAGE depending on the protein
size. The
purified protein is then dialyzed 2X against phosphate-buffered saline (PBS)
in order to place it
into an easily workable buffer. The purified protein is stored at 4° C
or frozen at -80°.
The following alternative method may be used to purify B. burgdorferi
expressed in E
coli when it is present in the form of inclusion bodies. Unless otherwise
specified, all of the
following steps are conducted at 4-10°C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture is
cooled to 4-10°C and the cells are harvested by continuous
centrifugation at 15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein per unit
weight of cell paste
and the amount of purified protein required, an appropriate amount of cell
paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The
cells are
dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics,
Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then
mixed with NaCI
solution to a final concentration of 0.5 M NaCI, followed by centrifugation at
7000 x g for 15
min. The resultant pellet is washed again using 0.5M NaCI, 100 mM Tris, 50 mM
EDTA, pH
7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
hydrochloride (GuHCI) for 2-4 hours. After 7000 x g centrifugation for 15
min., the pellet is
discarded and the B. burgdorferi polypeptide-containing supernatant is
incubated at 4°C
overnight to allow further GuHCI extraction.
Following high speed centrifugation (30,000 x g) to remove insoluble
particles, the
GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract with
20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA by vigorous
stirring.
The refolded diluted protein solution is kept at 4°C without mixing for
12 hours prior to further
purification steps.
To clarify the refolded B. burgdorferi polypeptide solution, a previously
prepared
tangential filtration unit equipped with 0.16 ~m membrane filter with
appropriate surface area
(e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed.
The filtered
sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive
Biosystems). The
column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500
mM, 1000
mM, and 1500 mM NaCI in the same buffer, in a stepwise manner. The absorbance
at 280 mm
of the effluent is continuously monitored. Fractions are collected and further
analyzed by SDS-
PAGE.
Fractions containing the B. burgdorferi polypeptide are then pooled and mixed
with 4
CA 02304925 1999-12-17
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58
volumes of water. The diluted sample is then loaded onto a previously prepared
set of tandem
columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20,
Perseptive Biosystems) exchange resins. The columns are equilibrated with 40
mM sodium
acetate, pH 6Ø Both columns are washed with 40 mM sodium acetate, pH 6.0,
200 mM NaCI.
The CM-20 column is then eluted using a 10 column volume linear gradient
ranging from 0.2 M
NaCI, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH
6.5. Fractions
are collected under constant Azgp monitoring of the effluent. Fractions
containing the B.
bur~dorferi polypeptide (determined, for instance, by 16% SDS-PAGE) are then
pooled.
The resultant B. burgdorferi polypeptide exhibits greater than 95% purity
after the above
refolding and purification steps. No major contaminant bands are observed from
Commassie
blue stained 16% SDS-PAGE gel when 5 ~g of purified protein is loaded. The
purified protein
is also tested for endotoxin/LPS contamination, and typically the LPS content
is less than 0.1
ng/ml according to LAL assays.
I S 6(b). Alternative Expression and Purification liorrelia polypeptides in E.
coli
Tthe vector pQElO is alternatively used to clone and express some of the
polypeptides of
the present invention for use in the soft tissue and systemic infection models
discussed below.
The difference being such that an inserted DNA fragment encoding a polypeptide
expresses that
polypeptide with the six His residues (i.e., a "6 X His tag") covalently
linked to the amino
terminus of that polypeptide. The bacterial expression vector pQElO (QIAGEN,
Inc., 9259 Eton
Avenue, Chatsworth, CA, 9131 I ) was used in this example . The components of
the pQElO
plasmid are arranged such that the inserted DNA sequence encoding a
polypeptide of the present
invention expresses the polypeptide with the six His residues (i.e., a "6 X
His tag")) covalently
linked to the amino terminus.
The DNA sequences encoding the desired portions of a polypeptide of SEQ ID
NOS:1-
155 were amplified using PCR oligonucleotide primers from genomic B.
burgdorferi DNA. The
PCR primers anneal to the nucleotide sequences encoding the desired amino acid
sequence of a
polypeptide of the present invention. Additional nucleotides containing
restriction sites to
facilitate cloning in the pQElO vector were added to the 5' and 3' primer
sequences, respectively.
For cloning a polypeptide of the present invention, the 5' and 3' primers were
selected to
amplify their respective nucleotide coding sequences. One of ordinary skill in
the art would
appreciate that the point in the protein coding sequence where the 5' and 3'
primers begins may
be varied to amplify a DNA segment encoding any desired portion of a
polypeptide of the present
invention. The 5' primer was designed so the coding sequence of the 6 X His
tag is aligned with
the restriction site so as to maintain its reading frame with that of B.
bur~dorferi polypeptide.
The 3' was designed to include an stop codon. The amplified DNA fragment was
then cloned,
and the protein expressed, as described above for the pQE60 plasmid.
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
59
The DNA sequences of SEQ ID NOS:1-155 encoding amino acid sequences may also
be
cloned and expressed as fusion proteins by a protocol similar to that
described directly above,
wherein the pET-32b(+) vector (Novagen, 601 Science Drive, Madison, WI 53711)
is
preferentially used in place of pQE 10.
The above methods are not limited to the polypeptide fragements actually
produced. The
above method, like the methods below, can be used to produce either full
length polypeptides or
desired fragements therof.
6(c). Alternative Expression and Purification of Borrelia polypeptides in
E. coli
The bacterial expression vector pQE60 is used for bacterial expression in this
example
(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311 ). However, in this
example, the
polypeptide coding sequence is inserted such that translation of the six His
codons is prevented
and, therefore, the polypeptide is produced with no 6 X His tag.
The DNA sequence encoding the desired portion of the B. bur~dnr~feri amino
acid
sequence is amplified from an B. burgdorferi genomic DNA prep the deposited
DNA clones
using PCR oligonucleotide primers which anneal to the 5' and 3' nucleotide
sequences
corresponding to the desired portion of the B. bur~dorferi polypeptides.
Additional nucleotides
containing restriction sites to facilitate cloning in the pQE60 vector are
added to the 5' and 3'
primer sequences.
For cloning a B. burgdorferi polypeptides of the present invention, 5' and 3'
primers are
selected to amplify their respective nucleotide coding sequences. One of
ordinary skill in the art
would appreciate that the point in the protein coding sequence where the 5'
and 3' primers begin
may be varied to amplify a DNA segment encoding any desired portion of a
polypeptide of the
present invention. The 3' and 5' primers contain appropriate restriction sites
followed by
nucleotides complementary to the 5' and 3' ends of the coding sequence
respectively. The 3'
primer is additionally designed to include an in-frame stop codon.
The amplified B. burgldorferi DNA fragments and the vector pQE60 are digested
with
restriction enzymes recognizing the sites in the primers and the digested DNAs
are then ligated
together. Insertion of the B. burgdorferi DNA into the restricted pQE60 vector
places the B.
burgdo~feri protein coding region including its associated stop codon
downstream from the
IPTG-inducible promoter and in-frame with an initiating AUG. The associated
stop codon
prevents translation of the six histidine codons downstream of the insertion
point.
The ligation mixture is transformed into competent E. coli cells using
standard procedures
3_S such as those described by Sambrook et al. E. coli strain M15/rep4,
containing multiple copies
of the plasmid pREP4, which expresses the lac repressor and confers kanamycin
resistance
("Kanr"), is used in carrying out the illustrative example described herein.
This strain, which is
only one of many that are suitable for expressing B. burkdorferi polypeptide,
is available
commercially (QIAGEN, Inc., supra). Transformants are identified by their
ability to grow on
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated
from resistant
colonies and the identity of the cloned DNA confirmed by restriction analysis,
PCR and DNA
sequencing.
Clones containing the desired constructs are grown overnight ("O/N") in liquid
culture in
5 LB media supplemented with both ampicillin (100 pg/ml) and kanamycin (25
pg/ml). The O/N
culture is used to inoculate a large culture, at a dilution of approximately
1:25 to 1:250. The cells
are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
isopropyl-b-D-
thiogalactopyranoside ("IPTG") is then added to a final concentration of 1 mM
to induce
transcription from the luc repressor sensitive promoter, by inactivating the
lacI repressor. Cells
10 subsequently are incubated further for 3 to 4 hours. Cells then are
harvested by centrifugation.
To purify the B. l7urgdorferi polypeptide, the cells are then stirred for 3-4
hours at 4°C in
6M guanidine-HCI, pH 8. The cell debris is removed by centrifugation, and the
supernatant
containing the B. burgdorferi polypeptide is dialyzed against 50 mM Na-acetate
buffer pH 6,
supplemented with 200 mM NaCI. Alternatively, the protein can be successfully
refolded by
15 dialyzing it against 500 mM NaCI, 20% glycerol, 25 mM Tris/HCl pH 7.4,
containing protease
inhibitors. After renaturation the protein can be purified by ion exchange,
hydrophobic
interaction and size exclusion chromatography. Alternatively, an affinity
chromatography step
such as an antibody column can be used to obtain pure B. burgdnrferi
polypeptide. The purified
protein is stored at 4° C or frozen at -80° C.
20 The following alternative method may be used to purify B. burgdorferi
polypeptides
expressed in E coli when it is present in the form of inclusion bodies. Unless
otherwise
specified, all of the following steps are conducted at 4-10°C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture is
cooled to 4-10°C and the cells are harvested by continuous
centrifugation at 15,000 rpm
25 (Heraeus Sepatech). On the basis of the expected yield of protein per unit
weight of cell paste
and the amount of purified protein required, an appropriate amount of cell
paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The
cells are
dispersed to a homogeneous suspension using a high shear mixer.
The cells ware then lysed by passing the solution through a microfluidizer
(Microfuidics,
30 Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then
mixed with NaCI
solution to a final concentration of 0.5 M NaCI, followed by centrifugation at
7000 x g for 1_5
min. The resultant pellet is washed again using O.SM NaCI, 100 mM Tris, ~0 mM
EDTA, pH
7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
35 hydrochloride (GuHCI) for 2-4 hours. After 7000 x g centrifugation for 15
min., the pellet is
discarded and the B. burgdorferi polypeptide-containing supernatant is
incubated at 4°C
overnight to allow further GuHCI extraction.
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
61
Following high speed centrifugation (30,000 x g) to remove insoluble
particles, the
GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract with
20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA by vigorous
stirring.
The refolded diluted protein solution is kept at 4°C without mixing for
12 hours prior to further
purification steps.
To clarify the refolded B. burgdo~feri polypeptide solution, a previously
prepared
tangential filtration unit equipped with 0.16 ~m membrane filter with
appropriate surface area
(e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed.
The filtered
sample is loaded onto a canon exchange resin (e.g., Poros HS-50, Perseptive
Biosystems). The
column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500
mM, 1000
mM, and 1500 mM NaCI in the same buffer, in a stepwise manner. The absorbance
at 280 mm
of the effluent is continuously monitored. Fractions are collected and further
analyzed by SDS-
PAGE.
Fractions containing the B. bur,~dor~eri polypeptide are then pooled and mixed
with 4
volumes of water. The diluted sample is then loaded onto a previously prepared
set of tandem
columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20,
Perceptive Biosystems) exchange resins. The columns are equilibrated with 40
mM sodium
acetate, pH 6Ø Both columns are washed with 40 mM sodium acetate, pH 6.0,
200 mM NaCI.
The CM-20 column is then eluted using a 10 column volume linear gradient
ranging from 0.2 M
NaCI, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH
6.5. Fractions
are collected under constant A~~~ monitoring of the effluent. Fractions
containing the B.
burgdorferi polypeptide (determined, for instance, by 16% SDS-PAGE) are then
pooled.
The resultant B. burgdorferi polypeptide exhibits greater than 95% purity
after the above
refolding and purification steps. No major contaminant bands are observed from
Commassie
blue stained 16% SDS-PAGE gel when 5 ~g of purified protein is loaded. The
purified protein
is also tested for endotoxin/LPS contanunation, and typically the LPS content
is less than 0.1
ng/ml according to LAL assays.
6(d). Cloning and Expression of B. burgdorferi in Other Bacteria
B. bur~dorferi polypeptides can also be produced in: B. burgdorferi using the
methods of
S. Skinner et al., ( 1988) Mol. Microbiol. 2:289-297 or J. I. Moreno ( 1996)
Protein Expr. Purif.
8(3):332-340; Lactobacillus using the methods of C. Rush et al., 1997 Appl.
Microbiol.
Biotechnol. 47(5):537-542; or in Bacillus subtilis using the methods Chang et
al., U.S. Patent
No. 4,952,508.
7. Cloning and Expression in COS Cells
A B. burgdor~jeri expression plasmid is made by cloning a portion of the DNA
encoding a
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
62
B. burgdorferi polypeptide into the expression vector pDNAI/Amp or pDNAIII
(which can be
obtained from Invitrogen, Inc.). The expression vector pDNAI/amp contains: ( 1
) an E. coli
origin of replication effective for propagation in E. coli and other
prokaryotic cells; (2) an
ampicillin resistance gene for selection of plasmid-containing prokaryotic
cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a CMV promoter,
a polylinker, an
SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an
"HA" tag to
facilitate purification) followed by a termination codon and polyadenylation
signal arranged so
that a DNA can be conveniently placed under expression control of the CMV
promoter and
operably linked to the SV40 intron and the polyadenylation signal by means of
restriction sites in
the polylinker. The HA tag corresponds to an epitope derived from the
influenza hemagglutinin
protein described by Wilson et al. 1984 Cell 37:767. The fusion of the HA tag
to the target
protein allows easy detection and recovery of the recombinant protein with an
antibody that
recognizes the HA epitope. pDNAIII contains, in addition, the selectable
neomycin marker.
A DNA fragment encoding a B. burgdorJeri polypeptide is cloned into the
polylinker
region of the vector so that recombinant protein expression is directed by the
CMV promoter.
The plasmid construction strategy is as follows. The DNA from a B. burgdorferi
genomic DNA
prep is amplified using primers that contain convenient restriction sites,
much as described above
for construction of vectors for expression of B. burgdorferi in E. coli. The
5' primer contains a
Kozak sequence, an AUG start codon, and nucleotides of the 5' coding region of
the B.
burgdorferi polypeptide. The 3' primer, contains nucleotides complementary to
the 3' coding
sequence of the B. burgdorferi DNA, a stop codon, and a convenient restriction
site.
The PCR amplified DNA fragment and the vector, pDNAI/Amp, are digested with
appropriate restriction enzymes and then ligated. The ligation mixture is
transformed into an
appropriate E. coli strain such as SURET"~ (Stratagene Cloning Systems, La
Jolla, CA 92037),
and the transformed culture is plated on ampicillin media plates which then
are incubated to allow
growth of ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and
examined by restriction analysis or other means for the presence of the
fragment encoding the B.
burgdorferi polypeptide
For expression of a recombinant B. burgdorferi polypeptide, COS cells are
transfected
with an expression vector, as described above, using DEAF-dextran, as
described, for instance,
by Sambrook et al. (supra). Cells are incubated under conditions for
expression of B.
burgdorferi by the vector.
Expression of the B. burgdorferi-HA fusion protein is detected by
radiolabeling and
immunoprecipitation, using methods described in, for example Harlow et al.,
supra.. To this
end, two days after transfection, the cells are labeled by incubation in media
containing 35S-
cysteine for 8 hours. The cells and the media are collected, and the cells are
washed and the
lysed with detergent-containing RIPA buffer: 150 mM NaCI, 1 % NP-40, 0.1 %
SDS, 1 % NP-
40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. (supra ).
Proteins are
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
63
precipitated from the cell lysate and from the culture media using an HA-
specific monoclonal
antibody. The precipitated proteins then are analyzed by SDS-PAGE and
autoradiography. An
expression product of the expected size is seen in the cell lysate, which is
not seen in negative
controls.
8. Cloning and Expression in CHO Cells
The vector pC4 is used for the expression of B. burgdorferi polypeptide in
this example.
Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.
37146). The
plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
Chinese
hamster ovary cells or other cells lacking dihydrofolate activity that are
transfected with these
plasmids can be selected by growing the cells in a selective medium (alpha
minus MEM, Life
Technologies) supplemented with the chemotherapeutic agent methotrexate. The
amplification of
the DHFR genes in cells resistant to methotrexate (MTX} has been well
documented. See, e.b~.,
Alt et al., 1978, J. Biol. Chem. 253:1357-1370: Hamlin et al., 1990, Biochem.
et Biophys.
Acta, 1097:107-143; Page et al., 1991, Biotechnology 9:64-68. Cells grown in
increasing
concentrations of MTX develop resistance to the drug by overproducing the
target enzyme,
DHFR, as a result of amplification of the DHFR gene. If a second gene is
linked to the DHFR
gene, it is usually co-amplified and over-expressed. It is known in the art
that this approach may
be used to develop cell lines carrying more than l ,000 copies of the
amplified gene(s).
Subsequently, when the methotrexate is withdrawn, cell lines are obtained
which contain the
amplified gene integrated into one or more chromosomes) of the host cell.
Plasmid pC4 contains the strong promoter of the long terminal repeat (LTR) of
the Rouse
Sarcoma Virus, for expressing a polypeptide of interest, Cullen, et al. (
1985} Mol. Cell. Biol.
5:438-447; plus a fragment isolated from the enhancer of the immediate early
gene of human
cytomegalovirus (CMV), Boshart, et al., 1985, Cell 41:521-530. Downstream of
the promoter
are the following single restriction enzyme cleavage sites that allow the
integration of the genes:
Bam HI, Xba I, and AsP 718. Behind these cloning sites the plasmid contains
the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high efficiency
promoters can also be
used for the expression, e.g., the human (3-actin promoter, the SV40 early or
late promoters or
the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
Clontech's Tet-Off and
Tet-On gene expression systems and similar systems can be used to express the
B. burgdorferi
polypeptide in a regulated way in mammalian cells (Gossen et al., 1992, Proc.
Natl. Acad. Sci.
USA 89:5547-5551. For the polyadenylation of the mRNA other signals, e.g.,
from the human
growth hormone or globin genes can be used as well. Stable cell lines carrying
a gene of interest
integrated into the chromosomes can also be selected upon co-transfection with
a selectable
marker such as gpt, 6418 or hygromycin. It is advantageous to use more than
one selectable
marker in the beginning, e.g., G418 plus methotrexate.
The plasmid pC4 is digested with the restriction enzymes and then
dephosphorylated
using calf intestinal phosphates by procedures known in the art. The vector is
then isolated from
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
64
a 1 % agarose gel. The DNA sequence encoding the B. burgdorferi polypeptide is
amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of
the desired
portion of the gene. A 5' primer containing a restriction site, a Kozak
sequence, an AUG start
codon, and nucleotides of the 5' coding region of the B. burgdorferi
polypeptide is synthesized
and used. A 3' primer, containing a restriction site, stop codon, and
nucleotides complementary
to the 3' coding sequence of the B. burgdorferi polypeptides is synthesized
and used. The
amplified fragment is digested with the restriction endonucleases and then
purified again on a 1 %
agarose gel. The isolated fragment and the dephosphorylated vector are then
ligated with T4
DNA ligase. E. coli HB 101 or XL-1 Blue cells are then transformed and
bacteria are identified
that contain the fragment inserted into plasmid pC4 using, for instance,
restriction enzyme
analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used for
transfection. Five
~g of the expression plasmid pC4 is cotransfected with 0.5 ~,g of the plasmid
pSVneo using a
lipid-mediated transfection agent such as LipofectinT"" or LipofectAMINE.T""
(LifeTechnologies
Gaithersburg, MD). The plasmid pSV2-neo contains a dominant selectable marker,
the neo gene
from Tn5 encoding an enzyme that confers resistance to a group of antibiotics
including 6418.
The cells are seeded in alpha minus MEM supplemented with 1 mg/ml 6418. After
2 days, the
cells are trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus
MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml 6418.
After about
10-14 days single clones are trypsinized and then seeded in 6-well petri
dishes or 10 ml flasks
using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM,
800 nM).
Clones growing at the highest concentrations of methotrexate are then
transferred to new 6-well
plates containing even higher concentrations of methotrexate ( 1 ~M, 2 ~M, 5
~tM, 10 mM, 20
mM). The same procedure is repeated until clones are obtained which grow at a
concentration of
100-200 ~M. Expression of the desired gene product is analyzed, for instance,
by SDS-PAGE
and Western blot or by reversed phase HPLC analysis.
The disclosure of all publications (including patents, patent applications,
journal articles,
laboratory manuals, books, or other documents) cited herein are hereby
incorporated by reference
in their entireties.
The present invention is not to be limited in scope by the specific
embodiments described
herein, which are intended as single illustrations of individual aspects of
the invention.
Functionally equivalent methods and components are within the scope of the
invention, in
addition to those shown and described herein and will become apparant to those
skilled in the art
from the foregoing description and accompanying drawings. Such modifications
are intended to
fall within the scope of the appended claims.
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
89
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
91
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
92
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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
93
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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
94
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
96
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
97
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
98
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
99
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
100
h h O _ - N M _
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
101
d' C~ t'~O ~ ~ ~ M N V
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SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
102
V7 ~1' N M ~ ~' ~ 00 00 N M
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W
a
m
a
SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
103
~o N o ~_ ~ ~t as
~ ~
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N
W
a
d
E
SUBSTITUTE SHEET (RULE 26)
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
104
TABLE 3.
Borrelia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
Contig ORF ID Start (nt)Stop (nt)
ID
1 1 2330 1134
1 2 3317 2934
1 8 11375 13021
1 9 11673 11386
1 10 12925 13629
1 I1 13538 14146
1 17 25212 24700
1 18 25782 25357
1 19 26115 25870
1 21 27308 27051
1 22 29628 30458
1 29 40696 41217
1 30 41201 41992
1 31 42542 41985
1 32 42593 42982
1 34 44234 44031
1 38 48041 47079
1 41 49318 49617
1 43 53234 51810
1 50 59737 58208
1 58 68227 67733
1 65 79757 80404
1 66 81516 80401
1 7S 89552 88353
1 82 93338 92766
1 85 95207 95854
1 104 108788 108621
1 105 109764 108943
1 108 112003 111599
1 113 114317 115846
1 114 114522 114316
I 119 118439 118927
I 121 119802 119599
1 125 125688 123967
1 129 128594 129235
1 135 136116 135259
1 136 136558 136298
1 139 139149 139559
1 141 140573 140121
1 143 141738 141412
1 145 142218 142060
I 146 142686 142342
1 154 150528 149074
I 158 153832 153981
i 163 158277 158474
CA 02304925 1999-12-17
WU 98/58943 PCT/US98/12764
105
Borrelia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
1 171 168052 166205
1 176 _ 171038
171592
~
1 186 179607 180089
1 189 182345 182046
1 191 182567 182773
1 199 192561 192716
1 205 196592 197476
1 218 207717 206752
1 219 207733 208437
1 221 209337 208915
1 222 209712 209335
1 231 217179 216025
1 238 223660 223418
1 240 224720 225724
1 242 227006 227275
1 248 231761 231501
1 251 232973 233308
1 252 233669 234004
1 254 235115 235456
1 258 241824 242198
1 261 248009 247773
1 269 256846 255872
1 276 265430 265158
1 279 266582 266298
1 281 268474 268280
1 286 274157 274384
1 292 280495 280274
1 294 281344 281042
1 298 287276 285714
1 303 292943 292644
1 304 293273 293037
1 30S 294965 294648
1 308 299427 298699
1 309 299051 - 299212
1 326 320375 319785
1 327 320425 321036
1 331 324198 324413
1 339 332785 332459
1 341 333503 334138
1 342 334116 334739
1 343 334880 335446
1 350 342916 342443
1 351 344789 342897
1 363 357596 356931
1 367 361065 360859
1 370 362519 362196
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
106
Borrclia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
1 374 366905 366114
1 377 368632 369537
1 378 369928 370560
1 379 370532 371353
1 382 375028 373193
1 383 375102 375542
1 387 378677 378198
1 400 394952 394722
1 401 396247 394937
1 403 397569 398327
1 406 399103 399294
1 436 416160 416570
1 445 424660 423950
1 446 425181 424642
1 450 428559 428200
1 451 428933 428619
1 455 432590 431628
1 461 437823 438092
1 463 438690 438313
1 466 440749 440222
1 470 441568 441350
1 471 442039 441614
1 472 442216 442037
1 473 442666 442262
1 476 445202 445017
1 493 462106 462519
1 494 462893 462549
1 504 482111 481035
1 505 481552 481800
1 509 483249 483668
1 512 484864 485157
1 516 489171 488527
1 519 492989 492375
1 520 493626 492997
1 521 494169 494864
1 524 497185 497385
1 525 497674 499254
1 527 500251 501294
1 528 501281 502156
1 558 533912 533667
1 568 541267 541491
1 571 544436 544257
1 572 544565 545068
1 578 549603 551198
1 580 551508 551657
1~ 581 552337 551513
CA 02304925 1999-12-17
WO 98/58943 PCT1US98/12764
107
Borrelia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
1 585 556051 557271
1 590 ~~~ 561342561139
1 591 561825 561520
1 592 562536 563360
i 596 565758 566519
1 599 568389 568682
1 602 568680 568856
1 605 570829 571167
1 609 576170 577093
1 612 581549 581091
1 614 582910 584013
1 619 589384 588674
1 624 592665 ---593465
1 626 594542 595405
1 672 631642 632175
1 677 636650 636892
1 678 637059 638078
1 681 640861 640412
1 686 644887 645207
1 689 649716 649961
1 690 650436 650735
1 691 650733 651056
1 693 653303 653689
1 705 664733 664918
i 707 665979 666770
i 718 679155 678391
1 721 680664 681047
1 722 681523 - 681849
1 724 681809 682171
1 727 682853 - - 683272
1 734 687648 688067
1 739 691613 --- 692290
1 751 707290 -- - - --X07718
1 763 719197 718904
1 764 720030 719257
1 769 722198 722482
1 783 733736 - 734647
1 785 735554 -736618
1 787 737124 -- 739184
1 792 742924 744801
1 799 753128 752655
1 811 766129 765980
1 812 766438 767772
1 815 770062 -- 769790
1 818 771890 - 772282
1 ~ 831 ~ 7882191 --. 788836
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
108
Borrelia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
l 832 788824 789615
1 838 793566 793414
1 840 794295 794119
1 844 796774 796586
1 852 803096 802908
1 858 809371 809970
1 864 816108 816497
1 865 816672 817283
1 866 817281 817838
1 872 823841 824836
1 876 828191 828739
1 877 828749 829147
1 879 831328 831714
1 880 831698 833005
1 885 836201 835677
1 890 841171 840590
1 891 840594 840860
1 899 849453 850148
1 902 851608 852687
1 918 862867 863109
1 920 864292 864705
1 923 865660 865346
1 925 868212 869273
1 928 871012 872580
1 933 878576 879166
1 939 884338 883268
1 940 884999 884325
1 949 892388 892924
1 957 900141 899296
1 958 900534 900139
1 959 901526 900510
1 962 902383 903258
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
109
0oOw0 v~ t~-~7-~n W O ~ --v0 N t~ O t1- M O
w,, O~O~I'~00 ~O I~-I~ '~ I~~D ~O~ I~ ~O ~D M I~~D
"fl
O O'ctN o0 0o v0 ~'~v~tt .--~.-. O O O O O
.
