CA2304925A1 - Borrelia burgdorferi polynucleotides and sequences - Google Patents

Abstract

The present invention provides polynucleotide sequences of the genome of Borrelia Burgdorferi, polypeptide sequences encoded by the polynucleotide sequences, corresponding polynucleotides and polypeptides, vectors and hosts comprising the polynucleotides, and assays and other uses thereof. The present invention further provides polynucleotide and polypeptide sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use.

Description

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--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 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).

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 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 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 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 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 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.

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 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 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.

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 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, 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 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 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:1-155, 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.

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.

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 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, 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.

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 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.

WO 98!58943 PCT/US98/12764 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 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.

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 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 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 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 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).

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, 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 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.

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) 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 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 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.

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 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.

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 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 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 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, 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 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 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.

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 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

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:

( 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.

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.

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 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.

Thirty-two reactions are loaded per AB373 Scquencer each day, for a total of 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 WO 98/58943 PCT/US98/t2764 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.

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 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 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 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 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 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 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.

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 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.

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 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 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 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.

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SUBSTITUTE SHEET (RULE 26) TABLE 3.
Borrelia burgdorferi - Putative coding regions of novel proteins not similar to know proteins Contig ORF ID Start (nt)Stop (nt) ID

i 163 158277 158474 Borrelia burgdorferi - Putative coding regions of novel proteins not similar to know proteins 1 176 _ 171038 ~

Borrclia burgdorferi - Putative coding regions of novel proteins not similar to know proteins 1~ 581 552337 551513 Borrelia burgdorferi - Putative coding regions of novel proteins not similar to know proteins 1 590 ~~~ 561342561139 i 596 565758 566519 i 707 665979 666770 i 718 679155 678391 1 ~ 831 ~ 7882191 --. 788836 Borrelia burgdorferi - Putative coding regions of novel proteins not similar to know proteins l 832 788824 789615 0oOw0 v~ t~-~7-~n W O ~ --v0 N t~ O t1- M O

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M M M ~ ~ ~f' 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 7 5464 ~ 3869 Borrelia burgdorferi - Putative coding regions of novel proteins not similar to know proteins L. _ __ 8 5187 5699 Borcelia burgdorferi - Putative coding regions of novel proteins not similar to know proteins 11 ( 9 7248 7685 Borrelia burgdorfcri - Putative coding regions of novel proteins not similar to know proteins 11 13 _ 10154 Borrelia burgdortveri - Putative coding regions of novel proteins not similar to know proteins Borrelia burgdorferi - Putative coding regions of novel proteins not similar to know proteins 103 1 _ 301 2 (1) GENERAL INFORMATION:

(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:

(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:

AGGATCATTTTTAGCTATTAGCAGAGAAGTGTTTTT'PACCAAAGTTAGACATAATGAACT 2760 AATATT.TAATAAAAGGCAAAGCTATAAACACCATATACTTATTTTATTATTTTTTTCATT 5340 AAAAAATAGGGTATTCTTGGTGAATCGTTTTAAAAGGGGGTATAGTAAGCTAAP~AAACTC5460 AAA.AATAAAAATGATTTATTAAGAATTATTAGTAACTTATAAAAACTTTATAAGTTACAT 5700 AAATATGAAAAAA.AAAAATTTATCAATTTACATGATAATGCTAATAAGTTTATTATCATG 5880 WO 98/58943 PCT/US98/12?64 ltil AAA.AAGAGGC GATATTGAAAATCTTCATACTCAATTAAATAGTGGACTTAGCGAGAGAGC 6480 AGAGACGAGT AAATAAAAAAATATAT'PTTAAAGGCTAATAACTTAAAATCAAAGTCTTCT 6840 TCTTTTTTAT TAAAAATATGTTTACTAAACAGAGC.'TCAAAAATGACTATATTTAGTATCT 6960 AAATCGGCAA GAGGAGCCATTATTGGC'ATAGTGAGACTAGCATGTCCTGATGAAGATGGA 7740 CAAA.ACTTTT TTCAAACGACCTATTTCCGAAAATATATTTATTGGATTTAAATATTCTTC11220 AACACAA.AAA ACACTACTAGAAAGCTCTTTAATAAAAAATCCTTCTAATGTAGAATATCG11580 WO 98/58943 PCT/US98/12'I64 CTTTTATAGGAGCAA.AAAAGTTCCAAGAAATATAAATTGTAAAATTATTTCTCCAAATAG14280 TAACAGGTTCTAGACCAATTTGGTCCATATACCC'PTTAAAATATTTAAAAGTCTTAGTAT14520 ATCTAAAAGGAACTTCAAACCCAAATACATCTCCTTTGTTAAGATTGGAAAAACTAAAA.A16320 TAACATAATCTTTATTAACACTGGGTGCTAAATTTTCTGCAAA.AAAATAAGTTAAATATC 16560 GTCAAATTTA AGCTTATCAATAAGCT'PTTTTTTAGACAATCTCTCACCCGAAATAATTTT20580 TTCAAAATCA TAGCCAACATTTTTAT'PTTTATTATTAAGTTCCTTTTCTAAAATATTTTG20640 TTGACGTCTT CTATTAGTCTCCTCAA'rTGCCTCCC'.GCATAGCTAAACTAATTTTGTCGTA20760 AGTAGTAGAT CTTAAAAATCCCACCT'rATCAGCATCTAATATTGCAACAAGAGATACTTC20880 AGTAGTAATT AAAACCCGCTCTTTAAGAGCCACTC'.TTTTTTGAATTTCGCTGTAAAGATC21120 TTCCATTTGC CCATCAGAGTGCCTAG'PAATAATTTCAGGATCAACAAGACCTGTTGGACG21180 AATTATTTGG TCAACAACCACACTACTTTTCTCA'I'TCTCTTCAACACCCGGGGTTGCAGA21240 AATTACAAAA TAAGATTTAGCAA.AAAGAGTAAAACTATTTGTAGCTCCTAAATTTTTTTT21720 TTTATCTCTT TCTAGATTCATTAAAG"_'TCTCTCATAATAAAGCTCTACAAAAATATCTGA219OO

TCAGAAATTA AAGAATATAAATACCCCATAAACGCAATAAAATT~_'ATACTTGAAAACGAA24120 AAA.AACAGCA ATAGTTGAAGGCCTTGCATCAAGCATAGTGCF,AAAAAAAATAAGTAGCAA24960 TAGAGGCGAG TTTGAAGATCGTTTAAATAATATAATTAAGTATATTGAAAAAAACAAAA.A25080 ATTCCAAACA ATTACCGTAAAAGAGCCTGATGAAAAAGATaCACTAA.AAATAATCGAAAA25320 WO 98/58943 PCT/US98/127b4 AAATATCCTTACaAAGGAAAATGTAGAAGAAATTTGCAAAAACTACTTAAACACCCTTAA 26280 ATTGCTCCTT TTGGAGAAGAAGACTACAGAATAC'PCAAAAAACACACAAATGTCGATATA27540 ATTTGGATAT GCTCAAAAACAGGAAAAAAAATTTGCATTGGATCAA.AAAAAGAGCTTGAA27960 ACTCTTACAA TCCTGGGAACTGCTCT'rTTTGAAAACACAGCATTCAAAAACGTTATTGTA28260 GAAAAAGAGA