O OO\O~ 00 00 CO 00 00O~J 0000 00 00 00 00 0000
3
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pp
> . U O p O O bU U bO O by
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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
147
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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
148
O O I~ ~ M ~ ~ M
N t~ N MN ~ M ~ M M M N M N N
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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
149
M M M N ~..
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CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
TABLE 6. 150
Borrclia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
Contig ID OKF ID Start (nt) Stop (nt)
2 4 2730 3554
2 5 3559 3410
2 7 5464 ~ 3869
2 13 10502 9999
2 17 13800 13576
2 19 15368 15204
2 28 21155 21400
2 50 41944 42186
2 58 53786 52911
2 59 54816 53773
61 57393 55813
2 63 57882 57682
2 65 60898 60203
2 66 61441 62070
67 62078 62692
2 70 65896 66540
2 74 70203 69910
2 78 71818 71399
80 72956 74032
2 81 73515 73267
2 90 92181 92525
2 91 92968 92555
2 108 109872 110057
2 112 112408 112812
2 113 112858 113037
2 114 113035 113460
2 115 113506 113724
2 119 114325 114852
3 6 3279 4079
3 8 5156 6019
3 54 42256 42789
3 59 47264 47506
3 60 47673 48692
3 63 51475 51026
3 70 60330 60575
3 71 61050 61349
3 72 61347 61670
3 74 63917 64303
3 86 75347 75532
3 88 76593 77384
3 99 89769 89005
3 102 91278 91661
3 103 92137 92463
3 105 92423 92785
3 108 93467 93886
3I 115 98262 98681
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
151
Borrelia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
3 121 102227 102904
3 126 111308 110055
4 6 3751 4179
4 7 4218 5042
4 19 16115 15516
4 20 17028 16075
4 21 17379 17092
4 22 17735 17397
4 24 19243 18785
4 25 18942 19196
4 26 20677 19259
4 27 19431 19751
4 29 21376 20876
4 30 21899 21423
4 31 22918 21845
4 33 23951 23553
4 37 26253 25627
4 38 26991 26332
4 39 28181 26931
4 40 29175 28522
4 43 30605 30342
4 45 34906 33548
4 48 35750 35932
3 2102 1527
5 5 2656 2393
5 7 3460 2900
5 10 6544 5645
5 40 25278 24322
5 41 25235 25600
5 42 25665 25276
5 44 25881 25663
5 47 27883 27410
5 48 28351 27881
5 49 29028 28324
5 50 29454 29026
5 56 32199 31666
5 57 32571 32200
5 58 32826 32569
5 60 32913 33245
5 61 33766 33575
5 62 34173 33742
5 64 35514 34861
6 2 954 1181
6 3 1590 1763
6 5 3400 3954
6 7 4691 5218
L. _ __ 8 5187 5699
6T
CA 02304925 1999-12-17
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152
Borcelia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
6 11 6498 5983
6 12 6975 6727
6 14 7978 7448
6 15 8479 7976
6 22 15106 15636
6 27 19999 18842
6 28 20036 20668
6 29 21814 20690
6 30 20949 21269
6 35 24136 23630
6 37 25697 26248
7 8 8100 7792
7 10 8145 8288
7 11 9374 8517
7 12 9771 9325
7 13 9652 10185
7 14 10163 9765
7 15 10517 10173
7 16 11363 10524
7 17 11904 11392
7 18 12495 11902
7 19 13516 12473
7 20 12807 13154
7 22 15149 14697
7 24 15855 15046
7 25 15503 15826
7 26 16638 15853
7 27 19344 16636
7 31 19473 19727
7 32 20067 19675
7 33 20762 20049
7 34 21136 20738
7 36 22975 23406
7 40 26667 25870
8 3 2907 4118
8 5 5898 6059
8 6 7399 8313
8 13 15645 15899
8 14 17281 16331
8 15 16905 17111
4 3211 3684
10 6 3857 4456
10 8 5982 5599
10 11 8038 7802
10 14 10255 10100
11 7 5688 5828
11 ( 9 7248 7685
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153
Borrelia burgdorfcri - Putative coding regions of novel proteins not similar
to know proteins
11 10 7672 8028
11 13 _ 10154
9642
12 1 101 370
12 2 982 680
12 3 1390 1115
12 4 1528 1388
12 5 1913 1431
12 11 7308 6616
14 2 3588 3328
14 4 4657 4815
14 9 7981 8511
15 1 1 327
15 2 325 1077
15 3 1478 657
15 4 2360 1758
15 5 2839 2507
15 9 3922 3743
15 10 4145 3900
1S 11 4112 4270
15 13 7677 6127
15 14 7852 7709
1S 15 8052 7825
15 16 8222 7857
16 2 1733 1936
16 3 1905 2063
16 6 5212 4220
16 7 8903 8505
17 2 1500 1709
17 5 4097 4660
17 7 6344 6189
18 1 1635 - 2465
18 2 2509 3306
18 3 3332 4390
18 5 4933 4727
18 7 6353 7084
18 8 7098 7625
20 7 4700 4557
22 4 2175 1228
22 5 2132 2314
22 6 2829 2173
22 8 3254 3601
22 9 4408 4169
22 10 4875 4402
22 11 5343 4873
23 2 2283 1537
23 3 3564 2617
25 6 3677 4147
CA 02304925 1999-12-17
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154
Borrelia burgdortveri - Putative coding regions of novel proteins not similar
to know proteins
26 7 4251 3889
28 2 732 1739
29 3 310 885
31 1 28 195
32 3 935 1603
32 4 1637 2332
37 2 1379 1059
42 4 2708 2388
44 2 1734 1159
44 4 2942 2532
47 4 2336 2115
50 1 908 120
52 4 674 501
56 1 152 1465
56 2 611 459
56 3 1479 2150
58 3 1691 1329
58 5 1867 2046
59 2 2018 1044
61 1 1 657
61 3 1389 1907
62 4 1115 1345
63 1 663 325
63 2 769 446
63 3 1759 1013
65 1 472 903
65 2 901 1236
67 1 387 4
67 2 979 401
67 3 1482 961
68 2 451 612
69 3 840 574
71 1 363 4
72 1 586 933
73 1 300 4
73 2 824 279
73 3 1396 1145
79 1 22 1119
82 1 701 303
82 2 1188 775
84 1 331 134
84 2 983 348
87 1 277 2
87 2 1136 267
96 1 434 57
96 2 748 557
97 2 976 659
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
I55
Borrelia burgdorferi - Putative coding regions of novel proteins not similar
to know proteins
103 1 _ 301 2
103 2 886 299
105 1 36 509
106 1 425 3
106 3 761 600
112 1 416 799
113 1 685 59
118 1 1 489
118 2 487 753
120 2 299 691
124 1 1 630
127 1 702 322
135 1 287 3
135 2 649 407
136 1 1 645
140 2 619 332
145 1 1 480
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
(1) GENERAL INFORMATION:
156
(i) APPLICANT: Human Genome Sciences, Inc. et al.
(ii) TITLE OF INVENTION: Borrelia burgdorferi Polynucleotides and
Sequences
(iii) NUMBER OF SEQUENCES: 155
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Human Genome Sciences, Inc.
(B) STREET: 9410 Key West Avenue
(C) CITY: Rockville
(D) STATE: Maryland
(E) COUNTRY: USA
(F) ZIP: 20850
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.50 inch, l.4Mb storage
(B) COMPUTER: HP Vectra 486/33
(C) OPERATING SYSTEM: MSDOS version 6.2
(D) SOFTWARE: ASCII Text
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: Herewith
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
157
(A) NAME: Brookes, A. Anders
(B) REGISTRATION NUMBER: 36,373
(C) REFERENCE/DOCKET NUMBER: PB370PCT
(vi) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (301) 309-8504
(B) TELEFAX: (301) 309-8512
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 910715 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE SEQ ID 1:
DESCRIPTION: NO:
ATATAATTTTTAATTAGTATAGAATATGTTAAACTTTACCCTTGAATTTTTCTACTCTAT 60
TTGTATATTCTATAGAAAAAACGATTAGAATTAAACAAAGCCATAACTGAACCAACGGTA 120
ATTAGTAGATAAAGGGATCAAAATATTTTTTATTGCAGCAAGAATACCTTGGTATATTAG 180
AAAAACCAAAAGTCATAGTCAAATCATCTTTTGATAACAATCCCCAAATCTATAATTTAT 240
TATGAAATTAATTGCTCCCTTGAAAAGATTAGTTTTTAAAACTACAAGACTACTATCAAT 300
CACTATCAGATAGATTAAAACAACCTTTACAAGAAAAAAATCTTACTACTATTTTATTGT 360
AAATGTATTATAAAATAAGTTCATGCAAAAACTTACAATTTTTCACAACAAACTACAATA 420
AAATCATGTAAACAAACAATTTCTTTGAAAATTAAGCAAATTTATAAATATAAATTATAA 480
AGATATATATTTTTATATGATCAATAATAAAAATTAATAGGATACTTATTTGGAAAAATT 540
ATTGAAAAAACAATAAGCATGAATTGCCACAATAAGCTAATTGTCACTTAATAATTCTTG 600
TTTACTAGACCACATTAGTATAAACTCAAATATTGGCTACTATAATATAGGGGCTTTATA 660
CGCCACATGTTTAATGATAACATAAGAAAATATTGCAATAATAAAAAGATTGAAATATCT 720
TTATTAGAAAAGAATCTCGATAATTTAGAAAACAGAATAAAAATCATAACTAATAAATAT 780
AACGTTGAAAAAAATATATTCAAACTTTAACTATACAATTAATTACACCTTAAAAATGCG 840
TTACATAAAAATTAAGGACTACTATAAATAGAAAACACCACATAACCTACAGACTCTAAA 900
GGAATAATTAAATCCTCATATTTCAGTTCTCCAAAAGTTTAAATAGGGGCCTTTTACTTT 960
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158
TCTTGATTAGCATATACATTATTAAAGGCATCTTCTTGGGCACTATCCTAAACTTTTTTA 1020
CATTATTATTATTTTATTCTTTATTATTACAAGATAATTCAAGAATCTAGATTACAAGAT 1080
ATCAATCCTGCCATTAGTAGTTCAATAAAACATTTAGAATATTTATACATTATTTAATGT 1140
ATTTTTTTCATTTTTGAAATAATATTGTTATAACTTAACTTAATAAGATATTTGATTTCT 1200
TCAACTTGAGAATCCGATGTACATAGAATCTGAACATCTCCTCTGCCCCATTTGCCAATA 1260
TTCTTAATATATCTAGTAAAACCCTCTTTTAAAATTATTTGATCTAGAGCAACAGTAATA 1320
GTAATATTAATTTTATTTACCCCAGGTCTAAAGCTAAAATCTACAAAATATCCGCCCTGT 1380
ACTTTAAATCCTGTATAGCACTGTGTTTCAACTTTCTCAATTTCATTAAAATTTAAAACA 1440
AAAATAAAATCTTCTAATTCTTTATATATTGCTTTCATATCGGAATTTAATTTTTCAAAT 1500
TTTTTTAAATTTTCGGTTTTAATATTATTATCTTTTATACCAGAATCTGTGTCATCTTCT 1560
ATGTCACTTTTCTTGCTGTTTACTAATACATCGCTTTTTTTTTCATCAAAAAACATACTA 1620
AAAATATTTTTAATAATATCATTAAATATTTTATCTGAATATGTTTTTTTAAAACCAATT 1680
TTAGCTTTAAAAAAATCAAGCAAATCAACACTTGGATTTTTTGTTTCCTTTTTTAAATAA 1740
GCTGAAAATTTGTCTGTATATTTTTTTTCTAATGCAAAAGATCTAGCCTCTTCAACATTC 1800
AAAGAATTTCTAGAAAACTTTTTAAGATATTCAAAATCCTTAGATGTTAATTTTTCTAAA 1860
TTAACAACCATAAAAGGCTCATTGTCTAACAAATTATCTTTATCTAGGTCAGTATAGAAT 1920
CTATATTCTATGCCATCTGTTAATATACCAAATTCAACTCTCTTTGCTTGAGAACGAATA 1980
TTTTCAAAATAAGGTTTTAATTGCTTTAGATGATTTTCAAGCTTTTCCCTGCTATTATGA 2040
TATTTGGCCTCTATTAAAATAGTGGGTTCTTCATCCTTTTTTGTTGGATAAATAACATAA 2100
TCAACCCTTTTTAGTCCATCTTTAAGAATATCTGCCTTCTCTTCAACTTTAACAATTGAA 2160
ATATCAGTATGATCATAGCCCATCGCATCTAAAAATGGATCAATAAGATTTTGTCTTGTT 2220
TGTGCTTCATTTTCAATAAGATCCTTATCCTTTTGAATTTTTCTACTTACAGCTTTTATT 2280
GAATTTTCAAAATTTATATCTTTGTATTCATTTGGCATAATTATATTTTACCAATAAAAT 2340
TAAAAATTAATAATTCTAAAAATAAATTTCCAAAATGTTGTCTATTTTAAACTCTTAACT 2400
GATACCTTAATTCTTTTTTCTACCTAATTTTTTAGTTTAAAATCTTATTTTTTAATTTTA 2460
TTATTTTTTCCTTACCTTATTTATACTAAAATTTTTAGTATTTAGCGAATAATTTTCATA 2520
TCCTTTTATTAAAGACAAAATATGATTTTCTCTTTTTTGTTTTTTAATACCTTAAAATCA 2580
CTAAGCAAAGTAATAAAGTCTTCTTTGGTTAATGAATAAAAGACTAGCTATAATAAAATT 2640
ATTTTATTTTTCTTTACTAAATTCAAAATGCTCTAAATAAAGCAAATTAGAGAAATTCAA 2700
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AGGATCATTTTTAGCTATTAGCAGAGAAGTGTTTTT'PACCAAAGTTAGACATAATGAACT 2760
AGCCAAAATTTCTTCTTTGGGTTGAGGCATTGGACATTGACAAAGAAATGATTTTACAAT 2820
GTCGGTATTTTAAAACAAATCTTCTAATCATAAAATCAAATACAGTGCATTGAAAATAGA 2880
TATAATAAACAATTTTTTATAAAAAGATATTGGTATTTTCTCACAATTCATATCTATTTT 2940
ATAGAAACACAATAATAATTTTTAGGAGATAAAGTGCTAATCATGGTTCTTTCATTTGTA 3000
TTGCTTGCAATTCTTCTATAAAATATTCTTTCATTTGGGTACTGATCATCTTTAGTTAAG 3060
ATTTTTTCTAAATCTTCTTTATATCCTATCCATAAAAGCTTATAACCTTCTTTTACATAA 3120
TCATAAGTAAAAAATCTTAAATTAAATTGATAGATATTAGCCCCAGAATAAAGAAATATA 3180
AAGTTTTCATTATTATATTCCTTTAATAAAGATTTGCGATTCTTTATACTTGGATCTGGC 3240
CCTTTTTTAAAATTAATATCTTCTTTACTAAGAATACTAAATGAACTAAATATTTTGTTT 3300
AATTTGGCCCATGTTTAATTCAATTCCTTTATAAGGATTTTCTTTGCAGTCTTTTAAGTC 3360
TCTAGTTATTCCTTAATAATATTATCACTACTTTGAATAACAAATTTTGCTTTAAAATTT 3420
AATGTAAAAGTTTATTACTACGAGGAAATATCGCAAATTTAAAACTTGAATGCATATCTT 3480
AAAACCTTTTTTTGTTTTCAAACTGATAAATAAGTTAAGTTTATAATTACTAAATATATG 3540
CTTTCTTAGCAAGCTAAGACCAAATATCACAATAGAAGTAATTCTCAATAAACAAAATAC 3600
AAAAAGTAGTTATCATATCGTCTTTAACCTTAAATAAGGTTGCTATAAACAACCAAGATA 3660
TTTAATTTCTTTTAAAACCCTTATTCAATCTTTTTAAGCATAGGATCTTATAATTATAAG 3720
AATATAATTTTATTTACATCTCTATATTAATAGAAAGATGCAAATATGTGATCAAATTGT 3780
TATTTTTGTAATATGGAATAGTCCTTTATAGGGACGCTTAATGCTCTATACTTAAGATTG 3840
GAATTCTCTATGAAAATATATACTCGCTACCCATGTAAAGCTGACTTATTTTAGCACGTA 3900
TCGCTTAAACAATTATATTTATATTATCTTTTATAAAGTTAATTTTTTCTTGTAGATTAT 3960
TTTTTAATAAAAAAGGCACAAATTACCACAACAAGTTCCAGTATAAATTAATAGTTCTTA 4020
TCTCAACACTAAAGTACATAAACATCAAATATCAAAAATATATAAGAACAACATACTACA 4080
TTGTTTTAATGAAAACCTTAAAAGGAATGGTTAAACTCTCATTAAGCTAAAACCAATGCA 4140
AAAATATCTTTATAAATTAGCAAAAGAACTAAAAGTCACAAACAACTACCATAAAAATTT 4200
GGTAGTAAATTCTGGAACTGAAATTTACTATAAACTCAATTATTCTAAAAAAAATATTGC 4260
CTTAAATTAAAGAATGCCTTAAAAAAACAAAATGCTCTGATTTAAACCTATACCCAAAAT 4320
ACAAATTTACTAAAGAAGAAGATATAGATTTAGAGAAGATCTTAATAATAAAAATATTAA 4380
TATAAAAGTTGCTCAGTATGCTAAAGGCAAAGAGTTTAAGTCAAGTTTAGAAATTACAAA 4440
GAGTAAAACTATAAACTTCCTTTAAGAATGAAAATTTATTTTTATACTTACTTGGCTTAA 4500
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TATTAAGATTTTTTTATTCTTTTCATAATAATCTCTTCTATCACTTAACATTTTGCTATA 4560
CAAAAATCTTACACATCTAAATACTTTTTAAAAAAATTTGATTAGTGTTAGAATATATTC 4620
TATATTTATAAACTTTATTAGCACTCATAATTTTACTAAATTAATATATTATATTTAATT 4680
TATTTTTAAAATTTATCTCCATTTACCAAAAAAACTAAAATAAAACTCTCCAAACTTATA 4740
AATAAAAAAATAAGGCAAAACCCCAACAAACTCAAGATCTATAATACAAAAATACAATAT 4800
AAGAATCCCAAGCTTAAAAACAACCCCCTAAAATCTTTTTTTATTGGCGTTTTTAAATAA 4860
TGGTAATAAAGAATTCCAATCAACACGATCCCCCCTACAACTTTTCAAACCCTATAGCTT 4920
GGCTTTTTATATTATTTTTAAATTTACATGTCACAACAATAGATAATGCATAAAATAAGT 4980
ATTAATAAAACAAATACATTTATAGAACCTATACAATTATTGAGCATATGGCTAGTACTA 5040
AAAATGAAAATGTACAAGATAATATGCTATTAATAAAAATTAATGGCTACTAAAACTTTT 5100
GAATCCACATTTTTTCTTTAAAAAAATTCTAAATTATTAAAATAAATAGAAATTAAAATT 5160
ACCAAAAATATTATTATAGTAATAAATATGTAAAGCTATTTTTATTAAAACTGATAATAA 5220
AAATATAATAGCTAAAATAACATAAATTAACTTTAAATTATATCAAAGACTTAGATTTAA 5280
AATATT.TAATAAAAGGCAAAGCTATAAACACCATATACTTATTTTATTATTTTTTTCATT 5340
TTATTTAAATTAATTTAAATAAGACTCAATCAAATAATCAATCAAACATATTGGGTGAAG 5400
AAAAAATAGGGTATTCTTGGTGAATCGTTTTAAAAGGGGGTATAGTAAGCTAAP~AAACTC5460
TTATTAAAGAGGATGTTTATAGACTTAAAAGTCTAATTCAATATGAAAGAGGCTTTTTAA 5520
AGCTAAAAATGTTAAAGAAAATCAAATTAAGCAACAAGATGGTTTTGTTTCTATAAATAG 5580
TTTTAAAGAATATATACATTTGCACATACCCTTCATTATAACATCTACTAATTACACAAT 5640
AAA.AATAAAAATGATTTATTAAGAATTATTAGTAACTTATAAAAACTTTATAAGTTACAT 5700
AGTCAAAAATATAAAAAPTTAAAACAAAAAATTAACGATATGGAAAAATTGTATTTTATA 5760
GAAATAGAAATATATTTGCATTAAACAACTATGAATTTATAAAGATTCTAGTAGGAGAGA 5820
AAATATGAAAAAA.AAAAATTTATCAATTTACATGATAATGCTAATAAGTTTATTATCATG 5880
TAATACAAGTGACCCCAATGAATTAACTCGTAAAAAAATGCAAGACAAGAACGTGAAAAT 5940
TTTAGGATTTTTAGAGAAAATTCAAGCAGATAATAAAGAAATTGTTGAAAAACATATAGA 6000
AAAAAAAGAAAAACAAATGGTGCAGGCTGCTTCTGTAGCACCTATTAATGTAGAGAGTAA 6060
TTTCCCATATTATCTTCAAGAAGAAATAGAGATAAAAGAAGAAGAGTTGGTTCCAAATAC 6220
TGATGAAGAAAAGAAGGCAGAGAAGGCAATTAGCGATGGGAGTCTTGAATTTGCTAAATT 6180
AGTTGATGATGAAAATAAACTTAAAAATGAATCTGCGCAATTAGAATCTAGTTTTAATAA 6240
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TGTTTATAAA GAAATCTTAGAACTTGCAGATTTAATACAAGCAGAGGTGCATGTTGCAGG 6300
AAGGATAAAT AGCTATATAAAAAAAAGAAAGACCACTAAAGAAAAAGAATATAAGAAGAG 6360
AGAAATTAAG AATAAGATAGAAAAACAGGCTCTAATTAAGTTGTTCAATCAGTTATTAGA 6420
AAA.AAGAGGC GATATTGAAAATCTTCATACTCAATTAAATAGTGGACTTAGCGAGAGAGC 6480
ATCTGCAAAA TACTTTTTTGAGAAAGCCAAAGAAACTTTAAAAGCTGCTATTACTGAAAG 6540
ATTAAATAAC AAACGTAAAAATCGGCCATGGTGGGCAAGAAGAACACATAGTAATTTAGC 6600
AATACAGGCA AAAAATGAGGCAGAGGATGCTTTAAACCAATTAAGTACTTCTTCTTTTAG 6660
GATACTTGAA GCAATGAAAATAAAGGAAGATGTAAAACAGCTTCTTGAAGAAGTAAAATC 6720
TTTTCTAGAT TCTTCAAAGAGCAAAATCTTTTCTAGTGGCGATAGATTATATGATTTTTT 6780
AGAGACGAGT AAATAAAAAAATATAT'PTTAAAGGCTAATAACTTAAAATCAAAGTCTTCT 6840
GTTAAAGGAA GACTTTTTTATAATTTTATTTAAATAACGAAAAGCTTGATAGTTAAAAAA 6900
TCTTTTTTAT TAAAAATATGTTTACTAAACAGAGC.'TCAAAAATGACTATATTTAGTATCT 6960
CTATAAAAGA ATTTTTCAATATTTTAAAAAATTTATAGATAAACATAATCTAAAACCATG 7020
CATTAATACA AACCTAAAACATACTTGGTCACTTGTAAAAGTAAATTGTATCTAACTTTT 7080
TTTATTTATT GAATATACGTAAAAATTCTTTATAATTTCTATTTTAAAACGCTGCTATTT 7140
AGCAATACAA TAAAAGGCATTACAGATTGCAATCAAACAAACTAAAGTTTAAATAAAATA 7200
TTACCCTCTG TTCTAATCCTATCAAACAAGGTAATAAATTCTTTAAATTTCTAAAAGCCT 7260
AAACTTTAAA AGAACTTGTCGAAAATAATATTTCTCTTAAAAAAGGTTCTAATCTTTTAT 7320
TTATAAGAAC TTTTATACTATTATAAAAATGTATCTTGCCTTGATATATTTGTATTCTTT 7380
ATAAATCAAG CCTTCTACTTTTTTTAAGAATATTTCTATTTTTTATAAACTAGTTTTCTA 7440
CAATAGAAAA GAAATAACCCAAAGCCCTAAAAACTTAAATAAATGTTAGCTATAATAACT 7500
AAAATAGAGA TAAAAAACTCAATCATAAATAATGGTAAAACAAACTTAAACCACGTACCA 7560
TAACTCAATC TGGATATCCCCAATACAGCCATTATAACTCCGCTGGTAGGTGTTATCAAA 7620
TTAATAAGCC CAGATGCAGTCTGCATGGCAATAACAACTGAAGCTCTTGGAATTGACAAA 7680
AAATCGGCAA GAGGAGCCATTATTGGC'ATAGTGAGACTAGCATGTCCTGATGAAGATGGA 7740
ACAACAAATC CTATAAATATTTGAATAATTTCATTCAATATGATAAAAAGGGGTCTTGGA 7800
AGATTGTATA AAAAATTAGTAGCAGCATTTAACATAGTATCTGTAATCAACCCATCATCA 7860
CATACTATCA TAACACCTCTAGCAAGTCCAATAACAAGAGCAGCGGTTAGCAGACTTTCA 7920
GAACCTTTCA CAAACGCATCCCACATTTCAGTTTCACCTAATTTACAAATAAAAGCCGAT 7980
ATAATAGCAA CTCCAAGATACAACATTGTCATTTCTTGCATCCACCAACCAAGATTAACA 8040
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ATGCTAAATATCAAAATCAATATCATAAATCCAAATAAAAGTAAAACTAATTTATGAGCA 8100
AAAGTAAACTCAAGAGCATTCTGAGCATTATCTCCGGTAGAAAGTCCATCTTTTTTAACA 8160
AAATATTGATAATGTTCATCTTTTTGAGAATACACAAGCGATTTTGAGGGATCCTTTTTA 8220
ATTTTAGACGCATAAACACAAACATAGGTTATAGCAGCCAATACTGATACAAAATAAAGA 8280
ACAATTCTAAAATAAAATCCATCCTGCAAGCTAATAGAAGCTATTGCAGATGCAATTCCT 8340
GTCGCAAATGGATTTACAGTAGAAGCCATAGTTCCCACTCCAGCTCCTAAAGCAATAATA 8400
GCCGCTCCAACAAGACTATCATAACCCAAAGCTACTATCAAGGGAATCATAACAAAATAA 8460
AAAGGAAGGGTCTCTTCACTCATTCCGGTTACAGTTCCACCAATTGAAAAAATAAACATT 8520
AACAAAGGAATAAGCAACTTATCTTTGTGCCCCAACTTCTTGATTAAAAAATAAATTCCC 8580
ACATCTATTGCTCCAGTTTTCATAATAATCCCATAAGCACCCCCAACAATTAAAACAAAA 8640
ACAATAACTTCAACTGCATGTTCCATCCCCTTTGACATTGCGGTTAAAATAGTCATAATA 8700
GGATGTAAAAATCCCCTAGAGCCTCGATCTACATATTGATAAGTTCCAGCAACAATTATT 8760
TCCCTTTTAGATCCATCACCCATTTGCTTAAATTCTTTATCAAACTTACCGGCAGGAATC 8820
ACATACGTTAAAATGGTAACAAATACAATTAAAGAAAATATTATTGTAAAACTACTTGGC 8880
ATTTTGATCATAACGTTTCTCCTAAATAATTTCATAAATTTAATTTCACATAAAAAATAC 8940
TGTTATCCCAAAGTTGATACCATAATAGCTTTAATGGTATGCACCCTATTTTCAGCCACA 9000
TCAAAAACAACTGAATTTTTACTTTCAAAAATTTCTTCTGTAACTTCAATTCCATCAAGT 9060
CCGTATTTATCAAAAATATCCTTACCAATCACAGTGTTTAAGTCATGAAAAGCAGGCAAG 9120
CAATGCATAAATATTGCATCATCTTTTGCCATGCACATTATCTCTTTATTAACCTGATAA 9180
GCCTTTAGAAGATTTATTCTATCTTCCCAATTACTCTCCCCCATAGATACCCACACGTCT 9240
GTATACACAACATCAGCACATTTAACAGCCTCTTCTTTAGAATCTGTAATTGTAATTTTA 9300
CCCCCACTCTCTAGGGCTAAAGACCTAGCCTTAAGCGTCAAATCGGGGTCTGGAAAAAGC 9360
TCTTTGGGAGCAAAAATTCTAAAATCAAGCCCCATAATAGCACAGCCTTTCAATAAAGAA 9420
TTAGCAACATTCCCCCTACCATCGCCACAAAACACTATTTTAATCCCTTTCAAACTCCCC 9480
TTATGTTCTTTTATTGTCATTAAATCGGCTAGTATTTGGGTTGGGTGAGAAATATCTGTC 9540
AATCCATTGTAAACAGGAACATTAGAATAATTCGCCAAACATTCAACAGTCTGTTGAGAA 9600
AAGCCTCTAAATCCAATAGCATCATACATGCGTCCCAAAACTCTAGCGGTATCTATCATA 9660
GACTCTTTTGAGCCCATTTGATTACCCTTAGATCCCAAATAAGTAATATTTGCCCCTTGA 9720
TCATAGGCTGCGATCTCAAAAGCACACCGGGTCCTTGTTGAATCTTTCTCGAAAATTATA 9780
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ACTATATTTT TACCTTTAAGTTTTTGCACTTCAATTCCTGCATATTTTGACTTTTTTAAA9840
TTAATCGATA AATCAAGTAAATATTTAATATCTTTGCTTGTAAAATCTAAAAGATTTAAA9900
AAGCTCCTAT TTCGTAAATTATACATCAACCACCAACCTTTACAATCAAGTTTTTAAAAA9960
CTCATTTAAC TCATGCTTAAACATGCTTAAATATTAAATATCCTCTCTTACTAAAGACAT10020
AGACATGCAT CTTGGCCCACCACGACCCCTTGAAAGCTCGCTAGACGGAATTCTGTGAAC10080
TTTAATACCA TTTTCTTCAAACAGCTTATTAGTTACATGATTTCTAGAATAAGCAATTAC10140
TTCTCCTGGA GCTATCGCCAAAACATTAGCACCATCATTCCATTGTTCTCTTGCACCATG10200
TATTAAATCT CCACCCGCACATTTTATTATGTCAATTTTTCTGCCTAAATAAAAGCTCAA10260
AACATCTTTA AGCTTGGCTTTTTCTTTTTTAATATTAATTTTATTAGAATTTGAATTGTA10320
AGTTAAAACA TAAATTGAGAAATACATATCATCACTTGTAAAACTTGTAAAAACGCTATA10380
ATCAATTTGG GTAAAAACTGTGTCTAAGTGCATATAGGCTCTGTTTTTTGGAATTTTAAA10440
AGCCAAAATT GTGCTAAATGGAGCCTTATTTTTAAAAAGACTAGCAGCTAGTTTTTCTAC1050C
AGACCCCGCT TCTGTTCTTTCTGAGATTCCAATAACCAAAAGATCTTTATTTAAAACAAA10560
CTCATCCCCA CCTTCCAAAGAAGTTTCTTCCCATCTATTAAACCAAATTGGAACATTTTC10620
TTTGTAAGCG GAATGATATTTAAAAATATACTCTGCAAATATTGTCTCTCTACGTCTAAC10680
CTTGGTATAC ATTTTATTTATTGTAATTCCATTGCCAATACTGGCAAAAGGATCTCTGGT10740
AAATAAAACA TTGGGCATAGGATCAATAACAAAAAGACTTGAACCATTAACCCAATCATC10800
AAGCGAAAAT TCACAATCTTTAAGCTCTTCTCTTGCAACGCCGGAAATCATTTTAGAAAC10860
CATATTATCA ACGGTTAAATTAGAAAAATAATCTTTTAAAATATTAATTACACCATCTGT10920
TTTTATTTCT GCTTCCAGAATAAATTGAGATATAAATTTATTTTTGAGCGCTACAGAAGA10980
AGCAAGAACT TCACTAACAAGATCCTCAACATACTCAATTTCAACTGAATTATCTTTTAA11040
AATATTTACA AAAACTTCATGCTCTTGTCTTGCAACTTTAAGATAAGGAATATCATCAAA11100
TAAAAAATTT TTCATAATCAAGGGTGTCAAATTTTCTAATTCTTCTCCTGGCCTATGAAG11160
CAAA.ACTTTT TTCAAACGACCTATTTCCGAAAATATATTTATTGGATTTAAATATTCTTC11220
TTCCATCGAT TTCCCCCTTTATGAAAATTGTCATATATTAAAATACTATAGTTTATATTA11280
AAAAACATCA ACTATTTTTAATAATATTAAAAATATAATATAAATATAAAAAATTGAAAA11340
AATAAAAGTT CTAAAAAACTTCAAATCAAAAACATAAACAAAAAATTATGCTAAAATACT11400
AATCATGAAG AATATTAATAGATTAATATTATTAATATTAACTACACACACTTTATTATT11460
CTCTTGTGCC TTAATTGCAGATAATAAGTCAAAAAATTTAAGCACATCAGAAATCATATT11520
AACACAA.AAA ACACTACTAGAAAGCTCTTTAATAAAAAATCCTTCTAATGTAGAATATCG11580
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AATACCAATATCCAGTATCCAAGAAATTTTAAACAATAACAATGATTCTTTTTTAATAAA 11640
AAAAACAGCAGCAAAAATCAAAATAAGCCCTCAAAAACTTGAAGAAATAAAAAACTATCT 11700
AAATGCTTATAAAAATTATCTAAATAATGAAACAGAATGGATAAAGTTTATAGATCAAAG 11760
TAGCGTCAATGGAAATTTAACAATTAAAATTGATACTGCTTTTGAAAAAAAAACAAATTT 11820
TAATCATACAAATTCAGATAATGAAAATTTAACAGAACTAATAGAACTACAAATGCATCT 11880
GGAAAAAGAAATTTTAAACTTAATTGAGCAAACATTTCATGATAAAAATTTAGGATATAT 11940
ACAATTAAGTCACATCAACTCATTCTTTCCTCAAGAAAATATAAACTCAATAACAAAAGA 12000
AATAATAGATGGAAAAGAATATATTGCACCGCACATAATAGCAAATCAATTATTAAAAAT 12060
AAAAGATAAAAAATATTTTGAACAATTTATGCACTTTTTAAAAGTTGAAAACAGCAAAAT 12120
AAAAACAATAATTGAAAAACAAAAAATTTCAGATCTTCACAATGAACTGTATTATTCAAA 12180
ACAATCCCCGCCCAGAAGAAGAAAAAGGTCAACTGCCGATTCCGATAATAACAATAAATA 12240
CGATATAATACCAAAAATAATAGACCCAAATACAGGCATTGAAATAACTCCTAAAAATTT 12300
AAGATCTATTTTATCAAATGGCGACATAATACTAATAAAACCAAAAATAGATTGGACAGA 12360
ATTTTTTTATTTTTGGCAACATGTGGGAATATTTGATGAAGAAAAATATGAAGCCACTAA 12420
AAAAATTGCATTCAATGGAATTGATAGCTTTGATATAAAATCAATAATTACAAGCAATCA 12480
AATCAAATTCGATACAGCATCTACTCAAGGTTCAGGATACGAAAAGCTTTCAACATACGT 12540
ACAATCAAGAATATTAAAAATATTCTCACCAATAACAGACATAAGAACAATTCAAAAAGC 12600
TATTAATTTTGGAAGAAGTAGATACATTGACAATAACTTTGGATATATGGTTCCATTAAT 12660
ATCCTCTAATTTATGGACAGATTCATTCAATCTTGAAGAAATTCACAACAAAACCTATTG 12720
CTCTTTAATGGTTGATAGAATATATAAAATAGCAGGACTTAATGTATCAAGAAATTACGA 12780
AATTTCGGGAATAATTACTCCTGGAGAAATAAATGCAGCAGCTTACAATTTTTACATGTC 12840
TTATACGATTGCAGGAATACTTCCAAGCGTGCTTCCAAAAAGGCTCATTAAACCAACATT 12900
AAAAGAAAAATTCATTGGTTACAATAAAGAAATAGTAGATGCAATAGAATTAAAAAAATC 12960
GAAAGAAAAAATTTTTGGGAGAGCTTGCAACATTACAAATCTCTGGTGCTCAGGAAGTTA 13020
ATACTACCCATGAACAAATATTATGCCTGTATTTTGATTAACAGCAATGAAAAAATTATT 13080
TTCAAATCCTGGGAAGAATGCAAAACCGCTATTAAAGGAAAAAACAATAAAATAAAAAGC 13140
TTCAAAACAATAGAACAAGCTCAAAATTGGCTATTTAATAATGAGAATAAAATTCACCAT 13200
CACCCAAATGGAATATATTTTGATTCTGGAACGGGAAGAGGAAAGGGCATAGAAATTAGA 13260
GTTGTAAACGAAAAAAGAATTTCAATATTGGATAAAATCTTAGATAAATCCTTGATTAAT 13320
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GAATATGGAAATTATTATGTCAAAAATTTTCAAGGAATTAGCAATAATTTTGGGGAACTG13380
CTTGCCCTATATACAGCTCTCAAAATAGCATTAAAAGAAAATATAATAAACATATTTGGG13440
GACAGTAAATTAATAATTGACTATTGGTCAAAAGGAATCTATAATAGCAAAAAATTAACA13500
CAAATTACTATTAATTTAATCAAAAAGACAACTGAACTAAGGAAAAAATTTGAAGAACAA13560
GGTGGAAAAATTTCTTTTATTCCAGGAAATGAAAATATTGCAGATCTTGGTTTTCATAAA13620
ACTAAGTAGAAATATTGTCAAAAAATACATAAAAACAATATTTCTGATTTCAATGGTTTA13680
TTTTTATTGTTGTACGACAATAAAAATAAACCATGATTATGAAACTGATTTTAAAGTTCT13740
AGAATCTCCCTCTAAATACATCAATATAGATGTAATTAAAGCTACAAATGAATATATTTA13800
TATTCAAATTACAAACAATAGCTTAGACGTAGTAAAAATAAATTGGCAAAACACTAGTCT13860
TAACAACGATAAGATCGTCTTAAAAAAAGAAGATCTTACAATAAACAATGAAACAGGGTA13920
TAAAAATAAATACAGAGAGTTTTTTATTGGTCCTAAAACTTCATTTAAATTTAAAGTATA13980
TCCACTAAAAATTCATTCTAAAAACAAAAATAGCAATAACTTAAGCTCAACTATTAAATA14040
TCCGTCTATTTTTAAGCTCAACATAACAAAAGTAGGAATTGAAGCAAAAAAAACAATAAA14100
TGTTTTAATAACAAGAACTACAAAAATTAATATTACTAATAAATGAAAATCATTATTATT14160
TTTTTGTTTTTCTATTAATAATAAACTCGTAGTTTGTATCTTTGTTGTTTTCAAATGACG14220
CTTTTATAGGAGCAA.AAAAGTTCCAAGAAATATAAATTGTAAAATTATTTCTCCAAATAG14280
GAGAATAAATCCCATCAGATCCCCTTAATAAATCTGGTTCTAAATTATATTCTCCAACAA14340
ATTTCCAATCATAAAAATTGAATTTAAAGCCTGATGAAAATTTTTTAATTTTAAAAAGTG14400
AATCTTTTCTGTCTTGAGAATTAAAGAAATTGAAAGATTTTGATAAATCAACAAAGAAAT14460
TAACAGGTTCTAGACCAATTTGGTCCATATACCC'PTTAAAATATTTAAAAGTCTTAGTAT14520
TAATAGATAAAGTAGAAAAGTAAATTTCTAAAAATTCTGTATATTTAAACTTCAAAGTCA14580
ATGCAGATCGAAGTTCATTATCCGTAAATTTCTGCAAATTTATTTTCCAACCAACATCTA14640
CCCCCAAGGTAAAAGAAAGCTTATTGTCAAAAAAAGTTAAAACGTACAATTCCTTTTTGT14700
AACTAGAATCTAAAGAATATGGAACAAGTTTGGTTGTAGTACCAATCTTGGAAAAATCTC14760
CTTTTAAAGGATCATAATTATATTCAAAGTCGTCTTTCATAGCAAACAAAAATTGAAAAT14820
CAAAAACATTAAGCTTAAAAGAAAGTTCAGAAACTCTATTTATCAAAGGATCATAGGCGA14880
CTAAAAAACTAAATTTAAAATAATCCAAATATCTCGGCTCAATTTTATAATACAAAGCAG14940
GAGACATTTCTAAATTTTTATAAGGCGATGATGGTTTTTGAGGCTCCAAAGGACTTTGAA15000
CAGCAGAAATTCCAGAGTTTTTCATAGCATCTTCTTTAAACTTTTTATAATATTTAATTC15060
CAATCCCAGCTTCTTGTAGCAAATAAGGAAAATCTAAAGAAAGTTTAAGCTCAGAAGAAG15120
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CTTTAATATCTTCAAAACTATTTTTTAATTCACCTGAAAGCTCAGTAGTAAAATAATCAT 15180
AATCATAAATTAAAGAGGCTGTTAAACTTTGATAAAAAGTTTCCGGATCAGATAAAAAAA 15240
TACTACTATTCTTATTAACCAAAGATTTTACATCAGAATCATATTTTTTATTAAATGAAT 15300
ATAAAGTAGCCTTATTTTCAAACTTTAAAGTACTTCTAGAAAATAAAGGATATCTAATAA 15360
AAGGAAGCAAGTTTAAATTTATTTGGTTAATAATAGAGTGCTCACTTTTTTTATCTTTAT 15420
CTTCAACTTTAAAATCTTTATTTAAAGGACTATACTCAATAGTATTAAGATATAATAAAT 15480
TTTCAAAAGTAATTAAACGATTGTAAAAATCAGCATGAATTTTTATATCCGTTTTATTTT 15540
TTATATCAAATAAATAATTTTTTATTTCATAATTAAAGTCCTTTGGACTTGTTATGCCAT 15600
AATTATCAAAAAAAACATTATTTCTTAAATAAGGATTAATGCCAAACCTAATAAAAAAAG 15660
AATCGGATTGATCAATATTTTTTAAAGTAATTGGTTCTGGAGGAATATATAAATCTTTGG 15720
TTAATTCTGTTGTTTTTTTAGTATTTTTCTCCTTCACACTCTTTTTATCATTATCTTTAT 15780
CTTCTAGATTTTTAATTTCTGGGCGCATTATCATTTCTTTAGTATCAGCTGGAAATGTCC 15840
ATTGGTTATTGTAAAGATCTTTTTGAAAATTCAAATCAATATATGGAGCATAAATTCTCT 15900
CCAAATAAAACCATTTTCTTGTAGGATCATTAACATCTTTTGGTTTCTCTAAAGGAGATT 15960
TAACATAAAGATTTTCATAGCCCGACAATTTAAAACTTAAACCTAAATTATTTAATTTAT 16020
AATCTAAAATCGAACCGTCATTAAATGTTCGCTTATAAAAAGAAGATAAATTCCAATCAA 16080
AAGTGCTAATGCTAGTTTGCTCTTTAACCGAATCTTTATCTAAATTTAAAAGAGAAAAAA 16140
ATGTAGCACTTTCTATCCTATCTCTAAAATCAATATTAACATACGGGTCAGAATAGTGCT 16200
CTAAAACAACCGAGAAAAGTGCATCACTTAAAAGAAATTCTGTTTTAAATTTAAATAAAT 16260
ATCTAAAAGGAACTTCAAACCCAAATACATCTCCTTTGTTAAGATTGGAAAAACTAAAA.A16320
GAGATTGTTTTAAAGTCCTATTATCAAAAGGATAATATCCTCCATCGTAACTATAAACAT 16380
TCCTGGTAAAACCCAATCCAAAATTTCCTTCCAAAGTTTTAAAATGCCCCAAAGTATTGC 16440
CCAAATTAAAATCAATTCCAGAATAAAATCCCAGATTAGCATAAATGTCAAAAATAAGCT 16500
TAACATAATCTTTATTAACACTGGGTGCTAAATTTTCTGCAAA.AAAATAAGTTAAATATC 16560
CATTTCTTATATAAGGTTTTTTACCCGAATTATAAACAGAATTGAAATCAAAATCCAAAA 16620
AAGAAGAATCTTCACTTGAAGATTTATTACCAAAAAGATAAACGGTATTAAAAACAGAAA 16680
AACCTTTTCGTGGATTTAGACCTAAAGATGGATTAAAAAACAAACTATCTCCCGGTCTGA 16740
AAAAAAAAGGAATATAAAATACTGGAACTCTTCCCATGTAAAATATGGCATTTAAAAACC 16800
CAAAATCTCCCGAGGGCAATGCCCATATTTTAGAAGCCTTGATTGAATAGTAAGGCTCTG 16860
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GAATTTTACT AGTTGTTGCAAAAGCTTGTTCCAAAATGGTAACATCATTGTCTATCTTTT16920
TTAAAACCTT TCCTCCAAACGAAAGAATATGATCTATTTGATTTTTTTGCATTTTTTTTT16980
GAAGAATACC ATTTTTTAATAAAAAATTTTGAGAATCAAAATCGACAAGAAATTCATTGC17040
CATAAAAATA AAGCTTTTCATTGGTATCCATATCAAGAATATATTCAACATTTCCAATAG17100
CATAAAGTTT TTTAGAGTTCTTATTAAGGACTATTCTGTCGCCTTTAATATTGTGCTTTT17160
TATTTTCTTT AATATCTTCAACCAAGATATTAACTCTTCCTTCAAAAATAATACTTTCAT17220
CTTTAGTAAG TCCATAAGTGAAATTTTCAAGATTATCTGCAGTTTCAATGATTATTTTAT17280
ATCTACCAGA TCCGGCAAGTCCCTTTCCTTTGATAAAAAGCTCAGGATCTATTCCAAACT17340
TTTTTAAAAG CAATTCTCGTATTTTTGAAACATCTGTTTCTTTTAAACCCTCTTTTAAGG17400
CCCATTTTTT TAAATCCTCATCGGTTGAAAGCTCAAGTTCTCTTAAATAAGATTTTTGAC17460
TTAAAGTTAG CTTATCCCTTTTTTTAGAATTTTCATCATCTATAGTCTGGGCAAAAATTG1752C
CATTAGAAAA TGTTAAAAAAATTAAAAATACTATAAAAGATTTTTTAAAAACATTCCTGT17580
ATAGGAATTC TCGCATTTTGCAACCTCTTCAGGAATACCAGAAACAACGATATTTCCCCC17640
TGCCAACCCA CCATCAGGACCCAAATCTATTATATAATCTGCCTGTTTAATTACATCCAA17700
ATTATGCTCT ATTAGTACAACTGTATTACCATTGGAAACTAACCGCTGCAAAACCTCTAA17760
CAACTTCTTT ATGTCATCAAAATGCAGCCCAGTTGTTGGTTCATCAATAATATAAAAGGT17820
TTTACCCGTG CTCTTTTTACTTAACTCAAAAGCCAACTTAATGCGCTGAGCTTCTCCTCC17880
TGATAAAGTT GTTGCAGATTGTCCTAATTTAATATATTCAAGTCCAACTTCAATTAAAAA17940
TTTTAAATAA TGACTAATTTTTGGGACATTCTCAAAAAATTTACTTGCCTCAAAAACACT18000
CATCTCTAAA ACATCATGTATATTTTTTCCTTTGTATCTAACTTCTAAAGTTTCTTCATT18060
GAATTTTTTA CCCTTACATAAATCACAAGGAACAAAAACATCTGGTAAAAAATGCATTTG18120
AATATTAAGA TACCCATCTCCTTGACATTTCTCACACCTTCCACCTTTAACATTAAAAGA18180
AAATCTGCCG GCTTTAAAACCCCTTGACTTTGCATCTGGAAGCTTGGCAAAAAGCTCCCT18240
AATTTCTGTA AAAAATCCAACATAAGTTGCTGGGTTTGATCTTGAAGTTCTCCCTATTGG18300