ACAAGAGAATTTGTAAGGCAAATACAAAATTTAAGAAAAGAAAAAA.ATTTTGATGTTAGC 29460 GF~AAAAAAAATAAACCTTGCCGATGACATATTTACACTAATAGGAATTGAAAAATGTTAA 29640 AATCAGTTCTTGGGCTTATAAGCAATTTATACTTTAGCTATAAAAA.AGAAAATAACGATT 29880 WO 98/58943 PCT/iJS98/12764 AGAATTAATA ATATTAACTAAAATA'PTTACTTGTTCGTCAGAAGTAGCACCTGTGCTTAT 31320 GCCAATATCA ATCGCTAAATAATGT'rTCATATTTATCCTCAAGGCCTAATTGTACATAAA 31440 TGATCATAAA TTTTCTATAGCAATA'PAAGAAATTTTAGAAATCTTGTTTATAACTATTTG 31500 ATTTAAAAGC TTGATTTTATACTTT'PCAATATCTCTGGATAAAAATTCAAGCTTAGAACT 32100 TTCCAAAGAA GAATAATCATTAAAA(_'TTAAATTTAAATTAAGTTTTAACAATAAATCCTT 32460 AAACTATTTG CTCATCAGAATCATATTCAGTTAT'PAAAAAAAAATTAAACCGTGAGAGAA 35220 ATTTCTATAG ATTATAACAATGGATTTAATCCAATTTACATTATTCCCAGCAATAAAA.AA35820 AAAGATAAAATAACAAAAATAATAA.AAA.ATAAATATGGGAAAGAGTATATAGAGGAAAAA 36660 ATAGCAATAGGAAAACTAGATAAAATAAAATATCTAAGTAAATTTTATAATGGCAA.AAAA37320 GGAAAACCTAAAAAATTAAAAATAATAAAAGCAAAAAA.AGCATCAACTAAAAAAATTGAC 37740 AAAAAATATAAAGAAGCTAATGATTTTTTAAAAAAAATAAACCP~AAAAAAGATCAAAAAT40980 CAA.AAAATAAAAAACGAAATCATTTCGCTAAAATTAAGAATAAATGAAGATAATATTAAT41040 ATGTTCACTA TCCTCATCAATTATCAAAGCTCCTTTTAATGATAAA.AAATCGCCCAAACT45360 ATTTAAAA.AA ACATTGCCTCTGCTAATATATTTTGAAATCAAATTTCTTATATCAAGATC45540 ACAACAGATCGAATATAATTTTTCTTCCTAGCAAAGAACCATCCGCTTTTP,AAAAAAACT47460 GATGGAAAA.A CGGGGTGAAAAAACCAAATATTTAAACCAAAGAATAAAATGGACAAATAA49080 ACTTCTATAA GTTTGGCACCACCTATTAATTGGGCAGAAATCAAAAACATTGF,AAAAAAA49980 ATGTATCAAG TTTTACTAATTACTAAAATAAAAAAACAAACCAAAAAAA.AATTATACTGG50400 ATTATAAAAATAATATTGAATGAATTTAATCAAAAAGACTACCTAAAAAATAAAA.AAATA51180 ACAGGCTTAT TTTCTGTAATTTCGGGC'.AACAAAGGCGGACTAGGAAGAGCTGGAGAATTA53160 GGAA.AAAACC AAACATTGAATAAAAAAGCAGACCTAAAAGAAAACATTTCAAAACACTCG53700 AATCAGACCA AATAAACATTAAAAAAGACACTGGAA'rAAAAATTAACAATAAAATTTTAA53820 CA 02304925 1999-12-17 , TAAAACCTTT AAAATGTTGG
CTTCCAGGCA
AAACCAATCA

TTACAAAGCCCATTATTCTTGAAAAA.AAAGTGGAAATCATGACCAAAATTGTAGAAACAA 54720 GAAAACACCCGAAATCCGCCCGAATCGGTTAAAAA.ATTTTTATTCCAAATTGTAAAATTA 55620 AGGAAGATTT

TCCCAAATAA TTATATATATTATTTCCTAACAATCC.'TATAAACAAAAAAATTGATAATCA55920 AAA.AAAATAT AGTTGTAAAAGAACTTGCTATATATATATGCAAATAGCCTAGATCCGCTA559$0 TGAAAAGCAG GTTCATGGACAAAAAAGTAAGAATCiATGCTTTTTATAAAAAGCTTATCTA56940 GGAATATTTT CTATTAGAATACATTCCCATTGAAAAAGCTAAAA.AAATAAAAAATAAAAC57180 GTTACTATTT

AGCTATTAAT CTTTTTGACAGTTCCTTCATAAATTTC'.GCCTACCTTTGGCTCTCTTACAA 60060 ' GGCGTTTATC GCCATCAATATCATCCA'rTATTTGTTCATCAATGCTTGTACCAAGAGTAG 60840 TATTTCGCAG CAAAGTTATGGCTTTATC'.TCTATTAAGCTTTCCCTTAACAAAACAAGCTT 61140 WO 98/58943 PCT/US98/i2764 TTGCTGAAGC AAAGCTAATATCTGTT'PCGGTTATTACCCCTGCTGATGAATGCACAACTC64140 w CCCCGGCTTG AGGCATTGAAGAAAATCCTAAAACACTAATGGCTTTAGCGGGTCCAACGC64440 TTCAAATTGC TCCTCCTCAACAACAT(:TTGCATAACTTTATCAAACATTTCGAGTGTTTC 66660 AGCTTGGCTT TCCATAAAATGTGGTGGCACTTGAC~CATAATTACTCTTTGTAACTTCAAA 70080 AAATAACCCC CTATTATTAGTTTTTC'rAATCTTTAGGTTATTAATCTTGCTAAGCAATCT 70140 ATCAAGCTTT AAGTCATAACCTCTTT'PAATAAGTTCATCAGGTGCACTTGAAATTGCACT 70260 ATCAAAATTA TGCTTGTCAAATAACTTTTTTACCCiTAAAAAATACAGAAAGAGCTTTTTC 70380 P.AA.AAATTCA ACATGATCTAGCCTGGTATTAATCTCAGAAATATTTAAAATTGGATTTAA 70560 TATTCCAAGA CTGGAAGTAGATAAATC'TATATAAGAAAACGAATAATAATCTTTATAATC 71100 CAGTGCTATT GTGGTAAAGAATATATGGATTATTTTTTTTCATAATCAAAGAAA.ACCCAT71760 ATAATTAGAAAAAGTATACCCGGTAAACGCTCCAAAACTAAGTTTaAGCAAAGAATAATT 73020 ATCAGGAACTTCCCTCTTGCCAGAAAA.AATAGGCCCATTAATATCCTGATAAGCAGTATT 73140 TAACAAATCA ACAAATTCCCCTCTTA'PCTTTTGCGAAGGAATAATTTCTGTAAAAATATA 73680 ATTCACCTTC TCAACAAGTTCGCTATTCCAAAAAACTCTACTGCTATCAGAAAAA.ACAAC74340 TAATTAACCA AAA.AAAATGGAGAATCAAACTCAATACCCaAATTGACAACaAAATTTAAA 74820 TCTTTTGAAA AAGGAGACATTTGTTCTTTCAAA.AAACCAAAGCCCCCACTAATAAAAACA 74880 CTTATTGTAA CAAGCAGTTTATCATTTCTATCCTT'AATAGTATTTGTAGCCTCCGCTTTT 75300 GCTTGTAAAAAAGAAATATTACTATCTGGCCAAtGAAAAGTAACACCGCTGTCAAAATTT 75540 TTATCCAATAATCCTAAATTCATAGAAAACTGAACGGGAACGCCCAAAA.AAGTTAATTTA 76680 TmTGCCTCAAGATATCCACTCAAAGAATAATTTTTAAAATCATTCTTTTTAAAATTTAGC 76740 AAGTCTTGAAATTTAAGCAAAAAATAACTTCCATATTCATCGGAATTAACCCCTAA.AAAA76860 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 . ~- ~
--

Claims (21)

What Is Claimed Is:

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of:
(a) any one nucleotide sequence of SEQ ID NOS:1-155; or (b) a nucleotide sequence complementary to any one of the nucleotide sequences in (a).
(c) a nucleotide sequence at least 95% identical to any one of the nucleotide sequences of SEQ ID NOS:1-155; or, (d) a nucleotide sequence at least 95% identical to a nucleotide sequence complementary to any one of the nucleotide sequences of SEQ ID NOS:1-155.