TTTTTGATTT ATTTGAATAATTTTATCGATTTTTTCATACCCAACAATATCTTTAAAGCC18360
ATCACAATAC TTTTCATTAAGCTTTAATCTACTATCAAGAGCTGGATATAACACCTCGTT18420
AAGTAAAGTA CTTTTTCCGCTACCAGAAACACCTGTTATTACGGTAAAAACTCCCAAAGG18480
GATACTTAAG TCTATATTTTTAAGATTATTTTTATTAGAGCCCAAAAGCAAAATTTCTCC18540
CTTATCTGCC TTTCTTCTAGAGCTTGGAACATCTATTTTAAACTTGCCGCTAAGATATTG18600
ACCAGTTAAA CTATTTTTGCTATTTAAAATATCAATCAAGGCTCCCTTTGCAACTATTTC18660
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CCCTCCAAGAATTCCAGCACCAGGACCCATATCAATAATATAGTCCGCAGTACGCAAAGT18720
TTGCTCATCATGTTCAACAACAATTACAGTATTACCAAGATTTTTAAGATTAACAAGAGT18780
AGAGATTAATTTTTCATTATCTCTTTGATGAAGACCAATACTTGGCTCATCAAGAACATA18840
AATAACACCCGAAAGTGCTGATCCTATTTGAGTAGCAAGCCTAATACGCTGAGCCTCGCC18900
ACCAGATAGACTACCTGATATTCTATTTAAATATAAATAAGAAAGGCCAACATCAATTAA18960
AAATTTAAGCCTACTTTTAATTTCCTTTAAAATTTCTTTAGATATTTTTTCGTCCACCAT19020
ATCAAGCTGCAAGTTTTCAAAAAATACATAAGAATCAAATACTGACAAATTGGTAAGATC19080
TTGAATGTCTTTTCCATTAATTTTCACAGTTAAAGCTCCAACGCTTAGGCGTTTACCTTT19140
GCATGAATTACATATTTTTTTAGACATCAAATTTTCGTAAAAAATTTTAGTACTCTCTGA19200
TTCTGTTGCAAGATATCGCCTTTTTAAAAGGGGCAAAAGTCCTTCAAATGTTTTAGAATA19260
ATGAAATCCTCCATCTAGCTCTTTTGCTTCCATTTCTTTGGACTGGTAAATAAAATCTAT19320
TTTTTCATTTGAGCCGTATAAAATCTGTTTAAGAACTTTATCTGGAATGTCTTTTATGGG19380
AGTATTTAAGTCAAAATTATAATGTTTAGCAAGTCCTTTAAAAATAGCCACAGACCAAGA19440
TGAACTTGTCTTAAACGTAAGAAAAGCATCATCATTAAAAGAAAGACTAGTATCAGGACA19500
AATGCTCTCAAAATCAAACTCAAGTGTAACGCCAAGACCAGAGCACTCACTGCAAGCACC19560
AAATGGACTATTAAATGAAAAAAGTCTGGGTTCTATAAGAGGAAGTGAAAATCCACACAA19620
AGGACAACTGTTGTGCTCTGTAAATAGTTTGTCTATTTTTTCCAAATCATTATCAATTTC19680
CACTCTTAAATATCCATTAGAAACAGCAAGAGAAGTCTCAATAGATTCCGCAAGTCTAAC19740
TCGAACATTATTACCAAGCTTAATCCTATCAACTATAATTTCAATGGTATGTTTTTTATT19800
TTTATGTAAATTTAAATTAAGTGCATCTTCTATTAAATAATCTTCAGAATTTATCCTAAC19860
TCTATTAAAACCTTGATTTAATATTTTTTCTAAAACCTTTTTATGAGAGCCTTTAGACCC19920
CCTTACAATTGGTGCAAAAAGTATAACCTTGGATCCTTCAGAATAACTTAAAATAGTATT19980
AACTATTTTATCTAAAGATTGCTCTTCTATTAATCTACCATCATTTGGACAGTATGCTTT20040
ACCAATTTTTGCAAATATTAGTCTATAGTAATCATAAATCTCAGTAATTGTTCCAACAGT20100
AGAGCGGGGATTATTGCTTATTGTTCTCTGCTCAATAGCTATAGAAGGAGAAAGTCCATC20160
TATATAATCAACATTGGGTTTTTTCATTACACCTAAAAACTGCCTTGCATAAGCTGAAAC20220
AGATTCCATATACCTTCTTTGCCCTTCTGCAAAAATAGTATCAAAAGCCAGAGAAGACTT20280
GCCAGAGCCACTCTTGCCAGATATTACAACTAAACCATCTTTTGGAATATCTACATCAAC20340
ATTTTTTAAATTATGTTCTTTTGCTCCTCTGACAATAATTTTTTTTTTCAAACTTTTTTC20400
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CAAAAATTAC ACCTCTCTTTTTTTATTACGAGCTATACTAATTTTGCTACTAAGCTCTTT20460
TATTTTATCT CTTAAAACAATTGCGTCTTCAAATCTTTCATCATTAACAGCTTCTTCTAA20520
GTCAAATTTA AGCTTATCAATAAGCT'PTTTTTTAGACAATCTCTCACCCGAAATAATTTT20580
TTCAAAATCA TAGCCAACATTTTTAT'PTTTATTATTAAGTTCCTTTTCTAAAATATTTTG20640
AATCTTTTTA ACAATTGTCTTAGGAGTAATATTATTTTTTTTATTATAATCAATCTGAAT20700
TTGACGTCTT CTATTAGTCTCCTCAA'rTGCCTCCC'.GCATAGCTAAACTAATTTTGTCGTA20760
ATACATTATT ACAAGTCCATTAGAATTTCTAGCAGCCCTACCAATTGTTTGTATTAATGA20820
AGTAGTAGAT CTTAAAAATCCCACCT'rATCAGCATCTAATATTGCAACAAGAGATACTTC20880
TGGAATATCT AAGCCCTCTCTAAGCAGGTTAATCCCAACAATAACATCGATTTCAGATTT20940
TCTAAGCAAC GAAATAACTTCCACTCTCTCAAGGGTATCAAGCTCTGAATGTAAATATTT21000
TGCCCTTACG CCAAGATTTACCAAATATTCAGTCAAATCCTCAGACATTTTTTTTGTCAA21060
AGTAGTAATT AAAACCCGCTCTTTAAGAGCCACTC'.TTTTTTGAATTTCGCTGTAAAGATC21120
TTCCATTTGC CCATCAGAGTGCCTAG'PAATAATTTCAGGATCAACAAGACCTGTTGGACG21180
AATTATTTGG TCAACAACCACACTACTTTTCTCA'I'TCTCTTCAACACCCGGGGTTGCAGA21240
TACAAACACA ACCTGATTAATTAATGCTTCAAATTCATCATATTTAAGAGGTCTGTTTTC21300
AAGCGCTGCA GGAAGTCTAAACCCAAAGTTAACAAGATTTAATTTTCTAGAATGATCTCC21360
ATTATACATT CCCCTAAATTGAGGCAATGTAACATGAGATTCATCTACAAATAATAAGTA21420
ATCTTTCGGA AAAAAATCAAAAAGACAATAAGGTCTTTCCATTGTACTTCCACTCAAATA21480
TTTAGAATAA TTTTCAATGCCCGAACAAAACCCTGTTTCTCTAAGCATTTCCAAATCATA21540
CTCTACCCTC TGTTTGAGTCTCTCGGCTTCTACAAGTTTGCCATTGTCTTTAAAATATTG21600
ACATTGAAGA CTTAAATCATGAGATATTTTGGGTATCGCTTCTAATACATTTTCATAAGG21660
AATTACAAAA TAAGATTTAGCAA.AAAGAGTAAAACTATTTGTAGCTCCTAAATTTTTTTT21720
AGAAAATGAA CTAACTCTATATATTTCAACAATTTCATCAAAATCTAAACAAATTCGATA21780
AGCAAACTCT CCATGTTCACTGCTAGGCCAAATTTCAACAATATCTCCCTTAATCGAAAA21840
TTTATCTCTT TCTAGATTCATTAAAG"_'TCTCTCATAATAAAGCTCTACAAAAATATCTGA219OO
TATTTCTTTA ATAGAAATCTTTTGACCTACAAAAAATTCTCGTGCTGATTTTTTGAAAAA21960
ATCTGGAGAT CCAAGAGCATAAATTGAAGATACGGTTGCAACAACAATTACATCTCGTCT22020
TTTAGCAAGA GACGTTACCGTTCTTATTCGCTTAATTTCTATCTCAGTATTAATAGTGGC22080
TTCTTTTTCA ATAAATAAATCTTTTGAAGGAACATAAGATTCTGGCTGATAATAATCATA22140
ATAAGAAACA AAATACTCAACAGCATTATTTGGAAAAAAATCTTTAAACTCTCTATAAAG22200
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CTGTGCTGCTAATGTTTTGTTGTGACTGACAACTAAGGCAGGCCTGTTTAGATCTTTTAT 22260
TATATTTGCAATTGTAAAAGTCTTTCCACTGCCTGTAACACCTTTTAAAGTTTGATATTT 22320
ATTTCCAAGCAAAATAGAATTTTCAATCTCTTTTATTGCCTTAGGCTGATCCCCAGCAGG 22380
AAGATATTCTGACTTCAAAAAAAAATCTATCATTAATTTAACGACCAAAATTTAATACAC 22440
ATTCTTATAAATTATATGATAATAAATTCTATATCAAGTATATAATTCATTATAAATCAA 22500
TATAATTTAATTAATCTTTGTTTAATAAAATAAAAGGAAATATTGATGCTAAAAATCGAA 22560
GCTAAAAGAAAATTGAAAAATTATATTCTTCTTGAAGAAGATATGCATTTTAAAGAAGAA 22620
GCAATAAAAATTCAAAAAACAAATAATTCAACAGAAATTTTAAATAGATTTTACAAAGAT 22680
CTAGAATTTGGCACTGCTGGAATAAGGGGAATCATTGGAGCTGGAACATGTTACATGAAC 22740
ACATATAATATAAAAAAAATAAGCCAAGGAATATGCAATTACATACTTAAAATAAACAAA 22800
AACCCTAAAGTTGCAATAAGCTATGATTCAAGATATTTTTCAAAAGAATTTGCTTACAAT 22860
GCTGCTCAAATTTTTGCCTCAAATAATTTTGAAACATATATATATAAAAGTTTAAGACCT 22920
TCCCCACAACTATCTTATACAATAAGAAAATTTGACTGTGATGCTGGCGTTATGATAACA 22980
GCAAGTCATAATTCAAAAGAATATAATGGATATAAAGCATATTGGAAAGGTGGAATCCAA 23040
ATAATACCACCTCATGACACACTAATAACTAATGAAATTAAAAATACAAAAAACATAATA 23100
AATACAATTACCATAAAAGAAGGCATTGAAAAAGGGATCATCAAAGAACTTGGCAATGAA 23160
ATAGACGAAGAGTATGTGAAAGCAATAAACAAAGAATTGCCTGATTTTGAAAAGAATAGC 23220
AAAGAAACAAACTTAAAAATAGCCTACACAGCATTACATGGCACCGGTGGGACCATAATA 23280
AAAAAACTCTTTGCAAATAGCAAAATACGGCTTTTTTTAGAAAAAAATCAAATACTACCA 23340
AACCCTGAATTTCCAACAATAAATTATCCTAATCCAGAAAAACAAACATCAATGCTTAAA 23400
GTAATAGAGCTTGCAAAAAAAAAAGATTGTGACATTGCCCTTGCAACAGATCCAGATGCC 23460
GACAGAATAGGGATTGCATTTAAAGATCAAAACGAATGGATATTCTTAAACGGAAATCAA 23520
ATATCATGCATTTTAATGAACTATATACTCTCAAAAGAAAAAAATCCTAAAAATACATTT 23580
GTAATATCATCGTTTGTAACAACACCAATGCTAGAAAAAATTGCAAAAAAATATGGTTCT 23640
CAAATTTTTAGAACTTACACAGGATTTAAATGGATAGGAAGCTTAATTAATGAAATGGAA 23700
AAAAATGAACCAAATAAAAAATTTGCTTTTGCATGCGAAGAAAGTCATGGATATCTAATA 23760
GGAAGAAAGGTTAGAGATAAGGATGCATTTTCAGCCATAAAAGGAATTTGTTCTTTAGCA 23820
CTTGACTTAAAAGCCAAACAACAAACAATTAAGGATTATCTTGAAAAGATATACAAAGAA 23880
TTTGGATATTATGAAGAATTTAATATAGAAAAAAACTTTGAGGGGGCCAATGGAGAAATT 23940
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CAAAGAGAAA AGTTAATGCTAAAACTAAGAAAAGAACAAAAAGTACAATTTGCAGGAATT24000
AAAATAATTG AAAAATTAGACTATAAAACTCTTAAAAAGATTAACTTTAAAAATGAAATT24060
TCAGAAATTA AAGAATATAAATACCCCATAAACGCAATAAAATT~_'ATACTTGAAAACGAA24120
ATTGCAATAA TTGTAAGACCCTCTGGAACAGAGCCGAAAATTAAATTTTACATATCTGTA24180
AAACTAGAGT ATAAGGAAAAACATAAAATATTTGATATAATAAATGCAATAAAGATGGAG24240
ATAAAAAAAT ATTAACATAACAGAAAATTTAATAAATTTGGTAGAAATAGACTCAAAAGA24300
AATTGCAAGA AAAAATAAAAATAAAGAGGTTTCAATTTGGCACTTATTAATGTCTATAAT24360
TACCACTCCC AAAAAATCCGAAATAAAATTTATAGATAGCAAAACTCTAAAAAACATTAA24420
ACAAGAAGTT ATATCTGAAATAGATAAATTAGAGAAAATTTTAATAGAAAAAAACGAAAT24480
AATTATTCCC AAAATCAATAAAGAAATCTTTGCTCTCATAAAAGAAGCTAAAAAGGAATT24540
TAAATCCAAA CCTTTAATAGGGGCAAAAGAAATTTTTTATCAAATATTAAAAAATAAAAA24600
ACTTCTTAAA AAACATAAACTAAGTAAATCTAGCTTTAACTTTAAAGATCAAAATATATT24660
AGAATACATG GAAAAAAATAAAATAAGATTAATTGAAACCTACAAAGAATTTGATGAAGA24720
AATACGACTT GAAAATGAGCACTTTGAAATTGGAAAGTATGTCAAAAATTTAACAGCACT24780
TGCAAAAGCC AAAAAATTAGACCCCTTGGTTGGAAGAGAAGCAGAGATTAAAACTCTTAC24840
AAATATACTC TTGAGAAGAAATAAAAATAGTGCAATGCTAATAGGCGAACCTGGTGTGGG24900
AAA.AACAGCA ATAGTTGAAGGCCTTGCATCAAGCATAGTGCF,AAAAAAAATAAGTAGCAA24960
ACTACAAGAC AAAACAATTCTAATGCTTAAGGTTTCAAACTTGGTATCGGGAACAAAATA25020
TAGAGGCGAG TTTGAAGATCGTTTAAATAATATAATTAAGTATATTGAAAAAAACAAAA.A25080
CACAATCATA TTTATTGACGAAATACACACTCTAATAGGAGCTGGAAACTCTGAAGGAGC25140
TCTTGATGCA TCAAATATACTAAAACCATCACTTTCTAGAGCTGAAATACAAATTATTGG25200
CGCAACTACT TACAATGAATATCGAAAATATATTTCAAAAGACAAAGCATTCGCCAGAAG25260
ATTCCAAACA ATTACCGTAAAAGAGCCTGATGAAAAAGATaCACTAA.AAATAATCGAAAA25320
TATTGCAAAA AATTTTGAAGACTATCATGGAGTGATCTATGAAAAAAGCGCGCTTTTAAA25380
TATAGTAAAA CTTTCATCCAAATATCTAATAAATAAAAGATTTCCAGATAAAGCAATAGA25440
TATAATAGAC ATTGCCGGCGCAATTAAAAAGGAAGAACTTACAAAAGACAACATCATAAC25500
ATCAGATGAT ATACAAAAGGCAATAAATGAAATATTATCTATTAAAACAGCAAATAACAC25560
TAAAGAAGAA ATTTTAGAATTAAAAGAAATAGAAAGCGAAATAAATAAAAAGGTGATCGG25620
ACAAAAACAT GCGGTAAGCGAACTTATCAAAGAAATTATTAAAGTCAAACTTGGACTTAA25680
TGACGATTCT AAGCCTTTAACTTCAATATTGTTAATAGGATCAAGTGGATGTGGAAAAAC25740
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TGCTTTAACTGATGAAATATCTAAAAAAATTATCAAAGATCAAAATTCAGTATTAAAACT 25800
AGATATGTCAGACTATAAAGAAGAAAACTCTATTTCAAAATTAATTGGCACAAATCCAGG 25860
ATACGTAGGCTACTCTGATGGAGGCATTCTGACAAATAAATTAAGACATTCATTTGAAAC 25920
TTTAATATTGTTTGAAAATATTGAAAATGCCCACAGCTCTGTATTAAACCTAATAAGTCG 25980
AATGCTTGAAAACGGAGAACTTATTGACAGCAAAGAAGATAAAATACTATTTAAAAACAC 26040
AATTATAATAATGACTACAAACATTGGATCTAGAATGCTTCTTGGAGAAAAAAATATTGG 26140
ATTCAACAAAAATCAACAAAAAAGCTTAGAAACAAAAAGCTTTAAAGAAGAAATAAACCA 26160
AGATCTTGAAAAAAGATTTAAATTATCCTTTTTAGACAGAATTCAAAAAAAAATCATCCT 26220
AAATATCCTTACaAAGGAAAATGTAGAAGAAATTTGCAAAAACTACTTAAACACCCTTAA 26280
AACAAAATTTCACTCTAAAGGAATCGAGATAGAAATAAAAAAAGATGTTGACAAATTCAT 26340
AACCACAAAATACTATAAAAAAAATTCAGGAGCAAGAAGCGTAATTGCTGCAATAAAGGG 26400
GAAAATAGAAGAAAATATTATCACCAAAATAGCTGAAAATCAAAACATAAATAAAATAAC 26460
GATTTATTTAGAAAAAGAAAAAATAATAATAGAATAAAGAGGAATTATAATATGTTTAAA 26520
AAAGTAGAAAACAAGGCAAATTTTCCTAAAATAGAAGAAAAAATATTAAAATTTTGGAAT 26580
GACAATAAGATCTTTGAAAAATCAATAAAGCAGAGAGAAGGATGTGAAGAATTTACATTT 26640
TATGACGGACCGCCTTTTGCAACAGGACTTCCTCATTTTGGACATTTTGTTCCAAACACA 26700
ATAAAAGACATAATTCCAAGATATCAAACAATGCAAGGCAAGTATGTTAAAAGAAATTTT 26760
GGATGGGATACTCACGGACTACCTGTTGAATACGAAGTAGAAAAAAAATTGGGAATTTCT 26820
GGAAAATACGAAATAGAAAATTATGGCATTGAAAATTTTAACAAAGAATGCAGAAAAATA 26880
GTACTTAGATATACAGAAGAATGGAAAAATATAATCTTGAGACTTGGACGATGGGTAGAT 26940
TTTGAAAAGGGTTACAAAACCATGGATATAAGCTTCATGGAATCCGTGTGGTGGGTATTT 27000
AAAAATCTTTATGAAAAAGGTTTAATCTACGAAAGTTACTATGTACTACCCTATTCCCCA 27060
AAGCTTGCAACTCCGCTTTCAAATTTCGAAGTGAATCTTGGAGAATATAAAGAAGTCAAT 27120
GACCCATCATTAACAATAAAATTTAAAATAAAAGATAAAAACGAATACTTACTAGTGTGG 27180
ACAACCACCCCCTGGACATTGCCCTCAAACCTTGGAATTGCAGTAGGACAAGAAATAGAA 27240
TATTCTAAAATTTTTGACAAAACAAAAGAAGAGATTTTAATACTTGGATCAAAAAAGCTT 27300
AATAGCTATTACGATGATGAAAATTCATATACTATTATAGAAAAATTCAAAGGCAGCAAG 27360
CTTGAAGGCATAGAATATGAACCTATTTTTAACTACTTTTTAGAACAAAAAGATAAGGGG 27420
GCTTTCAAGGTACACACAGCTGATTATGTTACAACTGACGATGGAACAGGAATTGTTCAT 27480
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ATTGCTCCTT TTGGAGAAGAAGACTACAGAATAC'PCAAAAAACACACAAATGTCGATATA27540
ATAGACCCCT TAGATGCTGAATGTAAATTTACAAATCAGGTAAAAGATTTTAAAGGACTT27600
TTTGTAAAAG ATGCTGATAAAAAAATAATAGAAAACCTAAAATTACGCAATTTTTTATTC27660
AAAAGAGAAA ATTATCTACACAGGTATCCATTTTGTTATAGAACAAACTGCCCAATTATT27720
TACAGACCAA TAAGTTCGTGGTTTGTAAATGTAGAAAAAATAAAAACCAAACTTTTAGAG27780
GTAAATGAAA AAATTAATTGGATGCCAGCCCATTTAAAAAAAGGAAGATTTGGAAAATGG27840
TTAGAAAATG CAAAAGATTGGGCAATAAGCAGAAACAGATTTTGGGGAAATCCAATTCCA27900
ATTTGGATAT GCTCAAAAACAGGAAAAAAAATTTGCATTGGATCAA.AAAAAGAGCTTGAA27960
AACCTATCTG GCCAAAAAATCGAAGACTTACATAAAGACCAAATAGATAAAATAACCTGG28020
CCAAGCAAAG ACGGTGGCAAATTTATCAGAACAAGCGAGGTTCTCGATTGTTGGTTTGAA28080
TCTGGAGCAA TGCCTTACGCAAGCAACCATTATCCATTCACAAATGAAATTAATTTTAAA28140
AATATATTTC CTGCTGACTTTATTGCAGAAGGTCTAGATCAAACAAGAGGATGGTTTTAT28200
ACTCTTACAA TCCTGGGAACTGCTCT'rTTTGAAAACACAGCATTCAAAAACGTTATTGTA28260
AATGGACTTG TGCTTTCAAGCGATGGAAGAAAAATGTCAAAATCCTTTAAAAATTATACA28320
GACCCAATGC AAGTAATAAACACCTTCGGAGCTGATGCTTTAAGGCTTTATTTAATAATG28380
AGCCCTGTAG TTAAAGCTGATGATTTAAAATATAGCGACAATGGAGTAAGAGACGTTCTT28440
AAAAATATAA TAATACCCATTTGGAACGCTTATTCATTTTTCACAACTTATGCAATAATT28500
GATAAATTCA AACCTCCAAAAAATCTCAGCCTGGCTAAAAACAATAACCTTGACAAATGG28560
ATCATAAGCG AACTTGAAAGTCTAAAAAAAATACTAAATACAGAAATAGACAAATACAAT28620
CTAACAAAAT CAATAGAATCTTTACTTGAATTTATAGATAAATTAAACAATTGGTACATA28680
AGAAGATCAA GGCGAAGATTTTGGAAATCAGAAAACGATAAAGACAAAAATGATGCCTAC28740
GAAACATTAT ATTATGCAATCAAAACTTTAATGATTTTACTTGCACCTTTTATTCCATTT28800
ATAACAGAAG AGATTTATCAAAATTTAAAAACTGATGAAGACAAACAATCAATACACCTT28860
AACGATTATC CAAAAGCAAATGAAAATTTCATTAACAAAACAATTGAAGAGAAAATAAAT28920
CTCGCAAGAA AAATAACTTCAATGGCAAGATCACTCAGATCATTGCACAATATAAAAATA28980
CGCATGCCTA TTAGTACGATATATATCGTCACAAAAAATCAAAATGAACAAAATATGCTA29040
ATGGAAATGC AAGAAATAATATTAGATGAAATAAATGCAAAAGAAATGAAAATAAAAGCT29100
AACGAAGAGG AGCTTATAACTTACAAAGCAAAAGCAAACTTTAAAGAACTTGGGAAAAAG29160
CTTGGAAAAG ATATGAAAGCGGTATCTACTGAAATTAGCAAGCTAAAAAATGAAGACATA29220
ATAAAAATAA TAAATGGAACATCCTACGAGATAAAAGTAGCCAATGCAAAGCATTATTTA29280
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TCATTAAATGATATAATATTAGAAAGAGAA ACTTAAAAGTAATAAATGAA 29340
GAAAAAGAGA
GAATCCATTACAATAGGAATAGACTCACTAATCACTAAAGAGTTGTACTTGGAAGGGCTG 29400
ACAAGAGAATTTGTAAGGCAAATACAAAATTTAAGAAAAGAAAAAA.ATTTTGATGTTAGC 29460
GATAGAATAAATTTATACATAGAAAATAATGAAACTTTGAAAGAAATGCTAAATAAATTT 29520
GAAAAATACATTAAAACTGAAACATTAGCCTTAAATATCATATTAAACAAAAGTAAGCTA 29580
GF~AAAAAAAATAAACCTTGCCGATGACATATTTACACTAATAGGAATTGAAAAATGTTAA 29640
AAACATTAACAAAAATAATTACCATTTCATGCCTCATAGTGGGATGCGCAAGCCTGCCTT 29700
ACACTCCTCCAAAACAAAATCTAAATTACTTAATGGAACTTTTACCTGGCGCAAATTTAT 29760
ACGCCCATGTAAATTTAATTAAAAACAGGTCTATTTATAACTCTTTAAGCCCTAAATATA 29820
AATCAGTTCTTGGGCTTATAAGCAATTTATACTTTAGCTATAAAAA.AGAAAATAACGATT 29880
TTGCTCTACTAATAATGGGTAATTTCCCAAAAGATATTTTCTGGGGAATTCATAAAAATA 29940
GAAATACAGAATCAATAGGCAATATATTTACAAATCCAAAATGGAAACTTAAAAATTCAA 30000
ATATATACATTATTCCAAACAAAGCTAGAACTAGCATTGCAATAACCCAAAAAGATATAA 30060
CCGCAAAAGACAATAATATGCTAACAACAAAATATATTGGGGAAATAGAAAAAAATGAAA 30120