2. An isolated nucleic acid molecule of claim 1 comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a) or (b) of claim 1.

3. An isolated nucleic acid molecule of claim 1 comprising a polynucleotide which encodes an epitope-bearing portion of a polypeptide in (a) of claim 1.

4. Computer readable medium having recorded thereon the nucleotide sequence depicted in SEQ
ID NOS: 1-155, a representative fragment thereof or a nucleotide sequence at least 95% identical to a nucleotide sequence depicted in SEQ ID NOS: 1-155.

5. A method for making a recombinant vector comprising the step of inserting an isolated nucleic acid molecule of claim 1 into a vector.

6. A recombinant vector produced by the method of claim 5.

7. A host cell comprising the vector of claim 6.

8. A method of producing a polypeptide comprising:
(a) growing the host cell of claim 7 such that the protein is expressed by the cell; and (h) recovering the expressed polypeptide.

9. An isolated polypeptide comprising a polypeptide selected from the group consisting of:
(a) a polypeptide encoded by an ORF of any one sequence of SEQ ID NOS:1-155;
(b) a polypeptide encoded by an ORF of any one sequence of SEQ ID NOS: 1-155 except the N-terminal residue;

(c) a fragment of the polypeptide of (a) having biological activity; and (d) an epitope-bearing fragment of the polypeptide of (a).

10. An isolated antibody specific for the polypeptide of claim 9.

11. A polypeptide produced according to the method of claim 8.

12. An isolated polypeptide comprising an amino acid sequence at least 95%
identical to a sequence selected from the group consisting of an amino acid sequence of any one of the polypeptides in Table 1.

13. An isolated polypeptide antigen comprising an amino acid sequence of an B.
burgdorferi epitope shown in Table 4.

14. An isolated nucleic acid molecule comprising a polynucleotide with a nucleotide sequence encoding a polypeptide of claim 9.

15. A host cell which produces an antibody of claim 10.

16. A vaccine, comprising:
(1) one or more B. burgdorferi polypeptides selected from the group consisting of a polypeptide of claim 9; and (2) a pharmaceutically acceptable diluent, carrier, or excipient;
wherein said polypeptide is present, in an amount effective to elicit protective antibodies in an animal to a member of the Borrelia genus.

17. A method of preventing or attenuating an infection caused by a member of the Borrelia genus in an animal, comprising administering to said animal a polypeptide of claim 9, wherein said polypeptide is administered in an amount effective to prevent or attenuate said infection.

18. A method of detecting Borrelia nucleic acids in a biological sample comprising:
(a) contacting the sample with one or more nucleic acids of claim 1, under conditions such that hybridization occurs, and (b) detecting hybridization of said nucleic acids to the one or more Borrelia nucleic acid sequences present in the biological sample.

19. A method of detecting Borrelia nucleic acids in a biological sample obtained from an animal, comprising:

(a) amplifying one or more Borrelia nucleic acid sequences in said sample using polymerase chain reaction, and (b) detecting said amplified Borrelia nucleic acid.

20. A kit for detecting Borrelia antibodies in a biological sample obtained from an animal, comprising (a) a polypeptide of claim 9 attached to a solid support; and (b) detecting means.

21. A method of detecting Borrelia antibodies in a biological sample obtained from an animal, comprising (a) contacting the sample with a polypeptide of claim 9; and (b) detecting antibody-antigen complexes.