TGTTTTTTTGGATTCAAGATCCAACATTATTGCTCCCAAACCAAATAGTAAGCAGCAAAA 30180
ATTTAATTCCCTTTAGCAGTGGAACTTTGTCTATAAACAGCTTAAATCAAGAAGAATATA 30240
TTTTTAAATCCTTAATCAAAACAAATAATCCACCAATACTAAAAATATTGTCAAAAAAGT 30300
TAATTCCAACCGTCTTGACAAACATGACAAACCTCACAATATCAAGCCACATAAAGACCA 30360
CAATAAAAGACCAAAATACGGTTGAAATAGAATTTAATATTCAAAAATCTAGTGTTGAAA 30420
GCCTTATAGAAAAACTAGCTTCAAATATTCAAACCTAAAATTTCTGCCACTCCACTAAAA 30480
TGAGGTATTATTTTGATTTTTGCAAGTAAAATAATGAAACAAAGCTCCAATTTTACCAGA 30540
TTCATTATTAAATTTAGTAGGCTCAAGTGCTACAAGATTTTTTATATTATTATTATTATC 30600
AAAAGCCATTTCTAAAGACCATAAATTTTCTAATTTTTCATATATTCTATCTATTAAATC 30660
GGGTCTTGCGCTTATTCCTCCTCCGATCAAAATTTTTTCAGGATTCAAAATAAAAGTTAA 30720
ATTAAAAATACCAAATGACAAATTCTCAAAAAATCTATCAACTTCATTTTTGGCATGAAT 30780
ATTCCCATTCTCAGCTAGATCAAAAACAAATTCTCCTGAAACCTCTTTTAAAGGTTTTCC 30840
TAATCGCATAGCAACTCTTTTTCTTAAAGCCGAAACAGACGCAATGGATTCCCATTTGCA 30900
ATTAAAGGGAATATTGTTGCTAATACCTCCAGTAATCATAAATCCAACCTCTCCGGACAT 30960
AAAAGAATTTCCTCTTAAAAGCTTGCCATTTGCAAAAATTCCAGCACCAATTCCTGTGCC 31020
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AAGAGTTATA GCAATAAAATTATTAGAGTCAATAGCATTACCCTTAAATTTTTCTGCTAA 31080
GGCTACACAA TTAGCATCATTTTCAATCTCTGTACTTACTCCGGTTAAAGATTCTAATCG 31140
CTCTTTTAAA GGATAATTAACAAATCCAGAAATAGCATTTACCCTAAGAACATTTCCCTT 31200
AAGATCAACA AACCCAGGAATACAAATTGCAACTCCCGCAATATCACTTGATTCTTTGTA 31260
AGAATTAATA ATATTAACTAAAATA'PTTACTTGTTCGTCAGAAGTAGCACCTGTGCTTAT 31320
TTCATTTTTA TCAAAAAAAACACCGCTTGAATCTGAAAGCGAATATTTGGTACTAGTCCC 31380
GCCAATATCA ATCGCTAAATAATGT'rTCATATTTATCCTCAAGGCCTAATTGTACATAAA 31440
TGATCATAAA TTTTCTATAGCAATA'PAAGAAATTTTAGAAATCTTGTTTATAACTATTTG 31500
CGCTTTAATC ATCTCCTTCAAATAAGAAACATGGGAAATTATGCCAATTTGTCGCCCAGT 31560
CATCATTTGA AACTTAGAAAGCTTAGGCATAACTTGAGCCAAAGTATCTTCATCAAGATT 31620
GCCAAAACCT TCATCTAGAAAAAAAGCCTCTATTTTTAACTCACTATCCCTTATTTTATC 31680
AGATAAAGCT AAGGACAAAGCTAAAGACACAAGAAATTTCTCACCCCCAGACAAAGTTTT 31740
TACCGTTCTT ATTTTATTAACATCTTTTTTGTCTTCAATTAAAAAATCAAACTCTTTGCT 31800
CTCTTTGTTC GTTTTGAGCTCAAAATCAGGAAAAATCCACCTTAAATACTTTTCATTTGC 31860
CAGTCTTAAA ATATCATTAATTAAAAAAGTTTGAACATAATATTTCAATCCAGAAGATCT 31920
AATAACGACC TTCCTTAATACATCTAGCTTATCTTTCCTTTCTTTAGCAAGATTTAATTC 31980
ACCCCTTAGC GAGTCTAAATTAATTTTTTGTTGATTAATCTTTTTTTGAAGAGTTTGAAA 32040
ATTTAAAAGC TTGATTTTATACTTT'PCAATATCTCTGGATAAAAATTCAAGCTTAGAACT 32100
AATTGATAAA CTCAATTTTTGCAAAAAAGACAGACTATTCTTATTAATTTGCTCTAAGTC 32160
AGTTGTTGAT TCAAAAGAAAATGAACTAAAAAAAACATTTTTTAAATTTGAAATTAGATT 32220
AATAAAATTA TTTTGCTCTTCATTTAATCTCGCTTTTAAAGTCAATATAGATTCCTTTGT 32280
AAATTTAATT TGTGTTTCGGTTTTAATTTTTAAATCTTCTAAATTTTTAAGATTTAAAAC 32340
AAGCATATTC CATTCATTTTCCACACTTTTCTGCTTAGCCAAAACAACATTAAATTCCCT 32400
TTCCAAAGAA GAATAATCATTAAAA(_'TTAAATTTAAATTAAGTTTTAACAATAAATCCTT 32460
AATCTTTAAA AGATTTTGATCAAAATTTTTATTTTTCAAAGAAATTTCAATTTTTAAATC 32520
TTTTTTCTTA GCCTTAAATTGTTCAAGCTTTTCTAATTTATTTTCAAATGCTAAAATTTT 32580
TTCTCTGTCA AAATAATTTATGTATTTGTCAAATAAATTTTTTCCAATCAATCTTAAAAT 32640
TTCAGCATTA TTCTTTTCAAACTCCAAAGCATTAACCTCTTGTTTAGAGATTTCTTCTTG 32700
TCTACAAGAT AGCTGATATTTAAGCTCATCAATTTGATTTTGTATCAAATGAAGCTTTGA 32760
ATTTAAAGCG TAAAGCTCTTTCAAACTGCAAAGCGATAATTTATCTTTATGCTGATAATT 32820
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TTTATATTCAGCCTCAATATATTTTAGCTTCTCTTTATCACTCTCAATAGAAAAATTTTT32880
ATCATCAAGATATTTTAACAACTCTTTATACAAGTCAATCTTAATAATATTTTTTTCCTT32940
ATTTTTATTAAAATCTTCTTTGCCAGTAGATTTTAATAAAAATTCTAGCCTATCTCTATA33000
TTTTGAAATCAACTCATCATTAAAAGTTTGGAACAATTTTAATGCTTCATAGTAAACATA33060
TTTGTCAAAATCAAAATTAGATTTCTCAGAAGAAATGCTTTTAATCTCATCATTTTTTTT33120
GCTCTGAGATCTTAATAATTCTTTTTTTTCATCCTCAAGACTATTTTTCCTTAAAAGCAA33180
GCTTTGATAATCATTCTCAACAAGTTTTAAATTAAAAAGATTGCAATTTTTATTATAAAG33240
CTCCTTAACATAATTAAAATTAAAATCATTGCTATACAAATCTTTAATTTCTTCTAAATT33300
ATTCATCACTTTTGAAAGTTCTAAATCAAGCAATCCAATATCATTAAACAATTCGCCTTG33360
TACAGCAACTAAATTTTTCAAACTCCAAAAATCTGAACATAAATAAACTTTTCGATCCAA33420
ATCCAAATTTTCTTTTAATTTCTTTTGAAGGGAATGATCCTTCTCTAAAGAATTTAAATA33480
TTTAATCTGAGAAGATAATTGATCGTTTAGAGAAGACATTTCAATTTCCAAGCCCAAATA33540
TCTCTCATTAGACGCTATTGCCTGATTACATAAAGAAATAGCTCTTCTAATATTTTCAAG33600
ATCAATTTCTAATCGATCAATATCAACCAAATCCAAATAACCTTTAAGGGATTTACACTC33660
ACTCTCATCATAATCAAGAATTGATTTTTCATAAGATTCAGAATTCAACAATTTATCTAT33720
ATTAAACTTTGTGCGCTCAAAATCACTTTTTAAATAAAATTCCAAATTATCATATTTTTT33780
CAAATTAAAAATATTATCAATTATTGCAGCTTTCTCTTTGGGAGTTGACGTTAAAAATTC33840
TTGAAAATTGCCTTGTGGTAAAATTACAGTTTGACAAAATTGGTTAAAGTCTAATCGACA33900
AAGACTTTTAATATGCTCTAAAACATCGGTTCGACCCTCTATAATCCTATTATCAAAAAA33960
ACAATTAAGCAACATGCTCTTAGGAGTCTCTATATTTTTTACATTAAGCTCAACAAAAGA34020
TTCATAAATCTTCCCAGAAATAGTAAACGTTAATTTAACATAAGCGCTAGTCTCGCCTTT34080
TGATATAATATCTACAATTTTTTTTCCAAGTCTGTAAACACGAGCATATAGTGCCAAAGT34140
TATGCAATCTAAAATGGTGCTTTTGCCTGATCCAGTATTACCAGAAATTAAAAAAATGCC34200
CGATTGTCTTAAAAGAAGTGTATCGAAATTCAACTCATGTTCGCCTTTATAAGAAGCAAT34260
ATTTTTAAATATGAGCTTATTTATCCTCATATTCACCCAAATATCCGTTAGCTAAAACCT34320
CGTTAAAAAGAGAAATAAGCTCTTCTTCCTTAAATTTGATATCCCTAATAACACCGTTCT34380
CAAAATCCCATCTCAACTTTTTCTCAAAAAAATATTTTTCATCCATTTCAAGCACTTCAA34440
GTTCTCCAATAAAGTTGGAATCGTCTTGCAAATCCTGACTTGAGGGTAAAGAATAGGAAA34500
TAGAAACTAAATTCATAAAATTAAGCCTTGCTAAATCATAAATAGACTCCTCAGCGCTAG34560
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TATCAACTGC CTCATTAAGTTCAATTTTCAAATAAATAGTAAAAGACTCTTCTTTTTTGG 34620
AATTAGCCAA AAAATCAAGAACTTCATTTAAAGAACCTTTAGCAAAGATTAATTTATTGA 34680
AAATCGGCAC CGGAAATGCTTCTTGCAAGATTAATTTATTGTCATTAAAATGTAAAACGT 34740
TTATGTATTT ATCACAGGTCTCATTAAATGAATATTGCATAGGAGATCCTGAATAAACAA 34800
TATTATCTCT TAGTTTCATGAACTTATGAATATGCCCAAGAGCAACATAAGAAAAACCAT 34860
TTCCAAAAAC ATTAAAAGGGATAATATAACTACCTCCCAAGGTGTCAATCTTTTTACTGC 34920
TGCCAAAAAA AGAATGCGCCATTAATATCTTAGGAATTCCTTTATACTTGTTTTCTAAAA 34980
AATTAGATAA ATTTGATATTTTTTCTCTGTAGGCATTTTCTAAATTTTCAAGAAATAGTT 35040
TGCTGGAATA CTGATCTTCCAGTCCAAAAATATTGTCAAAATTTTGACCTAAAATAAGCC 35100
TTTCATTTAT ATGCGGAAGACAAACAACAATAAACTTAAGATTTCCATTATCTTTTAATA 35160
AAACTATTTG CTCATCAGAATCATATTCAGTTAT'PAAAAAAAAATTAAACCGTGAGAGAA 35220
GTTTTTTATT TATACTCAAATAATCCTTTTTGTCATGATTTCCAGAAATAACCACACACC 35280
ATTTACAAGA AGTAAAAGAAAGTTCATAAAAAAAATTATTCACTAATCTTTGCTCTTCAA 35340
ACCCAGGCCT TTTGGAATCATAAACATCCCCGGCAACAAGTAAAAGATCTATATTTTCTT 35400
TTTTAATAAA TTCTAAAAGAAAATATAAAAAATTTTTCTGCTCCTTAAGAATTGAAAAAT 35460
TTTCAATTTT TTTTCCAATGTGCCAATCTGAAGTATGCAGAATTTTATAATTGCTCACAA 35520
AATACTCTCA CTTTTTTTAATTCTTAAATTATATTTATATATTATAATACAATATATAAA 35580
CATAGGGAAT TTATGCAAAATAAAAAGTTGATAATAGTTGAATCGCCAACAAAAGCCAAA 35640
ACAATAAAGA AGTTTCTCGATGAATCATTTCTAGTAGAAGCATGCATTGGACATGTAGTA 35700
GATCTACCAA ACAACGCAAAAGAAATCCCAAAAGAATATAAAAAATACGAATGGGCAAAT 35760
ATTTCTATAG ATTATAACAATGGATTTAATCCAATTTACATTATTCCCAGCAATAAAA.AA35820
CCAATTGTAT CAAAACTAAAAAAATTAGTAAAAACAATAAATGAAATATATCTTGCAACC 35880
GACCAAGACA GAGAAGGAGAAACTATAGCATTTCACTTAAAAGAAGTATTAAAAATCAAA 35940
AACTACAAAC GGATGATATTTCATGAAATCACAGAAACCGCAATAACTGAATCACTAAAA 36000
AATACTAGAA ATATAGACATGAACCTTGTTAATGCCGGGGAAGCTAGAAGAATATTGGAC 36060
CGACTATACG GGTATACAATCTCTCCACTACTTTGGAAAAAAGTAGCTTATGGACTTTCT 36120
GCTGGGCGAG TACAATCTGTTGGATTAAAATTATTAATAGAGAAAGAAAAAACTAGAATA 36180
AATTTCAAAA AGGCAAATTATTATTCAATTTTACTTCAATGTAAACACGAGAAAAAAAAC 36240
TTGTTGCTTG AAGCAAAATTAGAAGAAATTGACGGCAAAAATATAGCAGAGGGTAAAGAC 36300
TTTGTAAATG AAACTGGAAAACTTAAAAATATTGCCAAAACAACAATAATAACCCAAGAT 36360
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TTAATGATAGAGCTTGAAAAAGAATTAAAAAATGGACAAAAAATTGAATTAATTTCAATA 36420
GAAACTAAAAAAATAAAAATACCTCCTCCAAAGCCATTTACCACCTCTACACTTCAACAA 36480
GAAATAAATAAGCGTCTTAAAATTGGAACAAAGCAAATCATGCAACACGCTCAAAAACTT 36540
TACGAACACGGATACATTACCTATATGAGAACAGACTCTCATAATATTGCTAAAATTGCA 36600
AAAGATAAAATAACAAAAATAATAA.AAA.ATAAATATGGGAAAGAGTATATAGAGGAAAAA 36660
GATAGAATTTATGAAAAAGAAAAAATGGCTCAAAATGCACATGAGGCAATAAGGCCTTCT 36720
GAAATATTTATTCCAAATGAAACCATAGAAATAGAAAGCAAAACCGCTAAAGAAATTTAC 36780
AAAATAATATGGGATAGAACCATTATTTCTGGAATGAAAGATGCAATAAAAGAAAATATA 36840
AAACTGACTTTTAAATATAAAAACTTAATTTTCAGATCAAGTTTTACAAAAATAATTTTT 36900
GATGGATTTCTTAAACACACTAAAGAACAAGATGAACATCTTAACATAAATTTTGACTTA 36960
ATTAAAAAGGGAGATACATTTTCCATAGTTAAAATGAAAACAAGTGAGCACGAAACAAAG 37020
GCTCCATTTAGATACACAGAAGCGTCTCTTGTGCAAAAAATGGAAAAAGAAGGAATAGGT 37080
CGTCCCTCGACCTATTCTACAATTATATCAACACTTTTAGAAAGAGAATATGCATTCAAA 37140
CTTAACAACACATTAATGCCAACTATAAAAGGCGCTGCTGTAATAAATCTTCTTGAGAAA 37200
TATTTTCCAGTACTCATTGAACTAAATTTCACCTCTAATATGGAAGAAAAATTAGACAAA 37260
ATAGCAATAGGAAAACTAGATAAAATAAAATATCTAAGTAAATTTTATAATGGCAA.AAAA37320
GGACTAAAAGATACAGTAATGCAACTAGAGCCTAAAATTGATTCCTCTGAATTTAGAACC 37380
GTTATTGAAAGTCAAAAAATAGAAAATAAAAATAGCATTAATTACACAATAAACATTGGT 37440
AAATATGGGCCTTATTTGATATTCAAAGGACATAATTACTCAATTAATGCAAAAACTCCA 37500
TTAGAAAATTTGTACAAAAAAGATGAAATAGAAAAAATAATAAATGAAAAAGAGCTAAAA 37560
CCCAATATACTTGGGGTTGATCCTTTAACAGGACTTAATGTGATCTTTAAAAATACAATT 37620
TACGGAAACATTGTTCAACTTGGAGAAGATACCCATGCCCCTCAAGAATATACAAAAAAA 37680
GGAAAACCTAAAAAATTAAAAATAATAAAAGCAAAAAA.AGCATCAACTAAAAAAATTGAC 37740
CCTGAAAACATAACATTGGAGCTTGCTTTAAAATTGCTCTCACTGCCAAAACCAATTGGC 37800
AAACATCCCCAAACCAATGAACAAATCATTGCTGCAACTGGTGTTTTTGGGGATTATATT 37860
AAAACTGAAAGCGGAAGCATTGCTTGCTCGCTAAAAAAAGATTTAAAAGCATATGACATA 37920
ACACTAGACAAGGCCATCAGCCTACTCAACGAAAGAGCCAATAAAGTGGGTATAATCGTT 37980
AAAACAATCACATTTTCTAAAAACAAAATTGGCAACAAAATATATATTTACAAAAAAAAC 38040
GACAAATTTTATGCTAAAATTAAAAGAAAGAAGATTGATTTACCTGATAACATTAATCTT 38100
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GAAGAAATAA ATGAGAAATATGTATTCAGCTTGTTATAAATATGAATGATTTCAAACTCC 38160
CAATTTATAA ATACAAAGATGAATTAATTAAAGTACTAAAAAACCACAATGTTTTAATTG 38220
TAGAAAGTCC AACAGGTAGCGGAAAAACCACCCAACTACCAAGAATAATATATGAAGCGG 38280
GTTTTGCAAA ATTAGGAAAAATTGGAGTAACTCAACCAAGAAGAATAGCTACAGTATCAA 38340
TAGCTGAATA TATTGCCAAGCATATTGGCGTAAATGTTGGAGAAGAAGTTGGCTATAAGA 38400
TAAGATTTGA AGAAATTACAAGCCCAAAAACCAAAATCAAATTAATGACTGACGGAGTGC 38460
TTCTGCAAGA GCTAAAAAAAGATACACTGCTTTATGAATATGATGTAATAATAATAGACG 38520
AAGCACACGA AAGAAGTTTAAACATTGATTTTATATTGGGTCTTATCAAAGACATTTCAA 38580
GGAAAAGGGA TGATTTTAAAATCATAGTTTCGTCTGCTACAATAAACACAAAAATATTTT 38640
CAAAATATTT TAATAATGCACCGGTTGTTAGTATTGAAACTATCACTTACCCAGTACAAA 38700
TAATATACAA TCCTCCTCTTTTAAACACATCAAAAGGAATGATATTAAAAATAAAAGAAA 38760
TTGTCTTAAA CGTAATAAAAGAAAAAAAAGCGGGAGATATTCTTATATTTTTATCTGGAG 38820
AGAAAGAAAT AAAAGAAACTATAAAAGAATTACAAGAATTAAACTCAAAAAAAAATTTAA 38880
TAATATTTCC TTTATACGGCAGAATGCCCAAAGAAGCTCAAGAGCAAATATTTATGACTA 38940
CTCCTAAAAA TAAAAGAAAAATAATAGTGTCAACAAACATAGCAGAAACTTCAATCACAA 39000
TTGAAAATAT TAAAATAGTAATAGATAGTGGAAAAGTTAAAACAAATAAATTCCAAACAA 39060
AAACTCATAC CTATTCGCTCCAGGAAGTTCCAATTTCAAAATCATCAGCAACTCAAAGAG 39120
CTGGTCGAGC AGGAAGACTTTCAAAAGGAACTTGCTACAGACTTTACAAAAGAGAAGATT 39180
ATCAATTAAG AGAAGATTATCAAAAAGAAGAAATATATAGAACAGACCTATCTGAGGTAG 39240
TGTTGAGAAT GGCAGATATTGGAATTAGAGATTTTACCCACTTTGACTTTATCTCAAAAC 39300
CATCAACGCA TTCGATTCAAACTGCAAGCAAAATATTAAAATCTCTGGATGCTATAAACA 39360
ATAAAAACGA ACTTACAGAAATTGGGAAATATATGATACTATTCCCATTAATACCAGCAC 39420
ATTCAAGAGC ATTAGTCGAAGCAATGATAAATTACCCACAAGCGATCTATCAAACCACAA 39480
TAGGTCTATC ATTTTTATCCACAAGTGGAATTTTTCTACTACCCCAAAATGAAGAAATGG 39540
AAGCTAGACA AGCTCACTTAAAATATAAAAATCCAATGGGAGATTTAATTGGGTTTGTTA 39600
ATATCTTTGA AGATTTTAAAAAAGCTCTAAATAAAGAAGCTTTCACAAAGGAAAATTATT 39660
TAGATCTACA AGGACTTGAAGAGATAGCAAATGTGCAAATGCAGCTTGAAAACATTATTA 39720
GCAAATTAAA TATACCAATAATACAAAAAGGTGTTTTTGACAACGAAGGATATTTAAAAT 39780
CAATAATGAG AGGAATGAGGGATTATATTTGCTTTAAAACTTCAAAAAAGAAATATAAAA 39840
CCATCAAGGC TCAAAACGTAATAATTCATCCTGGATCACTTATTAGCACCGATTCTGTGA 39900
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AATATTTTGTTGCAGGAGAAATTATAGAAACTACAAAAATGTATGCAAGATCTATTGGTG39960
TCTTAAAAAAAGAATGGATTGATGACATTATCCTTAATGAAGAGTTTAAACATAACGACA40020
TATCTAGCAAAGAGAACCAAATAACAAATACCGGGCAGACAAAAATTATCAATGAAATCA40080
AAATAGGGAAAAAAATTTTCAAAGCGGAATACAAAAATAACATTTATGTAATAAAAATTA40140
ACCTAGAAACGCTAAAAGAAATAATTTTTAAAAACGAACTAAACAATCAAAATAATGAAG40200
ATCTCAAAAAAATTAAAATACAATTGATGCATAAAAATATAACGGTTTTTAACAACAAAA40260
AATTTTTAGAAACTATAGAAATAGTCAAAAACATGGGAAAAGATTGGCATTGTATAAAAA40320
AATATGAAACAAAGAATGTAAACATTGACGAACCTGAAAAAATGAAAAATCTTTTAGAAT40380
GCACAATGCAATTTATAAGCTTTCCCCCCAAAAAAAACGCTCTTTTTTTATCGTTGGAAA40440
CAGATTATTCTGGAAATTTTAGACTAAAACCCAAACAAAATTTCATAATGGCAATAGAAG40500
AATCTATAGAAAGCATAAAAAGCCTTATAGAAAACAAAGAATACATACAAAAGTTACATT40560
TTATAAAAAAATTAATAAATAAGGTTTACAAAAAATTAAATTACTTTTTTTAAAAACTAA40620
ACTTTGAAAGCCTTGTTATAATATAAAATATAATAATCAAATATTATTCAAAGTTAACAG40680
CAATGAAGTTTATAATAAATTATGAACTGGCTATCCTTTTTTTATGTTTTATTATTTTTA40740
TTAATTTTTCCTTTTGAATTACAGAGTAATAATAAAGAAAATATAGAAAATTTAATAAAG40800
CTACATATGCTTTATGATTTAACCAATAACCTGTCAAAAGAATTAGAAACAATAAATAAA40860
ATTAAAAATTTTGACTTAGAACAACATTATCTGCTAATTACAAAATATTATCTAAAAATA40920
AAAAAATATAAAGAAGCTAATGATTTTTTAAAAAAAATAAACCP~AAAAAAGATCAAAAAT40980
CAA.AAAATAAAAAACGAAATCATTTCGCTAAAATTAAGAATAAATGAAGATAATATTAAT41040
GAAGAAGAAATCAAAAAAATTTTAAATAACGAAAAAAATATAGATGTCAAAATAATTTAT41100
CAAATATTCAGTCTTATAAAATTTAAAAATAAAAAATTAGCAAATAAAATTAAAAACATA41160
ATACTAACAAACTATCCCAAAAGCATTTATTCTTATAAAATAAAAAGAAATGAATAAAAA41220
AATATTAACACTGCTAGTATTGATTTTAAGTATTTCATCAGTACTAATGCTGTCCAAATC41280
AATCACCAAAAAATCCAAATACAAAATTATTAGGGATTATTTCATAAACAGCAATTATGT41340
TCTGGTGAAAATTGAAAATAAAGATCTAAAATTTACCATATCAAAACCTATTTACGACAA41400
AAAGCTAAATAATTACTTCTTTAAAGGCCAAACAACAAGCCATTTCTTAATTTCTAACAA41460
TGTTGACATTGCAATTAACACAAGTCCATACGAAGTTAAACAAAACATGTTTTTCCCAAA41520
AGGACTATACATATATAATAAAAAAATGATTTCAAAACAAATAAATAACTACGGAGAGAT41580
TGTAATAAAGCACAACAAAATTATATTAAATCCCAAGGAAGACGAAATAGAAAACTGCGA41640
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TTATGGATTT AGCGGATTTTTTGTTTTAATCAAAAACGGAAAGTATAAAAAAAATTTTAA41700
AGAAACAAGG CACCCAAGAACAATAATAGGAACTGATAAAAATAACAAGCATTTATTTCT41760
TGTTACAATA GAAGGAAGGGGTGTCAATAATAGCAAAGGGGCCTCTCTTAATGAAGCTAT41820
TGATTTTGCA TTAAGCTACGGCATGACTAACGCTATTAATCTAGACGGGGGGGGCTCAAG41880
CACTCTTGTT GTAAAATCAAATAACGCTCCTTACAAATTAAACTTCACAGCAAACATCTT41940
TGGACAGGAA AGACCTGTCCCATTTCATTTAGGAATAAAACTTCCTAATTGAAAAATCTC42000
CAACCGATAT TAAATCCAAGCATAATCTCAGTTGTTAACCCAGAAAAATTTTTATAATTA42060
GAAAATGGAG AAATAGAAAGCATAAACAAAGGCCTAATATATAAACCATCAAGATCGGGA42120
ATAAAAAGAT CAGCAGCAAGCCCCATGCTTAAAAAAAAGAGATTGAAGTTATTAGAGCTT42180
AAATCAAAAG CATATTTAAACCCAATTAATGGGAAAAGCATTCTAACCTGCTCTTTGAAA42240
ACCATTGGAT ATGTTCCATAAAGCCCAAGCGAGAAATATCTCCCATTGTGAGTAACAACA42300
AAAGCCTCTT TGTAAGACATTTCAAAAAGTACATAATTTGCATCAAAAAATAAATTCAAA42360
TTAATCCCAT GATCTGCTCTGGTAAAATTTGGAGCAAATTTAGTGGCGCCTGTTTTATCA42420
GTATAATTAG TAAATTGATAAGAAAAACCTCCACCAAAAGAAAGTGGATAAGAAACAATT42480
AAATTATTAG AAGAGATGAGAAATAAAATAAAAAAAAGATATTTCTTCATTAACAATCCT42540
TAAAAATTCT AAAAAATACTATATTATTATAGTAACACACTAAAGTAGTATATAAAAAAT42600
CTGGGAAATT ATGAATACAAAAACATTATATTTAATATCCTTAATTCTTTTAGCTTGCAA42660
TAAAAATAAC AAAATTCCTCTCATTCAAAAATTAGATTTGCCCAAAAGCAGCATTCTTGG42720
CTTTAGCAAT AAAATGGGCATAATAATAAAAGATTATGCTTTTCTTAGTAAAAGCACTAA42780
GAAAAATAGC GAATTGGATTATGATTACGCAATTCTACTCAGAAAAGACGAAGTCGTAAA42840
AATTGAAAAA ACACTAGAAAAAACAGAGCGCTATGGAATTGAAGGAAATTGGATCCTAGT42900
CAATTACAAG GGAACTAAAAGATACATCTTTAGCAAAGACATCAATATAGTCAACAATTT42960
AATAATTGAT CATTCTAAATAGCTTTACTACATAACCGGACAAAAGTCCGATCAATGTAA43020
TAAATTACTT ATTTTTTTTCTTATGTCTATTTTTTCTTCTTTTTTTCTTCCTTTTATGGG43080
TAGAAATTTT TTTTAATTTTCTTTTTCTTCCGCAAGGCACTCCCTAAATCTCCTTATCAA43140
CCTTAAAAAT TAAATTCAAAATGTTATCTAAATCATCCTCTGGATGAAGCCATAAAACAT43200
CAGAAATTTT GGCAAAAAAGGTCATTTGCCTTTTTGCATATAAAAACGAATTTTTGTTTA43260
TTAAACCTAT TATATCATTTAAACCATAGCAAGGTCTACTTTTCCATAACAAAAACTCAT43320
TATAGCCTAT TCCTTTAAAAGCCGGAGTATTTTCATTGTAACCCTTGCTAAATAAACCTT43380
TAATCTCGGA AAGTAGTCCACTATTAAGCATTTCATTAATTCTTATTGATATTCTGGTTT43440
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TCAAATCTTCAAAAGATCTCTTAAGGCCTATAATCACAATATTTTTAAATTCGCTACTTT 43500
GTTTTTTTTGAAATTGGCTAATAGGAATTCCTGTTTGATAGTAAACCTCAAGCGATCTTT 43560
TAATGCGATAAATATCATTCTTATTTAACATATTAAATCTGATGGGATCTACATTTTTTA 43620
ATTCTTTTAAAAGATAAGATTTACCCTTAAGCTCTAAAAGATTGTTTACATAAATTCTTA 43680
TTTTAGAAGTAACCAAGGGTGTTGAAGGAAATCCATCCTTTAAATGCTTAAAATAAAAAG 43740
CAGTACCTCCTACAAATATAGGAATTTTTTTTTTCTGTCTTATTTCTTTTACTATTTTTA 43800
AAGCTTGTTCGTAAAAAATTCCAATAGTATAATCCTTTTCGGGATCTAAAAAATCTACTA 43860
AATGATGCTTTATATGTTTCATTAAATTTTTACTTGGCTTTGAAGAAGCTATATTAAACT 43920
CTTTATAAACTTGAATAGAGTCAACATTAATAATTTCTGCTTTATTTTTTGGAAAATGAA 43980
ATAAAATATTGCTTTTGCCCACAGCTGTAGGGCCAAAAATAAAAACTACTCTATCTTCCT 44040
TCAATTGAATATCTAAATTTACCAGTCAAAACGTCATTAAAAGCTTGCCCTAATATTTTA 44100
TCATCAAAAATAGCGTGTTCTGCTAAAGAAATATTGTCTATAATTTGCTCAGTGCGCATT 44160
ATAGTTGCAACAACAAGTTCATAAAAATTTCCATCAAAATCTTGTATTTTTTTTAAAGGC 44220
ACTTTAGTCATTTAATCTCCTTATAAACCAAACATATTAAATAAGTTTAGCTATTTTTTT 44280
TCAAGTTTCTTTTATCAATTTCATCTCTAATTAATTCAAAAACTTCATTTGGATTAATAT 44340
TTGTAACATCAATCACTAAATCGGTCTCTGAAAAATAATCATCAATATCTATATTGTAAA 44400
TAGCTAAATATCTTTTTTTGTCATTTTCATCTCTAATAAAAGTACTGCTTAAAACGTCAG 44460
AATACATGCCCCCCTCTCTAGTCATTATTCTCTCAGCTCTAACTTCCATTTTAGCATAAA 44520
GATATATTTTTAAATCAGCACTCTTAGAAATCCAAATAGCAAGACGAGATGCAAGCACTG 44580
TATTATTTTTTCTAGAAAGCACAGACAATCTATTATCAAGGTATTTATCCCAATAATAAT 44640
CATTTCTGCCTATTATCTCTTTTTCATAAAACTCTGAAAAAGGAATATTATGCTCTCTTG 44700
CAATATCATGAAAAGTATAATTAATAAACTCAAGACCGTAATGTTTGGCAATCATCCCGC 44760
TTACAGTAGTATTGCCACAACCACTCTTACCAGAAAGTGCTATTTTCATTCAACATTCCT 44820
TATTTGCTCTTTTATTTTTTCTAAATTTAACTTCATATTCAAAATCAAATTCTTAATATC 44880
AAGATCAACCGCCTTATTACTCATGGTTGTTATTTCTCTGTGCATTTCTTGAGAAATAAA 44940
TTCAAGAGCTTTACCACATATCTCATATTCAAGATTTTTATAAAAAGTTTCTATATGAGA 45000
ATCTAAGCGCATGATCTCTTCATTAATGTCTAGACGAATTGCCATTTTAGCTGCCTCTTC 45060
TGCAATATTTAAATCTCTAAATTCATCCATTAATTTAGAAATATTTTCTTTAATACTTGC 45120
AAATAATTTGACATTTATATCACTGCAAGCATCTTTAACAATTTTAAGGTCCCGCTCTAT 45180
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TAACACAAGG GTTGACACTATGTCTGACTTGGTATTTTCTCCTTCAAAACTTCTTCCATT45240
ATTGTAATGT AATAAAGCTTCTTCTAGAACACCTTTAAACAACCCATAAATCTCTTCTTG45300
ATGTTCACTA TCCTCATCAATTATCAAAGCTCCTTTTAATGATAAA.AAATCGCCCAAACT45360
TAGTTCGTTT TTAATATTAAGATTGGTATGTGCCAAAGAATCTCTAAGCCTAGAAATAGC45420
CTCAATATAA TTGGGATTAATCGTAAAATTCACACTAGGAACCAATTCTTTATATCCTAC45480
ATTTAAAA.AA ACATTGCCTCTGCTAATATATTTTGAAATCAAATTTCTTATATCAAGATC45540
ATAGCCAGAA AAAATTTCTGGTAACCTAAATTTAAATTCTAAAAACTTTCCATTATAAGA45600
TTTCAAATTA ACACTAAACATATAGTTACCAATTATCTTTTCCAAATAAAAAAATCCCGT45660
CATGCTTTTC AATATAACACCCTTACAGAAAATCGTGTAAAATAAAATTATTTCCAGCGC45720
CCATTGTAAT AAACAAGTCTCCAGATATTAATAAACTTTTTATAAAATTAATAGAGTCTT45780
TAACATCCTT AAAAAAATAAGTATTCTTATTTATTTTTTTAATATTTAAAAACAATTTAA45840
CAGAAAGTTC ATCTGGATTAAAATTTTCCCTATTTGAAAGATATATATTGTGCAAAATTA45900
ATATATCGGC AGCACTTAGAACTTCAACAAAATCGGCAAAAAATTCTTTTGTTCTTGTAA45960
AGGTATGAGG CATAAAATCCAAAATTATACGTTTATTCTTATAAAAATTTTTAATACCAA46020
AAAGAGTATT TTTAATTTCCCTAGGATGATGAGCATAATCGTCCATGTAAATCACTCCAT46080
TTTCCTCTTT AACAACTTCAACCCTTCTTTTTATACCGCTATAATTTTTTGCAATTCTCT46140
TTATTGCTTC TTCAAAATCAAAAATTGATTTCCCATTACTTTCTAAGAAAAGATTTAAAG46200
CCAAAAGCGC TGCTGAAAAATTTAATACATTATGAAATAAAACAGTCTTAAGCTCAACAT46260
TTAACAAGCC TAAAAAAGAAAAACAAAAATATTCACTCCTAACTGCAATATTACTTATTT46320
GAAAATCAGA TAAATCTCCAGACCCATAGCTAAAAATACTTATATCTTTTCTGTTGATTT46380
GCCTTTTAAT TTTAAGCAAATTATTATCATCGGAATTAATTATCAATATTCCATTTTTCT46440
TTAAATTATT AATATACTGTAAAAAAGCCTCTTCAAGAGCCTCATAATTTTTAAAAAAAT46500
CAACATGCTC GTAGTCAACATTGGTTAAAATAAGCATATTAGGGCTAAAATTCAAAAAAT46560
GTTTCTTATA TTCACAAGTTTCAACAATAAAAATATTGCTAATACCTGCTATTGCAGAAT46620
TATCTTTAAA ATCTTTAACACTTGACCCCACAATAACATTGGGATTTAATCCTAATTTAT46680
TAAAAAGAAC ACCTAAAAACGCCGTAGTGGTAGTTTTACCATGAGAACCTGCAATTCCAA46740
TGCTATAGTA CTTTCTAGAAAGCTCTCCAAGAGCCTCAGGATAAGATAAAATAGGTATAT46800
TTAATTCTTT TGCCTCAAGTAAAACTTGCAAACCATCCTTATTATAGGCTGAAGAATATA46860
CTATTAAATC AAAAGACCTATCAAGCTGTTTTAATGAAAACTCATAAATATTATCATAAT46920
AAGATATTTT ATTATTACTTAAAATTTCATCGGTATAAAATTTATCAGAAACATCTACCC46980
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CTTCTACACAATACCCTTTTGAATTTAAAAAACAAGCCAGAGAACAAGCCCCACTTCCCT 47040
TTATTCCTACAAAAAAAATATTATTCAAATCGTCAAAATCAACCTTCATAGCTCTCTTCT 47100
AAATAATCTTTTTTGTCATAAAATTTATGTATAACTATATCGATAGATAATATATTAATT 47160
ATTGTATACTTAAGTACGTTCGTAATAATATAGGTTCTAAGCATTTCTGTATTATTAAGC 47220
AAGTTATTTCCAAGAGGAATATTTAAAAACAGTTTAAAAAGGTAATTGTTTCCATCTTTT 47280
TTGATTTTAAGATCATAAACAATATAATTTCTATCATACTCAGAAACACAATGTTCAATA 47340
ATTTGCCTTACAGCTCTCTTAGAAATAGATAAAACCCCTTCTTCATAAAAATGGGGCCTT 47400
ACAACAGATCGAATATAATTTTTCTTCCTAGCAAAGAACCATCCGCTTTTP,AAAAAAACT47460
TTAATTGAGTTTAATAGCAAATTGGGCCTAATAGACGTTATTTCAAAAGCAGCTGCTGGT 47520
ACAACATGCTCCCCCATTTGCCTTGAAATTCTTGCTTTTTCTATTTCCTGCCTGGTTGAA 47580
ACATCTGTTATGTAAATAATCTTAAAAACATTTGGCAACAAAAGTTTTGAAATTATTTTA 47640
TCTATCATTTTAAGACTTGTTCCTAATATTAATATTTTATTAAATTCTTCTTTTGCAAGC 47700
ACTTCAAGCATTTCTCTGCAATGAGCATCATCTTCAAATACAGATCGCCTTACCGCTTTA 47760
AAAACATTATCTTCAAACTTAGCAGAACTTCCGGCAATAATTTTCATATTTTTAATTAAA 47820
ACACCATCATCAATTATTAAAGGTATTGAATATTTATCTGCTACCAAATGCGATCTAAAG 47880
CTCTTGCCAGTTCCAGCAGATCCTACTAATGCATACACCTTAACCCTAACAAATTTATGC 47940
TTAATGCTAATTGCAAAGTCTTTAACCTTGCTTAATATTTTTTTTAAAAAAAAATCACTT 48000
GAAAAATTCTGAAAATTCATAGACATCAAAAATATCTTTTACTTGCTTTAAAAATCTAAA 48060
CTAGGCTTAGAGCAAAATTCATTCTTTTTAAAGAATGTTCCATTAAATTTTACCAAAAAA 48120
GCATGTAAAAAGTAATCGCTTTTCTTGAATTTATTACAATATTTCTTGTCATTAATTAAA 48180
GGATGATTGTTAAAGGAACACTGAGACCTTATTTGATGGGTAAAGCCTGTTTCAATAACA 48240
ATCTCGACAAGAGTAGCTCTTTTGCAAGATAATATTGGATTAACCTTTGTAATTGCATTA 48300
ACAAAATTTTTATCTTCTAAAACAAAAGTTTTTCTCAACCTTTTATTTCTAAATAAATGA 48360
TTTTTATAAACAACAGGAGACTTAACCTCGCCTAAAAGTATTGCAAAATATTTTTTAATT 48420
ATAGATCCACCACTAAATGCCTCACTTAGCTTTCTTGCAGTATTTATATTTTTTGCAAAA 48480
ATAATAATACCAGAAGTATTTCTGTCAAGCCTGTGAACTGCCGAAGGCTTAAAGCTTAGG 48540
GATCTTAAATTTTGACTTAAAAGATAAGAATTCACTAAAAAATCAAGAGAATTTTTACCT 48600
CCATGAACTAAAATACCTTTTTGCTTATCTAAAACAAGTAAGTCACTGTCTTCATAAATT 48660
ATTCTTTTTCGAATATATTGAAAATCAATATTGCTTTTAAAGCATTTATCCGTGGTTAAG 48720
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TTCAAATTTT GGGCTAAAGATTTGTACAAATAAATTTTATCACCTTTGCAAACTCTGCAT48780
GAAAAATGTG ATTTTAAACCATTTAGCCTAATGTCACCTTTTCTAATATGTTTTATTATA48840
CTCGCTTTAG AAAAATTTAAAATTTTAATTAAAATTGAATCTAGTCGCTTGCCATTATCA48900
TTAGCAAGCA CTTCTAAAAAAATATATTTATCCAAACGCAAAAAATACCCCTAACAAACC48960
TTACTATTTT TTTTACAAAAAAAATTAACTACTAAAAATGTAAATATAGAAACAAAAAAT49020
GATGGAAAA.A CGGGGTGAAAAAACCAAATATTTAAACCAAAGAATAAAATGGACAAATAA49080
AATATTAACC CTAAAAACATAGAAGCAAAAGCCGCTATTTTGCTTACAAAATTTAAATAA49140
AGTCCAAAAA CAATAATAGGGAAAAACGAAACTTCCAAAGCTCCAAAGGCAAAAATATTA49200
ATAAAGAATA AAAAATTGGGAGGAAAGAGAGAAAATATAAGTATTATTAAAATAAAAAAA49260
ATATTAGAAA TCATTATTATTCTGCCAATCTTTACATCTTCTTTTAAATCTTCTTTATAA49320
ATAAATATTG ACTTTATTAAAACAGATGTTATTAATAGCAAATTTGAATCCACTGTAGAC49380
ATTATTGCAG ATAAAAGACCTATAAAAAACATAAAACAAGAAAAAGGATTTAAAACTTTT49440
AAAGCCACAT TTAAAACAACTTTATCATTTGGACTTAAATCTGGAAAAAGAATAATAGCA49500
AAAAACCCTA TTAAATGCATCAAAACAATTAAAAAGCTAATAATAAAAGTAGAAATGGGA49560
AGAGAAAATT TTATAGCATTCTCATCTTTAAATGCTATAAAATTATTAATAATCTGAGGC49620
TGCCCTAGTA TTCCTATTCCTATTAATATCCAAAAAGAAATTATATATTGTGGCTTTAAG49680
TCAGCATTTG AAGGAAGTAAAAGGCTTTTATCTAAGCTAGACGTTGCTGTTTTGAATAAA49740
TTATTAATAC CCCCTCCCAAATCTAGCATCTTGGAAAACAAAATAACGGATGAAACTAGC49800
ATTAAAAATC CTTGAATCAAATCCGTATAAGCTACTGCCTTAAAGCCGCCAAAAAATACA49860
TAAATAAAAA CCAAGAAGGCAAAAAAAGTAAGACCAACTACGTAATCAATACCCCAAAAA49920
ACTTCTATAA GTTTGGCACCACCTATTAATTGGGCAGAAATCAAAAACATTGF,AAAAAAA49980
ATCAATACAA ATCCACTCATTAACGCCAAAAAATCACTTTCATATCTATGCCTAATATAA50040
TCAATAATAT TAATTGCATTAATTTTTTTTGATTCGCGATTTAATCTCTGACCAACAATA50100
ATAAAAACAA TTAAAGTTGTAGGAATTTGTATGGTAGCTAATAATATAAAAGATAATCCA50160
TACTTATAAA CAGCAGAGGGACCGGAAATAAAACTACTAGCACTAATATAGCTAGAAGAA50220
AATAACAAAG CCATAACAATAAAATTAATATTTCGATTTGCAAGAAAATATTTATTTAAT50280
AACAAAAACC TACCTCTATTTCTTTTTTTAAGAAAATCTAAAAATAAAAAAATATCAATA50340
ATGTATCAAG TTTTACTAATTACTAAAATAAAAAAACAAACCAAAAAAA.AATTATACTGG50400
GGAATAAAAT TCCTGACAAAAAAAACCACAAAGGAATATTAAATATAGTAGTTGATGTGT50460
CAATAAAATA GGCAAAACAAAACCACAATACAAACATAAAAACATACAATAATATAGCGT50520
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ACAAAATCCCATTTCTCATTTAAAACAACCTTAACAAAAGGACATCAAAATTTATAAATT50580
CAAATATAATGTATATTATATAATATATATTATATGGATAAAAATAAACATATATTAATT50640
GGTATATGTGGGGGCATAGCCTCTTACAAGTCAGTTTACATAGTTTCCAGTTTAGTTAAA50700
TTAGGATACAAAGTTAAAGTTATAATGACACAAAATGCAACTAAATTTATTACTCCATTA50760
ACTTTAGAAACCATTTCTAAGAACAAAATAATTACTAATTTATGGGATTTAGACCACAAT50820
GAGGTGGAGCATATAAAAATTGCAAAATGGGCACACCTAATTCTTGTTATTCCTGCTACC50880
TACAACACAATATCTAAAATTGCATCAGGAATTGCTGATGATGCATTAACTACAATAATA50940
TCTGCAAGCACGGCTCCTACTTATTTTGCAATAGCAATGAATAATATAATGTATTCAAAC51000
CCTATTTTAAAAGAAAATATAAAAAAGCTTAAAACTTATAATTATAAATTCATTGAACCT51060
GATAAAGGATTTTTAGCTTGCTCATCAAATGCTTTAGGGCGCCTTAAAAATGAAGACAAA51120
ATTATAAAAATAATATTGAATGAATTTAATCAAAAAGACTACCTAAAAAATAAAA.AAATA51180
CTTATAACAGCATCCAGAACTGAAGAATTAATAGATCCAATTCGCTATTTCTCAAATACA51240
TCAACGGGAAAAATGGGGTTTTGCTTAGCACAAGAGGCTGTCAAACTAGGAGCTCAAGTT51300
ACAATTATTACAGGACCAACCAATGAAAATGATCCTGAAGGGGTCAACATTATAAAAATA51360
AAAACTGCAATGGAAATGTACAAGGAAGCTCTCAAAATATATAATAAATTTGAAATAATA52420
ATTGGAGCCGCAGCTGTTGCCGATTTTAAACCCAAACACATTTTCAATAGTAAAATTAAA51480
AAAAATAAAATCAATAGATTATATATAAAATTAGTAAAAAATCCCGACATAATCCAACAC51540
ATAGGACACAATAAGCTTAAAAACCAAATTGTTATTGGATTTTGCGCTGAGAATTCTAAA51600
AATTTAATTCAAAAAGCTAAAGAAAAATTAAAAAAGAAAAACTTGGACTTTATCATTGCA51660
AATGAACTTAAATATTTTGGTTCAAAATTAAACAAAGTTTATATAATAAATAAACAAAGC51720
ATAAAAGAACTGCCAGAAATGGAAAAATCAGAAGTAGCTAAAGAAATTTTAAAAATTTTA51780
TACTAATATGCTTAATAGTTTATTAATAATCAATAATCTTTTAAAAGCATTTTAATATAT51840
TCGGAGTTCGTTTCACTTTTAATTTTTTTAAGCTTATCAGAAAGCGATGAAGATTTATTA51900
TAAACAGCTCCTTTTAATGCTAAATGCCTTAAATCAATATTTTTGCTGTCAATGATTTTA51960
GAATAAAAATTATCAAAATTACCCTTTTTACCAGCCAACATTGAAGCAACGCCCCTTAAA52020
ACATTTGAGGGTCTATTAATATTTTCTTTATTAACAATTTCTAAAGCAATTGACAATGCT52080
TTTAGAGAATCCTTATCTAAAAGGTAACTAAACATTGAAATTTTAAAATTATTGTCAATC52140
TTAAAATCAAACATAATGTTTTTTATCTCAATATTCCCAAGATCCATATCAATTAAGGCC52200
TTAGCAGAAGCCTCCCTAACTTTAAGAGATGGATCGCTTTTAAGCTTATAAATCAAAATA52260
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TCCTTTGCAG AAGAGTCCCTATGTCCTTTGATTGCATTAATAGCTTTAAACCTAATATTA52320
TCATCAGAAT CTCTTAAAAATCCTTGTAAAATCTCTTTAGACTTTAAAGAAGGATCTTTG52380
GACAAAGAAG CAATAATAGCTAATTTAACATTTAAATTATTGTTATTACTCTGAAGATAC52440
AAATCAGCAT TTTCAGTTACTTTATCTGAAGCAAGATATGACAACGCTTCGATTGCAGCA52500
GCCTTAATTG ATGGGCCCTCGTAATTATCTAGCGAAATTTCATAAATTCTATCCTGATAA52560
TCAACAGCGG ACATTTTTCCAAGAGCAATAAGTATTTCTCTTCTAGCCCCATCATTTCCA52620
GAATATTTTT CAAAAACTTCCATCATGTTTTTAGAATACTCAAGAGAATTAAGCTCTCCT52680
AAATAATAAG CTGCAATAGATACCACATTGCCCTCTTTATTTTCAAGAATGTCAATAAGA52740
GTTTTTTTTA ATTTTTCTTTATCATCAAACTCCTTAAGATACGAAATTGCCAAGCCAAAT52800
AAAGCGTTTG AATATCTTTTACTCTCATAATTTTCAAGAATATAATTTGCTGTATCAATG52860
CCCCCCGAAT ACTTAAGAGAAATAAACAATTCAAGTATTTCCCTTTTAAGCTCAGCATTA52920
AAAGTTTTCT CAAGTCTTTTTTTAAGAGAAAAATTATATTGACTATCGCTTGATTTTTTA52980
AGAGCTTTTA TAATGCTTGTCACTTGACTATCAAGCCCATAAAGAATTGTATCGTTAACA53040
TACTTACCAT CTAAACCAACATTAGAAAAATTCTCTCCCTTAGAAGAATTTTCTCTCTCA53100
ACAGGCTTAT TTTCTGTAATTTCGGGC'.AACAAAGGCGGACTAGGAAGAGCTGGAGAATTA53160
ACATTTTGAG CATACACATTAAAAATAAGTAAAAAAAATAAAAAATAAAAGTATTTCATA53220
AAGCATCCCT TCTAATATATCTAAAAAGCTTATTTATTCCTAAAACAGAATAACAAATAA53280
ACAAAACAAA AATACTAACAATTCCAGCTGCCATTAAAAAATAAAGATTTTTAAAACTAA53340
ACCCCACATC CCACTGAAACTTTTCAAAAAAGAAATAAATTGCATATAAAGGAAAAAGTG53400
TAATAATTGA CTTTAAAAGAACAAATAAAATTTCAATTAAATCAATTTTAACTCCTCTTT53460
TCAATATTAT AAAATAAAAAACAATTACACAAATCATAAAAGAAATAGATTGAGCTAATG53520
CTAAAGCGTT CAAACCATAATAATTAATACCAAAAACAGATATTGCAATATCAAGAATAG53580
AAAATAAAAC ACTCAAATAAAACGGTGTTTTTGCATCACGAATAGAAAAATAATATTTTT53640
GGAA.AAAACC AAACATTGAATAAAAAAGCAGACCTAAAAGAAAACATTTCAAAACACTCG53700
CTGTTTTTTG AGTATCATAAATAGAAAACTTGCCTCCCATAAGAAATAAATTTAAAATAT53760
AATCAGACCA AATAAACATTAAAAAAGACACTGGAA'rAAAAATTAACAATAAAATTTTAA53820
TTCCATCTAC TAAAAGGGCATTTAATTTTATATTATTCCCCAAAACAGCATGCTCTGCCA53880
TTTTGGGGAA AATCACTGTTGCAATAGAAATATAAAAAATTCCTACAGGAAGCTGATAAT53940
AAACTACAGC ATTACTAAGGATAGAAACACTTCCTATCTCAAGAGTAGATGCTAATGCAA54000
ATGAAATCTG CTGAGTAATAATTGAAATGGGAAAATCCAAGAATCATACGAAGCCATCTG54060
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GTTAAAAATT TCTCTGAAAT 54120
TAAAACCTTT AAAATGTTGG
CTTCCAGGCA
AAACCAATCA
TAAGGCAATTTGCAAACGGAATTAAAAATTGTAAAAACCCCCCAAAAATTACGCCAATAA 54180
CAGCACTATATATTCCAAAACGACCATAAAATAAGAATATGCTCAATATTATTCCAAAAG 54240
AAAGCATAATGGGCGAAAACGAAGGAATGAAAAAAATTTTATATGAATTTAGAACAGACA 54300
CGAAGATTGATGATAGGCTTATTAGTAAAATATATAATACCAAATAACCAAATACAGAAC 54360
TTGCAAAAATTAAGTTTTCTCCCCTATAATAAGATATAAAATACATAATAGGCTTTGCAA 54420
AAATAATCATAACTAAAACAATTAACCCAATAGAAATAATGTTAAAGGTTATGACAGTTC 54480
TGAAAAAAGAAACAGCTTTTTCGTGCGATTTGTTTTTTTCATGTGTAAATTCAGGCAAAA 54540
AAGCCGAGGTCATCGCGCCCTCTGAAAGAATTTTGCGCAAATTATTAGGAATATTGAAAA 54600
CATAGTTAAAAATATCAGCATCAAGATTTGCACCAAAATAATAAGAGAAAATCTTTATCT 54660
TTACAAAGCCCATTATTCTTGAAAAA.AAAGTGGAAATCATGACCAAAATTGTAGAAACAA 54720
CATATTTATTCATCGAAATTTTCCTCTTCATACTTTTTTAAATATGAAGTTCTAAAATTT 54780
AAAAAATCATCATTTAGAATTGCGGCTCTGATCTTTGAAATCAATCGAAACATATAGTGG 54840
ATATTATGTTCACTTGCCAAAACTATTCCAAAAAGCTCTTTCGATTTTATTAAATGTCTT 54900
AAATATCCTCTTGAATACCTTTTACATAAAGTACAGATGCAATTTTTCTCTACCTTAGAA 54960
GTATCATCCTTATACTCCTTTCTACCAATGCACATAATCCCATTATCTGTCAAAAGAGAC 55020
CCATGCCTAGTAATTCTTGCGGGATTAAAGCAATCAAAAATATCAATGCCATAATATATG 55080
GCATTAAGTATGTAATGGGGAGTGCCAATACCCATTACATACCTTGGTTTTTCTTTTGGT 55140
ATCAACAAAAAACTATATTCAAGGATTTCTAAATATTTCTCCCTTGGTTCTCCAACAGAA 55200
ATGCCTCCAATGGCAATACCTGGGCTGTCTAATTCCAATATATCATTGATACTTCTTTTC 55260
CTTAAATCTTTAAAAAAATTTCCTTGAGTTATTAAAAATAAAAGCCCGTTGTATCCCTCT 55320
TTTCTGTTTTTAGAAGATTTGAACGTGCTGCTAGCCCAATTGGTTGTAATATTTGTATAT 55380
AAATTGGCTTCATTATAATCAATCCCATAAGAACTGCAAATGTCAAGTGGCATAATAATA 55440
TCACTGCCAAAAATTTCTTGCATAGCAAATATTCCCTCGGAAGTAAAATAATGGTACGAT 55500
CCATCTATATGAGATTTAAAATGCACACCTTTTAGATCAATTTTTCTCAGATCAGAAAAA 55560
GAAAACACCCGAAATCCGCCCGAATCGGTTAAAAA.ATTTTTATTCCAAATTGTAAAATTA 55620
TGAAGACCAACATATTTTTCAACAGTTTTAATTCCCAGCCTTAAATATAAATGATAAGTA 55680
TTTGCAAGCATCAAATTACATTCTAACTTCTCAAGAACAGCATGTTTTAACCCTTTCATT 55740
GCCCCCAAAGTACCAACTGGCATAAAACAAGGAATATCTACTCTACCATG 55800
AGGAAGATTT
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AAAAATCCAA CCCTTGCATTAAAATGCTTATCATTCTTGATTACACTAAACATATAAATA55860
TCCCAAATAA TTATATATATTATTTCCTAACAATCC.'TATAAACAAAAAAATTGATAATCA55920
AAA.AAAATAT AGTTGTAAAAGAACTTGCTATATATATATGCAAATAGCCTAGATCCGCTA559$0
AAAAATTAAA AATTACAATAGAAATAACATAAACAACAGCAAAAGCAATGCTATTTAATA56040
GGCTGAGTAT AAAAATATTTTTTTTAAGAGCAAGAGCAATAAACCCAACAGTAAAGCTAA56100
GAAGAATCAA TCTAAATGAAAAGAATATCCTATTTAATAAATCAAAAAATGCATCAGAAT56160
AATTTAAACG TTCAGCTTTGAGAAAACTTATCCAATTAATAAGTTTAGTAAAATTTAACG56220
CCTTTGATGA GAGCATCACAGTTCTTATGTAATCGGGCGCCAGCTTAATAATTCCTGTCC56280
CATCAAGAAC ATCGTAGGCGTTCTCCTTAATTTTTTTACCAACCTTAACAAACTCTCTAA56340
TACCATAAAG CCTCCATTTATTATCTTTCCATTCGGCTTTATTTATATCGTACCTTGTTT56400
GAAACTCATC TTTATTGTCTTTAATTATAATCATCAAGTTAGCAAAAGTATTCTCATCAA56460
TATCATAAGA TTTGATATTATAAATTTCTCTAGCAAAATCCCTTATTATTATAGTTTTAT56520
CCCCAGATCT ACTGTCGCCAATGCTATTCTTAATAAGAACATCTCTTCTTGCTATAGTAT56580
CTATTACCAA ATAATTATCAAAAAAGAAAAGAACAACTGAAATAAATATACTAATTAAAA56640
TAATTGGTTT TAATATCCTGGTAAGTGGAACTCCACAACTAAAAAGACCTATTATTTCAT56700
TTCTCATAGA AAGATTGCCAATAAGATTCGAAATAGCAAAAAGAAAAGATAAAGCCACCC56760
CATCTGAGAA TGCCTTTGGCAAATATAAATAATAAATATAAAGAATATCCTTAAGGCCAA56820
TATTCTTTTC AAGATAGTTAAGAAGATTAACAAACAAATCACCAAGCATAATTAAAATCA56880
TGAAAAGCAG GTTCATGGACAAAAAAGTAAGAATCiATGCTTTTTATAAAAAGCTTATCTA56940
TTTTCATTTT TTTAACAATCTCAAAAAGAGAATTGCTCCTGCAATAATTAAAATTAAATT57000
AGGCAAAATA GTAACAATAATAGGACTTGGTGCATACTGCACAGTATAAACTTTTCCACC57060
AATAAACATT ACCCAATAAAAAACACAAACAATAATTGAAATTACAAGTTCAAGAATAAT57120
GGAATATTTT CTATTAGAATACATTCCCATTGAAAAAGCTAAAA.AAATAAAAAATAAAAC57180
TGAAAGTGGT AAACTAATTTTTTGATAAAATTCAAGATTAAACAGAGCCAAATTTTGCTT57240
CATGCTTCTA TCTTGATAAGGTTTGAAATTTAAATTTAAATTATACATATAGTTTAAATT57300
TTCAAAAACA TAAGATTCATCCACATAATAGTTTTGATTGTATAAATAATTTAAATAAAG57360
ATTTGAAAAA TTTAAACTTAAAAAGTCTCCTTCTAGATTATTTTTTATATTTGAATCTGC57420
AATTAAATTA TTTTGCTTTTTAATTAATTTTATAACATCTCTCATGCTCATTTGTGAAGG57480
AGTTACATAA TTTAATAAAAAACTATCACTAAATGTAACCTGATCGATTGAATATTTCAT57540
CTTATCTGCA TAAAAATAATCATAAAATCCACTCTCACTGTCTGTTAAGGCAATAGATAG57600
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AACATCATTTAAAATAAAATACACTTGAAAATTTTCTTTTCTAATATCAAGATTTTTTGC57660
CATAAATATTCTATCAAAACCCTTAAGCCCAGTGTTATCAAAAAAAGTTACATTTTTATA57720
ACCATTTTCCGATTTCTCACCAGAAACAAAAATCAAATCTCCATATTGTTTGCTTGAATA57780
AGGCTTTAATACCAAATGGGGAACTTCTTCTTTTATTTCATTAAAAATTTTTAATCTGCC57840
AATAGATCCAAGTGGAAGTAAAATATCATTGGATATAAAAGATACAAAAGCAATAACTAT57900
TCCCAATTTAAAAAATGGGACAAGTAAATCAAAAATTGATATGCCAATTGAACGAAAAGC57960
TAAAATTTCATTGTGAAGCTTGAATTTATGAATAGTAAGAATTACTGAAATCAAAGAAGC58020
AAAAGGGGGAGAAAGCGCAATAACCATAGGAAGAGAATATATAATAAAAATAAAAGCCTT58080
AAAAAAGGGAACATAATTTTGAAGAAGTATTCTCATAAAGAATAAAATTTGATTTATAAA58140
AAATACGAAAAAGAAAAATAAAAACGTAATTAAAAAATATTTAAAAAATTCAGCAATTAT58200
GTAAGACTCATAACTGTTTTTTAATATTTTCATCTACAAAAACCAAACCGTTATTAATAG58260
TGCCCAGTATGACATAATTTTCAAATCTAGAAACTTTTATTAAATTCAAATTAAGAAGCC58320
CATTATTGGGTCCAAAATAATCCCAGTTGTCATTTTCAGAATCATAAATCAATAACCCAT58380
GATCAAAGGTTGCAAACAATAGCTTTTTATCTTTAATCTCCATATCCATAAAATAATTAA58440
CATCAATATTATTGGCAATAACGTGCTTTTTGTAACTATTTTTATTTAAATTTAATTCAA58500
AAAGACCCCCACCATATGTTCCAACAAAATAACTATCTTTATATTCTTTTATAAAATTAA58560
TATTTTTTTCATTATCATTTTTGCTAAAAAAATCCAAATGTTCAATCTTTTTCAAATTAT58620
CGACATTAACACTATAAATAGCCTTGTCAACTGTTCCAACTAATAATAAATTTTTTAAAC58680
TATCAAAGCAGAGTGAAGAAATTTTATTAGATCCAAGCGGTATATTTTTCCAATTTTTTA58740
AATCATAAAACCATAATCCAGAATTTAGAGTGCCAACAAATATTCCATTTTTAACAGCAA58800
GCAAAACTTGTACATTGCTAAAATCAGCATTACCGGGAACATTTATTTGCTTTAAATCCC58860
CATCAACATCATCTATATAATAAACAACATTTTTACCACCAATATAAATTGTTCCATTAT58920
AATCCGCAAAACCCCTAATGCCATTTAAAAAAATGCTTTTTTTATCCTTAAGATAGACTC58980
TACAATCATTTTTTTTAATATTATATCTTAAAAGCCCTCCCAATATATTAGTTACAAATA59040
TATTGTCATTAAAGACAAATGTATCAAAAACGCTGTTGTCAAGAAATCCTAAAGACTCTA59100
AGTTTAAATGACTTACTCCAAGTTTATTACTTAAAAAGCCATAAATCTCTCTATCATAAC59160
CTGAGATAGAAAATTCATTAATCTCTTTAAATGCAGAAATTGCATCAGATTTTTCTTTAA59220
CAAGATATTTTAATTCAGCTAATCTTAAACTAGCATGAGAATATTTATAGTCTTTTAAAA59280
ATAGATCAAAATTATATTCGGAAAGATCATAAAAACCATTCTCATAATTTACATATCCAA59340
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AAAACAAATT CGCTTTAGCTAGCAATTCTCTGCTGTGCTGATTCTCATTA 59400
GTTACTATTT
TATTCAAATA ATAATTTGTCAAGCCAATATTTTTTTTAGCATAACTTTCCCTGGCTTTTT 59460
TAAAATAAAA ATTATTTTCTTTTAAAAAAGCATCACTAAGAAAAAAAGATTCATTATCTT 59520
TTAATCCTAT CAAATAGTCTCTTTTAAATTTACCCTCATTACTCCCAAGAACAACATTAT 59580
TATCATCAAT AATGACTGCTTCTTTTTTCTTCTCTACAATATCACTAATATGAGAATCTT 59640
GAATAGATCT ATCTGTAGTAAGGCAGGAAAAAAACAAGAAAAAACATATAAGACAACCTT 59700
TAAATAAATT ATTCAAAACAAATTTCATATTATTTTAATAATCTTTATCTCTAACAATTT 59760
CAAGATCAAT TTTTCCAAACTTGTCTATATCAATTATTTTGACCTTAATTCGCTGACCTT 59820
CTTCTAATTT TGGGGGTCGAACTAACCCCGCATTACCTCTTATATTCTCTCCACCGCCAC 59880
CAAATCTAGA ATATCTATTACTATTCCCAAATCTTCCAGAACCATACTTACTGTCTCTGG 59940
GTTTCAAACG AGTACTTAAAAATCCTTCCTTTGCAGGAGTAAGTTCAATAAAAGCCCCAA 60000
AGCTATTAAT CTTTTTGACAGTTCCTTCATAAATTTC'.GCCTACCTTTGGCTCTCTTACAA 60060
TACTCTCTAT TCTTTCTTTAGCTTTTTGCATCTTAAAATCATCATCCCCGAAAAGAATGA 60120
TTTTTCCATT CTGCTCAATTTGAACCTTAACTTCAAATTCATCTGTTATAGCCTTAACAG 60180
TTTTTCCAGT AGATCCTATCACAAGAGATATCTTGTCAATGTCAATTTGAAGTTGAACAA 60240
TTTTAGGAGC ATACTTAGATATACCAACTCTTGAATTAGAAATTACAGTATTCATAATAG 60300
ATAATATATG TATTCTACCTATTCTTGCTTGCTCAAGAGCATCTCTCATTAAATCTTTAG 60360
'
TAACATTTTC AATCTTAATATCCATTTGAAATCCAGTAATTCCATTTTTTGTACCGGCCA 60420
CTTTAAAGTC CATATCACCTAGATGATCTTCTTCTCCAAGAATATCACTTAAAACTACAT 60480
ATTTATCCCC TTCGCTAATAAGCCCCATGGCTATCCCCGCAACCTGCCCTTTAACAGGAA 60540
CCCCTGCTGA CATTAAAGACATGCTCCCAGCACAAACAGTAGCCATTGAAGAAGATCCGT 60600
TAGACTCTAA AACCTCAGAAACTACCCTAATGGTATAAGGAAAATCATTTTTTCCAGGAA 60660
CCATTGATTC TAAAGCTCTTTGAGCTAAATGACCATGGCCAATCTCGCGCCTGCCAGTCA 60720
TTAGTCTACC GGTCTCACCAACTGAAAATGGGGGAAAATTGTAGTGGAGCATAAAATTAA 60780
GGCGTTTATC GCCATCAATATCATCCA'rTATTTGTTCATCAATGCTTGTACCAAGAGTAG 60840
TTACCGCTAA AGCTTGCGTCTCTCCCCTTGTAAAAAGCGCAGATCCATGCGTTCTACTTA 60900
AAATATCAAC TTCTGAGATAATATCTCTTATCTCATTAGGAGTTCTGCCATCTGTTCTAA 60960
TATTATCGTT AAGAATAGAGCTTCTAACAATCTCCTTCTCAAAATCATCAAAAGCCTTAT 61020
GAAAAAGAGA TTCATTGCTATCAGTCAATTTCTCAAGAGAAGAAAAGTACTCATAAGATT 61080
TATTTCGCAG CAAAGTTATGGCTTTATC'.TCTATTAAGCTTTCCCTTAACAAAACAAGCTT 61140
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CTTTAAGATCAGCATAAACAAAATCCCTAAGCTCATCTTTAAATTCAAATATTTTTTCTT 61200
CAAAAGCTAAAGGAAGTTTTTCCTTCTTGCCTACAATATCTAAAAATTCTTTTTGAGCAT 61260
TACAAATTTGCTTAATATATTCATGAGCACCATCTATTGCTGAGAGCAAAATATCCTCAC 61320
CAACCTCATTAGCACCACCTTCTACCATAGTAATTCCATTTAAACTTCCGGCAACAACAA 61380
TATCAAGATCAGAATCATGAATCTCTTCAAACGAAGGGTTTACTATAAACTTACCATTCA 61440
AATAAACCATTCTAACAGCTGCAATTGGACCATTAAACGGAATATCTGACAAAAAAACTG 61500
CCGTAAAAGCAGCATTCATTCCAACAATATCAGGAGGATTAAGCTGATCTGTAGCTAAAG 61560
TTGTAGGAATTACTTGAATTTCTCGACCAAATCTTTTATCAAAAAGAGGTCTCATCGGCC 61620
TGTCTATTAGTCTGGAAACAAGTATTTCTTTATCCTTTGGCTTTCCTTCTCTTTTGATAA 61680
ATCCTCCCGGAATTTTACCGGCTGCATAATATTTCTCATTATATTCAACAGAAAGCGGAA 61740
CAAAATCTAAATCTTCTCTCACGTTACTCGAGCAACAAACAGTTGCAAGAACCGAAGATC 61800
CACCATAAGTTGCAAGAACCGATCCATTAGCCTGTTTAGCCATAAATCCGGTCTCAAACA 61860
CTAACTCGTCTCTGCCTATTTTCAACTTTAATATTTTCCTCAAAATTCAACCTCTTTTTA 61920
TTTTCTAAGACCAAGTTTAGATATCAACATCCTATAAGCTTCTAAATCTTTTTTCTGGTA 61980
ATACCGCAATAAACTTCGCCTTTGCCCTACTAACTTTAACAAGCCTCTTTTTGAACTATG 62040
ATCTTTTTTATTTATCTTTAAATGTTCAGTTAAATACTTTATTCTACCTGTAATAAGTGC 62100
AATCTGAACCCCAACAGAACCAGTATCACTTTCATTTTTTCCAAATTCAGAAACTATTTT 62160
TTGCTTTTGCTTTTTATCTATCATAAAGCAACTCCTATACCATTATAGCAAAGCTCTAAC 62220
AAACCTCTTGCCATAATTTAAAATAACTACTACGATAGATTATAATATTTTTTCTTAAAA 62280
ATAACAAAAGCAATTTATCCTTTTCGGGTTATTTTTAATAATAAATTATTAAATTGTTTA 62340
AAAAAACAAATATAAAATTAAAATGTCATAATATATTTATAATTAAAATATAATGACATA 62400
TTTATATTTATTCAAACCCACTCCTTGAATTACTGCTAATATTTTCTCTTCTCTGGATTT 62460
TAAAATTTTAAATTCATTAATATTGATTTCAATTTCAAAATAAACACCATTTTTAACAAG 62520
ATTTATCTTATTAGAATCAATGTAAACCTTTTCAAAACTTTTTAAAGATTCTAGACTAAT 62580
TAAGGAAGCTTTACTCAAATTTTCACACAATGTGGAATCTTTTAATCTAAACATACCTAC 62640
TTTAGTCCTTTTTAAATTACTAACATACGCGCAAGAATTTAGAGAATATGCCAAATCTCT 62700
TGCAATACTCCTAATATAAGTACCTTTTGAACAGCTAATTTTCAAACTAAGCAAAGAAGA 62760
ACTAAAATCATAACTTAATCTTTGAATATTATAAACAGTGACTTTTCGTTTTTTAATTTC 62820
AAAAAACTTTCCATTCAAAGCAAGTTTATAGGCTCTGCTGCCATCAATATGAACAGAAGA 62880
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
193
AAATCTAGGA GGACTTTGATAAATCTCTCCTACAAAATCTTTAAGCTTTAAATCTATATC62940
CTCTACATTA GGAATATAATCTGTTTTACTAACTATTCTTCCATTCGGATCAAGGGTATC63000
TGTTTCTAAT CCAAATCTGAATTCTGCTACATACTCTTTATCTAAAGAAGTAAAATAACC63060
TGAAAGCTTT GTGTATTTTCCCACAAGACAAACCAAAATTCCACTTGCAAATTTATCAAG63120
TGTGCCAGCA TGCCCAACACGATTTGTATTAAAATATTTTTTTATAGGGAAAAGAGTTTC63180
AAAAGAAGTT TTACCTTGTTCTTTATTAATTAAAAGGAATCCATTTTCCAAATTTAATTC63240
TCTCTTGTAG TATTTAATCCTTCAATTAACTTATTAACATAAAATGATTTGGAAAGAGAA63300
TCATCCTTAA CAAATAATAATTTGGGAGTGCTTCTAACTTTAATTCGCTTAATAATTTGA63360
CTTTGAATAA ATCCCTTAGCATTATTTAAAGCTTTAACTGCATTGTCCAAAGAAGCACCT6342C
TCCTTAATAG AGCCCATAAACACTTTAGCATTTATTAAATCTTTTGAAAATTCTACTTTA63480
ACCACGGTTA AAAATGAATGAATTCTGGGATCTTTAATCCCCCCACTTACTATTAAATTG63540
CCGATTTCTT GAGCAATAAAACTTTCAAGTTTAAACTTTTTAATATTCTTATACATAAAC63600
ACATATAAAT AAAACAATACTAAGTTTTAAAAGATTTTTTAACCTTTTTTACCTCAAATG63660
CTTCAATTAT ATCTCCTTCTTTAATATTAGCATAATTATCAATCATAATACCACACTCAT63720
ATTGCTCAGC AACTTCTTTAACATCATCTTTAAATCGCTTTAAAGATGAAATTTTGCCGG63780
AATGAATCTG TAAACCATCTCTCATTACATTAGTAATCGCATCTCGCTTTATTAGCCCCC63840
GAGAAACATA ACAACCGGCTATTACCCCTATTTTAGGAACATTTATTACAGCTCTCACTT63900
CAGCAAAGCC AATAAACTGCTGCTCAACATCTGGCTCAAGCATTCCTTCAAGAACTGACC63960
TAACATCATT TATAGCATCATAAATAACATTGTACTTTCTAATCTCAACTTTTTCCTGAT64020
CTGCTAGTAC CTGAGCTTTTGCAGTAGGCCTTACATGAAATCCAATAACAATAGCATCGC64080
TTGCTGAAGC AAAGCTAATATCTGTT'PCGGTTATTACCCCTGCTGATGAATGCACAACTC64140
TTACTCGAAC CTCATCGTTTGTTAATTTTTCAAGAGAATTCTTTAAAGCTTCCACTGAGC64200
CTTGAACATC TGCTTTTAAAATTATTTTAAGCTCTTTAAGCGCTCCTTCTTTAATTGAAT64260
CATAAAGATT CAACATAGTAACTTTCTTTACATTTTTGGAAGATTCATATTTTTTAAGAT64320
CTTGTCTTTT AGAACTGATCAATTTTGCTTCTTTTTCAGTTTTAGTTACTTGAAAAGGAT64380
w CCCCGGCTTG AGGCATTGAAGAAAATCCTAAAACACTAATGGCTTTAGCGGGTCCAACGC64440
TCTTAACAGA AACACCCTTTTCGCTAATTAATGCCTTAACTTTACCATAGCACGCTCCAC64500
CCACAAAAGA ATCTCCCACATAAAGCGTTCCATCCTCAATAATAACAGAACAAACTATTC64560
CGCGCCCCAA ATCAATCTTGGCATCAAGCACTTTTCCAATAGCTCTTTTGGATGGATTTG64620
CCTTTAACAA CATCATATCTGACTGTAAAAGAATCATATCAAGTAGTTCAGAAATTCCTA64680
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
194
TATTTTTAAGAGCAGAAATCATCACAAAAATAGTATCTCCCCCCCAATCCTCAGATACTA 64740
AACCGTATTCTGAAAGCTGGTGTTTAATCTTATCGGGATTTGAATCTGGTAAATCAATCT 64800
TATTTATAGCAACAATAATTGGAACATTTGCCTCTTTTGCATGATTGATAGCCTCAATGG 64860
TTTGGGGCATAACACCATCAATTGCTGACACAACAAGAACAACAATATCTGTAACTTGAG 64920
CCCCACGACTTCTCATCATAGTAAAAGCTTCATGACCAGGAGTATCTAAAAATGTTATTT 64980
CTCGATCATTATAAACAATAGTATAAGCTCCAATATGCTGAGTAATACCACCGGACTCTG 65040
TTTGATTTATATCTATATTTTGAAGCACAGAAAGTAGTTTGGTTTTGCCATGATCAACAT 65100
GACCCATTATTGTAATAACAGGAGGCTTTTCAACTCTTTTGCTTTGATCTTCCACTTCTT 65160
CTTCTATAACCGTTTCATCATAAATAGAGACAACATTAACTTTTGAACCATATTCTTCAA 65220
CTAAAATAGTTGCAGTATCAGAATCTATCTTTTCATTAATAGTAACCATTACGCCCAAAG 65280
CCATTAATTTAGCAATCAAATCAGAAGATTTTAAATTCATCTTTCTTGCAAGATCAGAAA 65340
CAGTAATGCTACCCATAATGTCAATTGACTTTGGAATAGGGTTGGCTAAATTTTCTCTCT 65400
TCTTTTTCTGAAGTTGTTCAAAAACTTTTTGTTCAATTGTTTTGCTCTCAGTTTCTGCTT 65460
TTTTTCTCTTATAGCTTTTTTGACTCTCTTGTTGCTGTTTTTTTTTCTCGCCAAGCTTAC 65520
GATTTAACTCTTTACTATTCTCAGAATCCGCTGCAGGTGTGCTGCTAACAATAGCGGGAA 65580
CTTTAGTTTTTATAAGTCTTCTAAAAGACATAGAAGTAGTAGTATATTTATTTTGAGAAT 65640
TATTTTTGGCAACATATGTTTTCTTTACTGAACCTTGATATTGAAAGGATAAGCTGTCTC 65700
TGTTTTGTGAATATCCACCAGTTCTGTTATCTCTGTTTTGTGAATATCCACCAGTTCTAT 65760
TGTCTCTGTTTTGTGAATATCCACCAGTTCTATTGTCCCTGTTTTGTGAATATCCACCAG 65820
TTCTATTGTCCCTGCTTTGTGAATATCCACCAGTTCTGTTGTCTCTGCCTTGTGAATATC 65880
CACCTCTATTATCCCTATTTTGTGAATACCCACCAGTTCTGTTGTCTCTGTTTTGTGAAT 65940
ACCCACCAGTTCTGTTGTCTCTGTTTTGTGAATACCCACCAGTTCTGTTGTCTCTGTTTT 66000
GGGAATATCCACCAGCTCTATTGTCCCTATTTTGTGAATATCCACCAGCTCTATTGTCCC 66060
TATTTTGTGAATACCCGCCAGTTCTATTGTCTCTACTTTGCGAATATTCAGCCTTATTGC 66120
TGTTATTATGCAAATCAACAAAGCTATTTGAATCATTTTTAACGCTTAAATCATTATATG 66180
TTACAATTTTTACTACCTTCTTTTTCAACTTAATAATCTTAACTTTTTTGCCATCTTCAT 66240
TTTTAATATCATCAATATTTTTCGACAAACCTACTCCTCCTCAAACTCAAAACTAAGCCC 66300
TATTTTACAACCCGGACAGGAGGTCATATTTTCATTAATAACAACACCGCATTCAGGACA 66360
AAGAAGCTCTTCATCTTCTTCTACCTTTTCCATAGACTCATCATTGTCATTAGCAATTAT 66420
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
195
TATCATCCCC TCTTTTAATATTTTGTTAATTTCTTCTTGTTTTTCATAACTTACACCAAG 66480
ATTAAAAAGC ACTCCCTCATCTGCTTGTAAAAAATTGTTAATATCATCAAACCCCTCTTT 66540
TGATAAATTA GAAATCACAGAAGGATCAAGCAATTTAAGATCACTTATTTTACTAATCTC 66600
TTCAAATTGC TCCTCCTCAACAACAT(:TTGCATAACTTTATCAAACATTTCGAGTGTTTC 66660
TTGCTTAAAC TCCGAATTAGCCTTCATTTCTGCAAATTGACTGCTAGTTTTAACATCAAT 66720
AGCCCAGTCA AGAAGTCTATTAGCAAGTCTAACATTTTGACCCATTTTACCTATAGCAAG 66780
AGAAAGCTGG TCATCACTAACAACCACTAAAGCTTTATGTAAATCCTCGTCAAGAATATA 66840
AACATGTTCT ATCTTTGAAGGAGTCAAAGAATCCTTTATAAATTCTTTAATATCTTTACT 66900
ATAGGGAATA ATATCAATTTTTTCTCCTTCAAGTTCCTTAATTATAGATTGAATTCTGAC 66960
TCCTTTTTGT CCTATACAAGGACCAACAGGATCAATCTCTTCTTTTTCAGAATAAACAGC 67020
GACTTTGATT CTGTAACCAGGATCGCGAACTATTTTATGAATCTTAATAATACCTTCTTC 67080
AATTTCTGGA ATTTCAAGCGCTAAAAGCTCTTCAATAAACTTTGGATGGGTCCTAGAAAG 67140
AATAACTTCA ATACCATTTTTACCCTTTTTGACATTATAAACTAAAACTCTAATCTTATC 67200
ATTAAGGTTA TAAACTTCTCTTGGCGATTGATATTTCTTGGGAATTATACCATCCGTATT 67260
ACCAAGATTA ACATAAAGATCACCATTTCTATTTTGTTGAACGTACCCAATAACAACCTT 67320
ATTCAACTTG CTTTTAAATTCTGATAAAATCTCATTATCCTCAATTCCTTGCAGGTCATT 67380
TTTGGTTCTT TGTTTTGCAACCTGAATAGAAAGCCTATCAAAAACTTTGGGATTAATTTC 67440
AATGTAAGCA TAATCACCTTCTACAATATTTTCCTTTGAGATATCTTTTTCTAATATTTC 67500
AAGCAAAGAA TCTTTTACCTCTTTTACAATTTTCTTTTTTGCATAAACAGACAAATCTCC 67560
CGTATCATCA TCAAACTTAATAAAAGCATTCTCATTGCTTCCAAAATACTTCTTATAAGC 67620
TATTAATACT GATTCTTTAATTGTTTTTCTAATAGAATCTATACTCATGCCACGATCATT 67680
TGCAATATTT ACAATCATATGCCCCGTGCCCTTTATCATCCAAACTTCCTCCTTAAACTA 67740
ATCTAGCCTT TTTAACATCACTATAAAAAACATTTACTTCTTTGCTATCTGTTTTAAAAA 67800
TAAAACTTTT TGGCTTTGACTCTAATATAAAACCCTCTTCAAATTCATTATCCAACATCA 67860
ACTTAATCTT TTTACCTTCAAAAATTTTAAACTCTCTGTCACTTTTTATTTTTCTATCTA 67920
TTCCTGGAGT AGAAAGCTCTAAAGTAAAACCATATTTAAGATTTGCTTCTAAAATTAATA 67980
AAATCATTTT ATGCAAATCAGTCAAAAPATCAATATCTAAGGAAAAATTTTTACTATAAA 68040
GAACTATTTG AATTTTTCCATTATTTTTATTTCTAAAGATATTAATTTCTAATATCTCAA 68100
CATTTAACCG CCCTGTTAAATCTTTTATCAAATTAAAAACTTCATTATTTTTGTCAAAAT 68160
ACTTAATCAA CTGTATTCCTAAAAATAATAAGGTTCTTTTAAAGAACCTTATTAAATACA 68220
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
196
TAAACTAAACCTTAAGTCAAGGTTAACACTTAGCAAAAATAATGTCAACATTAAACCAAA 68280
TTATAAGATTTGGCCAAAGAAAGCTTTATCACCTTATAAGCTCCCAATTGCATAATAAGA 68340
TCTTCACAAATGCACATAGATGCTCCTGTGGTAACAATATCATCAAGTAAAACAATCTTT 68400
TTAAACTGAAAATTTTTATATTTTGATCTTAATTTAATCTTATTTTCAAGATTTTTAAAT 68460
CTAAGATTCCCTTTCATTAACTTCTGGCTTTTTCCATACTTTCTTGAAAAAATATTTATA 68520
TAATTAAAACCAAAACGGCTTAACAAAATACCAATGTATTCCATATGATCAAAACCATAA 68580
AATAATTTTCTTTTAAAACTACAAGGAACAGTTACTATTTGATCAAAATCAATATTATTT 68640
AAACATTCAGCAATTCCACTTGCCAAAAATCTACCAATTGACTTTTGAGCATCCCTTTTA 68700
TAAGACAAAATTAAAGATTTGTAATGCTCTTTATATTCAAAAAAATAAATCAAATTCTCA 68760
TCAAATTTAATGTTAAAATTAAAAAGTGACTTACATTGGTCACAAAGAGCATTAGAAGAT 68820
ACATACCTTTTTCCACAAAAGACACAAAAAGGCAAAAATATACTCTTTAAAACATTTAAA 68880
TAGCTCATACTAACTGGACTGAGAAATAACCTTTAAAACAATTTGATTTAACAGCTCAAT 68940
TGATTGAAAAGGAGTAATATTATTAATATCTATGTTAGAAATAAAATTTTTTAACTCTAA 69000
ATACTCATTTAATTTAATATGAATATCAGTGTCATTTTTCAAGATCTCTTTATCATTACC 69060
ATCAGAAGAAACATGGGGAAGAAACTCTAAACAAGAGTTGCCCTCTCGGCCCACCAAACT 69120
TTCTAGAATAACATTAGCTCTATCTATTACCCTTAAGGGAAGTCCTGCTATGCGAGCAAC 69180
ATAAATACCATAAGAATTAAGAGATGGCTTTTCTTCAACTTCTCTTAAGAAAACAAGATC 69240
GTTGCCCTGCTTTTCAATTTTCATTGAAAGATTAATAAAAGCCTGATGATTAATAGACGA 69300
CAATTCATGAAAATGTGTGGCAAACAAACTTCTAGCTTTAATATACTCTAAAATATACTC 69360
TATAATAGAATAAGCAATAGCAAGCCCATCATTTGTGCTAGTACCTCTTCCAACTTCATC 69420
CATAATTATTAAACTCTTTTCTGTTGCATTCCTTAAAATGTTGGCTGTTTCATTCATTTC 69480
AACTAAAAAAGTGGATTCCCCTTTGGCAATGTTATCACTTGCTCCAATCCTGCAAAAAAT 69540
TTTATCTGTAATACCTATTAAAGCTTTAGAAGCTGGCACAAAAGAGCCTATATGCGCCAT 69600
TAAAGTAATTAAAGCCACCTGACGCAAATAGGTTGATTTACCTGCCATATTAGGTCCAGT 69660
AATTAAACAAAAATACTTTTCTTTATTAATTCTTACAAAATTTTCAGTAAAGATTTCAGT 69720
ATTTTTAGTGTAGTGCTCAACAACAGGATGCCGAGACTTTTCAAGAAGAATTTCTTTACC 69780
AGATGTCAATACAGGCCTTTTATATTCATTTTTTTTTGCCAAATAACCAAAGTTAACAAC 69840
TAAATCAATATATGCAAAAAATTCTGCAACCTTTTTAAGAACTTTATTATGCATAACAAC 69900
ATTTGATGCTATTTCATCAAAAATTTCCTGTTCAAAAGCAACCACATTATCTTCAGCATT 69960
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
197
ATTAATATCC ACCTCAAGAGAAATAAGTTTTTCTGTTTTATATCTTTTTGAAGAATTTAA 70020
AGCTTGGCTT TCCATAAAATGTGGTGGCACTTGAC~CATAATTACTCTTTGTAACTTCAAA 70080
AAATAACCCC CTATTATTAGTTTTTC'rAATCTTTAGGTTATTAATCTTGCTAAGCAATCT 70140
CTCTGATTCA AGATATTGATCAATATATTTATTTGCATTAATCTTTAAATCTTTTAAGTT 70200
ATCAAGCTTT AAGTCATAACCTCTTT'PAATAAGTTCATCAGGTGCACTTGAAATTGCACT 70260
ATTTATCAAA AAATAAACTTTAGAAATACTATCCTCTTCAAATTTATCAAAATTCCAATA 70320
ATCAAAATTA TGCTTGTCAAATAACTTTTTTACCCiTAAAAAATACAGAAAGAGCTTTTTC 70380
AATAAATAAA AAATCTTTTTTAATATATCTTTTCATTTGAATCCTAGATATTATTCTCTC 70440
AATATCCCAT ATATTAATAAAAGTTTCTCTTAAAGTCACAGTCAAGCTAATATTTTTGCA 70500
P.AA.AAATTCA ACATGATCTAGCCTGGTATTAATCTCAGAAATATTTAAAATTGGATTTAA 70560
AATAAATTCT CTTAAAAGTCTCTTTCCCATTGCAGTTTTGCAATCATTTAACACAGAATA 70620
TAATGAATAT TGAGAAGAAAAATCATTATTATTTTTTACAAGTTCAAGATTAACTTGAGT 70680
TACGTCATCA AGAAACATGTACGAAGAATCATTATTGATATCTATTTTATCAATATTACT 70740
TAATAAATTT TTTAAATTATTTTTTATATGATTTATAATAAGAAAAATTGAAATGTAATA 70800
GGGCTTTTCC TCATCAAATCCAAGAGAGCTCAATCCAAGTATGTTAAAATGCTCCTTTAT 70860
TGTTTTTATT GCAATATCCTTATCAAGATGCCAAGTAGGAACTCTGTTAATTAAAAATCT 70920
ACTAAGATTA AGCTTCTCTGAGTATTCATAATAAAAATTTTCAGAAACTATTATCTCTTT 70980
AGGAGAGTAT TTCTCAAGATCCCTTTTAAGTTTTTCAAAAAAACCATTCTCATAAAACAT 71040
TATTCCAAGA CTGGAAGTAGATAAATC'TATATAAGAAAACGAATAATAATCTTTATAATC 71100
ACTAATAGCA ACTAAATAGTTATTAATATCATCATTTAAAAAATCTTCATCAATAATAAC 71160
GCCTGGGGTT ATTACCTCAACAACCTCTCTTTCTAAAGGCCCCCCAGAAGTAGAATTGGA 71220
CGCTTGTTCA CAAATTGCAACCTTTTTATCAAATAAAATTAATTTCCTTATATATTCTTT 71280
ACTGGTATGA TAAGGAACCCCACACATTGGAACATTTTCTCTTTTTGTCAACGTTAAATT 71340
AAGAAGCTTG CTTACCTCAATTGCATCATCAAAAAACATTTCATAAAAACTTCCTACTCT 71400
GAAAAAAAGA ACAGCATCTTTATATTTTTTCTTGATATCTAAATACTGCCTTATCATTGG 71460
GGTAACATTT TTTTCCATATGCTTCCTAAATAATATTGAATTACAATTGATATTATAAAA 71520
TAAATATAAT TCAATTAAAAAGAAAGAATATAAAATAATAAAAAGACCATAAAAAAAATA 71580
TTTTACGCAA TTAAACGCTATTTAATTATTAAAAAGCCTAATGTTTTAAATTTAATTAAC 71640
TTTAAGGGTT TTTATTGTCCTTTTCTAAAAGATGCTTAACAACATCGTTTGTTATATCAA 71700
CAGTGCTATT GTGGTAAAGAATATATGGATTATTTTTTTTCATAATCAAAGAAA.ACCCAT71760
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
198
TAATTTCTGCAACATATTGAATACCACGAAGTATTTTACTTAAAGATTCACTATTATTAT 71820
TTAAACTATTAATATTGGCCAATCTCTGCTGTTCTAAATTATTCTTTGCTAAACTAGACA 71880
CTCTCTTTAGCTCATCAACTTTCAAATTATATTGATTTCCAAAAGATCTTGCATTATCTA 71940
AATCATTATCAGCAATCGATTTATCATACATATGTTTTAAATTCTTAAGCTCTAAATTTA 72000
ACGTATTTATTTGTTCTTGATACCGATTTTTTATTTGATCAAGATTAGCTTTCAATTGAG 72060
GATTTAAAACTTCAATCACAATTCTATCAAAATCAACAATTCCTATCTTAATAACATCTA 72120
TCGAAAAAACATTAAAAGATAATAAAAAAAACAATGGTAAAATCAAAAAAAACACAAATT 72180
TCTCCATAGCATTAATCAATATCTCATCTCAATTCCTAAGAAAAATTTAAATCCAGAATA 72240
ATATTTGTAATAGCTGTTAACTTTATCATTGTCAAAATAAAAAGGATAAGCTATTACAAA 72300
AGACAGCGGCAATTGAGGTAAAAGACTTCTAATTCCAGTTCCCCAGCTAAAAGCAAAACT 72360
ACTAAAAGGTCTAAACAAAGAATTTTCTTGCCCTTCTAAAGAATAGGAAGCAAAATCTAT 72420
AAAAAAAGCATCCCAAACTAAAATATTTTTTAACAAAGGAATAGATATCTGCACAGTATT 72480
TACAAAAGAACTGTAAATATTTTTCAAAATCCCCCAACCTCTAGCCTGCATAAAATTTTC 72540
ACTAAGAATTATGTGGTGATGGGGTTGAATTTCAATTTCAAAACCATTACCAAGAGGAGG 72600
TAATATATTTGAATAGACACTCCTTAAGGTCAAAATAATATCAAAATAAGGAGTAAACAC 72660
ATCCTCATATCCCAAAAGAGAAAAATATCTCTCAAAAGTTGTAGAAGATTTAATAAAATG 72720
GCTCTGACCAAATAAAAATCCACCAAAAAAATCAAACTGTTGCTTAAGTAAAAATCCATT 72780
ATTAGATAAAGAGGTAGAATTTCTTGTATCCCAAGCCGCGCTCAAACTAAGAGAATTTTC 72840
AAATCTAAAAGTTTTATAATTGTCTCTTAAATAATAATTTGAAGGTCTGTTAACCTCATT 72900
ATCATAAAAAACATATTTTAAAGCAGTTTGCAAAGTGCCAAGAAGGGTTTGTTTGCCAAG 72960
ATAATTAGAAAAAGTATACCCGGTAAACGCTCCAAAACTAAGTTTaAGCAAAGAATAATT 73020
CATAGCATTAAAATCGGAAAAGCTTTTAGCATCTCGATATTCTTCCCAACTTGTAAATGG 73080
ATCAGGAACTTCCCTCTTGCCAGAAAA.AATAGGCCCATTAATATCCTGATAAGCAGTATT 73140
AACGGAATGTGAAAAATCTATAAATCCACCTACGGTCCATCTTTTTTGAAAAAACCAATT 73200
ATCTCTAAATGTCAAACTAAGACTTTGCTCTAAAAAAGATAAATTTAGTCTTGCTGCAAA 73260
ATAATAGCCTTCGCCTAAAAAATTAGAAAGCTCCCACTGCCCAAATACTGAGAATGGAAA 73320
TGAAGAATTTGAATTGCCTCCAAAATTCATACCAAATCCAAAATTACTTGTTGCTCGCTC 73380
CTCAATGTTTAAATTTATTTTCATAAGCCCTTCTGTATTGCCTGGAACAATATCAGGAAT 73440
TACATTTGAAAAATAACCAAGCTGCTGTAAATTTGCCATACCCATCTTAAACTTGTCCAA 73500
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
199
ACTAAAAACA TCTCCCTCTTGAAGAGGAATCTCTCTAAGTATTACATGCGAAGCTGTATT 73560
TTTATTTTTA GAAACAGTAATAGACTCAATATGAGCTTTATCCTTTTCTAAAATTTTAAT 73620
TAACAAATCA ACAAATTCCCCTCTTA'PCTTTTGCGAAGGAATAATTTCTGTAAAAATATA 73680
CCCTTCTCTA AAATAACTTTCCTTAATTTTGACAAAATCCTGCTCAAATTTAGAATCATT 73740
AAAAATATCA CCTTCGCTAAAGGTAATAAAACTTTTTAATTCTTCCAAACTAAAAACTGA 73800
ATTACCAGAA ATTTCAAGCTTTCCAAATCTAAAAACATTGCCTTCTGAAAGAAAATATTT 73860
CAAAAAAACT TCCTTTTCTAGTCTTTTAGAATCTTTAAGGGAATCTTTAATATCAACAGT 73920
GCTATTGATA ATCTTAACATCAATATATCCATTATTTTTATAAAAAGACTCTAATTGACG 73980
CTTGTCTTTA TCAACATTACTTTTTAAATATTTACCATCTGAGAAAAGAGACACTACTCT 74040
TGATGCTAAA GATTTCCTCAAGGTACTGCTTTTAAAGCTTAAATTTCCTTCAAAGTCAAT 74100
CCCCTTAACA ACATATTTGGGTCCAGCTACTATATTAAAAATAATATCAACTAAATTTCC 74160
TTCTTCTTTG ATTTCAAAATTTGCAGAAACCTCAAGATATCCCATGTCTTTATACATCTC 74220
TTCAAGCTTG CCAATACCTTTATTAACACTTGCAAGATTTAAAGGCTCATTGGTTTTAAT 74280
ATTCACCTTC TCAACAAGTTCGCTATTCCAAAAAACTCTACTGCTATCAGAAAAA.ACAAC74340
AGAATTAACT AAAGATTTTTCTTTTACAATAAATGTAATAAAAAGATCCTCACCATCTAT 74400
TTTAAATATA GGCTTAATAAGCCCAGAAAAATAATCAAGAGAATAAAGATCAATTTGCAA 74460
TTTATCAAAA ATTTCATTAGAATATGACACGCCAATGTAAGGTTTTAAAATATTAATAAA 74520
ATCTCTCTCC TTCTTATTCTTAAGTCCTTCAAAATTAATACCCTTTATTATTTTCCCCTT 74580
GTAATTTTCA ACTTGACCAAAACTAAAAACAACAAAAAATATTAAAAAACTTACAAAAAA 74640
CAAACCTCTA ATTGAACCCATCTTAACCTCTTAACAATTTAATATTTAAATTTCCAAGAA 74700
ATGCCTATAT TATTTCCTATTCCATCTAAACCTTTTTTCATAAAATTGTAATCAAACTCA 74760
TAATTAACCA AAA.AAAATGGAGAATCAAACTCAATACCCaAATTGACAACaAAATTTAAA 74820
TCTTTTGAAA AAGGAGACATTTGTTCTTTCAAA.AAACCAAAGCCCCCACTAATAAAAACA 74880
CCCTCAACAA GATATTTGCCTACCTTAACACTTGTATTGTCAAGAACATCAACAAAAGTA 74940
GGATTCCCGA TTTTGAAAAAATTACTATTAATAGAATTTTTCAATATATCTGTCTTTATA 75000
CTCAACAAGT CTAAGTTTAATACAGAACGCATATAATCTTCAATGGGTTGAATTAAAAAA 75060
TCAAGAGCAA TGTCACTTACTATTCCAATTGCCATTTCAGCAGCATTAGTCCCTGCCGAT 75120
CGCAATCCTC CCTCATACCCTCCTATTGTTGAGCCTGAAAGCAAATATTTAATTTCCTGC 75180
TCATTTCTAG AAGGATAAGACATAAACTCAATTTTCCATAAACTTAAAGGACTATCAATG 75240
CTTATTGTAA CAAGCAGTTTATCATTTCTATCCTT'AATAGTATTTGTAGCCTCCGCTTTT 75300
CA 02304925 1999-12-17
WO 98/58943 PCT/US98/12764
200
ATCCATGGATCAAATTTAGCCCTACTCTCATTAAAGGATATATAAGAGCCGCTTTTAAAA 75360
ATAAATTTTTTATTATTATAATTAACAAAACCACTTGCAATATTCAAATCTCCCTTAATA 75420
ATAAAATCATCTGTTTTTGTGTCAGATTTTATTACAAGTTTATCCCCTCTTGAAATAGTA 75480
GCTTGTAAAAAAGAAATATTACTATCTGGCCAAtGAAAAGTAACACCGCTGTCAAAATTT 75540
ATCTCAAGATCAGTTAAAATATCAAAATCTAGAAGATTAATATCAGTTTGCAATCTTTTT 75600
GCTCGTTTGAAAGGATTTATTAATAAATCAACAACAGAACTTTCAAGAGAATAAACCCAA 75660
GCATTTGAAATATTTAAAATGCCTTTAAACATAATTTCATCAGCATTTCCTTCAATTGAA 75720
AAATCGCCTAAAGCATAGCCTGTAAAGCTTAAGGCAATTTTTTCAAATTTAATAGGAACT 75780
CCCGTTCTACCAGTCACATTAATATCTATTTTGTAATAATCAATAATAGTATCACTTAAA 75840
AAATTTAAATTTAAACTAGTAGAAACAAAAATCTTGGAATATCGATCTAGGTTAAACTCA 75900
TTACTAAAAATGATTTTATTATCCTGAATTGCAACTGGCATATCAAATATTTCTAAAGCT 75960
CTATCACCACCAAATTTTCTAGAAGCTCTTAAATACTCAGTGCTAATTGATCCTTTTTGA 76020
ATATTTAAACTTCCATTAATATTAGGATTATACAAATCCCCATCAATATCGAATTCACCA 76080
TTTAAAACAAGATCGTAAAGAATAAAATGATTGTCAACATTAAATAAAGAATGTGAATCC 76140
AAAAAATCTTTGGTGATTATTTTTGAATCAAATTTAATATCTCTCACATTTCCAAGAATT 76200
TTATTTTTAATTATTTTGCCTGACGAATTAAAACTAAGGGGCAAATAATCTTTTAAAATA 76260
AAGCTATATTCCCCACTGCCATAATGATATAAAACATTAACAAGGTCATAATCAATTGAA 76320
GCCATATTAAATTTTTCAAAATCATTGCTAAATTCAATCGTAAGATTGGATAAAGATTCA 76380
TTTTTATACTTAATGTCTCTAAAGAAAAATTCGCCCTCTGTTTTGGTTTTCAAAACTCCA 76440
AATTGCTCTTCAACTCCACTTTGGAAATTTCTAAAATTTGCATTAAACCTGTAAGAAAAC 76500
AAGTCGCTATTAAAAACTATATTAGATACCCCTATAGAACTGCTTAGGTCATATCTTAAG 76560
CTTCCGGTAAGAAACTCTTTTTTATTGCGCTTGGCTTTTATGTCATATATGTTAAGCTTA 76620
TTATCCAATAATCCTAAATTCATAGAAAACTGAACGGGAACGCCCAAAA.AAGTTAATTTA 76680
TmTGCCTCAAGATATCCACTCAAAGAATAATTTTTAAAATCATTCTTTTTAAAATTTAGC 76740
AAAAAATTACCATTAACCTTTCCCTCTAAAAGGGAGAAAGAATTAAAATTGTAAAAATCT 76800
AAGTCTTGAAATTTAAGCAAAAAATAACTTCCATATTCATCGGAATTAACCCCTAA.AAAA76860
TATCTCTCTGAATTTAAGCTAGAAAATAGATTCAAACTTCCATTAAAATTATCTTTTAAA 76920
TTAAATTGTCCATATCCTTGCAAATTTGAAAGTTTGTTTATAAGTTTAACATCAGAAATG 76980
TACAATTTATCATATTCATATAAGCCCTTAAAACCAAAGTTGAAATTATAGGCAGGTGTT 77040
DEMANDES OU BREVETS VOLUMtNEUX
LA pRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET
COMPREND PLUS D'UN TOME.
CECI EST LE TOME ~ DE
NOTE: Pour les tomes additionels, veuillez contacter !e Bureau canadien des
f revels
JUMBO APPLICATIONSIPATENTS .
THIS SECT10N OF THE APPL1CAT10NlPATENT CONTAINS MORE
THAN ONE VOLUME
THlS fS VOLUME OF -
__
' NOTE: For additional volumes-phase-contact the Canadian Patent Office . ~- ~
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